Volume 115, Issue 10; March 13, 2007,pp.1177-1332

Issue Highlights Issue Highlights Circulation 2007 115: 1177

Editorials Aging and Sinoatrial Node Dysfunction: Musings on the Not-So-Funny Side Haris M. Haqqani and Jonathan M. Kalman Circulation 2007 115: 1178 - 1179, doi:10.1161/CIRCULATIONAHA.106.685248 Therapeutic Gene Regulation: Targeting Transcription Frank J. Giordano Circulation 2007 115: 1180 - 1183, doi:10.1161/CIRCULATIONAHA.106.685255

Original Articles Arrhythmia/Electrophysiology Declining Into Failure: The Age-Dependent Loss of the L-Type Calcium Channel Within the Sinoatrial Node Sandra A. Jones, Mark R. Boyett, and Matthew K. Lancaster Circulation 2007 115: 1183 - 1190; published online before print March 5 2007, doi:10.1161/CIRCULATIONAHA.106.663070

Remote Magnetic Navigation to Guide Endocardial and Epicardial Catheter Mapping of Scar-Related Ventricular Tachycardia Arash Aryana, Andre d’Avila, E. Kevin Heist, Theofanie Mela, Jagmeet P. Singh, Jeremy N. Ruskin, and Vivek Y. Reddy Circulation 2007 115: 1191 - 1200; published online before print February 12 2007, doi:10.1161/CIRCULATIONAHA.106.672162

Cardiovascular Surgery Adjustable, Physiological Ventricular Restraint Improves Left Ventricular Mechanics and Reduces Dilatation in an Ovine Model of Chronic Heart Failure Ravi K. Ghanta, Aravind Rangaraj, Ramanan Umakanthan, Lawrence Lee, Rita G. Laurence, John A. Fox, R. Morton Bolman, III, Lawrence H. Cohn, and Frederick Y. Chen Circulation 2007 115: 1201 - 1210; published online before print March 5 2007, doi:10.1161/CIRCULATIONAHA.106.671370

Coronary Heart Disease Renal Insufficiency Following Contrast Media Administration Trial (REMEDIAL): A Randomized Comparison of 3 Preventive Strategies Carlo Briguori, Flavio Airoldi, Davide D’Andrea, Erminio Bonizzoni, Nuccia Morici, Amelia Focaccio, Iassen Michev, Matteo Montorfano, Mauro Carlino, John Cosgrave, Bruno Ricciardelli, and Antonio Colombo Circulation 2007 115: 1211 - 1217; published online before print February 19 2007, doi:10.1161/CIRCULATIONAHA.106.687152

Heart Failure Direct Myocardial Effects of Levosimendan in Humans With Left Ventricular Dysfunction: Alteration of Force-Frequency and Relaxation-Frequency Relationships Michael M. Givertz, Costa Andreou, Chester H. Conrad, and Wilson S. Colucci Circulation 2007 115: 1218 - 1224; published online before print March 5 2007, doi:10.1161/CIRCULATIONAHA.106.668640 Muscarinic Modulation of the Sodium-Calcium Exchanger in Heart Failure Shao-kui Wei, Abdul M. Ruknudin, Matie Shou, John M. McCurley, Stephen U. Hanlon, Eric Elgin, Dan H. Schulze, and Mark C.P. Haigney Circulation 2007 115: 1225 - 1233; published online before print March 5 2007, doi:10.1161/CIRCULATIONAHA.106.650416

Genetics Use of a Constitutively Active Hypoxia-Inducible Factor-1 Transgene as a Therapeutic Strategy in No-Option Critical Limb Ischemia Patients: Phase I DoseEscalation Experience Sanjay Rajagopalan, Jeffrey Olin, Steven Deitcher, Ann Pieczek, John Laird, P. Michael Grossman, Corey K. Goldman, Kevin McEllin, Ralph Kelly, and Nicolas Chronos Circulation 2007 115: 1234 - 1243; published online before print February 19 2007, doi:10.1161/CIRCULATIONAHA.106.607994 Prevalence of Desmin Mutations in Dilated Cardiomyopathy Matthew R.G. Taylor, Dobromir Slavov, Lisa Ku, Andrea Di Lenarda, Gianfranco Sinagra, Elisa Carniel, Kurt Haubold, Mark M. Boucek, Debra Ferguson, Sharon L. Graw, Xiao Zhu, Jean Cavanaugh, Carmen C. Sucharov, Carlin S. Long, Michael R. Bristow, Philip Lavori, Luisa Mestroni for the Familial Cardiomyopathy Registry and the BEST (Beta-Blocker Evaluation of Survival Trial) DNA Bank Circulation 2007 115: 1244 - 1251; published online before print February 26 2007, doi:10.1161/CIRCULATIONAHA.106.646778

Imaging Incremental Value of Strain Rate Imaging to Wall Motion Analysis for Prediction of Outcome in Patients Undergoing Dobutamine Stress Echocardiography Charlotte Bjork Ingul, Ellen Rozis, Stig A. Slordahl, and Thomas H. Marwick Circulation 2007 115: 1252 - 1259; published online before print February 26 2007, doi:10.1161/CIRCULATIONAHA.106.640334

Molecular Cardiology Moderate Pulmonary Arterial Hypertension in Male Mice Lacking the Vasoactive Intestinal Peptide Gene Sami I. Said, Sayyed A. Hamidi, Kathleen G. Dickman, Anthony M. Szema, Sergey Lyubsky, Richard Z. Lin, Ya-Ping Jiang, John J. Chen, James A. Waschek, and Smadar Kort Circulation 2007 115: 1260 - 1268; published online before print February 19 2007, doi:10.1161/CIRCULATIONAHA.106.681718 Identification of a Novel Polymorphism in the 3'UTR of the L-Arginine Transporter Gene SLC7A1: Contribution to Hypertension and Endothelial Dysfunction Zhiyong Yang, Kylie Venardos, Emma Jones, Brian J. Morris, Jaye Chin-Dusting, and David M. Kaye Circulation 2007 115: 1269 - 1274; published online before print February 26 2007, doi:10.1161/CIRCULATIONAHA.106.665836

Vascular Medicine Pulmonary Arterial Hypertension Is Linked to Insulin Resistance and Reversed by Peroxisome Proliferator–Activated Receptor- Activation Georg Hansmann, Roger A. Wagner, Stefan Schellong, Vinicio A. de Jesus Perez, Takashi Urashima, Lingli Wang, Ahmad Y. Sheikh, Renée S. Suen, Duncan J. Stewart, and Marlene Rabinovitch Circulation 2007 115: 1275 - 1284; published online before print March 5 2007, doi:10.1161/CIRCULATIONAHA.106.663120

Contemporary Reviews in Cardiovascular Medicine Endothelial Function and Dysfunction: Testing and Clinical Relevance John E. Deanfield, Julian P. Halcox, and Ton J. Rabelink Circulation 2007 115: 1285 - 1295, doi:10.1161/CIRCULATIONAHA.106.652859

Congenital Heart Disease for the Adult Cardiologist Coronary Artery Anomalies: An Entity in Search of an Identity Paolo Angelini Circulation 2007 115: 1296 - 1305, doi:10.1161/CIRCULATIONAHA.106.618082

AHA/ACC/HRS Scientific Statements Recommendations for the Standardization and Interpretation of the Electrocardiogram: Part I: The Electrocardiogram and Its Technology: A Scientific Statement From the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society Endorsed by the International Society for Computerized Electrocardiology Paul Kligfield, Leonard S. Gettes, James J. Bailey, Rory Childers, Barbara J. Deal, E. William Hancock, Gerard van Herpen, Jan A. Kors, Peter Macfarlane, David M. Mirvis, Olle Pahlm, Pentti Rautaharju, and Galen S. Wagner Circulation 2007 115: 1306 - 1324; published online before print February 23 2007, doi:10.1161/CIRCULATIONAHA.106.180200

Recommendations for the Standardization and Interpretation of the Electrocardiogram: Part II: Electrocardiography Diagnostic Statement List: A Scientific Statement From the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society: Endorsed by the International Society for Computerized Electrocardiology Jay W. Mason, E. William Hancock, and Leonard S. Gettes Circulation 2007 115: 1325 - 1332; published online before print February 23 2007, doi:10.1161/CIRCULATIONAHA.106.180201

Images in Cardiovascular Medicine Huge Pericardial Hemangioma Imaging Abdel-Rauf Zeina, Ghassan Zaid, Dawod Sharif, Uri Rosenschein, and Elisha Barmeir Circulation 2007 115: e315 - e317, doi:10.1161/CIRCULATIONAHA.106.647289 Cardiovocal Syndrome Associated With Huge Left Atrium Okan Gulel, Diyar Koprulu, Zafer Kucuksu, Mustafa Yazici, and Senem Cengel Circulation 2007 115: e318 - e319, doi:10.1161/CIRCULATIONAHA.106.649814 Infarction-Like Electrocardiographic Changes Due to a Myocardial Metastasis From a Primary Lung Cancer Panagiotis Samaras, Frank Stenner-Liewen, Stefan Bauer, Gerhard W. Goerres, Lotta von Boehmer, Nina Kotrubczik, Rolf Jenni, Christoph Renner, and Alexander Knuth Circulation 2007 115: e320 - e321, doi:10.1161/CIRCULATIONAHA.106.650762

Correspondence Letter by Pischon et al Regarding Article, "Adiponectin and Coronary Heart Disease: A Prospective Study and Meta-Analysis" Tobias Pischon, Matthias B. Schulze, and Eric B. Rimm Circulation 2007 115: e322, doi:10.1161/CIRCULATIONAHA.106.666677 Response to Letter Regarding Article, "Adiponectin and Coronary Heart Disease: A Prospective Study and Meta-Analysis" Naveed Sattar, Lynne Cherry, A. Michael Wallace, Goya Wannamethee, Julia Tchernova, Nadeem Sarwar, John Danesh, and Peter H. Whincup Circulation 2007 115: e323, doi:10.1161/CIRCULATIONAHA.106.671289

Corrections Correction Circulation 2007 115: e324, doi:10.1161/CIRCULATIONAHA.107.181837 Correction Circulation 2007 115: e325, doi:10.1161/CIRCULATIONAHA.107.181838

European Perspectives European Perspectives Circulation 2007 115: 37f - 42f

Circulation JOURNAL

OF THE

AMERICAN HEART ASSOCIATION

Issue Highlights Vol 115, No 10, March 13, 2007

REMOTE MAGNETIC NAVIGATION TO GUIDE ENDOCARDIAL AND EPICARDIAL CATHETER MAPPING OF SCAR-RELATED VENTRICULAR TACHYCARDIA, by Aryana et al. Catheter mapping of ventricular tachycardia is challenging because of tissue heterogeneity in diverse scar-related disease substrates as well as limitations in maneuverability and accessibility to critical regions of the ventricular endocardium and epicardium. In this issue of Circulation, Aryana and colleagues report their clinical results obtained by using a remote magnetic navigation system combined with electroanatomic mapping to identify areas of interest related to ventricular tachycardia, while using a limited amount of fluoroscopy exposure. Although the majority of epicardial and endocardial ventricular sites could be safely and effectively mapped remotely, because of current limitations of the magnetic navigation catheter, most patients required additional manual ablation using an irrigated catheter to ablate fully the ventricular tachycardia substrates. See p 1191.

RENAL INSUFFICIENCY FOLLOWING CONTRAST MEDIA ADMINISTRATION TRIAL (REMEDIAL): A RANDOMIZED COMPARISON OF 3 PREVENTIVE STRATEGIES, by Briguori et al. Exposure to radiocontrast media can lead to transient and permanent renal failure in high-risk patients such as those with preexisting moderate or severe renal failure. Current methods to prevent contrast-induced nephrotoxicity include volume expansion with saline and limitation of contrast volume. Other strategies have been proposed, including N-acetylcysteine (NAC), sodium bicarbonate infusion, and ascorbic acid. The study by Briguori et al was a 2-center, randomized, double-blind study of 3 strategies to prevent contrast-induced nephrotoxicity in 326 patients with moderate chronic renal disease undergoing coronary or peripheral angiography. Moderate renal failure was defined as a creatinine concentration of 2 to 8 mg/dL. Patients were randomized to saline plus NAC, sodium bicarbonate infusion plus NAC, or saline plus ascorbic acid plus NAC. Contrast-induced nephropathy defined as an increase in creatinine concentration by 25% occurred in 9.9%, 1.9%, and 10.3%, respectively. The sodium bicarbonate infusion plus NAC produced significantly better results than the other regimens (P⫽0.019). Other measures of contrast-induced nephrotoxicity showed similar results. In addition, similar results were seen in high-risk subgroups such as those with diabetes. These findings support the use of volume expansion with sodium bicarbonate plus NAC as a more effective strategy in preventing contrastinduced nephrotoxicity than saline plus NAC or saline plus ascorbic acid and NAC. See p 1211.

USE OF A CONSTITUTIVELY ACTIVE HYPOXIA-INDUCIBLE FACTOR-1␣ TRANSGENE AS A THERAPEUTIC STRATEGY IN NO-OPTION CRITICAL LIMB ISCHEMIA PATIENTS: PHASE I DOSE-ESCALATION EXPERIENCE, by Rajagopalan et al. Critical limb ischemia is a serious manifestation of peripheral atherosclerosis associated with limb loss in a considerable number of patients. Although surgery and percutaneous transluminal angioplasty may provide improvement in some patients, in others, amputation is the only option. Several systems in the body sense hypoxia, among them the hypoxia-inducible factor-1␣. Such ischemia-sensing systems activate protective pathways in the body to limit tissue damage. In the study by Rajagolpalan et al, therefore, the authors tested whether a constitutively active transcription factor of hypoxia-inducible factor-1␣ transfected with an adenovirus would be safe and potentially ameliorate symptoms and improve outcome in patients with critical limb ischemia and no other options. No serious adverse events of this treatment were noted, but peripheral edema disease progression and ischemia were still common. In approximately half of the hypoxiainducible factor-1␣ treated patients, however, complete pain resolution was noted at 1 year and ulcer healing in almost one third. Although very preliminary, this study suggests that this novel gene therapy approach in critical limb ischemia should be pursued further and may, if confirmed in larger studies, offer a treatment for those patients with no other options. See p 1234.

Visit http://circ.ahajournals.org: Images in Cardiovascular Medicine Huge Pericardial Hemangioma Imaging. See p e315.

Cardiovocal Syndrome Associated With Huge Left Atrium. See p e318. Infarction-Like Electrocardiographic Changes Due to a Myocardial Metastasis From a Primary Lung Cancer. See p e320.

Correspondence See p e322.

Editorial Aging and Sinoatrial Node Dysfunction Musings on the Not-So-Funny Side Haris M. Haqqani, MBBS; Jonathan M. Kalman, MBBS, PhD

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n the century since the discovery by Keith and Flack of the sinoatrial node in the mole heart, a detailed mosaic of its cellular, anatomic, and electrophysiological properties has emerged. The human sinus node has been found to be anatomically constant and well localized, occupying an approximately 10-mm subepicardial region on the sulcus terminalis at the superior cavo–atrial junction.1 Histologically, its ultrastructure of central P cells (likely corresponding to the leading pacemaker site) and outer transitional zone merging with surrounding atrial myocardium have been well characterized.1 Great progress also has been made in defining the ionic mechanisms responsible for the sinoatrial action potential and its spontaneous pacemaker activity, including important contributory roles for ICa,L, Ik, and the funny current, If.1 This morphologically discrete, unifocal sinus node is not the exclusive force behind clinical sinus rhythm, however. Detailed animal and human mapping has demonstrated that normal cardiac pacemaker activity is widely distributed in the right atrium. In the human atrium, the pacemaker complex extends for up to 75 mm along the long axis of the sulcus terminalis and precaval band.2 At times, even left atrial pacemakers may be active during normal sinus rhythm.2 Graduated differential sensitivity to adrenergic and vagal inputs exists along the integrated pacemaker complex such that superior sites tend to dominate during periods of sympathetic drive, whereas inferior sites are activated by increased parasympathetic tone. Increasing the complexity, each sinus beat may have multicentric origin, and the nature of conduction out of the node also seems to be variable in response to autonomic tone.2 The presence of a diffuse pacemaker complex in humans is supported not only by mapping studies but also by experience with catheter ablation of the sinus node. To achieve successful reduction of sinus rate, frequently extensive ablation along a large segment of the pacemaker complex is required.3 Furthermore, the difficulty in achieving successful long-term reduction in sinus rate after ablation may be viewed as a testament to the nonlocalized, redundant nature of the atrial pacemaker complex.3

related permanent pacing, and its prevalence is projected to increase as the population ages.5 It is important to note that the characterization of an extensive atrial pacemaker complex has changed our conception of the pathophysiology underlying and necessary for the development of sinus node dysfunction (SND). Sanders et al6 have demonstrated that patients with clinical SND do have evidence of diffuse atrial structural remodeling involving most regions of the right atrium. Many of these changes were particularly marked along the long axis of the crista terminalis, where extensive fractionated electrograms, double potentials, and slowed conduction were observed together with a marked reduction in voltage amplitude. This latter observation may be a marker for regional fibrosis, but histological corroboration was lacking. This structural remodeling was frequently associated with caudal migration of atrial pacemaker activity to the inferior region of the sulcus terminalis and a localized unicentric site of the earliest activity. These observations suggest that development of a diffuse atriopathy is necessary for clinical SND to be manifest. Indeed, similar but less marked atrial remodeling has been shown in association with human aging in patients without structural heart disease or atrial arrhythmias, who do not (yet) have clinical manifestations of SND.7 In that study, by Kistler et al,7 aging was associated with conduction slowing and voltage loss that, although diffuse, was particularly marked around the region of the crista terminalis, together with electrophysiological evidence of a decrease in sinus node reserve. The study by Kistler et al7 has demonstrated that subclinical loss of sinus node function in an otherwise healthy, aged population can be accompanied by widespread changes in the atrial electrical substrate. However, it is not understood why these manifestations are silent in the majority of people, whereas in others, they result in the development of clinical SND. The presence of other factors such as ischemia, cardiac failure, or other causes of atrial stretch and remodeling may play an important contributory role. Reduction in sinus node reserve has been shown to occur in congestive cardiac failure8 and in the presence of chronic right atrial stretch associated with an atrial septal defect.9 Both of these conditions also demonstrate the type of diffuse atrial structural remodeling described above. Furthermore, the diffuse atriopathy of aging provides a mechanism not only for SND but also for the commonly associated atrial arrhythmias and, in particular, atrial fibrillation, which will develop in up to 50% of patients with SND.10 In addition, the development of atrial fibrillation may further exacerbate SND. This effect seems to be secondary to high atrial rates,11 and at least partial reversal of this adverse sinus node remodeling may occur after successful catheter ablation of atrial fibrillation12 or atrial flutter.13

Article p 1183 Sick sinus syndrome, first described as a clinical entity almost 4 decades ago,4 is the most common indication for bradycardiaThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Department of Cardiology, Royal Melbourne Hospital, and The University of Melbourne, Melbourne, Australia. Correspondence to Jonathan M. Kalman, MBBS, PhD, FACC, Department of Cardiology, Royal Melbourne Hospital, Grattan St, Parkville, Victoria, Australia 3050. E-mail [email protected] (Circulation. 2007;115:1178-1179.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.685248

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Haqqani and Kalman Whereas clinical investigation of SND can only focus on the gross electrophysiological and structural changes detectable with mapping techniques, the study by Jones et al14 in this issue of Circulation provides an important insight into the molecular mechanisms underlying the senescent loss of sinoatrial node function. In a guinea pig sinus node model, the authors show progressive downregulation of sinus node ICa,L expression from an early age, commencing in the central zone at the leading pacemaker site. This process continued with increasing age until peripheral sinus node zones were also involved, ultimately reducing the depolarization reserve and excitability of the pacemaker complex. Previously, these investigators have also demonstrated increasing electrical disconnection of the guinea pig sinus node with aging, due to loss of connexin-43 expression in both the central and peripheral zones of the sinus node.15 The 2 studies together provide a mechanism both for age-related reduction in sinus automaticity and impaired sinus node conduction. Perhaps surprisingly, in the context of clinical mapping studies, the authors found no changes in collagen content or signs of fibrosis associated with aging in this guinea pig sinus node model. Similarly, Alings et al16 found that although the relative volume of collagen in the human sinoatrial node increases from childhood to adulthood, no further increase occurred once adulthood had been reached. Other studies have shown that in humans, aging is associated with increasing conduction anisotropy in the atrium that, histologically, was associated with increasing collagen.17 The development of generalized atrial fibrosis does not necessarily implicate the sinus node region, and further studies in humans are necessary to settle this issue. Although the study by Jones et al14 does not address downregulation of ICa,L at other atrial sites, this has been documented in a senescent cellular canine model.18 Such global reduction in Cav1.2 suggests, again, a generalized process of senescent electrical and structural remodeling, of which SND and atrial fibrillation are the 2 most visible clinical manifestations. Interestingly, Jones et al14 describe a negative chronotropic effect of nifedipine on the leading pacemaker site; this was more marked in the aged sinus nodes. This result is concordant with previous reports of the effects of topical nifedipine in isolated sinoatrial node preparations, underscoring the importance of the ICa,L in action potential upstroke in this zone of the sinus node. The clinical implications of this observation are unclear, however. One clinical report found no adverse effect on sinus node function with clinical doses of nifedipine given to patients with SND after pharmacological autonomic blockade.19 Similar data exist for the closely related dihydropyridine Cav1.2 antagonist amlodipine.20 The study by Jones et al14 adds greatly to our current conceptualization of SND. It increasingly seems that, apart from their clinical association, SND, atrial fibrillation, and aging are fundamentally connected at a mechanistic level. Changes in sinoatrial ion channel expression and development of widespread atrial structural remodeling may both play roles in the development of these common conditions in the aging heart.

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Disclosures Professor Kalman is supported by research grants from St Jude Medical and Medtronic, Inc. H. Haqqani reports no conflicts.

References 1. Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc Res. 2000;47:658 – 687. 2. Boineau JP, Canavan TE, Schuessler RB, Cain ME, Corr PB, Cox JL. Demonstration of a widely distributed atrial pacemaker complex in the human heart. Circulation. 1988;77:1221–1237. 3. Man KC, Knight B, Tse HF, Pelosi F, Michaud GF, Flemming M, Strickberger SA, Morady F. Radiofrequency catheter ablation of inappropriate sinus tachycardia guided by activation mapping. J Am Coll Cardiol. 2000;35:451– 457. 4. Ferrer MI. The sick sinus syndrome in atrial disease. JAMA. 1968;206: 645– 646. 5. Kusumoto FM, Goldschlager N. Cardiac pacing. N Engl J Med. 1996;334: 89 –97. 6. Sanders P, Morton JB, Kistler PM, Spence SJ, Davidson NC, Hussin A, Vohra JK, Sparks PB, Kalman JM. Electrophysiological and electroanatomic characterization of the atria in sinus node disease: evidence of diffuse atrial remodeling. Circulation. 2004;109:1514 –1522. 7. Kistler PM, Sanders P, Fynn SP, Stevenson IH, Spence SJ, Vohra JK, Sparks PB, Kalman JM. Electrophysiologic and electroanatomic changes in the human atrium associated with age. J Am Coll Cardiol. 2004;44:109 –116. 8. Sanders P, Kistler PM, Morton JB, Spence SJ, Kalman JM. Remodeling of sinus node function in patients with congestive heart failure: reduction in sinus node reserve. Circulation. 2004;110:897–903. 9. Morton JB, Sanders P, Vohra JK, Sparks PB, Morgan JG, Spence SJ, Grigg LE, Kalman JM. Effect of chronic right atrial stretch on atrial electrical remodeling in patients with an atrial septal defect. Circulation. 2003;107: 1775–1782. 10. Lamas GA, Lee K, Sweeney M, Leon A, Yee R, Ellenbogen K, Greer S, Wilber D, Silverman R, Marinchak R, Bernstein R, Mittleman RS, Lieberman EH, Sullivan C, Zorn L, Flaker G, Schron E, Orav EJ, Goldman L. The mode selection trial (MOST) in sinus node dysfunction: design, rationale, and baseline characteristics of the first 1000 patients. Am Heart J. 2000;140:541–551. 11. Hadian D, Zipes DP, Olgin JE, Miller JM. Short-term rapid atrial pacing produces electrical remodeling of sinus node function in humans. J Cardiovasc Electrophysiol. 2002;13:584 –586. 12. Hocini M, Sanders P, Deisenhofer I, Jais P, Hsu LF, Scavee C, Weerasoriya R, Raybaud F, Macle L, Shah DC, Garrigue S, Le Metayer P, Clementy J, Haissaguerre M. Reverse remodeling of sinus node function after catheter ablation of atrial fibrillation in patients with prolonged sinus pauses. Circulation. 2003;108:1172–1175. 13. Sparks PB, Jayaprakash S, Vohra JK, Kalman JM. Electrical remodeling of the atria associated with paroxysmal and chronic atrial flutter. Circulation 2000;102:1807–1813. 14. Jones SA, Boyett MR, Lancaster MK. Declining into failure: the agedependent loss of the L-type calcium channel within the sinoatrial node. Circulation. 2007;115:1183–1190. 15. Jones SA, Lancaster MK, Boyett MR. Ageing-related changes of connexins and conduction within the sinoatrial node. J Physiol. 2004;560:429 – 437. 16. Alings AM, Abbas RF, Bouman LN. Age-related changes in structure and relative collagen content of the human and feline sinoatrial node: a comparative study. Eur Heart J. 1995;16:1655–1667. 17. Spach MS, Dolber PC. Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopic level in human cardiac muscle: evidence for electrical uncoupling of side-to-side fiber connections with increasing age. Circ Res. 1986;58:356 –371. 18. Dun W, Yagi T, Rosen MR, Boyden PA. Calcium and potassium currents in cells from adult and aged canine right atria. Cardiovasc Res. 2003;58: 526 –534. 19. Kouvaras G, Chronopoulos G, Nikolaou P, Sofronas G, Cokkinos D. Effect of nifedipine on the sick sinus syndrome. Angiology. 1989;40:450 – 457. 20. Vetrovec GW, Dailey S, Kay G, Epstein A, Plumb V. Haemodynamic and electrophysiological effects of amlodipine, a new long-acting calcium antagonist. Postgrad Med J. 1991;67(suppl 5):S60 –S61. KEY WORDS: Editorials 䡲 aging 䡲 arrhythmia 䡲 ion channels 䡲 remodeling 䡲 sinoatrial node 䡲 sick sinus syndrome

Editorial Therapeutic Gene Regulation Targeting Transcription Frank J. Giordano, MD

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alternative splicing—Vegf121, 165, 189, and 205—and additional minor splice variants also have been described. At least 2 of the major Vegf-A splice variants, Vegf121 and Vegf165, have been tested clinically as single agents in gene therapy trials for human peripheral arterial disease and coronary artery disease. It is now clear that the alternative Vegf-A splice variants are not biologically equivalent or redundant.5,6 Furthermore, blood vessels induced in response to single Vegf splice variants seem to be more permeable, and perhaps less mature, than those induced by activating the native Vegf-A gene with consequent expression of multiple Vegf splice variants.7,8 The clinical relevance of these differences is not yet clear, but they do illustrate how the determination of biological complexity does not stop at the primary DNA sequence. In the study reported by Rajapogolan et al1 in this issue, the authors injected an adenovirus that encodes a stable and active form of the hypoxia-inducible transcription factor HIF-1␣ into the skeletal muscles of patients with severe peripheral arterial disease, in an effort to stimulate the growth of new blood vessels. HIF-1␣ is a basic helix-loop-helix transcription factor that regulates the expression of a wide repertoire of genes in response to decreased oxygen tension. When oxygen levels within a tissue or cell decrease, HIF-1␣ levels increase. HIF-1␣, as a heterodimer with HIF-1␤ (aryl hydrocarbon nuclear transferase), then binds to specific sequences within the regulatory regions of these genes and alters their transcription.9 One of the genes regulated by HIF-1␣ is the Vegf-A gene. Thus, an expected, desired effect of this HIF-1␣ gene therapy approach is activation of the endogenous Vegf-A gene and consequent expression of all the major Vegf-A splice variants. This represents one potential advantage of this transcriptional approach to therapeutic angiogenesis. Another important potential advantage of using this transcriptional approach is that it takes advantage of a biological pathway that has evolved naturally as a mechanism whereby tissues can adapt to decreased oxygen availability, including the ability of those tissues to grow new blood vessels. HIF-1␣ can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in adaptive responses to hypoxia, including a significant number of genes involved in regulating vascular growth and reactivity. It is likely that the full complement of angiogenesis-associated genes regulated by HIF is not yet known, and thus “flipping the HIF switch,” as in the approach by Rajagopolan and colleagues, may induce the expression of factors that have not yet been identified as contributors to the processes of angiogenesis and vascular remodeling. Thus, using this type of approach may unleash the therapeutic power of genes and

n this issue of Circulation, Rajagopolan et al1 report the first clinical results of a gene therapy approach predicated on transcriptional activation of a patient’s own genes. This represents a second-generation gene therapy methodology for cardiovascular disease and is important for a number of reasons. We have witnessed in the past decade the primary sequencing of the human genome.2,3 One of the initial reactions to this milestone accomplishment was surprise at the relatively small number of definitive genes that are encoded by human DNA. Although the exact number is still uncertain, estimates as low as 23 299 have been made. In comparison, the genome of the worm Caenorhabditis elegans encodes approximately 19 000 genes, and the genome of the common fruit fly encodes approximately 18 000 genes, raising the question of how such significant differences in biological complexity and diversity are engendered by so few genes. A complete set of answers to this question is not currently in our grasp, but some crucial aspects are understood and are relevant to the clinical trial discussed here.

Article p 1234 One manner in which the biological effect of a finite number of genes is amplified is by alternative splicing. This is the process whereby a single gene encodes a number of alternative proteins by simply including or excluding specific exons within the coding sequence of that gene during mRNA transcription and maturation. The number of alternative splice variants that exist as transcriptional products of the human genome is not known, but this process alone likely increases the number of unique proteins that can be encoded by the genome 2- to 3-fold, possibly more. Interestingly, there are known human cardiovascular diseases caused by inappropriate splicing, including a cardiomyopathy and sudden death syndrome in children caused by deficient production of wild-type very-long-chain acyl-CoA dehydrogenase, the result of exclusion of an exon attributable to abnormal splicing.4 Relevant to the study by Rajagopolan et al are the alternative splice variants of the vascular endothelial growth factor gene (Vegf-A). There are 4 major alternative Vegf-A proteins encoded by the same Vegf-A gene because of The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, Conn. Correspondence to Frank J. Giordano, MD, Cardiovascular Medicine, Yale University School of Medicine, 333 Cedar St, 3FMP, New Haven, CT 06520. E-mail [email protected] (Circulation. 2007;115:1180-1182.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.685255

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Giordano biological pathways we do not yet understand, without a requisite requirement for knowing how it all fits together and works. The potential disadvantage of this approach is that HIF-1␣ has also evolved to regulate the expression of genes involved in other biological processes associated with adaptation to hypoxia, including genes that control cellular metabolism, glucose uptake, erythropoiesis, apoptosis, the cell cycle, and other processes. This biological permissiveness may theoretically lead to undesired clinical effects. Thus, the fact that HIF-1␣ gene therapy seems safe at all dose ranges tested is quite comforting and represents one of the major contributions of the study conducted by Rajagopolan and colleagues. In a broader, more general sense, the HIF-1␣ clinical study by Rajagopalan et al1 highlights the importance of gene regulation, which is likely as significant to human health as are the gene mutations that have been the focus of human genetics to data and that are now the subject of intense, high-throughput, genome-wide searches. Genes are essentially our cellular instruction set for how and when to build specific proteins. How those instructions are read is of crucial biological importance. Every human cell type has the same genomic DNA sequences, yet some become retinal rods, whereas others become contractile cardiac myocytes. Exactly what determines in each specific cell type which genes are read, how they are read, and what proteins result is not yet clearly understood, but it involves a variety of processes that cardiovascular clinicians and scientists will undoubtedly be hearing much more about in the future, including DNA methylation, histone acetylation, gene silencing, transcriptional control, and other related topics. Many of these come under the general designation of epigenetics, the manner in which the function of genes is determined by factors other than their base sequences.10 An intriguing example of how epigenetics may affect cardiovascular health is the hypothesis that environmental factors within the womb during pregnancy may lead to alterations in DNA methylation or chromatin structure in fetal DNA, leading to long-term changes in gene expression that translate into a higher lifetime risk of cardiovascular disease in the offspring of these pregnancies. These epigenetic determinants of disease risk would not be found by searching for gene mutations, and it is prudent to consider these types of epigenetic disease determinants as we continue our search for the causes and best therapies for cardiovascular disease. The first clinical trials of therapeutic induction of blood vessel growth began over a decade ago, and to date we have still not managed to develop an approach that yields the same tremendously robust, angiographically discernible collateral vessel development that occurs spontaneously in many fortunate patients. Still, the field remains quite promising, and the potential clinical benefits are of sufficient magnitude to warrant continued research and development in this area. We must, however, keep in mind that the biological control of blood vessel growth is quite complex and that therapeutically recapitulating this complex biology is, understandably, a difficult challenge. The HIF-1␣ approach by Rajagopolan and colleagues is a significantly different approach than those applied clinically to date, and it represents an important new

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direction in the field. Whether HIF-1␣ therapy will prove superior to the specific single-gene/single-protein approaches that have been clinically tested to date is not answered by this phase I study, but it will become evident in future efficacy trials. Irrespective of whether HIF-1␣ gene therapy proves clinically efficacious, it represents advancement in our thinking about how best to mimic human biology. Other approaches applying gene regulation as a therapeutic strategy are also in development, including the use of de novo engineered transcription factors targeted to specific genes7; these, too, may significantly advance the field. Defining the human genome sequence was a major accomplishment. Appropriately, we are moving forward past this identification of the instruction set and toward understanding how the instruction set defines biology and how it might be used therapeutically.

Sources of Funding Dr Giordano is supported by National Institutes of Health grants HL075616 and HL64001.

Disclosures Dr Giordano is a consultant for Edwards Lifesciences.

References 1. Rajagopalan S, Olin J, Deitcher S, Pieczek A, Laird J, Grossman PM, Goldman CK, McKellin K, Kelley R, Chronos N. Use of a constitutively active hypoxia-inducible factor-1␣ transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase 1 dose-escalation experience. Circulation. 2007;115:1234 –1243. 2. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C, Yao A, Ye J, Zhan M, Zhang W, Zhang H, Zhao Q, Zheng L, Zhong F, Zhong W, Zhu S, Zhao S, Gilbert D, Baumhueter S, Spier G, Carter C, Cravchik A, Woodage T, Ali F, An H, Awe A, Baldwin D, Baden H, Barnstead M, Barrow I, Beeson K, Busam D, Carver A, Center A, Cheng ML, Curry L, Danaher S, Davenport L, Desilets R, Dietz S, Dodson K, Doup L, Ferriera S, Garg N, Gluecksmann A, Hart B, Haynes J, Haynes C, Heiner C, Hladun S, Hostin D, Houck J, Howland T, Ibegwam C, Johnson J, Kalush F, Kline L, Koduru S, Love A, Mann F, May D, McCawley S, McIntosh T, McMullen I, Moy M, Moy L, Murphy B, Nelson K, Pfannkoch C, Pratts E, Puri V, Qureshi H, Reardon M, Rodriguez R, Rogers YH, Romblad D, Ruhfel B, Scott R, Sitter C, Smallwood M, Stewart E, Strong R, Suh E, Thomas R, Tint NN, Tse S, Vech C, Wang G, Wetter J, Williams S, Williams M, Windsor S, Winn-Deen E, Wolfe K, Zaveri J, Zaveri K, Abril JF, Guigo R, Campbell MJ, Sjolander KV, Karlak B, Kejariwal A, Mi H, Lazareva B, Hatton T, Narechania A, Diemer K, Muruganujan A, Guo N, Sato S, Bafna V, Istrail S, Lippert R, Schwartz R, Walenz B, Yooseph S, Allen D, Basu A, Baxendale J, Blick L, Caminha M, Carnes-Stine J, Caulk P, Chiang YH, Coyne M, Dahlke C, Mays A, Dombroski M, Donnelly M, Ely D, Esparham S, Fosler C, Gire H, Glanowski S, Glasser K, Glodek A, Gorokhov M, Graham K, Gropman B, Harris M, Heil J, Henderson S, Hoover J, Jennings D, Jordan C, Jordan J, Kasha J, Kagan L, Kraft C, Levitsky A, Lewis M, Liu X, Lopez J, Ma D, Majoros W, McDaniel J, Murphy S, Newman M, Nguyen T, Nguyen

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N, Nodell M, Pan S, Peck J, Peterson M, Rowe W, Sanders R, Scott J, Simpson M, Smith T, Sprague A, Stockwell T, Turner R, Venter E, Wang M, Wen M, Wu D, Wu M, Xia A, Zandieh A, Zhu X. The sequence of the human genome. Science. 2001;291:1304 –1351. 3. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, StangeThomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D,

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Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ; International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409: 860 –921. Orii KO, Aoyama T, Souri M, Orii KE, Kondo N, Orii T, Hashimoto T. Genomic DNA organization of human mitochondrial very-long-chain acyl-CoA dehydrogenase and mutation analysis. Biochem Biophys Res Commun. 1995;217:987–992. Grunstein J, Masbad JJ, Hickey R, Giordano F, Johnson RS. Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature. Mol Cell Biol. 2000;20:7282–7291. Maes C, Carmeliet P, Moermans K, Stockmans I, Smets N, Collen D, Bouillon R, Carmeliet G. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev. 2002;111:61–73. Rebar EJ, Huang Y, Hickey R, Nath AK, Meoli D, Nath S, Chen B, Xu L, Liang Y, Jamieson AC, Zhang L, Spratt SK, Case CC, Wolffe A, Giordano FJ. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med. 2002;8:1427–1432. Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS, Arbeit JM. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha. Genes Dev. 2001;15:2520 –2532. Giordano FJ. Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest. 2005;115:500 –508. Cho KS, Elizondo LI, Boerkoel CF. Advances in chromatin remodeling and human disease. Curr Opin Genet Dev. 2004;14:308 –315.

KEY WORDS: Editorials 䡲 angiogenesis 䡲 gene therapy 䡲 hypoxia 䡲 molecular biology 䡲 vasculature



genes

Arrhythmia/Electrophysiology Declining Into Failure The Age-Dependent Loss of the L-Type Calcium Channel Within the Sinoatrial Node Sandra A. Jones, PhD; Mark R. Boyett, PhD; Matthew K. Lancaster, PhD Background—The spontaneous activity of pacemaker cells in the sinoatrial (SA) node controls heart rate under normal physiological conditions. Clinical studies have shown the incidence of SA node dysfunction increases with age and occurs with peak prevalence in the elderly population. The present study investigated whether aging affected the expression of Cav1.2 channels and whether these changes could affect pacemaker activity, in turn leading to age-related SA node degeneration. Methods and Results—The SA node region was isolated from the right atrium of guinea pigs between birth and 38 months of age. Immunofluorescence studies showed Cav1.2 protein was present as punctate labeling around the outer membrane of atrial cells but was absent from the center of the SA node. The area lacking Cav1.2-labeled protein progressively increased from 2.06⫾0.1 (mean⫾SEM) mm2 at 1 month to 18.72⫾2.2 mm2 at 38 months (P⬍0.001). Western blot provided verification that Cav1.2 protein expression within the SA node declined during aging. Functional measurements showed an increased sensitivity to the L-type calcium blocker nifedipine; SA node preparations stopped beating in 100 ␮mol/L nifedipine at 1 day old, compared with 30 ␮mol/L at 1 month and 10 ␮mol/L at 38 months of age. Furthermore, the amplitude of extracellular potentials declined within the center and periphery of the SA node during aging. Conclusions—The present data show Cav1.2 channel protein decreases concurrently with reduced spontaneous activity of the SA node with increased age, which provides further evidence of mechanisms underlying the age-related deterioration of the cardiac pacemaker. (Circulation. 2007;115:1183-1190.) Key Words: calcium 䡲 ion channels 䡲 pacemakers 䡲 sinoatrial node 䡲 aging 䡲 proteins 䡲 electrophysiology

D

cardiomyopathies, often associated with structural remodeling and fibrosis of the SA node.7–9 Healthy aging, however, is associated with an intrinsic decline in pacemaker function in the absence of such structural remodeling, which implicates a progressive change in the cellular properties of the constituent cells of the SA node.10 A number of ion channels, including the high-voltage activated L-type Ca2⫹ current and low-voltage activated T-type Ca2⫹ current, contribute to the pacemaker activity of the SA node.11,12 However, it was the L-type calcium current rather than the T-type current that was determined to be responsible for the upstroke of the action potential and, thus, depolarization of the SA node.13 L-type Ca2⫹ currents are conducted via CaV1.2 channels, which consist of a poreforming ␣1-subunit in association with ␤- and ␣2␦-subunits.14 The ␣1-subunit contains the voltage sensor, the selectivity filter, and binding sites for all known calcium channel blockers and possesses a molecular weight of ⬇200 kDa. L-type calcium channels are sensitive to dihydropyridines,

ysfunction of the sinoatrial (SA) node progressively increases with age regardless of gender, with the highest incidence occurring within the elderly population (ⱖ65 years old), who by 2040 will account for 30% of the Western population.1,2 Clinical observations of sinus dysfunction range from rhythm disturbance to bradycardia, sinus pauses, sinus arrest, and arrhythmias, which without medical intervention can result in sudden death.3 Sinus node dysfunction may account for ⬎50% of pacemaker implants in the United States.4

Editorial p 1178 Clinical Perspective p 1190 The apparent causes of sinus node dysfunction considered across the entire age range can be intrinsic or extrinsic in origin. Extrinsic factors known to induce dysfunction include hypoxia, digitalis, and antiarrhythmic agents such as ␤-blockers and calcium antagonists.5,6 Intrinsic factors include congenital abnormalities, myocarditis, dystrophies, and

Received September 6, 2006; accepted December 15, 2006. From the Institute of Membrane and Systems Biology (S.A.J., M.K.L.), Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom, and School of Medicine (M.R.B.), University of Manchester, Manchester, United Kingdom. The online-only Data Supplement, consisting of expanded Methods and a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/ CIRCULATIONAHA.106.663070/DC1. Correspondence to Dr Sandra A. Jones, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.663070

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such as nifedipine, which selectively block the ␣1-subunit of the CaV1.2 channel. Spontaneously beating cells from the SA node have been characterized in terms of their pharmacological responses to nifedipine. Peripheral SA cells only exhibit moderate negative chronotropism in the presence of nifedipine, compared with central nodal cells where spontaneous activity was abolished.15,16 Therefore, the L-type Ca2⫹ current has been demonstrated to play an obligatory role in pacemaking within the center of the SA node. The cardiac action potential originates from the center of the SA node at the leading pacemaker site and propagates to the periphery (the border of the SA node with the surrounding atrial muscle), then across the remaining heart tissue, assisted by the presence of specialized cellular junctions. Myocardial tissue possesses desmosomes that are responsible for the intercellular adhesion of the cardiac myocytes, consisting of desmoplakin and desmin protein, and electrical junctions or “gap junctions” that predominantly consist of connexin43 (Cx43) protein, which facilitates propagation of the action potential through the heart.17 The location of the center of the SA node can be identified either by mapping of action potentials across the SA node18,19 or by the absence of Cx43 protein via anti-Cx43 antibodies. Studies of humans and rodents (including the hamster, rabbit, rat, and guinea pig) have failed to detect Cx43 protein within the center of the SA node, and consequently, it was concluded that Cx43 protein was absent from the center of the SA node.18,20 –23 Our previous studies of the guinea pig found that Cx43 protein expression in the center of the SA node was absent, and during aging, the area without Cx43 protein increased to encompass the center and periphery of the SA node in the elderly animals.24 The resultant electrical disconnection of the SA node in the elderly animal will contribute to the increasing incidence of SA node dysfunction in the elderly; however, other electrophysiological changes may also contribute to the developing dysfunction. Our hypothesis is that expression of the Cav1.2 channel may decline with age, resulting in a depression of excitability of the tissue and failure of the pacemaker ability of the SA node, due to a suppression of the upstroke of the SA node action potential. Additionally, subsequent changes in intracellular calcium handling are likely to occur: A reduction in calcium influx is likely to lead to reduced intracellular calcium and content of the sarcoplasmic reticulum, which may have important implications for pacemaking and the ability of the heart to respond to ␤-adrenergic stimulation.25 Hence, we have investigated expression of the Cav1.2 channel within the SA node and the susceptibility of pacemaking to L-type calcium channel blockade with age to determine whether changes in ICa,L are implicated in the age-related degeneration of the SA node.

Methods For detailed materials and methods, please see the online-only Data Supplement.

Sample Acquisition Healthy tricolored guinea pigs were studied at ⬍1 day of age, at 1 month of age (young), at 18 months of age (adults), at 26 months of age (old), and at 38 months of age (senescent).

Extracellular Electrode Recording The SA node preparation was maintained in Krebs Ringer solution at 37°C. Extracellular modified bipolar electrodes were used to measure the intrinsic heart rate and monitor changes when subjected to incremental doses of nifedipine from 0.3 to 100 ␮mol/L.

Immunofluorescence Frozen SA node sections were labeled with antibodies to Cav1.2 and Cx43 protein according to protocols described previously.26

Analysis of Protein Expression Tissue samples (50 ␮g total protein per lane) were separated by electrophoresis under reducing conditions by 10% SDS-PAGE, followed by transfer to nitrocellulose membrane, and subsequently probed by antibodies as described previously.26

Statistical Analysis Data are expressed as mean⫾SEM, and statistical differences were assessed by ANOVA with subsequent pairwise ad hoc analysis made with a Holm-Sidak comparative test. Differences and correlations were taken as significant if P⬍0.05, and n corresponds to the number of animals. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Expression of Cav1.2 Protein Immunofluorescent-labeled Cav1.2 protein was detected by confocal imaging of guinea pig right atrial tissue sections at high magnification. Regions where Cav1.2 protein was expressed as a punctate pattern around the outer membrane of myocytes were classified as positive Cav1.2, and those regions where labeling could not be detected were concluded to be lacking Cav1.2 protein (Figure 1A).

Expression of Cav1.2 Protein Within the SA Node Declines With Age Sections across the right atrium taken at 0.5-mm intervals, perpendicular to the crista terminalis, were labeled with anti-CNC1 to permit 2D assessment of Cav1.2 protein distribution with age (n⫽5 right atria per age group). Confocal images of immunofluorescent-labeled tissue sections were converted into representational schematics in which the region lacking Cav1.2 protein was highlighted in blue (Figure 1B). To indicate the location of the SA node by the absence of Cx43 protein, adjacent sections were labeled with antiCx43 (highlighted in red). To illustrate this further, the right atrium of a 1-month-old animal was examined, and the schematic images of adjacent sections are shown at 0.5-mm intervals: the region lacking Cav1.2-labeled protein (left, Figure 1C) and the region lacking Cx43-labeled protein (right, Figure 1C). This procedure was repeated for each atrium examined in every age group. Those regions lacking labeled Cav1.2 protein and Cx43 protein were taken from sections and mapped to the photograph of the intact right atrium. These areas lacking Cav1.2- and Cx43-labeled protein were outlined, and examples are shown for a 1-month-old animal in Figure 1D and for a 26-month-old animal in Figure 1E. Figures 1D and 1E illustrate that the length of the area lacking Cav1.2-labeled protein increased from 2.0 mm in the 1-month-old animal to 7.0 mm in the 26-month-old animal,

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Figure 1. Expression of Cav1.2 protein within the SA node declines with age. A, High magnification of a right atrial tissue section labeled with anti-CNC1 antibody. Left, Positive-labeling of Cav1.2 protein observed as punctate labeling around the outer membrane of atrial cells; right, Cav1.2-labeled protein was absent from the SA node region. B, An example of a low-magnification confocal image of a right atrial section labeled with anti-CNC1 antibody showed Cav1.2 protein was absent from the SA node region but present in the surrounding atrial muscle. Schematic image shows the region lacking Cav1.2 protein in blue, with the remaining tissue that expresses Cav1.2 protein in white. C through E, Sections at 0.5-mm intervals through the right atria were labeled with anti-CNC1 antibody to exhibit the regions lacking Cav1.2 protein, which were mapped to the original photograph of the intact right atria. C, Schematic images of sections across the right atria of a 1-month-old animal illustrate the region’s absence of Cav1.2 protein (blue) within the SA node; the SA node was identified by a lack of Cx43 protein expression (red). D and E, At every 0.5-mm interval, the exact location of the regions lacking Cav1.2 protein and of Cx43 protein within the section were mapped to the original photograph of the intact SA node. The total area determined to lack Cav1.2 protein was outlined in blue, illustrated by the sample intact SA nodes for animals of 1 month (D) and 26 months (E) of age. F, Mean area⫾SEM lacking Cav1.2 protein expression vs age (n⫽5; ANOVA, P⬍0.001; linear regression: y (mm2)⫽0.4355x⫹0.0805; R2⫽0.91), and mean area⫾SEM lacking Cx43 protein expression vs age (n⫽5; ANOVA, P⬍0.0001; linear regression, y (mm2)⫽1.2007x⫹1.9099; R2⫽0.98).

and in addition, the area lacking Cav1.2-labeled protein was seated within the area lacking Cx43-labeled protein. Staining of tissue sections with aniline blue and fuchsin red to observe collagen and myocyte content, respectively, revealed no changes in collagen or myocyte content or distribution from the 18- to the 38-month-old animal. With advancing age, no signs of fibrosis or myocyte loss were observed (see online Data Supplement). The changes observed from the adult to the oldest animals studied therefore reflect changes in protein expression by the constituent cells. The area lacking labeled Cav1.2 protein increased with age from 2.0⫾0.1 mm2 at 1 month to 6.2⫾0.4 mm2 at 18 months, 9.5⫾0.9 mm2 at 26 months, and 18.7⫾2.2 mm2 at 38 months (n⫽5; ANOVA, P⬍0.001; Figure 1F). Linear regression to the total data set indicated a significant and proportional correlation between the area lacking Cav1.2 protein and age (linear regression: y (mm2)⫽0.4355x⫹0.0805, where x is the

age in months; R2⫽0.91). Overall, the area lacking Cav1.2 protein increased 9-fold during aging. The area lacking Cav1.2 protein was seated within an area also lacking Cx43 protein, known to be a marker of the SA node. This area lacking Cx43 increased from 3.5⫾0.6 mm2 in the 1-monthold animal to 47.6⫾2.0 mm2 in the 38-month-old animal (n⫽5; P⬍0.0001; linear regression, y⫽1.2007x⫹1.9099; R2⫽0.998). A significantly correlated linear relationship exists between the increasing area lacking protein for both Cav1.2 and Cx43 versus progressive age. Further analysis of the linear regressions, however, determined a significant difference in the slope of the 2 regressions (P⬍0.002), which indicates that the rate of Cav1.2 protein loss from tissue with age was less than that of Cx43 protein. This shows a scenario in which advancing electrical disconnection of the tissue precedes a loss of an additional ion channel (CaV1.2) critical for main-

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Figure 3. Sensitivity of the SA node to nifedipine increases during aging. The spontaneously beating SA nodes from animals 1 day, 1 month, and 38 months of age were subjected to increasing applications of nifedipine at 0.3, 1, 3, 10, 30, and 100 ␮mol/L. Recordings of SA node activity were made 40 minutes after each application. Sensitivity to nifedipine increased significantly with age. The dose of nifedipine required to stop SA node activity decreased from 100 ␮mol/L at 1 day old to 10 ␮mol/L at 38 months of age (n⫽5; ANOVA, P⬍0.01). Figure 2. Confirmation of antibody specificity and the decline of Cav1.2 protein expression within the SA node with increased age. A, Specificity of the antibody anti-CNC1 to determine Cav1.2 protein expression was confirmed by the absence of the 200-kDa band in the presence of the supplied control antigen. B, A typical blot illustrates the expression of desmin (53 kDa) and Cav1.2 (200 kDa) proteins across the examined age range. C, For every animal from each age group, Cav1.2 protein expression in the SA node region was expressed as a percentage of the paired atrial muscle sample. Data shown mean⫾SEM. Cav1.2 protein expression fell in the SA node region vs age (n⫽5; ANOVA, P⬍0.01; y⫽⫺1.8363x⫹67.024; R2⫽0.81).

taining the electrical function of the tissue. The additive effect of these 2 changes could lead to failure of the SA node in the elderly.

Western Blot Analysis of Cav1.2 Protein Expression To confirm our findings, paired samples of right atrial muscle and SA node from each animal were analyzed for Cav1.2 protein expression by Western blot (Figure 2). Specificity of the anti-CNC1 antibody was confirmed, as preadsorption with the supplied control peptide resulted in no band (Figure 2A). Illustrative membranes of desmin (53 kDa) and Cav1.2 (200 kDa) protein expression within SA node tissue across the age range are shown in Figure 2B. Uniform bands of desmin expression indicated equal protein loading. Cav1.2 protein expression within the SA node was expressed as a percentage of Cav1.2 protein expression within the paired right atrial muscle. Cav1.2 protein expression significantly declined from 88⫾8% at 0.03 month (1 day) to 50⫾6% at 1 month, 24⫾2% at 18 months, 15⫾1% at 26 months, and 5⫾1% at 38 months of age (n⫽5 right atria per age group; ANOVA, P⬍0.001). With linear regression, a significant negative correlation between age and Cav1.2 expression was established (y⫽⫺1.8363x⫹ 67.024; R2⫽0.81), confirming an age-dependent loss of Cav1.2 protein from the SA node.

Intrinsic Heart Rate Shows Increasing Sensitivity to Nifedipine With Age The intrinsic heart rate, the spontaneous rate at which action potentials manifest in the autonomically denervated SA node, was measured on the endocardial surface at the leading pacemaker site (LPS) of the preparations, then each preparation was subjected to incremental doses of nifedipine to block ICa,L (Figure 3). The rate of generated spontaneous action potentials in the absence of nifedipine significantly decreased with age from 249⫾13 bpm at 1 day to 177⫾5 bpm at 1 month and 152⫾5 bpm at 38 months of age (n⫽5; ANOVA, P⬍0.01). The 1-day-old animal showed the lowest sensitivity to nifedipine compared with the other ages studied; spontaneous activity of SA nodes from 38-month-old animals was halted by nifedipine 10 ␮mol/L, a 10-fold lower concentration than required to cause cessation of the SA node in 1-day-old animals. Overall, the SA node preparations from the 38-month-old animals stopped beating spontaneously at the lowest concentration of nifedipine compared with the other ages studied. This increased sensitivity to nifedipine indicates that these oldest SA node preparations contained the fewest Cav1.2 channels across the age range and were highly sensitive to further loss of functional channels. Clinically, such a blockade of these channels might occur when calcium antagonists are used to treat high blood pressure or arrhythmias. The presented results suggest that in the elderly, even low doses of such drugs could induce SA node dysfunction.

Extracellular Potentials Decrease in Amplitude With Age The peak negative deflection of extracellular potentials recorded by surface-positioned bipolar electrodes has been shown to correlate with the upstroke of the action potential in the underlying tissue. A small, slow depolarization is associated with a small deflection, and the reverse is true for large,

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Figure 4. Extracellular potentials decrease in amplitude with age. A through E, For each age group, peak extracellular potentials were recorded from the earliest site of SA node depolarization or LPS, central region (ⱕ2 mm from the LPS), peripheral region (ⱕ2 mm from the crista terminalis), and atrial muscle (for each site within an age group, n⫽5; ANOVA per age group, P⬍0.01). #Not significant (P⬎0.05). F, Age produced a significant decline in amplitude of the recorded deflections in central and peripheral regions of the SA node (n⫽5; ANOVA, P⬍0.001).

rapid depolarizations.12 Peak extracellular potentials were recorded at 4 sites across the SA node preparations: the earliest site of SA node depolarization, known as the LPS; the central region of the SA node (recorded at ⬇25% of the distance between the LPS and crista terminalis); the peripheral region of the SA node (recorded at ⬇75% of the distance between the LPS and crista terminalis); and on the right atrial muscle. Data are shown comparing the amplitude of the potential recorded at the 4 sites for each age group (Figures 4A through 4E; n⫽5; ANOVA, P⬍0.001) and the changes observed with increasing age (Figure 4F). The 1-day-old SA node preparations exhibited significant differences in peak amplitude between all sites examined (Figure 4A). For the 1- and 18-month-old SA node preparations, significant differences in amplitude were determined between all sites, with the exception of LPS to center (Figures 4B and 4C). At the ages of 26 and 38 months, no differences

were exhibited in the amplitude between the sites of the LPS, center, and periphery of the SA node preparations; however, significant differences among these sites existed when they were compared with the atrial muscle site (Figures 4D and 4E). For further analysis, data were summarized for the individual sites by increasing age. A significant decline in the amplitude of the recorded extracellular potential was revealed at the central and peripheral sites of the SA node during progressive aging (Figure 4F; n⫽5, ANOVA, P⬍0.001). The other examined sites, the LPS and atrial muscle, did not exhibit significant changes in potential amplitude with increasing age.

Discussion Four conclusions can be drawn from the present study: (1) from 1 month of age onward, Cav1.2 protein cannot be

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detected at the center of the SA node, and (2) this area lacking labeled Cav1.2 protein progressively increases in size during aging. Functionally, (3) sensitivity of the SA node to nifedipine increases during aging, and (4) the amplitude of the extracellular potential, which correlates to the upstroke of the action potential (principally determined by ICa,L), decreases with age at the center and periphery of the SA node. Overall, the present data provide strong evidence that expression of the Cav1.2 channel, and, consequently, ICa,L, significantly declines with age within the SA node, resulting in suppressed excitability, which contributes in turn to failure of the SA node in the elderly. We have previously reported that the guinea pig SA node contains an area lacking Cx43 protein that continues to progressively increase in size as the animal ages, which leads to slowed conduction of the action potential within the area deficient in Cx43 protein.24 The present study furthers this observation, showing that Cav1.2 protein was not detectable in an area within the SA node of 1-month-old animals and that this area lacking Cav1.2 protein (2.0⫾0.1 mm2) significantly increased in a linear manner that correlated with age, to 18.7⫾2.2 mm2 at 38 months (Figures 1B through 1E). This area lacking Cav1.2 protein always lies within the expanding area lacking Cx43 protein. However, the loss of Cav1.2 protein occurs at a rate slower than the loss of the Cx43 protein (Figure 1E). Analysis by Western blot confirmed these observations (Figure 2). Seisenberger et al,27 using the Cav1.2 channel knockout mouse model, elegantly demonstrated the specific effect of the absence of Cav1.2 protein from cardiac tissue. The knockout mice die of sudden death regardless of age, owing to the absence of Cav1.2 channels necessary for cardiac rhythm generation and muscle contraction.27 However, it is not just through genetic manipulations that remodeling of calcium channel expression can be shown to occur and lead to cardiac dysfunction. Anyukhovsky et al28 induced atrial arrhythmias in adult and elderly dogs. The atria of the latter were found to have a shorter action potential duration, slowed conduction, and increased dispersion.28 On further analysis, it was ascertained that the right atrial cells (from unidentified locations) of elderly dogs contained 50% less ICa,L than their adult counterparts.28 This finding has a parallel in cases of human atrial fibrillation, in which a shortened action potential duration, decrease in ICa,L amplitude, and decrease in ICa,L density have all been noted.29,30 We now additionally propose that within the SA node, an age-related decline in expression of the L-type calcium channel causes suppression of action potential formation and propagation, leading to failure of the SA node as a pacemaker. Such changes are likely to increase the probability of atrial fibrillation, leaving questions about whether the changes observed in calcium channel expression in the many elderly patients who suffer from this most common of arrhythmias are a consequence of the arrhythmia or a consequence of an aging effect that has contributed to precipitation of the arrhythmia. It is widely accepted that aging does not affect in vivo heart rate because of autonomic compensatory mechanisms; however, aging does significantly reduce the intrinsic heart rate.31 Previously, we reported that the intrinsic heart rate in our

guinea pig model drops from 249⫾13 bpm at 1 day to 152⫾5 bpm in the 38-month-old animal.24 In the present study, however, we tested the sensitivity of the intrinsic heart rate to the commonly deployed calcium antagonist nifedipine. Nifedipine is known to selectively block CaV1.2 channels; therefore, ICa,L can be blocked with no effect on ICa,T, INa, IK, or If in the guinea pig SA node.16 Application of incremental doses of nifedipine to the SA node resulted in the slowing of spontaneous activity, followed by the complete termination of beating. At 1 day old, the animal’s SA node exhibited the least sensitivity to nifedipine. The highest sensitivity was observed in SA nodes from the oldest animals (38 months). These data parallel the progressive age-correlated decline in expression of Cav1.2 channels from the 1 day old to the oldest animals (38 months of age) and highlight the changing sensitivity of the SA node pacemaker to pharmacological and potentially autonomic influences. Such changes in pharmacological sensitivity highlight that further consideration should be given to adjusting dose according to age, particularly in the case of elderly patients. Previous work has shown that the amplitude of deflection of recordings of extracellular potential correlates closely with the amplitude of the action potential in the underlying tissue.19 The present data agree with this previous work: The LPS and central regions of the SA node display small extracellular potentials that correspond with their small, slow action potentials. However, with progressive aging, the central region of the SA node shows an additional decline in the amplitude, as does the periphery region of the SA node until the whole of the SA node displays comparable electrical activity throughout. Yamamoto et al19 showed that blockade of the L-type calcium channel could produce such notable reductions in the amplitude of extracellular potentials. From the data presented, we believe the major contributor to the effect observed in the present data is a dramatic decline in the expression and hence recruitment of the L-type calcium channels in the aged tissue. The role of calcium in the generation of spontaneous activity within the SA node is controversial but ubiquitously acknowledged as being of significant importance. Maneuvers that increase intracellular calcium and calcium fluxes via Cav1.2 lead to increases in spontaneous activity and suppression of calcium fluxes or levels by buffering, or appropriate blockers decrease or halt SA node activity. In the intact structure, such manipulations have a particular fascination because they are also often associated with movements of the LPS within the SA node structure. This “pacemaker shift” presumably reflects the complex heterogeneity of the nodal structure and balance of multiple ionic fluxes and electrotonic interactions that regulate its activity. Previous work has found the expression of the proteins involved in regulation of calcium handling, including Cav1.2, to be lower in the center than in the periphery of the SA node in adult rats.32 Functionally, in the rabbit SA node, the cells at the center of the SA node have been shown to have smaller, slower calcium transients with each beat than cells from the periphery.33 The age-related changes in the expression of Cav1.2 reported will lead to significant disruption of the normal heterogeneity of the SA node, which is likely to lead to a reduced ability of the

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node to respond to physiological stimuli (such as adrenergic stimuli, responsiveness to which is reduced in the elderly) and to maintain robust function as the leading pacemaker of the heart, a function preserved by the graded heterogeneity of the SA node structure.34 As the area of tissue lacking Cx43 and Cav1.2 expands to consume the SA nodal region, heterogeneity declines, electrical depolarization is suppressed, and connectivity and communication across this complex tissue falter. This is what we propose heralds the age-related failure of the SA node as a viable pacemaker for the heart. In sinus node dysfunction associated with dystrophies, myopathies, and ischemia, structural changes are well documented and are likely to be an important factor in the creation of sinus dysfunction.35 The role of structural changes in creating sinus dysfunction in the case of healthy aging is controversial. Some studies have suggested that a loss of cells within the node throughout life together with associated fibrosis could be responsible for dysfunction of the aged SA node.36 Interpretation of such studies is complicated by the heterogeneous nature of the nodal structure, and few have mapped changes that occur throughout the SA node in detail. Detailed evidence, where available, shows that structural remodeling of the sinus node occurs during development into adulthood, involving increases in collagen content. The size and collagen content of the SA node then remain stable into old age, which suggests that progressive structural remodeling in the elderly does not underlie the increasing incidence of sick sinus syndrome.10 Other investigators also found no association between collagen and fibroblast content and conduction times of the cardiac action potential across the SA node, nor spontaneous activity of the SA node.37 We have also found no evidence of structural remodeling and growth of the sinus node from the adult animal to the elderly animal24 or evidence of fibrosis (see online Data Supplement). The conclusion of the data from such studies is that age-dependent fibrosis and structural remodeling are not responsible for the progressively increasing incidence of sinus node dysfunction with healthy aging. We conclude that the described changes in the properties of the cells that constitute the SA node lead to age-dependent deterioration in the ability of this vital structure to serve as a stable cardiac pacemaker.

Conclusions Data shown are consistent with our hypothesis that expression of Cav1.2 protein within the SA node continues to decline during aging and that this in turn increases the susceptibility of the node to L-type calcium channel blockers such as nifedipine, symptomatic of the loss of Cav1.2 channels from the SA node. In addition, it is shown that electrical activation of the tissue was reduced across the whole of the SA node, including the central and peripheral regions, in the elderly animal, which implicates a loss of ICa,L in the agerelated degeneration of the SA node. Consequently, the loss of Cav1.2 channels in conjunction with a depletion of Cx43 protein depresses conductivity and hence increases the potential for dysfunction of the SA node with age and the prevalence of arrhythmogenic activity, as is observed clinically. The results of the present work indicate that methods to restore normal calcium regulation to the SA node and

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connectivity to the atrial tissue are likely to produce effective treatments for SA node dysfunction. Additionally, this work indicates that care should be taken in prescribing the commonly used calcium antagonists to the elderly, in whom the tolerance of the SA node is reduced and the risk of potentially precipitating SA node dysfunction is increased.

Sources of Funding This work was funded by a project grant awarded to Dr Jones from the British Heart Foundation (PG/02/006/13486).

Disclosures None.

References 1. Hartel G, Talvensaari T. Treatment of sinoatrial syndrome with permanent cardiac pacing in 90 patients. Acta Med Scand. 1975;198: 341–347. 2. Lakatta EG, Sollott SJ. Perspectives on mammalian cardiovascular aging: humans to molecules. Comp Biochem Physiol A. 2002;132:699 –721. 3. Mandel WJ, Jordan JL, Karagueuzian HS. Disorders of sinus function. Curr Treat Options Cardiovasc Med. 1999;1:179 –186. 4. Rodriguez RD, Schocken DD. Update on sick sinus syndrome, a cardiac disorder of aging. Geriatrics. 1990;45:26 –30. 5. Linker NJ, Camm AJ. Drug effects on the sinus node: a clinical perspective. Cardiovasc Drugs Ther. 1988;2:165–170. 6. Alboni P, Baggioni GF, Scarfo S, Cappato R, Percoco GF, Paparella N, Antonioli GE. Role of sinus node artery disease in sick sinus syndrome in inferior wall acute myocardial infarction. Am J Cardiol. 1991;67: 1180 –1184. 7. Benson DW, Wang DW, Dyment M, Knilans TK, Fish FA, Strieper MJ, Rhodes TH, George AL Jr. Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A). J Clin Invest. 2003;112:1019 –1028. 8. Segawa I, Kikuchi M, Tashiro A, Hiramori K, Sato M, Satodate R. Association of myotonic dystrophy and sick sinus syndrome, with special reference to electrophysiological and histological examinations. Intern Med. 1996;35:185–188. 9. Inoue S, Shinohara F, Sakai T, Niitani H, Saito T, Hiromoto J, Otsuka T. Myocarditis and arrhythmia: a clinico-pathological study of conduction system based on serial section in 65 cases. Jpn Circ J. 1989;53:49 –57. 10. Alings AM, Abbas RF, Bouman LN. Age-related changes in structure and relative collagen content of the human and feline sinoatrial node: a comparative study. Eur Heart J. 1995;16:1655–1667. 11. Brown HF. Electrophysiology of the sinoatrial node. Physiol Rev. 1982; 62:505–530. 12. Hagiwara N, Irisawa H, Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol. 1988;395:233–253. 13. Doerr T, Denger R, Trautwein W. Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp. Pflugers Arch. 1989;413:599 – 603. 14. Catterall WA. Structure and regulation of voltage-gated Ca2⫹ channels. Annu Rev Cell Dev Biol. 2000;16:521–555. 15. Kodama I, Nikmaram MR, Boyett MR, Suzuki R, Honjo H, Owen JM. Regional differences in the role of the Ca2⫹ and Na⫹ currents in pacemaker activity in the sinoatrial node. Am J Physiol. 1997;272: H2793–H2806. 16. Verheijck EE, van Ginneken AC, Wilders R, Bouman LN. Contribution of L-type Ca2⫹ current to electrical activity in sinoatrial nodal myocytes of rabbits. Am J Physiol. 1999;276:H1064 –H1077. 17. Jongsma HJ, Wilder R. Gap junctions in cardiovascular disease. Circ Res. 2000;86:1193–1197. 18. Trabka-Janik E, Cooms W, Lemanski LF, Delmer M, Jalife J. Immunohistochemical localization of gap junction protein channels in hamster sinoatrial node in correlation with electrophysiologic mapping of the pacemaker region. J Cardiovasc Electrophysiol. 1994;5:125–127. 19. Yamamoto M, Honjo H, Niwa R, Kodama I. Low-frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc Res. 1998;39: 360 –372.

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20. van Kempen MJ, Fromaget C, Gros D, Moorman AF, Lamers WH. Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res. 1991;68:1638 –1651. 21. Oosthoek PW, Viragh S, Mayen AE, van Kempen MJ, Lamers WH, Moorman AF. Immunohistochemical delineation of the conduction system, I: the sinoatrial node. Circ Res. 1993;73:473– 481. 22. ten Velde I, de Jonge B, Verheijck EE, van Kempen MJ, Analbers L, Gros D, Jongsma HJ. Spatial distribution of connexin43, the major cardiac gap junction protein, visualizes the cellular network for impulse propagation from sinoatrial node to atrium. Circ Res. 1995;76:802– 811. 23. Oosthoek PW, van Kempen MJA, Wessels A, Lamers WH, Moorman AFM. Distribution of the cardiac gap junction protein, connexin43 in the neonatal and adult human heart. In: Marechal G, Carraro U, eds. Abstracts of European Conference on Muscle Contraction and Cell Motility. Oxford, UK: Springer Netherlands; 1990:85–90. 24. Jones SA, Lancaster MK, Boyett MR. Ageing-related changes of connexins and conduction within the sinoatrial node. J Physiol. 2004;560: 429 – 437. 25. Lakatta EG, Maltsev VA, Bogdanov KY, Stern MD, Vinogradova TM. Cyclic variation of intracellular calcium: a critical factor for cardiac pacemaker cell dominance. Circ Res. 2003;92:e45– e50. 26. Jones SA, Morton MJ, Hunter M, Boyett MR. Expression of TASK-1, a pH-sensitive twin-pore domain K⫹ channel, in rat myocytes. Am J Physiol. 2002;283:H181–H185. 27. Seisenberger C, Specht V, Welling A, Platzer J, Pfeifer A, Kuhbandner S, Striessnig J, Klugbauer N, Feil R, Hofmann F. Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse. J Biol Chem. 2000;275:39193–39199. 28. Anyukhovsky EP, Sosunov EA, Chandra P, Rosen TS, Boyden PA, Danilo P Jr, Rosen MR. Age-associated changes in electrophysiologic

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remodeling: a potential contributor to initiation of atrial fibrillation. Cardiovasc Res. 2005;66:353–363. Bosch RF, Zeng X, Grammer JB, Popovic K, Mewis C, Kuhlkamp V. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res. 1999;44:121–131. Van Wagoner DR, Pond AL, Lamorgese M, Rossie SS, McCarthy PM, Nerbonne JM. Atrial L-type Ca2⫹ currents and human atrial fibrillation. Circ Res. 1999;85:428 – 436. Di Gennaro M, Bernabei R, Sgadari A, Carosella L, Carbonin PU. Age-related differences in isolated rat sinus node function. Basic Res Cardiol. 1987;82:530 –536. Musa H, Lei M, Honjo H, Jones SA, Dobrzynski H, Lancaster MK, Takagishi Y, Henderson Z, Kodama I, Boyett MR. Heterogeneous expression of Ca(2⫹) handling proteins in rabbit sinoatrial node. J Histochem Cytochem. 2002;50:311–324. Lancaster MK, Jones SA, Harrison SM, Boyett MR. Intracellular Ca2⫹ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control. J Physiol. 2004;556:481– 494. Zhang H, Holden AV, Boyett MR. Gradient model versus mosaic model of the sinoatrial node. Circulation. 2001;103:584 –588. Segawa I, Kikuchi M, Tashiro A, Hiramori K, Sato M, Satodate R. Association of myotonic dystrophy and sick sinus syndrome, with special reference to electrophysiological and histological examinations. Intern Med. 1996;35:185–188. de Melo SR, de Souza RR, Mandarim-de-Lacerda CA. Stereologic study of the sinoatrial node of rats: age related changes. Biogerontology. 2002; 3:383–390. Opthof T, de Jonge B, Jongsma HJ, Bouman LN. Functional morphology of the mammalian sinuatrial node. Eur Heart J. 1987;8:1249 –1259.

CLINICAL PERSPECTIVE The present study focused on healthy aging and the high incidence of sinoatrial dysfunction in the elderly, hypothesized to be due to progressive changes in the properties of the constituent cells of the sinoatrial (SA) node. L-type Ca2⫹ currents conducted via CaV1.2 channels are responsible for the upstroke of the action potential and, thus, depolarization of the SA node. The present data show that expression of Cav1.2 protein within the SA node progressively declines during normal healthy aging, in association with an increasing sensitivity to the L-type calcium channel blocker nifedipine. The amplitude of electrical activation of the tissue was significantly reduced across the whole of the SA node in the elderly animal, thus implicating the loss of ICa,L in the age-related degeneration of SA node function. This loss of Cav1.2 channels in conjunction with a previously described depletion of Cx43 protein from the SA node (Cx43 is responsible for connectivity) would be predicted to lead to increasing dysfunction of the SA node with age and an increase in the incidence of arrhythmias, both events that are indeed observed clinically. The results of the present study suggest that therapies aimed at restoration of connectivity and normal calcium regulation to the SA node could provide an effective treatment for progressive age-associated SA node dysfunction. Additionally, the present work indicates that care should be taken in prescribing the commonly used “calcium antagonists” to the elderly, in whom tolerance of the SA node is reduced and the risk of precipitating SA node dysfunction is increased.

Remote Magnetic Navigation to Guide Endocardial and Epicardial Catheter Mapping of Scar-Related Ventricular Tachycardia Arash Aryana, MD; Andre d’Avila, MD; E. Kevin Heist, MD, PhD; Theofanie Mela, MD; Jagmeet P. Singh, MD, PhD; Jeremy N. Ruskin, MD; Vivek Y. Reddy, MD Background—The present study examines the safety and feasibility of using a remote magnetic navigation system to perform endocardial and epicardial substrate-based mapping and radiofrequency ablation in patients with scar-related ventricular tachycardia (VT). Methods and Results—Using the magnetic navigation system, we performed 27 procedures on 24 consecutive patients with a history of VT related to myocardial infarction, dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy, or sarcoidosis. Electroanatomic mapping of the left ventricular, right ventricular, and ventricular epicardial surfaces was constructed in 24, 10, and 12 patients, respectively. Completechamber VT activation maps were created in 4 patients. A total of 77 VTs were inducible, of which 21 were targeted during VT with the remotely navigated radiofrequency ablation catheter alone. With a combination of entrainment and activation mapping, 17 of 21 VTs (81%) were successfully terminated in a mean of 8.4⫾8.2 seconds; for the remainder, irrigated radiofrequency ablation was necessary. The mean fluoroscopy times for endocardial and epicardial mapping were 27⫾23 seconds (range, 0 to 105 seconds) and 18⫾18 seconds (range, 0 to 49 seconds), respectively. In concert with a manually navigated irrigated ablation catheter, 75 of 77 VTs (97%) were ultimately ablated. Four patients underwent a second procedure for recurrent VT, 3 with the magnetic navigation system. After 1.2 procedures per patient, VT did not recur during a mean follow-up of 7⫾3 months (range, 2 to 12 months). Conclusions—The present study demonstrates the safety and feasibility of remote catheter navigation to perform substrate mapping of scar-related VT in a wide range of disease states with a minimal amount of fluoroscopy exposure. (Circulation. 2007;115:1191-1200.) Key Words: ablation 䡲 catheter ablation 䡲 electrophysiology 䡲 magnetic resonance imaging 䡲 mapping 䡲 tachycardia 䡲 tomography

S

ignificant advances have been made in catheter ablation of scar-related ventricular tachycardia (VT). These advances are due in part to an improved understanding of the pathophysiology governing these tachyarrhythmias and to technological advances such as electroanatomic mapping (EAM) systems. This advancement has led to a paradigm shift in the strategy by which VT is mapped and ablated: substrate-based catheter ablation.1– 4 Instead of mapping during VT, this approach involves identifying and ablating the arrhythmogenic myocardium predominantly during sinus rhythm. Although effective, this approach is limited by the need for a degree of ventricular mapping accuracy and detail that requires advanced operator skill with catheter manipulation.

Clinical Perspective p 1200 When used in concert with a compatible EAM system, remote navigation technology may facilitate cardiac mapping and ablation independently of operator dexterity. The magnetic navigation system (MNS) uses highly flexible catheters equipped with small magnets embedded in the tip for catheter orientation with an external magnetic field. To date, this platform system has been used in mapping and ablation of accessory pathways in patients with AV nodal or AV reentrant tachycardia and in the treatment of atrial fibrillation.5–7 The present study examines the hypothesis that substrate-based endocardial and epicardial remote magnetic mapping and ablation can be safely and effectively performed in patients with scar-related VT.

Received October 26, 2006; accepted December 22, 2006. From the Cardiac Arrhythmia Service, Massachusetts General Hospital, and Harvard Medical School, Boston, Mass. The online-only Data Supplement, consisting of movies, is available with this article at http://circ.ahajournals.org/cgi/content/full/ CIRCULATIONAHA.106.672162/DC1. Correspondence to Vivek Y. Reddy, MD, Cardiac Arrhythmia Service, Massachusetts General Hospital, 55 Fruit St, GRB–109, Boston, MA 02114. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.672162

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Figure 1. View-synchronization of the MNS and EAM systems. Shown are the MNS navigation screen with integrated fluoroscopic images (A) and the EAM ventricular surface on the CARTO-RMT screen (B). The top-right panel on the MNS screen (A) is view-synchronized to the right panel on the EAM screen (B). To move the catheter in the direction shown on the EAM (yellow arrow), the operator simply moves the icon on the MNS screen in a similar fashion (orange arrow). If the catheter does not reach the desired location because of an obstacle such as a papillary muscle or chordae tendineae, the catheter is simply remotely withdrawn to free if of the obstacle(s) and re-advanced to the desired location.

Methods The present study was approved by the Massachusetts General Hospital Institutional Review Board Committee and performed according to institutional guidelines.

Patient Population Between November 2005 and October 2006, a total of 27 procedures were performed on 24 consecutive patients with a history of scar-related VT. The cause of VT substrate was diverse: post– myocardial infarction (MI), dilated cardiomyopathy, arrhythmogenic right ventricular (RV) cardiomyopathy, hypertrophic cardiomyopathy, and sarcoidosis. Twenty of 27 procedures (74%) were performed under general anesthesia; the remainder were done under only moderate sedation. In 18 procedures (67%), intra-aortic balloon pump counterpulsation was used for either prophylaxis against worsening heart failure or hemodynamic stabilization during arrhythmia induction and mapping. All intra-aortic balloon pumps were removed immediately at the completion of the procedure.

Remotely Guided MNS The remotely controlled catheter navigation system used in the present study consists of 2 independent but communicating components: the Niobe Stereotaxis MNS (Stereotaxis, Inc, St Louis, Mo) and a prototype EAM system (CARTO-RMT, Biosense Webster, Inc, Diamond Bar, Calif). The MNS uses 2 large magnets positioned on either side of the procedure table to generate a composite magnetic field for directional catheter orientation, as described previously.5–7 The CARTO-RMT EAM platform is a prototype magnetic localization system similar to the standard CARTO system8; the major important difference is its ability to localize the ablation catheter without interference from the MNS magnetic field. This EAM system can localize both standard CARTO and the specialized CARTO-RMT catheters. Accordingly, EAM can be performed either in the conventional fashion with manual catheter manipulation or remotely with the MNS. The EAM system and the MNS communicate in a unidirectional fashion. Three-dimensional locations can be prescribed on the EAMs for transfer to the MNS. The MNS then calculates the vector required of the magnetic field to orient the catheter in this direction. If the catheter fails to move to the desired location because of obstacles in its path (eg, trabeculae, chordae tendineae, papillary muscles), the operator can simply withdraw the catheter remotely to free it of the obstacle(s) and then readvance to the desired location. Alternatively, the magnetic field can be remotely manipulated to incrementally manipulate the catheter. The communication between the CARTORMT EAM and the MNS provides a synchronized view of the heart so that free hand manipulations of this magnetic vector can be performed to iteratively move the catheter along the cardiac surface (Figure 1).

Cardiac Chamber Mapping and Radiofrequency Ablation A transseptal puncture was performed under the guidance of fluoroscopy and, if available, intracardiac echocardiography, and an 8.5F Mullins sheath was placed near the mitral valve plane. Intravenous heparin was administered just before transseptal puncture. Left ventricular (LV) substrate mapping was performed with a combination of transseptal and retrograde aortic approaches in all patients; endocardial RV mapping was performed with femoral venous access. In selected patients, epicardial mapping was performed with the percutaneous subxiphoid needle puncture technique.9 –11 Two types of catheters were used for mapping and ablation: a remotely guided, 4-mm-tip, quadripolar (RMT) catheter with 3 embedded magnets to align with the MNS-generated magnetic field (Navistar-RMT, Biosense-Webster, Inc) and a manually directed, externally irrigated, 3.5-mm-tip catheter (Thermocool, BiosenseWebster, Inc). For the last 15 procedures, the RMT catheter had a thermocouple embedded in its tip for temperature monitoring. Programmed stimulation included up to 3 extrastimuli and rapid pacing from 2 ventricular sites (right or left, depending on the location of the scar) to document cycle lengths and 12-lead ECG morphologies of all inducible VTs. In all patients, baseline ventricular substrate-based mapping was performed remotely with the MNS and the RMT mapping catheter. Mapping consisted of constructing 3-dimensional electroanatomic voltage maps of the chamber(s) of interest (LV, RV, and/or ventricular epicardial surface) during sinus rhythm or RV pacing, displaying peak-to-peak bipolar electrogram amplitude with a fill threshold of at least 15 and 20 mm for endocardial and epicardial maps, respectively. As previously described, a bipolar electrogram voltage amplitude ⬎1.5 mV was defined as normal myocardium in post-MI patients.11–14 For other pathologies, the amplitude scales were adjusted to best identify the diseased myocardial tissue.11–14 Fluoroscopy exposure times during ventricular chamber mapping were recorded in most patients. The chamber mapping time was defined as the time elapsed starting just after the RMT catheter was placed into the relevant chamber and ending after the chamber map was completed just before radiofrequency ablation was begun. After detailed mapping to fully define the scar borders, ventricular activation and entrainment mapping during sustained VT was performed. In a few selected patients with sustained hemodynamically stable VT, full-chamber activation maps were generated; most underwent partial activation mapping only. In all patients, a combination of entrainment, late potential, and pace mapping was used during substrate mapping. Briefly, if a hemodynamically stable VT was inducible (stable for even a few seconds), standard entrainment maneuvers were used to identify and ablate the critical pathway of the circuit. Typically, the RMT catheter was used to deliver these radiofrequency applications remotely along the endocardial and/or epicardial surfaces. Power titration was based on impedance monitoring or, when available, temperature monitoring to achieve 55°C to 65°C and 65°C to 85°C for endocardial and

Aryana et al TABLE 1. Patient

Remote Ventricular Substrate Mapping

1193

Clinical Characteristics of Patients Undergoing Remote Mapping and Ablation Age, y

LVEF, %

NYHA HF Class

VT Substrate

ICD

AAD

History

1

68

27

IV

MI

Yes

A, BB

Syncope, slow incessant MMVT, DCCV

2

68

34

III

DCM

Yes

BB

Near syncope, sustained MMVT, ICD storm

3

77

15

III

MI

Yes

A, BB

Near syncope, slow incessant MMVT, ICD storm, prior failed RFA

4

49

45

IB

MI

No

BB

Symptomatic sustained MMVT

5

75

42

II

MI

Yes

BB

MMVT degenerated into PMVT, ICD storm

6

49

45

II

MI

No

BB

Symptomatic MMVT

7

67

18

III–IV

MI

Yes

BB, M

Sustained MMVT, ICD storm, prior failed RFA

8

54

53

IB

ARVC

Yes

A, F, S

Recurrent MMVT, ICD storm, prior failed RFA

9

76

33

III

MI

Yes

BB, M

Recurrent MMVT, ICD therapy

10

47

30

IB

Sarcoidosis

Yes

M, S

Symptomatic sustained MMVT, ICD storm

11

62

30

III

MI

Yes

A, BB

Near syncope, sustained MMVT, ICD therapy

12

83

27

II

MI

Yes

A, BB

Syncope, recurrent MMVT, ICD storm

13

50

83

IB

HCM

Yes

S

Recurrent MMVT, ICD storm

14

31

52

IB

ARVC

Yes

BB, M

Symptomatic sustained MMVT, ICD storm

15

65

20

III–IV

DCM

Yes

A, BB, M

Symptomatic sustained MMVT, ICD storm

16

55

23

III–IV

MI

Yes

A, BB, M

MMVT, ICD therapy

17

69

32

II

MI

Yes

BB

Recurrent MMVT, ICD storm

18

64

55

II

MI

No

A, BB

Symptomatic MMVT

19

72

20

III–IV

MI

Yes

BB, S

Recurrent MMVT, ICD storm

20

53

42

III

DCM

No

A, BB, M

Symptomatic recurrent MMVT

21

21

70

IB

HCM

Yes

BB, M

Recurrent PMVT, ICD therapy

22

64

61

IB

ARVC

No

M, S

Symptomatic MMVT (below ICD detection zone), DCCV, prior failed RFA

23

62

20

IB

MI

Yes

BB, M

Incessant MMVT, ICD storm

24

81

15

III–IV

MI

Yes

A, BB

Symptomatic slow incessant MMVT (below ICD detection zone)

LVEF indicates LV ejection fraction; NYHA HF, New York Heart Association heart failure; AAD, antiarrhythmic drug; A, amiodarone; BB, ␤-blocker; F, flecainide; M, mexelitine; S, sotalol; MMVT, monomorphic VT; DCCV, external direct current cardioversion; DCM, dilated cardiomyopathy; RFA, radiofrequency ablation; PMVT, polymorphic VT; ARVC, arrhythmogenic RV cardiomyopathy; and HCM, hypertrophic cardiomyopathy.

epicardial lesions, respectively. If the arrhythmia(s) failed to terminate or remained inducible after ablation with the RMT catheter, a manual irrigated catheter was used to eliminate the VT. In addition, the manual catheter was used in most patients to ablate additional remaining putative target sites such as late potentials, pace map sites with a good QRS match to an inducible VT and a long stimulus to QRS time, and VT exit sites identified by pace mapping along scar borders. With the manual catheter, power was titrated to achieve an impedance fall of ⬇10%. The postablation stimulation protocol included at least as aggressive a stimulation protocol as that used to initially induce the VTs at the beginning of the procedure.

Follow-Up VT recurrence was identified by history and clinical symptoms and through device interrogation. All patients received anticoagulation with warfarin or aspirin after the procedure. Antiarrhythmic drug therapies that patients had been prescribed long term were either continued at the same or reduced dosage or, in selected cases, discontinued. Antiarrhythmic agents that had been initiated recently to control multiple/incessant VT were discontinued. Patients were seen in the implantable cardioverter-defibrillator (ICD) clinic for device interrogation at 1 to 2 months and every 3 months thereafter. All data are presented as mean⫾SD. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results

are shown in Table 1. The mean age of the study cohort was 61⫾15 years (range, 21 to 83 years). Twenty-one patients (87%) were men. The mean LV ejection fraction as assessed by echocardiography was 37⫾18% (range, 15% to 83%), with a mean LV dimension of 57⫾11 mm (range, 40 to 77 mm) at end diastole and 46⫾13 mm (range, 22 to 69 mm) at end systole. Eleven patients (46%) had advanced heart failure as defined by a New York Heart Association functional class III or IV. The origin of VT substrate was related to MI in 15 patients (62%), dilated cardiomyopathy in 3 (13%), arrhythmogenic RV cardiomyopathy in 3 (13%), hypertrophic cardiomyopathy in 2 (8%), and sarcoidosis in 1 (4%). Nineteen patients (79%) had an ICD. In addition, 19 patients (79%) were receiving antiarrhythmic drug therapy. All patients had symptomatic or recurrent monomorphic VT; ICD therapy or cardioversion was required in 19 cases (79%). Fifteen of 24 patients (62%) presented with either incessant VT or ICD storm, defined as ⱖ3 appropriate ICD therapies in a 24-hour period. Four patients (17%) had a history of previously failed VT ablation using a conventional approach (ie, not using remote navigation). All patients had evidence of at least a single VT morphology on a 12-lead ECG or stored ICD electrograms.

Patient Characteristics

Mapping Approach

A total of 27 procedures were performed on 24 consecutive patients with a history of scar-related VT. Patient characteristics

In 24 of 27 procedures, LV endocardial mapping was performed via both transseptal and retrograde aortic approaches.

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Circulation TABLE 2.

March 13, 2007 Results of Arrhythmia Induction and Fluoroscopy Usage Time During Mapping and Ablation Induced VTs

Patient

SVT, n

CLs of Induced VTs, ms

Fluoroscopy Time Using RMT Total Fluoroscopy Time, min

Endocardial, s

Epicardial, s

Total, s

1

1

585

22

N/A

N/A

2

0

···

13

N/A

䡠䡠䡠 N/A

3–1*

2

524, 405

24

N/A

䡠䡠䡠

N/A

䡠䡠䡠

3

N/A

3–2†

1

520

16

3

4

2

280, 220

16

N/A

5

1

236

30

N/A

䡠䡠䡠 N/A

N/A

6

0

295 (NSVT)‡

12

13

䡠䡠䡠

13

7

5

417, 420, 435, 473, 562

25

12

8

2

550, 232

15

105

䡠䡠䡠 31

136

9

1

330

24

41

䡠䡠䡠

41

10–1*

3

330, 326, 421

26

38

䡠䡠䡠

38

10–2†

1

540

16

20

䡠䡠䡠

20

11

5

482, 345, 406, 438, 470

26

44

䡠䡠䡠

44

12

7

470, 520, 412, 386, 540, 480, 440

27

18

13

0

···

14

52

䡠䡠䡠 33

85

14

3

380, 320, 262

15

40

0

40

15

8

304, 349, 358, 384, 389, 421, 426, 433

23

38

䡠䡠䡠

38

16

6

731, 482, 459, 445, 473, 132

31

19

䡠䡠䡠

19

17

6

360, 335, 520, 339, 328, 344

25

32

䡠䡠䡠

32

18–1*

3

384, 398, 417

33

17

3

365, 283, 208

29

32

䡠䡠䡠 49

17

18–2† 19

1

380

27

29

20

4

471, 316, 297, 365

13

15

21

0

···

22

4

351, 379, 330, 201

23

5

426, 304, 271, 337, 276

22

5

0

5

24

3

562, 712, 325

21

0

1

1

䡠䡠䡠 18

N/A 12

18

81 29 33

9

3

11

14

19

N/A

N/A

N/A

SVT indicates sustained VT; CL, cycle length; NSVT, nonsustained VT; and N/A, data not available. *First mapping and ablation procedure for this patient. †Second mapping and ablation procedure for this patient. ‡Induction of nonsustained VT only.

LV mapping was not performed in 2 arrhythmogenic RV cardiomyopathy patients (patients 8 and 14) and during the initial procedure on the patient with sarcoidosis (patient 10 –1). RV endocardial mapping was performed using a long sheath advanced to the approximate plane of the tricuspid valve for stabilization. Pericardial access was successful in 13 of 16 patients (81%), including 2 patients with prior cardiac surgery. Pericardial access could not be achieved in the remaining patients because of previous cardiac surgery (1 patient) or the presence of pericardial adhesions resulting from prior MI (1 patient) or sarcoidosis (1 patient).

Remote Substrate-Based Ventricular Mapping Maps of LV and RV endocardium and ventricular epicardium were constructed in 24, 10, and 12 procedures, respectively. The mean LV and RV chamber and pericardial space volumes were 220⫾104, 264⫾111, and 651⫾145 cm3, respectively. The mean total points collected during mapping of LV and RV endocardium and ventricular epicardium were

142⫾75, 76⫾73, and 123⫾37, respectively. Of these, the mean points collected with the RMT catheter were 111⫾67 (78%), 74⫾63 (97%), and 122⫾34 (99%), respectively. Remote mapping of these chambers required a mean duration of 84⫾44 minutes (48⫾18 seconds per point), 66⫾48 minutes (42⫾18 seconds per point), and 75⫾34 minutes (36⫾12 seconds per point), respectively. As shown in Table 2, the fluoroscopy times required to perform endocardial and epicardial remote mapping were 27⫾23 seconds (range, 0 to 105 seconds) and 18⫾18 seconds (range, 0 to 49 seconds), respectively; the total fluoroscopy time to complete remote mapping was 34⫾32 seconds (range, 1 to 136 seconds). The total procedural fluoroscopy time was 21⫾7 minutes (range, 9 to 33 minutes). Bipolar ventricular voltage amplitude maps were generated in all patients. Abnormal ventricular scar tissue, defined by low-voltage electrogram amplitude, was seen in all but 3 patients. Examples of ventricular EAMs constructed in patients with post-MI, arrhythmogenic RV cardiomyopathy, and hypertrophic cardiomyopathy–related VTs are shown in Figures 2 and 3.

Aryana et al

Remote Ventricular Substrate Mapping

1195

Figure 2. Full chamber remote activation mapping during VT. Electroanatomical activation (A) and voltage (B) maps were constructed during VT (cycle length of 524 ms), in a post-MI patient (#3) with an apical LV scar. The large area of normal voltage in panel B represents the abutting septal portion of the RV. The entire cycle length of the tachycardia was mapped. The insets in panel A represent the sequence of intracardiac ECGs as they traverse through electrical diastole (a facile visual representation of the VT is shown in a propagation map, Movie I, online-only Data Supplement ). The arrows point to entrainment sites (dark green). At each of these site (dark green points), except the last site (far left), entrainment with concealed fusion was noted with post-pacing intervals equal to the tachycardia cycle length; entrainment at the last site (far left) revealed again the tachycardia cycle length equaling the post-pacing interval, but manifest fusion was noted. Ablation at the constrained portion of the channel (red point) with the remote catheter did not terminate the rhythm, but manual irrigated ablation did terminate the rhythm, albeit a late termination (15 sec). While VT was not inducible at the end of the procedure, the patient presented with another VT that was also remotely mapped as traversing this channel, but in the opposite direction. During the second procedure, this channel was completely ablated with irrigated radiofrequency energy to eliminate VT; no VT recurred after this second procedure. Also shown are electroanatomical activation (C & D) and voltage (E) maps during VT (cycle length of 550 ms) in a patient with arrhythmogenic right ventricular cardiomyopathy (#8). Half of the tachycardia cycle length was mapped in this patient. A diastolic potential is seen in the inset; at this location, entrainment with concealed fusion and a post-pacing interval equal to 550 ms was observed. Endocardial ablation terminated the VT, but it was re-inducible; epicardial ablation at the opposite site completely eliminated the VT.

VT Induction and Catheter Ablation A total of 77 VTs were inducible during 23 of 27 procedures (85%), whereas in 4 patients (patients 2, 6, 13, and 21), sustained monomorphic VT could not be induced (Table 2). In 1 of these 5 patients (patient 6), nonsustained monomorphic VT was induced repeatedly and thus targeted for ablation. Although no VTs were inducible in the other 3, in 1 patient (patient 2), typical atrial flutter was induced repeatedly and eliminated by cavotricuspid isthmus ablation. During sustained hemodynamically stable VT, full-chamber activation mapping was performed in 4 patients during 5 procedures, and partial activation mapping was performed in the remaining. Entrainment mapping was performed in 20 procedures. Pace mapping and targeting of late and fractionated potentials were used in 20 and 23 procedures, respectively. Figures 4 and 5 illustrate examples of substrate mapping and ablation in patients with hemodynamically stable and unstable VTs. Of the 77 inducible VTs, 21 were targeted for ablation during VT with the remote catheter. Of these, a total of 17 VTs (81%) were successfully terminated during 15 procedures at a mean duration of 8.4⫾8.2 seconds (Table 3). In 2 procedures, the entire ablation was successfully completed with the remote catheter alone, whereas in 22 procedures, the

manual, irrigated catheter also was used to enhance procedural safety and efficacy. In concert with the latter catheter, 75 of 77 VTs (97%) were eliminated altogether. The mean total duration of radiofrequency delivery per procedure was 26⫾11 minutes. The mean total radiofrequency lesions delivered with the remote and manual catheters were 8⫾8 and 24⫾12 (total, 31⫾12), respectively. During 4 procedures (patients 3–1, 4, 9, and 17), arrhythmia termination did not occur with remote ablation but was achieved with the manual catheter. In 3 additional cases (patients 5, 10 –1, 12, and 15), termination of VT was performed solely with the manual catheter. In 2 cases, a “pop” occurred while radiofrequency energy was delivered with the standard remote catheter, but neither was associated with thromboembolism. Ventricular arrhythmias were inducible in 5 patients at the end of the procedure (Table 3). In 3 cases (patients 3–2, 4, and 22), this induced rhythm was ventricular flutter. Another patient (patient 15) was found to have 8 inducible VTs, with successful elimination of all but 1. In the final patient (patient 23), programmed stimulation resulted in only nonsustained polymorphic VT.

Complications The 30-day mortality from the procedure was zero. One patient (patient 7) with prior cardiac surgery who underwent

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March 13, 2007

Figure 3. Correlation of electroanatomical mapping with 3-dimensional imaging. Anterior-posterior (A) and posterior-anterior (B) views from an electroanatomical voltage map, and a delayed-enhancement cardiac magnetic resonance imaging image of the LV inferior scar (C) are shown from a patient with magnetic resonance imaging–related VT (#6). A late potential is shown in the inset. The hyperenhanced infracted tissue is visualized as white along the LV inferior wall, which is the same location as the infarct seen on the electroanatomical maps.18 Panel (D) shows an epicardial and panel (E) a mesh view of an epicardial map with an embedded endocardial map. On the other hand, in a patient with hypertrophic cardiomyopathy (#13), there was no significant amount of scarred tissue visualized during either epicardial (D) or endocardial (E) mapping; the lower electrogram amplitude region overlying the base and acute margins of the RV simply represent epicardial fat (there was a significant lack of late/double/fractionated potentials in this region).19 Consistent with this image, delayed-enhancement computer tomography imaging (F) revealed scattered intramural hyperenhanced regions consistent with no significant scar20 (magnetic resonance imaging could not be performed because of the patient’s ICD).

pericardial mapping with the manual catheter developed a loculated pericardial effusion in the posterior aspect of the left atrium with partial compression requiring surgical pericardial decompression. One patient (patient 22) who underwent RV epicardial mapping only (no LV endocardial mapping) developed transient right ulnar nerve palsy after the procedure. It was unclear whether the palsy represented an embolic stroke or was the result of prolonged immobilization under general anesthesia. Another patient (patient 23) developed uncomplicated bilateral lower-extremity deep venous thrombosis that was successfully treated with anticoagulation. Finally, 1 patient (patient 1) died more than a month after the procedure as a result of advanced, medication-refractory heart failure.

Follow-Up During follow-up, repeat VT ablation was required in 4 patients. One patient (patient 17) with prior cardiac surgery continued to have recurrent VT associated with ICD discharges and underwent successful epicardial ablation with a manual approach after minimally invasive surgical subxiphoid access to the pericardial space.15 The other 3 patients (patients 3, 10, and 18) presented with slow VT without ICD discharges and underwent successful repeat VT ablation with the MNS. During follow-up, inappropriate ICD discharges occurred in 1 patient (patient 9) as a result of atrial fibrillation. In toto, there were no VT events after a mean follow-up of 7⫾3 months (range, 2 to 12 months). On the other hand, if assessed after a single procedure only, procedural success was achieved in 20 of 24 patients (83%).

Discussion A number of advances have been made in recent years in catheter ablation of complex arrhythmias. Nevertheless, manipulation of ablation catheters during substrate-based mapping of scar-related VT requires adequate experience and manual dexterity and can be limited by the technical skill required for detailed mapping of ventricular myocardium. By obviating this skill requirement, remotely controlled magnetic/robotic navigation systems may enhance catheter-directed arrhythmia mapping and ablation. Magnetic navigation of cardiac catheters was first reported over a decade ago in a neonate with complex congenital heart disease in whom it was shown to enhance catheter guidance and manipulation.16 Since then, similar systems have been used safely in a variety of invasive cardiovascular procedures, including ablation of supraventricular cardiac arrhythmias.5–7 The present study demonstrates the safety and efficacy of endocardial and epicardial substrate mapping of VT with the MNS in the setting of a variety of cardiac pathologies. In addition to mapping, a subset of these patients also underwent ablation in the LV and RV chambers and the pericardial space. In toto, this remote navigation system proved capable of each of the 3 major components of substrate-based mapping and VT ablation: (1) delineating and identifying the diseased myocardium, (2) performing the necessary electrophysiological maneuvers required to identify the arrhythmogenic zones within the scar critical for VT maintenance, and (3) in a subset of induced VTs, delivering radiofrequency energy to terminate VT.

Aryana et al

Remote Ventricular Substrate Mapping

1197

Figure 4. Remote mapping and ablation of hemodynamically stable VT. Shown are the clinical slow VT at 585 ms (A), inferior views of the electroanatomical activation (B) and voltage (C) maps during VT, and a cardiac computed tomography scan showing a calcified LV inferobasal scar (D) from a patient with post-MI VT (#1). E, At the start of an attempt at entrainment from an inferior wall site deep within the scar (denoted by the black arrow in panel B), the first paced beat terminated the VT without manifest global ventricular capture. F, Just apical to this site (denoted by the red arrow in panel B), stable diastolic potentials are seen during VT; entrainment with concealed fusion and a post-pacing interval equal to 585 ms were observed at this location. G, During remote radiofrequency ablation at this site, the VT was eliminated in ⬍ 4 s of commencing energy delivery.

Delineation and Identification of Diseased Myocardium Accurate EAMs of the LV, RV, and ventricular epicardium could be constructed remotely in patients with a wide variety of disease states. The MNS-compatible RMT catheter offers several potential advantages over the conventional manual catheter during chamber mapping. Because its orientation is guided entirely by magnetic field and no deflection wires are required, the RMT catheter is softer than traditional deflectable catheters along its distal segment. This feature could result in several clinically significant benefits. First, it is possible that less endocardial trauma (a common occurrence during standard mapping, albeit of unclear clinical significance) would result from the use of an RMT catheter. In particular, the risk of remote cardiac perforation should be low. Second, the softer touch of the RMT catheter is likely to cause less deformation of cardiac chambers than manual mapping, potentially resulting in a more accurate rendering of cardiac chambers. Although the software for the CARTORMT system used in the present study did not support integration with 3-dimensional computer tomography/magnetic resonance imaging, it is possible that the registration process to perform image-guided therapy may be facilitated by a more precise rendering of the chamber volume. However, the absence of a comparative manual mapping group prevents us from making definitive conclusions on any these points.

Third, there was a minimal amount of fluoroscopy use during remote MNS mapping, ⬍1 minute in most cases, regardless of the mapping approach, endocardial or epicardial. This was related in a large part to use of the RMT catheter because it can be manipulated inside cardiac chambers with minimal concern for trauma. In addition, because the catheter tip can always be visualized in a real-time fashion by EAM, confirmation of position by fluoroscopy is rarely necessary. It is important to note that manual ablation was performed in most cases. Therefore, it is likely that additional reductions in total fluoroscopy will be realized once MNScompatible, irrigated radiofrequency ablation catheters become available for clinical use. This could result in a marked reduction in radiation exposure for both patients and operators during these complex procedures.

Identification of Arrhythmogenic Zones Required for VT To identify the arrhythmogenic zones within a scar, 4 mapping strategies commonly are used in clinical practice: activation mapping, entrainment mapping, late potential mapping, and pace mapping. Partial activation mapping was performed in all patients with sustained VT. Although not always required, full activation maps were generated during 5 procedures. Although the lack of comparative manual mapping precludes a definitive conclusion, our qualitative assess-

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March 13, 2007

Figure 5. Mapping and ablation of hemodynamically-unstable VTs. Four hemodynamically-unstable VTs were induced in a patient with dilated cardiomyopathy–related VT (#20). Remote epicardial voltage mapping (top) revealed a large low-voltage area without the presence of late or fractionated potentials, most likely representing epicardial fat.20 The remote endocardial voltage map (bottom) revealed an anterior-septal scar with associated fractionated and late potentials (inset). Pace mapping (right panel) from this area resulted in a QRS morphology similar to the first VT and with a delay between the stimulus to QRS. Irrigated radiofrequency ablation of late and fractionated potentials and good pace map sites resulted in successful elimination of all inducible VTs. The relationship of the endocardial and epicardial EAMs is better appreciated in Movie II in the online-only Data Supplement.

ment was that catheter-induced premature ventricular beats were less common than typically observed during manual mapping. This clinical observation is consistent with our prior experience of a marked reduction in premature ventricular beats during remote mapping compared with manual mapping in an experimental porcine model of healed MI.17 Electrogram stability is important during ventricular mapping to perform electrophysiological pacing maneuvers such as entrainment and pace mapping. In the present study, remote mapping demonstrated the requisite catheter stability to provide stable beat-to-beat electrogram morphology and consistent endocardial or epicardial ventricular capture during pacing. This was true whether performed during sinus rhythm or VT for entrainment or pace mapping, respectively. Detailed chamber mapping also was feasible during sinus rhythm to identify late potentials.

terminate. This is consistent with our previous observation and may in fact be related to the presence of epicardial fat serving as an insulating barrier to rapid and effective radiofrequency energy transmission to the target site. Although in 2 cases successful ablation was performed entirely with the RMT catheter alone, it should be emphasized that the manual irrigated catheter also was used in most the cases. The reason was the safety and efficacy limitations of standard 4-mm-tip ablation in left-sided cardiac chambers as a result of a higher thromboembolic potential, not a reflection of its maneuverability or stability. It remains to be determined whether the ablation process could have been completed entirely with an irrigated RMT catheter, a hypothesis that can be tested once this irrigated catheter becomes available for clinical use.

Study Limitations Delivery of Radiofrequency Energy to Terminate VT In a subset of the patient cohort, radiofrequency ablation with the MNS and RMT catheter could be performed safely and feasibly in the LV, RV, and epicardial space. The initial procedure proved successful in 20 of 24 patients (83%). Of the 4 patients with recurrences, 3 had a successful repeat ablation procedure with the MNS (2 requiring a combined endocardial/epicardial approach), whereas the fourth patient was ablated manually with a pericardial approach after surgical pericardial access.15 Seventeen VTs were successfully terminated in 15 procedures with radiofrequency application with the RMT catheter at a mean duration of 8.4 seconds. Of note, most terminations achieved by endocardial delivery of radiofrequency energy occurred in ⬍10 seconds (in 9 of 13), whereas most epicardial ablations took longer to

First, the fluoroscopic visual field was partially compromised during the procedures. With the magnets in place, it is usually not possible to fluoroscopically visualize the entire ventricular cavity. This was also evident during pericardial mapping, which generally requires a larger field of visualization. Nonetheless, this did not prove to be a major limitation because the need for fluoroscopy is greatly minimized with EAM. Second, the present study was not a randomized comparison of the safety and efficacy of remote and manual mapping and ablation. It was designed predominantly to address the feasibility of remote ventricular mapping. Therefore, definitive conclusions comparing these approaches cannot be reached without a formal comparative study. This includes assessments of catheter stability, premature ventricular beat frequency, and endocardial trauma. Third, although certain technical limitations of manual mapping are overcome

Aryana et al TABLE 3.

1 2 3–1* 3–2† 4 5 6 7 8 9 10–1* 10–2† 11 12 13 14 15 16 17 18–1* 18–2† 19 20 21 22 23 24 Mean SD

1199

Results of VT Ablation Time Duration and Location of Remote VT Termination

Patient

Remote Ventricular Substrate Mapping

RFA Lesions (by Catheter), n

VTs Terminated Remotely by RFA, ms

Time, s

Location

RMT

Manually Irrigated Catheter

585

3.9

LV

䡠䡠䡠 Not terminated 520 Not terminated

䡠䡠䡠 䡠䡠䡠 7.5

䡠䡠䡠 䡠䡠䡠 LV

4 3

25 28

䡠䡠䡠 3 4 2 0 1 2 12 18 3 5 4 30

䡠䡠䡠 20 22 20 28 18 31 0 23 27 7 24 21

䡠䡠䡠 18 6 8 3 1 9 4 6

䡠䡠䡠 0 44 33 56 27 30 23 15

䡠䡠䡠 24 10 8 8

䡠䡠䡠 14 36 24 12

䡠䡠䡠 䡠䡠䡠 435, 562 550 Not terminated

䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 2.7, 11.9 2

䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 LV EPI (RV)

䡠䡠䡠 540 470

䡠䡠䡠 䡠䡠䡠 1.4 0.9

䡠䡠䡠 䡠䡠䡠 RV LV

䡠䡠䡠 䡠䡠䡠 380

䡠䡠䡠 䡠䡠䡠 11.9

䡠䡠䡠 䡠䡠䡠 EPI (RV)

䡠䡠䡠 482 Not terminated 417 365 380 365

䡠䡠䡠 18.4

䡠䡠䡠 LV

䡠䡠䡠 31.6 9.3 16.4 2.7

䡠䡠䡠 LV LV LV LV

䡠䡠䡠 351 426 562, 712

䡠䡠䡠 13.8 1.8 3.6, 2.7 8.4 8.2

䡠䡠䡠 EPI (RV) LV LV

Arrhythmia Recurrence During Follow-Up Total RFA Time, min

Arrhythmias Inducible at End of Procedure

26

None

䡠䡠䡠 21 18 14 15 18 32 10 20 23 14 22 46

䡠䡠䡠 None VFL VFL None None None None None None None None None None None MMVT None None None None None None None VFL PMVT None

䡠䡠䡠 19 44 36 50 26 27 22 23 䡠䡠䡠 28 36 46 26 11

VT No No MMVT No No No No No No No MMVT No No No No No No MMVT No MMVT No No No No No No No

䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 RFA indicates radiofrequency ablation; MMVT, monomorphic VT; VFL, ventricular flutter; EPI, epicardial; and PMVT, polymorphic VT. *First mapping and ablation procedure for this patient. †Second mapping and ablation procedure for this patient.

with remote mapping, one must still master the other skills required to perform remote ventricular mapping, including maneuvering the somewhat complex software architecture of remote navigation. Fourth, although both retrograde and transseptal approaches were used during the present study, the transseptal approach was preferable because of the enhanced response of the catheter tip to remote advancement/ retraction. That is, during movement of the catheter during retrograde aortic mapping, the “slack” of the catheter along the arch of the aorta results in a relatively slow response time for movement of the catheter tip, a phenomenon less pronounced with transseptal mapping. This requires, however, that the operator be familiar with the transseptal puncture technique.

Conclusions The present study presents clinical evidence for the feasibility of remote catheter navigation to perform ventricular

ICD Event No No No No 䡠䡠䡠 No 䡠䡠䡠 No No Yes No No No No No No No No No 䡠䡠䡠 䡠䡠䡠 No 䡠䡠䡠 No 䡠䡠䡠 No No 䡠䡠䡠 䡠䡠䡠

substrate-based mapping in humans in a wide range of disease pathologies. The enhanced maneuverability of the RMT catheter permitted accurate mapping of difficult-toreach areas. The remote approach was safe and efficacious, and it was possible with a minimal amount of fluoroscopy time and radiation exposure to both patients and operators. By obviating the need for the advanced operator skill often required for detailed ventricular mapping, substrate-based VT mapping with this approach may become much more widespread and effective.

Sources of Funding This work was supported in part by a National Institutes of Health K23 award (HL68064) to Dr Reddy.

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Disclosures Dr Reddy has served as a consultant to Biosense-Webster, Inc. Dr Ruskin has served on the Medical Advisory Board of Stereotaxis, Inc. The remaining authors report no conflicts.

References 1. Marchlinski FE, Callans DJ, Gottlieb CD, Zado E. Linear ablation lesions for control of unmappable ventricular tachycardia in patients with ischemic and nonischemic cardiomyopathy. Circulation. 2000;101:1288 –1296. 2. Soejima K, Suzuki M, Maisel WH, Brunckhorst CB, Delacretaz E, Blier L, Tung S, Khan H, Stevenson WG. Catheter ablation in patients with multiple and unstable ventricular tachycardias after myocardial infarction: short ablation lines guided by reentry circuit isthmuses and sinus rhythm mapping. Circulation. 2001;104:664 – 669. 3. Reddy VY, Neuzil P, Taborsky M, Ruskin JN. Short-term results of substrate mapping and radiofrequency ablation of ischemic ventricular tachycardia using a saline-irrigated catheter. J Am Coll Cardiol. 2003; 41:2228 –2236. 4. Arenal A, Glez-Torrecilla E, Ortiz M, Villacastin J, Fdez-Portales F, Sousa E, del Castillo S, de Isla LP, Jimenez J, Almendral J. Ablation of electrograms with an isolated, delayed component as treatment of unmappable monomorphic ventricular tachycardias in patients with structural heart disease. J Am Coll Cardiol. 2003;41:81–92. 5. Faddis MN, Chen J, Osborn J, Talcott M, Cain ME, Lindsay BD. Magnetic guidance system for cardiac electrophysiology: a prospective trial of safety and efficacy in humans. J Am Coll Cardiol. 2003;42: 1952–1958. 6. Ernst S, Ouyang F, Linder C, Hertting K, Stahl F, Chun J, Hachiya H, Bansch D, Antz M, Kuck KH. Initial experience with remote catheter ablation using a novel magnetic navigation system: magnetic remote catheter ablation. Circulation. 2004;109:1472–1475. 7. Pappone C, Vicedomini G, Manguso F, Gugliotta F, Mazzone P, Gulletta S, Sora N, Sala S, Marzi A, Augello G, Livolsi L, Santagostino A, Santinelli V. Robotic magnetic navigation for atrial fibrillation ablation. J Am Coll Cardiol. 2006;47:1390 –1400. 8. Eldar M, Ohad D, Bor A, Varda-Bloom N, Swanson DK, Battler A. A closed-chest pig model of sustained ventricular tachycardia. Pacing Clin Electrophysiol. 1994;17:1603–1609. 9. Sosa E, Scanavacca M, D’Avila A, Oliveira F, Ramires JAF. Nonsurgical transthoracic epicardial catheter ablation to treat recurrent ventricular tachycardia occurring late after myocardial infarction. J Am Coll Cardiol. 2000;35:1442–1449.

10. D’Avila A, Scanavacca M, Sosa E, Ruskin JN, Reddy VY. Pericardial anatomy for the interventional electrophysiologist. J Cardiovasc Electrophysiol. 2003;14:422– 430. 11. Reddy VY, Wrobleski D, Houghtaling C, Josephson ME, Ruskin JN. Combined epicardial and endocardial electroanatomic-mapping in a porcine model of healed myocardial infarction. Circulation. 2003;107: 3236 –3242. 12. Callans DJ, Ren JF, Michele J, Marchlinski FE, Dillon SM. Electroanatomic left ventricular mapping in the porcine model of healed anterior myocardial infarction: correlation with intracardiac echocardiography and pathological analysis. Circulation. 1999;100:1744 –1750. 13. Wrobleski D, Houghtaling C, Josephson ME, Ruskin JN, Reddy VY. Use of electrogram characteristics during sinus rhythm to delineate the endocardial scar in a porcine model of healed myocardial infarction. J Cardiovasc Electrophysiol. 2003;14:524 –529. 14. Marchlinski FE, Garcia F, Siadatan A, Sauer W, Beldner S, Zado E, Hsia H, Lin D, Cooper J, Verdino R, Gerstenfeld E, Dixit S, Russo A, Callans D. Ventricular tachycardia/ventricular fibrillation ablation in the setting of ischemic heart disease. J Cardiovasc Electrophysiol. 2005;16: S59 –S70. 15. Soejima K, Couper G, Cooper JM, Sapp JL, Epstein LM, Stevenson WG. Subxiphoid surgical approach for epicardial catheter-based mapping and ablation in patients with prior cardiac surgery or difficult pericardial access. Circulation. 2004;110:1197–1201. 16. Ram W, Meyer H. Heart catheterization in a neonate by interacting magnetic fields: a new and simple method of catheter guidance. Catheter Cardiovasc Diagn. 1991;22:317–319. 17. Basu Ray I, Houghtaling C, McPherson C, Kastelein N, Ruskin JN, Reddy VY. Use of a remote magnetically-guided catheter for ventricular electroanatomical substrate mapping and ablation in a porcine model of healed myocardial infarction. Heart Rhythm. 2005;2:S158. Abstract. 18. Kim RJ, Fieno DS, Parrish TB, Harris K, Chen EL, Simonetti O, Bundy J, Finn JP, Klocke FJ, Judd RM. Relationship of MRI delayed contrast enhancement to irreversible injury, infarct age, and contractile function. Circulation. 1999;100:1992–2002. 19. Lardo AC, Cordeiro MA, Silva C, Amado LC, George RT, Saliaris AP, Schuleri KH, Fernandes VR, Zviman M, Nazarian S, Halperin HR, Wu KC, Hare JM, Lima JA. Contrast-enhanced multidetector computed tomography viability imaging after myocardial infarction: characterization of myocyte death, microvascular obstruction, and chronic scar. Circulation. 2006;113:394 – 404. 20. Abbara S, Desai JC, Butler J, Nieman K, Reddy VY. Mapping epicardial fat with multidetector computed tomography to facilitate percutaneous transepicardial arrhythmia ablation. Eur J Radiol. 2006;57:417– 422.

CLINICAL PERSPECTIVE Significant advances have been made in recent years in catheter ablation of scar-related ventricular tachycardia as a result of both (1) an improved understanding of its pathophysiology and (2) technological advances that aid in performing the procedure. In particular, substrate mapping, in which the ventricle is mapped predominantly during sinus rhythm, allows successful catheter ablation of virtually all ventricular tachycardias, regardless of their hemodynamic effect. Although highly effective, ventricular tachycardia ablation remains an uncommon procedure, in part because of the advanced operator skill required to perform detailed ventricular mapping. The present study provides clinical evidence for the safety and feasibility of remote catheter navigation in performing ventricular substrate mapping in a wide range of disease pathologies. Used in concert with a compatible electroanatomic mapping system, remote magnetic navigation technology proved capable of performing each of the 3 major components of substrate-based ventricular tachycardia ablation: (1) delineating and identifying endocardial and epicardial scarred tissue, (2) performing the necessary electrophysiological maneuvers required to identify those arrhythmogenic zones critical for maintaining tachycardia, and (3) delivering radiofrequency energy to terminate both endocardial and epicardial ventricular tachycardias. The enhanced maneuverability of the remotely navigated catheter using this system allowed accurate mapping of otherwise difficult-to-reach areas. Finally, the “soft touch” of the remotely guided catheter permitted this detailed ventricular mapping with minimal fluoroscopy use. Thus, by obviating the need for the advanced operator skill required for substrate mapping, remote navigation technology may result in more widespread and effective catheter ablation of ventricular tachycardia.

Cardiovascular Surgery Adjustable, Physiological Ventricular Restraint Improves Left Ventricular Mechanics and Reduces Dilatation in an Ovine Model of Chronic Heart Failure Ravi K. Ghanta, MD; Aravind Rangaraj, MD; Ramanan Umakanthan, MD; Lawrence Lee, MD; Rita G. Laurence, BS; John A. Fox, MD; R. Morton Bolman III, MD; Lawrence H. Cohn, MD; Frederick Y. Chen, MD, PhD Background—Ventricular restraint is a nontransplantation surgical treatment for heart failure. The effect of varying restraint level on left ventricular (LV) mechanics and remodeling is not known. We hypothesized that restraint level may affect therapy efficacy. Methods and Results—We studied the immediate effect of varying restraint levels in an ovine heart failure model. We then studied the long-term effect of restraint applied over a 2-month period. Restraint level was quantified by use of fluid-filled epicardial balloons placed around the ventricles and measurement of balloon luminal pressure at end diastole. At 4 different restraint levels (0, 3, 5, and 8 mm Hg), transmural myocardial pressure (Ptm) and indices of myocardial ˙ O2) were determined in control (n⫽5) and ovine heart failure (n⫽5). Ventricular restraint oxygen consumption (MV ˙ O2, and improved mechanical efficiency. An optimal physiological restraint level of therapy decreased Ptm and MV 3 mm Hg was identified to maximize improvement without an adverse affect on systemic hemodynamics. At this ˙ O2 indices decreased by 27% and 20%, respectively. The serial longitudinal optimal level, end-diastolic Ptm and MV effects of optimized ventricular restraint were then evaluated in ovine heart failure with (n⫽3) and without (n⫽3) restraint over 2 months. Optimized ventricular restraint prevented and reversed pathological LV dilatation (130⫾22 mL to 91⫾18 mL) and improved LV ejection fraction (27⫾3% to 43⫾5%). Measured restraint level decreased over time as the LV became smaller, and reverse remodeling slowed. ˙ O2, and the rate Conclusions—Ventricular restraint level affects the degree of decrease in Ptm, the degree of decrease in MV of LV reverse remodeling. Periodic physiological adjustments of restraint level may be required for optimal restraint therapy efficacy. (Circulation. 2007;115:1201-1210.) Key Words: heart failure 䡲 remodeling 䡲 surgery

V

entricular restraint is a nontransplantation surgical treatment for heart failure (HF) in which both ventricles are wrapped with material designed to mechanically constrain the ventricles.1,2 The intent is to provide passive end-diastolic support to constrain ventricular size without pathological diastolic restriction. Numerous studies have demonstrated that passive ventricular restraint may prevent or reverse left ventricular (LV) dilation and remodeling in HF.3– 8 The precise mechanics of this, however, are not known. Our hypothesis is that ventricular restraint decreases transmural myocardial pressure (Ptm) by pressure application on the epicardium at end diastole. This hypothesis has never been documented because current restraint devices do not allow for the measurement of restraint level or Ptm.9,10 In addition, definitive studies that evaluate the effect of alteration of

restraint level on ventricular mechanics have not yet been performed, and no criteria exist to optimize restraint therapy in a physiological manner to maximize LV performance.

Clinical Perspective p 1210 Our hypothesis is that ventricular restraint unloads the LV and reduces Ptm and myocardial oxygen consumption ˙ O2). We also hypothesize that restraint level affects (MV therapeutic efficacy, and that, as the LV size decreases, the effective restraint level will decrease as an indication of LV improvement. To evaluate these questions, we developed an adjustable fluid-filled balloon to quantitatively apply restraint to the entire epicardial surface of both ventricles. With this new technique— quantitative ventricular restraint (QVR)— both the direct measurement of Ptm and the quantitative application of ventricular restraint are possible.

Received October 20, 2006; accepted January 2, 2007. From the Division of Cardiac Surgery (R.K.G., A.R., R.U., L.L., R.G.L., R.M.B., L.H.C., F.Y.C) and Division of Cardiac Anesthesia (J.A.F.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass. Guest Editor for this article was Robert A. Kloner, MD, PhD. Correspondence to Frederick Y Chen, MD, PhD, Division of Cardiac Surgery, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.671370

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Methods Study Design Overview This study was divided into 2 parts. In part I, we evaluated the ˙ O2 immediate effect of ventricular restraint on Ptm and indices of MV in normal (n⫽5) and HF ovines (n⫽5). To produce failure, ovines underwent first (D1) and second (D2) diagonal coronary artery ligation. HF, defined as an LV ejection fraction (EF) ⬍35% and a 100% increase in LV end-diastolic volume (EDV), developed 2 months after ligation. All animals underwent placement of QVR balloons over the ventricular epicardium in a terminal study. The ˙ O2, systolic effect of ventricular restraint level on Ptm, indices of MV contractility, and systemic hemodynamics in normal and HF ovines was determined. We then identified an optimal restraint level that ˙ O2 and minimized maximized improvement in Ptm and indices of MV adverse effects on systemic hemodynamics. In part II, optimized QVR was applied in a 4-month longitudinal study in HF ovines to assess the long-term effect of restraint on LV EDV, EF, and the level of restraint itself. In this long-term study, 6 ovines underwent D1/D2 ligation and developed HF 2 months postligation. After the development of HF, 3 animals underwent QVR balloon implantation at the optimal restraint level and 3 animals underwent no device implantation as the control group. All animals were then followed up for an additional 2 months with serial echocardiography. In the QVR animals, restraint level was measured weekly. Throughout the study period, the QVR balloon volume was not adjusted. At termination, fluid was withdrawn from the QVR balloon and measured to verify that any changes in restraint level were not caused by a leak in the balloon. A total of 16 adult male ovines (30 to 40 kg) were used for this study. All animals received humane care in compliance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 86-23, revised 1996). The protocol was approved by the Institutional Animal Care and Use Committee at Harvard Medical School.

Quantitative Ventricular Restraint Balloons We designed and constructed a half-ellipsoidal balloon from medical-grade polyurethane sheets (Polyzen, Apex, NC; Figure 1). Each balloon was composed of two 1-mm-thick layers. An access line was placed between the 2 layers to allow pressure measurement inside the balloon lumen and the addition or withdrawal of fluid. The outer layer of the balloon was composed of a flexible but inelastic polyurethane layer. Because the outer layer of the balloon is inelastic but flexible, fluid introduced into the balloon lumen has only one direction of filling space—inward toward the epicardial surface. This creates a tighter wrap. Conversely, withdrawal of fluid from the balloon lumen results in a looser wrap. The balloon access line was connected to an implantable portacath (Bard Access Systems). The port was accessed with an 18-gauge Huber needle and connected to a Statham P10EZ pressure transducer (SpectraMed, Oxford, Calif). Measurement of the luminal pressure inside the balloon when the heart is largest in volume— end diastole—allows wrap tightness to be precisely quantified. At end diastole, the pressure inside the balloon is solely a function of heart volume plus the fluid volume and the mechanical properties of the outer layer. We previously verified with a dynamic testing procedure that the frequency response of the Statham transducer plus the cannula was adequate to measure fluctuations in balloon pressure to the required accuracy.11,12 In parts I and II, QVR balloons were implanted via a median sternotomy and pericardiotomy. The QVR balloon was placed over the heart to completely envelop both ventricles and secured to the atrioventricular groove. In part II, the QVR portacath was tunneled through the fifth intercostal space into the left anterior chest wall. A separate 3-cm chest wall incision was made to secure the port. The sternum and port incision were then closed in layers.

Figure 1. A, Photograph of half-ellipsoidal fluid-filled balloon for QVR. Restraint level may be quantified by measurement of luminal pressure via the attached portacath. Restraint level may be adjusted by a change in volume of fluid instilled in the balloon. B, Intraoperative photograph of balloon implantation. The QVR balloon is placed below the atria, around both ventricles, and secured to the heart along the arteriovenous groove. The portacath is tunneled through the left anterior chest wall. LA indicates left atrium; RA, right atrium.

diastole, the time point when the heart is largest in volume. To change restraint level, volume was added or removed from the balloon. Fluid was removed to lower the restraint level or added to raise the restraint level while balloon pressure was monitored in real time. To define an arbitrary maximum restraint level applied to a given subject without tamponade, we injected saline into the balloon until mean aortic pressure decreased by 10 mm Hg. We defined this restraint level as Pmax. At restraint levels higher than Pmax, tamponade physiology prevailed. We recorded data at 4 sequential restraint levels: 0 (baseline), 1/3 Pmax, 2/3 Pmax, and Pmax.

Quantitative Ventricular Restraint Individual restraint levels were defined by the maximum pressure applied by the balloon to the epicardium, given a constant volume of saline inside the balloon. Maximum balloon pressure occurred at end

Heart Failure Model A postinfarction ovine model of HF, described by Moainie et al, was used.13 This model includes many of the features of ischemic human

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TABLE 1. Baseline LV Size, Mechanics, and Energetics in Normal vs HF Ovines Normal Ovines (n⫽5)

HF Ovines (n⫽5)

P

LVEF, %

51.4⫾12.9

31.5⫾9.9

0.03

LV EDV, mL

61.1⫾6.6

123.5⫾23.4

0.005

Stroke volume, mL

31.9⫾11.1

37.7⫾10.7

NS

Cardiac output, L/min

3.12⫾0.89

3.09⫾0.58

NS

Echocardiography

Pressure-volume Mean Ptm, mm Hg

33.5⫾4.5

30.4⫾4.3

NS

EDPtm, mm Hg

5.7⫾1.8

9.4⫾2.0

0.02

Ees, mm Hg/mL

2.34⫾0.37

1.55⫾0.38

0.01

9.9⫾8.1

55.4⫾17.6

0.003

Volume axis intercept, mL Energetics Tension time index, mm Hg 䡠 s

18.5⫾3.4

19.4⫾4.6

NS

Stroke work, mm Hg mL

1642⫾609

1246⫾342

NS

571⫾215

1042⫾359

0.046

73⫾11.5

54⫾6.3

Potential energy, mm Hg mL/beat Mechanical efficiency, %

0.02

EDPtm indicates end-diastolic transmural myocardial pressure; Ees, end-systolic elastance; and NS, not significant.

dilated cardiomyopathy, which includes global increase in LV EDV and end-systolic volumes and sphericity index and reductions in systolic wall thickening and EF. A total of 11 ovines (5 in part I; 6 in part II) underwent D1/D2 ligation for this study. A left anterior thoracotomy was performed through the 4th intercostal space. D1 and D2 were identified and ligated with 4-0 polypropylene sutures. The thoracotomy was closed in layers. A single chest tube was introduced through a separate incision and removed before extubation. Serial echocardiography was performed on all ovines preinfarction and then weekly. HF, defined as an LV EF ⬍35% and a 100% increase in LV EDV, developed in all ovines 8 weeks postinfarction (Table 1).

Anesthesia and Postoperative Care For coronary ligation and balloon implantation, animals were presedated with Telazol (Wyeth)(4.4 mg/kg) and endotracheally intubated. Anesthesia was maintained with 1% to 2% isofluorane. A 16-gauge intravenous line was placed in the left external jugular vein for access and measurement of central venous pressure. Animals received magnesium (2 g intravenously), amiodarone (1.5 mg/kg intravenously), and lidocaine (3 mg/kg intravenously) before infarction and an infusion of amiodarone (0.01 mg/kg per min) and lidocaine (2 mg/min) for 60 minutes afterward. Animals received buphrenorphine (5 ␮g/kg intramuscularly every 12 hours for 2 days) for pain control and cefazolin (4 mg/kg intramuscularly every 12 hours for 2 days) for antibiotic prophylaxis.

Pressure-Volume Analysis In part I, 5 HF and 5 normal ovines were placed under general anesthesia and underwent QVR balloon placement. An electromagnetic aortic flow probe (Carolina Medical Electronics, King, NC) was placed to measure aortic flow. High-fidelity micromanometers (Millar Instruments, Houston, Tex) were placed in the LV and ascending aorta via the right and left femoral arteries. An 8F conductance catheter for LV volume measurement (Webster Laboratories, Baldwin Park, Calif) was placed via the right carotid artery.14 –16 A 20-mL balloon occluder was introduced into the inferior vena cava via the right femoral vein. All electrocardiographic and hemodynamic signals were digitized at 200 Hz.

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For each subject, all hemodynamic signals were recorded at 4 sequential ventricular restraint levels (0, 1/3 Pmax, 2/3 Pmax, and Pmax) over 20 beats. At each restraint level, a caval occlusion was performed with the balloon occluder. All data were collected with the ventilator off to avoid respiratory variations. All data were analyzed on a microcomputer with MATLAB (The Mathworks, Natick, Mass). End diastole was defined as the time point in the cardiac cycle that corresponded to the R-wave on the ECG. This point corresponds closely to the closing of the mitral valve. Begin-ejection was defined as the point at which aortic flow first becomes non-zero. End systole was defined as the point at which aortic flow fell to zero after the beginning of ejection. Hemodynamic signals were ensemble-averaged over 10 beats. Ptm across the heart wall was defined as the LV pressure minus the epicardial pressure (measured by the balloon). The transmural tension-time index (TtTI) was calculated by integration of Ptm with respect to time over the cardiac cycle. The end-systolic pressure-volume relationship was then calculated from the caval occlusion data, by the procedure of Kono et al.17 We then determined the transmural pressure-volume area (PtVA), stroke work (area circumscribed by the pressure-volume loop), potential energy (PtVA ⫺ stroke work), and mechanical efficiency (ratio of stroke work to PtVA) for each restraint level.18

Serial Echocardiography In part II, quantitative 2-dimensional transthoracic echocardiography (Cypress Acuson, Siemens Medical Solutions, Malvern, Pa) with a 3.5-MHz probe was performed preinfarction and then weekly in all animals throughout the study period. Parasternal long-axis images and short-axis images of the LV to the tips of the papillary muscles were obtained. LV EDV and end-systolic volumes were calculated by Simpson’s rule with the Cypress Acuson. LV EF was calculated as the ratio of the difference of EDV and end-systolic volume to EDV.19 All echocardiograms were analyzed by an investigator blinded to the treatment group and time.

Statistics All data are expressed as mean⫾SD. Means between groups were compared with the Student t test. Repeated measures data were assessed with a doubly multivariate repeated measures design to control for the interrelatedness of our outcomes. Statistics were performed with SPSS 12.0 (Chicago, Ill). Results with P⬍0.05 were considered significant. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Quantitative Ventricular Restraint By measuring balloon pressure via the portacath, real-time intraoperative and postoperative measurement of ventricular restraint level was possible. Representative intraoperative tracings for aortic flow, aortic pressure, LV pressure, epicardial (balloon) pressure, and Ptm are depicted in Figure 2. Epicardial pressure applied to the heart varied throughout the cardiac cycle, peaked at end diastole, decreased rapidly during systole after the onset of ejection, and then increased rapidly during diastole as blood filled the heart. Restraint level was adjusted by the injection or withdrawal of saline from the balloon lumen via the access port. Injected volume of saline varied from 0 and 360 mL as needed to attain the desired restraint level. The mean Pmax for all ovines in this study was determined to be 8.0⫾0.1 mm Hg. No difference was observed in Pmax between normal and HF ovines.

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Part I Baseline Differences in LV Mechanics and Energetics Between Normal and HF Ovines Left ventricular size, mechanics, and energetics observed in the normal and postinfarction HF ovine model are tabulated in Table 1. HF ovines had enlarged hearts with depressed contractility, elevated diastolic pressures, and impaired mechanical efficiency. HF ovines demonstrated depressed LV EF (⬍35%) and higher LV EDV (increase of 62.1 mL or 102%). Decreased contractility was manifested by a decreased end-systolic elastance (Ees) and a rightward shift in the end-systolic pressure-volume relationship. There was no statistically significant difference in stroke volume or cardiac output. Although there was no difference in mean Ptm, Ptm was elevated throughout diastole in HF ovines compared with controls. End-diastolic Ptm was 3.7 mm Hg (65%) higher in HF ovines compared with normal ovines. In HF ovines, potential energy was 471 mm Hg-mL/beat (82%) higher secondary to increased LV volume. HF hearts were mechanically less efficient than normal hearts. No statistically significant difference existed in stroke work, PtVA, or TtTI between HF and normal ovines. Immediate Effects of Ventricular Restraint on Left Ventricular Mechanics and Energetics The immediate effects of ventricular restraint on LV mechanics and indices of myocardial energetics in HF and normal ovines are illustrated in Figure 3. Transmural Myocardial Pressure Ventricular restraint decreased Ptm throughout the cardiac cycle for both HF and normal ovines. The greatest decrease in Ptm occurred during diastole, with maximal reduction at end diastole. Figure 3A demonstrates the percent reduction in mean Ptm as a function of restraint level for normal and HF ovines. In HF ovines, mean Ptm was significantly reduced by 7%, 19%, and 35%, respectively, at each of the 3 sequentially increasing restraint levels from baseline (P⬍0.05). Ventricular restraint reduced Ptm equally in normal and HF ovines. Figure 3B demonstrates the percent reductions in Ptm during systole, diastole, and at end diastole in HF ovines. The greatest reduction was noted at end diastole. EDPtm decreased by 27%, 30%, and 36%, respectively, for all 3 sequentially increasing restraint levels from the baseline (P⬍0.05).

Figure 2. Representative hemodynamic recordings of aortic flow, mean arterial pressure, LV pressure, balloon pressure, and transmural myocardial pressure are demonstrated above. Balloon pressure reaches a maximum at end diastole, rapidly falls during systole, and then rapidly rises during diastole. ED indicates end diastole; BE, begin-ejection; and ES, end systole.

Indices of Myocardial Energetics Ventricular restraint decreased TtTI and PtVA in both normal and HF ovines (Figure 3, C and D). In HF ovines, TtTI decreased by 12%, 19%, and 33%, respectively, for all 3 increasing restraint levels tested from the baseline (P⬍0.05). In HF ovines, PtVA decreased by 20%, 27%, 51%, respectively, for all 3 increasing restraint levels from baseline (P⬍0.05). Ventricular restraint reduced TtTI and PtVA equally in normal and HF ovines. In HF ovines, ventricular restraint improved mechanical efficiency (Figure 3E). Mechanical efficiency improved from 55% at baseline to 63% at Pmax (P⬍0.05). In normal ovines, ventricular restraint had no effect on mechanical efficiency (P⫽0.65).

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Figure 3. Short-term effect of ventricular restraint on LV mechanics and indices of myocardial energetics as a function of restraint level in normal and HF ovines. A, Mean percentage change in Ptm from baseline; B, mean percentage change in Ptm from baseline during systole, diastole, and end diastole in HF ovines; C, mean percentage change in TtTI from baseline; D, mean percentage change in PtVA from baseline; E, mechanical efficiency; F, Ees. *Statistically significant change (P⬍0.05) from baseline. †Statistically significant (P⬍0.05) difference between normal ovines (䡩) and HF ovines (䢇).

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March 13, 2007 Adverse Effects of High Levels of Restraint on Hemodynamics

Restraint Level

MAP, mm Hg

HR, bpm

SV, mL

CO, L/min

LV EDP, mm Hg

CVP, mm Hg

Baseline

51.5 (⫾5.9)

84 (⫾14)

37.7 (⫾10.7)

3.1 (⫾0.6)

10.6 (⫾4.6)

14.1 (⫾7.9)

1/3 Pmax

51.5 (⫾7.2)

86 (⫾13)

32.7 (⫾7.4)

2.8 (⫾0.5)

10.0 (⫾3.6)

13.3 (⫾6.9)

2/3 Pmax

48.5 (⫾7.2)*

87 (⫾11)

31.1 (⫾4.7)

2.7 (⫾0.6)

11.2 (⫾5.4)

15.5 (⫾7.1)

Pmax

43.0 (⫾6.9)*

87 (⫾10)

26.0 (⫾5.1)*

2.3 (⫾0.7)*

11.7 (⫾4.6)*

18.5 (⫾9.7)*

MAP indicates mean arterial pressure; HR, heart rate; SV, stroke volume; CO, cardiac output; and CVP, central venous pressure. *Statistically significant change from baseline (P⬍0.05).

End-Systolic Elastance In both normal and HF ovines, ventricular restraint had no immediate effect on Ees (Figure 3F). In HF ovines, Ees was 1.55⫾0.38 mm Hg/mL at baseline and 1.61⫾0.39 mm Hg/ mL at Pmax. In normal ovines, Ees was 2.54⫾0.37 mm Hg/mL at baseline and 2.37⫾0.39 mm Hg/mL at Pmax. Similarly, ventricular restraint had no effect on the volume-axis intercept in both normal and HF ovines. Thus, ventricular restraint did not affect contractility in the short term as measured by end-systolic pressure-volume relationship. Immediate Adverse Effects of Ventricular Restraint on Hemodynamics Mean aortic pressure, heart rate, stroke volume, cardiac output, LV end-diastolic pressure, and central venous pressure in HF ovines for each restraint level are summarized in Table 2. As expected, at high restraint levels, restrictive physiology prevailed as demonstrated by a decrease in mean aortic pressure and cardiac output with a concomitant increase in LV EDP and central venous pressure. At low restraint levels, however, aortic pressure and cardiac output were unchanged. Optimization of Ventricular Restraint Level Increasing levels of wrap tightness caused greater decreases ˙ O2. Very high levels of restraint in Ptm and indices of MV impaired systemic hemodynamics. This suggests that an optimal restraint level exists, where the reduction in Ptm and ˙ O2 is maximized while the effect on systemic hemodyMV namics is minimized. Ideally, this restraint level would correct pathologically elevated stress and strain seen in HF. Figure 4A and 4B illustrate the short-term effect of ventricular restraint on LV EDPtm and potential energy in HF ovines. The shaded regions in Figure 4, A and B, indicate the normal range of LV EDPtm and potential energy found in ovines. Figure 4C illustrates LV EDPtm and mean aortic pressure, normalized to baseline, for the 4 different restraint levels studied. At a restraint level of 1/3 Pmax (3 mm Hg), previously elevated LV EDPtm and potential energy were reduced and corrected to normal levels, whereas mean aortic pressure remained unaffected until restraint levels were greater than 1/3 Pmax. Thus, a restraint level of 1/3 Pmax (3 mm Hg) corrected abnormally elevated LV EDPtm and potential energy with no effect on mean aortic pressure. These data

suggest that a restraint level of 3 mm Hg is the optimal physiological restraint level for these HF ovines.

Part II Long-Term Optimized Ventricular Restraint Therapy QVR was then applied at the optimal therapeutic restraint level of 1/3 Pmax (3 mm Hg) in 3 HF ovines for 2 months and compared with controls that received no treatment over the same time period. Figure 5, A and B, demonstrates the effect of optimized restraint on LV EDV and LV EF in these ovines compared with controls. At the time of QVR balloon implantation, both groups demonstrated dilated cardiomyopathy, with increased LV EDV and depressed LV EF. Over the subsequent 2-month period, QVR significantly decreased LV EDV from 130⫾22 mL to 91⫾18 mL (⫺30%, P⬍0.05), and improved LV EF from 27⫾3% to 43⫾5% (P⬍0.05). In contrast, control animals developed increasing measures of HF, which included an increase of LV EDV from 113⫾19 mL to 168⫾7 mL (49%, P⬍0.05) and a decrease of LV EF from 33⫾2% to 19⫾8% (P⬍0.05). LV EDV was 77 mL lower (⫺90%, P⬍0.05) and LV EF was 24% higher (P⬍0.05) in QVR ovines compared with controls. In QVR ovines, LV EDV decreased 21 days after the initiation of QVR therapy (P⬍0.05), and LV EF improved 42 days after initiation of QVR therapy (P⬍0.05). Furthermore, restraint level decreased as the heart decreased in size (Figure 5C). As restraint level decreased, reverse remodeling slowed, as measured by the rate of change of LV EDV over time.

Discussion Previous studies have demonstrated the potential for ventricular restraint to prevent and reverse pathological LV remodeling.1– 8,20,21 To date, no study has evaluated LV mechanics in restraint or the effect of adjustable restraint. Current ventricular restraint techniques, such as the Acorn Cardiac Support Device (Acorn Cardiovascular, St. Paul, Minn) and the Paracor (Paracor Medical Systems, Sunnyvale, Calif), do not allow for either the quantitative adjustment of restraint level or the measurement of Ptm.3,10 A standard for wrap tightness does not exist. Surgeons are instructed to apply the Acorn Cardiac Support Device in such a way that it fits not too loosely and not too tightly, but “snugly.” Once wrapped at the initial procedure, the restraint wrap is constant and unchanging even as the heart undergoes reverse remodeling.

Ghanta et al

Figure 4. Optimization of ventricular restraint level. A, End-diastolic Ptm in HF ovines (䢇) at 4 different restraint levels. B, Potential energy in HF ovines at 4 different restraint levels. In A and B, the shaded region represents the range in normal ovines. C, Normalized end-diastolic Ptm and mean aortic pressure at the 4 different restraint levels. At the optimal level of restraint pathologically elevated end-diastolic Ptm and potential energy are corrected to the normal level with no affect on mean aortic pressure. *Statistically significant (P⬍0.05) change from baseline.

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To address these limitations, we used a half-ellipsoid, fluid-filled epicardial balloon. Restraint therapy ultimately acts on the heart via application of either shear stresses (tangential forces) or normal stresses (perpendicular forces) to the epicardium. Because neither is quantified with current restraint therapy, a separation of wrap mechanics from ventricular mechanics is neither possible nor measurable. The QVR balloon essentially introduces a fluid layer as the sole means to implement ventricular restraint. Because fluids by definition cannot sustain shear stresses, the QVR balloon affects ventricular restraint by normal forces only. Other restraint devices apply some degree of shear stress. Sheer stress does not contribute to reverse remodeling but may contribute to cardiac restriction. The QVR balloon thus provides a methodology to definitively understand and quantify the effect of ventricular restraint by measurement of those normal forces (the only stresses exerted) on the epicardium. With this technique, we demonstrated that ventricular restraint decreases Ptm throughout the cardiac cycle. The greatest reduction in Ptm occurred during diastole, with the maximum reduction at end diastole. The largest reduction in EDPtm occurred when restraint level was increased from 0 (baseline) to 1/3 Pmax (3 mm Hg). As restraint level increased, further decreases in Ptm were more pronounced during systole with relatively modest further reductions during diastole. Thus, at low restraint levels, ventricular restraint affected Ptm primarily during diastole. At high restraint levels, ventricular restraint acted primarily during systole to decrease Ptm, consistent with early restrictive physiology. Important concerns with restraint therapy are cardiac restriction and impairment of coronary blood flow. The maximum applicable restraint level will be limited by the restrictive effect of the restraint device, primarily on the lowpressure right ventricle. It is well known that elevated epicardial pressure impairs right ventricular filling and atrioventricular coupling. In the present study, high levels of restraint (8 mm Hg) decreased cardiac output and increased central venous pressure. At low restraint levels (3 mm Hg), no evidence of cardiac restriction was seen (Table 2). Previous studies have demonstrated that elevated pericardial pressure also decreases coronary blood flow through either a reduction of coronary perfusion pressure or an increase in coronary vascular resistance.22,23 This effect, however, primarily only occurs in low cardiac output states or very high pericardial pressures (⬎15 mm Hg). Although we did not measure coronary blood flow, at our low levels of epicardial pressure we found no evidence of significant ischemia, as contractility was not impaired and LV function improved. Excessive ventricular restraint may impair coronary blood flow and be an important concern, particularly in patients with underlying coronary artery disease and subclinical myocardial ischemia. Because wall stress is directly proportional to Ptm, these results suggest that ventricular restraint reduces wall stress. If ventricular restraint reduces wall stress, a major determinant

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Figure 5. Efficacy of optimized QVR. A, LV EDV in treatment ovines (䢇) and control HF ovines (䡩). B, LV EF in treatment and control HF ovines. C, Restraint level and rate of change of LV EDV (dEDV/dt) over a 2-month treatment period. *Statistically significant change (P⬍0.05) from time 0 (time of QVR balloon implantation). †Statistically significant difference (P⬍0.05) between treatment and control HF ovines.

˙ O2, one would expect to see a decrease in O2 uptake. In of MV ˙ O2 indices are acutely decreased this study, we show that MV secondary to a decrease in Ptm and LV unloading. Because of higher filling volumes, dilated cardiomyopathic hearts have higher potential energy (or internal work) compared with normal controls. These data show that in cardiomyopathy, ventricular restraint unloads the LV and decreases potential energy more significantly than stroke work. Mechanical efficiency is thus improved [stroke work⫼(stroke work ⫹ potential energy)] in cardiomyopathic hearts. Conversely, in normal hearts with typical LV loading, ventricular restraint decreases potential energy and stroke work equally and thus has no effect on mechanical efficiency. Comparing normal to cardiomyopathic hearts, we found that ventricular restraint decreased Ptm, TtTi, and PtVA equally at the levels we tested. We conclude that LV size, load, and contractility were not determinative factors in the ˙ O2 at the restraint levels we tested. reduction of Ptm and MV We also conclude that the mechanical effect of restraint is primarily determined by wrap mechanics and not by the contractile or mechanical properties of the heart itself. Standardization of wrap mechanics should thus allow for standardization of therapy for patients. An interesting question is whether optimization to the highest physiological restraint level may improve therapeutic efficacy. By application of optimal restraint over a 2-month period, we demonstrated a 30% reduction in LV EDV compared with baseline in ovines. Progressive LV dilatation was prevented and reversed, as LV EDV was 90% higher in controls after 2 months. In comparison, Saveedra et al found a 19⫾4% reduction in LV EDV over 6 months in a canine model of HF that used the Acorn Cardiac Support Device.8 Other studies in animal models demonstrated prevention rather than reversal of LV dilation.5,6 In human studies, modest improvements in LV EDV have been found.24 Reverse remodeling may be dependent on restraint level. At low restraint levels, ventricular restraint may provide simple containment. At such levels of restraint, progression of disease may be halted but reversal of dilatation may not necessarily be enabled. Our results suggest that at higher restraint levels ventricular restraint contains and unloads the LV, which leads to decreased LV size. The greater decrease in LV EDV seen in this study compared with previous studies may be caused by the optimized restraint level. The data demonstrate that restraint level is not constant as the LV remodels. We found that pressures measured in the QVR balloon decreased with time (Figure 5C). As the LV undergoes reverse remodeling and becomes smaller, the ventricular wrap stays constant. Our conclusion is that a smaller LV effectively loosens the ventricular restraint wrap and restraint level decreases. In addition, as the restraint level decreased, reverse remodeling slowed (as measured by the rate of change of LV EDV over time) (Figure 5C). This finding suggests that restraint level is an important determinant of reverse remodeling efficiency. To maximize therapeutic efficacy, periodic adjustment of wrap tightness to maintain the most effective physiological restraint level may be beneficial.

Ghanta et al With present devices, the restraint wrap is constant and unchanging, even as the heart undergoes reverse remodeling. With a quantitative technique, patient-specific restraint levels might be identified at the time of restraint device implantation. Postoperatively, restraint level could be measured and adjusted via a portacath to maintain the optimal level. Restraint level could be adjusted as the LV remodels. Periodic assessment of ventricular mechanics and filling in realtime as wrap tightness is adjusted noninvasively could be performed via echocardiography to ensure the most physiologically appropriate restraint level. This study has some important limitations. We used 16 ovines in this study (10 in part I and 6 in part II). Only 3 underwent long-term QVR implantation. Despite this small sample size, we still had sufficient power to identify a significant difference in LV EDV and LV EF in control versus QVR ovines. The analysis of part I was performed in a time-limited terminal experimental preparation under general anesthesia. General anesthesia diminishes compensatory adrenergic and neurohormonal responses, such as a compensatory rise in heart rate with a significant fall in blood pressure observed at marked levels of restraint. These responses may alter the optimal restraint level in conscious subjects. In addition, we used pressure-volume analysis to determine the short-term effects of restraint on LV mechanics, energetics, and mechanical efficiency. Although these techniques are widely validated and allow determination of the short-term effects of restraint, the effect of restraint on wall stress will change over time as the LV changes in size, shape, and mass. More sophisticated techniques, such as cardiac magnetic resonance imaging, that take into account 3-dimensional geometry will be required to quantitatively evaluate the long-term effects of restraint on wall stress. In summary, we have demonstrated a new quantitative technique for the measurement and application of adjustable ventricular restraint. With use of QVR in a postinfarction ovine model of HF, our results demonstrate that ventricular ˙ O2 and improves mechanical restraint decreases Ptm and MV ˙ O2, and mechanical efficiency. Improvements in Ptm, MV efficiency were dependent on wrap tightness. With QVR, an optimized therapeutic restraint level was identified to restore ˙ O2 and to physiology and maximize reductions in Ptm and MV minimize the effects on systemic hemodynamics. Optimized ventricular restraint reversed pathological LV dilation and improved LV function. Restraint level, however, decreased over time as the LV became smaller, and reverse remodeling slowed until a new steady-state LV volume was achieved. Clinically, interval adjustment of restraint to the optimal physiological level may be required to maintain maximum therapeutic benefit.

Sources of Funding This work was supported by Brigham and Women’s Hospital, Department of Surgery (Dr Chen), Cardiac Surgery Research Fund (Dr Cohn, Dr Bolman); by the American Association for Thoracic Surgery, Andrew G. Morrow Scholarship (Dr Chen); by the National Institutes of Health F32 National Research Service Award (Dr Ghanta); and by the Center for Integration of Medicine and Innovative Technology (CIMIT; Dr Chen).

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Disclosures The Brigham and Women’s Hospital has patent rights on the device described in this article.

References 1. Oh JH, Badhwar V, Mott BD, Li CM, Chiu RC. The effects of prosthetic cardiac binding and adynamic cardiomyoplasty in a model of dilated cardiomyopathy. J Thorac Cardiovasc Surg. 1998;116:148 –153. 2. Konertz WF, Shapland JE, Hotz H, Dushe S, Braun JP, Stantke K, Kleber FX. Passive containment and reverse remodeling by a novel textile cardiac support device. Circulation. 2001;104:I270 –I275. 3. Power JM, Raman J, Dornom A, Farish SJ, Burrell LM, Tonkin AM, Buxton B, Alferness CA. Passive ventricular constraint amends the course of heart failure: a study in an ovine model of dilated cardiomyopathy. Cardiovasc Res. 1999;44:549 –555. 4. Sabbah HN, Sharov VG, Gupta RC, Mishra S, Rastogi S, Undrovinas AI, Chaudhry PA, Todor A, Mishima T, Tanhehco EJ, Suzuki G. Reversal of chronic molecular and cellular abnormalities due to heart failure by passive mechanical ventricular containment. Circ Res. 2003;93: 1095–1101. 5. Pilla JJ, Blom AS, Brockman DJ, Ferrari VA, Yuan Q, Acker MA. Passive ventricular constraint to improve left ventricular function and mechanics in an ovine model of heart failure secondary to acute myocardial infarction. J Thorac Cardiovasc Surg. 2003;126: 1467–1476. 6. Cheng A, Nguyen TC, Malinowski M, Langer F, Liang D, Daughters GT, Ingels NB Jr, Miller DC. Passive ventricular constraint prevents transmural shear strain progression in left ventricle remodeling. Circulation. 2006;114:I79 –I86. 7. Acker MA. Clinical results with the Acorn cardiac restraint device with and without mitral valve surgery. Semin Thorac Cardiovasc Surg. 2005; 17:361–363. 8. Saavedra WF, Tunin RS, Paolocci N, Mishima T, Suzuki G, Emala CW, Chaudhry PA, Anagnostopoulos P, Gupta RC, Sabbah HN, Kass DA. Reverse remodeling and enhanced adrenergic reserve from passive external support in experimental dilated heart failure. J Am Coll Cardiol. 2002;39:2069 –2076. 9. Sabbah HN. The cardiac support device and the myosplint: treating heart failure by targeting left ventricular size and shape. Ann Thorac Surg. 2003;75:S13–S19. 10. Magovern JA. Experimental and clinical studies with the Paracor cardiac restraint device. Semin Thorac Cardiovasc Surg. 2005;17:364 –368. 11. Chen FY, Aklog L, deGuzman BJ, Laurence RG, Couper GS, Appleyard RF, Cohn LH, McMahon TA. New technique measures decreased transmural myocardial pressure in cardiomyoplasty. Ann Thorac Surg. 1995;60:1678 –1682. 12. Chen FY, deGuzman BJ, Aklog L, Lautz DB, Ahmad RM, Laurence RG, Couper GS, Cohn LH, McMahon TA. Decreased myocardial oxygen consumption indices in dynamic cardiomyoplasty. Circulation. 1996;94: II239 –II244. 13. Moainie SL, Gorman JH 3rd, Guy TS, Bowen FW 3rd, Jackson BM, Plappert T, Narula N, St John-Sutton MG, Narula J, Edmunds LH Jr, Gorman RC. An ovine model of postinfarction dilated cardiomyopathy. Ann Thorac Surg. 2002;74:753–760. 14. Gawne TJ, Gray KS, Goldstein RE. Estimating left ventricular offset volume using dual-frequency conductance catheters. J Appl Physiol. 1987;63:872– 876. 15. Baan J, Jong TT, Kerkhof PL, Moene RJ, van Dijk AD, van der Velde ET, Koops J. Continuous stroke volume and cardiac output from intraventricular dimensions obtained with impedance catheter. Cardiovasc Res. 1981;15:328 –334. 16. Steendijk P, Van der Velde ET, Baan J. Left ventricular stroke volume by single and dual excitation of conductance catheter in dogs. Am J Physiol. 1993;264:H2198 –H2207. 17. Kono A, Maughan WL, Sunagawa K, Hamilton K, Sagawa K, Weisfeldt ML. The use of left ventricular end-ejection pressure and peak pressure in the estimation of the end-systolic pressure-volume relationship. Circulation. 1984;70:1057–1065. 18. Suga H. Ventricular energetics. Physiol Rev. 1990;70:247–277. 19. Folland ED, Parisi AF. Noninvasive evaluation of left ventricular function: the ejection fraction. Compr Ther. 1979;5:47–54.

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20. Pilla JJ, Blom AS, Brockman DJ, Bowen F, Yuan Q, Giammarco J, Ferrari VA, Gorman JH 3rd, Gorman RC, Acker MA. Ventricular constraint using the acorn cardiac support device reduces myocardial akinetic area in an ovine model of acute infarction. Circulation. 2002;106: I207–I211. 21. Blom AS, Mukherjee R, Pilla JJ, Lowry AS, Yarbrough WM, Mingoia JT, Hendrick JW, Stroud RE, McLean JE, Affuso J, Gorman RC, Gorman JH 3rd, Acker MA, Spinale FG. Cardiac support device modifies left ventricular geometry and myocardial structure after myocardial infarction. Circulation. 2005;112:1274 –1283.

22. Abel FL, Mihailescu LS, Lader AS, Starr RG. Effects of pericardial pressure on systemic and coronary hemodynamics in dogs. Am J Physiol. 1995;268:H1593–H1605. 23. Klopfenstein HS, Bernath GA, Cogswell TL, Boerboom LE. Coronary artery hemodynamics in conscious dog during cardiac tamponade. Circ Res. 1987;60:845– 849. 24. Acker MA, Bolling S, Shemin R, Kirklin J, Oh JK, Mann DL, Jessup M, Sabbah HN, Starling RC, Kubo SH. Mitral valve surgery in heart failure: insights from the Acorn Clinical Trial. J Thorac Cardiovasc Surg. 2006;132: 568 –577, 577 e1– e4.

CLINICAL PERSPECTIVE Heart failure is a deadly epidemic with few therapeutic options. Ventricular restraint therapy is a promising nontransplantation surgical option for heart failure in which the heart is wrapped with passive material to prevent adverse dilatation. Numerous studies have demonstrated that restraint reverses left ventricular dilatation and promotes reverse remodeling in animal models and human patients. This includes the largest prospective trial of any surgical procedure for heart failure, the ACORN Clinical Trial, which involved 300 patients. Despite these promising results, the science behind restraint remains sparse. With current devices, restraint level is neither adjustable nor measurable. Surgeons’ instructions are to place the device on the heart “snugly.” Questions remain as to the exact levels that would give a salutary effect but would not cause any hemodynamic compromise by restriction or tamponade. Because restraint levels are currently not measurable, we lack a rational approach to the application of this therapy. The findings in this study establish the need for a measurable and adjustable technique to apply ventricular restraint. They show that improvements in ventricular mechanics and energetics correlate to the level of restraint therapy applied. As the heart shrinks, the restraint level also decreases and the rate of reverse remodeling slows. The clinical implications of these findings are that such a technique is necessary to optimize reverse remodeling and that periodic adjustments of restraint level may be required for continued benefit. Therapy may then be customized for each patient on the basis of rational criteria and decreased if restrictive effects are noted. Although further study is required, adjustable measurable restraint therapy may represent an important clinical option for patients with end-stage heart failure.

Coronary Heart Disease Renal Insufficiency Following Contrast Media Administration Trial (REMEDIAL) A Randomized Comparison of 3 Preventive Strategies Carlo Briguori, MD, PhD; Flavio Airoldi, MD; Davide D’Andrea, MD; Erminio Bonizzoni, PhD; Nuccia Morici, MD; Amelia Focaccio, MD; Iassen Michev, MD; Matteo Montorfano, MD; Mauro Carlino, MD; John Cosgrave, MD; Bruno Ricciardelli, MD; Antonio Colombo, MD Background—Volume supplementation by saline infusion combined with N-acetylcysteine (NAC) represents an effective strategy to prevent contrast agent–induced nephrotoxicity (CIN). Preliminary data support the concept that sodium bicarbonate and ascorbic acid also may be effective in preventing CIN. Methods and Results—Three hundred twenty-six consecutive patients with chronic kidney disease, referred to our institutions for coronary and/or peripheral procedures, were randomly assigned to prophylactic administration of 0.9% saline infusion plus NAC (n⫽111), sodium bicarbonate infusion plus NAC (n⫽108), and 0.9% saline plus ascorbic acid plus NAC (n⫽107). All enrolled patients had serum creatinine ⱖ2.0 mg/dL and/or estimated glomerular filtration rate ⬍40 mL · min⫺1 · 1.73 m⫺2. Contrast nephropathy risk score was calculated in each patient. In all cases, iodixanol (an iso-osmolar, nonionic contrast agent) was administered. The primary end point was an increase of ⱖ25% in the creatinine concentration 48 hours after the procedure (CIN). The amount of contrast media administered (179⫾102, 169⫾92, and 169⫾94 mL, respectively; P⫽0.69) and risk scores (9.1⫾3.4, 9.5⫾3.6, and 9.3⫾3.6; P⫽0.21) were similar in the 3 groups. CIN occurred in 11 of 111 patients (9.9%) in the saline plus NAC group, in 2 of 108 (1.9%) in the bicarbonate plus NAC group (P⫽0.019 by Fisher exact test versus saline plus NAC group), and in 11 of 107 (10.3%) in the saline plus ascorbic acid plus NAC group (P⫽1.00 versus saline plus NAC group). Conclusions—The strategy of volume supplementation by sodium bicarbonate plus NAC seems to be superior to the combination of normal saline with NAC alone or with the addition of ascorbic acid in preventing CIN in patients at medium to high risk. (Circulation. 2007;115:1211-1217.) Key Words: angiography 䡲 angioplasty 䡲 complications 䡲 contrast media 䡲 kidney 䡲 prevention

R

adiocontrast media can lead to a reversible form of acute renal failure that becomes apparent soon after the administration of the dye and is generally benign.1 Transient dialysis may be required, however, especially in high-risk patients.2,3 The optimal strategy to prevent contrast agent–induced nephrotoxicity (CIN) remains uncertain. The most recent guidelines4 recommend intravenous volume expansion with a saline solution, use of a low- or iso-osmolality contrast agent, and limits on the volume of contrast agent.

enges a wide variety of oxygen-derived free radicals. NAC may prevent CIN by stopping direct oxidative tissue damage and by improving renal hemodynamics.6 – 8 Recently, 2 additional antioxidant strategies have aroused considerable interest: volume supplementation by sodium bicarbonate9 and the administration of ascorbic acid.10 Both approaches should be effective because of their antioxidant properties. We hypothesized that a combination of different antioxidant compounds may give additive benefit in preventing CIN. To test this hypothesis, we performed a prospective, double-blind, randomized study comparing different combinations of antioxidant compounds in patients at medium to high risk for CIN undergoing iso-osmolar contrast agent administration during coronary or peripheral procedures.

Clinical Perspective p 1217 The generation of reactive oxygen species has been considered an important pathophysiological cause of CIN.5 N-acetylcysteine (NAC) is a potent antioxidant that scav-

Continuing medical education (CME) credit is available for this article. Go to http://cme.ahajournals.org to take the quiz. Received December 28, 2006; accepted January 5, 2007. From the Laboratory of Interventional Cardiology and Department of Cardiology, Clinica Mediterranea, Naples (C.B., D.D., A.F., B.R.); Laboratory of Interventional Cardiology, “Vita e Salute” University School of Medicine, San Raffaele Hospital, Milan (C.B., F.A., N.M., I.M., M.M., M.C., J.C., A.C.); and Institute of Medical Statistics and Biometry, University of Milan, Milan (E.B.), Italy. Correspondence to Carlo Briguori, MD, PhD, Interventional Cardiology, Clinica Mediterranea, Via Orazio, 2, I-80121, Naples, Italy. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.687152

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Methods Patient Population The present 2-center, randomized, double-blind study compared 3 different strategies for preventing CIN in patients with chronic kidney disease who underwent coronary and/or peripheral angiography and/or angioplasty from January 2005 to August 2006. During this time period, consecutive eligible patients scheduled for coronary and/or peripheral angiography and/or angioplasty were considered for enrollment. Individuals ⱖ18 years of age with stable serum creatinine concentration ⱖ2.0 mg/dL and/or or an estimated glomerular filtration rate ⬍40 mL · min⫺1 · 1.73 m⫺2 were considered eligible for the present study. Estimated glomerular filtration rate was calculated by applying the level-modified Modification of Diet in Renal Disease formula: (186.3⫻serum creatinine⫺1.154)⫻(age⫺0.203)⫻(0.742 if female).11 Exclusion criteria were serum creatinine levels ⱖ8 mg/dL, a history of dialysis, multiple myeloma, pulmonary edema, acute myocardial infarction, recent exposure to radiographic contrast within 2 days of the study, pregnancy, and administration of theophylline, dopamine, mannitol, or fenoldopam. The local ethics committee approved the study protocol, and all patients gave written informed consent.

Protocol After enrollment, patients were randomly assigned to 1 of the 3 following treatments: intravenous saline plus NAC administration (saline plus NAC group), intravenous sodium bicarbonate plus NAC administration (bicarbonate plus NAC group), or intravenous saline plus intravenous ascorbic acid plus NAC (saline plus ascorbic acid plus NAC group). All 3 therapies were instituted both before and after administration of the contrast agent. Isotonic saline (0.90%) was given intravenously at a rate of 1 mL/kg body weight per hour (0.5 mL/kg for patients with left ventricular ejection fraction ⬍40%) for 12 hours before and 12 hours after administration of the contrast agent.4,12,13 Patients allocated to the bicarbonate plus NAC group received 154 mEq/L sodium bicarbonate in dextrose and H2O, according to the protocol reported by Merten et al.9 The initial intravenous bolus was 3 mL · kg⫺1 · h⫺1 for 1 hour immediately before contrast injection. After this, patients received the same fluid at a rate of 1 mL · kg⫺1 · h⫺1 during contrast exposure and for 6 hours after the procedure. Patients allocated to the saline plus ascorbic acid plus NAC group received 3 g ascorbic acid intravenously 2 hours before followed by 2 g the night and the morning after the procedure.10 We used the intravenous infusion of ascorbic acid because of the low bioavailability after oral administration. All patients received NAC (Fluimucil, Zambon Group SpA, Milan, Italy) orally at a dose of 1200 mg twice daily on the day before and the day of administration of the contrast agent (total of 2 days).14 Diuretics were routinely withheld on the day of contrast injection. Serum creatinine, blood urea nitrogen, sodium, and potassium were measured the day before and 24 and 48 hours after administration of the contrast agent; additional measurements were performed in all cases of deterioration of baseline renal function. The risk score for predicting CIN was calculated according to Mehran et al.15 Urinary pH was measured at the time of enrollment and during treatment (the morning before contrast media administration in the saline plus NAC group, after infusion of the bolus when the patient spontaneously voided in the bicarbonate plus NAC group, and after the first dose in the saline plus ascorbic acid plus NAC group).

Contrast Agents Iodixanol (Visipaque, 320 mg iodine/mL, Amersham Health), a nonionic, iso-osmolar (290 mOsm/kg water) contrast agent, was used in all patients. Two different cutoffs were used to identify patients receiving a high-contrast load: ⱖ 140 mL14 and 5⫻ kilograms of body weight divided by serum creatinine (mg/dL), a weight- and creatinine-adjusted maximum contrast dose.16 This limit was converted to a dichotomous variable by dividing the actual amount of contrast received by the calculated maximum contrast dose to determine the “contrast ratio.” If the ratio was ⬎1, then the maximum contrast dose was considered exceeded.16

Study End Points The primary outcome measure was development of CIN, defined as an increase in the serum creatinine concentration ⱖ25% from the baseline value at 48 hours after administration of the contrast media or the need for dialysis.4 Additional efficacy end points included an increase in the serum creatinine concentration ⱖ0.5 mg/dL at 48 hours after contrast exposure and a decrease of estimated glomerular filtration rate ⱖ25% at 48 hours. Acute renal failure requiring dialysis was defined as a decrease in renal function necessitating acute hemodialysis, ultrafiltration, or peritoneal dialysis within the first 5 days after intervention.

Statistical Analysis Treatment assignment among the 3 groups was determined by randomization in a 1:1:1 ratio. To ensure that almost equal numbers of patients receive each of the 3 treatments, a randomization block was used (Plan Procedure of SAS, version 8.2, SAS Institute Inc, Cary, NC). The sample size was selected to demonstrate a reduction in the primary end point of CIN from 15% in the saline plus NAC group14,15 to 2% in the bicarbonate plus NAC group and/or saline plus ascorbic acid plus NAC group.9 With the use of a 2-sided ␹2 test with a significance level of 0.05, a total of 288 randomized patients gave the study 90% power. Continuous variables are represented as mean⫾SD or as medians (Q1 to Q3). One-way ANOVA test, the nonparametric Wilcoxon signed rank test for repeated measures, and the Mann-Whitney U test for nonrepeated measures were used to determine differences between normal and nonnormally distributed continuous variables, respectively. Categorical variables were reported as percentages and were analyzed by the ␹2 or Fisher exact test. Treatment comparisons for the primary end point (CIN) were performed with Fisher exact test. To test the impact of preventive therapy strategy (as defined by the study group) on the creatinine level at 48 hours, we used the ANCOVA model after transforming creatinine levels into natural logarithm (to overcome the problem of the nonnormal distribution). In the ANCOVA, we used as covariates the baseline log-creatinine level and the contrast nephropathy risk score. Familywise levels of P⬍0.05 were considered significant. Multiplicity issues resulting from the pairwise comparisons were approached with the Bonferroni adjustment (yielding a significance threshold of 0.025). Two-tailed unadjusted probability values are reported throughout this article. Data were analyzed with SPSS 11.0 (SPSS Corp, Chicago, Ill) for Windows. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Clinical Characteristics Between January 2005 and August 2006, 351 patients were randomized to the 3 groups of treatment (Figure 1). A total of 25 patients did not complete the study because they did not have contrast exposure (n⫽2) or they did not have serum creatinine evaluation 48 hours after contrast exposure (n⫽23). In all 23 patients, serum creatinine level was assessed within 1 week after contrast exposure; none have developed clinical renal failure, and the creatinine level available at follow-up did not reach the cutoff of an increase ⱖ25% from the baseline. Therefore, 326 were analyzed: 111 patients in the saline plus NAC group, 108 patients in the bicarbonate plus NAC group, and 107 patients in the saline plus ascorbic acid plus NAC group. One hundred seven patients had coronary angiography alone, 73 underwent ad hoc percutaneous coronary intervention, 96 had scheduled percutaneous coronary intervention, 21 had peripheral angiography, and 27 had peripheral angioplasty. The clinical and biochemical characteristics of the patients in the 3 groups

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Figure 1. Diagram showing the flow of participants through each stage of the trial.

are shown in Tables 1 and 2. There were no statistically significant differences between groups in the most important clinical and procedural characteristics. Baseline serum creatinine levels, estimated glomerular filtration rate, and incidence of diabetes mellitus were similar in the 3 groups. The total volume of intravenous hydration was lower in the bicarbonate plus NAC group compared with both the saline plus NAC and saline plus ascorbic acid plus NAC groups (bicarbonate plus NAC group, 1081⫾445 mL; saline plus NAC group, 1562⫾585 mL; and saline plus ascorbic acid plus NAC group, 1599⫾584; P⬍0.001). The amount of contrast agent administered was similar in the 3 groups (saline plus NAC group, 179⫾102 mL; bicarbonate plus NAC group, 169⫾92 mL; and saline plus ascorbic acid plus NAC group, 169⫾94 mL; P⫽0.69). A large volume of contrast dye (defined by both ⬎140 mL and contrast ratio ⬎1) was used in ⬎50% of patients in all 3 groups (Table 1). Mean contrast nephropathy risk score was ⬇10 in all 3 groups (Table 2). A high (ⱖ11) risk score occurred in 26 of 111 patients (24%) in the saline plus NAC group, in 39 of 108 patients (36%) in the bicarbonate plus NAC group (P⫽0.054 versus saline plus NAC group), and in 27 of 104 patients (26%) in the saline plus ascorbic acid plus NAC group (P⫽0.75 versus saline plus NAC group). Patients receiving sodium bicarbonate experienced urinary alkalinization. In contrast, in patients in the saline plus NAC group and saline plus ascorbic acid plus NAC group, nonsignificant changes in urinary pH were observed (Table 2).

Contrast Agent–Induced Nephrotoxicity Median serum creatinine concentration for all patients was 1.95 mg/dL (range, 1.80 to 2.28 mg/dL). In all 3 groups, the median serum creatinine concentration decreased significantly from baseline to 48 hours after contrast agent administration (P⬍0.05 for all; Figure 2). The rate of CIN (increase ⱖ25% of creatinine concentration) was significantly lower in the bicarbonate plus NAC group (2 of 108 patients, 1.9%) than in the saline plus NAC group (11 of 111 patients, 9.9%; P⫽0.019; Table 3). In contrast, the rate of CIN was not statistically different between the saline plus NAC group and saline plus ascorbic acid plus

NAC group (11 of 107, 10.3%; P⫽1.00; Table 3). The additional efficacy end points (ie, an increase ⱖ0.5 mg/dL in creatinine concentration and a decrease of estimated glomerular filtration rate ⱖ25% at 48 hours after contrast exposure) also were observed less often in the bicarbonate plus NAC group than in the saline plus NAC group and saline plus ascorbic acid plus NAC group (Table 3). Renal failure requiring temporary dialysis occurred in 1 patient in the saline plus NAC group (0.9%), 1 in the bicarbonate plus NAC group (0.9%), and 4 in the saline plus ascorbic acid plus NAC group (3.8%). A global significant interaction between treatment strategies was observed in the creatinine level 48 hours after adjustment for baseline creatinine level and risk score as covariates (F⫽3.85; P⫽0.022 by ANCOVA model). Subanalysis of the effectiveness of the 3 preventive strategies was performed according to the following variables: volume of contrast media, risk score, and diabetes mellitus. Rate of CIN was lower in the bicarbonate plus NAC group even in higher-risk subsets (including patients with contrast ratio ⬎1, risk score ⱖ11, or diabetes mellitus) (Figure 3).

Discussion The main result of the present study is that the combined administration of sodium bicarbonate plus NAC significantly reduces the risk of CIN in a medium- to high-risk population compared with sodium chloride plus NAC or sodium chloride plus ascorbic acid and NAC. Contrast media accounts for 10% of all causes of hospitalacquired renal failure.1–3 CIN causes a prolonged in-hospital stay and represents a powerful predictor of poor early and late outcome.1–3 Careful preprocedural stratification has been recommended. The risk score proposed by Mehran et al15 is simple to calculate and very useful for individual patient risk assessment. Most patients enrolled in the trial had a medium to high risk score. The mean risk score was ⬇10, with an expected 14% risk for CIN. Approximately 30% of our population had a risk score ⱖ11, with an expected ⱖ26% risk for CIN.15 Volume supplementation and the use of a limited amount of low- or isoosmolality contrast agents are the pivotal recommended strategies for CIN prevention.4 In patients at higher risk, additional

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TABLE 1.

Clinical Characteristics of the Patients in the 3 Groups Saline Plus NAC Group (N⫽111)

Bicarbonate Plus NAC Group (N⫽108)

Saline Plus Ascorbic Acid Plus NAC Group (N⫽107)

P

Age, y

71⫾9

70⫾9

69⫾8

0.14

Male, n (%)

90 (81)

95 (88)

84 (78.5)

0.17

Weight, kg

75⫾12

77⫾13

77⫾11

0.26

Height, m

1.66⫾0.8

1.67⫾0.9

1.67⫾0.6

0.22

27⫾3

27⫾4

28⫾4

0.10

Systolic

142⫾23

140⫾21

139⫾21

0.25

Diastolic

77⫾10

77⫾11

76⫾11

0.99

Body mass index, kg/m2 Blood pressure, mm Hg

Mean

100⫾12

98⫾10

97⫾12

0.55

LVEF, %

51⫾10

48⫾10

50⫾12

0.16

LVEF ⬍40%, n (%)

12 (11)

19 (18)

18 (17)

0.39

Systemic hypertension, n (%)

96 (86.5)

99 (92)

88 (82)

0.18

Diabetes mellitus, n (%)

61 (55)

53 (49)

63 (59)

0.35

Non–insulin requiring

31 (28)

24 (22)

30 (28)

䡠䡠䡠

Insulin requiring

30 (27)

29 (27)

33 (31)

27 (24.5)

39 (36)

31 (29)

䡠䡠䡠 0.14

ACE inhibitors

64 (58)

63 (59)

63 (59)

0.97

Calcium channel blocker

43 (38.5)

43 (40)

44 (41)

0.51

Angiotensin II receptor inhibitor

21 (19)

22 (21)

23 (21.5)

0.69

Diuretics

48 (43.5)

46 (42.5)

48 (45)

0.83

␤-Blockers

57 (51)

59 (55)

66 (62)

0.18

Statins

82 (74)

78 (72)

85 (79.5)

0.20

Peripheral chronic artery disease, n (%) Drugs, n (%)

Performed procedure, n (%) Coronary angiography

34 (30)

39 (36)

34 (32)

0.59

PCI

34 (30)

29 (27)

33 (31)

0.50

Coronary angiography and ad hoc PCI

30 (27)

18 (24)

25 (23.5)

0.18

Peripheral procedures

13 (12)

22 (20)

13 (12)

0.15

Iliac-femoral arteriography

6 (5.5)

9 (8.3)

6 (5.6)

䡠䡠䡠

Carotid artery angioplasty

4 (3.6)

6 (5.5)

3 (2.8)

䡠䡠䡠

Femoral artery angioplasty

3 (2.7)

4 (3.7)

3 (2.8)

䡠䡠䡠

Iliac artery angioplasty

0

3 (2.7)

1 (0.9)

Volume of contrast media, mL ⬎140 mL, n (%) Contrast ratio ⬎1, n (%)

179⫾102

169⫾92

169⫾102

䡠䡠䡠 0.69

70 (63)

56 (52)

57 (55)

0.21

61 (55)

59 (54.5)

66 (63.5)

0.34

Values are mean⫾SD unless otherwise indicated. LVEF indicates left ventricular ejection fraction; ACE, angiotensin-converting enzyme; and PCI, percutaneous coronary intervention.

efforts should be attempted. Use of NAC, although not recommended, is suggested in this subset of patients.4 The combined approach of sodium bicarbonate plus NAC allow us to satisfy the crucial requirement of volume supplementation and administer a potent antioxidant treatment.

Volume Supplementation Intravascular volume expansion is usually accomplished by isotonic saline.4 Volume supplementation prevents CIN mostly by the inhibition of arginine-vasopressin (via vagal inputs from the mechanoreceptors located at the AV junctions and by a direct effect of osmolality on the supra-aortic nuclei)

and the increase in medullary perfusion and regional PO2.5 In the present study, the total volume of intravenous hydration was lower in the bicarbonate plus NAC group compared with both the saline plus NAC group and saline plus ascorbic acid plus NAC group. This supports the concept that the mechanism of the effectiveness of sodium bicarbonate in preventing CIN is not likely to be a result of a volume expansion larger than that obtained by isotonic saline.

Antioxidant Therapy The adverse effects of contrast media on renal function may involve the generation of reactive oxygen species, which may

Briguori et al TABLE 2.

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Biochemical Characteristics of the Patients in the 3 Groups Saline Plus NAC Group (N⫽111)

Bicarbonate Plus NAC Group (N⫽108)

Saline Plus Ascorbic Acid Plus NAC Group (N⫽107)

P

Baseline

1.95 (1.69 to 2.26)

2.04 (1.88 to 2.36)

1.93 (1.82 to 2.16)

0.33

After 48 h

1.88 (1.54 to 2.36)

1.90 (1.67 to 2.29)

1.88 (1.53 to 2.32)

0.43

35⫾8

32⫾7

33⫾9

0.69

40 to 60, n (%)

35 (32)

25 (24)

27 (25)

䡠䡠䡠

20 to 40, n (%)

68 (62)

72 (78.5)

69 (67.5)

䡠䡠䡠

7 (6)

8 (7.5)

9 (9)

Serum creatinine (medians Q1 to Q3), mg/dL

eGFR, mL 䡠 min⫺1 䡠 1.73 m⫺2

⬍20, n (%) Contrast nephropathy risk score

9.1⫾3.4

9.5⫾3.5

9.3⫾3.6

䡠䡠䡠 0.21

Score ⱕ5

19 (17)

14 (13)

11 (10)

䡠䡠䡠

Score 6 to 10

66 (59)

55 (51)

69 (64.5)

䡠䡠䡠

Score 11 to 16

25 (23)

34 (31)

21 (19.5)

䡠䡠䡠

1 (1)

5 (5)

6 (6)

䡠䡠䡠

Baseline

73⫾29

81⫾31

78⫾34

0.23

After 48 h

67⫾30

64⫾23

72⫾39

0.36

Score ⬎16 Serum urea nitrogen, mg/dL

Serum sodium, mEq/L Baseline

140⫾4

140⫾4

141⫾4

0.44

After 48 h

140⫾4

140⫾3

141⫾4

0.39

Baseline

4.8⫾0.6

4.8⫾0.6

4.8⫾0.6

0.28

After 48 h

4.4⫾0.5

4.4⫾0.6

4.5⫾0.5

0.55

1703⫾746

1485⫾650

1604⫾746

0.42

Baseline

5.3⫾0.6

5.4⫾0.6

5.4⫾0.6

0.90

After treatment

5.6⫾0.8

6.6⫾0.9*

5.4⫾0.5

⬍0.001

Serum potassium, mEq/L

Urine volume, mL/24 h Urine pH

eGFR indicates estimated glomerular filtration rate. To convert serum creatinine to ␮mol/L, multiply by 88.4. *P⬍0.001 vs other groups.

play a role in the effects of various vasoconstrictors.5,17–19 Furthermore, medullary hypoxia promotes mitochondrial generation of reactive oxygen species.20,21 For this reason, clinical trials have been performed using various antioxidant compounds with the aim of lowering the occurrence of CIN by scavenging reactive oxygen species. Three antioxidant approaches have been investigated in most of the studies: NAC, sodium bicarbonate, and ascorbic acid.

NAC may prevent CIN by stopping direct oxidative tissue damage and by improving renal hemodynamics.6 – 8 The antioxidant effect of NAC seems to be dose dependent.14,22 Although not firmly recommended, NAC administration is suggested especially in high-risk patients.4 Free-radical formation is promoted by an acidic environment typical of tubular urine but is inhibited by the higher pH of normal extracellular fluid.23,24 It has been hypothesized that alkalin-

Figure 2. Serum creatinine concentrations before and after contrast administration in the 3 groups. Error bars indicate median.

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Circulation TABLE 3.

March 13, 2007 Contrast Agent–Enhanced Nephrotoxicity Saline Plus NAC Group (N⫽111), n (%)

Bicarbonate Plus NAC Group (N⫽108), n (%)

Saline Plus Ascorbic Acid Plus NAC Group (N⫽107), n (%)

P

Serum creatinine increase by ⱖ25%

11 (9.9)

2 (1.9)*

10 (10.3)†

0.010

Serum creatinine increase by ⱖ0.5 mg/dL

12 (10.8)

1 (0.9)‡

12 (11.2)†

0.026

eGFR decrease by ⱖ25%

10 (9.2)

1 (0.9)§

10 (10.3)†

0.018

eGFR indicates estimated glomerular filtration rate. *P⫽0.019, †P⬎0.05, ‡P⬍0.003, §P⬍0.009 vs saline plus NAC group.

izing renal tubular fluid with bicarbonate25 may reduce injury. At physiological concentrations, bicarbonate scavenges peroxynitrite and other reactive species generated from nitric oxide.26 Additional evidence of the effectiveness of an antioxidant strategy comes from the recent observation by Spargias et al,10 who investigated the impact of ascorbic acid in preventing CIN. Ascorbic acid is a potent, water-soluble antioxidant capable of scavenging a wide array of reactive oxygen species that can cause damage to macromolecules such as lipids, DNA, and proteins.27 In addition, ascorbic acid can regenerate other antioxidants, acting as a coantioxidant.27 Could the combination of different antioxidant compounds be more effective than a single agent? The results of the present study support this hypothesis within the boundaries of the trial design, which tested the combination of NAC and another antioxidant agent. Therefore, we can recommend that the combined prophylactic strategy of volume supplementation by sodium bicarbonate plus NAC should be used to prevent CIN in patients at medium to high risk undergoing coronary or peripheral procedures. The lack of a favorable protective effect of the combination of ascorbic acid plus NAC compared with NAC alone suggests additional and/or alternative mechanism(s) (other than antioxidant effect), which require further investigation. We may hypothesize that NAC and ascorbic acid work through similar pathways, whereas the protective action of bicarbonate may be different compared with NAC and therefore additive.

The higher amount of HCO3⫺ in the proximal convoluted tubule may buffer the higher amount of H⫹ as a result of cellular hypoxia and facilitate Na⫹ reabsorption through the electrogenic Na⫹/HCO3⫺ cotransporter.28

Study Limitations The results of the present study cannot be extended to patients at high or very high risk (score ⱖ16) for CIN. Furthermore, we did not test the combination of bicarbonate and ascorbic acid. It has been pointed out that the advantage of NAC administration might be based on a decrease in serum creatinine concentration, reflecting either an increase in creatinine excretion or a decrease in creatinine production.29 On the other hand, Izzedine et al30 reported that a therapeutic dose of NAC did not interfere with serum creatinine assays. We did not measure cystatin C, which seems to be a more reliable marker of renal injury.29

Conclusion The combined strategy of volume supplementation by sodium bicarbonate plus NAC seems to be superior to the association of normal saline plus NAC alone or plus ascorbic acid and NAC in preventing CIN in patients at medium to high risk.

Disclosures None.

Figure 3. Effect of the 3 preventive approaches in selected subsets according to volume of contrast media, risk score, and presence of diabetes mellitus. Large volume indicates contrast ratio ⬎1; higher risk, risk score ⱖ11. The symbols indicate the unadjusted odds ratios; horizontal lines, 95% CIs.

Briguori et al

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CLINICAL PERSPECTIVE The generation of reactive oxygen species has been considered an important pathophysiological cause of contrast agent–induced nephrotoxicity. We tested whether a combination of different antioxidant compounds may give additive benefit in preventing contrast agent–induced nephrotoxicity. Consecutive patients with chronic kidney disease (serum creatinine ⱖ2.0 mg/dL and/or estimated glomerular filtration rate ⬍40 mL · min⫺1 · 1.73 m⫺2) were randomly assigned to prophylactic administration of 0.9% saline infusion plus N-acetylcysteine (NAC; n⫽111), sodium bicarbonate infusion plus NAC (n⫽108), and 0.9% saline plus ascorbic acid plus NAC (n⫽107). Contrast agent–induced nephrotoxicity occurred in 11 of 111 patients (9.9%) in the saline plus NAC group, in 2 of 108 (1.9%) in the bicarbonate plus NAC group (P⫽0.019 versus saline plus NAC group), and in 11 of 107 (10.3%) in the saline plus ascorbic acid plus NAC group (P⫽1.00 versus saline plus NAC group). We can therefore recommend that the combined prophylactic strategy of sodium bicarbonate plus NAC should be used to prevent contrast agent–induced nephrotoxicity in patients at medium to high risk undergoing contrast exposure. The lack of favorable protective effect of the combination of ascorbic acid plus NAC compared with NAC alone suggests additional and/or alternative mechanism(s) (other than antioxidant effect), which require further investigation. We may hypothesize that NAC and ascorbic acid work through similar pathways, whereas the protective action of bicarbonate may be different compared with NAC and therefore additive.

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Heart Failure Direct Myocardial Effects of Levosimendan in Humans With Left Ventricular Dysfunction Alteration of Force-Frequency and Relaxation-Frequency Relationships Michael M. Givertz, MD; Costa Andreou, MD; Chester H. Conrad, MD, PhD; Wilson S. Colucci, MD Background—Enthusiasm for the development of Ca2⫹ sensitizers as inotropic agents for heart failure has been tempered by reports of impaired relaxation. Levosimendan, which increases myofilament Ca2⫹ sensitivity via Ca2⫹-dependent binding to troponin C, exerts positive inotropic and lusitropic effects in failing human myocardium in vitro. We sought to determine the direct effects of levosimendan on failing human myocardium in vivo, and in particular whether levosimendan exerts heart rate– dependent effects on systolic or diastolic function. Methods and Results—Ten patients with left ventricular dysfunction caused by nonischemic dilated cardiomyopathy (mean left ventricular ejection fraction, 27⫾2%) were instrumented with an infusion catheter in the left main coronary artery, a high-fidelity micromanometer-tipped catheter in the left ventricle, and a bipolar pacing wire in the right atrium. Inotropic (peak ⫹dP/dt) and lusitropic (Tau) responses were assessed during continuous intracoronary drug infusion in sinus rhythm followed by atrial pacing at 20, 40, and 60 beats per minute above the sinus rate. Under control conditions (intracoronary 5% dextrose in water), atrial-pacing tachycardia decreased Tau by 13% (P⬍0.05), but did not increase ⫹dP/dt. Intracoronary levosimendan (3.75 and 12.5 ␮g/min for 15 minutes each) increased ⫹dP/dt dose-dependently and decreased Tau over a range of heart rates, but did not alter the slope of the force-frequency or relaxation-frequency relationship. Conclusions—Myocardial calcium sensitization with levosimendan exerts mild inotropic and lusitropic effects in humans with left ventricular dysfunction, but does not alter the force-frequency or relaxation-frequency relationship. (Circulation. 2007; 115:1218-1224.) Key Words: calcium 䡲 contractility 䡲 heart failure 䡲 levosimendan 䡲 myocardium

D

espite optimal medical therapy, patients with chronic left ventricular (LV) systolic dysfunction often require hospital admission for symptoms of worsening congestion and/or systemic hypoperfusion.1 Positive inotropic agents currently available for the treatment of decompensated heart failure, which include dobutamine and milrinone, are limited by their tendency to increase heart rate and stimulate arrhythmias. These adverse effects in failing human myocardium are mediated primarily by an increase in intracellular calcium. Calcium-sensitizing agents, which act by directly increasing the sensitivity of the myofilament to calcium, may exert positive inotropic effects without proarrhythmia. The enthusiasm for the development of calcium sensitizers has been tempered, however, by reports of impaired relaxation,2 especially under hypoxic conditions or at higher stimulation frequencies, and reduced energy efficiency as a result of increased crossbridge cycling.

Clinical Perspective p 1224 Levosimendan enhances calcium sensitivity of the contractile apparatus via calcium-dependent binding to cardiac troponin C.3–5 In skinned fibers from normal guinea pig papillary muscles, levosimendan causes concentration-dependent, direct positive inotropic effects without impairment of relaxation. Hasenfuss et al6 studied the effects of levosimendan on failing human myocardium in vitro and demonstrated both positive inotropic and lusitropic effects over a range of twitch frequencies. Furthermore, there was no frequency-dependent rise in diastolic tension with levosimendan. In muscle strips with weak inotropic responses, the increase in intracellular calcium was significantly higher with milrinone than with levosimendan, which suggests different modes of action. In dogs with pacing-induced heart failure, levosimendan improved both systolic and diastolic function without a change in heart rate or myocardial oxygen consumption.7 Improve-

Received October 6, 2006; accepted January 2, 2007. From the Cardiomyopathy Program and Cardiovascular Section (M.M.G., C.A., W.S.C.), Boston University Medical Center, Boston, Mass; and Cardiology Section (C.H.C.), VA Boston Healthcare System, Boston, Mass. Guest Editor for this article was Martin M. LeWinter, MD. Correspondence to Michael M. Givertz, MD, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail [email protected] © 2007 American Heart Association, Inc.

ttp://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.668640

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Givertz et al ment in systolic function without an increase in myocardial oxygen consumption has also been demonstrated in healthy volunteers8 and patients with cardiovascular disease.9 In normal myocardium, an increase in heart rate results in an increase in contractile force, which is reflective of the fact that a positive relationship exits between force and frequency. The force-frequency relationship, originally described in the frog heart more than 120 years ago, has subsequently been confirmed in cardiac muscle strips and isolated ventricular myocytes from most mammals, including humans, and in the intact normal human heart.10,11 In contrast, the failing human heart has a force-frequency relationship that is attenuated, flat, or even inverted.12,13 The mechanism responsible for the attenuated force-frequency relationship in failing myocardium is incompletely understood, but may involve abnormalities in calcium handling that worsen at higher frequencies.14 The effect of increased calcium sensitization on the forcefrequency relationship is unknown. Therefore, we sought to determine the direct effects of levosimendan on failing human myocardium in vivo, and in particular whether levosimendan exerts heart rate– dependent effects on systolic or diastolic function. To avoid systemic effects of levosimendan, we infused levosimendan into the left main coronary artery, a technique that we have used previously to selectively demonstrate direct myocardial effects of vasoactive agents in heart failure.15,16

Methods Study Population The study population consisted of 10 men (mean age, 59⫾4 years) who underwent diagnostic cardiac catheterization for evaluation of chronic LV systolic dysfunction. All patients were in sinus rhythm and were found to be free of significant coronary artery disease at the time of cardiac catheterization. Hypertension was felt to be a contributing factor to heart failure in 4 patients, and alcohol was considered a contributing factor in 3 patients; 3 patients were diagnosed with idiopathic dilated cardiomyopathy through exclusion of coronary artery disease or other known causes of dilated cardiomyopathy. Patients were in New York Heart Association functional class I (n⫽1), II (n⫽2), III (n⫽5), and IV (n⫽1) with a mean LV ejection fraction of 27⫾2%. Cardiovascular medications consisted of angiotensin-converting enzyme inhibitors (n⫽9), diuretics (n⫽7), digoxin (n⫽4), ␤-blockers (n⫽1) and other vasodilators (n⫽5). Medications were not administered on the morning of the study. The study protocol was approved by the Research and Development Committee of the Boston VA Medical Center, and all patients provided written informed consent.

Hemodynamic Measurements Before the experimental protocol, all subjects underwent routine diagnostic left and right heart catheterization via the femoral approach. Coronary angiography was performed with nonionic contrast media, and the research protocol was begun a minimum of 20 minutes after completion of the diagnostic catheterization. Detailed methods used for hemodynamic measurements and intracoronary drug infusions have been described previously.15,16 A 6F L4 Judkins catheter (Cordis, a Johnson & Johnson Company, Miami Lakes, Fla) was advanced from the right femoral artery to the left main coronary ostium for intracoronary drug infusion. A 7F high-fidelity micromanometer-tipped pigtail catheter (Millar Instruments, Houston, Tex) was advanced from the left femoral artery and positioned in the LV for measurement of LV pressure. A 5F bipolar pacing wire was advanced from the right femoral vein to the right atrial appendage for atrial pacing. Femoral artery pressure was monitored from a 7F

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sidearm sheath (Cordis) present in the right femoral artery. After instrumentation, an additional 5000 U of heparin was administered intravenously. Hemodynamic measurements included heart rate, LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and LV developed pressure calculated as LVSP⫺LVEDP. LV contractility was assessed as the peak rate of rise of LV pressure (⫹dP/dt), and LV relaxation was measured as the time constant of isovolumic pressure decay (Tau) by the method of Weiss.17 Each measurement was obtained as the mean of at least 40 consecutive beats. ECG and LV pressure were continuously monitored throughout the protocol, and they were digitally recorded with a Macintosh personal computer. Hemodynamic data were analyzed off-line with PowerLab software (AD Instruments, Colorado Springs, Colo).

Drug Infusions and Pacing Protocol As a control solution, 5% dextrose in water (D5W) with heparin (1 U/mL) was infused into the left main coronary artery at a rate of 2 mL/min for 5 minutes with a Harvard pump, and baseline hemodynamics were recorded in normal sinus rhythm during the fifth minute. Right atrial pacing was then initiated at a rate of 20 beats per minute (bpm) above the baseline sinus rate for 2 minutes, and hemodynamics were measured during the second minute. Atrial pacing was then increased to 40 and 60 bpm above the baseline sinus rate. The pacing protocol was terminated if there was development of atrioventricular block or evidence of myocardial ischemia (eg, angina, ST changes, or increased LVEDP). To reestablish baseline conditions, the pacemaker was turned off, and D5W continued to be infused into the left main coronary artery for at least 5 minutes until all hemodynamics had returned to baseline values (⫾10%). Levosimendan was then infused into the left main coronary artery at rates of 3.75 and 12.5 ␮g/min for 15 minutes each. These rates were chosen to achieve intracoronary steady-state levosimendan concentrations of 30 and 100 ng/mL, respectively, with an assumption of a left coronary blood flow rate of 125 mL/min.18 In patients with acute decompensated heart failure, levosimendan exerted sustained hemodynamic effects when administered intravenously at mean doses that ranged from 0.14 to 0.26 ␮g/kg per min, and achieved plasma concentrations of ⬇60 ng/mL and 120 ng/mL, respectively.19 During the tenth minute at each infusion rate, hemodynamics were recorded in normal sinus rhythm. Right atrial pacing was then performed successively at 20, 40, and 60 bpm above the baseline sinus rate, with hemodynamic measurements and pacing end points as described for the control condition.

Statistical Analysis The hemodynamic variables of primary interest were LV peak ⫹dP/dt and Tau. Each variable was analyzed separately with analysis of variance with a repeated measures model to assess the effects of treatment and atrial pacing. Each dose of levosimendan was compared with the control by use of the Dunnett-Hsu test. A test for linear dose response was performed with a repeated measures analysis of covariance. All analyses were performed with SAS statistical package version 6 (SAS Institute, Inc, Cary, NC), and data are presented as mean⫾SEM. Statistical results were considered significant if the probability of obtaining the results by chance was 0.05 or less. All tests were 2-tailed. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Baseline Hemodynamics and Response to Intracoronary Levosimendan in Sinus Rhythm Baseline hemodynamics revealed chronic heart failure with an average heart rate of 76 bpm, and LVSP and LVEDP of 131 mm Hg and 32 mm Hg, respectively (Table 1). Chronic LV systolic dysfunction was associated with marked impairment in both contractility and isovolumic relaxation with a

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TABLE 1. Hemodynamic Response to Intracoronary Levosimendan in Normal Sinus Rhythm Control HR, bpm LVSP, mm Hg

Levo at 3.75 ␮g/min

Levo at 12.5 ␮g/min

76⫾5

76⫾4

76⫾4

131⫾5

134⫾5

135⫾5

LVEDP, mm Hg

32⫾3

30⫾3

29⫾3

LVPdev, mm Hg

100⫾5

104⫾6

106⫾6*

LV peak ⫹dP/dt, mm Hg/s

769⫾57

802⫾59*

826⫾62*

72⫾6

72⫾7

65⫾5*

Tau, ms

Levo indicates levosimendan; HR, heart rate; and LVPdev, left ventricular developed pressure. *P⬍0.05 vs control.

mean LV peak ⫹dP/dt of 769⫾57 mm Hg/s and Tau of 72⫾6 ms, respectively. In sinus rhythm, intracoronary levosimendan at 3.75 and 12.5 ␮g/min caused modest dose-dependent increases in LV peak ⫹dP/dt of 4.5⫾2.0% and 7.4⫾2.8%, respectively (P⬍0.05 versus control for both), despite nonsignificant reductions in LVEDP (Figure 1). Interpatient variability in the inotropic response to high-dose levosimendan was evidenced by a range in the percent change in LV peak ⫹dP/dt from ⫺9% to 21% (Figure 2A). Tau was unchanged after low-dose levosimendan but decreased by 8.6⫾3.6% after intracoronary levosimendan at 12.5 ␮g/min (P⬍0.05 versus control). Interpatient variability in the lusitropic response to high-dose levosimendan was also observed, and the percent change in Tau ranged from ⫺25% to 15% (Figure 2B). Intracoronary levosimendan had no effect on heart rate or LVSP (Table 1).

Effect of Intracoronary Levosimendan on the Force-Frequency Relationship Under control conditions (intracoronary D5W), atrial pacing to a maximal heart rate of 133⫾6 bpm resulted in reductions in LVSP from 131 mm Hg to 119 mm Hg (P⬍0.05) and LVEDP from 32 mm Hg to 22 mm Hg (P⬍0.05) without a change in LV developed pressure (Table 2). Atrial pacing resulted in a modest increase in LV peak ⫹dP/dt at 20 and 40 bpm above the baseline sinus rate, but there was no difference

Figure 2. Positive inotropic and lusitropic responses to intracoronary levosimendan (Levo) at 12.5 ␮g/min. Shown are the percent changes in LV peak ⫹dP/dt (A) and Tau (B) for individual numbered subjects in normal sinus rhythm. *P⬍0.05 versus control.

in contractility at the highest pacing rate compared with the baseline sinus rate (800 mm Hg/s versus 769 mm Hg/s, P⫽NS), consistent with an attenuated force-frequency relationship. Levosimendan at 3.75 and 12.5 ␮g/min resulted in mild, dose-dependent, positive inotropic effects during atrialpacing (Table 2, Figure 3). Specifically, LV peak ⫹dP/dt increased by 6% and 8%, respectively, at 20 bpm above the baseline sinus rate and by 6% and 9%, respectively, at 40 bpm above the baseline sinus rate. However, levosimendan had no effect on the slope of the force-frequency relationship (Figure 3). Figure 1. Relationship between changes in LV peak ⫹dP/dt and LVEDP with intracoronary levosimendan (Levo) at 3.75 and 12.5 ␮g/min in normal sinus rhythm.

Effect of Intracoronary Levosimendan on the Relaxation-Frequency Relationship Under control conditions, atrial pacing resulted in a gradual decrease in Tau from 72 ms during baseline sinus rhythm to

Givertz et al

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TABLE 2. Hemodynamic Response to Intracoronary Levosimendan During Atrial Pacing at 20, 40, and 60 bpm Above the Baseline Sinus Rate Pacing Level B

B⫹20

B⫹40

B⫹60

HR, bpm Control

76⫾5

95⫾5†

115⫾6†

133⫾6†

Levo at 3.75 ␮g/min

76⫾4

96⫾5†

116⫾5†

133⫾6†

Levo at 12.5 ␮g/min

76⫾4

97⫾5†

116⫾5†

133⫾6†

LVSP, mm Hg Control

131⫾5

131⫾4

126⫾6†

119⫾4†

Levo at 3.75 ␮g/min

134⫾5

133⫾5

126⫾5†

117⫾4†

Levo at 12.5 ␮g/min

135⫾5

132⫾4

127⫾5†

119⫾5†

LVEDP, mm Hg Control

32⫾3

28⫾3†

24⫾3†

22⫾3†

Levo at 3.75 ␮g/min

30⫾3

26⫾2†

21⫾3†

19⫾3†

Levo at 12.5 ␮g/min

29⫾3

24⫾3†

19⫾3†

18⫾3†

LVPdev, mm Hg Control

100⫾5

103⫾4

102⫾5

97⫾5

Levo at 3.75 ␮g/min

104⫾6

107⫾6

105⫾5

98⫾5

Levo at 12.5 ␮g/min

106⫾6*

109⫾5

108⫾5†

101⫾5

LV ⫹dP/dt, mm Hg/s Control

769⫾57

817⫾65†

833⫾66†

800⫾64

Levo at 3.75 ␮g/min

802⫾59*

867⫾72*†

880⫾67†

832⫾66

Levo at 12.5 ␮g/min

826⫾62*

886⫾72*†

907⫾67*†

861⫾74

Tau, ms

Figure 3. Effect of intracoronary levosimendan (Levo) at 3.75 ␮g/min (䡲) and 12.5 ␮g/min (’) compared with control (intracoronary D5W, 䡩) on LV peak ⫹dP/dt during baseline sinus rhythm (B) and atrial pacing at 20, 40, and 60 bpm above sinus rhythm (B⫹20, B⫹40, B⫹60). *P⬍0.05 versus control.

artery while heart rate was altered with right atrial pacing. We found that levosimendan exerts modest positive inotropic and lusitropic effects over a range of heart rates, but does not alter the slope of the force-frequency or relaxation-frequency relationship. These novel in vivo data provide the first look at the direct myocardial actions of levosimendan not confounded by systemic vascular effects and extend prior in vitro findings from failing animal and human myocardium.

Control

72⫾6

69⫾6

65⫾5

63⫾6†

Levo at 3.75 ␮g/min

72⫾7

62⫾5

59⫾5†

62⫾6†

Inotropic Effects of Levosimendan

Levo at 12.5 ␮g/min

65⫾5*

59⫾4*†

58⫾5†

56⫾6*†

Levosimendan is a pyridazinone-dinitrile derivative that enhances calcium sensitivity of the myofilaments via calciumdependent binding to troponin C.20 Discovered by screening for compounds with high affinity to the troponin complex, levosimendan stabilizes troponin C in a conformation that

B indicates baseline; Levo, levosimendan; HR, heart rate; and LVPdev, left ventricular developed pressure. *P⬍0.05 vs control; †P⬍0.05 vs B.

63 ms at 60 bpm above the baseline sinus rate (P⬍0.05), consistent with a positive relaxation-frequency relationship (Table 2). Compared with control, low-dose levosimendan had no significant effect on Tau during atrial-pacing tachycardia, whereas levosimendan at 12.5 ␮g/min resulted in mild positive lusitropic effects over a range of heart rates (Table 2, Figure 4). Specifically, Tau decreased by 14%, 11%, and 11% at 20, 40, and 60 bpm above the baseline sinus rate. However, levosimendan had no effect on the slope of the relaxation-frequency relationship (Figure 4).

Safety At the intracoronary doses used, levosimendan was well tolerated in all patients without hypotension or proarrhythmia. No patients developed evidence of myocardial ischemia (eg, angina, ST segment changes, or increased LVEDP) during atrial pacing tachycardia.

Discussion In the present study, we assessed the direct myocardial effects of levosimendan in patients with heart failure by administration of subsystemic doses of drug into the left main coronary

Figure 4. Effect of intracoronary levosimendan (Levo) at 3.75 ␮g/min (䡲) and 12.5 ␮g/min (’) compared with control (intracoronary D5W, 䡩) on Tau during baseline sinus rhythm (B) and atrial pacing at 20, 40, and 60 bpm above sinus rhythm (B⫹20, B⫹40, B⫹60). *P⬍0.05 versus control.

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triggers contraction.3 Multiple laboratory studies have shown that levosimendan exerts positive inotropic effects in both normal and failing myocardium with a median effective dose of ⬇0.1 ␮mol/L.3 Subjects in this study had chronic dilated cardiomyopathy with marked impairment in systolic function (average LV peak ⫹dP/dt, 769 mm Hg/s). Levosimendan exerted modest dose-dependent effects on ⫹dP/dt over a wide range of heart rates (mean, 76 to 133 bpm), with an average 8% increase from baseline. In addition to calcium sensitization, levosimendan has other pharmacological properties that may have contributed to the positive inotropic (and lusitropic) effects we observed. Initial in vitro studies demonstrated potent and selective inhibition of phosphodiesterase type III.21 However, subsequent experiments showed that with lower doses of levosimendan, similar to those used in this study, phosphodiesterase III inhibition does not contribute to positive inotropic effects.22,23 Levosimendan also causes vasodilation via opening of adenosine triphosphatase–sensitive K⫹ channels.24 This effect may contribute to coronary9 and systemic25 vasodilation with the intravenous administration of levosimendan. The direct effects of K⫹-channel opening on myocardial contractility remain unclear, although 1 study in an animal model of diabetic cardiomyopathy suggested a negative inotropic effect.26

Lusitropic Effects of Levosimendan Unlike other agents that increase calcium sensitization via calcium-independent mechanisms (eg, MCI-154, EMD57033), levosimendan does not impair myocardial relaxation in vitro.6,27 Pagel et al7 showed that levosimendan had no deleterious effect on myocardial relaxation in normal dogs and improved resting diastolic function in dogs with heart failure. Additional animal data show that levosimendan attenuated the increase in LVEDP and decreased Tau during exercise.28 In failing and nonfailing human myocardium, Brixius et al27 demonstrated a positive lusitropic effect of levosimendan, in part mediated by cAMP. Activation of K⫹ channels also improves relaxation in failing myocardium.29 Our in vivo human data show that levosimendan improves both cardiac contractility and relaxation in patients with heart failure. To our knowledge, this is the first catheterization laboratory– based study to assess changes in isovolumic relaxation in response to levosimendan. Previous studies that used intravenous administration of levosimendan have been confounded by systemic effects, which include reduction in LV preload and afterload.9 Improvement in diastolic function, even if modest, might contribute to favorable effects of levosimendan in patients with acute heart failure regardless of ejection fraction. In acute coronary syndromes, levosimendan improves diastolic function30 and causes an upward and leftward shift of the pressure-volume relationship that is consistent with improved contractile function of stunned myocardium.31

Interpatient Variability As previously demonstrated in our laboratory with intravenous32 and intracoronary dobutamine,15,32 we observed significant interpatient variability in the inotropic and lusitropic

responses to intracoronary levosimendan in heart failure. In explanted failing human myocardium, Hasenfuss et al6 also demonstrated wide variability in the inotropic response to levosimendan with the peak increase in twitch tension ranging from ⫺35% to ⫹80%. One explanation for the interpatient variability that we observed may be varying etiologies of heart failure. However, in the study be Hasenfuss et al,6 etiology (ischemic versus nonischemic) did not explain the variability in the inotropic response. Other explanations for interpatient variability include differences in duration of heart failure and alteration of myocardial gene expression, variability in phosphorylation of troponin I, which may alter the affinity of levosimendan to troponin C, and differences in cardiac medications, resting hemodynamics, or both.

Force-Frequency and Relaxation-Frequency Relationships The force-frequency relationship is attenuated, flat, or inverted in failing myocardium.10,12 Notably, the degree to which this relationship is blunted in heart failure correlates with the reduction in peak functional capacity.33 Underlying cellular and molecular mechanisms are not well understood, but frequency-dependent impairment in calcium handling by the sarcoplasmic reticulum has been implicated.14,34 In the present study, we observed no change in the slope of the force-frequency relationship with myocardial calcium sensitization. Although ⫹dP/dt improved modestly in a dosedependent manner over a range of heart rates, the percent change from baseline did not increase at higher rates. This upward shift of the force-frequency relationship is similar to the frequency-independent changes observed by Hasenfuss et al6 with levosimendan in explanted failing human myocardium. Others, however, have demonstrated improved forcefrequency in vitro with the use of lower concentrations of levosimendan27 and in exercising dogs with heart failure.28 Our present findings, like those of Hasenfuss et al,6 are not surprising because levosimendan increases myofilament calcium sensitivity by acting distal to calcium but does not appear to affect calcium handling by the sarcoplasmic reticulum. Few investigators have demonstrated correction of the force-frequency abnormality in heart failure. Schwinger et al35 demonstrated partial reversal of the negative forcefrequency relationship in failing human myocardium with low-dose isoprenaline, and similar in vitro effects have been observed with gene transfer of sarcoplasmic reticulum calcium ATPase.36 Myocyte “recovery” after mechanical circulatory support is also associated with improved forcefrequency.37 Most recently, biventricular pacing was shown to improve the inotropic response to increased heart rates in patients with advanced heart failure and ventricular conduction delay.13 The effect of chronic neurohormonal blockade (eg, with angiotensin-converting enzyme inhibitors and ␤-blockers) on the force-frequency relationship is unknown. The relaxation-frequency relationship is not as well studied, but may have important functional significance in both normal and failing hearts. We observed a ⬇13% reduction in Tau with atrial pacing tachycardia in both the control state and after intracoronary levosimendan. Our intact human data

Givertz et al with levosimendan are nearly identical to ex vivo studies in failing human myocardium.6 This positive lusitropic effect has not been observed with other calcium sensitizers,38 and may be caused in part by phosphodiesterase III inhibition that leads to activation of protein kinase A and phosphorylation of phospholamban. Without control subjects, we cannot comment on whether the relaxation-frequency relationship in heart failure is preserved; however, we previously demonstrated preservation of the lusitropic response to ␤-adrenergic receptor stimulation in patients with severe heart failure,15 and others have shown preserved relaxation-frequency relations in adults39 and children40 with myocardial hypertrophy.

Study Limitations By infusing levosimendan directly into the left main coronary artery, we were able to avoid changes in loading conditions that might confound the interpretation of ⫹dP/dt and Tau. In the present study, levosimendan increased ⫹dP/dt despite a nonsignificant reduction in LV filling pressure. We cannot exclude the possibility that levosimendan caused coronary vasodilation and increased myocardial blood flow; however, doses used in our study were 10- to 100-fold lower than those used by Michaels et al.9 The use of ␤-blockers was low and this may have affected the response to intracoronary levosimendan; such an interaction was shown previously with dobutamine and enoximone.41 Last, we cannot comment on the acute cellular actions of levosimendan or the myocardial effects of levosimendan given at higher doses, for longer duration, or during physiological tachycardia (eg, with exercise).

Conclusions In summary, the present study demonstrates that levosimendan exerts positive inotropic and lusitropic effects over a range of heart rates in patients with LV systolic dysfunction. These effects are therefore independent of heart rate and systemic vascular actions of the drug. In addition, levosimendan does not alter the slope of the force-frequency or relaxation-frequency relationship. This hemodynamic profile is consistent with a primary site of action that is distal to calcium handling proteins that are involved in abnormal calcium homeostasis in failing myocardium.

Sources of Funding This work was supported in part by a grant-in-aid from the American Heart Association, Massachusetts Affiliate (to Dr Givertz), an American College of Cardiology/Merck Fellowship Award (to Dr Andreou), and by Orion Pharma, Espoo, Finland.

Disclosures Dr Colucci has received grant support from Abbot for the Randomized Multicenter Evaluation of Intravenous Levosimendan Efficacy (REVIVE) trial.

References 1. Nohria A, Lewis E, Stevenson LW. Medical management of advanced heart failure. JAMA. 2002;287:628 – 640. 2. Hajjar RJ, Gwathmey JK. Calcium-sensitizing inotropic agents in the treatment of heart failure: a critical view. Cardiovasc Drugs Ther. 1991; 5:961–965.

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3. Lehtonen L. Levosimendan: a calcium-sensitizing agent for the treatment of patients with decompensated heart failure. Curr Heart Fail Rep. 2004;1:136 –144. 4. Haikala H, Nissinen E, Etemadzadeh E, Levijoki J, Linden IB. Troponin C-mediated calcium sensitization induced by levosimendan does not impair relaxation. J Cardiovasc Pharmacol. 1995;25:794 – 801. 5. Edes I, Kiss E, Kitada Y, Powers FM, Papp JG, Kranias EG, Solaro RJ. Effects of levosimendan, a cardiotonic agent targeted to troponin C, on cardiac function and on phosphorylation and Ca2⫹ sensitivity of cardiac myofibrils and sarcoplasmic reticulum in guinea pig heart. Circ Res. 1995;77:107–113. 6. Hasenfuss G, Pieske B, Castell M, Kretschmann B, Maier LS, Just H. Influence of the novel inotropic agent levosimendan on isometric tension and calcium cycling in failing human myocardium. Circulation. 1998;98: 2141–2147. 7. Pagel PS, McGough MF, Hettrick DA, Lowe D, Tessmer JP, Jamali IN, Warltier DC. Levosimendan enhances left ventricular systolic and diastolic function in conscious dogs with pacing-induced cardiomyopathy. J Cardiovasc Pharmacol. 1997;29:563–573. 8. Ukkonen H, Saraste M, Akkila J, Knuuti MJ, Lehikoinen P, Nagren K, Lehtonen L, Voipio-Pulkki LM. Myocardial efficiency during calcium sensitization with levosimendan: a noninvasive study with positron emission tomography and echocardiography in healthy volunteers. Clin Pharmacol Ther. 1997;61:596 – 607. 9. Michaels AD, McKeown B, Kostal M, Vakharia KT, Jordan MV, Gerber IL, Foster E, Chatterjee K. Effects of intravenous levosimendan on human coronary vasomotor regulation, left ventricular wall stress, and myocardial oxygen uptake. Circulation. 2005;111:1504 –1509. 10. Faggiano P, Colucci WS. The force-frequency relation in normal and failing heart. Cardiologia. 1996;41:1155–1164. 11. Endoh M. Force-frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance. Eur J Pharmacol. 2004;500:73– 86. 12. Feldman MD, Alderman JD, Aroesty JM, Royal HD, Ferguson JJ, Owen RM, Grossman W, McKay RG. Depression of systolic and diastolic myocardial reserve during atrial pacing tachycardia in patients with dilated cardiomyopathy. J Clin Invest. 1988;82:1661–1669. 13. Vollmann D, Luthje L, Schott P, Hasenfuss G, Unterberg-Buchwald C. Biventricular pacing improves the blunted force-frequency relation present during univentricular pacing in patients with heart failure and conduction delay. Circulation. 2006;113:953–959. 14. Pieske B, Kretschmann B, Meyer M, Holubarsch C, Weirich J, Posival H, Minami K, Just H, Hasenfuss G. Alterations in intracellular calcium handling associated with the inverse force-frequency relation in human dilated cardiomyopathy. Circulation. 1995;92:1169 –1178. 15. Parker JD, Landzberg JS, Bittl JA, Mirsky I, Colucci WS. Effects of beta-adrenergic stimulation with dobutamine on isovolumic relaxation in the normal and failing human left ventricle. Circulation. 1991;84: 1040 –1048. 16. Hare JM, Givertz MM, Creager MA, Colucci WS. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of beta-adrenergic inotropic responsiveness. Circulation. 1998;97: 161–166. 17. Weiss JL, Frederiksen JW, Weisfeldt ML. Hemodynamic determinants of the time-course of fall in canine left ventricular pressure. J Clin Invest. 1976;58:751–760. 18. Landzberg JS, Parker JD, Gauthier DF, Colucci WS. Effects of intracoronary acetylcholine and atropine on basal and dobutamine-stimulated left ventricular contractility. Circulation. 1994;89:164 –168. 19. Kivikko M, Lehtonen L, Colucci WS. Sustained hemodynamic effects of intravenous levosimendan. Circulation. 2003;107:81– 86. 20. Pollesello P, Ovaska M, Kaivola J, Tilgmann C, Lundstrom K, Kalkkinen N, Ulmanen I, Nissinen E, Taskinen J. Binding of a new Ca2⫹ sensitizer, levosimendan, to recombinant human cardiac troponin C: a molecular modelling, fluorescence probe, and proton nuclear magnetic resonance study. J Biol Chem. 1994;269:28584 –28590. 21. Boknik P, Neumann J, Kaspareit G, Schmitz W, Scholz H, Vahlensieck U, Zimmermann N. Mechanisms of the contractile effects of levosimendan in the mammalian heart. J Pharmacol Exp Ther. 1997;280: 277–283. 22. Haikala H, Kaheinen P, Levijoki J, Linden IB. The role of cAMP- and cGMP-dependent protein kinases in the cardiac actions of the new calcium sensitizer, levosimendan. Cardiovasc Res. 1997;34:536 –546. 23. Kaheinen P, Pollesello P, Hertelendi Z, Borbely A, Szilagyi S, Nissinen E, Haikala H, Papp Z. Positive inotropic effect of levosimendan is

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24.

25.

26.

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28.

29.

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31.

32.

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correlated to its stereoselective Ca2⫹-sensitizing effect but not to stereoselective phosphodiesterase inhibition. Basic Clin Pharmacol Toxicol. 2006;98:74 –78. Yokoshiki H, Katsube Y, Sunagawa M, Sperelakis N. Levosimendan, a novel Ca2⫹ sensitizer, activates the glibenclamide-sensitive K⫹ channel in rat arterial myocytes. Eur J Pharmacol. 1997;333:249 –259. Slawsky MT, Colucci WS, Gottlieb SS, Greenberg BH, Haeusslein E, Hare J, Hutchins S, Leier CV, LeJemtel TH, Loh E, Nicklas J, Ogilby D, Singh BN, Smith W; on behalf of the Study Investigators. Acute hemodynamic and clinical effects of levosimendan in patients with severe heart failure. Circulation. 2000;102:2222–2227. Brown RA, Petrovski P, Savage AO, Ren J. Influence of ATP-sensitive K⫹ channel modulation on the mechanical properties of diabetic myocardium. Endocr Res. 2001;27:269 –281. Brixius K, Reicke S, Schwinger RH. Beneficial effects of the Ca2⫹ sensitizer levosimendan in human myocardium. Am J Physiol Heart Circ Physiol. 2002;282:H131–H137. Tachibana H, Cheng HJ, Ukai T, Igawa A, Zhang ZS, Little WC, Cheng CP. Levosimendan improves LV systolic and diastolic performance at rest and during exercise after heart failure. Am J Physiol Heart Circ Physiol. 2005;288:H914 –H922. Kawabata H, Ryomoto T, Ishikawa K. Role of cardiac ATP-sensitive K⫹ channels induced by angiotensin II type 1 receptor antagonist on metabolism, contraction and relaxation in ischemia-reperfused rabbit heart. Jpn Circ J. 2001;65:451– 456. De Luca L, Sardella G, Proietti P, Battagliese A, Benedetti G, Di Roma A, Fedele F. Effects of levosimendan on left ventricular diastolic function after primary angioplasty for acute anterior myocardial infarction: a Doppler echocardiographic study. J Am Soc Echocardiogr. 2006;19: 172–177. Sonntag S, Sundberg S, Lehtonen LA, Kleber FX. The calcium sensitizer levosimendan improves the function of stunned myocardium after percutaneous transluminal coronary angioplasty in acute myocardial ischemia. J Am Coll Cardiol. 2004;43:2177–2182. Colucci WS, Wright RF, Jaski BE, Fifer MA, Braunwald E. Milrinone and dobutamine in severe heart failure: differing hemodynamic effects and individual patient responsiveness. Circulation. 1986;73:III175–III-183.

33. Hajjar RJ, DiSalvo TG, Schmidt U, Thaiyananthan G, Semigran MJ, Dec GW, Gwathmey JK. Clinical correlates of the myocardial force-frequency relationship in patients with end-stage heart failure. J Heart Lung Transplant. 1997;16:1157–1167. 34. Gwathmey JK, Slawsky MT, Hajjar RJ, Briggs M, Morgan JP. Role of intracellular calcium handling in force-interval relationships of human ventricular myocardium. J Clin Invest. 1990;85:1599 –1613. 35. Schwinger RH, Bohm M, Muller-Ehmsen J, Uhlmann R, Schmidt U, Stablein A, Uberfuhr P, Kreuzer E, Reichart B, Eissner HJ. Effect of inotropic stimulation on the negative force-frequency relationship in the failing human heart. Circulation. 1993;88:2267–2276. 36. del Monte F, Harding SE, Dec GW, Gwathmey JK, Hajjar RJ. Targeting phospholamban by gene transfer in human heart failure. Circulation. 2002;105:904 –907. 37. Dipla K, Mattiello JA, Jeevanandam V, Houser SR, Margulies KB. Myocyte recovery after mechanical circulatory support in humans with end-stage heart failure. Circulation. 1998;97:2316 –2322. 38. Bohm M, Morano I, Pieske B, Ruegg JC, Wankerl M, Zimmermann R, Erdmann E. Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium. Circ Res. 1991;68: 689 –701. 39. Inagaki M, Yokota M, Izawa H, Ishiki R, Nagata K, Iwase M, Yamada Y, Koide M, Sobue T. Impaired force-frequency relations in patients with hypertensive left ventricular hypertrophy: a possible physiological marker of the transition from physiological to pathological hypertrophy. Circulation. 1999;99:1822–1830. 40. Banerjee A, Mendelsohn AM, Knilans TK, Meyer RA, Schwartz DC. Effect of myocardial hypertrophy on systolic and diastolic function in children: insights from the force-frequency and relaxation-frequency relationships. J Am Coll Cardiol. 1998;32:1088 –1095. 41. Metra M, Nodari S, D’Aloia A, Muneretto C, Robertson AD, Bristow MR, Dei CL. Beta-blocker therapy influences the hemodynamic response to inotropic agents in patients with heart failure: a randomized comparison of dobutamine and enoximone before and after chronic treatment with metoprolol or carvedilol. J Am Coll Cardiol. 2002;40:1248 –1258.

CLINICAL PERSPECTIVE Positive inotropic agents currently approved for the treatment of acute decompensated heart failure increase heart rate and stimulate arrhythmias by increasing intracellular calcium. Calcium-sensitizing agents were developed as an alternative and potentially safer means of inotropic support. Enthusiasm was tempered, however, by reports of impaired relaxation and the identification of more complex mechanisms of action that may contribute to adverse effects. Levosimendan increases myofilament calcium sensitivity by binding to troponin C and, in failing myocardium, has been shown to exert positive inotropic and lusitropic effects. In the present study, we demonstrated that subsystemic doses of levosimendan administered directly into the left main coronary artery increased cardiac contractility and relaxation over a range of heart rates dose-dependently, but did not alter the slope of the force-frequency or relaxation-frequency relationship. Although these in vivo mechanistic data do not shed additional light on the clinical controversy that surrounds levosimendan, they are notable in the demonstration of a remarkable consistency with in vitro findings in failing human myocardium, and they emphasize the importance of interpatient variability in hemodynamic responses to vasoactive therapy in heart failure. Additional studies are warranted to determine the functional significance of the heart rate–independent effects of levosimendan on failing myocardium, and to elucidate the variability in gene or protein expression that may contribute to differential patient responses in heart failure. With further advances in the field of pharmacogenomics, variability in the short- and long-term toxicity of cardiotonic agents may also be identified.

Muscarinic Modulation of the Sodium-Calcium Exchanger in Heart Failure Shao-kui Wei, MD; Abdul M. Ruknudin, PhD; Matie Shou, MD; John M. McCurley, MD; Stephen U. Hanlon, MD; Eric Elgin, MD; Dan H. Schulze, PhD; Mark C.P. Haigney, MD Background—The Na-Ca exchanger (NCX) is a critical calcium efflux pathway in excitable cells, but little is known regarding its autonomic regulation. Methods and Results—We investigated ␤-adrenergic receptor and muscarinic receptor regulation of the cardiac NCX in control and heart failure (HF) conditions in atrially paced pigs. NCX current in myocytes from control swine hearts was significantly increased by isoproterenol, and this response was reversed by concurrent muscarinic receptor stimulation with the addition of carbachol, demonstrating “accentuated antagonism.” Okadaic acid eliminated the inhibitory effect of carbachol on isoproterenol-stimulated NCX current, indicating that muscarinic receptor regulation operates via protein phosphatase– induced dephosphorylation. However, in myocytes from atrially paced tachycardia-induced HF pigs, the NCX current was significantly larger at baseline but less responsive to isoproterenol compared with controls, whereas carbachol failed to inhibit isoproterenol-stimulated NCX current, and 8-Br-cGMP did not restore muscarinic responsiveness. Protein phosphatase type 1 dialysis significantly reduced NCX current in failing but not control cells, consistent with NCX hyperphosphorylation in HF. Protein phosphatase type 1 levels associated with NCX were significantly depressed in HF pigs compared with control, and total phosphatase activity associated with NCX was significantly decreased. Conclusions—We conclude that the NCX is autonomically modulated, but HF reduces the level and activity of associated phosphatases; defective dephosphorylation then “locks” the exchanger in a highly active state. (Circulation. 2007;115: 1225-1233.) Key Words: calcium 䡲 electrophysiology 䡲 heart failure 䡲 receptors, adrenergic, beta 䡲 sodium

T

he cardiac Na-Ca exchanger (NCX), a protein found in the sarcoplasmic membrane, acts as the major Ca2⫹ efflux path and is an important Ca2⫹ handling protein regulating intracellular Ca2⫹ in excitation-contraction coupling in the heart. The NCX plays an important role in pathological states as well, acting as a major Ca2⫹ entry site during ischemia/reperfusion.1 In heart failure (HF), the NCX is significantly upregulated in both human2 and animal models,3,4 which may significantly impair cardiac contractility by reducing sarcoplasmic reticular Ca2⫹ content via premature Ca2⫹ efflux.5 It may also promote unstable repolarization with early and/or delayed afterdepolarizations, triggering fatal ventricular arrhythmias.6

Clinical Perspective p 1233 Cardiac output is controlled on a beat-to-beat basis by the interaction of the sympathetic and parasympathetic nervous systems. Cardiac sympathetic stimulation activates the ␤-adrenergic receptor (␤-AR), whereas parasympathetic stimu-

lation acts on cardiac muscarinic receptor (M-2) systems. These signaling systems interact to regulate cardiac contractility and rate by modulating critical effector proteins7 via “accentuated antagonism,”8 a phenomenon in which the effect of sympathetic stimulation is rapidly reversed by parasympathetic stimulation despite continued application of the initial stimulus. Accentuated antagonism allows rapid increases in cardiac output in response to physiological challenge while preventing toxicity from excess adrenergic tone.9 In HF both ␤-AR and M-2 cardiac responsiveness are depressed, contributing to lost cardiac reserve, increased arrhythmic susceptibility,10 and death. However, the underlying cellular mechanism of these phenomena is far from clear. Some studies have attributed this loss of autonomic responsiveness to the desensitization of ␤-AR receptors and/or downregulation of signal transduction in HF.11,12 However, recent work suggests that the L-type Ca2⫹ channel and ryanodine receptor are tonically phosphorylated (“hyperphosphorylated”) at baseline in failing human myocytes,13–16 which could contribute to depressed

Received July 25, 2006; accepted December 29, 2006. From the Division of Cardiology, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Md (S.W., M.S., J.M.M., S.U.H., M.C.P.H.); Department of Microbiology and Immunology, University of Maryland, School of Medicine, Baltimore (A.M.R., D.H.S.); Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore, Md (A.M.R.); and Division of Cardiology, Walter Reed Army Medical Center, Washington, DC (E.E.). The online-only Data Supplement, consisting of figures, is available with this article at http://circ.ahajournals.org/cgi/content/ full/CIRCULATIONAHA.106.650416/DC1. Correspondence to Mark C.P. Haigney, MD, Division of Cardiology, Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD 20814. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.650416

Angelini TABLE 4.

Coronary Artery Anomalies

1301

Pathophysiological Mechanisms and Coronary Anomalies (Functional Classification) Proof of Action

Pathophysiological Mechanism Misdiagnosis

Coronary Anomaly “Missing” coronary artery

Certain

“Hypoplastic” coronary artery Myocardial ischemia, primary (fixed and/or episodic)

x

Ostial stenosis

x

Coronary fistula

x x

Muscular bridge Myocardial ischemia, secondary (episodic)

Increased risk of fixed coronary atherosclerotic disease

Secondary aortic valve disease

x

Tangential origin (ACAOS) intramural course

x

Myocardial bridge, plus spasm and/or clot

x

Coronary ectasia (plus mural clot)

x

Coronary fistula (plus mural clot)

x

Coronary fistula

x

ALCAPA

x

Coronary ectasia

x

Muscular bridge (proximal to)

x

Coronary aneurysm (ostial)

x

Coronary fistula

x

ALCAPA

x

Increased risk of bacterial endocarditis

Coronary fistula

Ischemic cardiomyopathy (hibernation)

ALCAPA

x

Volume overload

Coronary fistula

x

ALCAPA

x

Ectopic ostia (tangential)

x

Unusual technical difficulties during coronary angiography or angioplasty

Complications during cardiac surgery

Unlikely

x

Ostial atresia

ALCAPA

Possible

x

x

Split left coronary artery

x

Coronary fistula

x

Ectopic ostia and proximal course

x

Muscular bridge

x

ALCAPA indicates anomalous origination of the left coronary artery from the pulmonary artery. Adapted from Angelini P et al10 with permission from Lippincott, Williams & Wilkins. Copyright 1999.

tion), changes in the aortic pressure (as at the onset of hypertension or aortic regurgitation), or a rapid weight gain, especially in patients who receive negative chronotropic agents, which increase the stroke volume if the cardiac output remains essentially unchanged. Moreover, a treadmill stress test, which should be transformed into an adenosine test because of an inadequate effort or chronotropic response, may be the most accurate predictive test for ACAOS because it associates an increased cardiac output with nonphysiological bradycardia. Unfortunately, though, such a hybrid protocol is a potential cause of sudden death, specifically in ACAOS carriers, and should generally be avoided or at least closely monitored in a hospital environment. When a carrier of ACAOS dies suddenly, in the absence of other lethal cardiovascular conditions, a low cardiac output and bradycardia or asystole typically occur early after extreme exercise, after which syncope and/or death ensues. Terminal ventricular fibrillation may also occur as a manifestation of critical ischemia or of reperfusion arrhythmia.30 –32

Both the anomalous right and left coronary arteries can be responsible for sudden death, although the risk has not been adequately quantified in specific studies. Most likely, predisposing factors include the severity of baseline stenosis, the specific conditions at the time of the crisis, and the myocardial territory at risk.7,33 Additionally, one must realize that the possible manifestations of ACAOS include not only sudden death but also dyspnea, palpitations, angina pectoris, dizziness, and syncope.4,10,12,26,32 Whereas sudden death is usually associated with extreme exercise in young adults,34 the other manifestations of ACAOS are more frequently seen in older adults (in our experience, specifically women) and are related to the onset of hypertension. Interestingly, Cheitlin33 claimed that sudden death is seen only in young patients, possibly because of progressive hardening of the aortic wall in adults. During aortic valve replacement, an intramural ectopic coronary artery can also be liable to critical worsening of extrinsic compression by the prosthetic ring, as recently reviewed by Morimoto and colleagues.35

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Figure 4. Images obtained from a 42-year-old man with severe angina of recent onset, who had recently developed hypertension. A, Selective angiogram of the RCA in the left anterior oblique projection. In this view, the proximal course appears wider than the more distal vessel, and it originates next to the LCA (which is also ectopic), high above the left aortic sinus (LS). B, IVUS image of the distal RCA. The cross-sectional area is 10.8 mm2, and the shape is circular. C, IVUS image of the proximal segment of the RCA, whose lumen is severely compressed laterally (minimal diameter, 1.5 mm; maximal diameter, 3.8 mm; cross-sectional area, 4.2 mm2; area stenosis, 61%). The LCA had also milder ostial stenosis. D, IVUS of the proximal RCA after stent angioplasty (3.5 ⫻ 12 mm; postdilated at 18 atm). The shape has become round, and the area has expanded to match that of the distal normal vessel.

Outlines for Diagnostic and Treatment Protocols In carriers of ACAOS, the clinical histories are consistent in only 1 aspect: Either these patients die suddenly (typically at a young age and after extreme exertion), or they have no characteristic presentation. Most patients are asymptomatic for a large portion of their lives, and an atypical chest-pain syndrome is the most common reason they are referred for coronary angiography, which is when the diagnosis is typically made. The milder cases are more likely to be identified fortuitously (because of a falsely positive stress test and/or coincidental atherosclerotic disease). The fact that CAAs include many different entities and that no single observer or group has collected a large enough series to clarify the natural prognosis of each entity may contribute to our difficulty in the clinical identification of these lesions, especially the ones that could lead to angina or sudden cardiac death.21 For most types of coronary anomalies, the fundamental clinical approach could be: “Do not bother to look for these innocent anomalies, but be prepared to recognize them as benign if one is accidentally found,

typically at coronary angiography.” However, for a few CAAs that are possibly or predictably malignant (fundamentally, ACAOS), we should establish solid diagnostic screening protocols, especially for athletes and other young individuals subjected to extreme exertion.9,10,29,33 As noted above, ACAOS patients can succumb to sudden cardiac death, usually but not necessarily at a young age, possibly even at the newborn stage.36 Retrospectively reviewed, only a few persons reported to have died of ACAOS had significant symptoms, usually atypical chest pain, dyspnea, syncope, or their equivalents, before the final event.5–7,9,10,13–15 A specific workup protocol is indicated mostly for athletes and military personnel with these symptoms. In view of the fairly rare nature of ACAOS, it would not seem practical or cost-effective to extend the indications for such a workup to all schoolchildren on a routine basis. Nevertheless, larger prospective studies are needed before this decision can become final.37–39 In patients with suspected ACAOS, testing should sequentially include electrocardiography, Holter monitoring (basi-

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March 13, 2007 carbachol did not result in a significant inhibitory effect on isoproterenol-stimulated INCX in HF cells (Figure 2B2). This is the first direct evidence of blunted M-2 regulation of the NCX current in HF. Our previous study in a ventricularly paced HF model demonstrated that increased basal INCX and reduced ␤-AR responsiveness are due to hyperphosphorylation of NCX in HF, but the mechanism of hyperphosphorylation and the interaction with the muscarinic system were not elucidated. Possible explanations for the failure of muscarinic modulation in HF include a decrease in M-2 receptor number, downregulated signal transduction, and/or altered protein phosphorylation state. These alternatives are investigated in the following experiments.

Altered Receptor Number and Downregulated Signal Transduction Are Excluded for Explaining Blunted ␤-〈R and M-2 Regulation of NCX Current in HF

Figure 2. INCX responsiveness to isoproterenol and carbachol in control and failing myocytes. A1, A representative current tracing of INCX from a control myocyte in the basal state (blue), after exposure to isoproterenol (2 ␮mol/L, red), and after isoproterenol plus carbachol (5 ␮mol/L, green). Isoproterenol markedly increased outward and inward INCX, whereas carbachol reversed the increase in INCX induced by isoproterenol, consistent with “accentuated antagonism.” A2, The mean data of peak outward current (at ⫹70 mV; the number of independent experiments is shown in parentheses) as described in A1, confirming that isoproterenol (ISO) significantly increased INCX (*P⬍0.01, isoproterenol vs basal) and that carbachol (CCH) significantly reversed isoproterenol stimulation (#P⬍0.05, carbachol vs isoproterenol). B, INCX from failing myocytes (format analogous to that in A). B1, In cells from failing animals, basal INCX is significantly increased compared with control myocytes, whereas isoproterenol induced a significantly smaller further increase. Unlike in control cells, carbachol had no effect on the isoproterenol-stimulated current, demonstrating failure of muscarinic-accentuated antagonism. B2, The mean data of peak outward current (at ⫹70 mV; the number of independent experiments is shown in parentheses) in myocytes from failing animals as described in B1, confirming the failure of carbachol to reverse the effect of isoproterenol. *P⬍0.01.

Blunted ␤-AR and M-2 Regulation of the NCX Current in HF

Recent evidence suggests that ␤-AR stimulation increases NCX activity in mammalian myocytes and that M-2 stimulation can modulate this effect.17–20 Figure 2A1 shows a representative tracing of INCX from control myocytes in basal conditions, in the presence of the ␤-AR agonist isoproterenol (2 ␮mol/L/L), and in the presence of isoproterenol plus the M-2 agonist carbachol (5 ␮mol/L). Isoproterenol markedly increased outward and inward INCX in control myocytes, and carbachol significantly inhibited the isoproterenol-stimulated effect. The mean population data of peak outward current density (at ⫹70 mV) reveals that isoproterenol increased INCX ⬇300% in controls, whereas carbachol almost completely reversed it (P⬍0.01; Figure 2A2). However, in failing myocytes basal INCX was significantly increased compared with control myocytes, whereas isoproterenol manifested blunted stimulation, increasing the peak INCX to a significantly lesser extent (Figure 2B2). The application of the M-2 agonist

We found no evidence of a reduction in either muscarinic or ␤-adrenergic receptor protein expression in HF, suggesting that receptor number was not changed drastically in our model and pointing to a defect in signal transduction (see online-only Data Supplement). Stimulation of the cardiac muscarinic receptor (mainly M-2) is thought to activate soluble guanylyl cyclase, leading to an increase in intracellular cyclic GMP (cGMP). This increase in cytosolic cGMP could either activate cGMP-dependent protein kinase G (PKG) to phosphorylate effectors or activate a protein phosphatase to dephosphorylate PKA-induced phosphorylation. To identify the altered steps resulting in blunted ␤-AR and M-2 regulation of INCX in HF, we exposed failing cells to 8-Br-cAMP and 8-Br-cGMP to directly stimulate PKA and PKG. In control myocytes, cAMP (1 mmol/L) significantly increased INCX in a manner similar to that of isoproterenol, whereas cGMP significantly reversed this effect; cGMP also reversed the effect of isoproterenol (Figure 3). In failing myocytes, however, both cGMP effects were blunted, suggesting that the impaired ␤-AR and M-2 regulation is not due to reduced receptor number or uncoupling of the ␤-AR and M-2 signaling pathways. Rather, the defect must be downstream from the generation of cAMP and cGMP.

Muscarinic System Antagonizes ␤-AR Stimulation via Protein Phosphatase

Figure 4A shows that okadaic acid (1 ␮mol/L) significantly reversed the carbachol inhibition of isoproterenol-stimulated NCX current in control myocytes. This result suggests that a protein phosphatase is a crucial component for M-2 regulation of NCX in myocytes and argues for dephosphorylation as opposed to phosphorylation as a mechanism for M-2 modulation of the NCX. Furthermore, if a protein phosphatase is the mediator of M-2 modulation in the control state, it follows that infusion of exogenous protein phosphatase enzyme should reverse isoproterenol stimulation in HF in a manner similar to that of carbachol in the controls. In a separate experiment, dialysis of PP1 (10 U/mL) through the intracellular solution significantly inhibited INCX in basal and isoproterenol-stimulated conditions in failing myocytes but had no significant effect on basal current in control myocytes (Figure 4B). These data suggest that M-2 stimulation inhibits isoproterenol-stimulated

Wei et al

Figure 3. INCX responsiveness to isoproterenol and cGMP in control and failing myocytes. Format is analogous to that in Figure 2. In control myocytes, either 8-Br-cAMP (2 mmol/L) or isoproterenol (ISO) (2 ␮mol/L, applied in separate cells) significantly increased INCX (*P⬍0.01), and 8-Br-cGMP (2 mmol/L) significantly reversed it (#P⬍0.05), similar to the effect of carbachol. However, in failing myocytes agonist responses were blunted to both 8-Br-cAMP and isoproterenol, and 8-Br-cGMP failed to significantly reduce the INCX, showing that the failure of muscarinic effect is not due to depression of cGMP but is due instead to a downstream defect.

INCX through activation of a protein phosphatase and that excessive protein phosphorylation in HF results in both increased basal activity and decreased ␤-AR responsiveness. A unifying hypothesis explaining these phenomena, as well as the failure of the NCX to respond to M-2 stimulation in HF, would be that the protein phosphatase associated with the NCX is significantly downregulated or inhibited in HF. To test this hypothesis, we measured protein phosphatase levels and activity in protein precipitated with the NCX complex.

Protein Phosphatases Associated With NCX in HF To test whether the protein phosphatases associated with the NCX are decreased in HF, we examined PP1- and PP2aassociated NCX with a specific antibody directed against PP1 and PP2a in NCX proteins immunoprecipitated by NCX antibody. The NCX proteins were immunoprecipitated by NCX antibody from control and failing heart tissues. The proteins in the NCX complex were separated with the use of PAGE and transferred to nitrocellulose membranes. These membranes were immunoblotted with PP2a antibodies, and the same blot was reprobed with NCX antibody after the membrane was stripped of antibodies from a previous experiment. Figure 5A is a representative Western blot of PP1 and PP2a in control and failing heart samples, showing that PP1 protein is significantly reduced in HF. Surprisingly, PP2a expression appears significantly increased in failing heart muscle compared with control in absolute terms (Figure 5B;

NCX Regulation in Heart Failure

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Figure 4. The muscarinic modulation of NCX activity appears to act via a protein phosphatase. A, A representative current tracing showing that protein phosphatase inhibition by okadaic acid (OKA) (1 ␮mol/L) eliminates the inhibitory effect of carbachol (CCH) on isoproterenol (ISO)-stimulated INCX in control myocytes (left). The mean peak outward current density in the basal state, in the presence of isoproterenol, isoproterenol plus carbachol, and isoproterenol plus carbachol plus okadaic acid are shown in the right panel (P⬍0.05, ISO⫹CCH vs ISO or ISO⫹CCH vs ISO⫹CCH⫹OKA; n⫽7), indicating that the muscarinic inhibition operates through activating a protein phosphatase. B, The effects of intracellularly applied PP1 (10 U/mL) on INCX in myocytes from control and failing (HF) pigs. PP1 significantly depressed basal INCX (HFBasal) current and after isoproterenol stimulation (HFISO) [*P⬍0.05, PP1(⫹) vs PP1(⫺) exposed NCX current in HF]. PP1 had no significant effect on unstimulated control myocytes, suggesting that the NCX is not phosphorylated significantly at baseline.

P⬍0.05). However, the total amount of NCX protein was increased in failing hearts by ⬇40% (Figure 5B; P⬍0.05). After normalization for the amount of NCX protein from control and failing hearts, group analysis confirmed that the mean amount of PP1 in failing hearts was 33% that in control hearts (P⬍0.01), but PP2a levels were not altered with respect to NCX. To test whether this relative shift in protein expression would alter phosphatase activity, we assessed protein phosphatase activity associated with the NCX and in bulk myocardium with para-nitrophenyl phosphate and malachite green methods. Protein phosphatase activity associated with the NCX was significantly depressed in failing heart compared with control, but no significant difference existed between protein phosphatase activity in bulk myocardium between failure and control states (Figure 6). These results provide direct evidence that protein phosphatase activity associated with the NCX is downregulated in HF.

Discussion

This is the first study to investigate the interaction of ␤-AR and M-2 regulation of the NCX in HF. The principal findings are as follows: (1) muscarinic receptor stimulation in control

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Figure 5. Identification and quantification of protein phosphatases associated with NCX protein. The NCX macromolecular complex was immunoprecipitated from normal and failure heart extracts with the use of NCX antibody and Protein A Sepharose beads. The proteins in the NCX complex were separated with the use of PAGE and transferred to nitrocellulose membranes. These membranes were immunoblotted with PP2a antibodies, and the same blot was reprobed with NCX antibody after the membrane was stripped of antibodies from a previous experiment. A shows PP1 protein (top) in the complex immunoprecipitated by NCX antibody, PP2a (middle), and the NCX protein itself in the complex (bottom) in representative control (C) and HF (F) animals. B, The top panel shows the average amount of PP1 protein (⫾SEM) associated with the NCX complex from 6 HF and 5 normal animals. There was significant reduction of PP1 enzyme associated with NCX in HF (**P⬍0.01). There was a slight (but significant, *P⬍0.05) increase in the PP2a enzyme amount associated with NCX in the HF compared with normal hearts (middle). NCX protein (bottom) measured by Western blot was modestly but significantly increased in HF hearts compared with controls (P⬍0.05).

myocytes significantly reverses ␤-AR stimulation of the NCX via activation of a protein phosphatase; (2) in HF, the NCX is “locked” in a relatively high activity state and is insensitive to both ␤-AR and M-2 regulation; (3) the common pathway of these alterations is downregulated protein phosphatase activity resulting in defective NCX dephosphorylation in HF; and (4) the profile of protein phosphatases associated with the NCX is significantly changed in HF with a reduction in PP1 but no change in PP2a. These results extend our previous findings that HF results in hyperphosphorylation of the NCX by providing a mechanism for an increased phosphorylation state, and they further explore the impact of this phenomenon on the autonomic modulation of the exchanger. Furthermore, the use of atrially paced animals addresses the concern that NCX hyperphosphorylation may represent an epiphenomenon of ventricular pacing.

Modulation of the NCX by the ␤-AR and Muscarinic Systems Controversy exists regarding whether the cardiac NCX is modulated by PKA.29,30 The cAMP-dependent Cl⫺ current (CFTR) and the Ca-activated Cl⫺ current are reported to be Ni2⫹ sensitive and have reversal potentials similar to those of NCX.31 Lin et al32 have suggested that these conductances could contaminate our recordings of NCX, especially during stimulation of the ␤-AR, causing an overestimation of the effect of isoproterenol on the NCX. In the present study, we found no effect of significantly reducing extracellular chloride on the bidirectional, Ni2⫹-sensitive current in pig ventricular myocytes. Additionally, we found that the current was Ca2⫹ dependent, which is consistent with the NCX but not CFTR. Our result is consistent with other reports that although the CFTR conductance is highly represented in small animals, it is nearly or

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Figure 6. Protein phosphatase activity in normal and failing hearts. The protein phosphatase activity associated with NCX protein complex and in the heart extract was determined with the use of para-nitrophenyl phosphate and phosphopeptide (KRpTIRR) as substrates. A, The amount of general phosphatase activity was reduced in the NCX complex–associated phosphatases (n⫽6; *P⬍0.01), but the activity was not significantly different in the heart extracts in HF compared with control pigs. B, The serine/threonine phosphatase activity associated with the NCX was also significantly reduced in HF animals compared with controls (C).

completely absent in large animals such as dogs or humans.33 Further underscoring the significance of interspecies differences, Ginsburg and Bers34 found no evidence of isoproterenol stimulation of NCX in meticulously controlled experiments in nonfailing rabbits. Although we have no experience with this model, one wonders whether differences in phosphatase activity may account for differences in response to isoproterenol; in such a case, an alternative approach using a phosphatase inhibitor might have yielded evidence of NCX augmentation. Our findings, furthermore, are consistent with those of other groups who found significant increases in NCX current after stimulation with isoproterenol.17,18,20 The exchanger has been reported to be associated with the regulatory R-1 complex comprising PKA, AKAP, PP1, protein kinase C, and PP2a,21 an important piece of evidence suggesting autonomic regulation. Nevertheless, the PKA phosphorylation site on the exchanger has not been identified. The rat cardiac NCX1 sequence presents 5 probable sites, of which 3 are intracellular (threonine at 74 and 618; serine at 389). Methodological differences in the assessment of NCX activity may also contribute to conflicting results. Caffeine superfusion during voltage clamp is a time-honored alternative method of detecting and quantifying the exchange current, but for purposes of studying changes induced by phosphorylation of the exchanger, the ramp method may be superior because it should be less sensitive to changes in sarco/endoplasmic reticular Ca2⫹-ATPase function and sarcoplasmic reticular calcium load. Further evidence that the NCX is regulated by the autonomic nervous system in a manner that is relevant to human disease is accumulating. Although stimulation of the M-2 receptor by carbachol in the absence of preceding ␤-AR stimulation has been shown to induce an indirect increase in the NCX current via subsarcolemmal Na⫹ gain,35 the present study confirms the observations of Zhang et al20 that NCX activity is strongly depressed by muscarinic agonists administered during ␤-AR stimulation, but the mechanism of signal transduction was not previously elucidated. Investigating the L-type Ca channel, Jiang et al36 reported that M-2 modulates ␤-AR stimulation via PKG-induced L-type channel phosphorylation, whereas Shen and Pappano37 have suggested that the primary response of

cGMP is to activate protein phosphatases to dephosphorylate PKA-induced phosphorylation. To examine these putative mechanisms, we reasoned that if the muscarinic effect is modulated by phosphorylation of a separate site on the NCX in the presence of isoproterenol, then application of a nonspecific protein phosphatase inhibitor, ie, okadaic acid, should increase (or at least not inhibit) muscarinic modulation of the current by allowing further accumulation of phosphorylation. Alternatively, if M-2 stimulation results in activation of an NCX-associated protein phosphatase, inhibition of that phosphatase by okadaic acid should reverse the effect of M-2 stimulation and restore the current. We found that okadaic acid indeed reversed the carbachol effect, consistent with activation of a phosphatase as the mechanism of muscarinic modulation of the NCX. Finally, Katanosaka et al38 have reported that the C-terminus of the A␤ of calcineurin, a phosphatase mechanistically implicated in cardiac hypertrophy, binds to the cytoplasmic loop of the cardiac NCX in hamsters. Prolonged phenylephrine exposure, which would stimulate the ␣-adrenergic system and act as a model of hypertension, resulted in inhibition of Na⫹o-dependent Ca2⫹ efflux from isolated myocytes in a manner that was prevented by inhibition by calcineurin. This is consistent with differential phosphatase modulation by distinct pathological signaling mechanisms.

Blunted ␤-〈R and M-2 Regulation in HF Recent studies have suggested that elevated tonic phosphorylation (hyperphosphorylation) of calcium handling proteins such as the L-type Ca channel and the ryanodine receptor might contribute to increased basal Ca2⫹ permeability and reduced responsiveness to ␤-AR stimulation. In our previous study, we also found that the NCX is hyperphosphorylated in HF, suggesting that downregulated protein phosphatase might be the underlying mechanism. In the present study, we have demonstrated reduced PP1 protein and protein phosphatase activity associated with the NCX, providing a mechanism for NCX hyperphosphorylation in HF. The present study differs somewhat from our previous work in that these animals were atrially paced into HF. This change should avoid artifactual changes in myocyte phenotype due to abnormal depolarization;

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however, the magnitude of the increase in the basal current due to HF appears to be 25% less than we found previously despite similar increases in NCX expression. The most likely explanation is that rapid ventricular pacing induces a more severe HF phenotype because of the introduction of intraventricular and atrioventricular dyssynchrony that would not be appreciated by echocardiograms performed in sinus rhythm. In the present study, we found that the M-2 receptor agonist carbachol loses its ability to inhibit ␤-AR stimulation on the NCX in HF. In contrast to the decreased numbers of ␤-AR receptors found in HF, the number of M-2 receptors is either unchanged or increased in HF.39 We found that application of cGMP fails to recover M-2 inhibition of ␤-AR signaling, confirming that the defect of muscarinic regulation is not at the M-2 receptor and/or Gi protein and must be downstream of cGMP. However, dialysis with PP1 significantly depressed both NCX current in basal conditions and after isoproterenol stimulation in HF. Furthermore, the protein phosphatase inhibitor okadaic acid eliminates carbachol inhibition of isoproterenol-stimulated INCX, indicating that M-2 regulation of the NCX is dependent on protein phosphatase activity. On the basis of these findings, we suggest that both ␤-AR and M-2 dysfunction in HF are due to downregulation of protein phosphatase, principally PP1.

Downregulated Protein Phosphatase Associated With NCX in HF The effect of HF on protein phosphatase expression and activity is clearly complex. Neumann et al40 have reported that cardiac PP1 protein and mRNA levels were increased in HF, resulting in decreased phospholamban phosphorylation and depressed sarcoplasmic reticulum–ATPase function, whereas other groups have found decreased local protein phosphatase expression. Marx et al14 found that the protein levels of both PP1 and PP2a associated with ryanodine receptor were reduced in HF. In the present study, we found that the NCX associates with protein phosphatases in a macromolecular complex in pig hearts, similar to findings in the rat. In contrast to the results of Marx et al (with respect to the ryanodine receptor), however, we found that PP1 expression is decreased, whereas PP2a associated with the NCX is unchanged in HF. Despite this shift in phosphatase profile associated with the complex, we found that total protein phosphatase activity associated with NCX is significantly depressed in HF compared with control (whereas total myocardial activity is unchanged). These results suggest that PP1 might play a principal role in NCX dephosphorylation in the control state, whereas PP2a is predominant in HF. Although it is generally believed that PP1 and PP2a have similar activity in terms of dephosphorylation of serine/threonine sites, the structure, activity, and regulation of these isoforms are distinct.41,42 PP1 activity is modulated by the inhibitor protein I-1, which is itself activated by PKA phosphorylation. PP2a can dephosphorylate I-1,43 but little is known about the regulation of PP2a itself. A recent report has suggested that the enzyme is inhibited by elevated [Ca2⫹]I,44 whereas overexpression of PP2a has been shown to result in reduce cardiac contractility.45 The present study suggests that PP2a activity is not modulated by muscarinic stimulation, resulting in failure of dephosphorylation of the NCX in response to M-2 stimulation. The effect of this shift in phosphatase

isoform expression on NCX regulation and the HF phenotype merits further investigation.

Conclusions Fifty percent of the deaths in patients with HF are sudden and presumably arrhythmic. Increased NCX activity in HF is likely to enhance depolarizing current, particularly when associated with spontaneous calcium release from the sarcoplasmic reticulum, which could result in afterdepolarizations triggering fatal ventricular arrhythmia. Furthermore, excess NCX activity has been tied to depression of the systolic calcium transient in HF, linking the NCX to mechanical pump dysfunction as well. The present study shows that in HF, the NCX is “locked” in a relatively high activity state and insensitive to both ␤-AR and M-2 regulation, which could reduce cardiac contractile reserve and increase susceptibility to arrhythmia. The common pathway of these alterations is downregulation of associated protein phosphatase activity, resulting in defective NCX dephosphorylation in HF. Dephosphorylation of the NCX represents a possible therapeutic target for reducing arrhythmia and pump dysfunction in HF.

Sources of Funding The present study was supported in part by grants from the Department of Defense (CO83OD to Dr Haigney; C083QF to Dr McCurley), the National Institutes of Health (HL62521 to Dr Schulze), the National Institutes of Aging (AG-020823 to Dr Ruknudin), and the American Heart Association (0265463U to Dr Wei; 9730173N to Dr Ruknudin).

Disclosures None.

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29. Condrescu M, Gardner JP, Chernaya G, Aceto JF, Kroupis C, Reeves JP. ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger. J Biol Chem. 1995;270:9137–9146. 30. Collins A, Somlyo AV, Hilgemann DW. The giant cardiac membrane patch method: stimulation of outward Na(⫹)-Ca2⫹ exchange current by MgATP. J Physiol. 1992;454:27–57. 31. Xue L, Jo H, Matsuoka S. Ni2⫹ inhibits both Na-Ca exchange and cAMP-dependent Cl⫺ currents in guinea-pig ventricular cells. Biophys J. 2003;86:613a. Abstract. 32. Lin X, Jo H, Sakakibara Y, Tambara K, Kim B, Komeda M, Matsuoka S. Beta-adrenergic stimulation does not activate Na⫹/Ca2⫹ exchange current in guinea pig, mouse, and rat ventricular myocytes. Am J Physiol. 2006;290:C601–C608. 33. Du XY, Finley J, Sorota S. Paucity of CFTR current but modest CFTR immunoreactivity in non-diseased human ventricle. Pflugers Arch. 2000;440:61–67. 34. Ginsburg KS, Bers DM. Isoproterenol does not enhance Ca-dependent Na/Ca exchange current in intact rabbit ventricular myocytes. J Mol Cell Cardiol. 2005;39:972–981. 35. Saeki T, Shen JB, Pappano AJ. Carbachol promotes Na⫹ entry and augments Na/Ca exchange current in guinea pig ventricular myocytes. Am J Physiol. 1997;273:H1984 –H1993. 36. Jiang LH, Gawler DJ, Hodson N, Milligan CJ, Pearson HA, Porter V, Wray D. Regulation of cloned cardiac L-type calcium channels by cGMPdependent protein kinase. J Biol Chem. 2000;275:6135– 6143. 37. Shen JB, Pappano AJ. On the role of phosphatase in regulation of cardiac L-type calcium current by cyclic GMP. J Pharmacol Exp Ther. 2002; 301:501–506. 38. Katanosaka Y, Iwata Y, Kobayashi Y, Shibisaki F, Wakabayashi S, Shigekawa M. Calcineurin inhibits Na⫹/Ca2⫹ exchange in phenylephrine-treated hypertrophic cardiomyocytes. J Biol Chem. 2005;280: 5764 –5772. 39. Le Guludec D, Cohen-Solal A, Delforge J, Delahaye N, Syrota A, Merlet P. Increased myocardial muscarinic receptor density in idiopathic dilated cardiomyopathy: an in vivo PET study. Circulation. 1997;96:3416 –3422. 40. Neumann J, Eschenhagen T, Jones LR, Linck B, Schmitz W, Scholz H, Zimmermann N. Increased expression of cardiac phosphatases in patients with end-stage heart failure. J Mol Cell Cardiol. 1997;29:265–272. 41. Luss H, Klein-Wiele O, Boknik P, Herzig S, Knapp J, Linck B, Muller FU, Scheld HH, Schmid C, Schmitz W, Neumann J. Regional expression of protein phosphatase type 1 and 2A catalytic subunit isoforms in the human heart. J Mol Cell Cardiol. 2000;32:2349 –2359. 42. duBell WH, Lederer WJ, Rogers TB. Dynamic modulation of excitationcontraction coupling by protein phosphatases in rat ventricular myocytes. J Physiol. 1996;493:793– 800. 43. Gupta RC, Neumann J, Watanabe AM, Sabbah HN. Inhibition of type 1 protein phosphatase activity by activation of beta-adrenoceptors in ventricular myocardium. Biochem Pharmacol. 2002;63:1069 –1076. 44. Palanivel R, Veluthakal R, Kowluru A. Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulinsecreting INS-1 cells. Am J Physiol. 2004;286:E1032–E1041. 45. Gergs U, Boknik P, Buchwalow I, Fabritz L, Matus M, Justus I, Hanske G, Schmitz W, Neumann J. Overexpression of the catalytic subunit of protein phosphatase 2A impairs cardiac function. J Biol Chem. 2004;279: 40827– 40834.

CLINICAL PERSPECTIVE The sodium-calcium exchanger is the most important protein for removing calcium from the cytosol of cardiac muscle cells. Although calcium is needed to drive systolic contraction, excess calcium is associated with arrhythmias and cell death. Despite the importance of the sodium-calcium exchanger in maintaining calcium homeostasis, the mechanisms controlling the activity of the exchanger are poorly understood. Sympathetic nervous system stimulation of the sodium-calcium exchanger by ␤-adrenergic agonists increases its activity in healthy pigs. In the present study in cardiac myocytes from healthy and heart failure pigs, the effect of agonists of the parasympathetic nervous system was tested and found to reverse the stimulatory effects of isoproterenol or cAMP analogues. In heart failure, however, the exchanger fails to respond to either sympathetic or parasympathetic nervous system activity but instead remains “locked” in a high activity state. This failure of modulation may contribute to increased myocyte calcium loss in heart failure, contributing to poor systolic contractile performance and arrhythmogenesis.

Genetics Use of a Constitutively Active Hypoxia-Inducible Factor-1␣ Transgene as a Therapeutic Strategy in No-Option Critical Limb Ischemia Patients Phase I Dose-Escalation Experience Sanjay Rajagopalan, MD; Jeffrey Olin, DO; Steven Deitcher, MD; Ann Pieczek, RN; John Laird, MD; P. Michael Grossman, MD; Corey K. Goldman, MD, PhD; Kevin McEllin, MS; Ralph Kelly, MD; Nicolas Chronos, MD Background—Critical limb ischemia, a manifestation of severe peripheral atherosclerosis and compromised lowerextremity blood flow, results in a high rate of limb loss. We hypothesized that adenoviral delivery of a constitutively active form of the transcription factor hypoxia-inducible factor-1␣ (ie, Ad2/HIF-1␣/VP16 or HIF-1␣) into the lower extremity of patients with critical limb ischemia would be safe and might result in a durable clinical response. Methods and Results—This phase I dose-escalation program included 2 studies: a randomized, double-blind, placebocontrolled study and an open-label extension study. In total, 34 no-option patients with critical limb ischemia received HIF-1␣ at doses of 1⫻108 to 2⫻1011 viral particles. No serious adverse events were attributable to study treatment. Five deaths occurred: 3 in HIF-1␣ and 2 in placebo patients. In the first (randomized) study, 7 of 21 HIF-1␣ patients met treatment failure criteria and had major amputations. Three of the 7 placebo patients rolled over to receive HIF-1␣ in the extension study. No amputations occurred in the 2 highest-dose groups of Ad2/HIF-1␣/VP16 (1⫻1011 and 2⫻1011 viral particles). The most common adverse events included peripheral edema, disease progression, and peripheral ischemia. At 1 year, limb status observations in HIF-1␣ patients included complete rest pain resolution in 14 of 32 patients and complete ulcer healing in 5 of 18 patients. Conclusions—HIF-1␣ therapy in patients with critical limb ischemia was well tolerated, supporting further, larger, randomized efficacy trials. (Circulation. 2007;115:1234-1243.) Key Words: angiogenesis 䡲 gene therapy 䡲 growth substances 䡲 hypoxia 䡲 peripheral vascular disease ritical limb ischemia (CLI) affects ⬇2% of patients ⱖ50 years of age with documented peripheral arterial disease and carries a poor prognosis for life and limb. The 1-year mortality rate in patients with CLI is ⬇25% and may be as high as 45% in those who have undergone amputation.1,2 Limb loss in CLI is a manifestation of advanced systemic atherosclerosis and is a consequence of marked impairment in tissue perfusion in the lower extremities. The current standard of care includes peripheral bypass grafting and percutaneous approaches to improve lower-extremity blood flow; however, a substantial number of patients are ineligible for these treatments or experience short-lived improvements.1 Moreover, results to date from both recombinant protein and gene-based formulations of single growth factors, with several exceptions, have been disappointing.3– 6 One potential explanation is that the use of a single angiogenic cytokine

C

Editorial p 1180 Clinical Perspective p 1243 may be insufficient to generate adequate and durable neovascularization. A combination of ⱖ2 cytokines, acting by differing mechanisms, may act synergistically to achieve a more robust and durable biological response.7,8 Alternatively, the use of a relevant transcription factor may allow the initiation of a coordinated series of cellular events, all of which may conspire to recapitulate vessel growth and/or physiological events to normalize cellular hypoxia. 9 The transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) used in the present study is one such factor that may normalize intracellular oxygen levels by increasing the synthesis of multiple proangiogenic cytokines (eg, vascular endothelial growth factors, angiopoi-

Received December 13, 2005; accepted December 18, 2006. From Ohio State University, Section of Vascular Medicine (S.R.), Columbus; Mount Sinai School of Medicine (S.R., J.O.), New York, NY; Cleveland Clinic Foundation (S.D.), Cleveland, Ohio; St Elizabeth’s Medical Center, Boston, Mass (A.P.); Washington Hospital Center, Washington, DC (J.L.); University of Michigan, Ann Arbor (P.M.G.); Ochsner Clinic Foundation, New Orleans, La (C.K.G.); Genzyme Corp, Cambridge, Mass (K.M., R.K.); and Atlanta Cardiology Research Institute, P.C., Atlanta, Ga (N.C.). Dr Deitcher is currently at Nuvelo Corp, San Carlos, Calif. Correspondence to Sanjay Rajagopalan, MD, Section of Vascular Medicine, 473 W 12th Ave, Division of Cardiovascular Medicine, Ohio State University, Columbus, OH 43210-1252. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.607994

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Rajagopalan et al etins, endothelial nitric oxide synthase) and genes that facilitate survival of ischemic tissue (eg, metabolic pathways that favor glucose over fatty acid metabolism). This phase I dose-escalation study represents the first report on the safety of a modified, constitutively active form of HIF-1␣ (Ad2/HIF-1␣/VP16) in patients with advanced atherosclerosis and tissue ischemia.

Methods The study protocols and informed consents were approved by the Institutional Review boards and Institutional Biosafety committees of all participating institutions. The study design and study protocols, approved by the US Food and Drug Administration (FDA), were performed in accordance with FDA regulations CFR Title 21,10 applicable International Conference on Harmonization guidelines,11 and National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules.12 The randomized study was initiated in October 1999 and completed in June 2004 (ie, after 12 months of follow-up data on the last patient enrolled).

Selection Criteria for Inclusion in the Trial CLI patients between 21 and 85 years of age with no options for surgical or endovascular revascularization and total or subtotal occlusion of at least 1 main artery in a limb confirmed by angiography were recruited to the study from 5 centers in the United States. CLI was defined as Rutherford category (RC) 4 or 5 present for a minimum of 4 weeks without response to conventional therapies, with lack of further revascularization options confirmed by both the investigator and an independent reviewer at the institution. Patients willing and able to discontinue other nonhealing ulcer treatments at least 3 days before treatment and to give written informed consent participated. Exclusion criteria included contraindications to growth factor therapy that have been published previously (eg, history of cancer within 5 years, active diabetic retinopathy),13–15 inflammatory arteritides, RC6 status, prior successful lower extremity arterial surgery, angioplasty, or lumbar sympathectomy during the 2 months before screening. Patients who had participated in other experimental protocols within 30 days of enrollment or who had ever been enrolled in a similar vascular endothelial growth factor or fibroblast growth factor adenoviral or plasmid gene therapy protocol were excluded.

Study Design This phase I program consisted of 2 dose-escalation safety studies: a randomized, double-blind, placebo-controlled (RDBPC) design and an open-label extension (open-label) design. There was no intent to conduct hypothesis-driven comparisons of safety or efficacy between the treatment groups. Figure 1 describes the flow of patients through the studies by dosing cohort. The first study was placebo controlled to ensure objectivity of initial safety evaluations by investigators and an independent Data Safety Monitoring Board. On the basis of results of preclinical safety and bioactivity testing, 5 dosing cohorts were evaluated, increasing from 1⫻108 to 1⫻1010 viral particles (vp) in [1/2] log increments. The Data Safety Monitoring Board reviewed safety data from each dosing cohort before treatment of the nexthigher-dosing cohort and monitored safety data throughout the study. A total of 28 patients were enrolled in the RDBPC study with a 3:1 ratio of HIF-1␣ to placebo randomization (ie, 21 patients received HIF-1␣ and 7 received placebo): the 1⫻108-, 3⫻108-, and 3⫻109-vp groups comprised 4 patients each (3 HIF-1␣ and 1 placebo), and 6 subjects receiving HIF-1␣ and 2 receiving placebo were prospectively allotted to the 1⫻109- and 1⫻1010-vp groups (in which detectable bioactivity had been predicted on the basis of preclinical studies). In the RDBPC study, patients were designated as having experienced treatment failure by the investigator on the basis of prospectively defined criteria encompassing a blinded assessment of the onset or worsening of symptoms that originally qualified them for the study such as worsening rest pain, delayed ulcer healing, and

HIF in Critical Limb Ischemia

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development of osteomyelitis. Patients adjudicated as treatment failures were unblinded, and if the patient had been receiving placebo and still met the original entry criteria, he/she was eligible to receive the highest dose of HIF-1␣ deemed safe by the Data Safety Monitoring Board as part of an open-label extension study. Of the 7 HIF-1␣–treated patients who met treatment failure criteria, 4 proceeded to major amputation, whereas 3 placebo patients met treatment failure criteria in the RDBPC trial and rolled over to receive active HIF-1␣. (Another 3 HIF-1␣–treated patients underwent amputation before being classified treatment failures.) The open-label study was modified during its course on the basis of the safety data accrued to that point and new, supportive preclinical toxicity data. The modified open-label study was expanded to include treatment of 3 patients each with doses of 3⫻1010, 1⫻1011, and 2⫻1011 vp. (Patient 38, in screening at the end of the study, received 1⫻1010 vp.) A maximal dose of 2⫻1011 vp was chosen on the basis of additional preclinical toxicity data, in consultation with FDA and continuing Data Safety Monitoring Board review.

Study Assessments Patients returned for posttreatment follow-up on days 3, 7, 14, 21, 30, 45, 60, and 90; at 6 months; and at 1 year. Safety variables included adverse event reports and changes from baseline in physical examinations, clinical laboratory evaluations, adenoviral antibody titer measurement, retinal eye examinations, and examinations of the index limb to assess rest pain, ulcer status, and RC.16 Observations related to limb status included changes in ischemic rest pain, healing of ischemic ulcers, and ankle brachial index (ABI). Additionally, 3-dimensional gadolinium contrast– enhanced and/or 3-dimensional time-of-flight magnetic resonance angiography (MRA) were performed to detect changes in vascularization. Maximal intensity projections in similar orientations were used to compare pretreatment and posttreatment studies. An increase in the number of visible vessels or an increase in the intensity or apparent size of a previously visible vessel was considered an improvement. An independent reviewer blinded to patient treatment assignment scored the MRA data according to predefined specifications.

Ad2/HIF-1␣/VP16

Ad2/HIF-1␣/VP16 is a recombinant, replication-deficient adenovirus with an insert containing the DNA-binding and dimerization domains from the HIF-1␣ subunit, as well as a herpes virus VP16 transactivation domain to enable constitutive activation.17–19 Ad2/ HIF-1␣/VP16 is propagated in human 293 cells, a permanent cell line of primary human embryonal kidney cells that were immortalized with sheared fragments of human type 5 adenovirus DNA. The bulk substance was purified with column chromatography, filtration (for vector concentration), and final sterile filtration. The resulting preformulated drug substance subsequently underwent final dilution in formulation buffer consisting of phosphate-buffered saline with 10% sucrose. Ad2/HIF-1␣/VP16 is manufactured by Genzyme Corp (Cambridge, Mass).

Procedures for Administering Ad2/HIF-1␣/VP16

In all but 1 dose cohort, the total dose of Ad2/HIF-1␣/VP16 or placebo (ie, phosphate-buffered saline with 10% sucrose) was administered as a single treatment of 10 direct intramuscular injections with a volume of 100 ␮L for a total dose of 1.0 mL given into a single limb. In the 2⫻1011-vp cohort of the open-label study, the total dose of Ad2/HIF-1␣/VP16 consisted of twenty 100-␮L direct intramuscular injections of 1⫻1011 vp to achieve a total dose of 2⫻1011 vp given into a single limb for a total volume of 2.0 mL. The placement of the injections was at the discretion of the investigator and based on patient anatomy and the location of the occluded artery or arteries within the affected limb.

Statistical Analysis All patients receiving ⱖ1 HIF-1␣ or placebo injections were included in safety analyses and limb status observations. By-

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Open-Label

Total (both studies ) Initally Treated, n=3

Initially Treated, n=3 Treated as Rollover, n=1

Placebo Treated as Rollover, n=1

Placebo, n=1

Initially Treated, n=3

Initally Treated, n=3

Placebo, n=1

Placebo only, n=1

Initially Treated, n=6

Initally Treated, n=6

Placebo, n=2

Placebo only, n=2

Initially Treated, n=3

Initally Treated, n=3

Placebo, n=1

Placebo only, n=1

Initially Treated, n=6 Initally Treated, n=7 Placebo Treated as Rollover, n=2

Initially Treated, n=3

Initally Treated, n=3

Initially Treated, n=3

Initally Treated, n=3

Initially Treated, n=3

Initally Treated, n=3

3 x 10 10 vp dose group

Treated as Rollover, n=2

1 x 10 11 vp dose group

Initially Treated, n=1

2 x 10 11 vp dose group

1 x 10 10 vp dose group

3 x 10 9 vp dose group

1 x 109 vp dose group

3 x 108 vp dose group

1 x 108 vp dose group

RDBPC

Placebo, n=2

Total Enrollment (Unique Subjects ) N=38 Patients treated initially: n = 31 Patients given placebo only: n= 4 Patients treated as rollover patients: n= 3 Figure 1. Patient disposition by dose group in phase I studies of peripheral arterial disease for treatment of CLI.

group summary statistics were displayed for actual data reported at assessed time points, including descriptive statistics (sample count, mean, median, SD, minimum, and maximum) for continuous variables and frequencies and percentages for categorical

variables. No hypothesis testing between groups was planned or conducted in these phase I dose-escalation studies. All statistical analyses were conducted in a validated SAS system (SAS Institute Inc, Cary, NC).

Rajagopalan et al TABLE 1.

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Patient Demographics and Baseline Characteristics

Age, median (range), y

Treated* (n⫽34)

Placebo* (n⫽7)

66 (39–87)

67 (46–80)

All† (N⫽38) 66 (390–87)

Gender, n (%) Male

21 (62)

7 (100)

25 (66)

Female

13 (38)

0 (0)

13 (34)

Diabetics‡, n (%)

12 (35)

3 (43)

14 (37)

Current/previous tobacco user, n (%)

28 (82)

5 (71)

31 (82)

Previous bypass or percutaneous intervention, n (%)

30 (88)

5 (71)

32 (84)

Rest pain only, n (%)

16 (47)

4 (57)

18 (47)

Weeks, median (range) Ischemic ulcers, n (%) Weeks, median (range)

21 (4–208) 18 (53) 12 (1–50)

12 (5–32) 3 (43) 28 (24–30)

16 (4–208) 20 (53) 12 (1–40)

Lower limb artery, total occlusion per patient§, n (%) 4/4

15 (44)

3 (43)

3/4

8 (24)

2 (29)

9 (24)

ⱕ2/4

11 (32)

2 (29)

11 (29)

ABI, n (%)

24 (71)

5 (71)

26 (68)

ABI储, median (range)

0.36 (0.17–0.57)

0.39 (0.31–0.47)

18 (47)

0.38 (0.17–0.57)

*Data obtained from the 3 rollover patients during the placebo period and the subsequent treatment period are included in the placebo and treatment columns, respectively (ie, the 3 patients are counted in both columns). †The total represents unique patients; ie, each rollover patient is counted only once. The value used for this calculation was the first report (if evaluable). ‡One patient, patient 20, was first diagnosed with diabetes during the study and is therefore counted among the diabetic patients. Patient 20 was in the placebo group. §The 4 arteries referred to as sites of potential occlusion are the anterior tibial, tibioperoneal trunk, posterior tibial, and peroneal arteries. 储Patient values were not included if calcified, noncompressible vessels or other factors precluded accurate measurements of ABI.

All authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Patient Baseline Information Table 1 summarizes the baseline characteristics of the patients in the Ad2/HIF-1␣/VP16 phase I program, including demographics, diabetes status, time spent in the baseline RC class before the study, and the total number of arteries occluded at baseline. In addition, at baseline, 34% of individuals were on acetylsalicylic acid, 47% were on an angiotensin-converting enzyme inhibitor, 63% were on warfarin, 32% were on clopidogrel, and 58% were on statins. During the study, 47% of individuals were on acetylsalicylic acid, 58% were on an angiotensin-converting enzyme inhibitor, 68% were on warfarin, 45% were on clopidogrel, and 68% were on statins. Figures 2 and 3 present the baseline ulcer and rest pain status of individual patients in the study. Of the 34 patients treated with HIF-1␣ in both studies, 32 began the study with rest pain, 18 began with ulcers, and 16 began with both rest pain and ulcers. Of the 7 patients who received placebo treatment, 6 had rest pain, 3 had ulcers, and 2 had both rest pain and ulcers.

Safety End Points Adverse Event Profile All but 1 HIF-1␣ patient experienced adverse events in either the RDBPC study or the open-label study. Table 2 summarizes the adverse events occurring in at least 10% of patients by World Health Organization Adverse Reactions Terminology preferred term. Most of these events were mild or moderate in severity and were not serious. None was judged to be related to treatment. A total of 21 unique patients experienced serious adverse events: 17 patients who received HIF-1␣ only, 3 placebo patients, and 1 patient who had adverse events both as a placebo and as a treated (rollover) patient. The serious adverse events that occurred were consistent with the patients’ extensive disease and comorbidities. Deaths and Amputations Five patients died while during the course of the present study, 3 from the HIF-1␣ group and 2 from the placebo group. No death was judged to be related to active treatment (Table 3). The median time to death was similar in the treatment and placebo groups. In the RDBPC study, 7 HIF-1␣ patients and 3 placebo patients met treatment failure criteria; another 3 HIF-1␣ patients underwent amputation before meeting treatment failure criteria. The distribution of time to

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Rest Pain (Nights per Week of Rest Pain) Dose (vp)

Pt

Baseline

1x108

03*

7

04*†

7

06*

7

7

08*

4

0

0

10*

7

0

0

13*

7

7

0

3x109

18*

3

0

0

1x1010

22*

7

7

24*†

7

7

7

25*

7

0

0

28*

7

0

0

38



7

0

0

29



7

7

7

1x1011

32†

1

UNK

3

2x1011

36†

7

0

0

37†

7

7

4.5

04*†

7

14*,**

2

0

15*

7

0

Died, 11.4 Mo.

24*†

7

7

Early withdrawal; Rollover, 8.7 Mo.

3x108

1x109

3x10

10

Placebo

6 Mo. 2

1 Yr

Comment

0 Amputation, 1.2 Mo. Early withdrawal Early withdrawal

Died, 6.8 Mo.

Treatment Failure, 1.5 Mo.; Rollover, 2.3 Mo. 7

Abbreviations: Pt=patient; Mo.=month; UNK=unknown Orange =Lack of change from Baseline based on Rutherford Category; Red =Deterioration; Green =Resolution based on Rutherford Category

*Patient in the double-blinded study; †Patient in the open-label study; **Minor amputation or nonsurgical loss of toe Figure 2. Patients with rest pain only at baseline (RC4 patients): clinical outcomes.

treatment failure and/or amputation was similar in the HIF-1␣ and placebo groups. All 10 major amputations occurred in HIF-1␣ patients (29% of 34 treated patients): 7 patients in the RDBPC study and 3 in the open-label study, including 1 patient who had received placebo before receiving active HIF-1␣ (Table 3). Although 0 of the 7 placebo patients from the RDBPC study experienced a major amputation, there were 2 deaths and 3 treatment failures, with only 2 patients remaining in the placebo group at 1 year. Despite the presence of RC4 and RC5 patients in roughly equal proportions at baseline, the

RC5 patients constituted 90% of those who eventually required major amputations, 60% of those who died, and 71% of those who withdrew. In addition, although diabetics constituted only 37% of patients overall, they constituted 80% of the deaths in the trial. Potential Risk of Ad2/HIF-1␣/VP16 – Mediated Angiogenesis The adverse events described below are of special interest in the present study because of the theoretical risks of using a replication-deficient adenoviral vector and of the

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Figure 3. Patients with ulcers (u) and rest pain (rp) at baseline (RC5 patients): clinical outcomes.

constitutively active HIF-1␣ transgene. Dependent edema of the lower extremities and injection site reactions were the 2 most common events of interest and were noted in similar proportions of treated and placebo patients: 44% to 43% of each group for dependent edema and 24% to 29% for injection site reaction, respectively. There was no evidence for promotion of tumor growth, although some patients had a history of malignancy diagnosed and treated ⬎5 years previously. Vision disorders occurred in 29% of treated patients but no placebo patients; however, none of

the cases involved pathological choroidal neovascularization or new/active proliferative retinopathy. A variety of minor retinal findings, including drusen and A-V nicking, were reported. Flu-like symptoms occurred in 26% of the treated patients and no placebo patients. Of these 9 patients, only 2 experienced symptom onset within 72 hours of receiving Ad2/HIF-1␣/VP16 (range, 2 to 180 days; median, 15 days). As expected, a rise in antiadenoviral antibodies was observed in patients who had received Ad2/HIF-1␣/VP16. Antibody titers peaked at day

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TABLE 2. Adverse Events (by World Health Organization Adverse Reactions Terminology Preferred Term) That Occurred in at Least 10% of Patients Pooled HIF-1␣ Pooled Placebo Preferred Term

AEs, n Pts, n AEs, n

Total no. of patients

Limb and Surrogate Outcomes

All

34

䡠䡠䡠 74

7

33

6

䡠䡠䡠 435

39

Edema dependent

24

15

6

3

30

18

Disease progression NOS

17

12

1

1

18

13

41

Peripheral ischemia

17

11

1

1

18

12

Peripheral gangrene

10

10

0

0

10

10

Injection site reaction

8

8

2

2

10

10

Skin disorder

9

8

2

1

11

9

Cellulitis

Figures 2 and 3 summarize the clinical outcomes for each patient at baseline, 6 months, 1 year, and final disposition. Only 2 of the 7 placebo patients remained at 1 year: 1 patient had return of rest pain after reporting resolution at 6 months and a toe amputation, and 1 patient had continuing rest pain. At 6 months, limb status observations in HIF-1␣ patients included complete rest pain resolution in 12 of 32 patients alive with index limb and complete ulcer healing in 3 of 18 patients (3 of 9 alive with index limb). At 1 year, there was complete rest pain resolution in 14 of 32 patients (14 of 21 alive with index limb), complete ulcer healing in 5 of 18 patients (5 of 8 alive with index limb), and 5 cases of ulcer healing accompanied with resolution of rest pain. Figure 4 shows an example of ulcer healing at 1 year in a patient originally randomized to the placebo group and subsequently adjudicated as a treatment failure who went on to receive active therapy at 1⫻1010 vp. None of the patients in the 2 highest-dosing groups (1⫻1011 and 2⫻1011 vp) experienced either death or amputation. Complete ulcer healing occurred in patients given doses ranging from 1⫻109 to 2⫻1011 vp; ulcer healing occurred in all 3 RC5 patients given the 1⫻1011- to 2⫻1011-vp doses. ABI measurement was not available for all study patients because of arterial calcification, amputation, death, or early withdrawal. The median ABI for HIF-1␣ patients was 0.36 at baseline (n⫽24), 0.45 at 6 months (n⫽17), and 0.46 at 1 year (n⫽16). The median ABIs for placebo patients were 0.39 (n⫽5), 0.49 (n⫽3), and 0.48 (n⫽1), respectively. No correlation was seen between ABI and clinical outcomes, likely secondary because of the small number of patients. Complete and comparable baseline and 6- and 12-month MRAs were available in 18 (n⫽14 HIF-1␣, n⫽4 placebo) and 13 (n⫽11 HIF-1␣, n⫽2 placebo) patients, respectively. At 6 months, 7 HIF-1␣ patients showed improvement, whereas at 12 months, 2 HIF-1␣ patients showed improvement, with the remainder either worsening or showing no change. In the placebo group, 2 patients showed improvement at 6 and 12 months, respectively; the remainder worsened or

Pts, n AEs, n Pts*, n

䡠䡠䡠 361

Any AEs

supported by the sponsor’s preclinical toxicity data, however, and therefore is not planned.

11

8

1

1

12

9

Weight decrease

6

6

1

1

7

7

Skin ulceration

5

5

2

2

7

7

Postoperative pain

8

7

0

0

8

7

Nausea

6

6

1

1

7

7

Diarrhea

7

5

2

2

9

7

Melena

5

5

1

1

6

6

Fever

6

6

0

0

6

6

Anemia

5

5

1

1

6

6

Rhinitis

3

3

2

2

5

5

AEs indicate adverse events; Pts, patients; and NOS, nitric oxide synthase. Events were coded using the World Health Organization Adverse Reactions Terminology system. Multiple adverse events reported for a patient were counted once within a preferred term/body system. When a subject rolled over from placebo to active treatment, the subject is counted separately as a placebo and as a treated subject. *In this column, rollover patients are counted twice if they experienced adverse events during their involvement in both the placebo and treatment groups.

60 and then declined (data not shown; no dose-dependent trends were identified given the considerable between- and within-patient variability). Because of the small group sizes for each dose, no dose effect could be noted across the study cohorts. Consequently, a maximum tolerated dose was not established. Further dose escalation is not TABLE 3.

Treatment Failures, Amputations, and Deaths Treatment*

Treatment failure in double-blinded study, n (%)

7 (33)

Time to treatment failure, median (range), mo

1.7 (0.9–4.6)

Major amputations, n (%)

10 (29)

Time to major amputation, median (range)‡, mo

2.7 (0.8–4.7)

Deaths, n (%) Time to death, median (range), mo‡

3 (9) 6.7 (6.7–8.7)

Total Treated, n 21 䡠䡠䡠 34 䡠䡠䡠 34

Placebo* 3 (43) 1.5 (1.2–4.4) 0 (0) NA 2 (29)

Total Placebo, n 7

All†

Total Patients, n

10 (36)

28

䡠䡠䡠 7

1.6 (0.9–4.6)

䡠䡠䡠 38

䡠䡠䡠 7

2.7 (0.8–4.7)

10 (26) 5 (13)

䡠䡠䡠 38

8.6 (5.8–11.4) 6.7 (5.8–11.4) 䡠䡠䡠 䡠䡠䡠 䡠䡠䡠 Treatment failure data were collected in the double-blind study but not the open-label study. The definition of treatment failure is presented in Methods. *The number of treated patients includes rollover patients, ie, those initially assigned to the placebo group who later received treatment. †Each rollover patient is counted only once. ‡Times measured from the beginning of the study in which the event occurred to the first major amputation.

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Figure 4. Patient 27: complete resolution of an ulcer over a 1-year period after treatment (accompanied by rest pain resolution). A, A nonhealing ulcer at baseline in study 1. B, After placebo. Treatment failure 4.9 months after receiving placebo in study 1. C, Rollover to active treatment. Evidence of ulcer healing 6 months after receiving 1⫻1010 vp Ad2/ HIF-1␣/VP16. D, Complete ulcer healing 1 year after treatment on study 2. This patient, whose medical history included diabetes and a coronary artery bypass graft/aortic valve replacement procedure, died 14 months after treatment. The primary cause of death was sepsis, with secondary causes listed as liver disease and coronary artery disease.

showed no change compared with baseline. Improvement in MRA score did not correlate with improved clinical status, ABI, or the study injection sites.

Discussion The present phase I dose-escalation evaluation is the first clinical study to use a constitutively active formulation of the transcription factor HIF-1␣, which regulates the expression of specific genes involved in the response to hypoxia and wound healing.9,20 Ad2/HIF-1␣/VP16 appeared to be safe when delivered into skeletal muscle in the leg at doses ranging from 1⫻108 to 2⫻1011 in no-option CLI patients. An important point is that no safety problems emerged in this phase I program, including no evidence of malignancy or ocular neovascularization disorders related to the transgene, at least in the short term. Longer-term follow-up in a larger number of patients may be needed to firmly establish the safety of both vector and transgene. It is presumed that patients can receive only a single administration of adenoviral HIF-1␣/VP16 because of the generation of an adenoviral serotype-specific immune response.21 Adenoviral vectors have now been used in multiple clinical trials in cardiovascular disease through intramuscular, intramyocardial, and intracoronary routes, with no indication of hepatic or other systemic toxicity at the doses used in this trial.5,22–24 No dose-limiting side effects emerged in the present study at the maximum dose tested (2⫻1011 vp). Further dose escalations were not attempted because this was the highest dose supported by preclinical data and approved by the FDA for testing in humans. Additionally, further increases in dosing are associated with technical limitations with manufacturing adenoviruses at these high titers. Overall, the present study confirmed the high rate of progression of disease in advanced CLI (whether treated or

placebo), with amputation and mortality rates of 26% and 3% at 6 months and 26% and 13% at 1 year, respectively. Rates of amputation and mortality in patients treated with Ad2/HIF-1␣/VP16 were 29% and 0% at 6 months and 29% and 9% at 1 year. There was a high rate of crossover to active drug among placebo patients in the present trial, with most continuing on placebo experiencing disease progression or death at 1 year, similar to rates from randomized trials and meta-analyses of studies conducted in similar patient populations.1,25–27 In previous studies, 6-month rates of amputation ranged from 20% to 44%, and mortality rates ranged from 20% to 54%.1 Some of these differences are undoubtedly secondary to improvements in pharmacotherapy for atherosclerosis (eg, use of HMG CoA reductase inhibitors, angiotensin-converting enzyme inhibitors, and antiplatelet agents), the unique patient selection criteria in the trial, and improvements in interventional approaches. Improvement in limb status (ie, complete ulcer healing and/or resolution of rest pain) was observed in some of the 34 CLI patients treated with Ad2/HIF-1␣/VP16 in the 2 present studies. Complete resolution of rest pain occurred in 12 patients at 6 months (12 of 23 alive with index limb) and 14 patients at 1 year (14 of 21 alive with index limb). Complete ulcer healing occurred in 3 patients (3 of 9 alive with index limb) at 6 months and 5 patients (5 of 8 alive with index limb) at 1 year. Interpretation of the MRA results was confounded by the fact that a number of patients were unable to repeat the test either because they had experienced limb loss or for other reasons. Furthermore, we restricted evaluation to those patients who had comparable gadolinium contrast– enhanced MRA or timeof-flight MRAs, further reducing the total evaluable scans. The lack of correlation between clinical improvement and MRA-derived scores may therefore relate to some of these

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issues. Additional considerations include the use of higherresolution approaches (submillimeter), contrast-enhanced MRA below the level of the foot in an attempt to image collaterals, and standardization of “timing runs,” gadolinium contrast dose, and delivery at study sites to ensure comparability of data.

Conclusions These data provide encouraging initial evidence of a potentially important therapeutic approach in the treatment of vascular disease without evidence of serious toxicity. Additional, appropriately powered clinical trials are needed to evaluate the safety and efficacy of modified constitutively active forms of HIF-1␣ in peripheral arterial disease.

Acknowledgments The authors posthumously acknowledge Dr Jeffrey M. Isner for valuable contributions to the design and execution of this trial; Drs Mark A. Creager and Joseph Loscalzo for their contributions as members of the Data Monitoring Committee; Dr Iris Baumgartner for contributions in designing and performing the independent MRA review; Laura Emig for contributions to the management of the study; Annie Purvis for biostatistical support; and Monica Eiland, PhD, for assistance with manuscript preparation.

Source of Funding The present study was funded by a grant from Genzyme Corp, the manufacturer of Ad2/HIF-1␣/VP16.

Disclosures Drs Rajagopalan, Olin, Deitcher, Laird, Grossman, Goldman, and Chronos were primary investigators on this Genzyme-sponsored clinical study. In addition, Drs Rajagopalan, Olin, and Chronos received grant support and are on the Steering Committee or other scientific advisory boards for this clinical program. Dr Kelly and K. McEllin are employees of Genzyme Corp. A. Pieczek reports no conflicts.

References 1. Dormandy JA, Rutherford RB. Management of peripheral arterial disease (PAD): TASC Working Group: TransAtlantic Inter-Society Consensus (TASC). J Vasc Surg. 2000;31(pt 2):S1–S296. 2. Hirsch AT, Haskal ZJ, Hertzer NR, Bakal CW, Creager MA, Halperin JL, Hiratzka LF, Murphy WR, Olin JW, Puschett JB, Rosenfield KA, Sacks D, Stanley JC, Taylor LM Jr, White CJ, White J, White RA, Antman EM, Smith SC, Jr, Adams CD, Anderson JL, Faxon DP, Fuster V, Gibbons RJ, Hunt SA, Jacobs AK, Nishimura R, Ornato JP, Page RL, Riegel B. ACC/AHA 2005 practice guidelines for the management of patients with peripheral arterial disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (Writing Committee to Develop Guidelines for the Management of Patients With Peripheral Arterial Disease): endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation. 2006;113:e463– e654.

3. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation. 2002;105:788 –793. 4. Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS, Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER. The VIVA trial: Vascular Endothelial Growth Factor in Ischemia for Vascular Angiogenesis. Circulation. 2003;107:1359 –1365. 5. Rajagopalan S, Mohler ER 3rd, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, Blebea J, Macko J, Kessler PD, Rasmussen HS, Annex BH. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation. 2003;108:1933–1938. 6. Simons M, Ware JA. Therapeutic angiogenesis in cardiovascular disease. Nat Rev Drug Discov. 2003;2:863– 871. 7. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249 –257. 8. Cao R, Brakenhielm E, Pawliuk R, Wariaro D, Post MJ, Wahlberg E, Leboulch P, Cao Y. Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med. 2003;9:604 – 613. 9. Pajusola K, Kunnapuu J, Vuorikoski S, Soronen J, Andre H, Pereira T, Korpisalo P, Yla-Herttuala S, Poellinger L, Alitalo K. Stabilized HIF-1alpha is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer. FASEB J. 2005;19: 1365–1367. 10. US Food and Drug Administration. CFR Title 21 database search: April 2005. Available at: http://www.accessdata.fda.gov/scripts/cdrh/ cfdocs/cfcfr/CFRSearch.cfm?CFRPart⫽56. Accessed October 13, 2005. 11. International Conference on Harmonization (ICH), US Food and Drug Administration. Guidance for Industry, E6 good clinical practice: consolidated guidance: April 1996. Available at: http://www.fda.gov/ cder/guidance/959fnl.pdf. Accessed January 29, 2007. 12. National Institutes of Health, Recombinant DNA Advisory Committee. Guidelines for research involving recombinant DNA molecules: April 24, 2002. Available at: http://www4.od.nih.gov/oba/rac/ guidelines/guidelines.html. Accessed January 29, 2007. 13. Simons M, Bonow RO, Chronos NA, Cohen DJ, Giordano FJ, Hammond HK, Laham RJ, Li W, Pike M, Sellke FW, Stegmann TJ, Udelson JE, Rosengart TK. Clinical trials in coronary angiogenesis: issues, problems, consensus: an expert panel summary. Circulation. 2000;102:E73–E86. 14. Cao Y, Hong A, Schulten H, Post MJ. Update on therapeutic neovascularization. Cardiovasc Res. 2005;65:639 – 648. 15. Epstein SE, Kornowski R, Fuchs S, Dvorak HF. Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects. Circulation. 2001;104:115–119. 16. Rutherford RB, Baker JD, Ernst C, Johnston KW, Porter JM, Ahn S, Jones DN. Recommended standards for reports dealing with lower extremity ischemia: revised version. J Vasc Surg. 1997;26:517–538. 17. Jiang C, Lu H, Vincent KA, Shankara S, Belanger AJ, Cheng SH, Akita GY, Kelly RA, Goldberg MA, Gregory RJ. Gene expression profiles in human cardiac cells subjected to hypoxia or expressing a hybrid form of HIF-1 alpha. Physiol Genomics. 2002;8:23–32. 18. Vincent KA, Shyu KG, Luo Y, Magner M, Tio RA, Jiang C, Goldberg MA, Akita GY, Gregory RJ, Isner JM. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF1alpha/VP16 hybrid transcription factor. Circulation. 2000;102: 2255–2261. 19. Armentano D, Sookdeo CC, Hehir KM, Gregory RJ, St George JA, Prince GA, Wadsworth SC, Smith AE. Characterization of an adenovirus gene transfer vector containing an E4 deletion. Hum Gene Ther. 1995;6:1343–1353. 20. Vincent KA, Feron O, Kelly RA. Harnessing the response to tissue hypoxia: HIF-1 alpha and therapeutic angiogenesis. Trends Cardiovasc Med. 2002;12:362–367. 21. Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J. Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther. 1999;6:1574 –1583.

Rajagopalan et al 22. Mohler ER 3rd, Rajagopalan S, Olin JW, Trachtenberg JD, Rasmussen H, Pak R, Crystal RG. Adenoviral-mediated gene transfer of vascular endothelial growth factor in critical limb ischemia: safety results from a phase I trial. Vasc Med. 2003;8:9 –13. 23. Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman GW, Hachamovitch R, Szulc M, Kligfield PD, Okin PM, Hahn RT, Devereux RB, Post MR, Hackett NR, Foster T, Grasso TM, Lesser ML, Isom OW, Crystal RG. Angiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease. Circulation. 1999;100:468 – 474. 24. Grines CL, Watkins MW, Helmer G, Penny W, Brinker J, Marmur JD, West A, Rade JJ, Marrott P, Hammond HK, Engler RL. Angiogenic

HIF in Critical Limb Ischemia

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Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation. 2002;105:1291–1297. 25. Loosemore TM, Chalmers TC, Dormandy JA. A meta-analysis of randomized placebo control trials in Fontaine stages III and IV peripheral occlusive arterial disease. Int Angiol. 1994;13:133–142. 26. da Silva AF, Desgranges P, Holdsworth J, Harris PL, McCollum P, Jones SM, Beard J, Callam M. The management and outcome of critical limb ischaemia in diabetic patients: results of a national survey: Audit Committee of the Vascular Surgical Society of Great Britain and Ireland. Diabetes Med. 1996;13:726 –728. 27. Critical limb ischaemia: management and outcome: report of a national survey: the Vascular Surgical Society of Great Britain and Ireland. Eur J Vasc Endovasc Surg. 1995;10:108 –113.

CLINICAL PERSPECTIVE Development of agents that promote the formation of new, small-caliber vessels (angiogenesis) that have the potential for growth and remodeling into larger vessels over time (arteriogenesis) may represent an important therapeutic approach for individuals with severe limb-threatening peripheral arterial disease (ie, critical limb ischemia) who have exhausted other alternative forms of treatment. Here, we describe our experience in a gene-therapy, dose-escalation, phase I clinical trial with a constitutively activated form of the transcription factor hypoxia-inducible factor-1␣. Hypoxia-inducible factor-1␣ is a master switch gene that initiates and choreographs the expression of multiple proangiogenic genes in patients with critical limb ischemia. Although this phase I trial was not large enough to judge clinical efficacy in critical limb ischemia, the absence of major safety issues is reassuring. These data, along with the findings of clinical improvement in some patients, provided the rationale for a randomized, placebo-controlled trial of a hypoxia-inducible factor-1␣ transgene in a phase II study in subjects with advanced claudication symptoms that currently is ongoing in Europe and the United States. If this gene therapy approach were to prove safe and effective after subsequent, adequately powered phase III studies, it would provide clinicians with an alternative and/or an adjunctive approach to treating patients with refractory ischemic limb symptoms.

Prevalence of Desmin Mutations in Dilated Cardiomyopathy Matthew R.G. Taylor, MD, PhD; Dobromir Slavov, PhD; Lisa Ku, MS; Andrea Di Lenarda, MD; Gianfranco Sinagra, MD; Elisa Carniel, MD; Kurt Haubold, PhD; Mark M. Boucek, MD; Debra Ferguson, RN, MS; Sharon L. Graw, PhD; Xiao Zhu, BS; Jean Cavanaugh, MS, PA-C; Carmen C. Sucharov, PhD; Carlin S. Long, MD; Michael R. Bristow, MD, PhD; Philip Lavori, PhD; Luisa Mestroni, MD; for the Familial Cardiomyopathy Registry and the BEST (Beta-Blocker Evaluation of Survival Trial) DNA Bank Background—Desmin-related myofibrillar myopathy (DRM) is a cardiac and skeletal muscle disease caused by mutations in the desmin (DES) gene. Mutations in the central 2B domain of DES cause skeletal muscle disease that typically precedes cardiac involvement. However, the prevalence of DES mutations in dilated cardiomyopathy (DCM) without skeletal muscle disease is not known. Methods and Results—Denaturing high-performance liquid chromatography was used to screen DES for mutations in 116 DCM families from the Familial Dilated Cardiomyopathy Registry and in 309 subjects with DCM from the Beta-Blocker Evaluation of Survival Trial (BEST). DES mutations were transfected into SW13 and human smooth muscle cells and neonatal rat cardiac myocytes, and the effects on cytoskeletal desmin network architecture were analyzed with confocal microscopy. Five novel missense DES mutations, including the first localized to the highly conserved 1A domain, were detected in 6 subjects (1.4%). Transfection of DES mutations in the 2B domain severely disrupted the fine intracytoplasmic staining of desmin, causing clumping of the desmin protein. A tail domain mutation (Val459Ile) showed milder effects on desmin cytoplasmic network formation and appears to be a low-penetrant mutation restricted to black subjects. Conclusions—The prevalence of DES mutations in DCM is between 1% and 2%, and mutations in the 1A helical domain, as well as the 2B rod domain, are capable of causing a DCM phenotype. The lack of severe disruption of cytoskeletal desmin network formation seen with mutations in the 1A and tail domains suggests that dysfunction of seemingly intact desmin networks is sufficient to cause DCM. (Circulation. 2007;115:1244-1251.) Key Words: cardiomyopathy 䡲 desmin 䡲 genetics 䡲 heart failure

D

esmin-related myofibrillar myopathy (DRM) is a rare heritable myopathy affecting skeletal and cardiac muscle (OMIM #601419),1 caused by mutations in the desmin (DES) gene. Skeletal muscle weakness starting in the lower limbs and progressing to involve truncal, neck flexor, bulbar, and respiratory muscles without cardiac involvement has been reported most commonly.2–5 Cardiac manifestations in other families include restrictive cardiomyopathy, dilated cardiomyopathy, conduction system diseases, arrhythmias, and sudden death.6,7 The majority of the ⬎40 DES mutations reported cause DRM and are localized to the central rod domain of the desmin protein (Figure 1). Selected skeletal and cardiac muscle biopsies from affected patients have shown cytoplasmic aggregations of intermediate filaments, which are presumed to reflect a disruption of the assembly of desmin protein into a filamentous network.4,6 –14 Mutations in

␣-B-crystallin (CRYAB), dystrophin (DMD), and myotilin (TTID) cause similar morphological changes to skeletal muscle. Transfection of various DES mutations into cellular models has largely confirmed in vitro aggregation of desmin protein and disruption of the cytoplasmic filamentous desmin network.4,5,8,9,11,13–15

Clinical Perspective p 1251 Only 1 reported mutation (Ile451Met), located in the tail domain of the desmin protein and found in a study of 44 subjects with dilated cardiomyopathy (DCM), has been linked to DCM without skeletal muscle disease.16 The Ile451Met mutation is either a recurrent or founder mutation because a screen of 265 unrelated Japanese DCM cases for this specific mutation detected 3 additional cases (1.1%).12 Ile451Met mutations were also found in a family with classic DRM4 and in another family with slowly progressive skeletal

Received June 16, 2006; accepted December 29, 2006. From the University of Colorado at Denver and Health Sciences Center, Denver, Colo (M.R.G.T., D.S., L.K., E.C., K.H., D.F., S.L.G., X.Z., C.C.S., C.S.L., M.R.B., L.M.); Department of Cardiology, University Hospital, Trieste, Italy (A.D.L., G.S.); Division of Cardiology, The Children’s Hospital, Denver, Colo (M.M.B., J.C.); and Stanford University and the Cooperative Studies Program, Department of Veterans Affairs, Stanford, Calif (P.L.). Clinical trial registration information—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00000560. Reprint requests to Matthew Taylor, MD, PhD, 12635 E Montview Blvd, Suite 150, Aurora, CO 80010-7116. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.646778

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Figure 1. Desmin protein represented with precoil domain (PCD) and rod domains separated by linkers and flanked by non-helical head and tail domains. Mutations showing abnormal desmin aggregation in cellular studies are underlined. Mutations reported are identified by wild-type amino acid code/position/mutant amino acid; (mutations identified in current study in boldface italics). Arrows indicate phenotype reported for each mutation (up arrows-skeletal muscle only; down arrows- cardiomyopathy only; bi-directional arrows- both skeletal and cardiac muscle disease; *indicates restrictive cardiomyopathy; the R350P mutation causes skeletal and cardiac myopathy and occurs at the same site as our R350W mutation). ⌬-indicates deletion.

myopathy with no apparent cardiac involvement and incomplete penetrance evidenced by 3 adult healthy mutation carriers.5 Thus, the contribution of DES mutations to isolated DCM and whether mutations located outside the desmin tail region can cause DCM are incompletely understood. The largest previous population study of 63 cases of DCM did not identify any DES mutations.17 The aim of the present study was to comprehensively screen a large population of DCM cases for DES mutations with the use of 2 unique DCM cohorts: (1) subjects from 115 DCM families with familial and nonfamilial (sporadic) forms and (2) 309 DCM subjects from the Beta-Blocker Evaluation of Survival Trial (BEST).18,19

Methods Patient Cohorts The family-based cohort drew subjects from the Familial Cardiomyopathy Registry, a multicenter DCM study. Affected subjects from 116 DCM families were screened for DES mutations (1 subject from a recessive-appearing pedigree was unaffected). The diagnosis of DCM was made according to published criteria20; probands and available relatives were evaluated by the investigators, an evaluation that included a detailed history, physical examination, 12-lead ECG, and echocardiography; medical records were also reviewed in the case of unavailable or deceased relatives. Informed consent was obtained from Familial Cardiomyopathy Registry subjects under the institutional review board policies of the participating institutions. The 148 subjects were predominantly white and comprised 90 men and 58 women with an average age at study entry of 44 years. A second group of DCM subjects was screened from the BEST cohort, which was cosponsored by the National Heart, Lung, and Blood Institute and the Department of Veterans Affairs Cooperative Studies Program. BEST was a multicenter study of moderate to severe congestive heart failure (New York Heart Association class III-IV and ejection fraction of ⱕ35% at entry) comparing bucindolol with placebo.19 A subset of BEST subjects participated in a DNA bank, and we were granted approval to study 309 anonymous samples from DCM subjects. This cohort had an average age of 56 years and was divided into 213 men and 96 women, with 240 and 69 nonblack and black (non-Hispanic) subjects, respectively.

DES Mutation Screening Genetic screening was done with the use of denaturing highperformance liquid chromatography with a Transgenomic WAVE Fragment Analysis System (Transgenomic Inc, Omaha, Neb) followed by selective DNA sequencing of variants with abnormal denaturing high-performance liquid chromatography elution profiles on an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, Calif). The 9 DES exons and intron boundaries and the 5⬘ upstream enhancer sequence that modulates expression of the desmin protein21

were studied (primer sequences and conditions available on request). Wild-type control DNA was added to those samples from Familial Cardiomyopathy Registry families in which autosomal recessive inheritance or sporadic disease was suspected. This approach favors the formation of heteroduplexes in which homozygous mutations could potentially be present. This step was not done in the case of the BEST samples because inheritance data were not collected in that study. We used standard criteria for classification of a variant as a pathogenic mutation including the following: predicted alteration of amino acid sequence, location of the mutation at an evolutionarily conserved residue, predicted effects on protein secondary structure, segregation among affected family members, and absence of the variant in a collection of 300 predominantly white control chromosomes from healthy subjects.

Expression Studies The majority of DES mutations studied previously led to in vivo (muscle biopsies) or in vitro (cellular models) abnormal aggregation of cytoplasmic desmin protein. Because cardiac tissue was not available from our subjects, we used the in vitro approach to determine whether the mutations found by genetic screening had cellular phenotypes. Accordingly, we developed constructs for cellular expression in (1) SW13 cells (ATCC, American Tissue Culture Collection, Manassas, Va), a human adrenal cortex carcinoma cell line, that does not express the intermediate filaments desmin, vimentin, or keratin; (2) human coronary artery smooth muscle cells (Cambrex Bio Science Walkersville, Inc, Rutherford, NJ), which express desmin and vimentin; and (3) neonatal rat cardiac ventricular myocytes cultured as previously described.22,23 In the case of the myocyte experiments, cotransfection of desmin construct and green fluorescent protein (GFP) constructs were done (pmaxGFP; Amaxa, Inc, Gaithersburg, Md). Briefly, ventricular cells of 1- to 3-day-old rats were isolated by trypsin digestion, and 2⫻106 cells were transfected with 2 ␮g total DNA (1 ␮g GFP construct and 1 ␮g of each desmin construct) with the use of a Nucleofector device (Amaxa, Inc) according to the manufacturer’s instructions and with Rat Cardiomyocyte–Neonatal Nucleofector transfection kits (Amaxa Inc). Briefly, cells were pelleted and suspended in 100 ␮L of the appropriate buffer. DNA was added to the solution, and cells were electroporated with the neonatal rat ventricular myocyte–specific program. Cells were plated in a 10-cm2 slide coated with 1% gelatin. Media solutions were supplemented with HEPES (pH 7.5) to a final concentration of 20 mmol/L to buffer the media pH. Putative mutations were created in DES cDNA with a modified overlap extension approach,24 with the use of oligonucleotides containing the DES mutations. Polymerase chain reaction (PCR) fragments containing full-length desmin cDNA were inserted into pcDNA3.1/V5-His-Topo T/A cloning vector (Invitrogen, Carlsbad, Calif). Oligonucleotides with single-nucleotide mutations in combination with flanking primers (primers available on request) were used to generate PCR fragments from desmin cDNA clone (Gene Bank accession No. BC032116). PCR fragments without a mutation that overlapped mutagenized PCR products (overlap range, 243 to 613 nucleotides) were amplified with separate primers, and then

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Characteristics of DES Mutation Carriers Age at Diagnosis, y

Race

Gender

NYHA Class

Glu108Lys

60

White

M

Ser298Leu

45

White

F

Asp312Asn

35

Black

Arg350Trp

55

White

Val459Ile*

44

Val459Ile*

34

Mutation

Conduction System Disease

Muscle Disease

EF, %

LVEDD, cm

Status (Comments)

3

LAFB

None

33

NA

Living

3

LBBB

None

27

NA

Living

M

3

None

None

17

NA

Dead (age 45 y at last follow-up)

M

2

None

None

29

7.1

Living (EF 19% at 68 y)

Black

M

3

None

None

34

7.6

Living

Black

F

3

1° AVB

None

17

NA

Living

NYHA indicates New York Heart Association; EF, ejection fraction; LVEDD, left ventricular end-diastolic diameter; LAFB, left anterior fasicular block; LBBB, left bundle-branch block; AVB, atrioventricular block; and NA, not available. *Allele frequency of 1% of black (ethnically matched) controls.

full-length desmin cDNA was generated by extension of overlapping PCR fragments. All mutations were confirmed by sequencing. Plasmid DNA constructs were transfected into Escherichia coli (top10; Invitrogen Inc), isolated, and sequenced from single colonies to confirm the presence of the mutations. Transfection was performed with the FuGene 6 transfection reagent (Roche Diagnostics Corp, Indianapolis, Ind) according to the manufacturer’s instructions (3 ␮L FuGene was diluted in 97 ␮L Opti-MEM (Invitrogen), and 1 ␮g plasmid DNA was added; DNA-FuGene complex was formed at room temperature for 30 minutes and added to cell culture). Cells (SW13 and coronary artery smooth muscle cells) were grown on 8-cm2 slides to ⬇50% confluence and then transfected with 1 ␮g of plasmid DNA. After incubation for 48 hours, the slides were washed 3 times, for 10 minutes each, with PBS, and cells were fixed with an ice-cold mixture of 70% methyl alcohol and 30% acetone for 10 minutes. Slides were washed 3 times, for 10 minutes each, with PBS. Immunostaining with primary monoclonal mouse anti-desmin antibody (D1033; Sigma Inc, St Louis, Mo) at 1:1000 dilution in PBS overnight (⬇16 hours) at 4°C was followed by washing 3 times with PBS and incubation with secondary anti-mouse antibody FITC conjugate (F5387; Sigma) at 1:1000 dilution for 1 hour at room temperature. Slides were washed 3 times with PBS, and coverslips were mounted in drop of Mowiol. Fluorescent confocal microscopy with the use of an Olympus IX81 inverted motorized spinning-disk confocal microscope (Olympus America Inc, Center Valley, Pa) was used to evaluate the transfected cells for expression of desmin protein and its abilities to form an intracellular filament network. The Gln389Pro mutation reported by Goudeau et al25 to cause severe desmin network disruption was used as a positive control for our experiments. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results Mutation Screening Mutation screening detected 4 novel variants in 4 DCM subjects that predicted missense changes and met criteria for pathogenic mutations (Glu108Lys:c.408.G3 A; Ser298Leu:c.979.C3 T; Asp312Asn:c.1020.G3 A; Arg350Trp:c.1134.C3T; reference NM_001927.3) (Figure 1 and Table). The Glu108Lys mutation is the first pathogenic mutation reported in the highly conserved 1A rod domain. The mutations were absent in 300 control chromosomes and were unique among all the alleles screened in the study subjects. We also found a mutation in the tail domain (Val459Ile:c.1461.G3 A;) in 2 black BEST subjects of a total of 69 black subjects in the BEST cohort. This mutation is only 8 amino acids away from the only other mutation (Ile451Met)

linked to isolated DCM, which is also a recurrent mutation in unrelated families.4,5,12,16 The Val459Ile mutation was also absent in our standard controls but was heterozygous in 2 of 100 samples (200 chromosomes) from a population of black controls (Coriell Institute; human variation panel HD100A; http://www.coriell.org/). A sixth DES mutation of interest was found in our study at position 213. An alanine to valine (Ala213Val:c.730.T3 C; reference NM_001927.3) substitution was detected in a large familial dilated cardiomyopathy pedigree and in 6 unrelated BEST subjects. Three other groups have reported finding Ala213Val mutations, which have been proposed as lowpenetrant mutations.3,26,27 To determine whether this was a pathogenic mutation in our study, we analyzed DNA samples from 12 additional members of this pedigree. The 213Val mutation did not segregate with the disease phenotype because it was present in 4 healthy individuals and absent in 1 affected individual. We further analyzed 86 DNA samples from healthy controls without cardiomyopathy and detected the Arg213Val mutation in 2 samples. These data suggest that the Ala213Val is most likely a benign polymorphism (allele frequency ⬇1%), making it currently the only known amino acid polymorphism in the rod domain of desmin.

Phenotype Analysis The carriers of DES mutations in our study all had a pure DCM without any detected involvement of skeletal muscle disease (Table). Elevated creatinine kinase levels have been reported in some DRM patients.4,15,25 The creatinine kinase level was normal in the 1 subject for whom that information was available (Arg350Trp mutation). In contrast to typical observations in DRM, severe conduction system disease and arrhythmias were absent. The single mutation from the Familial Cardiomyopathy Registry cohort occurred in a male DCM subject who developed DCM at age 55 years. He had no evidence of skeletal myopathy and was the only affected member of his family (left ventricular ejection fraction 19% at age 68 years in 2004). His 2 children were clinically evaluated and were found to be normal; no DNA was available from the children. In this patient, the following DCM genes had already been screened negative: LMNA, MYH7, MYH6, TMPO, TNNI3, and SGCD.

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B

Figure 2. Immunofluorescence microscopy of transfected SW13 (A) and human coronary artery smooth muscle cells (B). The consequences of transfected mutations are shown for SW13 (no background desmin expression) and human coronary artery smooth muscle cells (constitutive desmin expression). The top 2 panels show pattern of wild-type desmin filamentous networks (top left panel shows detail). The desmin cytoskeletal network for the Glu108Lys mutation did not appear different from the pattern seen in wild-type cells. The remaining 4 mutations all showed evidence for aggregation of intracytoplasmic desmin protein, although the phenotype of Val459Ile was less severe. The Gln389Pro mutation reported by Goudeau et al24 served as a positive control. Magnification ⫻60.

Expression Studies Immunofluorescence microscopy analysis of transfected SW13 and human coronary artery smooth muscle cells and neonatal rat cardiac myocytes expressing our desmin mutations revealed obvious cellular phenotypes for mutations in the 2B rod domain (Ser298Leu, Asp312Asn, and Arg350Trp) (Figures 2 and 3) that mirrored the desmin disruption seen in the Gln389Pro-positive control. For these mutations, severe disruption of the normal desmin cytoskeletal architecture occurred in the majority of studied cells with clumping and aggregation of antibody-positive staining cytoplasmic pro-

tein. The mutations had a dominant phenotype in the human coronary artery smooth muscle cell lines in which the constitutively expressed desmin was unable to compensate for the presence of the introduced desmin mutations. Interestingly, the Glu108Lys mutation, located in a highly conserved region of the 1A rod domain, did not disrupt the assembly of desmin in SW13 or human coronary artery smooth muscle cells. Mutations in the 1A domain have not been reported previously, even mutations in the neighboring head domain are rare, and the effects on desmin network assembly have not been studied.

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March 13, 2007 Ile451Met tail mutation has also been reported in clinically unaffected adults, suggesting that the penetrance is incomplete.5 Overall, cellular studies and clinical findings suggest that the Val459Ile and the neighboring Ile451Met tail domain mutations are low-penetrant mutations that exert more modest pathogenic effects than the rod-domain mutations found in DRM.5,28

Discussion

Figure 3. Immunofluorescence microscopy of transfected neonatal rat cardiac myocytes, which constitutively express desmin. Cotransfections were done with GFP and mutant desmin constructs, as described in Methods. The desmin cytoskeletal network for the Glu108Lys mutation did not appear different from the wild-type cells. The remaining 4 mutations all show evidence for aggregation of intracytoplasmic desmin protein, although the phenotype of Val459Ile was less severe. The Gln389Pro mutation reported by Goudeau et al24 served as a positive control. Left column, GFP images; middle column, desmin images; and right column, merged images. Magnification ⫻60.

The Val459Ile mutation in the tail domain showed an intermediate phenotype with modest impairment of the desmin filamentous network affecting approximately half of the cells observed. This result is similar to another reported tail domain mutation (Ile451Met) in which only subtle in vitro effects on desmin assembly were noted.4 Our Val459Ile mutation was found in 2 unrelated black patients; similarly, the Ile451Met mutation has been reported in at least 6 unrelated families.4,5,12,14 The Val459Ile mutation was found in 2 apparently healthy black control subjects; likewise, the

Our screening of DES in 425 probands with DCM, the largest number to date, indicates that DES mutations account for 1% to 2% of DCM. Before our study, only 1 mutation linked to isolated DCM (tail domain, Ile451Met) had been reported.16 It had been suggested that the tail domain might be functionally important in cardiac tissue.29 Our results indicate that rod domain mutations are also capable of leading to isolated DCM. The low prevalence of DES mutations is in agreement with 2 smaller studies in DCM in which a screen of a combined 107 families yielded 1 mutation (Ile451Met).16,17 Another study of 265 Japanese DCM cases in whom only exon 8 was studied found 3 instances (1.1%) of the Ile451Met mutation.12 The detection of DES mutations in DCM is not restricted to familial forms of DCM, as evidenced by the 4 different mutations found in the BEST cohort, which was not a family-based study. Our study further demonstrates that DCM due to DES mutations can present without clinically recognizable skeletal muscle involvement. In addition, our data show that mutations in the rod domain can result in a DCM phenotype. Finally, the Ala213Val variant reported previously3,27,28 is likely a rare nonpathogenic polymorphism. Overall, DES mutations can cause a spectrum of phenotypes including skeletal myopathy, mixed skeletal-cardiac disease (“desmin-related myopathy”), and cardiomyopathy (DCM as well as hypertrophic or restrictive cardiomyopathies). Cardiac conduction disease may also be found in some cases. Of the 28 DES mutations we found in the literature in which cardiac status was clearly reported, only the Ile451Met mutation is described as causing an isolated DCM phenotype, although others have linked this mutation to DRM.4,5,16 The 5 novel mutations reported here had an apparently cardiacrestricted phenotype. Mild conduction disease was present in 2 of the 4 mutation carriers, and none of the carriers had evidence of clinical skeletal muscle disease, suggesting that the recognition of DES mutation carriers in the cardiology clinic setting on the basis of clinical data alone is a difficult task. Desmin is expressed early in cardiac development, is the major muscle-specific intermediate filament protein, and is highly expressed in heart tissue, accounting for 2% of cellular protein in myocytes.2,26,29 Desmin filament assembly is a complex process that proceeds stepwise, with desmin monomers first associating into parallel coiled-coil homodimers. After this, antiparallel staggered tetramers form, leading next to laterally associating unit length filaments that ultimately interact end to end to form the final desmin filament.26 The process of filament assembly depends on the central rod domain, whereas the tail domain is believed to be important for interactions between tetramers and elongation of higherorder filament structures.2,30,31 The collection of morpholog-

Taylor et al ically homogeneous disorders in which desmin architecture is perturbed is referred to as myofibrillar, desmin-related myopathies and is due to mutations in desmin as well as myotilin, dystrophin, and ␣-B-crystallin. Haploinsufficiency for desmin may potentially be less deleterious than singlepoint mutations (the bulk of reported mutations); this model is supported by observations in mice heterozygous for desmin-null mutations that have unremarkable skeletal and cardiac phenotypes.32 Three of the DCM-causing DES mutations we found (Ser298Leu, Asp312Asn, and Arg350Trp) are located in the 2B rod domain and caused severe disruption of desmin filament assembly. This domain of desmin is home to the majority of known DES mutations and is believed to be important for dimer-dimer interactions within the mature desmin filament as well as with correct filament assembly.4,33 The clustering of mutations in the 2B domain means that genetic analysis in suspected desmin-related myopathy could be initially targeted to this region as well as to the 1B rod region, where another clustering of mutations occurs. Cardiac conduction abnormalities are prominent features of mutations in the 2B domain, being reported in 7 of 10 previously described 2B mutations.4,6 –9,11,14,15,34 A left bundle branch block was present in the Ser298Leu carrier in our study, and normal conduction was found for the Asp312N and Arg350Trp carriers. In our cellular assay, all 3 2B domain mutations caused the same phenotype of the positive control, Gln389Pro.25 Notably, the introduction of a proline, not normally present in the desmin rod and able to cause kinks in protein structure, was the mutant residue in 8 of the 13 previously reported 2B rod domain mutations causing DRM.4,6 –9,11,14,15,34 On the contrary, the mutations we found are predicted to have less severe structural effects, an observation that might explain why overt muscle disease was absent in our subjects. The Arg350Trp mutation substitutes a nonpolar, hydrophobic residue for a basic hydrophilic arginine moiety in the conserved ␣-helical coil 2B domain of desmin. The GOR IV secondary structure analysis program states that a loss of ␣-helical structure in the 2B coil is predicted to occur because of this mutation.35 The same residue is involved in a family with skeletal, cardiac, and diaphragmatic muscle disease carrying a proline substitution at the 350 position.36 The Ser298Leu and Asp312Asn mutations introduce nonpolar amino acids and are predicted to result in additional ␣-helical structure by the nnpredict program.37 The Glu108Lys mutation found in our study is unique for 2 reasons. First, it is the only mutation found thus far in the 1A helix domain and in a location that is the most highly evolutionary conserved region of desmin and of all intermediate filaments. The Glu108 position is fully conserved among desmin, vimentin, neurofilament L protein, cytokeratins 8 and 18, and nuclear lamins A and B1.31 The fourth position of the repeating heptad unit is a “core” position, located on the concave side of the 1A domain, and is believed to affect dimer-dimer interactions.33 This mutation predicts the introduction of ␣-helix structure at the position of a random coil by the GOR IV program.35 Second, the lysine substitution did not interrupt the in vitro desmin network

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assembly. No other 1A helical domain mutations are known to exist for comparison. We suggest that the pathogenic effect of this Glu108Lys mutation occurs beyond the stage of desmin filamentous assembly, perhaps affecting dimer-dimer interactions rather than a fatal interruption of the actual assembly process. That not all DES pathogenic mutations will disrupt desmin network assembly has been suggested by others.3,5,26,38,39 The initial studies of the first DCM mutation, Ile451Met, were equivocal, and in a follow-up study it was reported that no effect on desmin filamentous network was observed.5 Most recently, the DRM mutations Ala213Val, Glu245Asp, Ala360Pro, Asn393Ile, Gln389Pro, and Asp399Tyr demonstrated normal filament assembly in SW13 cells.28,40 The authors of these studies concluded that the severity of the phenotype does not correlate directly with the in vitro cellular models. Other properties of desmin protein beyond mere network assembly could also be important in the expressed disease phenotype. Additional studies are needed to determine whether fundamental differences exist in desmin function between DCM and DRM DES mutations. The Val459Ile mutation was recurrent in 2 of our 69 black subjects (allele frequency of 1.4%) as well as present in 2 of 100 black control subjects (1%), and it was restricted to samples having black ethnicity. This finding could be compatible with a polymorphic variant (Fisher exact test, P⫽0.54), although an intermediate phenotype was shown in the transfection studies. This tail-domain mutation is 8 amino acids away from the other DCM-causing DES mutation (Ile451Met) reported in several studies. The in vitro findings of both of these tail mutations are less striking and appear to represent a less severe effect on desmin assembly.4,5,41 The Ile451Met mutation has also been linked to DRM and isolated skeletal muscle disease and has been found in ostensibly asymptomatic adults, observations perhaps explained by other genetic or environmental modifiers of the phenotype.4,5 Thus, the balance of the data would indicate that these are rare mutations with low penetrance. The association of the Val459Ile mutation to blacks is similar to observations for hereditary amyloidosis due to transthyretin mutations. In this condition, also characterized by abnormal cellular protein aggregation, the Val122Ile TTR mutation has a frequency of 3% in blacks and is associated with reduced penetrance and late-onset disease.42,43 Our study is limited by the possibility that some of the isolated DCM cases we studied will develop skeletal muscle involvement later in life. Formal neurological assessments were not done in the BEST cohort, none of the subjects in our study had indications for skeletal muscle biopsy, and we cannot exclude the possibility of mild skeletal muscle disease in the DES mutation carriers. In a previous report, a patient with a splicing site mutation predicting the deletion of amino acids 214 to 245 presented with second-degree atrioventricular heart block and DCM followed by muscle involvement developing over the next 10 years.11 We screened for mutations in the exons, promoter, and periexonic portions of the introns, but it is possible that our screen could have missed other intronic mutations. We were also limited by the lack of cardiac or skeletal muscle biopsy material in our patients and

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could not test whether our patients have desmin aggregation in myocytes, as has been demonstrated by others.6,12 Finally, an estimate of the penetrance of DES mutations is also not apparent from the literature because data are currently available predominantly from affected individuals, although we and others have found evidence of probable mutations in healthy adults.3,5 The effects of mutation status on lifetime penetrance as well as age-dependent manifestations of the disease will need to be explored in future family studies. In summary, we found that DES mutations can be implicated in 1% to 2% of DCM without skeletal muscle disease involvement. We have shown that DCM-causing mutations occur outside the tail domain and have reported the first mutation in the highly conserved region of the 1A rod domain. Disruption of desmin cytoskeletal networks was not reproducibly shown in the 1 previous DES mutation linked to DCM (Ile451Met). Three of our mutations located in the 2B helical rod domain clearly interfere with desmin network assembly, suggesting that similar pathogenic mechanisms exist between DCM and DRM. One of our mutations, Glu108Lys, occurred in the most conserved domain of desmin and did not visibly disrupt the pattern of intracytoplasmic desmin filaments. It is possible that this mutation exerts its effect by perturbing the desmin dimer-dimer interactions in the assembled network. Our finding of a second desmin tail mutation that is recurrent and low penetrant indicates that mutations in the less-conserved tail domain are perhaps better tolerated and may require interactions with other genetic or environmental factors to exert an effect. Finally, because clinical screening of DES is now available, clinicians evaluating patients with cardiomyopathy/myopathies need to be selective in their utilization of DES testing and critical in their evaluation of novel variants detected in such testing. Clinical mutation testing of DCM patients should initially focus on other genes in which the prevalence of mutations is higher, such as LMNA.44 – 47 We believe that specific testing for DES mutations should be included in a second-tier level of testing if mutations in more frequent DCM genes are not found.

Acknowledgments The investigators wish to acknowledge the generous contributions of the cardiomyopathy patients and families participating in the Familial Cardiomyopathy Registry.

Sources of Funding The present study was supported by grants from the National Institutes of Health (HL67915-01A1; HL69071-01), American Heart Association (0150453N), and Muscular Dystrophy Association (PN0007-056).

Disclosures None.

References 1. Online Mendelian Inheritance in Man (OMIM). Johns Hopkins University, Baltimore, Md. MIM Number: #601419: April 10, 2006. Available at http://www.ncbi.nlm.nih.gov/entrez/dispomim. cgi?id⫽601419. Accessed February 14, 2007. 2. Paulin D, Li Z. Desmin: a major intermediate filament protein essential for the structural integrity and function of muscle. Exp Cell Res. 2004; 301:1–7.

3. Goldfarb LG, Vicart P, Goebel HH, Dalakas MC. Desmin myopathy. Brain. 2004;127:723–734. 4. Dalakas MC, Park KY, Semino-Mora C, Lee HS, Sivakumar K, Goldfarb LG. Desmin myopathy, a skeletal myopathy with cardiomyopathy caused by mutations in the desmin gene. N Engl J Med. 2000;342:770 –780. 5. Dalakas MC, Dagvadorj A, Goudeau B, Park KY, Takeda K, SimonCasteras M, Vasconcelos O, Sambuughin N, Shatunov A, Nagle JW, Sivakumar K, Vicart P, Goldfarb LG. Progressive skeletal myopathy, a phenotypic variant of desmin myopathy associated with desmin mutations. Neuromuscul Disord. 2003;13:252–258. 6. Arbustini E, Pasotti M, Pilotto A, Pellegrini C, Grasso M, Previtali S, Repetto A, Bellini O, Azan G, Scaffino M, Campana C, Piccolo G, Vigano M, Tavazzi L. Desmin accumulation restrictive cardiomyopathy and atrioventricular block associated with desmin gene defects. Eur J Heart Fail. 2006;8:477– 483. 7. Olive M, Goldfarb L, Moreno D, Laforet E, Dagvadorj A, Sambuughin N, Martinez-Matos JA, Martinez F, Alio J, Farrero E, Vicart P, Ferrer I. Desmin-related myopathy: clinical, electrophysiological, radiological, neuropathological and genetic studies. J Neurol Sci. 2004;219:125–137. 8. Dagvadorj A, Goudeau B, Hilton-Jones D, Blancato JK, Shatunov A, Simon-Casteras M, Squier W, Nagle JW, Goldfarb LG, Vicart P. Respiratory insufficiency in desminopathy patients caused by introduction of proline residues in desmin c-terminal alpha-helical segment. Muscle Nerve. 2003;27:669 – 675. 9. Kaminska A, Strelkov SV, Goudeau B, Olive M, Dagvadorj A, Fidzianska A, Simon-Casteras M, Shatunov A, Dalakas MC, Ferrer I, Kwiecinski H, Vicart P, Goldfarb LG. Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy. Hum Genet. 2004;114:306 –313. 10. Munoz-Marmol AM, Strasser G, Isamat M, Coulombe PA, Yang Y, Roca X, Vela E, Mate JL, Coll J, Fernandez-Figueras MT, Navas-Palacios JJ, Ariza A, Fuchs E. A dysfunctional desmin mutation in a patient with severe generalized myopathy. Proc Natl Acad Sci U S A. 1998;95: 11312–11317. 11. Park KY, Dalakas MC, Goebel HH, Ferrans VJ, Semino-Mora C, Litvak S, Takeda K, Goldfarb LG. Desmin splice variants causing cardiac and skeletal myopathy. J Med Genet. 2000;37:851– 857. 12. Miyamoto Y, Akita H, Shiga N, Takai E, Iwai C, Mizutani K, Kawai H, Takarada A, Yokoyama M. Frequency and clinical characteristics of dilated cardiomyopathy caused by desmin gene mutation in a Japanese population. Eur Heart J. 2001;22:2284 –2289. 13. Schroder R, Goudeau B, Simon MC, Fischer D, Eggermann T, Clemen CS, Li Z, Reimann J, Xue Z, Rudnik-Schoneborn S, Zerres K, van der Ven PF, Furst DO, Kunz WS, Vicart P. On noxious desmin: functional effects of a novel heterozygous desmin insertion mutation on the extrasarcomeric desmin cytoskeleton and mitochondria. Hum Mol Genet. 2003; 12:657– 669. 14. Sjoberg G, Saavedra-Matiz CA, Rosen DR, Wijsman EM, Borg K, Horowitz SH, Sejersen T. A missense mutation in the desmin rod domain is associated with autosomal dominant distal myopathy, and exerts a dominant negative effect on filament formation. Hum Mol Genet. 1999; 8:2191–2198. 15. Sugawara M, Kato K, Komatsu M, Wada C, Kawamura K, Shindo PS, Yoshioka PN, Tanaka K, Watanabe S, Toyoshima I. A novel de novo mutation in the desmin gene causes desmin myopathy with toxic aggregates. Neurology. 2000;55:986 –990. 16. Li D, Tapscoft T, Gonzalez O, Burch PE, Quinones MA, Zoghbi WA, Hill R, Bachinski LL, Mann DL, Roberts R. Desmin mutation responsible for idiopathic dilated cardiomyopathy. Circulation. 1999;100:461– 464. 17. Tesson F, Sylvius N, Pilotto A, Dubosq-Bidot L, Peuchmaurd M, Bouchier C, Benaiche A, Mangin L, Charron P, Gavazzi A, Tavazzi L, Arbustini E, Komajda M. Epidemiology of desmin and cardiac actin gene mutations in a European population of dilated cardiomyopathy. Eur Heart J. 2000;21:1872–1876. 18. A trial of the beta-blocker bucindolol in patients with advanced chronic heart failure. N Engl J Med. 2001;344:1659 –1667. 19. The BEST Steering Committee. Design of the Beta-Blocker Evaluation Survival Trial (BEST). Am J Cardiol. 1995;75:1220 –1223. 20. Mestroni L, Maisch B, McKenna WJ, Schwartz K, Charron P, Rocco C, Tesson F, Richter A, Wilke A, Komajda M. Guidelines for the study of familial dilated cardiomyopathies. Eur Heart J. 1999;20:93–102. 21. Li Z, Marchand P, Humbert J, Babinet C, Paulin D. Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice. Development. 1993;117:947–959.

Taylor et al 22. Mariner PD, Luckey SW, Long CS, Sucharov CC, Leinwand LA. Yin Yang 1 represses alpha-myosin heavy chain gene expression in pathologic cardiac hypertrophy. Biochem Biophys Res Commun. 2005; 326:79 – 86. 23. Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells: crossstriations, ultrastructure, and chronotropic response to isoproterenol. Circ Res. 1982;50:101–116. 24. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77:51–59. 25. Goudeau B, Dagvadorj A, Rodrigues-Lima F, Nedellec P, Casteras-Simon M, Perret E, Langlois S, Goldfarb L, Vicart P. Structural and functional analysis of a new desmin variant causing desmin-related myopathy. Hum Mutat. 2001;18:388 –396. 26. Bar H, Strelkov SV, Sjoberg G, Aebi U, Herrmann H. The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation? J Struct Biol. 2004;148:137–152. 27. Bowles NE, Jimenez S, Vatta M, Chrisco M, Szmuskovicz J, Capetanaki Y. Familial restrictive cardiomyopathy caused by a missense mutation in the desmin gene. Pediatr Res. 2002;51. Abstract. 28. Bar H, Mucke N, Kostareva A, Sjoberg G, Aebi U, Herrmann H. Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages. Proc Natl Acad Sci U S A. 2005;102: 15099 –15104. 29. Paulin D, Huet A, Khanamyrian L, Xue Z. Desminopathies in muscle disease. J Pathol. 2004;204:418 – 427. 30. Herrmann H, Haner M, Brettel M, Muller SA, Goldie KN, Fedtke B, Lustig A, Franke WW, Aebi U. Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains. J Mol Biol. 1996;264:933–953. 31. Strelkov SV, Herrmann H, Geisler N, Wedig T, Zimbelmann R, Aebi U, Burkhard P. Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly. EMBO J. 2002;21:1255–1266. 32. Li Z, Colucci-Guyon E, Pincon-Raymond M, Mericskay M, Pournin S, Paulin D, Babinet C. Cardiovascular lesions and skeletal myopathy in mice lacking desmin. Dev Biol. 1996;175:362–366. 33. Strelkov SV, Herrmann H, Aebi U. Molecular architecture of intermediate filaments. Bioessays. 2003;25:243–251. 34. Goldfarb LG, Park K-Y, Cervenacova L, Gorokhova S, Lee H-S, Vasconcelos O, Nagle JW, Semino-Mora C, Sivakumar K, Dalakas MC. Missense mutations in desmin associated with familial cardiac and skeletal myopathy. Nat Genet. 1998;19:402– 403.

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35. Garnier J, Gilbrat JF, Robson B. GOR method for predicting protein secondary structure from amino acid sequence. Method Enzymol. 1996; 266:540 –553. 36. Bar H, Fischer D, Goudeau B, Kley RA, Clemen CS, Vicart P, Herrmann H, Vorgerd M, Schroder R. Pathogenic effects of a novel heterozygous R350P desmin mutation on the assembly of desmin intermediate filaments in vivo and in vitro. Hum Mol Genet. 2005;14:1251–1260. 37. NNPREDICT Protein secondary structure prediction. In. 38. Rogers KR, Eckelt A, Nimmrich V, Janssen KP, Schliwa M, Herrmann H, Franke WW. Truncation mutagenesis of the non-alpha-helical carboxyterminal tail domain of vimentin reveals contributions to cellular localization but not to filament assembly. Eur J Cell Biol. 1995;66: 136 –150. 39. Kaufmann E, Weber K, Geisler N. Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues. J Mol Biol. 1985;185:733–742. 40. Bar H, Kostareva A, Sjoberg G, Sejersen T, Katus HA, Herrmann H. Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation. Exp Cell Res. 2006;312:1554 –1565. 41. D’Adamo P, Fassone L, Gedeon A, Janssen EA, Bione S, Bolhuis PA, Barth PG, Wilson M, Haan E, Orstavik KH, Patton MA, Green AJ, Zammarchi E, Donati MA, Toniolo D. The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies. Am J Hum Genet. 1997;61:862– 867. 42. Yamashita T, Hamidi Asl K, Yazaki M, Benson MD. A prospective evaluation of the transthyretin Ile122 allele frequency in an AfricanAmerican population. Amyloid. 2005;12:127–130. 43. Jacobson DR, Pastore RD, Yaghoubian R, Kane I, Gallo G, Buck FS, Buxbaum JN. Variant-sequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans. N Engl J Med. 1997; 336:466 – 473. 44. Burkett EL, Hershberger RE. Clinical and genetic issues in familial dilated cardiomyopathy. J Am Coll Cardiol. 2005;45:969 –981. 45. Daehmlow S, Erdmann J, Knueppel T, Gille C, Froemmel C, Hummel M, Hetzer R, Regitz-Zagrosek V. Novel mutations in sarcomeric protein genes in dilated cardiomyopathy. Biochem Biophys Res Commun. 2002; 298:116 –120. 46. Kamisago M, Sharma SD, DePalma SR, Soloman S, Sharma P, McDonough B, Smoot L, Mullen MP, Woolf PK, Wingle ED, Seidman JG, Seidman CE. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N Engl J Med. 2000;343:1688 –1696. 47. Taylor MR, Robinson ML, Mestroni L. Analysis of genetic variations of lamin A/C gene (LMNA) by denaturing high-performance liquid chromatography. J Biomol Screen. 2004;9:625– 628.

CLINICAL PERSPECTIVE Desmin-related myofibrillar myopathy is a cardiac and skeletal muscle disease caused by mutations in the desmin gene. Mutations cluster near the center of the protein (2B domain) and typically cause skeletal muscle disease, which usually precedes any overt cardiomyopathy. Previously, only 1 desmin mutation leading to a pure cardiac phenotype had been reported, and the overall prevalence of desmin mutations in dilated cardiomyopathy was unknown. We screened the desmin gene for mutations using denaturing high-performance liquid chromatography in 425 unrelated patients and detected 5 novel mutations in 6 individuals for an overall prevalence of 1.4%. None of the individuals had any overt signs of skeletal muscle disease. The Glu108Lys mutation we detected is the first reported pathogenic mutation in the highly conserved 1A domain of the desmin protein. Expression of the 3 2B domain mutations in cell lines, including neonatal rat cardiac myocytes, severely disrupted the normal cytoplasmic desmin network. One mutation in the tail domain had a milder effect on desmin architecture, and the mutation in the 1A domain did not visibly affect desmin staining, suggesting that dysfunction of seemingly intact desmin networks is sufficient to cause dilated cardiomyopathy. Overall, the results show that desmin mutations are a relatively rare cause of dilated cardiomyopathy, can present in the absence of overt muscle disease, and mechanistically disrupt desmin cytoskeletal architecture in the majority of cases.

Imaging Incremental Value of Strain Rate Imaging to Wall Motion Analysis for Prediction of Outcome in Patients Undergoing Dobutamine Stress Echocardiography Charlotte Bjork Ingul, MD; Ellen Rozis, MD; Stig A. Slordahl, PhD; Thomas H. Marwick, MD, PhD Background—Wall motion score at dobutamine stress echocardiography is an independent predictor of mortality. We sought to determine whether quantification of DSE by strain rate imaging was incremental to wall motion score for predicting outcome. Methods and Results—In 646 patients undergoing dobutamine stress echocardiography for the evaluation of known or suspected coronary disease, customized software was used to automatically measure peak systolic strain rate (SRs) and end-systolic strain (Ses) in 18 segments. Results were expressed as the number of abnormal segments and the mean SRs and Ses per patient. All-cause mortality was identified over 7 years of follow-up (mean, 5.2⫾1.5 years). Contributions of clinical, wall motion, and SRs and Ses data to outcome were analyzed with Cox models, which also were used to define cut points for SRs and Ses. Ischemia (new or worsening wall motion abnormalities) was detected in 45%, and 39% had a previous myocardial infarction. In patients with no ischemia, annualized mortality without and with previous myocardial infarction were 2% and 3% compared with 5% in patients with ischemia. Peak wall motion score index, mean SRs, segmental Ses, and segmental SRs were all predictors of mortality, but only segmental SRs (hazard ratio, 3.6; 95% CI, 1.7 to 7.2) was independently predictive. In sequential Cox models, the model based on clinical data (overall ␹2, 12.7) was improved by peak wall motion score index (18.4, P⫽0.002) and further increased by either segmental SRs (31.8, P⬍0.001) or mean SRs (25.7, P⫽0.009). Conclusions—Segmental analysis by SRs, derived from automated strain rate imaging analysis of dobutamine stress echocardiography response, offers prognostic information that is independent and incremental to standard wall motion score index. (Circulation. 2007;115:1252-1259.) Key Words: echocardiography 䡲 prognosis 䡲 stress

A

bnormal wall motion (WM) at dobutamine stress echocardiography (DSE) is an independent predictor of mortality in patients with known or suspected coronary artery disease (CAD), and risk increases in parallel with the extent of WM abnormality.1,2 Although DSE is used widely to assess CAD, its interpretation is demanding, and its accuracy depends on the experience and training of the reader.3 Despite technical advances and digital image processing and display, both its reproducibility and accuracy are dependent on image quality. DSE also is limited by its relative insensitivity for mild single-vessel disease, low sensitivity for recognizing the presence of multivessel coronary disease, and difficulty in detection of ischemia within areas of abnormal resting WM. A more objective technique could overcome many of these limitations.

2-dimensional echocardiography, and several studies have shown the technique to be feasible for DSE using either velocity or strain rate imaging (SRI).4 – 6 SR and strain can characterize regional myocardial deformation at rest and can quantify normal or abnormal regional function during DSE.5,7,8 These results may be compromised by many pitfalls, however, including reverberations, misalignment, and angulation. Indeed, none of the tissue Doppler– derived parameters are completely objective because the region of interest is set manually and therefore may be colored by the assessment of WM. To objectively analyze regional myocardial function on the basis of tissue Doppler, we have developed a quick and feasible automated SRI method that includes tracking the region of interest laterally by using the speckle pattern and axially by tissue Doppler.9 In the tissue Doppler method, SR is derived from the velocity gradient of 2 points at a fixed distance along the ultrasound beam, and strain is measured by the temporal integration of SR. The automated method for

Clinical Perspective p 1259 Quantification of DSE with tissue Doppler is less dependent on 2-dimensional image quality than quantification from

Received May 24, 2006; accepted November 10, 2006. From the University of Queensland, Brisbane, Australia (C.B.I., E.R., T.H.M.), and the Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway (S.A.S.). Correspondence to Thomas H. Marwick, MD, PhD, University of Queensland, Department of Medicine, Princess Alexandra Hospital, Brisbane, QLD 4102, Australia. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.640334

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Ingul et al TABLE 1.

Clinical Characteristics

Risk factors Diabetes mellitus (fasting glucose ⬎7 mmol/L)

127 (20)

Hypertension (BP ⬎140/90 mm Hg)

330 (52)

Hypercholesterolemia (total cholesterol ⬎4 mmol/L)

324 (51)

Smoking

148 (23)

Past cardiac history Previous MI

251 (39)

Angina

294 (46)

Chronic heart failure

28 (4)

Previous PTCA

67 (10)

Previous CABG

60 (9)

Drug therapy

␤-Blockers

256 (40)

Nitrates

203 (32)

Calcium channel blockers

157 (24)

Weight, kg, mean⫾SD

78⫾18

Height, cm, mean⫾SD

169⫾14

Values are n (%) when appropriate. BP indicates blood pressure; PTCA, percutaneous coronary angioplasty; and CABG, coronary artery bypass grafting.

measuring peak systolic strain rate (SRs) has been shown to increase the sensitivity of DSE compared with WM.8 The potential of SRI to predict mortality has not previously been studied. Therefore, the objective of the present study was to ascertain whether quantification of DSE by SRI is incremental to WM analysis for predicting the outcome of patients with known or suspected CAD.

Methods Patient Selection Between 1998 and 2000, 787 consecutive studies were performed using DSE and simultaneous tissue Doppler acquisition for evaluation of myocardial ischemia at Princess Alexandra Hospital (Brisbane, Australia). The clinical characteristics of these patients are listed in Table 1. The most common reasons for referral were evaluation after infarction (n⫽197, 25%), evaluation of chest pain or dyspnea (n⫽189, 24%), and preoperative noncardiac risk assessment (n⫽165, 21%). If a patient had ⬎1 DSE during the time period (n⫽17), only the first test was entered into the analysis. In all, 68 patients were excluded from the analysis because of incomplete acquisition or archiving problems (n⫽23) or technical problems precluding SR measurement, including low frame rate (⬍75 frames per second; n⫽25), poor image quality (n⫽5), or failure of the tracking algorithm (n⫽10). All-cause mortality was identified over 7 years of follow-up (mean, 5.2⫾1.5 years), which was completed by the beginning of 2006. Follow-up data were not available in 56 patients (7%). Thus, the final study sample consisted of 646 patients (241 women; age, 61⫾12 years).

Stress Protocol After a resting echocardiogram was obtained, a standard DSE protocol was performed with incremental dobutamine infusion rates of 5, 10, 20, 30, and 40 ␮g · kg⫺1 · min⫺1 every 3 minutes. Patients who did not achieve 85% of the age-predicted maximal heart rate (220⫺age) were given atropine in 0.3-mg increments up to 1.2 mg until target heart rate was achieved. Cardiac rhythm and blood pressure were monitored before and during the test. The stress test was terminated because of completion of the protocol, severe ischemia (severe angina, extensive new WM abnormalities, horizon-

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tal or downsloping ST-segment depression ⬎2 mm, ST-segment elevation ⬎1 mm in patients without prior myocardial infarction [MI]), systolic blood pressure ⬎240 mm Hg or ⬍100 mm Hg, serious ventricular arrhythmia, or patient intolerance of side effects. The test was considered nondiagnostic if the patient failed to achieve target heart rate without inducible WM abnormality.

Echocardiographic Acquisition Cine loops from LV apical views (4-chamber, 2-chamber, and long-axis views) were recorded simultaneously in both tissue second-harmonic mode and tissue Doppler mode (Vivid 5, GE Vingmed Ultrasound, Horten, Norway). Images were recorded at baseline, low dose, peak, and recovery in a digitized quad-screen format. The pulse repetition frequency was between 1 and 1.5 kHz, and the frame rate (both for tissue Doppler and B-mode) ranged from 75 to 133 per second (median, 90). The number of samples along the beam ranged from 102 to 258 (median, 150) for tissue Doppler and 188 to 486 (median, 420) for B-mode imaging. The number of beams ranged from 10 to 28 (median, 16) for tissue Doppler and from 20 to 87 (median, 41) for B-mode imaging. Echo data were stored digitally for subsequent offline analysis.

Echocardiographic Analysis Two dimensional gray-scale echo images, interpreted by an experienced observer, were scored as normal (score⫽1), hypokinetic (2), severely hypokinetic (2.5), akinetic (3), or dyskinetic (4). Infarction was recognized on the basis of severe hypokinesis, akinesis, or dyskinesis at rest. Ischemia was identified in the presence of new or worsening WM abnormality in ⱖ1 segments during DSE, and a study was identified as abnormal if there were scarred segments with or without ischemia. WM score index (WMSI), number of ischemic segments, and number of scarred segments also were compared. A negative study was characterized by a normal response in all segments.

SRI Analysis Automated measurements of SRI variables used a Matlab-based (MathWorks Inc, Natick, Mass) custom-made program described earlier by our group.9 All SRI measurements were obtained by a single observer blinded to all results (clinical and WM). Segments were tracked according to position, orientation, and length throughout the cycle, axially (along the ultrasound beam) by tissue Doppler data, and laterally by speckle tracking. The difference in acquisition time for B-mode and tissue Doppler imaging data was taken into account and compensated for by temporal interpolation in the speckle tracking algorithm. The segment was discarded if the speckle pattern region failed to track properly or if the gray-scale image showed regions with missing ultrasound data (dropouts) or large reverberations. The displacement of the kernel region was used to check the tracking; ideally, this should be 0 because the kernel returns to the same baseline position by the end of the cardiac cycle. A displacement ⬎2 mm indicated poor tracking. The timing of aortic valve closure was defined automatically with tissue Doppler imaging.10 For the purposes of the present study, SRI parameters were measured using velocity data. This method involved automatic placement of a region of interest in the center of the basal and mid segments at end diastole and the basal part of the apical segments to limit the effects of angulation toward the apex. The segment was tracked throughout the cardiac cycle as described. Longitudinal SR was calculated from the velocity gradient along the ultrasound beam, and strain was calculated as the temporal integral of SR corrected from Eulerian SR to Lagrangian strain, both angle dependent.11 The strain length (distance for velocity gradient calculation) was 10 to 15 mm; axial averaging of 1 mm and temporal averaging of 10 ms were used for the analysis. Peak SRs was determined as the maximal negative SR value during ejection; end-systolic strain (Ses) was defined as the magnitude of strain at aortic valve closure. Measurements were made in 18 segments from 3 apical views at peak stress, all blinded from WM and clinical results. SR and Ses results were expressed as the mean

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TABLE 2.

DSE Data

March 13, 2007

Rest Heart rate, bpm Rest systolic blood pressure, mm Hg Rest diastolic blood pressure, mm Hg Rate-pressure product, mm Hg/min

Stress

75⫾14

139⫾13

140⫾25

157⫾32

77⫾13

75⫾16

10 553⫾2827 21 330⫾4970

WMSI

1.26⫾0.41

1.38⫾0.46

Dobutamine dose, ␮g 䡠 kg⫺1 䡠 min⫺1

38⫾5

䡠䡠䡠

Atropine dose, mg

0.8⫾0.28

䡠䡠䡠

Chest pain during DSE, n (%)

150 (23)

䡠䡠䡠

ST-segment depression ⬎0.1 mV, n (%)

167 (26)

䡠䡠䡠

75 (12)

䡠䡠䡠

DSE stopped because of limiting side effects, n (%)

were used to estimate the univariable hazard ratio of event for each variable.13 The incremental value of SRI variables over WMSI and clinical variables was assessed in a series of Cox models in which the first model consisted of fitting the clinical data entered as a block (diabetes mellitus, previous MI, age) in the test group. Each echo variable (rest and peak WMSI, mean SRs, mean Ses, segmental SRs, segmental Ses) was then entered into separate models in combination with this clinical block. Model ␹2 was then compared for each Cox model. Analyses were performed using standard statistical software (SPSS version 12, SPSS, Chicago, Ill), and values of P⬍0.05 were considered statistically significant. The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Values are mean⫾SD when appropriate.

Results

value of all segments (mean SRs or mean Ses) or the number of abnormal segments at risk (segmental SRs or segmental Ses). The series were randomly divided, with half of the patients (n⫽320) used for defining the optimal cut points of patients at low and high risk of mortality with respect to SRs, Ses, WMSI, number of ischemic segments, and number of scarred segments. The number of abnormal segments at risk was defined as the number with an SR or Ses below the defined cut points. The remaining patients were used for testing these cut points.

Follow-Up Data All-cause mortality was the primary end point. Follow-up was obtained by review of the patient’s hospital or family practice chart or telephone interview with the patient or relative. Patients were censored at the time of percutaneous or surgical revascularization.

Statistical Analysis Categorical variables are expressed as proportions; continuous variables are expressed as mean⫾SD. ANOVA was used to compare the means between the 4 different groups with or without ischemia and previous MI, with Scheffé post hoc test used for multiple comparisons. Multiple measures per patient were corrected for within a general linear model. The McNemar test was used for comparison of categorical data for per-patient analysis of WM and SRI. KaplanMeier curves were used to estimate the survival function for time to death, and a log-rank test was used to compare differences between survival curves. The independent association of clinical WMS and SRI variables with outcome was assessed in a series of Cox models. Cut points for each SRI variable and peak WMSI were assessed from the derivation sample initially using a receiver-operating curve analysis12 to establish the approximate cut point and then entering this and adjacent cut points into a Cox model with the same clinical variables. Cox models

Dobutamine Echocardiography Adequate heart rate was achieved in 97% of the patients (n⫽640). In the remaining 21 patients, the protocol was terminated at a submaximal heart rate because of severe ischemia or side effects (hypotension, hypertension, ventricular arrhythmia, patient intolerance; n⫽15) or inability to attain target heart rate despite dobutamine and atropine (n⫽6). The test was nondiagnostic in 6 patients (1%), most commonly because of failure to reach target heart rate. Table 2 summarizes the hemodynamics and stress results for the 640 patients with adequate heart rate response. A completely normal test at rest and stress was found in 267 patients (42%). Resting WM abnormality was present in 251 patients (39%), and in 87 patients (14%), this was the only abnormality. Ischemia by WM was found in 286 patients (45%); ischemia alone was detected in 122 patients (19%) and ischemia with prior MI in 164 patients (26%). The majority (82%) of patients with ischemia had ⬎1 ischemic segment at peak stress. A total of 11 520 myocardial segments were analyzed with tissue Doppler. Automated analysis of SRs was possible in 93% of the segments compared with 87% for Ses. WM at peak identified 984 ischemic segments, 356 viable segments, and 979 scarred segments. Patients with a normal response to dobutamine and normal resting WM demonstrated significantly a higher magnitude of mean SRs and mean Ses compared with the patients with ischemia and prior MI (Table 3).

TABLE 3. Comparison of the Different Echocardiographic Variables in Patients With Maximal Stress Between the Patient Groups With or Without Ischemia and MI No Ischemia, No Prior MI (n⫽267)

Echo Variable at Peak

No Ischemia, Prior MI (n⫽87)

Ischemia, No Prior MI (n⫽122)

Ischemia, Prior MI (n⫽164)

All Patients (N⫽640)

WMSI rest

1.02 (0.14)

1.64 (0.40)*

1.02 (0.05)

1.86 (0.46)*

WMSI peak

1.02 (0.14)

1.60 (0.40)*

1.40 (0.24)*

1.63 (0.43)*

1.39 (0.46)

⫺2.53 (0.48)

⫺1.93 (0.57)*

⫺2.0 (0.44)*

⫺1.71 (0.52)*

⫺2.14 (0.60)

⫺13.7 (4.8)

⫺7.8 (4.5)*

⫺9.2 (4.7)*

⫺7.2 (4.4)*

⫺10.4 (5.0)

4.8 (3.5)

9.0 (4.1)*

8.6 (3.5)*

10.6 (4.0)*

7.6 (4.5)

5.0 (3.9)

10.2 (3.3)*

9.3 (3.5)*

10.7 (3.3)*

8.0 (4.4)

Mean SRs, s⫺1 Mean Ses, % Segmental SRs, s

⫺1

Segmental Ses, %

Values are expressed as mean (SD). *P⬍0.001 vs normal DSE without prior MI.

1.26 (0.41)

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Outcomes and Events During Follow-Up In total, 107 patients (17%) died during the follow-up period. Myocardial revascularization was performed in 51 patients: 31 had PCI (5%) and 20 had CABG (3%). MI occurred in 17 patients (3%). Figure 1 summarizes the mortality according to the presence of ischemia and previous MI. Most events occurred in patients with ischemia and previous MI, with an annualized event rate of 4.7% compared with 1.9% in the group with a normal stress test and no previous MI (P⫽0.004).

Clinical Predictors of Outcome By univariable analysis, the clinical predictors of death were age, diabetes mellitus, hypertension, and previous MI; all but hypertension (P⫽0.1) were independently predictive in the multivariable model. Additional nonsignificant clinical variables in the univariable analysis included gender (P⫽0.56), smoking (P⫽0.89), hypercholesterolemia (P⫽0.46), angina (P⫽0.62), and congestive heart failure (P⫽0.2).

Deformation and Predictors of Mortality The optimal cut point was approximated on the basis of receiver-operating characteristics curves, and this and adjacent values were then entered into a series of multivariate models, together with the same independent clinical predictors listed above. The optimal value was defined as the parameter that maximized the model ␹2 for prediction of mortality (Figure 2). These were an SRs ⬎⫺1.2 seconds⫺1 and Ses ⬎⫺3.5% for mean values and an SRs ⬎⫺1.2 seconds⫺1 and Ses ⬎⫺9% for segmental values. Mean SRs and segmental SRs provided stronger models than WMSI. The identification of low risk with mean quantitative parameters, including mean SRs ⬎⫺1.2 (93%) and mean Ses ⬎⫺3.5% (92%), was not significantly different from its allocation on the basis of peak WMSI ⬎1.7 (90%). However, definition based on ⬎10 segments with SRs ⬎⫺1.2 led more patients to be identified as low risk (95%; P⫽0.02 versus WMSI), whereas definition based on ⬎3 segments with Ses ⬎⫺9% led fewer to be identified as low risk (73%; P⬍0.001 versus WMSI). Ischemia by WM, number of ischemic segments, and peak WMSI were significant univariate predictors of death, as were all of the SRI variables at peak (except mean Ses) (Table 4). In a combined multivariable model including clinical and echocardiographic variables, the only significant independent predictor of death was segmental SRs at peak, with a hazard ratio of 3.6 (P⬍0.001) (Table 4).

Incremental Value of WM and SRI for Predicting Death A model using only clinical variables (diabetes mellitus, age, and previous MI) gave an overall ␹2 of 12.7. The addition of WMS at either rest or stress increased the power of this model. The addition of the number of abnormal segments based on SRs or Ses and mean SRs further increased the prediction from the combination of clinical and stress WM data (Figure 3), although the addition of mean Ses was not significant (P⫽0.3).

Figure 1. Kaplan-Meier survival curves according to DSE responses. A, Subdivision of all patients into those with no ischemia at peak DSE and those with ischemia at peak DSE. Numbers of patients at risk for 2, 4, and 6 years are indicated. B, Subdivision of patients with a normal DSE response into patients without prior MI and patients with prior MI. C, Subdivision of patients who develop ischemia at peak DSE into patients without prior MI and patients with prior MI.

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Figure 2. Model ␹2 values for a series of Cox models using different cutoff values for the WM, average SRs (systolic strain rate) and Ses (end-systolic strain), and number of segments above a threshold level of SRs and Ses.

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TABLE 4. Prediction of Mortality by Univariable and Multivariable Analysis of WM and SRI Variables at Peak Univariable Analysis Variable

Multivariable Analysis

Hazard Ratio (95% CI)

P

Hazard Ratio (95% CI)

P

Abnormal test

1.6 (0.9 to 2.7)

0.08

0.9 (0.4 to 1.8)

0.7

Ischemia by WM

2.1 (1.2 to 3.6)

0.005

1.7 (0.9 to 2.9)

0.08 0.07

Ischemic segments cut point ⬎1 segment, n

2.2 (1.3 to 3.7)

0.004

1.7 (1.0 to 2.0)

Scarred segments cut point ⬎1 segment, n

1.6 (0.9 to 2.7)

0.08

0.9 (0.4 to 1.7)

0.7

Peak WMSI cut point ⬎1.7

2.2 (1.2 to 3.8)

0.005

1.2 (0.6 to 2.2)

0.7

Mean SRs cut point ⬎⫺1.2 s⫺1

3.7 (2.0 to 6.7)

⬍0.001

1.8 (0.6 to 4.5)

0.3

Mean Ses cut point ⬎⫺3.5%

1.4 (0.6 to 3.1)

0.4

0.8 (0.3 to 1.8)

0.5

Segmental SRs cut point ⬎10 segments

3.3 (1.8 to 5.7)

⬍0.001

3.6 (1.7 to 7.2)

⬍0.001

Segmental Ses cut point ⬎3 segments

3.1 (1.4 to 6.8)

0.002

2.1 (0.8 to 4.8)

0.08

For the univariable analysis, each variable was run in a separate model. In the multivariable analysis, peak WMSI and SRI variables were run in separate models with both abnormal test and ischemia or both number of ischemic and number of scarred segments (these were covariates). The following clinical variables were included in the multivariable model: age, diabetes mellitus, and previous MI.

The incremental value of SR appears to be independent of whether WMS demonstrated scar or ischemia. In patients with abnormal WM at rest, mean peak SRs added incremental value to both rest and peak WMSI, increasing ␹2 from 4.2 to 10.0 (P⫽0.02) at rest and from 4.4 to 9.5 at peak (P⫽0.02). For the number of abnormal segments based on SRs, the increase was more modest, from 3.9 to 8.3 (P⫽0.03) at rest and from 3.4 to 8.3 at peak (P⫽0.03).

Discussion The present study demonstrates for the first time that the use of SRI provides additional information to WM analysis alone. Specifically, SRs was found to be an independent predictor of all-cause mortality in patients with known or suspected CAD, incremental to other variables.

Current Status of SRI SRI derived from high-frame-rate tissue Doppler data has been studied intensely over the last decade. Although the technique is limited by a number of technical challenges,14 it has been well validated as a means of accurate measurement of myocardial deformation15,16 and has been shown to quantify regional function in infarct populations and to demonstrate recovery of stunned myocardium.17,18 The incorporation of SRI into DSE has been most effective in measuring the low-dose response, where it has been effective in the detection of viable myocardium.19 –22 However, the use of SRI during peak-dose DSE is difficult because of signal noise, with only 1 published report suggesting incremental benefit.5 The use of both tissue Doppler and speckle techniques to track the myocardial wall and to perform an automated measurement of SRs appears to increase the sensitivity of DSE compared with expert conventional manual reading.8 Clearly, although the validation of the technique for identifying anatomic evidence of CAD is important, evidence that the findings are predictive of outcome would help to define the value of the technique. To date, however, no reports of the prognostic value of SRI have been published.

Prognostic Value of DSE

Figure 3. Incremental value of SRI variables in a series of Cox regression models predicting all-cause mortality. The clinical variables (C; diabetes mellitus, age, previous MI) were entered together, followed by separate models by combination of these with rest WMSI (rW) and peak WMSI (pW). Then, the clinical variables with peak WMSI were entered together, and each SRI variable added in separate analyses. sSes indicates segmental end-systolic strain; mSRs, mean peak systolic strain rate; and sSRs, segmental peak systolic strain rate.

The clinical markers of outcome in patients with CAD are well defined.23 Resting left ventricular function is well known to be of incremental value in predicting outcome.24,25 Although the risk of patients undergoing DSE exceeds that of those who are able to exercise, reflecting the association of events with reduced exercise capacity,26 a normal DSE carries a good prognosis, with an annualized event rate for cardiac death or infarction of 1.3% and all-cause mortality of 1.8%,25 similar to the all-cause mortality rate of 1.9% per year in the present study. Earlier studies have shown that abnormal WM analysis at both rest and stress are independently predictive of cardiac and all-cause mortality.1,2,24,27–30 Our findings support this previous experience, showing resting WM abnormality

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and ischemia at peak stress to add value to the usual predictors of outcome. However, even though conventional DSE is predictive of mortality, there are 2 important problems. First, it remains a subjective tool; its accuracy depends on the skills of the user.3 Second, although the extent and severity of abnormality at peak stress are predictive of outcome,31 WM abnormality is known to be relatively poor for distinguishing the true extent of CAD. Indeed, previous work has documented the incremental information obtained by averaging peak systolic velocity in patients with abnormal DSE.32 However, tissue velocity imaging has important limitations in the assessment of regional function, and the present study has shown that SR provides a solution not only to the problem of subjectivity but also to the need to quantify the extent of ischemic and infarcted tissue. Importantly, strain was inferior to peak SR, supporting the findings of diagnostic studies.5,8,33

Prognostic Value of Global Versus Regional Function Previous studies have shown that peak WMSI response to DSE is one of the strongest independent predictors of adverse cardiac outcomes, adding incremental prognostic information to both clinical data and resting left ventricular function.34 However, the incremental information provided by SRs to WMSI suggests that SRI provides an additional window to cardiac function. First, the use of SRI overcomes potential misinterpretations of WM analysis. For example, SRs may be less load sensitive than standard parameters such as ejection fraction or regional function. Second, SRs may provide information that WM cannot. In this respect, it is of interest that although the mean peak SRs for all segments and the use of peak SRs to categorize segments as normal and abnormal were both predictors of outcome, the segmental value of SRs was the only independent predictor of mortality. Mean SRs was a better predictor for patients with abnormal WM at rest.

Study Limitations The present prognostic study was set up in 1998, when tissue Doppler acquisitions were less sophisticated and informative than they are now. The frame rates for both tissue Doppler and B-mode images were low in some patients, and only 1 cine loop was stored. Nonetheless, only 5 patients were excluded because of bad image quality. We followed up patients for all-cause mortality in the belief that the definition of cardiac mortality is unreliable.34 Although it is possible that the predictors of cardiac mortality are different, the main cause of death in this elderly group can be expected to be cardiac, and we would expect the association of cardiac function with cardiac events to be even stronger. Coronary angiography or myocardial perfusion scintigraphy was not performed in most patients. An alternative design strategy would have been to study patients with defined coronary anatomy, thus linking the predictive capacity of WMS or SRs to anatomic evidence of CAD. However, this approach would not provide information relevant to normal patterns of patient selection.

Unfortunately, changes in medical therapy in the course of follow-up were not tracked in the present study. Although this lack of information is unfortunate, it is unlikely to have influenced the relative prognostic importance of deformation imaging and WM assessment. Finally, the current measurement of SRs is time consuming. Nonetheless, the adoption of an automated quantitative technique may overcome these limitations.

Conclusions In patients with known or suspected CAD, mortality can be predicted by the SRI response to DSE. Segmental analysis by SRs at peak appears to be the optimal approach that provides incremental value to WM analysis.

Sources of Funding The present study was supported by grants from the Norwegian University of Science and Technology, the Norwegian Research Foundation, and the National Health and Medical Research Council (project grant 210218), Canberra, Australia.

Disclosures Professor Marwick received research grant funding from General Electric Medical Systems that is unrelated to this work. The other authors report no conflicts.

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enhanced prognostic prediction using a simple risk score. J Am Coll Cardiol. 2006;47:1029 –1036. Poldermans D, Fioretti PM, Boersma E, Cornel JH, Borst F, Vermeulen EG, Arnese M, el-Hendy A, Roelandt JR. Dobutamine-atropine stress echocardiography and clinical data for predicting late cardiac events in patients with suspected coronary artery disease. Am J Med. 1994;97: 119 –125. Poldermans D, Fioretti PM, Boersma E, Bax JJ, Thomson IR, Roelandt JR, Simoons ML. Long-term prognostic value of dobutamine-atropine stress echocardiography in 1737 patients with known or suspected coronary artery disease: a single-center experience. Circulation. 1999;99: 757–762. Myers J, Prakash M, Froelicher V, Do D, Partington S, Atwood JE. Exercise capacity and mortality among men referred for exercise testing. N Engl J Med. 2002;346:793– 801. Biagini E, Elhendy A, Bax JJ, Rizzello V, Schinkel AF, van Domburg RT, Kertai MD, Krenning BJ, Bountioukos M, Rapezzi C. Seven-year follow-up after dobutamine stress echocardiography: impact of gender on prognosis. J Am Coll Cardiol. 2005;45:93–97. Elhendy A, Schinkel AF, Bax JJ, Van Domburg RT, Poldermans D. Prognostic value of dobutamine stress echocardiography in patients with normal left ventricular systolic function. J Am Soc Echocardiogr. 2004; 17:739 –743. Tsutsui JM, Elhendy A, Anderson JR, Xie F, McGrain AC, Porter TR. Prognostic value of dobutamine stress myocardial contrast perfusion echocardiography. Circulation. 2005;112:1444 –1450. Krivokapich J, Child JS, Walter DO, Garfinkel A. Prognostic value of dobutamine stress echocardiography in predicting cardiac events in patients with known or suspected coronary artery disease. J Am Coll Cardiol. 1999;33:708 –716. Hoffman R, Lethen H, Kuhl H, Lepper W, Hanrath P. Extent and severity of test positivity during dobutamine stress echocardiography. Eur Heart J. 1999;20:1485–1492. Marwick TH, Case C, Leano R, Short L, Baglin T, Cain P, Garrahy P. Use of tissue Doppler imaging to facilitate the prediction of events in patients with abnormal left ventricular function by dobutamine echocardiography. Am J Cardiol. 2004;93:142–146. Hanekom L, Moir S, Jeffries L, MacNab D, Marwick T. Selection of optimal strain rate imaging parameter for the diagnosis of coronary artery disease during dobutamine stress echocardiography: an angiographic comparison. Eur Heart J. 2005;26:210. Abstract. Lauer MS, Blackstone EH, Young JB, Topol EJ. Cause of death in clinical research: time for a reassessment? J Am Coll Cardiol. 1999;34: 618 – 620.

CLINICAL PERSPECTIVE Traditionally, echocardiography and other imaging modalities have provided the ability to measure radial motion of the myocardium on a regional or global basis. The development of strain and strain rate techniques has provided the ability not only to assess but also to quantify longitudinal motion (ie, ventricular shortening). Although these measurements have been validated against other methods and shown to be at least comparable to wall motion assessment, their incremental value has until now been unclear. In the present study of 646 patients undergoing dobutamine stress echocardiography for the evaluation of known or suspected coronary disease and followed up for all-cause mortality over 7 years, we sought to find out whether strain rate imaging was incremental to conventional wall motion scoring, which is itself a predictor of outcome. An automated method was used to measure peak systolic strain rate and end-systolic strain, and results were expressed as the number of abnormal segments and the mean systolic strain rate and end-systolic strain per patient. Segmental systolic strain rate was independently predictive of mortality, and this information was incremental to clinical, stress, and wall motion score data. The findings confirm that the evaluation of longitudinal myocardial motion using strain rate imaging is feasible and that it adds incremental information to the conventional assessment of radial function.

Molecular Cardiology Moderate Pulmonary Arterial Hypertension in Male Mice Lacking the Vasoactive Intestinal Peptide Gene Sami I. Said, MD; Sayyed A. Hamidi, MD; Kathleen G. Dickman, PhD; Anthony M. Szema, MD; Sergey Lyubsky, MD; Richard Z. Lin, MD; Ya-Ping Jiang, MD; John J. Chen, PhD; James A. Waschek, PhD; Smadar Kort, MD Background—Vasoactive intestinal peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, has been reported absent in pulmonary arteries from patients with idiopathic pulmonary arterial hypertension (PAH). We have tested the hypothesis that targeted deletion of the VIP gene may lead to PAH with pulmonary vascular remodeling. Methods and Results—We examined VIP knockout (VIP⫺/⫺) mice for evidence of PAH, right ventricular (RV) hypertrophy, and pulmonary vascular remodeling. Relative to wild-type control mice, VIP⫺/⫺ mice showed moderate RV hypertension, RV hypertrophy confirmed by increased ratio of RV to left ventricle plus septum weight, and enlarged, thickened pulmonary artery and smaller branches with increased muscularization and narrowed lumen. Lung sections also showed perivascular inflammatory cell infiltrates. No systemic hypertension and no arterial hypoxemia existed to explain the PAH. The condition was associated with increased mortality. Both the vascular remodeling and RV remodeling were attenuated after a 4-week treatment with VIP. Conclusions—Deletion of the VIP gene leads to spontaneous expression of moderately severe PAH in mice during air breathing. Although not an exact model of idiopathic PAH, the VIP⫺/⫺ mouse should be useful for studying molecular mechanisms of PAH and evaluating potential therapeutic agents. VIP replacement therapy holds promise for the treatment of PAH, and mutations of the VIP gene may be a factor in the pathogenesis of idiopathic PAH. (Circulation. 2007;115:1260-1268.) Key Words: cardiovascular diseases 䡲 genetics 䡲 hypertension, pulmonary 䡲 pathology 䡲 peptides 䡲 remodeling 䡲 vasculature

I

mammalian species in vitro9,10; neutralizes or attenuates the actions of endothelin and other vasoconstrictors11–13; reduces hypoxic pulmonary vasoconstriction in cats,14 newborn lambs, 15 Fawn-Hooded rats, 16 and rabbits with monocrotaline-induced pulmonary hypertension17; and inhibits the proliferation of pulmonary vascular smooth muscle from patients with IPAH.18 Furthermore, VIP is a cotransmitter of the physiological nonadrenergic, noncholinergic system of pulmonary vascular smooth muscle relaxation.19,20 Finally, VIP-containing nerves, normally plentiful in the pulmonary artery,21 were recently reported absent in pulmonary arteries from IPAH patients,18 and inhalation of the peptide had a beneficial therapeutic effect on those patients.18 Here we report that mice with targeted deletion of the VIP gene (VIP⫺/⫺) show hemodynamic, echocardiographic, anatomic, and histological changes in pulmonary hypertension that are not attributable to arterial hypoxemia or any significant cardiopulmonary disease.

diopathic (primary) pulmonary arterial hypertension (IPAH) is a relatively rare but highly fatal disease characterized by progressive PAH and increased thickening of smaller pulmonary arteries and arterioles, culminating in right ventricular (RV) failure.1–3 Considerable advances have been made in recent years in our knowledge of the pathophysiology, pathology, and genetic basis of the disease, and its treatment is now more successful.4 – 8 Much remains to be learned, however, about the pathogenetic mechanisms of the disease, particularly the interactions among multiple predisposing genes, and the influence of selected environmental factors.

Clinical Perspective p 1268 A variety of observations over the years have linked the neuropeptide vasoactive intestinal peptide (VIP) to the pulmonary and systemic circulation. With special reference to the pulmonary vascular bed and its alterations in IPAH, VIP relaxes pulmonary vascular smooth muscle from several

Received December 1, 2006; accepted January 2, 2007. From the Departments of Medicine (S.I.S., S.A.H., K.G.D., A.M.S., R.Z.L., Y.-P.J., S.K.), Pathology (S.L.), and Preventive Medicine (J.J.C.), State University of New York at Stony Brook; Department of Veterans Affairs Medical Center, Northport, NY (S.I.S., S.A.H., K.G.D., A.M.S., S.L., R.Z.L.); and Department of Psychiatry, University of California, Los Angeles, Los Angeles (J.A.W.). Correspondence to Sami I. Said, Pulmonary and Critical Care Medicine, SUNY Health Sciences Center, T–17– 040, Stony Brook, NY 11784. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.681718

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Methods Animals VIP⫺/⫺ mice, backcrossed to C57BL/6 mice, were prepared locally as described and genotyped to confirm the absence of the VIP gene.22,23 We mated homozygous (VIP⫺/⫺) males with homozygous (VIP⫺/⫺) females or, if necessary, with heterozygous (VIP⫹/⫺) females. For genotyping, we extracted DNA from 1-cm-long tail snips using a DNA isolation kit (Qiagen Inc, Valencia, Calif). DNA (100 ng) was subjected to polymerase chain reaction using primers to detect both VIP and the neomycin cassette. Control, wild-type (WT) C57BL/6 mice were from Taconic Labs (Germantown, NY). We examined animals ranging in age from 9 to 52 weeks. The entire study was approved by the institutional animal review committees.

Chemicals and Reagents VIP was from the Karolinska Institute (Stockholm, Sweden). All other chemicals, unless otherwise noted, were from Sigma Chemical Co (St Louis, Mo).

Hemodynamic Measurements Five VIP⫺/⫺ mice and 5 WT mice were anesthetized with ketamine (100 mg/kg) and fentanyl (0.05 mg/kg IP). A 1.4F 3-cm Mikro-Tip catheter (Millar Instruments Inc, Houston, Tex) was inserted through the right jugular vein and advanced to the right ventricle for digital recording of RV pressure. We also monitored left ventricular pressure in both groups of mice by direct catheterization via the carotid artery.

Echocardiographic Examination Five VIP⫺/⫺ and 5 WT mice were lightly anesthetized with pentobarbital (100 mg/kg IP). Echocardiographic examination was performed with a Vivid 7 (GE Medical Systems, Milwaukee, Wis) for mice equipped with a miniaturized high-frequency 13-MHz transducer. Evaluations were made offline, following the recommendations of the American Society of Echocardiography.24 RV and left ventricular size and function and pulmonary artery size were assessed in all animals.

Anatomic Assessment of RV Hypertrophy The heart was isolated and placed under a dissecting microscope. Attached vessels and both atria were dissected and removed. The RV wall was cut out, blotted, and weighed; then the left ventricular wall and septum (LV⫹septum) were treated the same way and weighed. The RV/(LV⫹septum) ratio was calculated in 6 male VIP⫺/⫺ mice and 5 male WT mice as an index of RV hypertrophy. To evaluate the differences in vascular pathology between male and female mice, we also assessed RV mass as measured by the mean RV/(LV⫹septum) weight ratio in 6 female VIP⫺/⫺ mice and 6 female WT mice.

Arterial Blood Gas Analysis To explore the possibility that the PAH in VIP⫺/⫺ mice was secondary to arterial hypoxemia, we measured arterial blood PO2 in samples collected directly from the carotid artery of 7 VIP⫺/⫺ mice. We confirmed these measurements by determining hemoglobin O2 saturation25 by a special mouse oximeter (Starr Life Sciences, Allison Park, Pa) applied to the shaved thigh.

Histological Examination and Morphometric Analysis For all histological procedures, the lungs were inflated to full capacity and fixed by intratracheal instillation of 1 mL 10% neutral buffered formalin, immersed in formalin overnight, and then embedded in paraffin. Sections (4 ␮m thick) were stained with hematoxylin and eosin or Masson’s trichrome stain for general morphology and morphometric analysis. Pulmonary arteries from 6 WT and 6 VIP⫺/⫺ mice were analyzed; measurements were taken of 4 separate vessels from each mouse and averaged to 1 set of values. Only arteries near smaller bronchi or terminal bronchioles, ⬇50 ␮m in diameter, were

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selected for analysis. We used the Image J program, version 1.34r (http://rsb.info.nih.gov/ij/), for measurement of total vessel area (␮m2), luminal area (␮m2), and inner circumference (␮m). Medial area (␮m2) was calculated as the difference between total and luminal areas. Standard medial thickness (␮m) was calculated as the ratio of medial area to inner circumference as described by Weibel.26 Average vessel diameter (␮m) was derived from total area measurements. For immunohistochemical detection of ␣-smooth muscle actin, paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 5 minutes. Immunostaining was performed with a mouse monoclonal antibody directed against ␣-smooth muscle actin (Sigma) in conjunction with an avidin/biotin-based kit designed to detect mouse primary antibodies in mouse tissue (Mouse-on-Mouse Peroxidase Kit, Vector Labs, Burlingame, Calif) used according to the manufacturer’s instructions. The primary antibody was at a final dilution of 1:1000. Color was developed by 3-minute incubation with diaminobenzidine (DAB Peroxidase Substrate Kit, Vector Labs), after which sections were washed, counterstained for 30 seconds with Hematoxylin QS (Vector Labs), dehydrated, and then mounted.

Progression of Vascular Pathology and Survival Rates Possible progression of the pathological lesions was evaluated by assessing the degree of RV hypertrophy in 9 male VIP⫺/⫺ mice 5 to 18 weeks of age, 9 mice 30 to 38 weeks of age, and 13 mice 51 to 143 weeks of age. Mortality rates were compared in 38 male VIP⫺/⫺ mice and 15 WT controls.

VIP Replacement Therapy Nine male VIP⫺/⫺ mice 4 to 12 weeks of age received VIP (15 ␮g IP in 0.2 mL phosphate-buffered saline) every other day for 4 weeks for a total of 14 injections, ending the day before examination. Another group of 9 male VIP⫺/⫺ mice of a similar age received 0.2 mL phosphate-buffered saline, without VIP, in the same manner and for the same duration. Our choice of the dosage, duration, frequency, and mode of administration of VIP was guided by protocols for related studies by other investigators.27 At the end of this treatment period, we evaluated the degree of RV thickening and vascular remodeling in smaller pulmonary arteries from the 2 groups of mice.

Gene Microarray Analysis RNA was isolated from lung samples from male VIP⫺/⫺ and WT mice and subjected to Affymetrix gene profiling (Expression Analysis, Durham, NC). The objective was to search for significant differences between the 2 groups in the expression of genes relevant to the pulmonary circulation. Genes of compounds that influence vasomotor tone, vascular smooth muscle proliferation, and collagen deposition were in special focus.

Statistical Analysis All summary data for continuous variables were expressed as mean⫾SEM. For continuous variables, 2-group comparisons were performed with both the parametric 2-sample t test and nonparametric Mann-Whitney test. When the statistical results were similar between the 2 approaches, probability values from parametric t tests were reported. When the results were different from each another, probability values from both parametric and nonparametric tests were reported. For mortality data, Kaplan-Meier curves were generated and compared through the use of the log-rank test. All analyses were performed with SPSS software (Stata Corp, Inc, College Station, Tex), and a 2-sided value of P⬍0.05 was regarded as statistically significant. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

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TABLE 1. Hemodynamics in Male VIP Knockout and Wild-Type Mice, RV Systolic Pressure Mouse Knockout 1 2 3 4 5 Mean SEM Wild-type 1 2 3 4 5 Mean SEM

Age, wk

RVSP, mm Hg

48 48 40 40 40

29.6 29.4 29.2 33.0 26.0 29.5* 1.1

32 32 44 44 44

18.4 22.4 16.0 15.4 9.1 16.3* 2.2

RVSP indicates right ventricular systolic pressure. * P⬍0.001.

Results Hemodynamic Evidence of RV Hypertension in VIP Knockout Mice

Mean RV pressure in VIP⫺/⫺ mice (n⫽5) was significantly elevated relative to that in WT mice (n⫽5) (29.5⫾1.1 versus 16.3⫾2.2 mm Hg; P⬍0.001). Systolic left ventricular pressure, however, was normal (96.1⫾6.0 mm Hg; n⫽7) and was not significantly different from that in WT mice (96.2⫾4.5 mm Hg; n⫽6; P⫽0.76; Tables 1 and 2).

Echocardiographic Confirmation of Pulmonary Arterial Thickening and RV Dilatation Echocardiographic analysis showed the wall of the main pulmonary artery in VIP⫺/⫺ mice (n⫽5) to be 0.24⫾0.02 mm

thick versus 0.16⫾0.02 mm in WT mice (n⫽5; P⫽0.013) and the pulmonary artery diameter to be wider in VIP⫺/⫺ mice (1.57⫾0.02 versus 1.24⫾0.14 mm; P⫽0.046 for 2-sample t test, P⫽0.16 for Mann-Whitney test). The area of the RV, a correlate of RV size, was greater in VIP⫺/⫺ mice than in WT mice: 8.47⫾1.08 mm2 during diastole and 5.99⫾1.01 mm2 during systole compared with 4.07⫾0.85 and 2.58⫾0.67 mm2, respectively, in WT mice (P⫽0.013 and P⫽0.022, respectively).

Anatomic Confirmation of RV Hypertrophy The RV/(LV⫹septum) weight ratio, used here as a measure of RV hypertrophy, was 0.34⫾0.01 in male VIP⫺/⫺ mice (n⫽6), significantly higher than in male control WT mice (0.21⫾0.01; n⫽5; P⬍0.001). The same weight ratio in 6 female VIP⫺/⫺ mice was 0.24⫾0.01, not significantly different from the corresponding value in 6 female WT mice (0.26⫾0.02), suggesting no significant RV hypertrophy in female VIP⫺/⫺ mice.

Histological and Morphometric Evidence of Thickened, Remodeled Pulmonary Arteries Comparing pulmonary arteries of similar diameter (45 to 50 ␮m), the medial wall was significantly thicker and the lumen was significantly narrower in VIP⫺/⫺ mice (n⫽6) than in control WT mice (n⫽6; Figure 1) (medial thickness, 14.45⫾2.86 versus 5.88⫾0.53 ␮m, P⫽0.030; ratio of medial area to total area, 0.68⫾0.04 versus 0.43⫾0.04, P⬍0.001). The most striking abnormality was a marked increase in the ratio of medial area to luminal area, which averaged 2.78⫾0.45 versus 0.83⫾0.13 (P⫽0.006). Numerous vessels were so severely narrowed they appeared almost totally occluded. In addition to the hematoxylin and eosin stain, which formed the primary basis for morphometric analysis (Figure 2), Masson’s trichrome stain demonstrated pronounced proliferation of medial smooth muscle and collagen (Figure 3), which was corroborated by ␣-smooth muscle actin immuno-

Figure 1. In small pulmonary arteries of comparable diameter (45 to 50 ␮m), media from male VIP⫺/⫺ mice were considerably thicker than media from WT control mice.

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Figure 3. Masson’s trichrome staining showing marked medial thickening of smaller pulmonary arteries from VIP⫺/⫺ mouse (B) vs WT mouse (A). Figure 2. Hematoxylin and eosin–stained sections of lungs from a male 6.6-month-old control mouse (A) and a male 7.3-monthold VIP⫺/⫺ mouse (B). Media of vessels marked by arrows are 5 and 17 ␮m wide, respectively.

staining (Figure 4). Little or no endothelial cell proliferation was observed.

Perivascular Inflammation Clusters of inflammatory cell, predominantly mononuclear, infiltrates were observed around smaller pulmonary vessels and airways (Figure 5).

No Hypoxemia to Explain the Pulmonary Hypertension Mean arterial oxygen tension (PaO2), measured in samples collected from the carotid artery from 7 VIP⫺/⫺ mice during air breathing, was 93.9⫾9.8 mm Hg compared with 93.1⫾9.1 mm Hg for WT mice (n⫽7; P⫽0.96). Confirming this normal finding, hemoglobin O2 saturation in VIP⫺/⫺ mice measured by direct oximetry was 96%.

No Systemic Vascular Pathology The renal arteries, examined as representative of systemic vascular beds, showed no evidence of vascular thickening such as that observed in the pulmonary arteries.

VIP Treatment Reduces RV Hypertrophy and Pulmonary Vascular Remodeling Nine male VIP⫺/⫺ mice that had been treated with VIP for 4 weeks showed considerably less RV hypertrophy than 9 control mice that had merely received buffer. The RV/ (LV⫹septum) ratio was 0.25⫾0.01 in the VIP-treated group (n⫽9), significantly lower than that in the buffer-treated group (0.34⫾0.01; n⫽9; P⬍0.001; Table 3) but not as low as in WT mice (0.21⫾0.01; n⫽5; P⫽0.002). In the same 2 groups of mice, the walls of smaller pulmonary arteries were proportionately less thickened in the VIP-treated than in the buffer-treated controls. Thus, the mean ratio of medial area to total area in smaller vessels from the VIP⫺/⫺ mice (n⫽9) was 0.59⫾0.06 compared with 0.74⫾0.03 in the buffer-treated mice (n⫽9; P⫽0.045 for 2-sample t test and P⫽0.065 for Mann-Whitney test; Table 3).

Progression of Pathological Lesions and Decreased Survival in Knockout Mice Despite the generally moderate severity of PAH and the lack of intimal proliferation, the pulmonary vascular pathology in the VIP⫺/⫺ mice showed evidence of being progressive; mice ⬎30 weeks of age had an RV/(LV⫹septum) ratio of

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Figure 4. ␣-Smooth muscle actin immunostaining of pulmonary arteries of comparable diameter from 2 male mice: WT (A) and VIP⫺/⫺ (B).

0.34⫾0.01 compared with 0.38⫾0.01 in mice ⬍30 weeks of age (P⫽0.047 based on 2-sample t test; P⫽0.026 based on nonparametric Mann-Whitney test). In addition, the VIP⫺/⫺ mice had a higher mortality rate relative to WT controls (P⬍0. 001 for log-rank test; Figure 6).

Gene Microarray Analysis Lungs from VIP⫺/⫺ mice showed significant alterations in the expression of several genes pertinent to pulmonary vascular tone and vascular remodeling. Genes for platelet-derived growth factor receptor ␤ polypeptide and platelet-derived growth factor-␤ polypeptide, procollagen type I, ␣1, endothelin receptor A, and angiopoietin 2 were upregulated by 1.8-, 1.3-, 1.3-, 1.4-, and 1.6-fold, respectively. On the other hand, the gene for adrenomedullin, a pulmonary vasodilator and antiproliferative peptide, was downregulated by 50%.

Discussion

Our results demonstrate that male VIP⫺/⫺ mice exhibit moderately severe PAH, with remodeled, muscularized pulmonary arterioles and smaller arteries, RV hypertension, and RV hypertrophy. Despite the presence of peribronchial cellular infiltrates and airway hyperresponsiveness in VIP⫺/⫺ mice, as

Figure 5. Hematoxylin and eosin–stained sections of lungs from a male WT control mouse (A) and a male VIP⫺/⫺ mouse (B) showing mononuclear inflammatory cell infiltrates around thickened vessels.

recently reported,23 no arterial hypoxemia or other signs of significant pulmonary or cardiac disease was present. The pulmonary vascular alterations in this experimental model closely resemble those in patients with moderately severe IPAH.2 Morphometric analysis showed that, for pulmonary arteries of comparable external diameter, vessels from VIP⫺/⫺ mice typically had markedly thickened medial layer and narrowed lumen, with a mean medial area/luminal area ratio 3.35 times that in WT mice. Complementary immunohistochemical studies revealed the medial thickening to result from accumulation of multiple layers of smooth muscle and collagen. The degree of medial thickening varied somewhat within the VIP⫺/⫺ mice, reflecting some degree of phenotypic heterogeneity. Endothelial cell proliferation, typically seen in advanced forms of the disease,28 was not observed in these mice, possibly reflecting the moderate severity of the pathological process or its relatively short duration. Alternatively, deletion of the VIP gene may result in partial expression of the pathological features of the human disease, other genetic defects being required for the missing features such as endothelial cell proliferation and the fuller expression of the disease process.

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Pulmonary Arterial Hypertension in VIP KO Mice

TABLE 2. Hemodynamics in Male VIP Knockout and Wild-Type Mice, LV Systolic Pressure Mouse

Age, wk

LVSP, mm Hg

Knockout 1

16

77.3

2

16

101.8

3

16

115.4

4

16

73.2

5

17

93.0

6

17

110.0

7

17

102.0

Mean

96.1*

SEM

6.0

Wild-type 1

16

92.1

2

13

107.0

3

13

78.6

4

13

107.0

5

15

92.3

6

15

100.3

Mean

96.2*

SEM

4.5

LVSP indicates left ventricular systolic pressure. *P⫽0.76.

Our observations were largely limited to male mice, both VIP⫺/⫺ and WT. Preliminary comparisons of lung sections from male and female mice confirmed the impression that medial thickening was less pronounced in female VIP⫺/⫺ mice than in their male counterparts. Similar gender differences have been reported in other models of PAH. In a study of mice with deletion of the endothelial nitric oxide synthase gene, structural evidence of pulmonary hypertension was

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evident in male but not in female adult mice.29 Further investigation of gender differences in the expression of the PAH phenotype in VIP⫺/⫺ mice and of the possible role of estrogen in such differences30,31 is clearly in order. A predominant expression of the pulmonary vascular abnormalities in male VIP⫺/⫺ mice, if confirmed, would be clearly different from human IPAH, which is more prevalent in women. In addition to the pulmonary vascular abnormalities in male VIP⫺/⫺ mice described here, lungs of the same mice showed perivascular and peribronchiolar inflammatory cell infiltrates (Figure 5). Coexistence of the 2 sets of findings is unlikely to be a mere coincidence; at least 2 groups of investigators have focused on the importance of inflammation as a factor in the pathogenesis of PAH.32,33 The VIP-related pituitary adenylate cyclase–activating peptide has VIP-like actions on the pulmonary circulation, such as vasodilation and inhibition of vascular remodeling.34 Mutant mice lacking the principal receptor for pituitary adenylate cyclase–activating peptide, the PAC1 receptor, present with severe pulmonary hypertension and RV failure, causing their death within the first postnatal weeks.35 Thus, deletion of either VIP or pituitary adenylate cyclase–activating peptide (or its main receptor) results in an experimental model of PAH. Although the 2 peptides are closely related both structurally and functionally and their actions are mediated by common receptors36 (of which PAC1 binds with pituitary adenylate cyclase–activating peptide with considerably greater affinity than with VIP), it is clear that neither peptide compensates for the absence of the other. It appears likely therefore that both peptides and their signaling pathways are probably required for the maintenance of normal hemodynamics of the pulmonary circulation. The mechanisms and pathways by which the absence of the VIP gene leads to expression of pathophysiological features of PAH are under investigation. The sequence of events probably begins with pulmonary vasoconstriction, which

Figure 6. Kaplan-Meier cumulative survival plot of 38 VIP-deficient male mice and 15 WT male mice showing considerably higher mortality rate in VIP⫺/⫺ mice (P⬍0.001).

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TABLE 3. Attenuation of RV Hypertrophy and Pulmonary Vascular Remodeling in Male VIP Knockout Mice by VIP Weight, g

Age, wk

RV Weight, mg

LV⫹Septum Weight, mg

RV/ (LV⫹Septum)

Pulmonary Vascular Thickness Medial Area/Total Area

1

24.8

8

22.6

83.7

0.27

0.68

2

20.8

8

18.3

67.4

0.27

0.47

3

19.9

10

22.6

84.7

0.27

0.43

4

21.2

10

18.9

86.2

0.22

0.35

5

22.5

13

18.2

74.9

0.24

0.50

6

24.0

13

20.6

82.9

0.25

0.50

7

23.6

13

21.7

83.9

0.26

0.89

8

22.2

13

17.8

76.0

0.23

0.73

9

23.8

16

19.8

83.0

0.24

0.74

Mean

22.5

11.6

20.1

80.3

0.25*

0.59†

SEM

0.6

0.9

0.6

2.1

0.01

0.06

Mouse VIP treated

Buffer treated 10

23.1

16

29.3

76.2

0.38

0.81

11

21.6

6

24.9

71.7

0.35

0.67

12

22.2

6

27.5

80.6

0.34

0.63

13

24.0

13

28.2

80.0

0.35

0.86

14

24.3

13

22.2

75.0

0.30

0.59

15

25.5

16

23.8

80.7

0.29

0.76

16

24.9

13

27.2

78.7

0.35

0.83

17

23.5

14

25.7

75.0

0.34

0.76

18

24.5

14

32.0

87.8

0.36

0.73

Mean

23.7

12.3

26.8

78.4

0.34*

0.74†

SEM

0.4

1.3

1.0

1.6

0.01

0.03

*P⬍0.001; †P⫽0.045 (t test), ‡P⫽0.065 (Mann-Whitney test).

causes increased pulmonary vascular resistance, leading to pulmonary and RV hypertension. Vascular remodeling would follow as an adaptive response to give the pulmonary vessels sufficient support to withstand the elevated pressure,37 and the process would culminate in RV hypertrophy and failure. Strong evidence has linked the pathogenesis of IPAH to mutations in the bone morphogenetic protein receptor-2 gene.4,38 Dysfunctional Smad signaling of transforming growth factor-␤39 probably accounts, at least in part, for the excessive smooth muscle cell proliferation in IPAH.40 Interactions between VIP and transforming growth factor-␤ have already been reported. Thus, transforming growth factor-␤, together with ciliary neurotrophic factor, synergistically induces VIP gene expression through the cooperation of Smad and other pathways.41 A variety of additional mechanisms may contribute to the smooth muscle cell proliferation and migration, a prominent feature in our experimental model. Preliminary data from gene microarray analysis suggest the involvement of other pathways with an established role in the pathogenesis of that disease.42 These include upregulation of endothelin and platelet-derived growth factor signaling,43 the angiopoietin/Tie 2 pathway,44 and collagen deposition. On the other hand, the gene for adrenomedullin, a pulmonary vasodilator and antiproliferative peptide, was significantly downregulated. VIP also has been demonstrated to induce the

biosynthesis of tetrahydrobiopterin,45 a critical cofactor in endothelial nitric oxide synthase function.46 Thus, the lack of the VIP gene may be expected to lead to decreased endothelial nitric oxide production. Because bone morphogenetic protein receptor-2 heterozygous mice show only mild pulmonary hypertension,47 many believe that “multiple genetic hits” are needed for the full expression of the disease.48,49 Our results suggest that a single hit may suffice for the expression of at least 1 experimental model of the disease; deficiency of the VIP gene alone resulted in the expression of a moderate PAH phenotype, although other gene alterations, secondary to the loss of the VIP gene as outlined above, may have contributed. Our findings also suggest the need for investigating the possibility that mutations in the VIP gene may be a factor in the pathogenesis of IPAH in humans. Unlike most other models of PAH, the VIP⫺/⫺ mouse expresses spontaneous PAH, including remodeled pulmonary vessels and RV hypertrophy, during normoxic breathing. In most other models, including those based on deletion of the endothelial nitric oxide synthase gene, vascular endothelial growth factor receptor-2 blockade, and platelet-derived growth factor pathway activation, frank expression of PAH and vascular remodeling requires the additional stimulus of hypoxia.50 Finally, the marked and highly significant attenuation of RV hypertrophy and of medial thickening after treatment

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with VIP suggests that the peptide may be capable of preventing or at least slowing the progression of the key pathological changes in PAH, thus reinforcing its potential therapeutic value in patients with IPAH.18

Acknowledgments We thank Maria Rienzi for help with the preparation of the manuscript and Tarek Abdel-Razek and Mathew Chin for assistance with the research.

Sources of Funding This work was supported by NIH grants HL–70212, HL– 68188 (to Dr Said), K08 HL071263 (to Dr Szema), and DK62722 (to Dr Lin), by an AHA grant (to Dr Lin), and by VA research funds.

Disclosures Dr Said is a consultant or on the advisory board at MondoBiotech. The other authors report no conflicts.

References 1. Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med. 2004;351:1655–1665. 2. Newman JH, Fanburg BL, Archer SL, Badesch DB, Barst RJ, Garcia JG, Kao PN, Knowles JA, Loyd JE, McGoon MD, Morse JH, Nichols WC, Rabinovitch MD. Rodman M, Stevens T, Tuder RM, Voelkel NF, Gail DB. Pulmonary arterial hypertension: future directions: report of a National Heart, Lung and Blood Institute/Office of Rare Diseases workshop. Circulation. 2004;109:2947–2952. 3. Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet. 2003;361: 1533–1544. 4. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, Morrell NW. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation. 2002;105:1672–1678. 5. Lee SH, Rubin LJ. Current treatment strategies for pulmonary arterial hypertension. J Intern Med. 2005;258:199 –215. 6. Machado RD, Pauciulo MW, Thomson JR, Lane KB, Morgan NV, Wheeler L, Phillips JA 3rd, Newman J, Williams D, Galie N, Manes A, McNeil K, Yacoub M, Mikhail G, Rogers P, Corris P, Humbert M, Donnai D, Martensson G, Tranebjaerg L, Loyd JE, Trembath RC, Nichols WC. BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension. Am J Hum Genet. 2001;68:92–102. 7. Newman JH, Wheeler L, Lane KB, Loyd E, Gaddipati R, Phillips JA 3rd, Loyd JE. Mutation in the gene for bone morphogenetic protein receptor II as a cause of primary pulmonary hypertension in a large kindred. N Engl J Med. 2001;345:319 –324. 8. Rubin LJ. Therapy of pulmonary hypertension: the evolution from vasodilators to antiproliferative agents. Am J Respir Crit Care Med. 2002; 166:1308 –1309. 9. Hamasaki Y, Mojarad M, Said SI. Relaxant action of VIP on cat pulmonary artery: comparison with acetylcholine, isoproterenol and PGE1. J Appl Physiol Respir Environ Exerc Physiol. 1983;54:1607–1611. 10. Saga T, Said SI. Vasoactive intestinal peptide relaxes isolated strips of human bronchus, pulmonary artery, and lung parenchyma. Trans Assoc Am Physicians. 1984;97:304 –310. 11. Boomsma JD, Foda HD, Said SI. Vasoactive intestinal peptide (VIP) reverses endothelin-induced contractions of guinea pig trachea and pulmonary artery. Biomed Res. 1991;12:273–277. 12. Hamasaki Y, Saga T, Mojarad M, Said SI. Vasoactive intestinal peptide counteracts leukotriene D4-induced contractions of guinea pig trachea, lung, and pulmonary artery. Trans Assoc Am Physicians. 1983;96: 406 – 411. 13. Said SI, Raza S, Berisha HI. Enhancement of systemic and pulmonary vasoconstriction by beta-amyloid peptides and its suppression by vasoactive intestinal peptide. Ann N Y Acad Sci. 1998;865:582–585. 14. Nandiwada PA, Kadowitz PJ, Said SI, Mojarad M, Hyman AL. Pulmonary vasodilator responses to vasoactive intestinal peptide in the cat. J Appl Physiol. 1985;58:1723–1728. 15. Toubas PL, Sekar KC, Sheldon RE, Pahlavan N, Said SI. Vasoactive intestinal peptide prevents increase in pulmonary artery pressure during hypoxia in newborn lambs. Ann N Y Acad Sci. 1988;527:686 – 687.

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16. Hamidi SA, Dickmann KG, Mathew S, Said SI. Pulmonary hypertension in Fawn-hooded rats: rapid induction with alveolar hypoxia, correlation with upregulation of endothelin receptors, and attenuation by vasoactive intestinal peptide. Proc Am Thorac Soc. 2005;2:A708. Abstract. 17. Gunaydin S, Imai Y, Takanashi Y, Seo K, Hagino I, Chang D, Shinoka T. The effects of vasoactive intestinal peptide on monocrotaline induced pulmonary hypertensive rabbits following cardiopulmonary bypass: a comparative study with isoproterenol and nitroglycerine. Cardiovasc Surg. 2002;10:138 –145. 18. Petkov V, Mosgoeller W, Ziesche R, Raderer M, Stiebellehner L, Vonbank K, Funk GC, Hamilton G, Novotny C, Burian B, Block LH. Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. J Clin Invest. 2003;111:1339 –1346. 19. Kubota E, Sata T, Soas AH, Paul S, Said SI. Vasoactive intestinal peptide as a possible transmitter of nonadrenergic, noncholinergic relaxation of pulmonary artery. Trans Assoc Am Physicians. 1985;98:233–242. 20. Said SI, Rattan S. The multiple mediators of neurogenic smooth muscle relaxation. Trends Endocrinol Metab. 2004;15:189 –191. 21. Dey RD, Shimosegava T, Said SI. Lung peptides and the pulmonary circulation. In: Said SI, ed. The Pulmonary Circulation and Acute Lung Injury. 2nd ed. Mount Kisco, NY: Futura Publishing Co; 1991;137–175. 22. Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Liu X, Waschek JA. Disrupted circadian rhythms in VIP- and PHI-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2003;285:R939 –R949. 23. Szema AM, Hamidi SA, Lyubsky S, Dickman KG, Mathew S, Abdel-Razek TT, Chen JJ, Waschek JA, Said SI. Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP. Am J Physiol Lung Cell Mol Physiol. 2006;291: 880 – 886. 24. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT, Sutton MS, Stewart WJ. Recommendations for chamber quantification: a report from the American Society of Echocardiography’s Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr. 2005;18:1440 –1463. 25. Grey LH, Steadman JM. Determination of the oxyhaemoglobin dissociation curves for mouse and rat blood. J Physiol. 1964;175:161–171. 26. Weibel ER. Principles and methods for the morphometric study of the lung and other organs. Lab Invest. 1963;12:131–155. 27. Delgado M, Abad C, Martinez C, Leceta J, Gomariz RP. Vasoactive intestinal peptide prevents experimental arthritis by downregulating both autoimmune and inflammatory components of the disease. Nat Med. 2001;7:563–568. 28. Loyd JE, Atkinson JB, Pietra GG, Virmani R, Newman JH. Heterogeneity of pathologic lesions in familial primary pulmonary hypertension. Am Rev Respir Dis. 1988;138:952–957. 29. Miller AA, Hislop AA, Vallance PJ, Haworth SG. Deletion of the eNOS gene has a greater impact on the pulmonary circulation of male than female mice. Am J Physiol Lung Cell Mol Physiol. 2005;289:L299 –L306. 30. Ahn BH, Park HK, Cho HG, Lee HA, Lee YM, Yang EK, Lee WJ. Estrogen and enalapril attenuate the development of right ventricular hypertrophy induced by monocrotaline in ovariectomized rats. J Korean Med Sci. 2003;18:641– 648. 31. Earley S, Resta TC. Estradiol attenuates hypoxia-induced pulmonary endothelin-1 gene expression. Am J Physiol Lung Cell Mol Physiol. 2006;283:86 –93. 32. Dorfmuller P, Perros F, Balabanian K, Humbert M. Inflammation in pulmonary arterial hypertension. Eur Respir J. 2003;22:358 –363. 33. Nicolls MR, Taraseviciene-Stewart L, Rai PR, Badesch DB, Voelkel NF. Autoimmunity and pulmonary hypertension: a perspective. Eur Respir J. 2005;26:1110 –1118. 34. Minkes RK, McMahon TJ, Higuera TR, Murphy WA, Coy DH, Kadowitz PJ. Analysis of systemic and pulmonary vascular responses to PACAP and VIP: role of adrenal catecholamines. Am J Physiol. 1992;263: H1659 –H1669. 35. Otto C, Hein L, Brede M, Jahns R, Engelhardt S, Grone HJ, Schutz G. Pulmonary hypertension and right heart failure in pituitary adenylate cyclase–activating polypeptide type I receptor– deficient mice. Circulation. 2004;110:3245–3251. 36. Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, Waschek JA. International Union of Pharmacology, XVIII: nomenclature of

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39. 40.

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receptors for vasoactive intestinal peptide and pituitary adenylate cyclaseactivating polypeptide. Pharmacol Rev. 1998;50:265–270. Stenmark KR, McMurtry IF. Vascular remodeling versus vasoconstriction in chronic hypoxic pulmonary hypertension: a time for reappraisal? Circ Res. 2005;97:95–98. Newman JH, Trembath RC, Morse JA, Grunig E, Loyd JE, Adnot S, Coccolo F, Ventura C, Phillips JA 3rd, Knowles JA, Janssen B, Eickelberg O, Eddahibi S, Herve P, Nichols WC, Elliott G. Genetic basis of pulmonary arterial hypertension: current understanding and future directions. J Am Coll Cardiol. 2004;43:33S–39S. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003;113:685–700. Yang X, Long L, Southwood M, Rudarakanchana N, Upton PD, Jeffery TK, Atkinson C, Chen H, Trembath RC, Morrell NW. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res. 2005;96: 1053–1063. Pitts RL, Wang S, Jones EA, Symes AJ. Transforming growth factor-beta and ciliary neurotrophic factor synergistically induce vasoactive intestinal peptide gene expression through the cooperation of Smad, STAT, and AP-1 sites. J Biol Chem. 2001;276:19966 –19973. Dickman KG, Hamidi SA, Szema AM, Said SI. Microarray analysis of pulmonary gene expression in mice lacking the vasoactive intestinal peptide (VIP) gene: confirmation of a role in asthma and pulmonary arterial hypertension (PAH). Eur Respir J. 2006;28(suppl):756S. Abstract.

43. Barst RJ. PDGF signaling in pulmonary arterial hypertension. J Clin Invest. 2005;115:2691–2694. 44. Dewachter L, Adnot S, Fadel E, Humbert M, Maitre B, Barlier-Mur AM, Simonneau G, Hamon M, Naeije R, Eddahibi S. Angiopoietin/Tie2 pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension. Am J Respir Crit Care Med. 2006;174:1025–1033. 45. Anastasiadis PZ, Bezin L, Gordon LJ, Imerman B, Blitz J, Kuhn DM, Levine RA. Vasoactive intestinal peptide induces both tyrosine hydroxylase activity and tetrahydrobiopterin biosynthesis in PC12 cells. Neuroscience. 1998;86:179 –189. 46. Khoo JP, Zhao L, Alp NJ, Bendall JK, Nicoli T, Rockett K, Wilkins MR, Channon KM. Pivotal role for endothelial tetrahydrobiopterin in pulmonary hypertension. Circulation. 2005;111:2126 –2133. 47. Beppu H, Ichinose F, Kawai N, Jones RC, Yu PB, Zapol WM, Miyazono K, Li E, Bloch KD. BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia. Am J Physiol Lung Cell Mol Physiol. 2004;287: L1241–L1247. 48. Machado RD, James V, Southwood M, Harrison RE, Atkinson C, Stewart S, Morrell NW, Trembath RC, Aldred MA. Investigation of second genetic hits at the BMPR2 locus as a modulator of disease progression in familial pulmonary arterial hypertension. Circulation. 2005;111: 607– 613. 49. Yuan JX, Rubin LJ. Pathogenesis of pulmonary arterial hypertension: the need for multiple hits. Circulation. 2005;111:534 –538. 50. Said SI. Mediators and modulators of pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol. 2006;291:L547–L558.

CLINICAL PERSPECTIVE Despite significant advances in our understanding of its pathogenesis and improvements in its prognosis, idiopathic pulmonary arterial hypertension remains an incompletely understood, incurable disease. In the present study, male mice lacking the gene for the vasoactive intestinal peptide, a vasodilator of pulmonary and systemic vessels and an inhibitor of vascular smooth muscle proliferation, showed features of moderately severe idiopathic pulmonary arterial hypertension. In addition to pulmonary hypertension, smaller pulmonary arteries were markedly thickened with medial accumulation of smooth muscle, and the right ventricle was hypertrophied. Confirming the cause-and-effect relationship between the vasoactive intestinal peptide gene deletion and the pulmonary vascular pathology, administration of vasoactive intestinal peptide (15 ␮g IP every other day for 4 weeks) attenuated the vascular remodeling and right ventricular hypertrophy. This experimental model, in which lesions resembling those of clinical idiopathic pulmonary arterial hypertension are expressed secondary to the loss of a single gene, should prove useful in exploring pathogenetic mechanisms of the disease, especially the interactions between genetic pathways, and in testing the efficacy of new investigational drugs. The ability of vasoactive intestinal peptide to ameliorate the pulmonary arterial hypertension pathology in these mice provides a solid rationale for its therapeutic potential in the human disease, as proposed in a recent clinical trial.

Identification of a Novel Polymorphism in the 3ⴕUTR of the L-Arginine Transporter Gene SLC7A1 Contribution to Hypertension and Endothelial Dysfunction Zhiyong Yang, PhD; Kylie Venardos, PhD; Emma Jones, BSc; Brian J. Morris, PhD; Jaye Chin-Dusting, PhD; David M. Kaye, MD, PhD Background—Endothelial dysfunction because of reduced nitric oxide bioavailability is a key feature of essential hypertension. We have found that normotensive siblings of subjects with essential hypertension have impaired endothelial function accompanied by altered arginine metabolism. Methods and Results—We have identified a novel C/T polymorphism in the 3⬘UTR of the principal arginine transporter, solute carrier family 7 (cationic amino acid transporter, y⫹ system), member 1 gene (SLC7A1). The minor T allele significantly attenuates reporter gene expression (P⬍0.01) and is impaired in its capacity to form DNA-protein complexes (P⬍0.05). In 278 hypertensive subjects the frequency of the T allele was 13.3% compared with 7.6% in 498 normotensive subjects (P⬍0.001). Moreover, the overall genotype distribution observed in hypertensives differed significantly from that in normotensives (P⬍0.001). To complement these studies, we generated an endothelial-specific transgenic mouse overexpressing L-arginine transporter SLC7A1. The Slc7A1 transgenic mice exhibited significantly enhanced responses to the endothelium-dependent vasodilator acetylcholine (⫺log EC50 for wild-type versus Slc7A1 transgenic: 6.87⫾0.10 versus 7.56⫾0.13; P⬍0.001). This was accompanied by elevated production of nitric oxide by isolated aortic endothelial cells. Conclusions—The present study identifies a key, functionally active polymorphism in the 3⬘UTR of SLC7A1. As such, this polymorphism may account for the apparent link between altered endothelial function, L-arginine, and nitric oxide metabolism and predisposition to essential hypertension. (Circulation. 2007;115:1269-1274.) Key Words: amino acids 䡲 endothelium 䡲 genes 䡲 genetics 䡲 hypertension 䡲 molecular biology 䡲 nitric oxide

T

he vascular endothelium plays a crucial role in the regulation of vascular tone, the modulation of vascular architecture, and the control of cellular adhesion. Endothelial dysfunction has been widely associated with various cardiovascular risk factors and disease states including hypertension, diabetes, smoking, atherosclerosis, and heart failure.1– 4 There is good evidence that the underlying degree of endothelial dysfunction confers incremental cardiovascular risk.5 Although in the context of diabetes and heart failure, for example, endothelial dysfunction is generally attributed to the underlying disease state per se, in hypertension and atherosclerosis it has been proposed that endothelial dysfunction may precede the onset of these conditions.

Clinical Perspective p 1274 Hypertension affects ⬇25% of the population in westernized societies and is a major risk factor for cardiovascular disease. Despite the enormous prevalence of hypertension, little progress has been made in the identification of the key underlying mechanisms, probably as a result of complex gene-environment interactions.6

Several studies, including our own, have observed endothelial dysfunction in the normotensive siblings of hypertensive individuals.7,8 These data suggest a genetic contribution from the pathway responsible for nitric oxide (NO) bioavailability. In particular, we noticed that in normotensive siblings of hypertensive subjects, endothelial dysfunction is accompanied by reduced arginine transport.8 In the present study, we report the discovery of a likely molecular mechanism responsible for this finding. This involved our identification of a novel polymorphism in the 3⬘UTR region of the solute carrier family 7 (cationic amino acid transporter, y⫹ system), member 1 gene (SLC7A1, previously “CAT-1”; chromosome 13q12-q14). The variant allele is associated with altered expression of SLC7A1, thereby explaining, at least in part, the pathophysiological observations.

Methods DNA Samples and Genotyping Twelve primer pairs corresponding to different intronic sites of human SLC7A1 gene (SLC7A1, GenBank accession numbers

Received September 19, 2006; accepted December 18, 2006. From the Wynn Department of Metabolic Cardiology (Z.Y., K.V., D.M.K.) and Vascular Pharmacology Laboratory (E.J., J.C.-D.), Baker Heart Research Institute, Melbourne; and School of Medical Sciences and Bosch Institute (B.J.M.), University of Sydney, Sydney, Australia. Correspondence to Dr David M. Kaye, Wynn Department of Metabolic Cardiology, Baker Heart Research Institute, PO Box 6492, St Kilda Rd Central, Melbourne, VIC 8008, Australia. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.665836

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NM_003045 for mRNA, NT_009799 for genomic sequence) were designed and used to resequence all of its exons. DNA and demographic details were obtained from 498 normotensive subjects (systolic/diastolic pressure of 121⫾1/74⫾1 mm Hg) and 278 hypertensive subjects (systolic/diastolic pressure of 160⫾2/98⫾1 mm Hg) whose parents both had the same blood pressure status as the subjects. The respective human ethics committees at each institution approved the present study. A 20-sample pool was used initially as polymerase chain reaction (PCR) template to detect possible polymorphisms by means of direct DNA sequencing. Each of the individual samples was then reassessed to confirm the polymorphisms. During this process, 1 novel single-nucleotide polymorphism (SNP) was found in the 3⬘UTR with the use of primers pri047 (5⬘-AGCTGTCTGGAGGTGACCAG-3⬘) and pri048 (5⬘GCCTGAGAGGGTTTGCTGT-3⬘) and PCR conditions of 94°C for 1 minute, followed by 35 cycles of 94°C, 60°C, and 72°C for 1 minute each, with an additional 8-minute extension at 72°C at the end of last cycle before 4°C for at least 2 minutes. PCR products were then gel-purified to remove excess primers and dNTPs before DNA sequencing performed at the Baker Heart Research Institute. The novel SNP, involving a C/T substitution, was genotyped with the use of 2 allele-specific primers designed in such way that the only difference between them was the polymorphism site at the very last nucleotide of their 3⬘ ends: 5⬘-GCAAGTGACGCACAGCCC-3⬘ (pri049) and 5⬘-GCAAGTGACGCACAGCCT-3⬘ (pri050). Two parallel PCRs were performed for each DNA sample. These contained both pri047 and pri048, plus either pri049 or pri050, under the same conditions of PCR as described above. After PCR products were run on a 1.5% agarose gel, DNA genotypes were called directly without the need for further DNA sequencing.

Reporter Gene Assays The reporter gene vectors pGL3-Basic and pGL3-TK (Promega, Madison, Wis) were used as background and positive control, respectively, for luciferase assays. Human DNA with homologous genotypes of the 3⬘UTR polymorphism (either CC or TT) was used as PCR template to generate allele-specific amplicons. Primer sets pri047-pri049 and pri047-pri050 were again used to generate 216-bp PCR products containing allele C and allele T, respectively. Other primers were also used to generate different sized allele-specific amplicons as well as “nonrelated” DNA from the last intron of SLC7A1. All DNA fragments were inserted downstream of luciferase gene at an XbaI site, immediately preceding the poly(A) sequence. pSV-␤-Galactosidase control vector (Promega) was used as internal control to correct for transfection efficiency among samples. Chinese hamster ovary cells were grown at 37°C under 5% CO2 in DMEM (GIBCO/BRL) supplemented with 2 mmol/L L-glutamine and 10% heat-inactivated FCS. At 50% to 70% confluence, cells were cotransfected with equimolar amounts of each reporter gene construct and 2 ␮g of internal control DNA (pSV-␤-Galactosidase control vector) by electroporation in 0.45-cm cuvettes. The electroporation conditions were 500 ␮F, 270 V. After incubation for 24 and 48 hours after transfection, cells were washed twice with PBS, harvested in the reporter lysis buffer provided in the luciferase assay system (Promega), and centrifuged at 13 000 rpm for 1 minute at 4°C. The supernatant was assayed for both luciferase and ␤-galactosidase activities. ␤-Galactosidase activity was measured colorimetrically with the use of Emax precision microplate reader (Molecular Devices, Sunnyvale, Calif). Luciferase activity was normalized to ␤-galactosidase activity to correct for differences in transfection efficiency. All of the assays were performed in triplicate, and the means of relative luciferase activity were plotted as percentage with respect to pGL3-TK. At least 3 independent experiments were performed for each reporter gene construct.

Electrophoretic Mobility Shift Assay Nuclear extracts from HeLa cells were purchased from Promega. Together with the primer pri047, either allele C-specific (pri049) or allele T-specific (pri050) primers were used to generate 216-bp 3⬘UTR DNA fragments as described above. After PCR, the 5⬘ phosphate groups of the DNA fragments were removed by alkaline

phosphatase before end-labeling with T4 polynucleotide kinase with the use of [␥-32P]ATP. The 32P end-labeled probes were preincubated with or without unlabeled competitors for 10 minutes in the presence of 1 ␮L of HeLa nuclear extracts, then incubated at room temperature for 20 minutes in 15 ␮L of binding solution (25 mmol/L HEPES buffer, pH 8, 50 mmol/L KCl, 0.5 mmol/L MgCl2, 0.5 mmol/L dithiothreitol, 2 ␮g poly[dI-dC]-poly[dI-dC], 10% glycerol). Competition assays were performed with 100-fold molar excess of unlabeled DNA fragments from relevant unlabeled PCR products or double-stranded oligonucleotides containing AP1, AP2, CREB, nuclear factor-␬B, OCT1, SP1, and TFIID binding recognition elements (Promega). The reaction mixture was then electrophoresed on a 4% polyacrylamide gel in ⫻0.5 TBE buffer. The gels were wrapped and exposed to Kodak BioMax MR films (Sigma-Aldrich, St Louis, Mo) for 12 to 16 hours. The autoradiography results were then quantified.

Generation and Genotyping of Transgenic Mice Overexpressing SLC7A1-GFP To establish the functional impact of an alteration in endothelial SLC7A1 expression in the range of that predicted by the reporter studies, we established an endothelial-specific Slc7A1 overexpressing transgenic mouse. Plasmid pT2BLacZpA1L7 containing mouse TIE2 promoter and longer enhancer fragment was a gift from Dr Thomas N. Sato (University of Texas Southwestern Medical Center at Dallas).9 mCAT-1-GFP was cloned into EcoRI–BamHI sites, followed by the insertion of TIE2 promoter fragment into the HindIII site of pBluescript KS(⫺) (Invitrogen, Carlsbad, Calif), resulting in the generation of plasmid pBSTIE2CAT-1gfp. The TIE2 longer enhancer fragment (⬇10.6 kb), released from the plasmid pT2BLacZpA1L7 by complete digestion with NotI and partial digestion with XbaI, was then inserted into pBSTIE2CAT-1gfp. All the cloning products, as well as the mCAT-1-GFP fusion, were confirmed by enzyme digestion and sequencing. The resulting plasmid, containing Slc7a1-GFP driven by TIE2 promoter and enhancer, was digested by SalI to remove the vector backbone. The remaining gene expression cassette was then gelpurified, resuspended in injection buffer (10 mmol/L Tris-HCl, pH 7.4, 0.1 mmol/L EDTA), and passed through a 0.45-␮mol/L filter (Millipore, Billerica, Mass) before microinjection into oocytes from C57B/L mice. To screen for positive transgenic mice, 4 primer sets, each with at least 1 primer binding to GFP sequences, were used to amplify DNA and mRNA from mouse samples. PCR using the 3 primer pairs, 5⬘-CTTTGCTCAGGGCGGACT-3⬘ (pri502) and 5⬘CTGACAGCAACTTGGACCAG-3⬘ (pri523), 5⬘-GTCCTCCTTGAAGTCGATGC-3⬘ (pri524); 5⬘-TCGTGACCACCCTGACCTAC-3⬘ (pri525), 5⬘-GATGTTGTGGCGGATCTTG-3⬘ (pri530); and 5⬘-GAGCAAGACCAAGCTCTCATTT-3⬘ (pri531), was performed by denaturation at 94°C for 3 minutes, followed by 35 cycles of 94°C for 30 seconds, 65°C for 30 seconds and 72°C for 2 minutes, and a final extension step at 72°C for 10 minutes. PCR involving primers 5⬘- CTAGTGGATCCTTACTTGTACAGCTCGTCCATGCC-3⬘ (pri508) and 5⬘-AAGCTTGAATTCACAGCAGATTCGCTCAGCACAATG-3⬘ (pri509) was performed under similar conditions except that the annealing temperature was 60°C and the extension time at each cycle was 3.5 minutes. Transgenic-positive mice were maintained by backcrossing to wild-type C57B/L and subjected to further PCR screening of their offspring.

Isolation and Fluorescence-Activated Cell Sorting of Endothelial Cells From Mouse Aorta Primary murine aortic endothelial cells were isolated from both wild-type and transgene-positive C57B/L mice (Baker Institute Animal Center, Melbourne, Australia) as described. Briefly, aortas were harvested, the adventitia was removed, and strips were placed lumen side down into Matrigel in culture medium ECCM (each 500 mL containing 200 mL of DMEM without FCS, 200 mL of Ham’s F12 media (Invitrogen), 100 mL of FCS, 15 mg of endothelial mitogen, 30 mg of heparin, and 1 mL of antibiotic/antimycotic). The isolated murine aortic endothelial cells s were further purified by fluores-

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cence-activated cell sorting (FACS) (model FACSAria, Becton Dickinson, Franklin Lakes, NJ) by utilizing their property of uptake of acetylated low-density lipoprotein (Ac-LDL). Briefly, murine aortic endothelial cells were incubated with DMEM containing 2 ␮g/mL Dil-Ac-LDL for 4 to 16 hours, were washed 3 times with PBS, and then were resuspended in FACS sorting buffer (each 100 mL S-MEM-Ca2⫹–free medium containing 2 mL of 0.5 mol/L EDTA, 2 mL of antibiotic/antimycotic, 1 mL of FCS). A 418-nm laser was used for excitation and 550 nm for emission, “scatter gates” were set to minimize the contribution of cell pairs, and “fluorescence gates” were chosen to eliminate the more highly fluorescent macrophages. The sorted cells were termed SLC7A1– murine aortic endothelial cells. They were maintained for up to 8 passages and used for experiments from passages 3 to 6.

Endothelial Function in Mouse Aortic Rings Aortic rings were prepared from wild-type and Slc7a1 transgenic mice (10 to 14 weeks old) as described previously. In brief, aortic ring segments (2 mm in length) were mounted into an isometric myograph (myograph model 610 mol/L, JP Trading, Copenhagen, Denmark). After a 30-minute equilibration period, each vessel was subjected to a passive length-tension stretch. This procedure enabled each vessel to be normalized to an internal circumference equivalent to 90% the transmural pressure of 100 mm Hg. Endothelial integrity was determined initially by the demonstration of at least 50% vasodilation to 1 ␮mol/L acetylcholine. Full concentration-response curves to acetylcholine (1 nmol/L to 100 ␮mol/L) were constructed with the use of vessels preconstricted with cirazoline at a concentration that achieved 70% of the contraction induced by KPSS (in mmo/L: KCl 124, KM2PO4 1.18, MgSO4 1.17, NaHCO3 25, CaCl2 2.5, EDTA 0.026, glucose 5.5 at pH 7.4).

Endothelial Arginine Transport and NO Production To examine the effect of endothelial Slc7a1 transgene expression, we compared the cellular uptake of [3H]L-arginine in wild-type and Slc7a1 transgenic aortic endothelial cells, isolated as above. [3H]LArginine was measured as described previously.10 In conjunction, the influence of SLC7A1 overexpression on NO production was determined with the NO fluorochrome 4-amino-5-methylamino2⬘,7⬘-dichlorofluorescein diacetate (DAF-FM, Molecular Probes, Eugene, Ore), as described previously.

Statistical Methods Data are presented as mean⫾SEM. Between-group comparisons were performed with the use of unpaired Student t tests for normally distributed data or ␹2 tests for categorical data. The authors had full access to and take full responsibility for the integrity of the data. All authors read and agree to the manuscript as written.

Results To discover potentially relevant SNPs in the exons of SLC7A1, we sequenced all 12 exons using pooled and individual DNA samples after amplification of each using flanking PCR primers. This identified a novel SNP located at nucleotide 2178 in the SLC7A1 3⬘UTR, 10 nucleotides (nt) downstream of the stop codon (nm_003045). Details of this SNP, subsequently referred to as ss52051869, have been placed online (www.ncbi.nlm.nih.gov/projects/SNP/snp_ss. cgi?ss ⫽52051869). To evaluate whether ss52051869 differentially affects SLC7A1 expression, we tested the effect of 3⬘UTR segments of contrasting genotype (CC versus TT) on luciferase reporter expression. Irrespective of genotype, the 3⬘UTR segment reduced luciferase expression. Notably, however, cells transfected with C allele construct (pGL3-TK-216-bp-Allele-C) had significantly higher luciferase activity than cells trans-

Figure 1. 3⬘UTR polymorphism of SLC7A1 modulates reporter gene expression. The insertion of major allele SLC7A1 3⬘UTR DNA fragments repressed luciferase production (P⫽0.005). The T allele produced greater repression than did the C allele, with cells transfected with pGL3-TK-216-bp-Allele-T having much lower luciferase activity than those transfected with pGL3-TK216-bp-Allele-C (P⫽0.048). RLU indicates relative light units.

fected with T allele construct (pGL3-TK-216-bp-Allele-T) (Figure 1). Constructs made with “nonrelated” DNA of similar size showed no alteration in reporter expression (data not shown). Furthermore, no inhibitory effects were seen when smaller DNA fragments were used, for example, a 114-bp DNA insert containing the polymorphism site (representing nt 2161 to 2174 of nm_003045), in the reporter gene assay (data not shown). We performed gel shift assays to investigate whether differences in allelic expression between C and T allele variants could be attributed to the differential binding of nuclear proteins. In these assays, 2 PCR amplicons corresponding to the sequence from nt 2161 to 2376 in the 3⬘UTR were 32P-labeled and allowed to interact with HeLa nuclear extracts. Both probes formed DNA-protein complexes and showed a similar pattern of migration on the gel. The allele C fragment, however, demonstrated much stronger binding than allele T (P⬍0.05) (Figure 2). To determine the binding specificity of these DNA-protein complexes, competition experiments were performed with 100-fold excess of unlabeled probes before the addition of nuclear extracts. DNAprotein complex formation was completely abrogated by unlabeled probe (Figure 2). To determine the identity of the nuclear proteins that bound, we performed additional competition electrophoretic mobility shift assays using the consensus oligonucleotides containing AP1, AP2, CREB, nuclear factor-␬B, OCT1, SP1, and TFIID binding elements. In particular, the preincubation of consensus oligonucleotides containing SP1, AP1, AP2, CREB, and TFIID binding elements resulted in abolition of the DNA-protein complexes, consistent with them being specific competitors (data not shown). Given the role of the 3⬘UTR in the regulation of mRNA expression, we determined whether there was a difference in ss52051869 allele frequency between hypertensive and normotensive subjects. In hypertensive subjects the frequency of the T allele was 13.3% compared with 7.6% in the normo-

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Figure 2. A, Electrophoretic mobility shift assays for 3⬘UTR SNP of SLC7A1. 32P-labeled 3⬘UTR DNA fragments containing different alleles of the polymorphism (probes “allele C” or “allele T ”) were mixed with or without HeLa nuclear extracts to investigate whether there was specific nucleotide-protein binding. Lane 1, allele C probe; lane 2, allele C probe plus HeLa extract; lane 3, excess unlabeled allele C DNA plus HeLa extract plus allele C probe; lane 4, allele T probe; lane 5, allele T probe plus HeLa extract; lane 6, excess unlabeled allele T DNA plus HeLa extract plus allele T probe. B, Quantification of electrophoretic mobility shift assay results for 3⬘UTR SNP of SLC7A1. Both allele C and allele T probes were able to specifically bind to nuclear extracts in an allele-specific manner, with the allele C probe exhibiting much greater binding than that for allele T (*P⫽0.0069). Group data (mean⫾SEM) are obtained from 3 separate experiments.

Figure 3. A, L-Arginine transport kinetic curves in aortic endothelial cells from Slc7a1 transgenic (open circles) and wild-type (closed circles) mice. #P⫽0.045. B, NO production (as measured by 4-amino-5-methylamino-2⬘,7⬘-dichlorofluorescein [DAF] fluorescence) by endothelial cells from Slc7a1 transgenic and wild-type mice. *P⬍0.001. RU indicates relative units.

arginine transport capacity in endothelial cells from wild-type and transgenic mice was in the range of that which might be expected for the effect of the major and minor SNP alleles on the basis of the reporter studies. In conjunction with the elevation of arginine transport, aortic endothelial cells from Slc7A1 transgenic mice also demonstrated a highly significant increase in NO production (Figure 3B). Having demonstrated the cellular effect of modest SLC7A1 overexpression, we next investigated the effect on vascular pharmacology in isolated aortic rings. Vascular rings obtained from Slc7A1 transgenic mice showed significantly greater sensitivity (EC50) to the endothelium-dependent vasodilator acetylcholine (Figure 4), whereas responses to the endothelium-independent vasodilator sodium nitroprusside were not altered (data not shown).

tensive subjects (P⬍0.001). Moreover, the genotype distribution observed in hypertensives differed significantly from that in normotensives, as shown in the Table. With regard to the TT genotype specifically, this was observed in 1 normotensive subject (0.2%) and 6 hypertensive subjects (2.2%). All genotype frequencies accorded with Hardy-Weinberg equilibrium. To evaluate the in vivo effect of altered SLC7A1 expression, we established endothelial-specific overexpression in a transgenic mouse. Full kinetic analysis of arginine transport in aortic endothelial cells from wild-type and Slc7A1 transgenic mice showed a significant increase in Vmax (2149⫾24 versus 1513⫾39 pmol/mg per minute, respectively; P⫽0.045), consistent with increased SLC7A1 expression (Figure 3A). Of note, the magnitude of the difference in

Discussion Intracellular L-arginine is derived predominantly from the extracellular milieu and transported principally via the type 1

Genotype and Allele Frequencies of 3ⴕUTR Polymorphism of SLC7A1 in Hypertensive and Normotensive Subjects Genotype Frequencies

Allele Frequencies

N

CC

CT

TT



P

C

T

␹2

P

Normotensive

498

423 (84.9%)

74 (14.9%)

1 (0.2%)

15.2

⬍0.001

920 (92.4%)

76 (7.6%)

11.6

⬍0.001

Hypertensive

278

210 (75.5%)

62 (22.3%)

6 (2.2%)

482 (86.7%)

74 (13.3%)

Group

2

Yang et al

Figure 4. Vasodilator responses to acetylcholine (ACh) in isolated aortic rings obtained from Slc7a1 transgenic and wild-type (wt) mice. *P⬍0.001.

cationic amino acid transporter SLC7A1.7 Clinical and experimental paradigms involving an extracellular deficiency of L-arginine or its transport have been shown to be associated with reduced endothelial function and NO production.11–13 Furthermore, the administration of L-arginine to hypertensive animals and humans has been shown to reduce blood pressure and to restore endothelial function in both hypertensive subjects and those with a genetic predisposition toward hypertension.8,14 The present study has found that a functional variant of the L-arginine transporter gene SLC7A1 is increased in frequency in subjects with essential hypertension and that, in experimental models, altered expression of SLC7A1 results in physiologically relevant changes in NO production and endothelial function. Although hypertension demonstrates significant familial aggregation, genomewide linkage studies have not provided consistent results, and in general the strength of various associations between putative loci of interest and blood pressure has been modest.15 At the same time, strong evidence supports the notion of environmental inputs into the subsequent development of hypertension, including overweight and elevated salt intake.16 In this context, acute intervention studies indicate that L-arginine may influence blood pressure.17 More generally, recent dietary intervention studies raise the possibility that combination diets rich in vegetables, nuts, and grains could exert influences on blood pressure by mechanisms beyond the effects of sodium reduction alone. In particular, the Dietary Approaches to Stop Hypertension (DASH) diet was shown to exert antihypertensive effects independent of sodium.18 As such, it has been shown that nuts alone improve endothelial function, and this may be accounted for by their significant L-arginine content.19 To establish the functional importance of relatively modest changes in SLC7A1 expression, we established an endothelial-specific transgenic mouse. Endothelial cells obtained from transgenic mice displayed a marked increase in NO under basal conditions, and isolated aortic rings from these mice showed evidence of increased endothelial function. It is acknowledged that in the generation of the Slc7A1 transgenic mice we did not specifically demonstrate the effect that might

Arginine Transporter Polymorphism and CVD

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be observed in the context of restoration of L-arginine transport in a clinical paradigm or by a “knock-in” mouse model of reduced L-arginine transport. Nevertheless, these experimental findings are directionally consistent with the expected expects of increased substrate availability for NO synthesis. In conjunction with these data, we showed recently that another well-recognized cardiovascular risk factor, cigarette smoking, significantly reduced L-arginine transport and NO production in endothelial cells.10 Taken together, the present study and our previous work strongly support the notion that genetic or environmental alterations in L-arginine transport have the capacity to directly influence NO production and thereby vascular tone. SLC7A1 is a high-affinity, low-capacity cationic amino acid transporter that facilitates uptake of arginine and lysine in mammalian cells. SLC7A1 is expressed almost ubiquitously, with the exception of adult liver, but its expression level varies considerably in different tissues and cell types.20 SLC7A1 expression can be modulated by a variety of stimuli including cell proliferation, growth factors, cytokines, certain hormones, microRNA, nutrients, and cellular stress, including amino acid deprivation.20 Interestingly, the 3⬘UTR of human SLC7A1 mRNA contains several potential target sites for miR-122, a liver-specific microRNA. Indeed, it was shown recently that activity of the endogenous SLC7A1 mRNA was translationally repressed by miR-122, and such repression could be reversed by the binding of HuR, an AU-rich element binding protein, to the 3⬘UTR of SLC7A1 mRNA.21 Our findings on the SNP in the 3⬘UTR of SLC7A1 mRNA are therefore consistent with the important regulatory role that the 3⬘UTR plays in controlling gene expression. In the present study, however, we did not directly correlate the genotype with SLC7A1 mRNA or protein expression because of the inability to obtain relevant vascular tissue or cells for such investigations. In conclusion, we have identified a functionally relevant SNP in the 3⬘UTR region of SLC7A1, the principal L-arginine transporter in humans. In the context of hypertension, this finding provides the basis for an interaction between a genetically programmed influence on vascular endothelial function and blood pressure with environmental factors, including diet and traditional cardiovascular risk factors.

Sources of Funding The present study was supported by grants from the National Health and Medical Research Council of Australia and the Atherosclerosis Research Trust (UK).

Disclosures None.

References 1. Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endotheliumdependent vascular relaxation in patients with essential hypertension. N Engl J Med. 1990;323:22–27. 2. De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. Endothelial dysfunction in diabetes. Br J Pharmacol. 2000;130: 963–974. 3. Thorne S, Mullen MJ, Clarkson P, Donald AE, Deanfield JE. Early endothelial dysfunction in adults at risk from atherosclerosis: different responses to L-arginine. J Am Coll Cardiol. 1998;32:110 –116.

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4. Kaye DM, Ahlers BA, Autelitano DJ, Chin-Dusting JP. In vivo and in vitro evidence for impaired arginine transport in human heart failure. Circulation. 2000;102:2707–2712. 5. Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000;101:1899 –1906. 6. Lifton R, Gharavi A, Geller D. Molecular mechanisms of hypertension. Cell. 2001;104:545–556. 7. McAllister AS, Atkinson AB, Johnston GD, Hadden DR, Bell PM, McCance DR. Basal nitric oxide production is impaired in offspring of patients with essential hypertension. Clin Sci. 1999;97:141–147. 8. Schlaich MP, Parnell MM, Ahlers BA, Finch S, Marshall T, Zhang WZ, Kaye DM. Impaired L-arginine transport and endothelial function in hypertensive and genetically predisposed normotensive subjects. Circulation. 2004;110:3680 –3686. 9. Schlaeger TM, Bartunkova S, Lawitts JA, Teichmann G, Risau W, Deutsch U, Sato TN. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc Natl Acad Sci U S A. 1997;94:3058 –3063. 10. Zhang WZ, Venardos K, Chin-Dusting J, Kaye DM. Adverse effects of cigarette smoke on no bioavailability: role of arginine metabolism and oxidative stress. Hypertension. 2006;48:278 –285. 11. Gold ME, Bush PA, Ignarro LJ. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun. 1989;164:714 –721. 12. Sobrevia L, Cesare P, Yudilevich DL, Mann GE. Diabetes-induced activation of system y⫹ and nitric oxide synthase in human endothelial cells: association with membrane hyperpolarization. J Physiol. 1995;489: 183–192. 13. Kamada Y, Nagaretani H, Tamura S, Ohama T, Maruyama T, Hiraoka H, Yamashita S, Yamada A, Kiso S, Inui Y, Ito N, Kayanoki Y, Kawata S,

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Matsuzawa Y. Vascular endothelial dysfunction resulting from L-arginine deficiency in a patient with lysinuric protein intolerance. J Clin Invest. 2001;108:717–724. Taddei S, Virdis A, Mattei P, Ghiadoni L, Sudano I, Salvetti A. Defective L-arginine–nitric oxide pathway in offspring of essential hypertensive patients. Circulation. 1996;94:1298 –1303. Wu X, Kan D, Province M, Quertermous T, Rao D, Chang C, Mosley T, Curb D, Boerwinkle E, Cooper R. An updated meta-analysis of genome scans for hypertension and blood pressure in the NHLBI Family Blood Pressure Program (FBPP). Am J Hypertens. 2006;19:122–127. Mullins L, Bailey M, Mullins J. Hypertension, kidney, and transgenics: a fresh perspective. Physiol Rev. 2006;86:709 –746. Lekakis JP, Papathanassiou S, Papaioannou TG, Papamichael CM, Zakopoulos N, Kotsis V, Dagre AG, Stamatelopoulos K, Protogerou A, Stamatelopoulos SF. Oral L-arginine improves endothelial dysfunction in patients with essential hypertension. Int J Cardiol. 2002;86:317–323. Sacks F, Svetkey L, WVollmer W, Appel L, Bray G, Harsha D, Obarzanek E, Conlin P, Miller E, Simons-Morton D, Karanja N, Lin P. Effects on blood pressure of reduced dietary sodium and the Dietary Approaches to Stop Hypertension (DASH) diet. N Engl J Med. 2001; 344:3–10. Ros E, Nunez I, Perez-Heras A, Serra M, Gilabert R, Casals E, Deulofeu R. A walnut diet improves endothelial function in hypercholesterolemic subjects. Circulation. 2004;109:1609 –1614. Hatzoglou M, Fernandez J, Yaman I, Closs E. Regulation of cationic amino acid transporters: the story of the CAT-1 transporter. Ann Rev Nutr. 2004;24:377–399. Bhattacharyya S, Habermacher R, Martine U, Closs E, Filipowicz W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell. 2006;125:1111–1124.

CLINICAL PERSPECTIVE

Hypertension currently affects ⬇25% of the population in westernized societies. Despite the high prevalence of hypertension and the long history of hypertension research, the pathogenesis of essential hypertension remains controversial. Most current theories suggest that essential hypertension results from a complex set of gene-environment interactions. One hallmark of hypertension is the presence of abnormal endothelial function. Interestingly, this phenomenon is commonly also observed in nonhypertensive siblings of individuals with hypertension. In the present study, we report, in hypertensive subjects, the increased presence of a novel polymorphism in a key gene responsible for the delivery of arginine into cells. We demonstrate that its presence can alter endothelial function and nitric oxide production, thereby indicating its potential role in the pathogenesis of hypertension. Identification of this gene polymorphism in hypertensives may in the future assist in the selection of certain antihypertensive interventions.

Vascular Medicine Pulmonary Arterial Hypertension Is Linked to Insulin Resistance and Reversed by Peroxisome Proliferator–Activated Receptor-␥ Activation Georg Hansmann, MD; Roger A. Wagner, MD, PhD; Stefan Schellong, BA; Vinicio A. de Jesus Perez, MD; Takashi Urashima, MD; Lingli Wang, MD; Ahmad Y. Sheikh, MD; Renée S. Suen, BSc; Duncan J. Stewart, MD; Marlene Rabinovitch, MD Background—Patients with pulmonary arterial hypertension (PAH) have reduced expression of apolipoprotein E (apoE) and peroxisome proliferator–activated receptor-␥ in lung tissues, and deficiency of both has been linked to insulin resistance. ApoE deficiency leads to enhanced platelet-derived growth factor signaling, which is important in the pathobiology of PAH. We therefore hypothesized that insulin-resistant apoE-deficient (apoE⫺/⫺) mice would develop PAH that could be reversed by a peroxisome proliferator–activated receptor-␥ agonist (eg, rosiglitazone). Methods and Results—We report that apoE⫺/⫺ mice on a high-fat diet develop PAH as judged by elevated right ventricular systolic pressure. Compared with females, male apoE⫺/⫺ were insulin resistant, had lower plasma adiponectin, and had higher right ventricular systolic pressure associated with right ventricular hypertrophy and increased peripheral pulmonary artery muscularization. Because male apoE⫺/⫺ mice were insulin resistant and had more severe PAH than female apoE⫺/⫺ mice, we treated them with rosiglitazone for 4 and 10 weeks. This treatment resulted in markedly higher plasma adiponectin, improved insulin sensitivity, and complete regression of PAH, right ventricular hypertrophy, and abnormal pulmonary artery muscularization in male apoE⫺/⫺ mice. We further show that recombinant apoE and adiponectin suppress platelet-derived growth factor-BB–mediated proliferation of pulmonary artery smooth muscle cells harvested from apoE⫺/⫺ or C57Bl/6 control mice. Conclusions—We have shown that insulin resistance, low plasma adiponectin levels, and deficiency of apoE may be risk factors for PAH and that peroxisome proliferator–activated receptor-␥ activation can reverse PAH in an animal model. (Circulation. 2007;115:1275-1284.) Key Words: apolipoproteins 䡲 glucose 䡲 hypercholesterolemia 䡲 hypertension, pulmonary 䡲 insulin 䡲 metabolism 䡲 PPAR gamma repressed by PPAR␥ are associated with insulin resistance3 and implicated in the pathobiology of PAH. These include interleukin-6,10,11 fractalkine,12,13 monocyte chemoattractant protein-1,14 endothelin-1 (ET-1),15–17 and the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA).18,19

A

lthough insulin resistance is associated with systemic cardiovascular disease,1–3 it has not been implicated as a predisposing factor in pulmonary arterial hypertension (PAH). Several findings, however, support such an association. Patients with idiopathic PAH have reduced pulmonary mRNA expression of peroxisome proliferator–activated receptor gamma (PPAR␥),4 a ligand-activated nuclear receptor and transcription factor that regulates adipogenesis and glucose metabolism.5–7 They also have reduced pulmonary mRNA expression of apolipoprotein E (apoE),8 a protective factor known to reduce circulating oxidized low-density lipoprotein and atherogenesis in the vessel wall.9 Deficiency of both PPAR␥ and apoE has been linked to insulin resistance and the metabolic syndrome.7,9 Elevated levels of several circulating factors that are normally

Clinical Perspective p 1284 Heightened signaling by platelet-derived growth factor-BB (PDGF-BB)/mitogen-activated protein kinase leading to smooth muscle cell (SMC) proliferation and migration is also a key clinical feature of pulmonary vascular disease.20 –22 With apoE deficiency, abundant oxidized low-density lipoprotein23 and PDGF-BB23 were shown to induce mitogen-

Received September 8, 2006; accepted December 29, 2006. From the Department of Pediatrics, Division of Pediatric Cardiology (G.H., S.S., V.A.D.J.P., T.U., L.W., M.R.), Department of Medicine, Division of Cardiovascular Medicine (R.A.W.), and Department of Cardiovascular Surgery (A.Y.S.), Stanford University School of Medicine, Stanford, Calif, and Department of Medicine, Division of Cardiology, University of Toronto, Toronto, Ontario, Canada (R.S.S., D.J.S.). The online-only Data Supplement, consisting of expanded Methods and tables, is available with this article at http://circ.ahajournals.org/cgi/content/full/ CIRCULATIONAHA.106.663120/DC1. Correspondence to Dr Marlene Rabinovitch, Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University School of Medicine, CCSR 2245B, 269 Campus Dr, Stanford, CA 94305-5162. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.663120

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activated protein kinase, transcription of growth-promoting genes (eg, cyclin D1), and subsequently proliferation and migration of systemic vascular SMCs.23,24 Interestingly, high glucose concentrations induce mitogen-activated protein kinase/phosphatidylinositol 3-kinase (PI3K)– dependent upregulation of PDGF receptor-␤ (PDGFR-␤) and potentiate SMC migration in response to PDGF-BB.24 In concert with PI3K, PDGFR-␤/mitogen-activated protein kinase-signaling also leads to SMC resistance to apoptosis.25 In systemic vascular SMCs, apoE and adiponectin,26 a PPAR␥ target in adipocytes,7 inhibit PDGF-BB–induced SMC proliferation and migration.23,27 ApoE internalizes the PDGFR-␤,28 –30 and adiponectin sequesters the ligand PDGFBB.31 Thus, in association with insulin resistance, reduced levels of apoE23 and adiponectin27 can be expected to enhance PDGF-BB signaling. In accordance with these observations, diabetic apoE-deficient (apoE⫺/⫺) mice show pronounced PDGF-BB signaling and neointimal thickening of the arterial vessel wall.32 We hypothesized that these factors would have similar effects on pulmonary arterial SMCs (PASMCs), consequently leading to PAH. PPAR␥ agonists are clinically used to make cells insulin sensitive, thereby obviating the detrimental effects of insulin resistance related to hyperlipidemia, inflammation, and mitogenesis in the vessel wall.33 Rosiglitazone, a PPAR␥ ligand of the thiazolidinedione class, enhances insulin-mediated glucose uptake and inhibits proliferation and migration of systemic SMCs induced by PDGF-BB.33,34 We therefore hypothesized that PAH would develop in apoE⫺/⫺ insulin-resistant mice but that the disease process would be attenuated or reversed by a PPAR␥ agonist (eg, rosiglitazone). In the present study, we show that apoE deficiency, in association with a high-fat (HF) diet, leads to both insulin resistance and PAH. Male apoE⫺/⫺ mice had more severe PAH (ie, higher right ventricular systolic pressure [RVSP], right ventricular hypertrophy [RVH], and enhanced peripheral PA muscularization) associated with insulin resistance and lower plasma adiponectin levels compared with female apoE⫺/⫺ mice. Because testosterone inhibits the secretion of adiponectin in adipocytes,35 we hypothesized that elevation of this vasoprotective adipocytokine may account for the less severe vascular phenotype in female apoE⫺/⫺ mice. We therefore treated male apoE⫺/⫺ mice with rosiglitazone and documented 8-fold-higher plasma adiponectin levels, improved insulin sensitivity, and complete regression of PAH, RVH, and abnormal PA muscularization. To further establish a direct link between apoE and adiponectin and SMC proliferation and survival, we treated murine (apoE⫺/⫺ and wildtype) PASMCs in culture with recombinant apoE and adiponectin. We showed that both proteins inhibit PDGF-BB– induced proliferation in apoE⫺/⫺ and wild-type PASMCs. Our data therefore suggest that insulin resistance and deficiency of apoE and/or adiponectin may be risk factors for PAH that can be reversed by PPAR␥ activation.

Methods Expanded Methods and Results sections are given in the online Data Supplement.

Experimental Design ApoE⫺/⫺ mice (B6.129P2-Apoetm1Unc/J) and C57Bl/6 control mice were obtained from Jackson Laboratories (Bar Harbor, Me). At 4 weeks of age, the mice were either continued on regular chow or switched to HF diet (Dyets No. 101511, Dyets Inc, Bethlehem, Pa) for a maximum of 21 weeks. For the nontreatment study, 15-weekold male and female mice (apoE⫺/⫺, C57Bl/6 controls) on either diet were studied. For the rosiglitazone treatment study, 15-week-old male mice (apoE⫺/⫺, C57Bl/6 controls) on HF diet were used. Half of the animals received rosiglitazone (GlaxoSmithKline, Research Triangle Park, NC) 10 mg/kg body weight per day PO incorporated into the food for 4 or 10 weeks. All protocols were approved by the Stanford Animal Care Committee.

Hemodynamic Measurements Measurements of RVSP and RV dP/dt were performed by jugular vein catheterization (1.4F, Millar Instruments Inc, Houston, Tex) under isoflurane anesthesia (1.5% to 2.5%) using a closed-chest technique in unventilated mice at 15, 19, and 25 weeks of age. Left ventricular (LV) end-diastolic pressure was determined by LV catheterization via the left carotid artery under isoflurane anesthesia. Systemic blood pressure was determined by the tail-cuff method in nonanesthesized mice. Measurements of cardiac output and function were performed by echocardiography.

RVH and LV Hypertrophy RVH was measured by the weight of the RV relative to LV⫹septum. LV hypertrophy was measured as absolute weight of the LV plus septum. LV dilatation was assessed by echocardiographic M-mode measurement of the LV end-diastolic inner diameter.

Lung Tissue Preparation Lungs were perfused with normal saline, fixed in 10% formalin overnight, and then either embedded in paraffin for standard histology or frozen for Oil-red-O staining. A subset of left lungs (approximately half) were barium infused via PA-inserted tubing to label peripheral PAs for morphometric analysis and micro– computed tomography (CT) imaging.

Morphometric Analysis Transverse left lung sections were stained by elastic van Gieson and Movat pentachrome stains. From all mice, we took the same full section in the mid portion of the lung parallel to the hilum and embedded it in the same manner. Muscularization was assessed in barium-injected left lung sections by calculating the proportion of fully and partially muscularized peripheral (alveolar wall) PAs to total peripheral PAs. All measurements were done blinded to genotype and condition.

Micro-CT Imaging A custom-built eXplore Locus RS120 Micro CT Scanner (GE Health Care, Ontario, Canada) was used to acquire nondestructing 3-dimensional images of barium-infused whole-lung specimens. Images were scanned at 49-␮m resolution and 720 views (70 kV [peak], 50 mAmps, 30-ms single image acquisition time) and reconstructed with the eXplore Reconstruction Utility, and volumes were viewed and rendered with the GE Health Care MicroView software.

Fasting Whole-Blood and Plasma Measurements We performed tail vein puncture in nonanesthetized, overnightstarved mice, followed by duplicate whole-blood glucose measurements with a glucometer (Freestyle/Abbott). Fasting blood plasma was obtained via retro-orbital bleeding or cardiac puncture. White blood cell count and hematocrit were assessed by the Stanford Animal Facility Laboratories. Hemoglobin A1c was measured by Esoterix (Calabasa Hills, Calif). Plasma ET-1 was measured by ELISA. Plasma ADMA levels were determined by high-performance liquid tomography at Oxonon Bioanalysis Inc (Oakland, Calif). All

Hansmann et al other plasma measurements were done in duplicate at Linco Diagnostics (St Charles, Mo).

Cell Culture Primary murine PASMCs were isolated from apoE⫺/⫺ and C57Bl/6 mice using a modified elastase/collagenase digestion protocol as previously described.36 Murine PASMCs were grown to 70% confluence and cultured in starvation media (DMEM, 0.1% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin) for 24 hours. Recombinant apoE (Chemicon International, Temecula, Calif) and adiponectin (BioVision, Mountain View, Calif) were added to quiescent cells 30 minutes before mitogenic stimulation with PDGF-BB (R&D Systems, Minneapolis, Minn).

Cell Proliferation Assays For cell counts, PASMCs were seeded at 2.5⫻104 cells per well of a 24-well plate in growth medium and allowed to adhere overnight. The medium was removed, and the cells were washed 3 times with PBS and incubated in starvation media for 24 hours, followed by PDGF-BB stimulation (20 ng/mL) for 0 and 72 hours. Cells were then washed with PBS, trypsinized, resuspended, and counted in a hemacytometer.

Statistical Analysis Values from multiple experiments are expressed as mean⫾SEM. Using the Kohmogorov-Smirnov test and larger data sets from previous studies, we could show that the measured values were approximately normally distributed. Statistical significance was determined using 1-way ANOVA, followed by Bonferroni’s multiple-comparison test unless stated otherwise. A value of P⬍0.05 was considered significant. The significance of our data also was confirmed by the nonparametric Mann-Whitney test. The number in each group is indicated in the column graphs and in the figure legends. For some of the metabolic measurements such as blood glucose, which did not require invasive blood draws, a larger number of animals could be assessed, resulting in minor unevenness in the numbers reported. All authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results ⴚ/ⴚ

ApoE and C57Bl/6 Mice on Regular Chow Have Similar RVSPs and RV Mass

First, we assessed 15-week-old male and female apoE⫺/⫺ mice on regular chow for the presence and severity of PAH. Values of RVSP, a measure of PAH, and of RV/LV⫹septum ratio, a measure of RVH, were similar in apoE⫺/⫺ and C57Bl/6 control mice. Moreover, no significant differences were observed in RVSP or RV/LV⫹septum ratio between genders of either genotype (Table 1).

ApoEⴚ/ⴚ Mice on HF Diet Develop PAH An 11-week HF diet treatment did not significantly increase RVSP in 15-week old C57Bl/6 mice (Table 1). In contrast, apoE⫺/⫺ mice on HF diet for the same duration developed PAH as judged by significant elevation in RVSP, with males having higher values than females (Figure 1A and Table 1). In addition, only male apoE⫺/⫺ mice on HF diet had RVH and enhanced peripheral PA muscularization (Figure 1B and 1C) compared with C57Bl/6 controls (P⬍0.001). A direct comparison revealed a more severe PAH phenotype in the male versus female apoE⫺/⫺ mice on HF diet in that RVSP (28.9 versus 24.9 mm Hg; P⫽0.0014), RVH (RV/LV⫹septum ratio, 0.41 versus 0.29; P⫽0.0093), and peripheral muscular-

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ization (47.8 versus 35.4%; P⫽0.0371) were significantly greater (Figure 1A through 1C, unpaired 2-tailed t test). RV systolic function (RV dP/dtmax) was augmented in apoE⫺/⫺ mice of both genders and reflected the elevated RVSP compared with C57Bl/6 controls (Table 1). RV diastolic function (RV dP/dtmin) was greater in both male (trend) and female (P⬍0.05) apoE⫺/⫺ mice compared with C57Bl/6 mice of the same gender. Systemic blood pressure, cardiac output, LV function (indicated by LV end-diastolic pressure of 2 to 5 mm Hg), and hematocrit were similar in both genotypes (Table 1). Thus, the elevation of RVSP in apoE⫺/⫺ compared with C57Bl/6 mice likely reflected an elevation in pulmonary vascular resistance.

PA Atherosclerosis in the ApoEⴚ/ⴚ Mice Does Not Cause Significant PA Stenosis Further studies were carried out to determine whether apoE⫺/⫺ mice on HF diet developed, in addition to neomuscularization of peripheral PAs, occlusive atheroma accounting for the elevated RVSP and RVH compared with C57Bl/6 mice. Micro-CT imaging of barium-injected lungs revealed an irregularly shaped main PA vessel wall in male and female apoE⫺/⫺ mice on HF diet but excluded PA branch stenosis as contributing to the RVSP elevation (Figure 1F and 1G). This feature was associated with nonocclusive atherosclerotic lesions only in large intrapulmonary arteries (diameter ⱖ500 ␮m) in apoE⫺/⫺ mice of both genders on HF diet (see Figure I in the online Data Supplement). Atherosclerotic lesions were neither seen in C57Bl/6 control mice on HF diet nor in mice of both genotypes on regular chow.

Insulin Resistance Is Associated With More Severe PAH in Male ApoEⴚ/ⴚ Mice We focused our attention on possible differences in the lipid profile and markers of insulin resistance associated with the more severe PAH phenotype in apoE⫺/⫺ versus C57Bl/6 mice, particularly in male versus female apoE⫺/⫺ mice. Higher plasma cholesterol (mainly non– high-density lipoprotein cholesterol) was observed in male and female apoE⫺/⫺ mice compared with C57Bl/6 controls on HF diet. In addition, apoE⫺/⫺ mice had moderately higher triglyceride levels that were similar in male and female apoE⫺/⫺ mice and independent of the diet (Tables I and II in the online Data Supplement). However, only in male apoE⫺/⫺ and not female apoE⫺/⫺ mice on HF diet did we observe features consistent with insulin resistance (ie, elevated fasting blood glucose and insulin levels), compared with C57Bl/6 controls (Figure 2). Because of the concordance of insulin resistance and the more severe PAH phenotype in male versus female apoE⫺/⫺ mice, we investigated whether the females had higher plasma levels of the insulin-sensitizing adipocytokines adiponectin and leptin. Although the HF diet resulted in a marked increase in adiponectin and leptin levels in control C57Bl/6 mice of both genders, this marked upregulation was absent in apoE⫺/⫺ mice (for adiponectin, see Figure 2A and 2B; for leptin, see Tables I and II in the online Data Supplement). It is therefore possible that the release of adiponectin and leptin from adipocytes in response to HF diet is to some extent apoE dependent. However, female mice of both genotypes had

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TABLE 1. RVSP and Heart Weight Measurements in C57Bl/6 (Control) and ApoEⴚ/ⴚ Mice on Regular Chow and Hemodynamic, Echocardiographic, and Heart Weight Measurements in C57Bl/6 (Control) and ApoEⴚ/ⴚ Mice on HF Diet Control Males

ApoE⫺/⫺ Males

Control Females

ApoE⫺/⫺ Females

P

n

Mice on regular chow RVSP, mm Hg

20.6⫾0.8

23.2⫾0.6

19.7⫾1.5

22.5⫾0.8

RV, mg

26.5⫾1.3‡

24.9⫾1.4†

17.9⫾0.9

18.1⫾0.7

4–5

RV/LV⫹S

0.26⫾0.01

0.30⫾0.01

0.26⫾0.01

0.27⫾0.02

LV⫹S, mg

101.0⫾2.7‡

84.6⫾4.3*

69.2⫾2.7

68.6⫾2.2

CM vs AM,* CM vs CF,‡ AM vs AF*

4–5

20.6⫾0.5

28.9⫾0.6‡

20.5⫾0.9

24.9⫾0.6†

AM vs CM,‡ AM vs AF,† AF vs CF†

4–5

1513⫾125*

AM vs CM,* AF vs CF*

4–5

AF vs CF*

4–5

CM vs CF,‡ AM vs AF†

4–5 4–5

Mice on HF diet Hemodynamics RVSP, mm Hg RV dP/dtmax, mm Hg/s

1132⫾82

RV dP/dtmin, mm Hg/s

⫺951⫾86

1754⫾62* ⫺1396⫾72

950⫾206 ⫺753⫾173

⫺1279⫾96*

Systolic BP, mm Hg

92⫾1.9

99⫾2.6

83⫾5.4

96⫾1.3

4–5

MAP, mm Hg

79⫾1.4

85⫾2.9

77⫾3.5

86⫾1.5

4–5

Diastolic BP, mm Hg

73⫾1.6

77⫾4.4

71⫾3.4

80⫾1.8

4–5

LVEDP, mm Hg

2.4⫾0.5

2.8⫾0.2

2.5⫾0.3

2.7⫾1.2

4–5

398⫾29.6

370⫾22.5

447⫾27

408⫾36

4–5

Echocardiography Heart rate, bpm

4–5

EF, %

74⫾0.7

77.6⫾5.4

70.2⫾2.4

85.7⫾2.6*

AF vs CF*

4–5

FS, %

37.5⫾0.6

42.0⫾6.2

34.3⫾1.9

51.2⫾2.9†

AF vs CF†

4–5

CO, mL/min)

30.0⫾2.1

29.1⫾5.5

24.3⫾2.2

24.7⫾3.8

4–5

LVIDD, mm

3.4⫾0.06

3.4⫾0.28

3.1⫾0.02

3.0⫾0.13

4–5

LVISD, mm

2.1⫾0.02

2.0⫾0.33

2.0⫾0.06

1.5⫾0.16

4–5

Heart weight RV, mg

19.5⫾1.3

33.0⫾4.0†

18.1⫾0.5

18.0⫾1.1

AM vs CM,† AM vs AF†

4

RV/LV⫹S

0.25⫾0.02

0.41⫾0.03‡

0.24⫾0.01

0.29⫾0.01

AM vs CM,‡ AM vs AF†

4

LV⫹S, mg

79.3⫾0.3

81.3⫾8.3*

74.8⫾1.4

60.9⫾2.0

AM vs AF*

4

47.1⫾0.8

44.5⫾0.9

42.4⫾2.6

44.8⫾0.8

4–5

2.1⫾0.5

1.4⫾0.4

2.3⫾0.6

1.2⫾0.2

4–5

Blood HCT, % WBC, 103 cells/␮L

Fifteen-week-old male and female mice on regular chow or HF diet for 11 weeks in normoxia. Statistically significant differences between C57Bl/6 (control) and apoE⫺/⫺ mice of either gender and between genders of the same genotype are indicated. Values are mean⫾SEM. CM indicates control males; AM, apoE⫺/⫺ males; CF, control females; AF, apoE⫺/⫺ females; S, septum; BP, blood pressure; MAP, mean arterial pressure; LVEDP, LV end-diastolic pressure (determined by left carotid artery/LV catheterization); EF, ejection fraction; FS, fractional shortening; CO, cardiac output; LVIDD, LV end-diastolic inner diameter; LVISD, LV end-systolic inner diameter; HCT, hematocrit; and WBC, white blood cell count. *P⬍0.05; †P⬍0.01; ‡P⬍0.001.

⬃50% higher plasma adiponectin levels than their male counterparts (Figure 2A and 2B). Gender differences in leptin levels were difficult to ascertain because of considerable variability in the individual values (Tables I and II in the online Data Supplement). Because testosterone inhibits the secretion of adiponectin in adipocytes,35 we hypothesized that higher adiponectin levels, in association with lack of insulin resistance, may account for the less severe pulmonary vascular phenotype in female apoE⫺/⫺ mice on a HF diet.

PPAR␥ Activation Elevates Plasma Adiponectin, Improves Insulin Sensitivity, and Reverses PAH On the basis of these data, we reasoned that the presence of PAH may be determined by the inability to sufficiently raise adiponectin levels in association with a HF diet (Figure 2A and 2B) and that the severity of the disease may be a function of the

degree of hyperinsulinemia and hyperglycemia (Figure 2C through 2F). We therefore hypothesized that treating the pulmonary hypertensive, insulin-resistant male apoE⫺/⫺ mice with a PPAR␥ agonist to increase plasma adiponectin and improve insulin sensitivity might arrest disease progression or reverse PAH. Thus, we treated 15-week-old male apoE⫺/⫺ and C57Bl/6 control mice on a HF diet with rosiglitazone 10 mg · kg⫺1 · d⫺1 incorporated into their food. The effects of both 4- and 10-week treatments on RVSP, RVH, and metabolic features were assessed. Four-week treatment with rosiglitazone resulted in much higher plasma adiponectin levels in C57Bl/6 control (5-fold) and apoE⫺/⫺ (8-fold) mice compared with untreated animals of the same genotype (Figure 3A). The higher plasma adiponectin in treated versus untreated apoE⫺/⫺ mice was associated with lower blood glucose (control level), indicating improved insulin sensitivity in apoE⫺/⫺ mice treated with rosiglitazone (Figure 3A, 3C, and 3E).

Hansmann et al

A

B

C

D

E

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RVSP in untreated C57Bl/6 and apoE⫺/⫺ mice over this period of time was not significantly different from the baseline values observed in the 15-week-old mice of the initial study (see Figure 1A compared with Figure 3B and Table 1 versus Table 2). In addition to the induction of plasma adiponectin and improvement of insulin sensitivity, a 4-week course of rosiglitazone treatment resulted in lower RVSP, RV mass (RV/LV⫹septum ratio), and percentage of muscularized arteries at the alveolar wall level that were similar to those in C57Bl/6 control mice (Figure 3B, 3D, and 3F). Because we started treatment at a time when the insulin-resistant male apoE⫺/⫺ mice already had elevated RVSP, RVH, and peripheral PA muscularization, our findings indicate that we had induced regression of PAH. Rosiglitazone given for 4 weeks caused no significant differences in systemic blood pressure, heart rate, LV systolic function (fractional shortening, ejection fraction), cardiac output, and RV systolic and diastolic function (Table 2). Other measurements such as hematocrit and white blood cell count also were similar in treated and untreated animals. There was, however, a tendency for rosiglitazone to cause mild LV dilatation and increased LV mass in both C57Bl/6 control and apoE⫺/⫺ mice (Table 2). All the features described above were sustained after a 10-week rosiglitazone treatment period, suggesting that the effect was not transient. However, at this time point, hematocrit was slightly lower in both control and apoE⫺/⫺ mice treated with rosiglitazone (Table III in the online Data Supplement). Decreased hematocrit and a tendency for LV dilatation and increased LV mass have been previously reported in the clinical setting as minor side effects of rosiglitazone treatment.37

Rosiglitazone Does Not Regulate Plasma ET-1 and ADMA in ApoEⴚ/ⴚ Mice

F

G

⫺/⫺

Figure 1. Pulmonary hypertension in apoE mice on HF diet in normoxia. Fifteen-week-old male mice on HF diet for 11 weeks. A, RVSP. B, RVH, measured as ratio of the weight of the right ventricle (RV) to that of left ventricle (LV) plus septum (S). C, Muscularization of alveolar wall arteries. Bars represent mean⫾SEM (n⫽4–5 as indicated in column graphs). *P⬍0.05; **P⬍0.01; and ***P⬍0.001. D and E, Representative photomicrographs of lung tissue (stained by Movat pentachrome) of 15-week-old male mice on HF diet showing a typical nonmuscular peripheral alveolar artery in a C57B1/6 mouse (D). A similar section in the apoE⫺/⫺ mouse shows an alveolar wall artery surrounded by a rim of muscle (E). F and G, Micro-CT imaging of barium-injected pulmonary arteries (PA). Representative irregularly shaped main PA wall is observed in apoE⫺/⫺ mouse on HF diet (arrow), but significant PA stenoses are excluded in C57B1/6 control (F) and apoE⫺/⫺ mice (G).

ET-1 and ADMA were measured in blood plasma after a 4-week treatment with rosiglitazone. Plasma ET-1 levels were lower in treated than in untreated C57Bl/6 mice. However, ET-1 levels were lower in apoE⫺/⫺ compared with C57Bl/6 mice and were not affected by rosiglitazone. ADMA levels were in the normal murine range, not different between genotypes, and not altered by rosiglitazone treatment (Table 2).

Recombinant ApoE and Adiponectin Inhibit PASMC Proliferation To support possible roles of apoE and adiponectin in protecting against the development of PAH, we showed that both proteins inhibit PDGF-BB–induced proliferation of PASMCs harvested from both C57Bl/6 control and apoE⫺/⫺ mice (Figure 4A and 4B).

Discussion Although a link between insulin resistance and systemic cardiovascular disease is evident in both clinical2 and experimental studies,3,33 this report is the first indication that there may be a possible link with PAH. If insulin resistance does contribute to the pathobiology of PAH in humans, it will be an extremely important relationship owing to the steadily increasing number of children, adolescents,1 and adults2 with

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March 13, 2007 Males

B

C

D

E

F

Females

Figure 2. Male but not female apoE⫺/⫺ mice develop insulin resistance and hypoadiponectinemia on HF diet. Plasma adiponectin (A,B), blood glucose (C, D), and plasma insulin (E, F). Overnight-starved 15-week-old male (A, C, E) and female (B, D, F) C57Bl/6 and apoE⫺/⫺ mice, on regular chow or HF diet for 11 weeks. Note that C57Bl/6 mice but not apoE⫺/⫺ mice of both genders upregulate their adiponectin levels when exposed to HF diet. Bars represent mean⫾SEM (n⫽5–10 as indicated in column graphs). *P⬍0.05; **P⬍0.01; and ***P⬍0.001.

the metabolic syndrome, which includes insulin resistance as a key element. ApoE⫺/⫺ mice of both genders on HF showed atheroma on histology and on micro-CT, affecting the large PAs in a nonocclusive manner. Similar features have been described in pathological specimens of adult patients with PAH.38 However, the cholesterol levels and the presence of atheroma were similar in male and female apoE⫺/⫺ mice, but only male apoE⫺/⫺ developed insulin resistance and severe PAH (ie, a combination of marked RVSP elevation, RVH, and enhanced peripheral PA muscularization). We therefore suggest that apoE deficiency, hypoadiponectinemia, and the related insulin resistance, rather than pulmonary atherosclerosis itself, were the major causes of PAH. The level of PAH seen in our model, with an RVSP baseline elevation of 7 to 9 mm Hg over controls for males on a HF diet in normoxia, was not based on LV dysfunction and is comparable to39 or even greater than40,41 that in the relatively few murine models of PAH described to date. The sole exception is the inducible vascular SMC dominantnegative bone-morphogenetic protein receptor II (BMP-RII) transgenic mouse. However, values in the control mice of this

BMP-RII model also were quite elevated, consistent with the relative “hypoxia” at Denver altitude.42 The likely mechanism by which apoE deficiency leads to the development of PAH appears to be facilitated by PDGF-BB signaling. From studies in systemic vascular SMCs, we know that PDGF-BB signaling in SMCs is suppressed when apoE binds to the low-density lipoprotein receptor–related protein (LRP), thereby initiating endocytosis and degradation of the LRP–PDGFR-␤–PDGF-BB complex.28 –30 We have shown here that PDGF-BB–induced proliferation also is suppressed by apoE in PASMCs, suggesting that a similar mechanism may be present in the pulmonary vasculature. In diabetic apoE⫺/⫺ mice, PDGFR-␤ signaling is increased in vascular SMCs, and systemic vascular disease can be reversed by the PDGFR tyrosine kinase inhibitor imatinib.32 We40 and others21 have shown that blockade of tyrosine kinase activity selective to the epidermal growth factor receptor40 or the PDGFR21 reverses experimental PAH and vascular remodeling caused by the toxin monocrotaline in rats. In association with insulin resistance, the male mice (apoE⫺/⫺ and C57Bl/6) had lower levels of adiponectin than

Hansmann et al

A

B

C

D

E

F

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Figure 3. Four-week treatment with the PPAR␥ agonist rosiglitazone reverses PAH, increases plasma adiponectin, and induces insulin sensitivity. Measurements of plasma adiponectin (A), blood glucose (C) and plasma insulin (E), RVSP (B), RVH (D), and muscularization of alveolar wall arteries (F). Nineteen-week-old male C57Bl/6 and apoE⫺/⫺ mice, all on HF diet for 15 weeks, were used. Bars represent mean⫾SEM (n⫽4 –16 as indicated in column graphs). *P⬍0.05; **P⬍0.01; and ***P⬍0.001.

the female mice. This finding is in keeping with a recent study demonstrating that testosterone inhibits the secretion of adiponectin in adipocytes.35 Adiponectin reverses insulin resistance26 and is independently associated with a reduced risk of type 2 diabetes mellitus in apparently healthy individuals.43 Moreover, the high-molecular-weight form of adiponectin binds PDGF-BB, thereby reducing PDGF-BB bioavailability31 and mitogenic postreceptor function in vascular SMCs.27 Adiponectin has been shown to be a transcriptional target of PPAR␥ in adipocytes.7 Because previous studies used the PPAR␥ agonist rosiglitazone to suppress intimal thickening in the apoE⫺/⫺ mouse,44 we reasoned that it might also be effective in preventing disease progression or reversing PAH. Indeed, treatment of male apoE⫺/⫺ mice with rosiglitazone increased plasma adiponectin, improved insulin sensitivity, and led to sustained regression of PAH, RVH, and abnormal muscularization of distal PAs. The source of adiponectin in our animal model is likely from visceral and subcutaneous as well as perivascular adipocytes.17 To further support a mechanistic relationship between apoE and adiponectin deficiency on the one hand and PAH with enhanced muscularization of peripheral arteries on the other, we showed that both recombinant apoE and adiponectin inhibited

PDGF-BB–induced proliferation of cultured murine wildtype and apoE⫺/⫺ PASMCs. In addition to elevating adiponectin levels and thus causing sequestration of PDGF-BB, PPAR␥ activation blocks PDGF gene expression45 and PDGF-BB–mediated systemic SMC proliferation and migration.33,34 This is likely to occur via inhibition of phosphorylated extracellular-regulated kinase nuclear translocation46 and/or induction of protein phosphatases47 that reduce phosphorylated extracellular-regulated kinase. Furthermore, PPAR␥ induces expression of LRP,48 the receptor necessary for apoE-mediated suppression of PDGF-BB signaling.28 –30 By blocking important survival pathways downstream of activated PDGFR-␤ (eg, PI3K),25 rosiglitazone could also induce apoptosis of proliferating vascular cells.33,49 We considered the possibility that PPAR␥ activation might impair the expression of ET-115 and the endogenous nitric oxide synthase inhibitor ADMA,18 both of which have previously been linked to clinical PAH16,19 and insulin resistance.17,18 However, although it is not surprising that rosiglitazone decreased ET-1 levels in the C57Bl/6 mice, we expected the apoE⫺/⫺ mice to have higher levels of ET-1 under control conditions. In the present study, however, we

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TABLE 2. Hemodynamic, Echocardiographic, Heart Weight, Hematocrit, and Other Blood Measurements in Male C57Bl/6 (Control) and ApoEⴚ/ⴚ Mice After Treatment With Rosiglitazone for 4 Weeks Parameter

Control

Control Rosi

ApoE⫺/⫺

ApoE⫺/⫺ Rosi

P

n

23.3⫾0.2

23.8⫾1.2

28.9⫾0.6‡

23.7⫾0.5‡

A vs C,‡ AR vs A‡

4–6

Hemodynamics RVSP, mm Hg RV dP/dtmax, mm Hg/s

1543⫾59

1762⫾170

1489⫾154

1668⫾155

RV dP/dtmin, mm Hg/s

⫺1207⫾104

⫺1575⫾198

⫺1665⫾147

⫺1569⫾128

4–6

103⫾3.4

95⫾2.4

108⫾4.0

100⫾2.3

7–8

MAP, mm Hg

85⫾3.3

78⫾2.6

90⫾4.2

80.6⫾3.2

7–8

Diastolic BP, mm Hg

75⫾3.3

70⫾2.8

81⫾4.4

71⫾3.9

7–8

Systolic BP, mm Hg

4–6

Echocardiography Heart rate, bpm

369⫾15.9

356⫾24.8

295⫾10.5*

334⫾16.6

EF, %

70.0⫾1.3

70.1⫾2.3

69.8⫾1.0

64.4⫾1.1

A vs C*

5–7 5–7

FS, %

34.2⫾1.0

34.5⫾1.8

34.1⫾0.7

30.2⫾0.8

5–7

CO, mL/min

39.0⫾3.2

48.7⫾4.6

32.0⫾2.6

40.8⫾2.5

LVIDD, mm

3.91⫾0.07

4.27⫾0.08*

3.97⫾0.11

4.20⫾0.06

LVISD, mm

2.59⫾0.04

2.80⫾0.11

2.63⫾0.10

2.96⫾0.07

5–7

LVPWd, mm

0.60⫾0.03

0.75⫾0.08

0.72⫾0.05

0.64⫾0.06

5–7

IVSd, mm

0.61⫾0.05

0.58⫾0.04

0.70⫾0.06

0.62⫾0.04

5–7

RV, mg

27.2⫾1.2

30.8⫾3.7

39.5⫾5.6

26.2⫾1.7

5–6

RV/LV⫹S

0.30⫾0.02

A vs C,† AR vs A‡

5–6

LV⫹S, mg

92.2⫾1.4

116.8⫾6.2*

82.5⫾6.4

103.5⫾3.7*

CR vs C,* AR vs A*

5–6

45.7⫾3.5

41.1⫾2.5

49.7⫾1.3

51.2⫾1.5*

AR vs CR*

5.5⫾2.2

3.8⫾1.2

2.0⫾0.4

3.6⫾1.0

5–7 CR vs C*

5–7

Heart weight 0.26⫾0.02

0.47⫾0.04†

0.25⫾0.01‡

Blood HCT, % WBC, 103/␮L ET-1, fmol/L

35.8⫾5.5

12.5⫾1.9

11.5⫾0.9*

14.5⫾1.4*

ADMA, ␮mol/L

0.24⫾0.02

0.29⫾0.05

0.22⫾0.02

0.21⫾0.02

5–7 5–7

A vs C,* CR vs C*

3–5 4–7

Nineteen-week-old male mice on HF diet for 15 weeks, untreated or treated with rosiglitazone for 4 weeks, in normoxia. Statistically significant differences between C57Bl/6 (control) and apoE⫺/⫺ mice of either group and between treated and untreated mice of the same genotype are indicated. ET-1 and ADMA were measured in blood plasma. Systemic blood pressure was measured at 24 weeks of age (ie, after treatment with rosiglitazone for 9 weeks). Values are mean⫾SEM. C indicates control; CR, control treated with rosiglitazone (Rosi); A, ApoE⫺/⫺ ; AR, ApoE⫺/⫺ treated with rosiglitazone; BP, blood pressure; MAP, mean arterial pressure; EF, ejection fraction; FS, fractional shortening; CO, cardiac output; LVIDD, LV end-diastolic inner diameter; LVISD, LV end-systolic inner diameter; LVPWd, LV end-diastolic posterior wall thickness; IVSd, end-diastolic interventricular septum thickness; S, septum; HCT, hematocrit; and WBC, white blood cell count. *P⬍0.05; †P⬍0.01; ‡P⬍0.001.

human PASMCs.50 Loss-of-function mutations in the BMPRII gene frequently occur in cases of familial and idiopathic PAH, and our preliminary results suggest that this would decrease endogenous PPAR␥ activity. Hence, a strategy

could not relate the reversal of PAH with rosiglitazone to a decrease in either ET-1 or ADMA plasma level. Recently, we made the interesting observation that BMP-2 induces nuclear shuttling and DNA binding of PPAR␥ in

A

B

Figure 4. Recombinant apoE (A) and adiponectin (B) inhibit PDGF-BB–induced (20 ng/mL) proliferation of murine PASMC harvested both from C57Bl/6 and apoE⫺/⫺ mice. Bars represent mean⫾SEM (n⫽3). *P⬍0.05; **P⬍0.01; and ***P⬍0.001.

Hansmann et al aimed at activating PPAR␥ could reverse the PAH phenotype. However, the inheritance pattern of BMPR-II is that of a dominant gene with low penetrance in that only ⬇20% of affected family members develop the disease.51 This underscores the importance of environmental modifiers like insulin resistance that could potentiate BMP-RII dysfunction. It also is possible that abnormalities in downstream effectors of BMP-2 such as PPAR␥ and its targets may contribute to the development of disease. The present study identified several new factors that may be involved in the pathogenesis of PAH and a potential treatment. We acknowledge that it is difficult to separate the effects of apoE deficiency and insulin resistance in our animal model and to extrapolate the potential impact of a successful experimental treatment to human disease. Therefore, it is important to examine whether insulin resistance, hyperlipidemia, impaired apoE function, and low adiponectin levels are risk factors for the progression of PAH in humans. It will also be of interest to investigate a relationship between molecular mechanisms that underlie insulin resistance, apoE and adiponectin levels, and known PAH pathways (eg, those downstream of BMP-RII). Taken together, these data might suggest a beneficial effect from the addition of PPAR␥ agonists to the treatment regimen for PAH patients.

9. 10.

11.

12.

13.

14.

15.

16.

Acknowledgments We thank Tim Doyle for help with micro-CT imaging and Grant Hoyt for excellent technical assistance.

17.

Sources of Funding

18.

This work was supported by NIH (1-R01-HL074186 – 01) and the Dwight and Vera Dunlevie Endowed Professorship (Dr Rabinovitch), a postdoctoral fellowship from the American Heart Association/ Pulmonary Hypertension Association (0425943H to Dr Hansmann), NIH grant HL876445– 03 (Dr Wagner), and a predoctoral fellowship from Boehringer Ingelheim Funds (S. Schellong).

19.

Disclosures

20.

None.

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27. Arita Y, Kihara S, Ouchi N, Maeda K, Kuriyama H, Okamoto Y, Kumada M, Hotta K, Nishida M, Takahashi M, Nakamura T, Shimomura I, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y. Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell. Circulation. 2002;105:2893–2898. 28. Boucher P, Gotthardt M, Li WP, Anderson RG, Herz J. LRP: role in vascular wall integrity and protection from atherosclerosis. Science. 2003; 300:329 –332. 29. Boucher P, Gotthardt M. LRP and PDGF signaling: a pathway to atherosclerosis. Trends Cardiovasc Med. 2004;14:55– 60. 30. Newton CS, Loukinova E, Mikhailenko I, Ranganathan S, Gao Y, Haudenschild C, Strickland DK. Platelet-derived growth factor receptor-beta (PDGFR-beta) activation promotes its association with the low density lipoprotein receptorrelated protein (LRP): evidence for co-receptor function. J Biol Chem. 2005;280: 27872–27878. 31. Wang Y, Lam KS, Xu JY, Lu G, Xu LY, Cooper GJ, Xu A. Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerizationdependent manner. J Biol Chem. 2005;280:18341–18347. 32. Lassila M, Allen TJ, Cao Z, Thallas V, Jandeleit-Dahm KA, Candido R, Cooper ME. Imatinib attenuates diabetes-associated atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24:935–942. 33. Marx NN, Duez HH, Fruchart JCJ-C, Staels BB. Peroxisome proliferatoractivated receptors and atherogenesis: regulators of gene expression in vascular cells. Circ Res. 2004;94:1168 –1178. 34. Law RRE, Goetze SS, Xi XXP, Jackson SS, Kawano YY, Demer LL, Fishbein MMC, Meehan WWP, Hsueh WWA. Expression and function of PPARgamma in rat and human vascular smooth muscle cells. Circulation. 2000;101:1311–1318. 35. Xu A, Chan KW, Hoo RL, Wang Y, Tan KC, Zhang J, Chen B, Lam M, Tse C, Cooper GJ, Lam KS. Testosterone selectively reduces the high molecular weight form of adiponectin by inhibiting its secretion from adipocytes. J Biol Chem. 2005;280:18073–18080. 36. Fouty BW, Grimison B, Fagan KA, Le Cras TD, Harral JW, Hoedt-Miller M, Sclafani RA, Rodman DM. p27(Kip1) is important in modulating pulmonary artery smooth muscle cell proliferation. Am J Respir Cell Mol Biol. 2001;25:652– 658. 37. St John Sutton M, Rendell M, Dandona P, Dole JF, Murphy K, Patwardhan R, Patel J, Freed M. A comparison of the effects of rosiglitazone and glyburide on cardiovascular function and glycemic control in patients with type 2 diabetes. Diabetes Care. 2002;25:2058 –2064. 38. Wagenvoort CA. Pulmonary atherosclerosis. In: Wagenvoort CA, Heath D, Edwards JE, eds. The Pathology of the Pulmonary Vasculature. Springfield, Ill: Charles C Thomas; 1964:58 –59. 39. Guignabert C, Izikki M, Tu LI, Li Z, Zadigue P, Barlier-Mur AM, Hanoun N, Rodman D, Hamon M, Adnot S, Eddahibi S. Transgenic mice overexpressing the 5-hydroxytryptamine transporter gene in smooth muscle develop pulmonary hypertension. Circ Res. 2006;98:1323–1330. 40. Merklinger SL, Jones PL, Martinez EC, Rabinovitch M. Epidermal growth factor receptor blockade mediates smooth muscle cell apoptosis

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and improves survival in rats with pulmonary hypertension. Circulation. 2005;112:423– 431. Long L, MacLean MR, Jeffery TK, Morecroft I, Yang X, Rudarakanchana N, Southwood M, James V, Trembath RC, Morrell NW. Serotonin increases susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ Res. 2006;98:818 – 827. West J, Fagan K, Steudel W, Fouty B, Lane K, Harral J, Hoedt-Miller M, Tada Y, Ozimek J, Tuder R, Rodman DM. Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ Res. 2004;94:1109 –1114. Spranger J, Kroke A, Möhlig M, Bergmann MM, Ristow M, Boeing H, Pfeiffer AF. Adiponectin and protection against type 2 diabetes mellitus. Lancet. 2003;361:226 –228. Phillips JW, Barringhaus KG, Sanders JM, Yang Z, Chen M, Hesselbacher SS, Czarnik AC, Ley K, Nadler J, Sarembock IJ. Rosiglitazone reduces the accelerated neointima formation after arterial injury in a mouse injury model of type 2 diabetes. Circulation. 2003;108:1994 –1999. Zhang J, Fu M, Zhao L, Chen YE. 15-Deoxy-prostaglandin J(2) inhibits PDGF-A and -B chain expression in human vascular endothelial cells independent of PPAR gamma. Biochem Biophys Res Commun. 2002;298:128–132. Goetze S, Kintscher U, Kim S, Meehan WP, Kaneshiro K, Collins AR, Fleck E, Hsueh WA, Law RE. Peroxisome proliferator-activated receptor-gamma ligands inhibit nuclear but not cytosolic extracellular signal-regulated kinase/mitogen-activated protein kinase-regulated steps in vascular smooth muscle cell migration. J Cardiovasc Pharmacol. 2001;38:909 –921. Wakino S, Kintscher U, Liu Z, Kim S, Yin F, Ohba M, Kuroki T, Schonthal AH, Hsueh WA, Law RE. Peroxisome proliferator-activated receptor gamma ligands inhibit mitogenic induction of p21(Cip1) by modulating the protein kinase Cdelta pathway in vascular smooth muscle cells. J Biol Chem. 2001;276:47650 – 47657. Gauthier A, Vassiliou G, Benoist F, McPherson R. Adipocyte low density lipoprotein receptor-related protein gene expression and function is regulated by peroxisome proliferator-activated receptor gamma. J Biol Chem. 2003;278:11945–11953. Bruemmer D, Yin F, Liu J, Berger JP, Sakai T, Blaschke F, Fleck E, Van Herle AJ, Forman BM, Law RE. Regulation of the growth arrest and DNA damage-inducible gene 45 (GADD45) by peroxisome proliferatoractivated receptor gamma in vascular smooth muscle cells. Circ Res. 2003;93:e38 – e47. Hansmann G, Rabinovitch M. Bone morphogenetic protein 2 (BMP-2) activates the transcription factor, peroxisome proliferator-activated receptor gamma (PPAR␥), in human pulmonary artery smooth muscle cells (HPASMC). Circulation. 2005;112:II-154. Abstract. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA 3rd, Loyd JE, Nichols WC, Trembath RC. Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension: the International PPH Consortium. Nat Genet. 2000;26:81– 84.

CLINICAL PERSPECTIVE Recent studies document an increased incidence of metabolic syndrome and associated insulin resistance in children, adolescents, and adults, placing them at risk for systemic cardiovascular disease. Insulin resistance also may be linked to pulmonary arterial hypertension (PAH) because reduced expression of apolipoprotein E (apoE) and peroxisome proliferator–activated receptor-␥ (PPAR␥), which is associated with insulin resistance, is observed in lung tissues from PAH patients. Decreased levels of apoE and the PPAR␥ target adiponectin may enhance platelet-derived growth factor-BB signaling in pulmonary artery smooth muscle cells in a manner similar to that observed in systemic smooth muscle cells. In keeping with these observations, we now report a novel animal model in which PAH is linked to insulin resistance and reversed by PPAR␥ activation. Male apoE-deficient (apoE⫺/⫺) mice on a high-fat diet do not upregulate the insulin sensitizers adiponectin and leptin (in contrast to control mice) but instead develop insulin resistance and severe PAH. Female apoE⫺/⫺ mice on high-fat diet have higher adiponectin levels and less severe PAH than male apoE⫺/⫺ mice. A 4-week treatment with the PPAR␥ agonist rosiglitazone led to an 8-fold increase in plasma adiponectin, improved insulin sensitivity, and complete regression of PAH in male insulin-resistant apoE⫺/⫺ mice. We also document that apoE and adiponectin inhibit platelet-derived growth factor-BB–induced proliferation of pulmonary artery smooth muscle cells in culture. Our data suggest that insulin resistance, low plasma adiponectin levels, and apoE deficiency may be risk factors for PAH that can be reversed by PPAR␥ activation. Hence, PPAR␥ agonists could be given consideration in the treatment of PAH patients, particularly those with documented insulin resistance.

Contemporary Reviews in Cardiovascular Medicine Endothelial Function and Dysfunction Testing and Clinical Relevance John E. Deanfield, MB, BCh, FRCP; Julian P. Halcox, MD, MA, MRCP; Ton J. Rabelink, MD, PhD

A

therosclerosis begins in childhood, progresses silently through a long preclinical stage, and eventually manifests clinically, usually from middle age. Over the last 30 years, it has become clear that the initiation and progression of disease, and its later activation to increase the risk of morbid events, depends on profound dynamic changes in vascular biology.1 The endothelium has emerged as the key regulator of vascular homeostasis, in that it has not merely a barrier function but also acts as an active signal transducer for circulating influences that modify the vessel wall phenotype.2 Alteration in endothelial function precedes the development of morphological atherosclerotic changes and can also contribute to lesion development and later clinical complications.3 Appreciation of the central role of the endothelium throughout the atherosclerotic disease process has led to the development of a range of methods to test different aspects of its function, which include measures of both endothelial injury and repair. These have provided not only novel insights into pathophysiology, but also a clinical opportunity to detect early disease, quantify risk, judge response to interventions designed to prevent progression of early disease, and reduce later adverse events in patients. The present review summarizes current understanding of endothelial biology in health and disease, the strengths and weaknesses of current testing strategies, and their potential applications in clinical research and patient care.

the remodeling of vascular structure and long-term organ perfusion.4 The pioneering experiments of Furchgott and Zawadzki first demonstrated an endothelium-derived relaxing factor that was subsequently shown to be nitric oxide (NO).5 NO is generated from L-arginine by the action of endothelial NO synthase (eNOS) in the presence of cofactors such as tetrahydrobiopterin.6 This gas diffuses to the vascular smooth muscle cells and activates guanylate cyclase, which leads to cGMP-mediated vasodilatation. Shear stress is a key activator of eNOS in normal physiology, and this adapts organ perfusion to changes in cardiac output.7 In addition, the enzyme may be activated by signaling molecules such as bradykinin, adenosine, vascular endothelial growth factor (in response to hypoxia), and serotonin (released during platelet aggregation).8 The endothelium also mediates hyperpolarization of vascular smooth muscle cells via an NO-independent pathway, which increases potassium conductance and subsequent propagation of depolarization of vascular smooth muscle cells, to maintain vasodilator tone.9 The endothelium-derived hyperpolarizing factors involved in this process are only partially understood (such as the cytochrome-derived factors and possibly C-type natriuretic peptide), and may differ between vascular beds. However, it is well recognized that Endothelium-Derived Hyperpolarizing Factor can compensate for loss of NO-mediated vasodilator tone, particularly in the microcirculation, and this appears important when NO bioavailability is reduced.10 Prostacyclin, derived by the action of the cyclooxygenase system, is another endothelium-derived vasodilator that acts independently of NO.11 Although it may contribute to some of the other regulatory roles of the endothelium, it appears to have a more limited role in the maintenance of vasodilator tone in humans. The endothelium modulates vasomotion, not only by release of vasodilator substances, but also by an increase in constrictor tone via generation of endothelin and vasoconstrictor prostanoids, as well as via conversion of angiotensin I to angiotensin II at the endothelial surface.12,13 These vasoconstrictor agents predominantly act locally, but may also exert some systemic effects and have a role in the regulation of arterial structure and remodeling.

Endothelium in Normal Vascular Homeostasis Although only a simple monolayer, the healthy endothelium is optimally placed and is able to respond to physical and chemical signals by production of a wide range of factors that regulate vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and vessel wall inflammation. The importance of the endothelium was first recognized by its effect on vascular tone. This is achieved by production and release of several vasoactive molecules that relax or constrict the vessel, as well as by response to and modification of circulating vasoactive mediators such as bradykinin and thrombin. This vasomotion plays a direct role in the balance of tissue oxygen supply and metabolic demand by regulation of vessel tone and diameter, and is also involved in

From the Vascular Physiology Unit (J.E.D., J.P.H.), UCL Institute of Child Health, London, UK; and the Department of Nephrology (T.R.), Leiden University Medical Center, Leiden, The Netherlands. Correspondence to Dr. John E Deanfield, Professor of Cardiology, Vascular Physiology Unit, Institute of Child Health, and Great Ormond Street Hospital for Children NHS Trust, 30 Guilford St, London WC1N 3EH, UK. E-mail [email protected] (Circulation. 2007;115:1285-1295.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.652859

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Figure 4. Images obtained from a 42-year-old man with severe angina of recent onset, who had recently developed hypertension. A, Selective angiogram of the RCA in the left anterior oblique projection. In this view, the proximal course appears wider than the more distal vessel, and it originates next to the LCA (which is also ectopic), high above the left aortic sinus (LS). B, IVUS image of the distal RCA. The cross-sectional area is 10.8 mm2, and the shape is circular. C, IVUS image of the proximal segment of the RCA, whose lumen is severely compressed laterally (minimal diameter, 1.5 mm; maximal diameter, 3.8 mm; cross-sectional area, 4.2 mm2; area stenosis, 61%). The LCA had also milder ostial stenosis. D, IVUS of the proximal RCA after stent angioplasty (3.5 ⫻ 12 mm; postdilated at 18 atm). The shape has become round, and the area has expanded to match that of the distal normal vessel.

Outlines for Diagnostic and Treatment Protocols In carriers of ACAOS, the clinical histories are consistent in only 1 aspect: Either these patients die suddenly (typically at a young age and after extreme exertion), or they have no characteristic presentation. Most patients are asymptomatic for a large portion of their lives, and an atypical chest-pain syndrome is the most common reason they are referred for coronary angiography, which is when the diagnosis is typically made. The milder cases are more likely to be identified fortuitously (because of a falsely positive stress test and/or coincidental atherosclerotic disease). The fact that CAAs include many different entities and that no single observer or group has collected a large enough series to clarify the natural prognosis of each entity may contribute to our difficulty in the clinical identification of these lesions, especially the ones that could lead to angina or sudden cardiac death.21 For most types of coronary anomalies, the fundamental clinical approach could be: “Do not bother to look for these innocent anomalies, but be prepared to recognize them as benign if one is accidentally found,

typically at coronary angiography.” However, for a few CAAs that are possibly or predictably malignant (fundamentally, ACAOS), we should establish solid diagnostic screening protocols, especially for athletes and other young individuals subjected to extreme exertion.9,10,29,33 As noted above, ACAOS patients can succumb to sudden cardiac death, usually but not necessarily at a young age, possibly even at the newborn stage.36 Retrospectively reviewed, only a few persons reported to have died of ACAOS had significant symptoms, usually atypical chest pain, dyspnea, syncope, or their equivalents, before the final event.5–7,9,10,13–15 A specific workup protocol is indicated mostly for athletes and military personnel with these symptoms. In view of the fairly rare nature of ACAOS, it would not seem practical or cost-effective to extend the indications for such a workup to all schoolchildren on a routine basis. Nevertheless, larger prospective studies are needed before this decision can become final.37–39 In patients with suspected ACAOS, testing should sequentially include electrocardiography, Holter monitoring (basi-

Angelini TABLE 4.

Coronary Artery Anomalies

1301

Pathophysiological Mechanisms and Coronary Anomalies (Functional Classification) Proof of Action

Pathophysiological Mechanism Misdiagnosis

Coronary Anomaly “Missing” coronary artery

Certain

“Hypoplastic” coronary artery Myocardial ischemia, primary (fixed and/or episodic)

x

Ostial stenosis

x

Coronary fistula

x x

Muscular bridge Myocardial ischemia, secondary (episodic)

Increased risk of fixed coronary atherosclerotic disease

Secondary aortic valve disease

x

Tangential origin (ACAOS) intramural course

x

Myocardial bridge, plus spasm and/or clot

x

Coronary ectasia (plus mural clot)

x

Coronary fistula (plus mural clot)

x

Coronary fistula

x

ALCAPA

x

Coronary ectasia

x

Muscular bridge (proximal to)

x

Coronary aneurysm (ostial)

x

Coronary fistula

x

ALCAPA

x

Increased risk of bacterial endocarditis

Coronary fistula

Ischemic cardiomyopathy (hibernation)

ALCAPA

x

Volume overload

Coronary fistula

x

ALCAPA

x

Ectopic ostia (tangential)

x

Unusual technical difficulties during coronary angiography or angioplasty

Complications during cardiac surgery

Unlikely

x

Ostial atresia

ALCAPA

Possible

x

x

Split left coronary artery

x

Coronary fistula

x

Ectopic ostia and proximal course

x

Muscular bridge

x

ALCAPA indicates anomalous origination of the left coronary artery from the pulmonary artery. Adapted from Angelini P et al10 with permission from Lippincott, Williams & Wilkins. Copyright 1999.

tion), changes in the aortic pressure (as at the onset of hypertension or aortic regurgitation), or a rapid weight gain, especially in patients who receive negative chronotropic agents, which increase the stroke volume if the cardiac output remains essentially unchanged. Moreover, a treadmill stress test, which should be transformed into an adenosine test because of an inadequate effort or chronotropic response, may be the most accurate predictive test for ACAOS because it associates an increased cardiac output with nonphysiological bradycardia. Unfortunately, though, such a hybrid protocol is a potential cause of sudden death, specifically in ACAOS carriers, and should generally be avoided or at least closely monitored in a hospital environment. When a carrier of ACAOS dies suddenly, in the absence of other lethal cardiovascular conditions, a low cardiac output and bradycardia or asystole typically occur early after extreme exercise, after which syncope and/or death ensues. Terminal ventricular fibrillation may also occur as a manifestation of critical ischemia or of reperfusion arrhythmia.30 –32

Both the anomalous right and left coronary arteries can be responsible for sudden death, although the risk has not been adequately quantified in specific studies. Most likely, predisposing factors include the severity of baseline stenosis, the specific conditions at the time of the crisis, and the myocardial territory at risk.7,33 Additionally, one must realize that the possible manifestations of ACAOS include not only sudden death but also dyspnea, palpitations, angina pectoris, dizziness, and syncope.4,10,12,26,32 Whereas sudden death is usually associated with extreme exercise in young adults,34 the other manifestations of ACAOS are more frequently seen in older adults (in our experience, specifically women) and are related to the onset of hypertension. Interestingly, Cheitlin33 claimed that sudden death is seen only in young patients, possibly because of progressive hardening of the aortic wall in adults. During aortic valve replacement, an intramural ectopic coronary artery can also be liable to critical worsening of extrinsic compression by the prosthetic ring, as recently reviewed by Morimoto and colleagues.35

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Methods for Clinical Assessment of Endothelial Function Technique (Outcome Measure)

Reproducible*

Reflects Biology

Reversible

Predicts Outcome†

Cardiac catheterization (change in diameter, change in coronary blood flow)

Noninvasive ⫺

Repeatable ⫺

⫹/⫺







Venous occlusion plethysmography (change in forearm blood flow)



⫹/⫺

⫹/⫺







Ultrasound FMD (change in brachial artery diameter)





⫹/⫺





⫹‡

PWA (change in augmentation index)





⫹/⫺







PCA (change in reflective index)





⫹/⫺







PAT (change in pulse amplitude)





⫹/⫺







⫹ indicates supportive evidence in literature; ⫺, insufficient evidence; FMD, flow-mediated dilatation; PWA, pulse wave analysis; PCA, pulse contour analysis; and PAT, pulse amplitude tonometry. *Reproducibility of PWA, PCA, and PAT has been less extensively investigated than FMD. †Studies that link PWA, PCA, and PAT to outcome have not yet been reported. ‡FMD is currently the standard for noninvasive assessment of conduit artery endothelial function because there is considerable clinical trial experience, validation, a firm link to biology, and association with cardiovascular events.

safe, noninvasive, reproducible, repeatable, cheap, and standardized between laboratories. The results should also reflect the dynamic biology of the endothelium throughout the natural history of atherosclerotic disease, define subclinical disease processes, as well as provide prognostic information for risk stratification in the later clinical phase. No single test currently fulfils these requirements, and a panel of several tests is therefore needed to characterize the multiple facets of endothelial biology. The advantages and disadvantages of the available methods are summarized in the Table.

Endothelial-Dependent Vasomotion Endothelial-dependent vasomotion has been the most widely used clinical end point for assessment of endothelial function. Testing involves pharmacological and/or physiological stimulation of endothelial release of NO and other vasoactive compounds, and often a comparison of vascular responses to endothelium-independent dilators such as nitroglycerine. Determination of local NO bioavailability not only reflects its influence on vascular tone, but also the other important functions of this molecule, which include thromboregulation, cell adhesion, and proliferation. Initial clinical studies of endothelial function were undertaken in the coronary circulation, and involved local infusion of acetylcholine with measurement of the change in vessel diameter by quantitative coronary angiography.41,42 This approach is a direct clinical analog of Furchgott and Zawadzki’s original experiment. Acetylcholine releases NO from vessels with an intact endothelium, which leads to vasodilatation, but causes vasoconstriction in subjects with endothelial dysfunction, as a result of a direct muscarinic smooth muscle vasoconstrictor effect. Doses that result in final blood concentrations in the range of 10⫺8 to 10⫺5 mol are the most appropriate for assessment of the physiological range of responses.43 Subsequently, these methods have been refined with use of the Doppler flow wires to measure resistance vessel function.44 Responses to a wide range of endothelial agonists that include substance P, adenosine, and bradykinin have also been measured, as well as physiological responses to cold-pressor testing and flow-mediated dilatation (FMD)

of proximal conduit arteries as a result of distal infusion of adenosine.45 In addition, use of specific NO antagonists such as L-NMMA has defined the contribution of NO to these vasomotor responses.46 These studies have provided important insights into the vascular effects of risk factors and the potential reversibility of endothelial dysfunction in response to interventions such as statins and angiotensin-converting enzyme inhibitors.47,48 Although these tests directly assess the coronary circulation, their invasive nature limits their use to patients with advanced disease, and precludes repeated testing during serial follow-up. Because endothelial dysfunction is a systemic process, however, a less invasive approach has been developed that utilizes the same principles of local infusion of pharmacological probes and measurement of changes in forearm resistance vessel tone by venous occlusion plethysmography.49 This has provided an opportunity to evaluate endothelial pathophysiology during the preclinical stage of disease by use of appropriate agonists and antagonists with construction of dose-response curves. A correlation between acetylcholine responses in the coronary circulation and in the forearm has been demonstrated. Venous occlusion plethysmography has been widely used, but it is invasive in that it requires arterial cannulation. This limits its repeatability, and prohibits its use in larger studies. Results are also difficult to standardize because baseline resistance vessel tone is variable, and testing protocols and set-up differ between research laboratories. The clinical relevance to atherosclerosis is also uncertain because microvascular pathophysiology does not necessarily reflect changes in the conduit arteries that are particularly predisposed to develop disease. For all these reasons, we reported in 1992 a noninvasive ultrasound-based test to assess conduit artery vascular function in the systemic circulation50 (Figure 4). In this method, brachial artery diameter is measured before and after an increase in shear stress that is induced by reactive hyperemia (FMD). When a sphygmomanometer cuff placed on the forearm distal to the brachial artery is inflated to 200 mm Hg and subsequently released 4 to 5 minutes later, FMD occurs predominantly as a result of local endothelial release of NO.51

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Figure 4. FMD of the brachial artery. A, Ultrasound probe held in stereotactic clamp with micrometer adjustment. B, Continuous measurement of brachial artery diameter (end-diastolic images obtained every 3 seconds), before, during, and after inflation and release of sphygmomanometer cuff on forearm. C, Relationship of FMD to coronary risk factors in 500 asymptomatic adults. Reproduced from Celermajer et al,21 copyright © 1994, with permission from the American College of Cardiology Foundation. D, Impact of diet and exercise on FMD in overweight Chinese teenagers over 6 weeks and 1 year. Reproduced from Woo et al54 with permission from Lippincott, Williams & Wilkins. Copyright © 2004, American Heart Association.

As in the coronary circulation, this brachial artery response can be contrasted to the endothelium-independent dilator response to sublingual nitroglycerine.50 This method is technically demanding, but can be standardized to yield reproducible results that correlate with coronary vascular endothelial function.52,53 Modern software development has allowed for continuous assessment of arterial diameter and blood flow throughout the whole protocol by use of accurate edge detection algorithms that can be manually edited. It is important to note that variations in technique, such as the position of the occluding cuff and duration of inflation, may produce results that are less representative of local NO activity. Brachial artery FMD has been studied widely in clinical research as it enables serial evaluation of young subjects, including children. It also permits testing of lifestyle and pharmacological interventions on endothelial biology at an early preclinical stage, when the disease process is most likely to be reversible.54 This test represents the gold standard for clinical research on conduit artery endothelial biology, and has opened up a new field of vascular epidemiology (see below). There are, however, practical challenges that need to be overcome before this technique could be suitable for use in routine clinical practice.52 These challenges include the need for highly trained operators, the expense of the equipment, and also the care required to minimize the effect of environmental or physiological influences, such as exercise, eating, caffeine ingestion, and important variations in temperature.

FMD is also determined, in part, by the magnitude of postischemic vasodilatation, which makes it also a measure of microcirculatory function.55 A number of alternative noninvasive approaches have been developed recently to study vascular biology in the peripheral circulation. These rely on the ability of the ␤2 agonist salbutamol to reduce arterial stiffness in an NO-dependent manner without significant reduction in blood pressure when given by inhaler at standard clinical doses.56 Changes in arterial stiffness can be measured with pulse wave analysis by radial artery tonometry or pulse contour analysis by digital photoplethysmography.56 The changes in augmentation index and reflection index are measured from the peripheral arterial waveform, and a central aortic waveform can be derived from pulse wave analysis data by a transfer function that has been validated in adults.57 Similarly, reactive hyperemia has been used to elicit changes in conduit artery pulse wave velocity and digital pulse volume that can be measured by oscillometry to identify limb arterial pulse pressure, wave form, timing, and also digital pulse amplitude tonometry.59,60 Several of these methods have been validated as measures of NO bioavailability. They have been shown to change with exposure to risk factors and with atherosclerotic disease, and may complement FMD testing.56 –59 The relative contribution of structural alterations in the vessel wall and endothelialdependent biology remains uncertain, however. Further validation is required, inclusive of a wider study of their

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reproducibility in different age groups and stages of disease, as well as clarification of their relationships with other established measures of endothelial function.

Circulating Markers of Endothelial Function A broader appreciation of the numerous functions of the endothelium can be obtained by study of the levels of molecules of endothelial origin in circulating blood. These include direct products of endothelial cells that change when the endothelium is activated, such as measures of NO biology, inflammatory cytokines, adhesion molecules, regulators of thrombosis, as well as markers of endothelial damage and repair. Many of these circulating markers are difficult and expensive to measure, and currently are only used in the clinical research setting. In this context, these measures can provide important information regarding mechanisms and severity of endothelial dysfunction in populations, and complement physiological tests of endothelial vascular control.61 As a result of biological and assay availability and variability, these factors currently have only a very limited role in the assessment of individual patients. Circulating levels of nitrites and nitrosylated proteins in part reflect endothelial generation of NO, but are difficult to measure and may not always represent endothelial NO production.62 Specifically, values may be confounded by the formation of adducts from other nitrogen-containing species, other sources of NO, and wide variation in dietary NO. Asymmetric dimethylarginine is an endogenously derived competitive antagonist of NO synthase. Levels are elevated in subjects with risk factors, such as dyslipidemia and hypertension, as well as in subjects with disease states associated with increased risk of atherosclerosis, such as diabetes and renal failure. Increased levels of asymmetric dimethylarginine are associated with a reduction in NO bioavailability in both animal and clinical studies.63 This increase in asymmetric dimethylarginine is, in part, caused by reduced activity of its breakdown enzyme dimethylarginine dimethylaminohydrolase, which is exquisitely sensitive to the altered cellular redox conditions that accompany risk factors and inflammation.64 Because asymmetric dimethylarginine levels have been linked to preclinical atherosclerotic disease burden and an adverse outcome, they may well prove to be a useful measure of endothelial status and a potential marker of risk in clinical practice.65 At present, however, the assay remains challenging and expensive. Endothelial cell activation leads to increased expression of inflammatory cytokines and adhesion molecules that trigger leukocyte homing, adhesion, and migration into the subendothelial space, which are processes fundamental to atherosclerotic lesion initiation, progression, and destabilization. Wellcharacterized molecules that can be measured in the circulation with commercial immunoassays include E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and P-selectin.66,67 Many of these molecules arise from multiple sources, which are not all clear, but E-selectin is probably the most specific for endothelial cell activation. Levels increase in association with cardiovascular risk factors, and have been associated with structural

and functional measures of atherosclerotic disease, as well as with adverse cardiovascular prognosis.68,69 Similarly, the procoagulant consequences of endothelial activation can be measured as a change in the balance of tissue plasminogen activator and its endogenous inhibitor, plasminogen activation inhibitor-1.70 Furthermore, von Willebrand factor, a largely endothelium-derived glycoprotein, is released into the circulation by activated endothelial cells. This agent has a function in further cellular activation as well as promotion of coagulation and platelet activation, and can be measured relatively easily.71,72 Appreciation that endothelial function reflects the net balance between injury and repair has led to the development of assays to quantify the detachment of mature endothelial cells and derived microparticles to represent the degree of damage, as well as determination of the number and functional characteristics of circulating endothelial progenitor cells to reflect the endogenous repair potential. Circulating endothelial cells that detach in the context of endothelial activation and loss of integrity can be measured in the circulation by both flow cytometry and a combination of magnetic bead selection and fluorescent microscopy.73 Mature circulating endothelial cells can be distinguished from circulating endothelial progenitor cells by virtue of their size and the expression of surface markers.74 The increased levels of circulating endothelial cells in patients with atherosclerotic disease and vascular inflammation suggests a direct relationship between the number of these cells in the peripheral circulation and the extent of endothelial injury. Endothelial microparticles are vesicles formed by the cell membrane after endothelial activation, and their composition can be used to characterize the status of the parent endothelial cell. Elevated circulating microparticles have been seen in a variety of conditions associated with endothelial activation or apoptosis.75,76 Their function is unclear, but they may not merely be markers of the state of the endothelium. They may also be diffusible mediators of molecules involved in cell signaling, and thus themselves may be proinflammatory.77 These observations are exciting, but progress in the understanding of their pathophysiological role as well as in quantification is required before measurement of endothelium-derived microparticles becomes part of clinical practice. Circulating endothelial progenitor cells can be characterized by the expression of characteristic surface markers, which are detectable by flow cytometry, but because a wide range of hematopoietic progenitor cells, which include abundantly present myeloid precursors, has the potential to adopt an endothelial phenotype, the specificity of these measurements is controversial.78 Further methods to characterize circulating endothelial progenitor cell biology include quantification of the potential to differentiate into an endothelial cell phenotype, as well as determination of functional characteristics, which include migration toward a chemical stimulus (eg, stromal cell– derived factor 1, vascular endothelial growth factor) adhesion, formation of vascular tubules, and the ability to attenuate ischemia in animal models.79,80 Thus, measurement of circulating endothelial cells and circulating endothelial progenitor cell levels provides a novel and exciting means to follow the determinants of endothelial

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Figure 5. Relationship between different measures of endothelial function and cardiovascular outcome. A, Intracoronary testing with acetylcholine in 308 patients referred for cardiac catheterization. Ach indicates acetylcholine; VC-Ach, vasoconstriction to Ach; VD-Ach, vasodilatation to Ach. Reproduced from Halcox et al85 with permission from Lippincott, Williams & Wilkins. Copyright © 2002, American Heart Association. B, FMD of the brachial artery in 199 patients undergoing vascular surgery. Reproduced from Gokce et al,100 copyright © 2003, with permission from the American College of Cardiology Foundation. C, Event-free survival in 519 patients with coronary disease according to levels of circulating CD34⫹KDR⫹ endothelial progenitor cells at enrollment. Reproduced from Werner and Nickenig81 with permission from the Foundation for Cellular and Molecular Medicine. Copyright © 2006.

injury and repair. Although the balance of these 2 cell populations has already been linked to other in vivo measures of endothelial function, and has been shown to be associated with future cardiovascular events,81 these novel measures still remain far from clinical use. Nevertheless, it is likely that important new insights into evolution of disease and potential treatment opportunities will emerge from this rapidly developing field.

Clinical Applications Current evidence suggests that endothelial function is an integrative marker of the net effects of damage from traditional and emerging risk factors on the arterial wall and its intrinsic capacity for repair. This endothelial-dependent vascular biology is critical, not only in the initiation and progression of atherosclerosis, but also in the transition from a stable to an unstable disease state with attendant risks (Figure 5). As a result, study of endothelial function in clinical research has emerged as an important end point that complements measurement of circulating risk factors, imaging techniques for structural arterial diseases burden (such as carotid intima media thickness, intravascular ultrasound, computed tomography), and traditional cardiovascular clinical outcomes. In patients with established atherosclerosis, disturbed vasomotion associated with endothelial activation may contribute to transient myocardial ischemia and angina pectoris.82 It is also associated with changes in plaque composition and biology, which may influence plaque stability.83 It should be appreciated that endothelial function, unlike measures of vessel wall morphology, has intrinsic biological variability, and thus a single measurement, much like blood pressure, may give only a snapshot and limited information. Nevertheless, several studies have shown that a single measurement of endothelial function in both the coronary and peripheral circulation can be of prognostic value in a number of different clinical cohorts, which includes patients with established coronary disease and those with atypical symptoms.84,85 A

number of issues, however, require further investigation in larger prospective studies. These include the incremental predictive value of this approach above other established risk markers, the applicability to the general population, and the most appropriate testing profile, which might include a combination of tests for circulating endothelial biomarkers and vasomotor responses. Strategies to reverse endothelial function have now been examined in a wide range of patients with vascular disease. Benefit has been shown with a number of pharmacological interventions, which include drugs that lower lipids and blood pressure, as well as with novel therapies based on new understanding of endothelial biology (eg, dietary supplementation with L-arginine).86 These have mostly,47,87 but not always,88 shown that recovery of endothelial function occurs in response to strategies known to reduce cardiovascular events. This adds support to the concept that restoration of endothelial function can restabilize the atherosclerotic disease process. Endothelial function testing in patients has also proven useful in the identification of new treatment approaches. Several classes of drugs have been shown to have direct actions on the endothelium that are independent of their effects on cardiovascular risk factors. Examples include the benefit of glitazones on endothelial function in nondiabetic coronary artery disease patients, and those of calcium antagonists in normotensive hypercholesterolemic subjects.89,90 Studies that examine the clinical impact of endothelial function have concentrated on patient cohorts with established coronary and peripheral atherosclerosis, and have mostly shown an independent prognostic impact on cardiovascular outcome. This suggests that vascular function testing may have a role in the clinical phase of atherosclerosis. The main value, however, of the study of endothelial function and its response to intervention may be earlier in the disease course. In advanced disease, a Pandora’s box of mechanisms has been opened, and outcome may depend on many factors, not all of which can be identified and modified. Indeed, in a small meta-analysis, Witte et al suggested that

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the association between FMD and cardiovascular risk was most obvious in lower-risk populations.91 The development of noninvasive measures to study endothelial function has been a major advance in the evaluation of large cohorts during the long preclinical stage of atherosclerosis. Several clinical reports have shown that endothelial dysfunction develops from the first decade of life in response to genetic and environmental risk factors, and this supports the wealth of experimental data that shows endothelial dysfunction to be on the causal pathway for the initiation and progression of atherosclerosis. We have recently studied endothelial function and measures of arterial stiffness in a cohort of ⬎7000 10-year-old children who are part of a large prospective population in which genetic and environmental influences on the emergence of functional vascular epidemiology are being investigated (Avon Longitudinal Study of Parents and Children).92 Others have also adopted this “functional vascular epidemiology” approach that uses combinations of structural and functional measures.93 In a large population of young adults (The Cardiovascular Risk in Young Finns Study), a strong inverse relationship has been shown between endothelial-dependent FMD and structural arterial disease (by carotid intima media thickness) after multivariable adjustment for traditional risk factors.94 This relationship was most striking in those with the worst FMD, which supports a protective role for the quiescent endothelial phenotype, as well as the complementary use of endothelial function testing and structural measurements for characterization of early disease. This relationship has not always been seen in other cohorts of older subjects.95 The importance of the management of not only the clinical phase of atherosclerosis but also early disease is becoming increasingly clear. A recent analysis of the Framingham study has highlighted the impact of early risk factor exposure. Risk factor status at age 50 years had a striking relationship with subsequent cardiovascular outcome and life expectancy in both men and women96 (Figure 6). The effect of genetic variants that alter levels of risk factor from childhood has also emphasized the importance of lifetime exposure and the greatly leveraged gains that can be anticipated from early intervention. In the Atherosclerosis Risk In Communities (ARIC) study, a polymorphism of the PCSK9 gene in black subjects resulted in a 28% reduction in LDL cholesterol levels from childhood and an 88% relative reduction in later cardiovascular events.97 This concept of lifetime risk management can be modeled to demonstrate the great potential for event-free life prolongation in individual subjects that can result from even modest reductions in risk factors introduced at an early stage.98 It is clear that scientific evaluation of the mechanism and triggers of initiation and progression of early arterial disease can only be performed by use of intermediate phenotype end points, and not by study of morbidity and mortality. This is likely to involve tests of endothelial function together with structural measures. There is a clear need for clinical testing strategies that refine risk assessment in individual subjects, and in particular identify those at high risk and their response to treatment. Although endothelial function testing has added enormously

Figure 6. Importance of risk factors and endothelial dysfunction in early life for atherosclerosis development and later cardiovascular outcome. A, Impact of cardiovascular risk factor profile at age 50 years on subsequent clinical events in the Framingham Study. Reproduced from Lloyd-Jones et al96 with permission from Lippincott, Williams & Wilkins. Copyright © 2006, American Heart Association. B, Association between risk factors and carotid IMT in young adults with enhanced, intermediate, and reduced FMD in the Cardiovascular Risk in Young Finns Study. RF indicates risk factor. Reproduced from Juonala et al94 with permission from Lippincott, Williams & Wilkins. Copyright © 2004, American Heart Association.

to the understanding of the atherosclerotic process, it is not yet suitable for screening or individual clinical decision-making. Endothelial function testing shows great promise in that it reflects important vascular biology, is associated with disease burden and outcome, and responds to interventions, but currently the available tests are too difficult, expensive, and variable for routine clinical use. Furthermore, as with other biomarkers used in research, the value in patients will depend on more information about the quantitative relationship between measures of endothelial function and outcome, not merely the associations shown in cohorts. In particular, the technical and biological variability of the various measures of endothelial function will need to be defined in large populations with and without disease. Very few currently fashionable biomarkers fulfill these stringent criteria for clinical use.99 Nevertheless, the ability to measure endothelial function noninvasively has already transformed understanding of the evolution of atherosclerosis. A comprehensive approach that involves measurement of genetic predisposition, risk factors,

Deanfield et al endothelial function, and structural arterial disease is likely to be the best way to evaluate new treatment strategies, particularly in the early preclinical phase of disease, during which better management should result in major public health benefits.

Sources of Funding Dr Deanfield has received grants from Sankyo Pharmaceuticals, the British Heart Foundation, Oak Foundation, British Medical Association, Aspreva Pharmaceuticals, Kidney Research Aid Fund, Medical Research Council, National Kidney Research Fund, Merck Sharpe and Dohme, and Sanofi-Aventis. Dr Halcox has received grant funding support from the British Heart Foundation. Dr Rabelink is supported by the Dutch Heart Foundation. The Vascular Physiology Unit has received research grant support from Sankyo Pharmaceuticals, Aspreva Pharmaceuticals, Sanofi-Aventis, and CORDA in the last 2 years.

Disclosures Dr Deanfield is on the speakers’ bureaus for Pfizer, Sanofi-Aventis, and AstraZeneca. Dr Halcox is on the speakers’ bureaus for Pfizer, Merck Sharpe & Dohme, and Sanofi-Aventis. Dr Rabelink is on the steering committee of the ROADMAP study and has served as a consultant to Merck, Bayer, and GlaxoSmithKline.

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marrow fraction and can be identified by endothelial NO synthase expression. Arterioscler Thromb Vasc Biol. 2006;26:1760 –1767. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593– 600. Deanfield J, Donald A, Ferri C, Giannattasio C, Halcox J, Halligan S, Lerman A, Mancia G, Oliver JJ, Pessina AC, Rizzoni D, Rossi GP, Salvetti A, Schiffrin EL, Taddei S, Webb DJ; Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension. Endothelial function and dysfunction. Part I. Methodological issues for assessment in the different vascular beds: a statement by the Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension. J Hypertens. 2005;23:7–17. Cox DA, Vita JA, Treasure CB, Fish RD, Alexander RW, Ganz P, Selwyn AP. Atherosclerosis impairs flow-mediated dilation of coronary arteries in humans. Circulation. 1989;80:458 – 465. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046 –1051. Okumura K, Yasue H, Matsuyama K, Ogawa H, Morikami Y, Obata K, Sakaino N. Effect of acetylcholine on the highly stenotic coronary artery: difference between the constrictor response of the infarct-related coronary artery and that of the noninfarct-related artery. J Am Coll Cardiol 1992;19:752–758. Drexler H, Zeiher AM. Endothelial function in human coronary arteries in vivo: focus on hypercholesterolemia. Hypertension. 1991;18: II90 –II99. Nabel EG, Selwyn AP, Ganz P. Large coronary arteries in humans are responsive to changing blood flow: an endothelium-dependent mechanism that fails in patients with atherosclerosis. J Am Coll Cardiol. 1990;16:349 –356. Goodhart DM, Anderson TJ. Role of nitric oxide in coronary arterial vasomotion and the influence of coronary atherosclerosis and its risks. Am J Cardiol. 1998;82:1034 –1039. Mancini GB, Henry GC, Macaya C, O’Neill BJ, Pucillo AL, Carere RG, Wargovich TJ, Mudra H, Luscher TF, Klibaner MI, Haber HE, Uprichard AC, Pepine CJ, Pitt B. Angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease: the TREND (Trial on Reversing ENdothelial Dysfunction) study. Circulation. 1996;94:258 –265. Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P. The effect of cholesterol-lowering and antioxidant therapy on endotheliumdependent coronary vasomotion. N Engl J Med. 1995;332:488 – 493. Joannides R, Bellien J, Thuillez C. Clinical methods for the evaluation of endothelial function: a focus on resistance arteries. Fundam Clin Pharmacol. 2006;20:311–320. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992;340:1111–1115. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, Luscher TF. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation. 1995;91: 1314 –1319. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R; International Brachial Artery Reactivity Task Force. Guidelines for the ultrasound assessment of endothelialdependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002;39:257–265. Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab S, Delagrange D, Lieberman EH, Ganz P, Creager MA, Yeung AC, Selwyn AP. Close relation of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol. 1995;26:1235–1241. Woo KS, Chook P, Yu CW, Sung RY, Qiao M, Leung SS, Lam CW, Metreweli C, Celermajer DS. Effects of diet and exercise on obesityrelated vascular dysfunction in children. Circulation. 2004;109: 1981–1986. Mitchell GF, Vita JA, Larson MG, Parise H, Keyes MJ, Warner E, Vasan RS, Levy D, Benjamin EJ. Cross-sectional relations of peripheral microvascular function, cardiovascular disease risk factors, and aortic stiffness: the Framingham Heart Study. Circulation. 2005;112: 3722–3728.

56. Hayward CS, Kraidly M, Webb CM, Collins P. Assessment of endothelial function using peripheral waveform analysis: a clinical application. J Am Coll Cardiol. 2002;40:521–528. 57. Wilkinson IB, Hall IR, MacCallum H, Mackenzie IS, McEniery CM, van der Arend BJ, Shu YE, MacKay LS, Webb DJ, Cockcroft JR. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol. 2002;22:147–152. 58. Nohria A, Gerhard-Herman M, Creager MA, Hurley S, Mitra D, Ganz P. Role of nitric oxide in the regulation of digital pulse volume amplitude in humans. J Appl Physiol. 2006;101:545–548. 59. Bonetti PO, Pumper GM, Higano ST, Holmes DR Jr, Kuvin JT, Lerman A. Noninvasive identification of patients with early coronary atherosclerosis by assessment of digital reactive hyperemia. J Am Coll Cardiol. 2004;44:2137–2141. 60. Naka KK, Tweddel AC, Doshi SN, Goodfellow J, Henderson AH. Flow-mediated changes in pulse wave velocity: a new clinical measure of endothelial function. Eur Heart J. 2006;27:302–309. 61. Smith SC Jr, Anderson JL, Cannon RO 3rd, Fadl YY, Koenig W, Libby P, Lipshultz SE, Mensah GA, Ridker PM, Rosenson R. CDC; AHA. CDC/AHA workshop on markers of inflammation and cardiovascular disease: application to clinical and public health practice: report from the clinical practice discussion group. Circulation. 2004;110:e550 – e553. 62. Rassaf T, Feelisch M, Kelm M. Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues. Free Radic Biol Med. 2004;36:413– 422. 63. Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol. 2004;24:1023–1030. 64. Boger RH, Sydow K, Borlak J, Thum T, Lenzen H, Schubert B, Tsikas D, Bode-Boger SM. LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases. Circ Res. 2000; 87:99 –105. 65. Boger RH, Maas R, Schulze F, Schwedhelm E. Elevated levels of asymmetric dimethylarginine (ADMA) as a marker of cardiovascular disease and mortality. Clin Chem Lab Med. 2005;43:1124 –1129. 66. Ridker PM, Brown NJ, Vaughan DE, Harrison DG, Mehta JL. Established and emerging plasma biomarkers in the prediction of first atherothrombotic events. Circulation. 2004;109:IV6 –IV19. 67. Rifai N, Ridker PM. Inflammatory markers and coronary heart disease. Curr Opin Lipidol. 2002;13:383–389. 68. Hwang SJ, Ballantyne CM, Sharrett AR, Smith LC, Davis CE, Gotto AM Jr, Boerwinkle E. Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study. Circulation. 1997;16:96:4219 – 4225. 69. Ridker PM, Hennekens CH, Roitman-Johnson B, Stampfer MJ, Allen J. Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men. Lancet. 1998;10:351:88 –92. 70. Vaughan DE. PAI-1 and atherothrombosis. J Thromb Haemost. 2005 3:1879 –1883. 71. Meyer AA, Kundt G, Steiner M, Schuff-Werner P, Kienast W. Impaired flow-mediated vasodilation, carotid artery intima-media thickening, and elevated endothelial plasma markers in obese children: the impact of cardiovascular risk factors. Pediatrics. 2006;117:1560 –1567. 72. Mannucci PM. Von Willebrand factor: a marker of endothelial damage? Arterioscler Thromb Vasc Biol. 1998;18:1359 –1362. 73. George F, Brisson C, Poncelet P, Laurent JC, Massot O, Arnoux D, Ambrosi P, Klein-Soyer C, Cazenave JP, Sampol J. Rapid isolation of human endothelial cells from whole blood using S-Endo1 monoclonal antibody coupled to immuno-magnetic beads: demonstration of endothelial injury after angioplasty. Thromb Haemost. 1992;67:147–153. 74. Goon PK, Boos CJ, Lip GY. Circulating endothelial cells: markers of vascular dysfunction. Clin Lab. 2005;51:531–538. 75. Werner N, Wassmann S, Ahlers P, Kosiol S, Nickenig G. Circulating CD31⫹/annexin V⫹ apoptotic microparticles correlate with coronary endothelial function in patients with coronary artery disease. Arterioscler Thromb Vasc Biol. 2006;26:112–116. 76. Diamant M, Tushuizen ME, Sturk A, Nieuwland R. Cellular microparticles: new players in the field of vascular disease? Eur J Clin Invest. 2004;34:392– 401. 77. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, Mallat Z. Circulating microparticles from patients with myocardial

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KEY WORDS: atherosclerosis factors 䡲 tests



endothelium



nitric oxide



risk

Congenital Heart Disease for the Adult Cardiologist Coronary Artery Anomalies An Entity in Search of an Identity Paolo Angelini, MD Coronary artery anomalies (CAAs) are a diverse group of congenital disorders whose manifestations and pathophysiological mechanisms are highly variable. The subject of CAAs is undergoing profound evolutionary changes related to the definition, morphogenesis, clinical presentation, diagnostic workup, prognosis, and treatment of these anomalies. To understand the clinical impact of CAAs, the fundamental challenge is the firm establishment, for a particular type of CAA, of a mechanism capable of interference with the coronary artery’s function, which is to provide adequate blood flow to the dependent myocardium. The present review focuses on anomalous origination of a coronary artery from the opposite sinus—the subgroup of CAAs that has the most potential for clinical repercussions, specifically sudden death in the young. For this subgroup, solid diagnostic screening protocols should be established, especially for athletes and other young individuals subjected to extreme exertion. Intravascular ultrasonography is the preferred means to evaluate the mechanisms responsible for ischemia in anomalous origination of a coronary artery from the opposite sinus and other potentially significant CAAs. Patients symptomatic of anomalous origination of a coronary artery from the opposite sinus may undergo medical treatment/observation, coronary angioplasty with stent deployment, or surgical repair. To be competent to advise CAA carriers, especially in the context of sporting or military activities, cardiologists should undergo specific training in these disorders. Only multicenter collaboration on protocols dedicated to CAAs can give rise to the large-scale studies needed to define the prognosis and optimal treatment of these disorders. (Circulation. 2007;115:1296-1305.) Key Words: coronary disease 䡲 death, sudden 䡲 diagnosis 䡲 heart defects, congenital 䡲 ischemia

T

group has concluded that a comprehensive and widely agreed-upon scheme to define and classify CAAs should initially consider all possible coronary anatomic variations independently from the clinical and hemodynamic repercussions of individual CAAs.10 Such a scheme should include 2 basic steps: (1) The normal coronary anatomy (Table 1) should be described in terms of quantitative and qualitative criteria, and (2) once the normal features have been excluded, the remaining features should be considered to define abnormality and should be used to generate a classification order.10 The basic issue in the definition of a normal coronary artery (and, hence, an anomaly) is the normal spectrum of variation. For example, whereas most experts agree that it is normal to have 2 coronary arteries (the right and the left), how should one consider the frequent presence of independent conal or infundibular branch ostia? This question leads to the next: How is a coronary artery differentiated from a smaller artery such as a conal branch?10 A further related question deals with the case of an absent left main stem: Is it normal to have a separate ostium for the circumflex and left anterior descending arteries? Such questions cannot be answered without an accepted solid criterion that defines the normal spectrum of variants. We have proposed that, when possible, one should use quantifiable criteria such as, “Any

he subject of coronary artery anomalies (CAAs) is undergoing profound evolutionary changes related to the definition, morphogenesis, clinical presentation, diagnostic workup, prognosis, and treatment of these anomalies.1–11 Initially, CAAs were the subject of anatomic discussions that centered around the description and classification of unusual morphologies.1 Eventually, the ischemic mechanisms of CAAs9,12–14 and the incidence of these anomalies in the normal human population were addressed in autopsied patients and coronary angiography populations.10 More recent studies have dealt with vexing questions related to pathophysiological mechanisms and clinical prognoses for different forms of CAAs.10,15 The present review focuses on anomalous origination of a coronary artery from the opposite sinus (ACAOS) with intussusception of the ectopic proximal vessel, which is the subgroup of CAAs that has the most potential for clinical repercussions, specifically sudden death in the young.

Definition of Coronary Anomalies Classification criteria for CAAs have been extensively discussed in the literature. Some authors prefer to categorize CAAs only as “major,” “severe,” “important,” or “hemodynamically significant” anomalies versus “minor” ones.10 Our

From the Department of Cardiology, Texas Heart Institute at St. Luke’s Episcopal Hospital, Houston, Tex. Correspondence to Paolo Angelini, MD, 6624 Fannin, Ste 2780, Houston, TX 77030. E-mail [email protected] © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.618082

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TABLE 1. Normal Features of the Coronary Anatomy in Humans Feature No. of ostia

Range 2 to 4

Location

right and left anterior sinuses (upper midsection)

Proximal orientation

45° to 90° off the aortic wall

Proximal common stem or trunk Proximal course Mid-course Branches Essential territories Termination

only left (LAD and Cx) direct, from ostium to destination extramural (subepicardial) adequate for the dependent myocardium RCA (RV free wall), LAD (anteroseptal), OM (LV free wall) capillary bed

LAD indicates left anterior descending artery; Cx, circumflex artery; RCA, right coronary artery; RV, right ventricular; OM, obtuse marginal artery; and LV, left ventricular.

form observed in ⬎1% of an unselected general population is normal.”11 The literature continues to entertain these and similar considerations while the field awaits a widely accepted endorsement by representative professional groups.10,16 Table 2 shows our group’s proposed comprehensive classification scheme. A basic principle of coronary classification should be that the nature and name of a specific coronary artery are assigned, not according to the site of origin or proximal course, but according to the dependent territory. Figures 1 and 2 show 2 complex CAAs that exemplify the methods used to describe any given complex case.10 Furthermore, 3 main coronary vessels (the left anterior descending, circumflex, and right coronary) (Figure 3) should probably be termed arteries, but the most distal vessels should be called coronary branches. We have proposed that a common proximal trunk, which joins 2 or 3 coronary arteries, should be named a mixed trunk. The only normally observed mixed trunk is the left main (common trunk or stem).10 The following criteria are proposed to define each coronary artery: 1. The right coronary artery (RCA) is the vessel that provides blood flow to the right ventricular free wall. It is not essential for the posterior descending branch to originate from the RCA (the most common pattern) or that the ostium of the RCA be located at the right anterior sinus of Valsalva (which is normal). 2. The left anterior descending artery is the vessel that provides blood flow to the anterior interventricular septum. It is not essential for the diagonal branch to originate from this vessel (as is normal). 3. The circumflex artery is the vessel that provides blood flow to the free wall of the left ventricle, on the obtuse margin of the heart.10

Incidence of Coronary Artery Anomalies Curiously, the literature shows that the overall incidence of CAAs is consistently mentioned by most authors, even the hundreds of them who report individual cases. This practice has led the nonspecialized audience to assume that CAAs, as

Figure 1. Angiograms from a 52-year-old man, in the left anterior oblique cranial (A) and right anterior oblique (B) projections. The patient had atypical chest pain and borderline nuclear stress test results. In these views, the whole coronary system is visualized from a single ostium, located at the right sinus. The right coronary artery (RCA) splits off from a short common trunk (CT), and continues into a terminal obtuse marginal branch (OM). The left main trunk (LM) crosses to the left off the CT, and courses intraseptally to give off a large septal branch (SB). The left coronary artery ends in the left anterior descending (LAD) and ramus (RM) branches. This is a case of clinically benign single coronary artery, which should more properly be called single coronary ostium because all the coronary arteries are present, though they are anomalous in their origin and course.

a whole, are a serious threat (not simply that some rare individual forms may be so).10 The greatest confusion in this regard is about myocardial bridges: Are they an anomaly, a pathological anomaly, or simply a normal feature of some coronary arteries in humans? The fact that such bridges are surely present in ⬎1% of the general population suggests that they may be a normal variant.10,17 Should only “severe” myocardial bridges be counted as pathological anomalies, and, if so, by what criteria?10 In one of the few prospective analyses to involve strict diagnostic criteria, which was performed in a continuous series of 1950 patients studied by coronary angiography, our group found that CAAs had a global incidence of 5.64% (Table 3), which is much higher than usually reported. Particularly noteworthy were the 0.92% incidence of anomalous origination of the RCA from the left sinus and the 0.15% incidence of anomalous origination of the left coronary artery from the right sinus (for a total incidence of 1.07% for ACAOS).10

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TABLE 2. Classification of Coronary Anomalies in Human Hearts A. Anomalies of origination and course

Continued (1) Posterior atrioventricular groove

1. Absent left main trunk (split origination of LCA)

(2) Retroaortic

2. Anomalous location of coronary ostium within aortic root or near proper aortic sinus of Valsalva (for each artery)

(3) Between aorta and pulmonary artery

a. High b. Low c. Commissural 3. Anomalous location of coronary ostium outside normal “coronary” aortic sinuses a. Right posterior aortic sinus b. Ascending aorta c. Left ventricle d. Right ventricle e. Pulmonary artery (1) LCA that arises from posterior facing sinus (2) Cx that arises from posterior facing sinus (3) LAD that arises from posterior facing sinus

(4) Intraseptal (5) Anterior to pulmonary outflow (6) Posteroanterior interventricular groove 5. Single coronary artery (see A4) B. Anomalies of intrinsic coronary arterial anatomy 1. Congenital ostial stenosis or atresia (LCA, LAD, RCA, Cx) 2. Coronary ostial dimple 3. Coronary ectasia or aneurysm 4. Absent coronary artery 5. Coronary hypoplasia 6. Intramural coronary artery (muscular bridge) 7. Subendocardial coronary course 8. Coronary crossing

(4) RCA that arises from anterior right facing sinus

9. Anomalous origination of posterior descending artery from the anterior descending branch or a septal penetrating branch

(5) Ectopic location (outside facing sinuses) of any coronary artery from pulmonary artery

10. Split RCA

(a) From anterior left sinus (b) From pulmonary trunk (c) From pulmonary branch f. Aortic arch g. Innominate artery h. Right carotid artery i. Internal mammary artery j. Bronchial artery k. Subclavian artery l. Descending thoracic aorta 4. Anomalous location of coronary ostium at improper sinus (which may involve joint origination or “single” coronary pattern) a. RCA that arises from left anterior sinus, with anomalous course

a. Proximal⫹distal PDs that both arise from RCA b. Proximal PD that arises from RCA, distal PD that arises from LAD c. Parallel PDs ⫻2 (arising from RCA, Cx) or “codominant” 11. Split LAD a. LAD⫹first large septal branch b. LAD, double (parallel LADs) 12. Ectopic origination of first septal branch a. RCA b. Right sinus c. Diagonal d. Ramus e. Cx C. Anomalies of coronary termination

(1) Posterior atrioventricular groove or retrocardiac

1. Inadequate arteriolar/capillary ramifications

(2) Retroaortic

2. Fistulas from RCA, LCA, or infundibular artery to:

(3) Between aorta and pulmonary artery (intramural)

a. Right ventricle

(4) Intraseptal

b. Right atrium

(5) Anterior to pulmonary outflow

c. Coronary sinus

(6) Posteroanterior interventricular groove (wraparound)

d. Superior vena cava

b. LAD that arises from right anterior sinus, with anomalous course

e. Pulmonary artery

(1) Between aorta and pulmonary artery (intramural)

f. Pulmonary vein

(2) Intraseptal

g. Left atrium

(3) Anterior to pulmonary outflow

h. Left ventricle

(4) Posteroanterior interventricular groove (wraparound)

i. Multiple, right⫹left ventricles

c. Cx that arises from right anterior sinus, with anomalous course

D. Anomalous anastomotic vessels

d. LCA that arises from right anterior sinus, with anomalous course

LCA indicates laft coronary artery; LAD, left descending coronary artery; RCA, right coronary artery; Cx indicates circumflex; and PD, posterior descending branch. Adapted from Angelini P et al10 with permission from Lippincott, Williams & Wilkins. Copyright 1999.

A group at the American Armed Forces Institute of Pathology18 recently reported some notable and groundbreaking statistics. In a continuous series of 6.3 million

18-year-old recruits who underwent intense military training for 8 weeks, the researchers identified 277 deaths unrelated to trauma. A review of the clinical and necropsy charts showed that, of 64 cardiac deaths, 21 (33%) were related to ACAOS

(1) Posterior atrioventricular groove (2) Retroaortic

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Figure 2. Angiograms from a 30-year-old man with a long-term history of crescendo angina, mainly at rest, accompanied by reversible ECG T-wave changes and lateral ischemia on the nuclear stress test. Angiograms in the right anterior oblique cranial (A) and straight (B) projections reveal a single coronary ostium and a prepulmonic course for the LM. View A was obtained after intracoronary administration of nitroglycerin; note the mildly narrowed mid LM with bridging collaterals (BCs). View B shows the effects of intracoronary infusion of acetylcholine (100 ␮g over 30 seconds) that reproduced typical angina and ECG changes. The LM stenosis has clearly increased in severity. C, IVUS image of the proximal LM (area, 8.9 mm2). D, IVUS image of the stenotic area after nitroglycerin administration (area, 6.1 mm2). This case shows the spastic potential of some anomalous vessels, in this instance at the level of a prepulmonic course. Calcium antagonists successfully abated the presenting angina syndrome.

of the left coronary artery (left-ACAOS) and that no other CAAs resulted in cardiac death. Although the authors did not specify, it is likely that none of these cases of left-ACAOS had been diagnosed before death (in an environment in which medical evaluations are routine). This is the first large-scale study of CAAs in which the denominator (all candidates at risk) was known, the setting of the clinical events was consistent (extreme physical training), and all the fatal events led to necropsy studies.18 In comparison, Drory and colleagues19 studied the incidence of CAAs in a continuous series of 162 patients with sudden unexpected death. The patients were ⬍40 years of age and underwent routine autopsy studies in Israel, where an autopsy is obligatory in such cases. The incidence of CAArelated sudden death was 0.6% (1 of 162 cases); taken together with the recent military recruit series,18 this result suggests that extreme exercise plays a powerful role in such deaths. In conclusion, the main interest of current clinical investigators seems to be to establish the incidence of those

individual types of CAAs that have become recognized for their clinical consequences.

Pathophysiological Mechanisms and Clinical End Points To understand the clinical impact of CAAs, the fundamental challenge is to firmly establish, for a particular type of CAA, a mechanism capable of interference with the coronary artery’s function to provide adequate blood flow to the dependent myocardium. Table 4 summarizes such mechanisms and the conditions under which they apply.10 Whereas some CAAs may cause occasional ischemia, others (eg, anomalous origination of the left coronary artery from the pulmonary artery) obligatorily cause ischemia, and yet others only predispose the patient to have a misdiagnosis or complications (clotting, spasm, or atherosclerotic buildup). The present review is limited to only 1 kind of coronary anomaly, ACAOS, which has recently been recognized as having serious prognostic implications in young individuals.5–7,9,12,20 In cases of ectopic origination of a CAA, only 1

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March 13, 2007 of the aorta and pulmonary artery, especially during exertion.22 Such a mechanism is unlikely, however, because at the site of closest aortopulmonary proximity the anomalous artery lies inside the aortic wall.26 In our more recent extensive experience with IVUS examination of CAAs, we have occasionally found an intramural aortic course in some type of ACAOS without an interarterial course. Specifically, only 2 patients had an unusual intramural anomalous “retroaortic” course: In 1 case, the left coronary arose from the posterior sinus27; in the other case, the circumflex artery arose from the right sinus. The reasons for our insistence on the intussusception of anomalous arteries are related to the following newly discovered mechanisms of stenosis (Figure 4):

Figure 3. Conceptual diagram that shows most of the possible paths (1 through 5) by which the RCA, left anterior descending artery (LAD), and circumflex artery (Cx) can potentially connect with the opposite coronary cusps. Paths: 1, Retrocardiac; 2, retroaortic; 3, preaortic, or between the aorta and pulmonary artery; 4, intraseptal (supracristal); 5, prepulmonary (precardiac). The aortic and pulmonary cusps are labeled according to their position in space: AL indicates antero-left; AR, antero-right; P, posterior; M, mitral valve; and T, tricuspid valve. Reproduced from Angelini et al10 with permission from Lippincott Williams & Wilkins. Copyright 1999.

specific abnormal course, traditionally called interarterial, or “between the aorta and pulmonary artery,” is associated with a severe prognosis.20 –25 Indeed, that anomaly has recently been observed, on intravascular ultrasound (IVUS) imaging, to consist of intramural proximal intussusception of the ectopic artery at the aortic-root wall.26 Never has an extramural course been observed with IVUS in such a scenario.21,26,27 The traditional terminology (between the aorta and pulmonary artery) implied that the aberrant artery was liable to a scissors-like mechanism, created by the close proximity TABLE 3. Incidence of Coronary Anomalies and Patterns, as Observed in a Continuous Series of 1950 Angiograms Variable Coronary anomalies (total) Split RCA

N (%) 110 (5.64) 24 (1.23)

Ectopic RCA (right sinus)

22 (1.13)

Ectopic RCA (left sinus)

18 (0.92)

Fistulas

17 (0.87)

Absent left main coronary artery

13 (0.67)

Circumflex arising from right sinus

13 (0.67)

LCA arising from right sinus

3 (0.15)

Low origination of RCA

2 (0.1)

Other anomalies

3 (0.27)

Coronary dominance patterns Dominant RCA Dominant LCA (circumflex) Codominant arteries (RCA, circumflex)

1641 (89.1) 164 (8.4) 48 (2.5)

LCA indicates left coronary artery. Adapted from Angelini P et al10 with permission from Lippincott, Williams & Wilkins. Copyright 1999.

1. Coronary hypoplasia. Our group has discovered that the intramural intussuscepted segment of the proximal ectopic artery is smaller in circumference than the more distal extramural vessel. With IVUS, we found it valuable to quantify this parameter with the hypoplasia index (ie, the ratio of the circumference of the intramural segment with respect to the circumference of the more distal segment).26,27 Arteries that arise congenitally inside the aortic media likely cannot grow normally either before or after birth. 2. Lateral compression. The cross section of the intramural segment is characteristically not circular but ovoid (Figure 4). The lateral compression results in a smaller area than that possessed by a circle of the same circumference.17 This parameter can be quantified with the asymmetry ratio (the ratio of the smallest to the largest diameter in an IVUS cross section).27 Additionally, our group has observed that the smaller diameter is further compressed during each systole, as manifested by pulsatile behavior observed with IVUS during the cardiac cycle. This liability to undergo intermittent worsening is most likely related to changes in stroke volume (and pulsatility of the ascending aorta) and to tachycardia, which is a behavior that becomes manifest during IVUS imaging when an experimental pharmacological challenge simulates exercise conditions.26 For instance, in 3 symptomatic patients with left-ACAOS, we found 49% to 70% area stenosis at baseline, which increased by 8% to 10% with stimulation.27 Such lesions are in the range of what the Coronary Artery Surgery Study (CASS) defined as critical stenosis of the left main coronary, which can cause sudden death.28 In this context, it is important to recognize the hemodynamic changes that occur during sports activities that involve maximal exertion; for example, from the resting state, the heart rate increases from 65 to 180 bpm, the cardiac output from 5 to 22 L/min, and the stroke volume from 77 to 122 cc.29 3. Stenotic segment length. With any coronary stenosis, the segmental length is another measure of severity. In ACAOS that involves the RCA, as well as in left-ACAOS, the length of the stenosis varies between 5 and 15 mm.26,27 In our series, all 3 of the aforementioned parameters showed great individual variability. It is likely that aortic wall distensibility (degree of cross-sectional area enlargement associated with a certain increase in pressure) is a further related variable that depends on intrinsic anatomic changes in the aortic wall (as in medial cystic necrosis or aortic dissec-

Angelini TABLE 4.

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1301

Pathophysiological Mechanisms and Coronary Anomalies (Functional Classification) Proof of Action

Pathophysiological Mechanism Misdiagnosis

Coronary Anomaly “Missing” coronary artery

Certain

“Hypoplastic” coronary artery Myocardial ischemia, primary (fixed and/or episodic)

x

Ostial stenosis

x

Coronary fistula

x x

Muscular bridge Myocardial ischemia, secondary (episodic)

Increased risk of fixed coronary atherosclerotic disease

Secondary aortic valve disease

x

Tangential origin (ACAOS) intramural course

x

Myocardial bridge, plus spasm and/or clot

x

Coronary ectasia (plus mural clot)

x

Coronary fistula (plus mural clot)

x

Coronary fistula

x

ALCAPA

x

Coronary ectasia

x

Muscular bridge (proximal to)

x

Coronary aneurysm (ostial)

x

Coronary fistula

x

ALCAPA

x

Increased risk of bacterial endocarditis

Coronary fistula

Ischemic cardiomyopathy (hibernation)

ALCAPA

x

Volume overload

Coronary fistula

x

ALCAPA

x

Ectopic ostia (tangential)

x

Unusual technical difficulties during coronary angiography or angioplasty

Complications during cardiac surgery

Unlikely

x

Ostial atresia

ALCAPA

Possible

x

x

Split left coronary artery

x

Coronary fistula

x

Ectopic ostia and proximal course

x

Muscular bridge

x

ALCAPA indicates anomalous origination of the left coronary artery from the pulmonary artery. Adapted from Angelini P et al10 with permission from Lippincott, Williams & Wilkins. Copyright 1999.

tion), changes in the aortic pressure (as at the onset of hypertension or aortic regurgitation), or a rapid weight gain, especially in patients who receive negative chronotropic agents, which increase the stroke volume if the cardiac output remains essentially unchanged. Moreover, a treadmill stress test, which should be transformed into an adenosine test because of an inadequate effort or chronotropic response, may be the most accurate predictive test for ACAOS because it associates an increased cardiac output with nonphysiological bradycardia. Unfortunately, though, such a hybrid protocol is a potential cause of sudden death, specifically in ACAOS carriers, and should generally be avoided or at least closely monitored in a hospital environment. When a carrier of ACAOS dies suddenly, in the absence of other lethal cardiovascular conditions, a low cardiac output and bradycardia or asystole typically occur early after extreme exercise, after which syncope and/or death ensues. Terminal ventricular fibrillation may also occur as a manifestation of critical ischemia or of reperfusion arrhythmia.30 –32

Both the anomalous right and left coronary arteries can be responsible for sudden death, although the risk has not been adequately quantified in specific studies. Most likely, predisposing factors include the severity of baseline stenosis, the specific conditions at the time of the crisis, and the myocardial territory at risk.7,33 Additionally, one must realize that the possible manifestations of ACAOS include not only sudden death but also dyspnea, palpitations, angina pectoris, dizziness, and syncope.4,10,12,26,32 Whereas sudden death is usually associated with extreme exercise in young adults,34 the other manifestations of ACAOS are more frequently seen in older adults (in our experience, specifically women) and are related to the onset of hypertension. Interestingly, Cheitlin33 claimed that sudden death is seen only in young patients, possibly because of progressive hardening of the aortic wall in adults. During aortic valve replacement, an intramural ectopic coronary artery can also be liable to critical worsening of extrinsic compression by the prosthetic ring, as recently reviewed by Morimoto and colleagues.35

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Figure 4. Images obtained from a 42-year-old man with severe angina of recent onset, who had recently developed hypertension. A, Selective angiogram of the RCA in the left anterior oblique projection. In this view, the proximal course appears wider than the more distal vessel, and it originates next to the LCA (which is also ectopic), high above the left aortic sinus (LS). B, IVUS image of the distal RCA. The cross-sectional area is 10.8 mm2, and the shape is circular. C, IVUS image of the proximal segment of the RCA, whose lumen is severely compressed laterally (minimal diameter, 1.5 mm; maximal diameter, 3.8 mm; cross-sectional area, 4.2 mm2; area stenosis, 61%). The LCA had also milder ostial stenosis. D, IVUS of the proximal RCA after stent angioplasty (3.5 ⫻ 12 mm; postdilated at 18 atm). The shape has become round, and the area has expanded to match that of the distal normal vessel.

Outlines for Diagnostic and Treatment Protocols In carriers of ACAOS, the clinical histories are consistent in only 1 aspect: Either these patients die suddenly (typically at a young age and after extreme exertion), or they have no characteristic presentation. Most patients are asymptomatic for a large portion of their lives, and an atypical chest-pain syndrome is the most common reason they are referred for coronary angiography, which is when the diagnosis is typically made. The milder cases are more likely to be identified fortuitously (because of a falsely positive stress test and/or coincidental atherosclerotic disease). The fact that CAAs include many different entities and that no single observer or group has collected a large enough series to clarify the natural prognosis of each entity may contribute to our difficulty in the clinical identification of these lesions, especially the ones that could lead to angina or sudden cardiac death.21 For most types of coronary anomalies, the fundamental clinical approach could be: “Do not bother to look for these innocent anomalies, but be prepared to recognize them as benign if one is accidentally found,

typically at coronary angiography.” However, for a few CAAs that are possibly or predictably malignant (fundamentally, ACAOS), we should establish solid diagnostic screening protocols, especially for athletes and other young individuals subjected to extreme exertion.9,10,29,33 As noted above, ACAOS patients can succumb to sudden cardiac death, usually but not necessarily at a young age, possibly even at the newborn stage.36 Retrospectively reviewed, only a few persons reported to have died of ACAOS had significant symptoms, usually atypical chest pain, dyspnea, syncope, or their equivalents, before the final event.5–7,9,10,13–15 A specific workup protocol is indicated mostly for athletes and military personnel with these symptoms. In view of the fairly rare nature of ACAOS, it would not seem practical or cost-effective to extend the indications for such a workup to all schoolchildren on a routine basis. Nevertheless, larger prospective studies are needed before this decision can become final.37–39 In patients with suspected ACAOS, testing should sequentially include electrocardiography, Holter monitoring (basi-

Angelini cally to document atrial or ventricular arrhythmias as nonspecific markers of ACAOS), and focused expert echocardiography (transthoracic and, if needed, transesophageal) with Doppler interrogation to identify the coronary origin and proximal course.40 – 42 In particular, the reported 0.17% incidence of ACAOS found by examination of a series of 2388 routine echocardiograms40 must be compared with the 1.07% incidence found at coronary angiography.10 The implication is that echocardiography is probably not as reliable a means to diagnose this disorder (especially if performed in adults and without specifically looking for ACAOS). The authors of the echocardiographic study37 reported that 1 of their negative results was followed by sudden death during follow-up observation, the diagnosis of ACAOS becoming apparent only at autopsy. If at least 2 normally located coronary ostia are identified with echocardiography, which is more often possible in children than adults, no further workup for ACAOS is probably required. If the coronary ostia are not clearly identified echocardiographically, however, or if an alternative method is needed, computed tomography or magnetic resonance imaging is recommended.21,41,43 These methods not only identify ACAOS more reliably than echocardiography, but also allow description of the dependent territory,21 which correlates with the prognosis, as discussed above. When ACAOS is identified in this manner, a further workup should include nuclear stress testing. Although the result is usually negative, this method is important both to evaluate effortinduced ischemia and scars and to establish a baseline for follow-up assessment in case of eventual intervention. Furthermore, selective coronary angiography is indicated more to rule out additional obstructive coronary disease of atherosclerotic origin than to evaluate the severity of congenital obstruction at the proximal ectopic vessel. The need for interventional treatment can be substantiated only by IVUS, as discussed above.26,27

Treatment Options Symptomatic carriers of ACAOS have 3 treatment options: medical treatment/observation, coronary angioplasty with stent deployment, and surgical repair. Despite the limitations of our current knowledge of such anomalies, intervention may be justified in some cases to prevent sudden death and improve the quality of life. Medical treatment (essentially with ␤-blockers) is probably as effective as restriction of activity (avoidance of severe exertion) in these patients.39 In a significant number of cases, right-ACAOS may not warrant intervention. Precise IVUS/clinical correlations should be prospectively obtained to establish acceptable selection criteria. Stent-angioplasty of the obstructed proximal intramural segment of a patient with right-ACAOS is technically feasible,26,44 and is probably justifiable in the presence of (1) disabling symptoms and/or a high risk of sudden death, (2) area stenosis more severe than 50% with respect to the distal normal vessel on IVUS, (3) a large dependent myocardial territory (more than a third of the total), and (4) reversible ischemia, as documented by a nuclear stress test.

Coronary Artery Anomalies

1303

Besides indicating the need for intervention, IVUS is also essential for proper deployment of a stent. We use IVUS data both to measure the length of the obstructed intramural RCA and to evaluate the cross-sectional area after stent deployment, aiming for a target luminal area similar to that of the distal vessel. Initially, timid dilatation of stents (for fear of aortic-wall dissection if excessively large balloon sizes were used) resulted in incomplete apposition along the longest diameter, some residual stenosis, and sometimes early postoperative restenosis. Presently, we feel confident that the immediate and late results are improved if full luminal restoration, to match the area of the distal vessel, is attained at the intramural segment and for about 4 mm beyond it. Apparently, only 1 group, in China, has reported the use of stent-angioplasty for left-ACAOS.45 In this case, the patient was a 14-year-old child with severe symptoms who received a stent at the left main trunk. The early results were favorable, but we prefer to postpone such experimental use of stents until stent-angioplasty is well established for the lower-risk indication of right-ACAOS. Our initial experience suggests that drug-eluting stents offer the best probability to avoid restenosis, but definitive data need to be collected regarding this off-label use of stents. Moreover, restenosis appears to be rare, and, if it does occur, is related to in-stent fibrocellular growth, not stent compression. Like many others, however, our group considers that left-ACAOS is generically, in itself, a solid indication for surgical intervention.27 Nevertheless, we continue to acquire IVUS data in these patients to further refine our treatment protocols. Despite the absence of objective studies, surgical treatment of ACAOS has been performed in large series of patients for several years.43,46 Surgical correction, which is especially recommended for left-ACAOS that involves a large territory at risk, may consist of (1) direct reimplantation of the ectopic artery at the aortic root (a technically difficult and unreliable approach); (2) unroofing of the intramural coronary segment, from the ostium to the exit point, off the aortic wall; or (3) osteoplasty, which creates a new ostium at the end of the ectopic artery’s intramural segment (Figure 5).27,43,46 – 48 Athletes and military personnel known to be ACAOS carriers should be advised by a specially trained cardiologist about permitted versus prohibited physical activities before and after intervention. Current guidelines issued by professional associations state that untreated carriers of ACAOS should not be involved in competitive sports or other strenuous activities.39 Treated patients should be reevaluated before being allowed to resume exercise at maximal capacity.

Conclusions Coronary artery anomalies should be regarded as an uneven diverse group of congenital disorders whose manifestations and pathophysiological mechanisms are highly variable. To be competent to advise CAA carriers, especially in the context of sporting or military activities, cardiologists should undergo specific training in these disorders. IVUS is the preferred means to evaluate the mechanisms responsible for ischemia in potentially significant CAAs, especially ACAOS.

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Figure 5. Diagram representation of a case of single coronary ostium at the right sinus. The LM runs intramurally inside the aortic-wall left sinus, just below the anterior aortic commissure, and takes off from the aorta at the center of the left cusp. At this point, a circle with stitches represents the newly created ostium after surgical repair. Cx indicates circumflex. Reproduced from Angelini et al27 with permission from Texas Heart Institute. Copyright 2006.

Clearly, this aspect of cardiology will not be able to develop fully without extensive collaboration between individual cardiologists and institutions.16 To further this goal, the Texas Heart Institute has established a Web site designed to promote multicenter collaboration on protocols dedicated to ACAOS patients (http://texasheart.org/Education/Resources/ caac.cfm). Only such efforts can give rise to the large-scale studies needed to define the prognosis and optimal treatment of individual forms of CAAs.

Acknowledgment For assistance in the preparation of this paper, the author thanks Virginia Fairchild, Senior Medical Editor, Texas Heart Institute at St. Luke’s Episcopal Hospital.

Disclosures Dr Paolo Angelini is an occasional expert witness in cases of coronary anomalies.

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AHA/ACC/HRS Scientific Statement Recommendations for the Standardization and Interpretation of the Electrocardiogram Part I: The Electrocardiogram and Its Technology A Scientific Statement From the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society Endorsed by the International Society for Computerized Electrocardiology Paul Kligfield, MD, FAHA, FACC; Leonard S. Gettes, MD, FAHA, FACC; James J. Bailey, MD; Rory Childers, MD; Barbara J. Deal, MD, FACC; E. William Hancock, MD, FACC; Gerard van Herpen, MD, PhD; Jan A. Kors, PhD; Peter Macfarlane, DSc; David M. Mirvis, MD, FAHA; Olle Pahlm, MD, PhD; Pentti Rautaharju, MD, PhD; Galen S. Wagner, MD Abstract—This statement examines the relation of the resting ECG to its technology. Its purpose is to foster understanding of how the modern ECG is derived and displayed and to establish standards that will improve the accuracy and usefulness of the ECG in practice. Derivation of representative waveforms and measurements based on global intervals are described. Special emphasis is placed on digital signal acquisition and computer-based signal processing, which provide automated measurements that lead to computer-generated diagnostic statements. Lead placement, recording methods, and waveform presentation are reviewed. Throughout the statement, recommendations for ECG standards are placed in context of the clinical implications of evolving ECG technology. (Circulation. 2007;115:1306-1324.) Key Words: AHA Scientific Statements 䡲 electrocardiography 䡲 computers 䡲 diagnosis 䡲 electrophysiology 䡲 intervals 䡲 potentials 䡲 tests

I

n the century since the introduction of the string galvanometer by Willem Einthoven,1 the electrocardiogram (ECG) has become the most commonly conducted cardiovascular diagnostic procedure and a fundamental tool of clinical practice.2,3 It is indispensable for the diagnosis and prompt initiation of therapy in patients with acute coronary syndromes and is the most accurate means of diagnosing intraventricular conduction disturbances and arrhythmias. Its in-

terpretation may lead to the recognition of electrolyte abnormalities, particularly of serum potassium and calcium, and permit the detection of some forms of genetically mediated electrical or structural cardiac abnormalities. The ECG is routinely used to monitor patients treated with antiarrhythmic and other drugs, in the preoperative assessment of patients undergoing noncardiac surgery, and in screening individuals in high-risk occupations and, in some

Other members of the Standardization and Interpretation of the Electrocardiogram Writing Group include Mark Josephson, MD, FACC, FHRS; Jay W. Mason, MD, FAHA, FACC, FHRS; Peter Okin, MD, FACC; Borys Surawicz, MD, FAHA, FACC; and Hein Wellens, MD, FAHA, FACC. The American Heart Association, the American College of Cardiology, and the Heart Rhythm Society make every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. Specifically, all members of the writing group are required to complete and submit a Disclosure Questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest. This statement was approved by the American Heart Association Science Advisory and Coordinating Committee on October 26, 2006, by the American College of Cardiology Board of Trustees on October 12, 2006, and by the Heart Rhythm Society on September 6, 2006. This article has been copublished in the March 13, 2007, issue of the Journal of the American College of Cardiology and in the March 2007 issue of Heart Rhythm. Copies: This document is available on the World Wide Web sites of the American Heart Association (www.americanheart.org) and the American College of Cardiology (www.acc.org). A single reprint is available by calling 800-242-8721 (US only) or writing the American Heart Association, Public Information, 7272 Greenville Ave, Dallas, TX 75231-4596. Ask for reprint No. 71-0389. To purchase additional reprints, call 843-216-2533 or e-mail [email protected]. Permissions: Multiple copies, modification, alteration, enhancement, and/or distribution of this document are not permitted without the express permission of the American Heart Association. Instructions for obtaining permission are located at http://www.americanheart.org/presenter.jhtml? Identifier⫽4431. A link to the “Permission Request Form” appears on the right side of the page. © 2007 American Heart Association, Inc., the American College of Cardiology Foundation, and the Heart Rhythm Society. 10.1161/CIRCULATIONAHA.106.180200

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cases, for participation in sports. As a research tool, it is used in long-term population-based surveillance studies and in experimental trials of drugs with recognized or potential cardiac effects. Indications for use of the ECG were summarized in a joint American Heart Association (AHA)/American College of Cardiology report in 1992.4 Because of its broad applicability, the accurate recording and precise interpretation of the ECG are critical. The establishment of and adherence to professionally developed and endorsed evidence-based standards for all phases of the ECG procedure is an important step in ensuring the high level of precision required and expected by clinicians and their patients.5 However, there has not been a comprehensive updating of ECG standards and criteria since 1978.6 –14 Since 1978, there have been many advances in the technology of electrocardiography; in the understanding of the anatomic, pathological, electrophysiological, and genetic information underlying ECG findings; and in the clinical correlations of ECG abnormalities. One of the most important changes in electrocardiography is the widespread use of computerized systems for storage and analysis. Many if not most ECGs in the United States now are recorded by digital, automated machines equipped with software that measures ECG intervals and amplitudes, provides a virtually instantaneous interpretation, and often compares the tracing to those recorded earlier by the same system. However, different automated systems may have different technical specifications that result in significant differences in the measurement of amplitudes, intervals, and diagnostic statements.15,16 For these reasons, the AHA initiated an updating of guideline statements for standardization and interpretation of the ECG. The project has been endorsed by the American College of Cardiology, the Heart Rhythm Society, and the International Society for Computerized Electrocardiology. The purposes of this project are as follows: (1) to review the status of techniques currently used to record and interpret the ECG and to identify opportunities for modification; (2) to simplify and unify the various descriptive, diagnostic, and modifying terminologies currently used in order to create a common and more easily applied lexicon; and (3) to identify the weaknesses of the descriptive, interpretative, and comparative algorithms and recommend changes that incorporate the newly recognized factors referred to above. The chairman (L.S.G.) was selected by the Electrocardiography and Arrhythmias Committee of the Council on Clinical Cardiology of the AHA. He formed an advisory group to assist in setting goals and to recommend other writing group members. The committee met on 5 occasions to discuss goals, identify specific areas that required updating, and review progress. A smaller working/writing group with a group leader was chosen for each topic. This is the first of 6 articles written in response to the AHA mandate. It is followed by a glossary of descriptive, diagnostic, and comparative statements that attempts to minimize repetitive and noninformative statements. Additional articles, to be published subsequently, will discuss the ECG interpretation of intraventricular conduction disturbances, abnormalities of ventricular repolarization, hypertrophy, and ischemia/infarction.

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The ECG and Its Technology The purposes of this statement are (1) to examine the relation of the resting ECG to its technology, (2) to increase understanding of how the modern ECG is derived and recorded, and (3) to promote standards that will improve the accuracy and usefulness of the ECG in practice. Special emphasis will be placed on the digital recording methods and computerbased signal processing that are used in current electrocardiographs to provide automated measurements that lead to computer-generated diagnostic statements. The writing group recognizes that technical details of the processing and recording of ECGs may be unfamiliar to clinicians. Accordingly, a major purpose of this document is to provide clinicians with insight into the generally missing link between technology and its consequences for clinical ECG interpretation. The evolution and application of ECG technology have profound clinical implications, as exemplified by the demonstration that measurements made by different automated ECG systems from reference ECG data can vary enough to alter diagnostic interpretation.15,17 Sensitivity and specificity of computer-based diagnostic statements are improving, but at the same time, it remains evident that physician overreading and confirmation of computer-based ECGs is required.15,16,18

Previous Standards and Reviews A number of recommendations for the standardization of ECG recording and guidelines for ECG interpretation in the computer era have appeared during the past several decades. The most recent comprehensive AHA recommendations for the standardization of leads and general technical requirements of ECG instruments were published in 1975.5 In 1978, task forces of the American College of Cardiology produced a collection of reports on optimal electrocardiography,7 which addressed standardization of terminology and interpretation,13 the development of databases,6 the quality of ECG records,12 computers in diagnostic cardiology,9 the use of ECGs in practice,10 cost-effectiveness of the ECG,11 and a discussion of future directions.14 In Europe, international common standards for quantitative electrocardiography (CSE) evolved from the work of Willems and colleagues.19 –22 The CSE studies were designed to reduce the wide variation in wave measurements obtained by ECG computer programs and to assess and improve the diagnostic classification of ECG interpretation programs.22 Given the expanding use of computer-based ECG systems and evolving technology, recommendations for bandwidth and digital signal processing standards during automated electrocardiography were formulated in 1990 by a committee of the AHA.23 In 1991, recommendations of the 1975 and 1990 AHA documents were incorporated into a summary document on diagnostic ECG devices that was developed by the Association for the Advancement of Medical Instrumentation (AAMI) and approved by the American National Standards Institute (ANSI).24 This document was reaffirmed by ANSI in 2001. Other statements have addressed related issues of ECG utilization and physician competence in interpretation of the ECG.16,18,25–27

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The ECG Signal and Its Processing Automated analysis of the digital 12-lead ECG involves signal analysis and diagnostic classification.28 Processing of the ECG occurs in a series of steps, each of which requires adherence to methodological standards. These steps include (1) signal acquisition, including filtering; (2) data transformation, or preparation of data for further processing, including finding the complexes, classification of the complexes into “dominant” and “nondominant” (ectopic) types, and formation of an average or median complex for each lead; (3) waveform recognition, which is the process for identification of the onset and offset of the diagnostic waves; (4) feature extraction, which is the measurement of amplitudes and intervals; and (5) diagnostic classification. Diagnostic classification may be heuristic (ie, deterministic, or based on experience-based rules) or statistical in approach.29

The ECG Signal The standard 12-lead ECG records potential differences between prescribed sites on the body surface that vary during the cardiac cycle; it reflects differences in transmembrane voltages in myocardial cells that occur during depolarization and repolarization within each cycle. The ECG was regarded by Einthoven et al30 as originating in a stationary, timedependent single-dipole source that can be represented by a vector, the heart vector. In this model, voltage in any lead was explained by projection of the heart vector onto the straight line that defined the lead axis. Burger et al31,32 expanded this concept by treating the lead axes as vectors. A lead vector, in addition to having a direction that is not the same as that of the lead axis, also has a length. Voltage in a lead is not merely the projection of the heart vector on the lead axis but also its projection on the lead vector times the length (ie, the “strength”) of the lead vector. Direction and strength of a lead vector depend on the geometry of the body and on the varying electric impedances of the tissues in the torso.31,32 Pairs of electrodes (or a combination of electrodes serving as 1 of the 2 electrodes) and the tracings that result from their use are known as leads. Placement of electrodes on the torso is distinct from direct placement on the heart, because the localized signal strength that occurs with direct electrode contact is markedly attenuated and altered by torso inhomogeneities that include thoracic tissue boundaries and variations in impedance. At any point in time, the electrical activity of the heart is composed of differently directed forces. Accordingly, the potential at any point on the body surface represents the instantaneous uncanceled electrical forces of the heart, where cancellation also is dependent on torso inhomogeneities. For further reading, see the comprehensive analysis of lead theory by Horacek in 1989.33 As electrodes move farther away from the heart, signal strength decreases together with lead strength. According to solid angle theory, signal magnitude can be related to both spatial and nonspatial factors.34 Nonspatial factors include the magnitude of transmembrane potential difference across a boundary within the heart. Spatial factors include the projected boundary of the difference in potential relative to the area of a sphere of unit size; this will increase with the absolute size of the area but decrease with distance of the electrode from

the heart. Simultaneously active wave fronts within the heart may confound the seeming simplicity of these models. The fundamental frequency for the QRS complex at the body surface is ⬇10 Hz, and most of the diagnostic information is contained below 100 Hz in adults, although lowamplitude, high-frequency components as high as 500 Hz have been detected and studied. The QRS of infants often contains important components as high as 250 Hz.35 The fundamental frequency of T waves is approximately 1 to 2 Hz.23 Filtering of the ECG signal to within the band between 1 to 30 Hz produces a stable ECG that is generally free of artifact, but this bandwidth is unacceptable for diagnostic recording because it produces distortions of both high- and low-frequency components of the signal. The high-frequency components of the ECG signal define the most rapidly changing parts of the signal, including Q waves and notched components within the QRS complex. Because QRS amplitude measurement depends on accurate detection of the peak of an R wave, an inadequate high-frequency response results in systematic underestimation of signal amplitude and in smoothing of notches and Q waves. On the other hand, an inadequate low-frequency response can result in important distortions of repolarization. Accordingly, the transfer functions of the filtering algorithms of analog and digital electrocardiographs have a major effect on the resulting ECG.

ECG Signal Processing Processing of the ECG signal by a digital electrocardiograph involves initial sampling of the signal from electrodes on the body surface. Next, the digital ECG must eliminate or suppress low-frequency noise that results from baseline wander, movement, and respiration and higher-frequency noise that results from muscle artifact and power-line or radiated electromagnetic interference.36 As a result, the ECG signal at the body surface must be filtered and amplified by the electrocardiograph. Digital filters can be designed to have linear phase characteristics, and this avoids some of the distortion introduced by classic analog filters. Once filtered, individual templates are constructed for each lead from data sampled generally from dominant complexes, from which amplitude and duration measurements are made. Global measurements are made from individual lead data or from mathematical combinations of simultaneously acquired individual lead data. Measurement error has an important effect on the accuracy of ECG diagnostic statements.37 Reference is made to the comprehensive analysis of technical factors that affect the ECG by Zywietz.38 In the present statement, factors that affect the processing of the ECG signal will be discussed in terms of technology, clinical implications, and recommendations.

Sampling of the ECG Signal Technology Direct-writing electrocardiographs, which were preponderant until the 1970s, recorded signals that were analog, that is, continuous, in nature. Nearly all current-generation ECG machines convert the analog ECG signal to digital form before further processing. Analog-to-digital conversion in modern digital ECGs generally occurs at the front end, such

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as the lead cable module. The initial sampling rate during analog-to-digital conversion at the front end is higher than the sampling rate that is used for further processing of the ECG signal. Oversampling was originally introduced to detect and represent pacemaker stimulus outputs, which are generally ⬍0.5 ms in duration. Front-end sampling has been performed at rates from 1000 to 2000 per second, but newer converters can routinely sample at 10 000 to 15 000 per second or even higher; other converters are adaptive in sampling rate, with output that is proportional to the energy detected. Clinical Implications The initial sampling rate used by the computer to transform the analog electrical signal to a series of discrete digital points (generally described in the unit of samples per second, or imprecisely as a sampling rate of x Hz) is most often many times greater than required for further processing of the ECG signal. This is known as “oversampling.” Pacemaker stimulus outputs are generally shorter in duration than 0.5 ms, and therefore, they cannot be reliably detected by ordinary signal processing technique at 500 to 1000 Hz. Accordingly, a primary benefit of oversampling is the detection of narrow pacemaker pulses. Pacemaker detection is not reliably or accurately performed in all current systems. Oversampling can also improve signal quality at the high-frequency cutoff. Separate from difficulties caused by pacemaker spike duration, the very small amplitudes of modern bipolar pacemaker stimulus outputs are often too small to be recognized on the standard ECG, a problem that requires resolution without introducing artificially enhanced pacemaker signals into the tracing. Recommendations Oversampling by a significant multiple of the upperfrequency cutoff is recommended to provide recommended bandwidth in the digitized signal. Manufacturers should continue to develop improved algorithms for the identification and quantitative presentation of pacemaker stimulus outputs and for their preservation during ECG storage and retrieval. Low-amplitude pacemaker stimulus outputs should not be artificially increased in amplitude to aid recognition, because this would distort the form of the recorded ECG. Instead, it is recommended that manufacturers incorporate a separate representation of detected pacemaker stimulus outputs into 1 row only of the standard output tracing that would aid the identification of atrial, ventricular, and biventricular pacing signals. The selected row might be a rhythm strip that accompanies the standard 3 rows of lead signals in 4 columns, or in the absence of a rhythm row, 1 of the standard rows might be selected for this purpose.

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introduces considerable distortion into the ECG, particularly with respect to the level of the ST segment.39,40 This distortion results from phase nonlinearities that occur in areas of the ECG signal where frequency content and wave amplitude change abruptly, as occurs where the end of the QRS complex meets the ST segment. Digital filtering provides methods for increasing the low-frequency cutoff without the introduction of phase distortion.23 This can be accomplished with a bidirectional filter by a second filtering pass that is applied in reverse time,41 that is, from the end of the T wave to the onset of the P wave. This approach can be applied to ECG signals that are stored in computer memory, but it is not possible to achieve continuous real-time monitoring without a time lag. Alternatively, a zero phase shift can be achieved with a flat step response filter,42 which allows the reduction of baseline drift without low-frequency distortion. Clinical Implications Low-frequency noise, such as that produced by respiration, causes the tracing to wander above and below the baseline. A low-frequency cutoff at 0.5 Hz, which was once widely used in ECG rhythm monitors, reduces baseline drift due to the generally lower frequency of respiratory motion but can result in marked distortion of repolarization that may produce artifactual ST-segment deviation.39 The 1975 AHA recommendations included a 0.05-Hz low-frequency cutoff for diagnostic electrocardiography.5 This recommendation preserves the fidelity of repolarization, but it does not eliminate the problem of baseline drift. Baseline drift suppression is necessary for coherent alignment of the sequential complexes that many modern ECG systems use in the formation of a representative PQRST complex, which is sometimes called a template; otherwise, baseline wander can distort template amplitudes. Newer digital filters can correct baseline drift while preserving the fidelity of ST-segment levels, and these digital methods obligate revision of prior standards required for analog filters. Recommendation To reduce artifactual distortion of the ST segment, the 1990 AHA document recommended that the low-frequency cutoff be 0.05 Hz for routine filters but that this requirement could be relaxed to 0.67 Hz or below for linear digital filters with zero phase distortion.23 The ANSI/AAMI recommendations of 1991, affirmed in 2001, endorsed these relaxed limits for low-frequency cutoff for standard 12-lead ECGs, subject to maximum allowable errors for individual determinants of overall input signal reproduction.24 These standards continue to be recommended.

High-Frequency Filtering Low-Frequency Filtering Technology The heart rate, in beats (cycles) per minute (bpm), when divided by 60 (seconds per minute) forms a lower bound for the frequency content in Hertz (Hz, cycles per second). In practice, this is unlikely to be lower than 0.5 Hz, which corresponds to a heart rate of 30 bpm; heart rates below 40 bpm (0.67 Hz) are uncommon in practice.23 However, with traditional analog filtering, a 0.5-Hz low-frequency cutoff

Technology The digital sampling rate (samples per second) determines the upper limit of the signal frequency that can be faithfully represented. According to the Nyquist theorem, digital sampling must be performed at twice the rate of the desired high-frequency cutoff. Because this theorem is valid only for an infinite sampling interval, the 1990 AHA report recommended sampling rates at 2 or 3 times the theoretical minimum.23 A series of studies have now indicated that data

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at 500 samples per second are needed to allow the 150-Hz high-frequency digital filter cutoff that is required to reduce amplitude error measurements to ⬇1% in adults.43,44 Greater bandwidth may be required for accurate determination of amplitudes in infants.35,45,46 The European CSE group recommended that waveforms should be recognized if they have amplitudes of at least 20 ␮V and durations of at least 6 ms.23 This implies a high-frequency response in the range of 150 Hz. A 2001 Dutch report showed that in order to keep amplitude errors ⬍25 ␮V in ⬎95% of the cases, a bandwidth up to 250 Hz is needed for pediatric cases and up to 150 Hz for adolescents.35 Clinical Implications The higher the frequencies contained in the filtered signal, the more accurate will be the measurement of rapid upstroke velocity, peak amplitude, and waves of small duration.44 Inadequate high-frequency response reduces the amplitude of QRS measurements and the ability to detect small deflections. Because digital ECGs have a temporal resolution in milliseconds and an amplitude resolution in microvolts, recommendations for the high-frequency response of ECGs have evolved over the years. A high-frequency cutoff of 100 Hz was considered adequate by the AHA in 1975 to maintain diagnostic accuracy during visual inspection of direct-writing tracings by electrocardiographers.5 Even so, it has long been recognized that higher-frequency components of the QRS complex are present47,48 and that these components may have clinical significance in patients with various forms of heart disease.49 –51 To measure routine durations and amplitudes accurately in adults, adolescents, and children, an upperfrequency cutoff of at least 150 Hz is required; an upperfrequency cutoff of 250 Hz is more appropriate for infants. An obvious consequence of these high-frequency recommendations is that reduction of noise by setting the highfrequency cutoff of a standard or monitoring ECG to 40 Hz will invalidate any amplitude measurements used for diagnostic classification.52 Recommendations The ANSI/AAMI standard of 1991, reaffirmed in 2001, recommended a high-frequency cutoff of at least 150 Hz for all standard 12-lead ECGs.24 The ANSI/AAMI document also details maximum allowable errors for individual determinants of overall input signal reproduction, which extend beyond the scope of the present report but are important guidelines for manufacturers.24 These most recent limits continue to be recommended for adolescents and for adults, with extension of the high-frequency cutoff to 250 Hz in children,35 subject to demonstration of fidelity testing by individual manufacturers according to standard methods.23 Electrocardiographs should automatically alert the user when a suboptimal highfrequency cutoff, such as 40 Hz, is used, and a proper high-frequency cutoff should automatically be restored between routine standard ECG recordings.

Formation of a Representative Single-Lead Complex Technology QRS waveform amplitudes and durations are subject to intrinsic beat-to-beat variability and to respiratory variability

between beats. Accordingly, the ANSI/AAMI standards recommend using the largest-amplitude deflection in each lead as representative of the magnitude for that measurement.24 Measurements from digitized records are more reproducible than those from analog tracings.53 Digital electrocardiographs can reduce or eliminate unwanted beat-to-beat variations within leads by forming “templates” for individual leads that serve as representative complexes. Willems et al54 have shown that programs that analyzed an averaged beat showed significantly less variability than programs that measured every complex or a selected beat; similar findings have been reported by Zywietz and colleagues.55 Single-lead average or median-complex templates may be derived from selected, accurately aligned complexes. One algorithm combines techniques to use the median values of several averaged cycles. Methods vary for the accurate alignment of normal PQRST complexes for these purposes but generally involve template matching and cross-correlation algorithms that exclude nondominant waveforms. Alignment is critical to the success of the measurement process that follows template formation. Noise, measured as RMS (root mean square) residual error in aligned representative complexes, can affect measurements of duration and compromise the tradeoff between sensitivity and specificity for infarction criteria, among other diagnoses.56 Residual error is reduced by incorporation of more complexes into the representative complex. Zywietz43 has demonstrated that noise levels in constructed complexes can be reduced to below 5 ␮V to allow deflections of 20 ␮V to be estimated with no more than 10% error. However, not all variability between complexes is due to noise, and a study using the CSE database has suggested that the diagnostic value of a representative complex may be improved under some circumstances by consideration of the classification of individual complexes.57 Although fidelity standards for other ECG features are contained in the 1990 AHA document,23 no fidelity standard exists for accuracy of representative beat construction. Clinical Implications Some biological beat-to-beat variation undoubtedly exists in the electrical activity of the heart, separate from respiratory variability, which is recorded in the surface ECG. For special purposes, such as the detection of QRS and T-wave alternans, it may be desirable to retain the ability to examine these beat-to-beat changes. For routine recording of the ECG, however, reduction of noise by formation of a single and stable representative complex for analysis of each lead results from exclusion of cycle-to-cycle change. Digital electrocardiographs can adjust for respiratory variability and decrease beat-to-beat noise to improve the measurement precision in individual leads by forming a representative complex for each lead. Automated measurements are made from these representative templates, not from measurement of individual complexes. Average complex templates are formed from the average amplitude of each digital sampling point for selected complexes. Median complex templates are formed from the median amplitude at each digital sampling point. As a result, measurement accuracy is strongly dependent on the fidelity with which representative templates are formed.

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Recommendations Digital electrocardiographs must provide beat alignment that allows selective averaging or formation of a representative complex with fidelity adequate for diagnostic ECG computer programs. Fidelity standards for construction of representative complexes need to be developed.

Global Measurement From Simultaneously Acquired Leads Technology Some, but not all, digital electrocardiographs utilize the time coherence of simultaneously acquired representative complexes to derive “global” measurements of intervals. Temporal superposition of complexes permits the earliest onset and latest offset of waveforms to be identified for measurement of intervals that are more accurate than can be obtained from single leads. This can be done by searching for the earliest and latest time points of rapid voltage change across temporally aligned individual complexes. Alternatively, a spatial vector magnitude may be created for multiple leads, as exemplified for 3 leads by (x2⫹y2⫹z2)1/2, and fiducial points may be determined from this magnitude function. An equally useful function can be derived as |⌬x|⫹|⌬y|⫹|⌬z|, where ⌬x is the amplitude difference between 2 consecutive samples in lead x, etc, which is a spatial velocity function. When only several selected representative complexes are included in the global measurement, intervals may still be underestimated if earliest onset and latest offset times are not detected. Conversely, global measurements may overstate intervals by inclusion of single-lead information that would not be visually accepted by a human overreader. Differences in measurements may also result from differences in the method of lead alignment or template formation and from differences in definition of waveform onset and offset by different algorithms of different manufacturers. The importance of this phenomenon is seen in determination of the QT interval, where different approaches to definition of T-wave offset can confound reproducibility.58,59 It is in this context that differences in ECG measurement performance of different computer-assisted analysis programs must be placed.15,17 Clinical Implications The capability for simultaneous 12-lead data acquisition by modern digital electrocardiographs obligates major reconsideration of measurement standards and reference values for intervals that were originally derived from analog, singlechannel recordings. When the vector orientation of any lead is approximately perpendicular to the heart vector during the initial or terminal portion of an ECG waveform, an isoelectric component of the initial or terminal component of the waveform will be recorded in that lead at that time. Because there can be no accurate time alignment of leads in singlechannel recordings, duration measurements from individual leads will in most cases fail to detect the earliest onset or the latest offset of waveforms. As a result, measurements from single leads will systematically underestimate durations of components of the PQRST complex.21 Simple demonstration of this phenomenon is seen in the measurement of QT

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dispersion that results from isoelectric components of the T wave in some leads of the normal ECG.60,61 Measurement from simultaneous leads provides a method for identification of the earliest onset and latest offset of waves that are used for duration measurements. Waveform measurements taken from temporally aligned lead information will be systematically greater than the corresponding measurements made from single leads or measurements averaged from several leads. P-wave and PR-interval durations, QRS duration, and QT interval in population studies will be greater when measured from temporally aligned multiple leads or from a spatial vector lead template than when measured from individual leads. In addition, global measurement can affect Q-wave durations that determine the ECG diagnosis of myocardial infarction. Accordingly, redefinition is required of population-based criteria for first-degree atrioventricular block, P-wave duration, Q-wave duration in infarction (relative to the earliest onset of the QRS complex), QRS duration, and QT intervals measured from simultaneous lead technology. Several studies of normal limits of ECG measurements derived from simultaneously recorded 12-lead ECGs have already been published.62– 66 Global measurement of the QT interval is desirable for routine electrocardiography, but global QT measurement remains problematic even when derived from temporally aligned complexes. This is due in part to differences in the currently available algorithms that are used to define and to identify the end of the T wave, which can affect measurements.59 Until reproducible methodology is established in this area, comparative analyses of ECGs must recognize the potential effect of different algorithms on resulting simultaneous lead measurements. Special situations, such as QT monitoring in drug trials, may continue to require alternative methods of QT measurement from single or multiple leads. Recommendations Global measurements of intervals should be obtained from time-coherent data in multiple leads to detect the earliest onset and latest offset of waveforms. For routine purposes, global measurements of P-wave duration, PR interval, QRS duration, and QT duration should be stated on the ECG report. A comparative study is needed of global measurements made by different methods from a reference standard. Differences in global measurement algorithms and methods should be minimized to promote standardization, but these differences must be accounted for in comparative studies within individuals and between individuals. Attention must be paid to definition of normal ECG ranges in children and adolescents, as well as in adults, with stratification for specific age groups, sex, and race. Where methods vary, algorithm-specific normal ranges for intervals need to be derived. With respect to QT interval, the end of the T wave as determined globally should match with a well-defined T-wave offset in at least 1 of its component individual leads. Alternative methods of QT measurement from single or multiple leads may be prescribed for special purposes such as drug evaluation, but it is inappropriate for studies involving serial comparison of the QT interval to use differing methods of QT measurement within trials.

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Data Compression for Transmission, Storage, and Retrieval of ECGs

signal,23,73 and they can be eliminated with newer methods of lossless compression (in which no loss of ECG data occurs).

Technology Digitized at 500 samples per second, 10 seconds of a single lead of ECG record requires ⬇10 kB of memory. Accordingly, 10 seconds of an uncompressed 12-lead ECG digitized at recommended standards would occupy about 80 to 100 Kb of memory, in addition to memory needed for template complexes and demographic data. Several methods of ECG data compression have been used to reduce processing time and to minimize the memory required for permanent data storage.67,68 Techniques include fast Fourier, discrete cosine, and wavelet transforms, as well as hybrid compression methods.69 –73 These methods can provide compression ratios of 8:1 to 10:1 with resulting root mean square errors that range from ⬍0.5% to ⬎2%.69,70,74 Compression ratio is generally inversely related to root mean square error, so that a recent algorithm was able to provide a 20:1 compression ratio but with a root mean square error of 4%.70 Because compression affects high-frequency components of the ECG to a greater extent than low-frequency components, at least 1 algorithm has used bimodal decimation of the signal in which QRS complexes are kept at 500 samples per second while the rest of the recording is compressed to lower sampling rates.75 Compression of data may occur before or after signal processing, but in either case, compression occurs before transfer of the signal to central storage systems and affects all retrieved records. Accordingly, the 1990 AHA report recommended that the fidelity of retrieved compressed data should be within 10 ␮V for corresponding samples.23 As computer networks increase transmission speed and storage capacity, lossless compression techniques may supersede other compression methods for some applications.

Recommendation Compression algorithms should perform in a manner that allows retrieved data to adhere to the fidelity standards established in the 1990 AHA statement with reference to the original signal.

Clinical Implications Compression of ECG data can speed transmission and retrieval of records that are stored in central databases and minimize memory required for storage. Algorithms based on a variety of mathematical transforms can compress data by a factor of ⬇8, with signal fidelity preserved within about a 2% overall error. However, the error may not be uniform throughout the ECG cycle. Data compression affects highfrequency (short duration) signals more than the smoother low-frequency signal. Therefore, compression has greater potential to alter measurements within the QRS complex, such as pacemaker spikes, Q-wave duration, and R-wave amplitude, than to alter other signals such as the ST segment and the T wave. In some cases, a noncompressed ECG taken at the bedside may differ from the tracing later retrieved from the stored, compressed file, which may also affect serial comparison of original and retrieved tracings when ECG waveforms are reanalyzed.76 Furthermore, differences in compression methodology may affect comparison of retrieved tracings from different manufacturers in the same way that different filters and different use of time-coherent templates affect measurements of the ECG signal. These differences will be minimal when compressed tracings adhere to established or newer standards of fidelity to the original

Standard Leads Location of Standard Limb and Precordial Electrodes Technology The standard 12-lead ECG5,24 consists of 3 limb leads (leads I, II, and III), 3 augmented limb leads in which the Goldberger modification of the central terminal of Wilson serves as a derived indifferent electrode that is paired with the exploring electrode (leads aVR, aVL, and aVF), and 6 precordial leads in which the Wilson central terminal serves as a derived indifferent electrode that is paired with the exploring electrode (V1 through V6). All leads are effectively “bipolar,” and the term “unipolar” in description of the augmented limb leads and the precordial leads lacks precision. Reference is made to the comprehensive study of lead systems for various types of electrocardiography by Macfarlane.77 Skin preparation by cleaning and gentle abrasion before electrode application can reduce noise and improve the quality of the recorded ECG.78 – 80 Historically, limb lead electrodes have been attached at the wrists and the ankles, with the patient in the supine position, generally with a pillow under the head. For routine 12-lead recording, the AHA statement of 1975 recommended placement of the 4 limb lead electrodes on the arms and legs distal to the shoulders and hips,5,81 and thus not necessarily on the wrists and ankles. Evidence exists that different placement of electrodes on the limbs can alter the ECG, a phenomenon that appears to be more marked with respect to the left arm electrode.81 Therefore, reevaluation of the magnitude of changes due to variation in limb electrode placement in clinical practice is required, as discussed below. Six electrodes are placed on the chest in the following locations: V1, fourth intercostal space at the right sternal border; V2, fourth intercostal space at the left sternal border; V3, midway between V2 and V4; V4, fifth intercostal space in the midclavicular line; V5, in the horizontal plane of V4 at the anterior axillary line, or if the anterior axillary line is ambiguous, midway between V4 and V6; and V6, in the horizontal plane of V4 at the midaxillary line. Clinical Implications Skin preparation and electrode placement have important effects on the ECG, and patient positional change, such as elevation and rotation, can change recorded amplitudes and axes. It has been widely accepted for many years that ECG amplitudes, durations, and axes are independent of the distal or more proximal location of the limb electrodes. As a result, routine recording of the ECG from the upper arm rather than from the wrist to “reduce motion artifact” has become popular and is facilitated by the development of disposable tab electrodes. However, one study has shown that electrode placement along the limbs can affect ECG voltages and

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durations, most importantly in the limb leads.81 Whether these differences are large enough to alter routine diagnostic criteria, such as voltage for left ventricular hypertrophy or Q-wave duration for inferior infarction, is unknown. Further confounding this situation is the variability in electrode placement that might have been present during the actual derivation of the diagnostic criteria involved, because studies during the past several decades have rarely described electrode placement in detail. From the time of their initial standardization by a joint committee of the AHA and the Cardiac Society of Great Britain and Ireland,82,83 the normal precordial electrode positions have been relatively horizontal in orientation. When precordial electrodes are positioned without reference to the underlying bony landmarks, the placement pattern often is erroneously vertical in orientation.84 Mapping data document the often profound alterations in waveforms that can result from precordial electrode misplacement.85,86 A common error is superior misplacement of V1 and V2 in the second or third intercostal space. This can result in reduction of initial R-wave amplitude in these leads, approximating 0.1 mV per interspace, which can cause poor R-wave progression or erroneous signs of anterior infarction.87 Superior displacement of the V1 and V2 electrodes will often result in rSr⬘ complexes with T-wave inversion, resembling the complex in lead aVR. It also has been shown that in patients with low diaphragm position, as in obstructive pulmonary disease,88,89 V3 and V4 may be located above the ventricular boundaries and record negative deflections that simulate anterior infarction. Another common error is inferior placement of V5 and V6, in the sixth intercostal space or even lower, which can alter amplitudes used in the diagnosis of ventricular hypertrophy. Precordial lead misplacement explains a considerable amount of the variability of amplitude measurements that is found between serial tracings.90 Some residual disagreement persists in current guidelines and texts on the standard for location of V5 and V6, with some sources retaining an early recommendation that these leads follow the course of the fifth intercostal space rather than the horizontal plane of V4. In addition, it is common to refer to the anterior axillary line as an anatomic marker for the placement of V5. These alternatives are discouraged because the course of the intercostal space is variable and the definition of an anterior axillary line only vague. Placement of precordial electrodes in women with large breasts remains problematic. Electrodes are most commonly placed beneath the breast, which should reduce amplitude attenuation caused by the higher torso impedance in women and, intuitively, would seem to favor reproducibility of positioning during routine practice. Conversely, one study has suggested that reproducibility of ECG measurements is slightly increased when electrodes are positioned on top of the breast.91 Another study using precisely ascertained electrode placement has suggested that precordial potential attenuation by the breast is very small.92 Yet another study has found attenuation only in V3 and an increase in voltage in V5 and V693 when electrodes are placed over the breast; this may result from V5 and V6 being correctly placed at the level of V4 rather than more inferiorly when V4 is positioned under the breast. Clearly, the magnitude of this effect in ordinary

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ECGs will depend greatly on the care with which electrodes are ordinarily placed and also on breast size, breast shape, and small changes in patient position. Similar considerations apply in relation to subjects with breast implants and in subjects who are obese. Recommendations Technicians and other medical personnel responsible for the recording of ECGs should have periodic retraining in skin preparation, proper electrode positioning, and proper patient positioning. All leads are effectively “bipolar,” and the differentiation between “bipolar” and “unipolar” in the description of the standard limb leads, the augmented limb leads, and the precordial leads is discouraged. Neither term should be used. Studies to clarify the effect of distal versus proximal limb lead electrode placement on ECG magnitudes and durations are required. Validity of test performance criteria for current diagnostic algorithms may be dependent on placement of limb leads in the same positions that were used for criteria development. Pending resolution of this issue, all ongoing studies used for criteria development must clearly document electrode placement with precision. The horizontal plane through V4 is preferable to the fifth intercostal interspace for the placement of V5 and V6 and should be used for placement of these electrodes. Definition of V5 as midway between V4 and V6 is conducive to greater reproducibility than occurs for the anterior axillary line, and this should be used when the anterior axillary line is not well defined. In the placement of V6, attention should be directed to the definition of the midaxillary line as extending along the middle, or central plane, of the thorax. For the time being, it is recommended that electrodes continue to be placed under the breast in women until additional studies using electrodes placed on top of the breast are available.

Derivation of the Standard Limb Leads and Relationships Among Leads Technology The 4 limb electrodes define the standard frontal plane limb leads that were originally defined by Einthoven. With the right leg electrode acting as an electronic reference that serves to improve common mode (unwanted noise) rejection, 3 pairs of electrodes exist. Within each pair, 1 electrode is established as the positive end of the lead in the sense that current flow toward that electrode is inscribed in an upward (positive) direction. The other electrode of the pair would inscribe the exactly opposite waveform. Lead I is defined as the potential difference between the left arm and the right arm (LA-RA), lead II is defined as the potential difference between the left leg and the right arm (LL-RA), and lead III is defined as the potential difference between the left leg and the left arm (LL-LA). In each case, net current flow toward the first electrode of the pair is defined as a positive voltage deflection in the recorded waveform. According to Kirchhoff’s law, the sum of the voltage gains and voltage drops in a closed circuit is equal to zero. Therefore, lead II⫽lead I⫹lead III at any instant in the cardiac cycle. This relationship is known as Einthoven’s law.

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Clinical Implications From 3 pairs of limb electrodes, 6 waveforms may be obtained, 3 of which are defined as the standard limb leads by establishing 1 of each pair as the electrode toward which net current flow will inscribe an upward (positive) voltage deflection on the ECG. The opposite waveforms, by definition, are mirror images of the standard limb leads. In this sense, the electrical activity defined by a lead pair can be examined from either perspective. Distinction of single electrodes from established “poles” is highlighted by selection of the LA electrode as the positive end of the LA-RA pair for lead I but not as the positive end of the LL-LA pair for lead III. Einthoven’s law indicates that any 1 of the standard limb leads can be mathematically derived from the other 2 leads. As a consequence, the 3 standard limb leads contain only 2 independent pieces of information. Even though limb lead placement is often represented in terms of the apices of an equilateral triangle, known as the Einthoven triangle, Einthoven’s law is entirely independent of any assumptions about geometric placement of the 3 electrodes. These considerations notwithstanding, redundant leads promote the appreciation of spatial morphological characteristics of the ECG and aid in its interpretation, such as calculation of axis, and consideration of the information from the perspective of both ends of the available leads can be clinically useful, particularly in the evaluation of ST-segment shifts during acute myocardial infarction. Recommendation Users should recognize the redundancy of information in the standard limb leads. Redundancy notwithstanding, the information contained in different perspectives from multiple leads can be used to improve recognition of ECG abnormalities.

Derivation of the Augmented Limb Leads and the Precordial Leads Technology An electrode potential can also be obtained as an average (or weighted average) of the potentials at 2 or more body surface locations, which creates a potential that is different from each of the contributing electrodes alone. Wilson and colleagues94 devised a central terminal based on the limb electrodes to serve as a new reference potential. The Wilson central terminal (WCT) is obtained as an average potential of the RA, LA, and LL electrodes, so that the potential at WCT⫽(RA⫹LA⫹LL)/3. Kirchhoff’s law does not require that the potential at WCT be zero or that it remain constant throughout the cardiac cycle. Potential differences between WCT and RA, LA, and LL, respectively, defined new frontal plane limb leads VR, VL, and VF. Wilson called these electrode pairs the “unipolar” limb leads. Wilson’s VR, VL, and VF leads had relatively low amplitudes because the potential at the exploring site was also included in the central terminal. By removing the single exploring potential from the central terminal, Goldberger produced the “augmented unipolar” limb leads, so-called because they mathematically are 50% larger in amplitude with respect to recordings that use the Wilson central terminal.95,96 The Goldberger central

terminals for the augmented limb leads are now obtained as (LA⫹LL)/2 for aVR, (RA⫹LL)/2 for aVL, and (RA⫹LA)/2 for aVF. Lead aVL therefore represents the potential difference between the left arm and the modified terminal of Goldberger and is given by LA⫺(RA⫹LL)/2, which can be reduced to (lead I⫺lead III)/2. Similarly, lead aVR is RA⫺(LA⫹LL)/2, which can be reduced to ⫺(lead I⫹lead II)/2, and lead aVF is LL⫺(LA⫹RA)/2, which can be reduced to (lead II⫹lead III)/2. These derived leads provide new vectorial perspective within the frontal plane. It should be noted that aVR⫹aVL⫹aVF⫽0 at any point in the cardiac cycle. The 6 standard precordial leads are based on potential differences between an exploring electrode on the chest wall and the original WCT. Each precordial lead, symbolized as Vi, represents the potential difference given by Vi⫺WCT. Clinical Implications The augmented limb leads and the precordial leads use a derived electrode to serve as the opposing electrode of the lead pair. Wilson made a reasonable assumption that the potential oscillations of his central terminal would be small compared with those of the exploring electrode and that his “unipolar” leads therefore would largely reflect the potential variation under the exploring electrode. Later investigators have often mistakenly taken this to mean that these leads reflect electrical activity only of cardiac regions in the vicinity of the exploring electrode. This fails to recognize that the potential at the exploring electrode is determined by all cardiac sources electrically active at a given instant of cardiac excitation and repolarization cycle. Even though the augmented limb leads provide vectorial insight within the frontal plane, each of these leads can also be mathematically derived from any 2 of the standard limb leads, as demonstrated above; accordingly, they do not contain new information but rather provide new views of cardiac electrical activity. This calculation is mathematically independent of any assumption about the equilateral nature of the Einthoven triangle. As a consequence, the 6 frontal plane leads, consisting of the 3 standard limb leads and the 3 augmented limb leads, actually contain only 2 independent measured signals. In practice, modern electrocardiographs measure potential differences for 2 pairs of limb lead electrodes and use these measurements to mathematically derive the third standard limb lead and each of the augmented limb leads. Although redundancy exists within the 6 frontal plane leads, visualization of multiple leads promotes appreciation of spatial aspects of the ECG that can be important to clinical interpretation. Unlike the mathematical relationships between the frontal plane limb leads, each of the precordial electrodes provides uniquely measured potential differences at the recording site with reference to the central terminal. Because the exploring precordial electrodes are not connected in a closed electrical loop like the extremity electrodes, the precordial leads are independent of each other; none can be calculated precisely from other information in the ECG. Therefore, the “standard” 12-lead ECG actually contains 8 independent pieces of information: 2 measured potential differences from which the 4 remaining limb leads can be calculated and the 6 independent precordial leads.

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Recommendations The augmented limb leads of the frontal plane and the precordial leads result from derived electrode pairs and should not be described as “unipolar.” Users should recognize the derived and redundant nature of the 3 augmented limb leads, but these are retained because multiple leads facilitate the clinical interpretation of the ECG.

Simultaneous Lead Presentation Technology With analog single-channel ECG recorders, each lead is recorded sequentially by means of a switching mechanism that connects applied electrodes in the prescribed combinations. Digital electrocardiographs are able to record the 8 channels of independent information simultaneously, with 4 of the limb leads being derived from the other 2. Alignment of separate channel writers must be precise to within 10 ms,24 and ideally less. The most commonly used output format involves lead separation based on rows and columns. For standard-sized paper, at 25 mm/s recording speed, four 2.5-second columns can be presented sequentially on the page, with no time disruption between different columns. Each column therefore represents successive 2.5-second intervals of a continuous 10-second record. In the most traditional simultaneous lead format, the first column records rows representing simultaneous leads I, II, and III; the second column records rows representing simultaneous aVR, aVL, and aVF; the third column represents simultaneous leads V1, V2, and V3; the fourth column represents simultaneous leads V4, V5, and V6. Additional rows may be available for 1, 2, or 3 leads of 10-second continuous recordings for rhythm analysis. Alternatively, additional rows may be utilized to present two 5-second recordings of 6 simultaneous limb leads and 6 simultaneous precordial leads, or 12 rows of simultaneous leads. Clinical Implications The major advantage of simultaneous lead acquisition is that it allows precise temporal alignment of waveforms from different leads, which results in spatial-temporal insights that have diagnostic value.97 By way of example, the temporal alignment of waveforms in aVR and aVL can aid in the diagnosis of fascicular block in the presence of infarction,98 whereas simultaneous views of P-wave and QRS waveforms in multiple leads can add information of value in the interpretation of arrhythmias and in the diagnosis of myocardial infarction.99 Recommendation Standard tracings obtained with digital electrocardiographs should provide accurate temporal alignment of multiple leads, with maximum misalignment of no more than 10 ms, and ideally as little as is practically feasible. The printed tracing may present temporally aligned groups of leads in different formats according to preference.

Alternative Information Format From Standard Leads Technology The Cabrera or orderly sequence reorients the frontal plane leads into a progressive anatomic array that extends logically

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and sequentially in the same way that the precordial leads progress sequentially from V1 through V6.100,101 With inverted aVR (⫺aVR or maVR) used to represent the signal between leads II and I, the sequence becomes, from right to left, III, aVF, II, ⫺aVR, I, and aVL, or from left to right, aVL, I, ⫺aVR, II, aVF, and III. In addition to improved spatial quantification of acute infarction, the Cabrera sequence facilitates calculation of the frontal plane axis.102 This presentation, when in sequence with the precordial leads, has also been termed the panoramic display.103 Clinical Implications Whether presented serially from single-channel recorders or in standard array from simultaneous-lead–acquisition devices, the sequence of limb lead presentation on ECG recordings is historical, not anatomic. Thus, whereas V1 through V6 progress leftward and slightly inferiorly across the precordium, the frontal plane limb leads follow no regular order that allows individual leads to be compared easily with anatomically directly adjacent leads. For example, lead aVF represents the potential difference from a vector perspective that is between lead III and lead II, but this is not easily appreciated from the standard array. Similarly, leads I and aVL are progressively counterclockwise, in the anatomic sense, from lead II. Lead aVR is often thought of as an intracavitary lead that looks toward the atria from the apex of the ventricles, but inversion of aVR can be considered to represent a perspective that lies anatomically within the counterclockwise progression from lead II to lead I.101 Use of inverted aVR has been reported to improve the diagnostic classification and estimation of risk associated with acute inferior and lateral myocardial infarction.104 Recommendations Routine use of the Cabrera sequence for display of the limb leads can be highly recommended as an alternative presentation standard. For display in a format of 4 columns of 3 leads, a left-to-right sequence (aVL to III) is logical because it is closer to traditional placement of limb lead I at the upper left. To maintain consistency, the left-to-right sequence is also recommended for horizontal display of the limb leads. However, it is recognized that the current limb lead array is so deeply entrenched in ECG tradition that change might take years to become generally accepted. At present, manufacturers should be encouraged to make this display available as a routine option in new electrocardiographs.

Alternative Lead Applications Torso and Other Modified Placement of the Limb Leads Technology Noise from motion of the arms and legs during ambulatory and exercise electrocardiography can be reduced by placement of the limb leads on the torso. In these diagnostic applications, 12-lead ECGs have been recorded with the Mason-Likar lead position,105 in which the arm electrodes are placed in the infraclavicular fossae medial to the deltoid insertions and the left leg electrode is placed midway between the costal margin and iliac crest in the left anterior axillary

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line. More recent applications of the Mason-Likar monitoring position place the arm electrodes over the outer clavicles.81,106 The precordial electrodes are placed in the standard positions. An alternative modification of limb lead placement developed for bicycle ergometry applies the arm electrodes to the upper outer arm and the leg electrodes to the anterior iliac crest.107 Torso limb leads are sometimes used to reduce motion artifact from the arms and legs during recording in infants. Clinical Implications Noise from motion of the limbs during routine ambulation and during exercise makes standard limb lead electrode placement impractical for ECG monitoring. Typical monitoring applications include bedside hard-wired or telemetered observation of rhythm and ST segments, quantitative ambulatory electrocardiography, and ECG recording during diagnostic exercise testing.108 Rhythm diagnosis is not adversely affected by monitoring lead placement; however, tracings that use torso electrodes differ in important ways from the standard 12-lead ECG. In addition to body position differences that affect the ECG,109 monitoring electrodes placed on the trunk do not provide standard limb leads, and distortion of the central terminal alters the augmented limb leads and the precordial leads.110,111 Tracings with Mason-Likar and other alternative lead placement may affect QRS morphology more than repolarization compared with the standard ECG; these differences can include false-negative and false-positive infarction criteria.81,112 Motion artifact of the limbs is a particular problem for routine recording in neonates, infants, and young children, in whom torso leads are sometimes used; the clinical significance of the resulting differences remains to be established. Recommendations ECGs recorded with torso placement of the extremity electrodes cannot be considered equivalent to standard ECGs for all purposes and should not be used interchangeably with standard ECGs for serial comparison. Evaluation of the effect of torso placement of limb leads on waveform amplitudes and durations in infants is required. Tracings that use torso limb lead placement must be clearly labeled as such, including 12-lead tracings derived from torso limb lead placement in neonates or in young children and during ambulatory and exercise electrocardiography in adults. Furthermore, tracings recorded in the sitting or upright position should not be considered equivalent to standard supine ECGs.

Reduced Lead Sets Technology It is possible to mathematically construct a synthesized 12-lead ECG from reduced lead sets. These syntheses can approximate but not duplicate the tracing obtained by the standard leads. The Frank lead system was devised as a lead set suitable for obtaining reproducible orthogonal lead information that could be used for vectorcardiography.5 The system involves 7 electrodes, 5 of which are applied at points in the horizontal plane that intersect the fifth intercostal space at the left sternal border: A at the left midaxillary line, C on the anterior left chest wall halfway between E and A, E at the

mid sternum anteriorly, I at the right midaxillary line, and M at the mid spine posteriorly. In addition, electrode H is placed at the junction of the neck and torso posteriorly, and electrode F is placed on the left foot. Orthogonal lead information is constructed from modeled weighting of lead voltages. The EASI lead system is a reduced 5-lead set that uses the E, A, and I electrodes from the Frank lead system and adds an electrode, S, at the top of the mid sternum, along with a ground reference electrode to provide orthogonally oriented signals.113 In addition to orthogonal data, transfer coefficients have been developed for the EASI lead system that produce synthesized 12-lead ECGs.114 Advantages of the EASI lead system for patient monitoring applications are the absence of limb electrodes, which allows the patient to move around without intolerable noise in the ECG signal, elimination of the need to determine intercostal spaces, and avoidance of the breast. Clinical Implications Because monitoring applications of reduced lead sets are widespread and 12-lead reconstruction algorithms are available in practice, it is important that the derived nature of these tracings is appreciated. The Frank lead system and other vectorcardiographic lead systems produce the orthogonal X, Y, and Z components of the heart vector. These can be combined into 3-dimensional vectorcardiographic loops displayed in 2-dimensional planes (frontal, horizontal, and sagittal); they can be directly examined as ECG voltage-time records as well. A number of transformations of orthogonal data can be used to produce a synthesized 12-lead ECG, but the generalized transfer coefficients used in these estimations are subject to individual variability in torso shape and heterogeneities of impedance. Patient-specific transformations derived from comparison with a baseline 12-lead ECG can improve the accuracy of subsequent synthesized tracings. Torso inhomogeneities also limit the fidelity of synthesized 12-lead tracings derived from EASI leads. An advantage of EASI leads is the relative anatomic simplicity of electrode placement. Tracings synthesized from the EASI leads have been shown to have useful correlative value with the standard 12-lead ECG115,116; however, it is recognized that these synthesized tracings can differ in interval duration and amplitude from the corresponding standard ECGs. Whether synthesized 12-lead tracings provide practical advantage and adequate reproduction of ST-segment shifts to be a substitute for standard tracings during acute ischemic syndromes is a matter of intense current investigation.117 Whether the accuracy of these transformations for the monitoring of repolarization changes can facilitate drug trials in ambulatory subjects is also under study. Recommendations Synthesized 12-lead ECGs are not equivalent to standard 12-lead ECGs and cannot be recommended as a substitute for routine use. All 12-lead tracings derived by synthesis from reduced lead sets must be clearly labeled as such. Although synthesized ECGs that use the EASI lead system may be demonstrably adequate for some purposes, such as monitoring of rhythm, they cannot be considered equivalent to

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standard 12-lead recordings or recommended at present as an alternative for routine use.

Expanded Lead Sets Technology Hybrid lead systems, incorporating 3 Frank leads with the standard 12 leads, can be used by some electrocardiographs. Expanded lead sets include the multiple-electrode arrays used for body surface mapping of the electrical activity of the heart. Torso arrays include wraparound electrodes in multiple horizontal and vertical lines. Details of these arrays are beyond the scope of the present report. Studies of body surface maps recorded from large electrode arrays have provided useful information about localization of ECG information on the thorax, but their complexity precludes their use as a substitute for the standard 12-lead ECG for routine recording purposes. Additional chest leads may be useful for investigation of acute infarction. Four additional precordial leads have been identified for use in this clinical setting (V3R, V4R, V5R, and V6R), each of which is placed on the right side in mirror image to the standard precordial placement of electrodes. Within this right-sided array of electrodes, standard V1 can be considered equivalent to V2R, and standard V2 can be considered equivalent to V1R. Examination of additional posterior chest leads has been proposed for the identification of ST-elevation events in the posterior wall, including V7 (at the posterior axillary line), V8 (below the scapula), and V9 (at the paravertebral border), each in the same horizontal plane as V6.118 –120 Clinical Implications Although acute right ventricular infarction can sometimes be recognized from ST-segment elevation in V1, studies dating from the early 1980s have demonstrated that additional right-sided precordial leads have value for the diagnosis of acute right ventricular infarction in patients with inferior infarction.121–123 In this setting, ST-segment elevation exceeding 0.1 mV in 1 or more of the right precordial leads is moderately sensitive and specific for right ventricular injury and has been associated with underlying right ventricular dysfunction124,125 and greater in-hospital complications.126 Acute infarction of the posterior wall of the left ventricle theoretically can be diagnosed from reciprocal ST-segment depression evident in precordial leads V1 through V3, and it appears that both the additional right-sided and additional posterior leads can be reconstructed from the standard ECG leads.127 (Alternate description of this territory as anatomically inferolateral rather than posterior will be discussed elsewhere.) Additional leads have not provided increased sensitivity for infarction in all studies128; however, STsegment elevation over the posterior left chest has been reported to be the only site of ST elevation found in some cases of posterior infarction.118 Recent guidelines for intervention in acute coronary syndromes differ in important ways for ST-elevation and for non–ST-elevation infarction.129 In this sense, anterior ST depression during infarction from a spatial vector perspective may be electrocardiographically equivalent to posterior ST elevation, but it may be quite different in terms of a literal interpretation of treatment

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guidelines that requires “ST elevation” in an intervention algorithm. Even so, ST elevation in posterior leads in acute posterior infarction is often ⬍1 mm in amplitude, and because of lead orientation, proximity effect, and torso inhomogeneity, it may not be equivalent in absolute magnitude to the ST depression present in anterior leads. ST elevation in 1 or more of the posterior leads has moderate sensitivity and high specificity for posterior wall infarction,130 but the value of these additional findings for the prediction of increased in-hospital complications is unresolved.126,131 Recommendations Because treatment of infarction may vary with right ventricular involvement, recording of additional right-sided precordial leads during acute inferior-wall left ventricular infarction is recommended. Routine recording of these leads in the absence of acute inferior infarction is not recommended. The use of additional posterior precordial leads can be recommended in settings in which treatment will depend on documentation of ST elevation during infarction or other acute coronary syndrome. Routine recording of these additional leads in the absence of an acute coronary syndrome is not recommended. As ST-segment vectors become increasingly used for improved diagnostic classification of myocardial infarction, the addition of a frontal plane ST-segment axis to the currently measured P-wave, QRS, and T-wave axes in the ECG header data is recommended.

Lead Switches and Misplacements Limb Lead and Precordial Lead Switches Technology Lead switches (or more correctly, electrode cable switches) occur when a dedicated lead wire and electrode combination is misplaced or when there is erroneous attachment of a dedicated lead wire to individually placed electrodes. Color coding of lead wires is a feature of manufacturing standards for electrocardiographs,24 but even so, it is possible to misconnect lead wires at the cable terminal. Time-coherent P-wave morphology can be used to clarify lead switches,132 and these principles should be applicable to computer algorithms. Computer algorithms that are adaptable to computerassisted electrocardiographs are capable of detecting lead switches.133–137 Clinical Implications Lead switches are really switches of the cable connections of 2 or more properly placed electrodes. This can result in erroneous pairing within the standard limb leads or within the pairing of an exploring lead with the central terminal. When an electrode that is switched involves the central terminal, all leads may be affected. Lead switches affect 2 or more of the standard leads, thereby distorting the ECG recording. Limb lead switches can result in false-positive and false-negative signs of ischemia.138 Some of these changes can be recognized by an alert technician or correctly interpreted by the reviewing physician, particularly when previous ECGs are available, whereas others may go unrecognized or require repeat recording of the ECG.139 Transposition of the left and

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right arm lead wires produces inversion of limb lead I, with a switch of leads II and III and a switch of leads aVR and aVL, whereas aVF remains unaltered. Because the central terminal is unaffected, there are no changes in the precordial leads. In normal situations, lead I is generally similar to V6 with respect to the morphology of the P wave and QRS direction. A clue to distinction of these findings from those present in a patient with mirror-image dextrocardia is that lead misplacement results in an important discordance between lead I and V6. As a corollary, the ECG in a patient with mirror-image dextrocardia may be “normalized” by purposely reversing the left and right arm lead wires and using mirror-image rightsided precordial leads. Transposition of the right arm and right leg lead wires is also easy to recognize, because lead II now records the nearly zero potential difference that exists between the 2 legs,140,141 which results in very low amplitude only in lead II, with inverted symmetry between standard lead I and lead III. Transposition of the left arm and left leg lead wires is more difficult to recognize because the main effects are an often subtle shift in axis and inversion of lead III; it can be suspected from changes in P-wave morphology in the limb leads,138 although the specificity of this approach has been challenged.137 Suspected lead switches may be confirmed by reference to a prior or subsequent tracing with correct lead placement. Transposition of lead wires to V1 and V2, to V2 and V3, or within all 3 leads can cause a reversal of R-wave progression that simulates anteroseptal wall infarction, but this artifact often can be recognized by distorted progression of the precordial P waves and T waves in the same leads. Recommendations Medical personnel responsible for the recording of routine ECGs should receive training on the avoidance of lead switches and guidelines for their recognition. Lead-switch detection algorithms should be incorporated into digital electrocardiographs along with alarms for abnormally high lead impedance, and suspected misplacements should be identified to the person recording the ECG in time to correct the problem. If not corrected before recording, a diagnostic statement alerting the reader to the presence of different types of lead switches should be incorporated into preliminary interpretive reports.

Lead Misplacement Technology ECG amplitudes and duration measurements vary with precordial lead placement, which often ranges widely from the recommended anatomic sites.84,142 The early work of Kerwin et al143 demonstrated that reproducibility of precordial lead placement to within 1 cm occurred only in about half of men and in even fewer women. Placement accuracy during routine electrocardiography appears to have decreased further with time. A recent study documented that fewer than two thirds of routinely applied precordial electrodes were applied within 1.25 inch of the designated landmark, but errors were not distributed randomly.84 A more vertical distribution of precordial electrodes than required resulted from superior misplacement of V1 and V2 electrodes in more than half of cases

and inferior-leftward misplacement of left precordial electrodes in more than one third. Clinical Implications Lead placement variability between recordings is an important reason for poor reproducibility of precordial ECG amplitude measurements.86,90,144 Reproducibility of duration measurements is generally better than reproducibility of amplitudes.145 It has been established that variation in precordial lead placement of as little as 2 cm can result in important diagnostic errors, particularly those that involve statements about anteroseptal infarction and ventricular hypertrophy. 142 Precordial lead misplacement can alter computer-based diagnostic statements in up to 6% of recordings.85 Recommendations Periodic retraining in proper lead positioning of the precordial leads should be routine for all personnel who are responsible for the recording of ECGs. Serial tracings in acute or subacute care settings should make use of some form of skin marking to promote reproducibility of lead placement when it is not possible to leave properly applied electrodes in place.

Computerized Interpretation of the ECG Technology Two computer-based processes are required for diagnostic digital ECG programs that provide diagnostic interpretation. The first stage is preparation of the signal for analysis by the processing methods discussed above. As discussed in prior sections of this statement, the fidelity of measurements used in diagnostic algorithms is determined by the technical issues that affect signal processing.9,23,28,42,146 These signalprocessing methods include signal preparation (sampling, filtering, and template formation), feature extraction, and measurement.147–151 Time-coherent simultaneous lead data and the construction of representative template complexes are critical to the reliability of feature extraction and measurement; global measurements of duration may be systematically smaller when time-coherent data are not used. The second stage of analysis applies diagnostic algorithms to the processed ECG. Diagnostic algorithms may be heuristic (experience-based rules that are deterministic) or statistical (probabilistic) in structure. Heuristic diagnostic algorithms were originally designed to incorporate discrete measurement thresholds into a decision tree or boolean combinations of criteria.152–155 Statistical diagnostic algorithms circumvent problems of diagnostic instability that are associated with small serial changes around discrete partitions by adding a probability statement to the diagnosis. These may be based on bayesian logic.156 Other statistical methods use discriminant function analysis, which can use continuous ECG parameters in addition to discrete variables to produce a point score.157,158 These algorithms tend to be more reproducible than earlier heuristic methods, even though they still may result in discrete thresholds for diagnostic statements. Neural nets differ from conventional discriminant function analysis in the way they are trained, in the resulting classifier, and in their derived decision boundaries.133,159,160 Statistical methods de-

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pend on a database of well-documented cases to find the optimal ECG parameters to use. Such a database must be large enough that the results are statistically reliable. The database must contain sufficient cases with varying degrees of abnormality, ranging from mild to severe cases, and a representative distribution of common confounding conditions.6,9,17,161 The statistics of well-documented populations have been used to develop diagnostic algorithms that no longer simply mimic the human reader.162 Similarly, it has also been shown that the addition of vector loop criteria (or the equivalent information deduced from simultaneous leads) improves 12-lead ECG diagnoses.97,98 Clinical Implications Given the potentially profound effects of technical factors on ECG measurements, it is not surprising that identical diagnostic algorithms might perform differently when applied to ECG signals that undergo processing by different methods. Adherence to methodological standards will minimize these differences, promote uniformity of measurement and interpretation, and facilitate serial comparison of tracings. Even with adherence to standards, small systematic differences in measurements might be expected between diagnostic instruments that use different processing methods, particularly with respect to diagnostically important global measures of QRS duration and QT interval. A 1985 study by the European CSE group demonstrated that measurement differences among 10 standard ECG systems could be large enough to alter diagnostic conclusions17; however, no recent studies have directly compared template and global measurements made with the current generation of commercially available standard ECG recording systems. Beyond the technical issues of measurement fidelity, evaluation of the performance of ECG programs is difficult.9,15,17,163 Programs may be compared with diagnoses of an expert cardiologist or consensus of expert cardiologists or with diagnoses ascertained by independent data. The CSE group evaluated 15 ECG and vectorcardiographic analysis programs against a reference database that included documented cases of ventricular hypertrophy and myocardial infarction,15 diagnoses that are strongly dependent on accurate measurement of amplitudes and durations and should favor computer analysis. Overall, the percentage of ECGs correctly classified by the computer programs

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(median 91.3%) was lower than that for the cardiologists (median 96.0%), whereas important differences in overall accuracy were found between different algorithms. Salerno et al18 reviewed 13 reports of computer ECG program performance and showed that these programs generally perform less well than expert readers with respect to individual diagnoses. Even so, this report found that computer assistance was able to improve the diagnostic performance of less expert readers. Recommendations Computer-based interpretation of the ECG is an adjunct to the electrocardiographer,164 and all computer-based reports require physician overreading. Accurate individual templates should be formed in each lead before final feature extraction and measurement used for diagnostic interpretation. Time-coherent data from multiple leads should be used to detect the earliest onset and latest offset of waveforms of global measurements used for diagnostic interpretation. Deterministic and statistical or probabilistic algorithms should be based on well-constructed databases that include varying degrees of pathology and an appropriate distribution of confounding conditions. Such algorithms should be validated with data that have not been used for development. Programs using complex diagnostic algorithms should document in reference material those measurements that are critical to the diagnostic statement, which might include synthesized vector loop or other novel measurements. Serial comparisons of sequential ECGs should be done by trained observers regardless of whether the ECG program provides a serial comparison. Assessment of the performance of different algorithms will be facilitated by use of a standardized glossary of interpretive statements.

Summary The present document outlines the relation of the modern digital electrocardiograph to its technology. Individual features of ECG processing and recording are considered in terms of their clinical implications. Recommendations focus on progress toward optimal use of the ECG. It is hoped that the standards set out in this document will provide a further stimulus to the improvement of ECG recording and interpretation.

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Disclosures Writing Group Disclosures

Writing Group Member Paul Kligfield

James J. Bailey Rory Childers Barbara J. Deal

Research Grant

Other Research Support

Speakers’ Bureau/ Honoraria

Weill Medical College of Cornell University

None

None

National Institutes of Health

None

University of Chicago

None

Northwestern University

None

Employment

Ownership Interest

Consultant/Advisory Board

None

Unilead (ECG electrode technology)– limited partner†

Philips Medical,* Mortara Instrument,* GE Healthcare,* Quinton Medical,* MDS Pharma Services,† Cardiac Science*

None

None

None

None

None

None

None

None

None

None

None

None

None

None

None

None

Other

Leonard S. Gettes

University of North Carolina

None

None

None

None

None

None

E. William Hancock

Stanford University Medical Center— retired Professor Emeritus

None

None

None

None

Philips Medical Systems,† Covance Diagnostics†

None

Jan A. Kors

Erasmus Medical Center

None

None

None

None

None

None

University of Glasgow

Cardiac Science,† Medtronic,† Heartlab,† Medcon,† Del Mar Reynolds,† Drayer†

None

None

None

Garhard Schmidt Consult,* Epiphany Cardiography,* IqTeq,* Cardiolex*

None

Peter Macfarlane

David M. Mirvis

University of Tennessee

None

None

None

None

None

None

Olle Pahlm

Lund University, Sweden

Philips Medical Systems*

None

None

None

None

None

Wake Forest University Medical School–retired

None

None

None

None

Philips Medical Systems†

None

Erasmus Medical Center

None

None

None

None

None

None

Duke University Medical Center

Medtronic,† Physiocontrol,† Welch Allyn†

None

None

None

None

None

Pentti Rautaharju

Gerard van Herpen Galen S. Wagner

This table represents the relationships of writing group members that may be perceived as actual or reasonably perceived conflicts of interest as reported on the Disclosure Questionnaire, which all members of the writing group are required to complete and submit. A relationship is considered to be “significant” if (1) the person receives $10 000 or more during any 12-month period, or 5% or more of the person’s gross income; or (2) the person owns 5% or more of the voting stock or share of the entity, or owns $10 000 or more of the fair market value of the entity. A relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *Modest. †Significant.

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Reviewer Disclosures

Employment

Research Grant

Other Research Support

Speakers’ Bureau/Honoraria

Ownership Interest

Consultant/ Advisory Board

Jonathan Abrams

University of New Mexico

None

None

None

None

None

None

Leonard S. Dreifus

Hahnemann University, School of Medicine

None

None

None

None

None

Merck Endpoint Committee None

Reviewer

Mark Eisenberg

Other

McGill University

None

None

None

None

None

University of California, San Francisco

None

None

St. Jude, Medtronic

None

None

None

Peter Kowey

Lankenau Hospital and Main Line Health

None

None

Medifacts

Cardionet

Medifacts

None

Frank Marcus

University of Arizona

None

None

None

None

None

None

Mayo Clinic

St. Jude Medical, Bard Electrophysiology

None

None

None

None

None

None

Nora Goldschlager

Thomas M. Munger

Robert J. Myerburg

University of Miami

None

None

None

None

None

David Rosenbaum

Case Western Reserve University

None

None

None

None

None

None

Richard Schofield

University of Florida

None

None

None

None

None

None

Samuel Shubrooks

Beth Israel Deaconess Medical Center

None

None

None

None

None

None

George Washington University

None

None

None

None

None

None

Cynthia Tracy

This table represents the relationships of reviewers that may be perceived as actual or reasonably perceived conflicts of interest as reported on the Disclosure Questionnaire, which all reviewers are required to complete and submit.

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(V7 to V9) in patients with acute inferior myocardial infarction: application for thrombolytic therapy. J Am Coll Cardiol. 1998;31:506 –511. Croft CH, Nicod P, Corbett JR, Lewis SE, Huxley R, Mukharji J, Willerson JT, Rude RE. Detection of acute right ventricular infarction by right precordial electrocardiography. Am J Cardiol. 1982;50:421– 427. Braat SH, Brugada P, de Zwaan C, Coenegracht JM, Wellens HJ. Value of electrocardiogram in diagnosing right ventricular involvement in patients with an acute inferior wall myocardial infarction. Br Heart J. 1983;49:368 –372. Lopez-Sendon J, Coma-Canella I, Alcasena S, Seoane J, Gamallo C. Electrocardiographic findings in acute right ventricular infarction: sensitivity and specificity of electrocardiographic alterations in right precordial leads V4R, V3R, V1, V2, and V3. J Am Coll Cardiol. 1985;6:1273–1279. Sinha N, Ahuja RC, Saran RK, Jain GC. Clinical correlates of acute right ventricular infarction in acute inferior myocardial infarction. Int J Cardiol. 1989;24:55– 61. Yoshino H, Udagawa H, Shimizu H, Kachi E, Kajiwara T, Yano K, Taniuchi M, Ishikawa K. ST-segment elevation in right precordial leads implies depressed right ventricular function after acute inferior myocardial infarction [published correction appears in Am Heart J. 1998; 136:5]. Am Heart J. 1998;135:689 – 695. Zalenski RJ, Rydman RJ, Sloan EP, Hahn K, Cooke D, Tucker J, Fligner D, Fagan J, Justis D, Hessions W, Pribble JM, Shah S, Zwicke D. ST segment elevation and the prediction of hospital life-threatening complications: the role of right ventricular and posterior leads. J Electrocardiol. 1998;31(suppl):164 –171. van Herpen G, Kors JA, Schijvenaars BJ. Are additional right precordial and left posterior ECG leads useful for the diagnosis of right ventricular infarct and posterior infarct? Also a plea for the revival of vectorcardiography. J Electrocardiol. 1999;32(suppl):51–54. Rosengarten P, Kelly AM, Dixon D. Does routine use of the 15-lead ECG improve the diagnosis of acute myocardial infarction in patients with chest pain? Emerg Med (Fremantle). 2001;13:190 –193. Braunwald E, Antman EM, Beasley JW, Califf RM, Cheitlin MD, Hochman JS, Jones RH, Kereiakes D, Kupersmith J, Levin TN, Pepine CJ, Schaeffer JW, Smith EE III, Steward DE, Theroux P, Gibbons RJ, Alpert JS, Faxon DP, Fuster V, Gregoratos G, Hiratzka LF, Jacobs AK, Smith SC Jr. ACC/AHA guideline update for the management of patients with unstable angina and non–ST-segment elevation myocardial infarction—2002: summary article: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on the Management of Patients With Unstable Angina). Circulation. 2002;106:1893–1900. Chia BL, Tan HC, Yip JW, Ang TL. Electrocardiographic patterns in posterior chest leads (V7, V8, V9) in normal subjects. Am J Cardiol. 2000;85:911–912, A10. Oraii S, Maleki M, Tavakolian AA, Eftekharzadeh M, Kamangar F, Mirhaji P. Prevalence and outcome of ST-segment elevation in posterior electrocardiographic leads during acute myocardial infarction. J Electrocardiol. 1999;32:275–278. Ho KK, Ho SK. Use of the sinus P wave in diagnosing electrocardiographic limb lead misplacement not involving the right leg (ground) lead. J Electrocardiol. 2001;34:161–171. Heden B, Ohlsson M, Edenbrandt L, Rittner R, Pahlm O, Peterson C. Artificial neural networks for recognition of electrocardiographic lead reversal. Am J Cardiol. 1995;75:929 –933. Heden B, Ohlsson M, Holst H, Mjoman M, Rittner R, Pahlm O, Peterson C, Edenbrandt L. Detection of frequently overlooked electrocardiographic lead reversals using artificial neural networks. Am J Cardiol. 1996;78:600 – 604. Edenbrandt L, Rittner R. Recognition of lead reversals in pediatric electrocardiograms. Am J Cardiol. 1998;82:1290 –1292, A10. Kors JA, van Herpen G. Accurate automatic detection of electrode interchange in the electrocardiogram. Am J Cardiol. 2001;88:396 –399. Heden B. Electrocardiographic lead reversal. Am J Cardiol. 2001;87: 126 –127. Abdollah H, Milliken JA. Recognition of electrocardiographic left arm/left leg lead reversal. Am J Cardiol. 1997;80:1247–1249. Peberdy MA, Ornato JP. Recognition of electrocardiographic lead misplacements. Am J Emerg Med. 1993;11:403– 405. Haisty WK Jr, Pahlm O, Edenbrandt L, Newman K. Recognition of electrocardiographic electrode misplacements involving the ground (right leg) electrode. Am J Cardiol. 1993;71:1490 –1495.

141. Castellanos A, Saoudi NC, Schwartz A, Sodi-Pallares D. Electrocardiographic patterns resulting from improper connections of the right leg (ground) cable. Pacing Clin Electrophysiol. 1985;8(pt 1):364 –368. 142. Herman MV, Ingram DA, Levy JA, Cook JR, Athans RJ. Variability of electrocardiographic precordial lead placement: a method to improve accuracy and reliability. Clin Cardiol. 1991;14:469 – 476. 143. Kerwin AJ, McLean R, Tegelaar H. A method for the accurate placement of chest electrodes in the taking of serial electrocardiographic tracings. Can Med Assoc J. 1960;82:258 –261. 144. Van Den Hoogen JP, Mol WH, Kowsoleea A, Van Ree JW, Thien T, Van Weel C. Reproducibility of electrocardiographic criteria for left ventricular hypertrophy in hypertensive patients in general practice. Eur Heart J. 1992;13:1606 –1610. 145. de Bruyne MC, Kors JA, Visentin S, van Herpen G, Hoes AW, Grobbee DE, van Bemmel JH. Reproducibility of computerized ECG measurements and coding in a nonhospitalized elderly population. J Electrocardiol. 1998;31:189 –195. 146. Draper HW, Peffer CJ, Stallmann FW, Littmann D, Pipberger HV. The corrected orthogonal electrocardiogram and vectorcardiogram in 510 normal men (Frank lead system). Circulation. 1964;30:853– 864. 147. Pipberger HV, Freis ED, Taback L, Mason HL. Preparation of electrocardiographic data for analysis by digital electronic computer. Circulation. 1960;21:413– 418. 148. Rikli AE, Tolles WE, Steinberg CA, Carbery WJ, Freiman AH, Abraham S, Caceres CA. Computer analysis of electrocardiographic measurements. Circulation. 1961;24:643– 649. 149. Pipberger HV, Stallman FW, Berson AS. Automatic analysis of the P-QRS-T complex of the electrocardiogram by digital computer. Ann Intern Med. 1962;57:776 –787. 150. Caceres CA, Steinberg CA, Abraham S, Carbery WJ, McBride JM, Tolles WE, Rikli AE. Computer extraction of electrocardiographic parameters. Circulation. 1962;25:356 –362. 151. Bonner RE, Schwetman HD. Computer diagnosis of electrocardiograms, II: a computer program for EKG measurements. Comput Biomed Res. 1968;1:366 –386. 152. Smith RE, Hyde FM. Computer analysis of the ECG in clinical practice. In: Manning GW, Ahuja SP, eds. Electrical Activity of the Heart. Springfield, Ill: Charles C Thomas; 1969:305. 153. Pordy L, Jaffe H, Chesky K, Friedberg CK. Computer analysis of the electrocardiogram: a joint project. J Mt Sinai Hosp N Y. 1967;34:69 – 88. 154. Pryor TA, Russell R, Budkin A, Price WG. Electrocardiographic interpretation by computer. Comput Biomed Res. 1969;2:537–548. 155. Bonner RE, Crevasse L, Ferrer MI, Greenfield JC Jr. A new computer program for analysis of scalar electrocardiograms. Comput Biomed Res. 1972;5:629 – 653. 156. Cornfield J, Dunn RA, Batchlor CD, Pipberger HV. Multigroup diagnosis of electrocardiograms. Comput Biomed Res. 1973;6:97–120. 157. Romhilt DW, Estes EH Jr. A point-score system for the ECG diagnosis of left ventricular hypertrophy. Am Heart J. 1968;75:752–758. 158. Okin PM, Roman MJ, Devereux RB, Pickering TG, Borer JS, Kligfield P. Time-voltage QRS area of the 12-lead electrocardiogram: detection of left ventricular hypertrophy. Hypertension. 1998;31:937–942. 159. Bortolan G, Willems JL. Diagnostic ECG classification based on neural networks. J Electrocardiol. 1993;26(suppl):75–79. 160. Heden B, Ohlsson M, Rittner R, Pahlm O, Haisty WK Jr, Peterson C, Edenbrandt L. Agreement between artificial neural networks and experienced electrocardiographer on electrocardiographic diagnosis of healed myocardial infarction. J Am Coll Cardiol. 1996;28:1012–1016. 161. Norman JE, Bailey JJ, Berson AS, Haisty WK, Levy D, Macfarlane PM, Rautaharju PM. NHLBI workshop on the utilization of ECG databases: preservation and use of existing ECG databases and development of future resources. J Electrocardiol. 1998;31:83– 89. 162. Warner RA, Ariel Y, Gasperina MD, Okin PM. Improved electrocardiographic detection of left ventricular hypertrophy. J Electrocardiol. 2002;35(suppl):111–115. 163. Bailey JJ, Itscoitz SB, Hirshfeld JW Jr, Grauer LE, Horton MR. A method for evaluating computer programs for electrocardiographic interpretation, I: application to the experimental IBM program of 1971. Circulation. 1974;50:73–79. 164. Laks MM, Selvester RH. Computerized electrocardiography: an adjunct to the physician. N Engl J Med. 1991;325:1803–1804.

AHA/ACC/HRS Scientific Statement Recommendations for the Standardization and Interpretation of the Electrocardiogram Part II: Electrocardiography Diagnostic Statement List A Scientific Statement From the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society Endorsed by the International Society for Computerized Electrocardiology Jay W. Mason, MD, FAHA, FACC, FHRS; E. William Hancock, MD, FACC; Leonard S. Gettes, MD, FAHA, FACC Abstract—This statement provides a concise list of diagnostic terms for ECG interpretation that can be shared by students, teachers, and readers of electrocardiography. This effort was motivated by the existence of multiple automated diagnostic code sets containing imprecise and overlapping terms. An intended outcome of this statement list is greater uniformity of ECG diagnosis and a resultant improvement in patient care. The lexicon includes primary diagnostic statements, secondary diagnostic statements, modifiers, and statements for the comparison of ECGs. This diagnostic lexicon should be reviewed and updated periodically. (Circulation. 2007;115:1325-1332.) Key Words: AHA Scientific Statements 䡲 electrocardiography 䡲 computers 䡲 diagnosis

T

vague terminology. Some statements that are commonly used by electrocardiographers but that do not provide diagnostically or clinically useful information are not included. Some statements have been excluded to reduce the size of the statement set, so long as their meaning is well represented by included terms. The Writing Group believes that the listing should be implemented as an available lexicon in report algorithms of the existing commercial electrocardiographs and that it should be used widely by ECG readers. The principal advantage of such use would be a worldwide improvement in uniformity of ECG interpretation. Such uniformity would promote better patient

his is the second of 6 articles designed to upgrade the guidelines for the standardization and interpretation of the ECG. The project was initiated by the American Heart Association and has been endorsed by the American College of Cardiology, the Heart Rhythm Society, and the International Society for Computerized Electrocardiography. The rationale for this upgrade and a description of the process are contained in Part I by Kligfield et al.1 The listing contained in the present statement seeks to present a limited set of ECG diagnostic statements that are clinically useful and that do not create unnecessary overlap or contain

Other members of the Standardization and Interpretation of the Electrocardiogram Writing Group include James J. Bailey, MD; Rory Childers, MD; Barbara J. Deal, MD, FACC; Mark Josephson, MD, FACC, FHRS; Paul Kligfield, MD, FAHA, FACC; Jan A. Kors, PhD; Peter Macfarlane, DSc; Olle Pahlm, MD, PhD; David M. Mirvis, MD, FAHA; Peter Okin, MD, FACC; Pentti Rautaharju, MD, PhD; Borys Surawicz, MD, FAHA, FACC; Gerard van Herpen, MD, PhD; Galen S. Wagner, MD; and Hein Wellens, MD, FAHA, FACC. The American Heart Association, the American College of Cardiology, and the Heart Rhythm Society make every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. Specifically, all members of the writing group are required to complete and submit a Disclosure Questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest. This statement was approved by the American Heart Association Science Advisory and Coordinating Committee on October 26, 2006, by the American College of Cardiology Board of Trustees on October 12, 2006, and by the Heart Rhythm Society on September 6, 2006. This article has been copublished in the March 13, 2007, issue of the Journal of the American College of Cardiology and in the March 2007 issue of Heart Rhythm. Copies: This document is available on the World Wide Web sites of the American Heart Association (www.americanheart.org) and the American College of Cardiology (www.acc.org). A single reprint is available by calling 800-242-8721 (US only) or writing the American Heart Association, Public Information, 7272 Greenville Ave, Dallas, TX 75231-4596. Ask for reprint No. 71-0390. To purchase additional reprints, call 843-216-2533 or e-mail [email protected]. Permissions: Multiple copies, modification, alteration, enhancement, and/or distribution of this document are not permitted without the express permission of the American Heart Association. Instructions for obtaining permission are located at http://www.americanheart.org/presenter.jhtml? Identifier⫽4431. A link to the “Permission Request Form” appears on the right side of the page. © 2007 American Heart Association, Inc., the American College of Cardiology Foundation, and the Heart Rhythm Society. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.180201

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care. Additional advantages would be facilitation of the establishment of a uniform teaching curriculum in electrocardiography, availability of a uniform glossary of terms for research application, and promotion of research to better validate diagnostic criteria for the specific terms in the limited lexicon. Although we recognize that each vendor of ECGs possesses a proprietary set of diagnostic statements and underlying criteria, we hope that this list of statements will be made available by each of them so that the reader can select it as the primary dictionary for use in interpreting all or some ECGs. We are also hopeful that the vendors will collaborate among themselves to align diagnostic criteria for this specific lexicon. This would not interfere with continued development of entirely independent, proprietary diagnostic software by each manufacturer.

statements could be made in comparing individual ECGs to ⱖ1 previous ECGs, the Writing Group recommends use of these 6 statements to convey clinically important information that could influence patient care by the attending physician while preserving brevity and uniformity. On the other hand, the Writing Group encourages readers to add uncoded text as needed to the report to more fully compare tracings. Tables 5, 6, and 7 establish rules for use of the primary, secondary, and modifier statements, alone or in combination. Table 8 is a set of commonly used statements that can, for the most part, be precisely reproduced by use of the primary and secondary statements and their modifiers. These statements are commonly used concatenations provided for the convenience of the reader.

Criteria for Diagnoses

Organization and Use Four lists are included within this document. The main listing (Table 1), “Primary Statements,” displays 117 primary diagnostic statements under 14 categories. The majority of the primary statements are nondescriptive and convey clinical meaning without additional statements. The second listing (Table 2), “Secondary Statements,” provides additional statements that can be used to expand the specificity and clinical relevance of both descriptive and other primary diagnostic statements. These secondary statements are divided into 2 groups. Those that are preceded by “suggests” invoke clinical diagnoses likely responsible for the ECG observation(s). Those that are preceded by “consider” are intended to propose at least 1, but sometimes ⬎1, potentially associated clinical disorder. This set of primary and secondary diagnostic statements constitutes what we might call the “core statement lexicon.” The third list (Table 3) contains adjectives that can be used to modify the diagnostic statements. None of the modifiers change the meaning of the core statement but rather serve to refine the meaning. The list contains general modifiers, which can be used with many of the core statements, and specific modifiers assigned to a specific category of statements. The fourth list (Table 4) is a short directory of comparison statements. It specifies 6 types of ECG changes that merit mention in the ECG interpretation and defines criteria to identify change within the 6 categories. Because so many

This listing does not specify diagnostic criteria for any of the statements. A single set of diagnostic criteria underlying the core statements would have great benefits for patient care and research. Although the Writing Group does not believe that a uniform criterion set can be achieved at this time, we encourage ECG vendors and electrocardiography researchers and experts to collaborate on the development of a universally acceptable criteria set and a means for perpetually refining it. Several of the chapters in this statement support specific criteria for some of the core statements.

Myocardial Infarction Terminology Advanced imaging techniques, including echocardiography2 and magnetic resonance,3,4 have demonstrated a need for change in existing terminology describing the cardiac location of myocardial infarction. New diagnostic statements for 6 common, distinct cardiac locations of myocardial infarction, documented by contrast-enhanced magnetic resonance, were recently recommended by a committee of the International Society for Holter and Noninvasive Electrocardiography.5 At the present time, the Writing Group considers the quantity of new data insufficient to recommend abandonment of existing terminology. Thus, traditional terms are listed in “Section M: Myocardial infarction” of the primary statement table (Table 1); however, we intend to revisit this issue when sufficient data have been developed.

Disclosures Writing Group Disclosures Employment

Research Grant

Other Research Support

Speakers’ Bureau/Honoraria

Ownership Interest

Consultant/ Advisory Board

Other

Covance Cardiac Safety Services

None

None

None

None

None

None

Leonard S. Gettes

University of North Carolina

None

None

None

None

None

None

E. William Hancock

Stanford University Medical Center

None

None

None

None

Philips Medical Systems,* Covance Diagnostics*

None

Writing Group Member Jay W. Mason

This table represents the relationships of writing group members that may be perceived as actual or reasonably perceived conflicts of interest as reported on the Disclosure Questionnaire, which all members of the writing group are required to complete and submit. A relationship is considered to be “significant” if (1) the person receives $10 000 or more during any 12-month period, or 5% or more of the person’s gross income; or (2) the person owns 5% or more of the voting stock or share of the entity, or owns $10 000 or more of the fair market value of the entity. A relationship is considered to be “modest” if it is less than “significant” under the preceding definition. *Significant.

Mason et al

Standardization and Interpretation of the ECG, Part II

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Reviewer Disclosures Reviewer

Employment

Research Grant

Other Research Support

Speakers’ Bureau/Honoraria

Ownership Interest

Consultant/ Advisory Board

Other

Jonathan Abrams

University of New Mexico

None

None

None

None

None

None

Leonard S. Dreifus

Hahnemann University, School of Medicine

None

None

None

None

None

Merck Endpoint Committee

Mark Eisenberg

McGill University

None

None

None

None

None

None

University of California, San Francisco

None

None

St. Jude; Medtronic

None

None

None

Peter Kowey

Lankenau Hospital and Main Line Health

None

None

Medifacts

Cardionet

Medifacts

None

Frank Marcus

University of Arizona

None

None

None

None

None

None

Thomas M. Munger

Mayo Clinic

St. Jude Medical, Bard Electrophysiology

None

None

None

None

None

Robert J. Myerburg

University of Miami

None

None

None

None

None

None

David Rosenbaum

Case Western Reserve University

None

None

None

None

None

None

Richard Schofield

University of Florida

None

None

None

None

None

None

Samuel Shubrooks

Beth Israel Deaconess Medical Center

None

None

None

None

None

None

George Washington University

None

None

None

None

None

None

Nora Goldschlager

Cynthia Tracy

This table represents the relationships of reviewers that may be perceived as actual or reasonably perceived conflicts of interest as reported on the Disclosure Questionnaire, which all reviewers are required to complete and submit.

References 1. Kligfield P, Gettes L, Bailey JJ, Childers R, Deal BJ, Hancock EW, van Herpen G, Kors JA, Macfarlane P, Mirvis DM, Pahlm O, Rautaharju P, Wagner GS. Recommendations for the standardization and interpretation of the electrocardiogram: part I: the electrocardiogram and its technology: a scientific statement from the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society. Circulation. 2007;115:–. 2. Bogaty P, Boyer L, Rousseau L, Arsenault M. Is anteroseptal myocardial infarction an appropriate term? Am J Med. 2002;113:37– 41. 3. Selvanayagam JB, Kardos A, Nicolson D, Francis J, Petersen SE, Robson M, Banning A, Neubauer S. Anteroseptal or apical myocardial infarction: a controversy addressed using delayed enhancement cardiovascular

magnetic resonance imaging. J Cardiovasc Magn Reson. 2004;6: 653– 661. 4. Bayes de Luna A, Cino JM, Pujadas S, Cygankiewicz I, Carreras F, Garcia-Moll X, Noguero M, Fiol M, Elosua R, Cinca J, Pons-Llado G. Concordance of electrocardiographic patterns and healed myocardial infarction location detected by cardiovascular magnetic resonance. Am J Cardiol. 2006;97:443– 451. 5. Bayes de Luna A, Wagner G, Birnbaum Y, Nikus K, Fiol M, Gorgels A, Cinca J, Clemmensen PM, Pahlm O, Sclarovsky S, Stern S, Wellens J, Zareba W; International Society for Holter and Noninvasive Electrocardiography. A new terminology for left ventricular walls and location of myocardial infarcts that present Q wave based on the standard of cardiac magnetic resonance imaging: a statement for healthcare professionals from a committee appointed by the International Society for Holter and Noninvasive Electrocardiography. Circulation. 2006;114:1755–1760.

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TABLE 1.

Primary Statements G. Ventricular tachyarrhythmias

A. Overall interpretation 1

Normal ECG

70

Ventricular tachycardia

2

Otherwise normal ECG

71

Ventricular tachycardia, unsustained

3

Abnormal ECG

72

Ventricular tachycardia, polymorphous

4

Uninterpretable ECG

73

Ventricular tachycardia, torsades de pointes

10

Extremity electrode reversal

74

Ventricular fibrillation

11

Misplaced precordial electrode(s)

75

Fascicular tachycardia

12

Missing lead(s)

76

Wide-QRS tachycardia

13

Right-sided precordial electrode(s)

14

Artifact

80

Short PR interval

15

Poor-quality data

81

AV conduction ratio N:D

16

Posterior electrode(s)

82

Prolonged PR interval

83

Second-degree AV block, Mobitz type I (Wenckebach)

B. Technical conditions

C. Sinus node rhythms and arrhythmias

H. Atrioventricular conduction

20

Sinus rhythm

21

Sinus tachycardia

84

22

Sinus bradycardia

85

23

Sinus arrhythmia

86

24

Sinoatrial block, type I

87

25

Sinoatrial block, type II

88

26

Sinus pause or arrest

89

27

Uncertain supraventricular rhythm

D. Supraventricular arrhythmias Atrial premature complex(es)

100

31

Atrial premature complexes, nonconducted

101

32

Retrograde atrial activation

102

33

Wandering atrial pacemaker

104

34

Ectopic atrial rhythm

105

35

Ectopic atrial rhythm, multifocal

106

36

Junctional premature complex(es)

107

37

Junctional escape complex(es)

108

38

Junctional rhythm

109

39

Accelerated junctional rhythm

110

40

Supraventricular rhythm

111

41

Supraventricular complex(es)

42

Bradycardia, nonsinus

AV block, varying conduction AV block, advanced (high-grade) AV block, complete (third-degree) AV dissociation

Aberrant conduction of supraventricular beat(s) Left anterior fascicular block Left posterior fascicular block Left bundle-branch block Incomplete right bundle-branch block Right bundle-branch block Intraventricular conduction delay Ventricular preexcitation Right atrial conduction abnormality Left atrial conduction abnormality Epsilon wave

J. Axis and voltage 120 121

50

Atrial fibrillation

122

51

Atrial flutter

123

52

Ectopic atrial tachycardia, unifocal

124

53

Ectopic atrial tachycardia, multifocal

125

54

Junctional tachycardia

128

55

Supraventricular tachycardia

131

56

Narrow-QRS tachycardia

F. Ventricular arrhythmias

2:1 AV block

I. Intraventricular and intra-atrial conduction

30

E. Supraventricular tachyarrhythmias

Second-degree AV block, Mobitz type II

Right-axis deviation Left-axis deviation Right superior axis Indeterminate axis Electrical alternans Low voltage Abnormal precordial R-wave progression Abnormal P-wave axis

K. Chamber hypertrophy or enlargement

Ventricular premature complex(es)

140

Left atrial enlargement

Fusion complex(es)

141

Right atrial enlargement

62

Ventricular escape complex(es)

142

Left ventricular hypertrophy

63

Idioventricular rhythm

143

Right ventricular hypertrophy

64

Accelerated idioventricular rhythm

144

Biventricular hypertrophy

65

Fascicular rhythm

66

Parasystole

60 61

Mason et al TABLE 1.

Standardization and Interpretation of the ECG, Part II

Primary Statements, Cont’d

L. ST segment, T wave, and U wave

TABLE 2.

1329

Secondary Statements

Suggests䡠 䡠 䡠 200

Acute pericarditis

145

ST deviation

201

Acute pulmonary embolism

146

ST deviation with T-wave change

202

Brugada abnormality

147

T-wave abnormality

203

Chronic pulmonary disease

148

Prolonged QT interval

204

CNS disease

149

Short QT interval

205

Digitalis effect

150

Prominent U waves

206

Digitalis toxicity

151

Inverted U waves

207

Hypercalcemia

152

TU fusion

208

Hyperkalemia

153

ST-T change due to ventricular hypertrophy

209

Hypertrophic cardiomyopathy

210

Hypocalcemia

211

Hypokalemia or drug effect

212

Hypothermia

213

Ostium primum ASD

214

Pericardial effusion

215

Sinoatrial disorder

154

Osborn wave

155

Early repolarization

M. Myocardial infarction 160

Anterior MI

161

Inferior MI

162

Posterior MI

163

Lateral MI

165

Anteroseptal MI

166

Extensive anterior MI

173

MI in presence of left bundle-branch block

174

181 182 183

Acute ischemia

221

AV nodal reentry

222

AV reentry

223

Genetic repolarization abnormality

224

High precordial lead placement

225

Hypothyroidism

Atrial-paced complex(es) or rhythm

226

Ischemia

Ventricular-paced complex(es) or rhythm

227

Left ventricular aneurysm

Ventricular pacing of non–right ventricular apical origin

228

Normal variant

229

Pulmonary disease

Atrial-sensed ventricular-paced complex(es) or rhythm

230

Dextrocardia

231

Dextroposition

Right ventricular MI

N. Pacemaker 180

Consider䡠 䡠 䡠 220

184

AV dual-paced complex(es) or rhythm

185

Failure to capture, atrial

186

Failure to capture, ventricular

187

Failure to inhibit, atrial

188

Failure to inhibit, ventricular

189

Failure to pace, atrial

190

Failure to pace, ventricular

AV indicates atrioventricular; MI, myocardial infarction.

CNS indicates central nervous system; ASD, atrial septal defect; and AV, atrioventricular.

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TABLE 3.

Modifiers

March 13, 2007

General

Myocardial infarction, cont’d

301

Borderline

332

Old

303

Increased

333

Of indeterminate age

304

Intermittent

334

Evolving

305

Marked

306

Moderate

340

307

Multiple

341

In a bigeminal pattern

308

Occasional

342

In a trigeminal pattern

309

One

343

Monomorphic

310

Frequent

344

Multifocal

312

Possible

345

Unifocal

313

Postoperative

346

With a rapid ventricular response

314

Predominant

347

With a slow ventricular response

315

Probable

348

With capture beat(s)

316

Prominent

349

With aberrancy

317

(Specified) Lead(s)

350

Polymorphic

318

(Specified) Electrode(s)

321

Nonspecific

General: conjunctions

Arrhythmias and tachyarrhythmias Couplets

Repolarization abnormalities ⱖ0.1 mV

360 361

ⱖ0.2 mV

302

Consider

362

Depression

310

Or

363

Elevation

320

And

364

Maximally toward lead

319

With

365

Maximally away from lead

322

Versus

366

Low amplitude

367

Inversion

330

Acute

369

Postpacing (anamnestic)

331

Recent

Myocardial infarction

TABLE 4.

Comparison Statements

Code

Statement

Criteria

400

No significant change

Intervals (PR, QRS, QTc) remain normal or within 10% of a previously abnormal value

401

Significant change in rhythm

New or deleted rhythm diagnosis

No new or deleted diagnoses with the exception of normal variant diagnoses HR change ⬎20 bpm and ⬍50 or ⬎100 bpm New or deleted pacemaker diagnosis 402

New or worsened ischemia or infarction

Added infarction, ST-ischemia, or T-wave-ischemia diagnosis, or worsened ST deviation or T-wave abnormality

403

New conduction abnormality

Added AV or IV conduction diagnosis

404

Significant repolarization change

New or deleted QT diagnosis New or deleted U-wave diagnosis New or deleted nonischemic ST or T-wave diagnosis Change in QTc ⬎60 ms

405

Change in clinical status

New or deleted diagnosis from Axis and Voltage, Chamber Hypertrophy, or Enlargement primary statement categories or “Suggests䡠 䡠 䡠” secondary statement category

406

Change in interpretation without significant change in waveform

Used when a primary or secondary statement is added or removed despite no real change in the tracing; ie, an interpretive disagreement exists between the readers of the first and second ECGs

QTc indicates corrected QT interval; HR, heart rate; bpm, beats per minute; AV, atrioventricular; and IV, intraventricular.

Mason et al TABLE 5.

Standardization and Interpretation of the ECG, Part II

General Use Rules

TABLE 6.

1331

Secondary–Primary Statement Pairing Rules

1

Secondary statements must be accompanied by a primary statement

Secondary Code

2

Modifiers must be accompanied by a primary statement

200

145–147

May Accompany These Primary Codes

3

A primary statement may be accompanied by nothing, by ⱖ1 modifiers, by ⱖ1 secondary statements, or by both.

201

21, 105, 109, 120, 131, 141, 145–147

202

105, 106, 145–146

4

Each secondary statement can accompany only certain primary statements (see Table 6)

203

109, 120, 125, 128, 131, 141, 143

5

Each general modifier can accompany only certain primary statements (see Table 7)

204

147

205

145–147

6

Each specific modifier can accompany only primary statements within its category

206

145–147

207

149

208

147

209

142

210

148

211

147–148, 150

212

14, 154

213

82, 105–106, 121

214

124

215

42, 131, 145–147

220

145–147, 151

221

55, 56

222

55, 56

223

148, 149

224

128

225

22, 24–26, 37, 38

226

145–147

227

145–147

228

80, 105, 128, 155

229

109, 120, 122–123, 125, 128, 131, 141, 143

230

128, 131

231

128

1330

Circulation

TABLE 3.

Modifiers

March 13, 2007

General

Myocardial infarction, cont’d

301

Borderline

332

Old

303

Increased

333

Of indeterminate age

304

Intermittent

334

Evolving

305

Marked

306

Moderate

340

307

Multiple

341

In a bigeminal pattern

308

Occasional

342

In a trigeminal pattern

309

One

343

Monomorphic

310

Frequent

344

Multifocal

312

Possible

345

Unifocal

313

Postoperative

346

With a rapid ventricular response

314

Predominant

347

With a slow ventricular response

315

Probable

348

With capture beat(s)

316

Prominent

349

With aberrancy

317

(Specified) Lead(s)

350

Polymorphic

318

(Specified) Electrode(s)

321

Nonspecific

General: conjunctions

Arrhythmias and tachyarrhythmias Couplets

Repolarization abnormalities ⱖ0.1 mV

360 361

ⱖ0.2 mV

302

Consider

362

Depression

310

Or

363

Elevation

320

And

364

Maximally toward lead

319

With

365

Maximally away from lead

322

Versus

366

Low amplitude

367

Inversion

330

Acute

369

Postpacing (anamnestic)

331

Recent

Myocardial infarction

TABLE 4.

Comparison Statements

Code

Statement

Criteria

400

No significant change

Intervals (PR, QRS, QTc) remain normal or within 10% of a previously abnormal value

401

Significant change in rhythm

New or deleted rhythm diagnosis

No new or deleted diagnoses with the exception of normal variant diagnoses HR change ⬎20 bpm and ⬍50 or ⬎100 bpm New or deleted pacemaker diagnosis 402

New or worsened ischemia or infarction

Added infarction, ST-ischemia, or T-wave-ischemia diagnosis, or worsened ST deviation or T-wave abnormality

403

New conduction abnormality

Added AV or IV conduction diagnosis

404

Significant repolarization change

New or deleted QT diagnosis New or deleted U-wave diagnosis New or deleted nonischemic ST or T-wave diagnosis Change in QTc ⬎60 ms

405

Change in clinical status

New or deleted diagnosis from Axis and Voltage, Chamber Hypertrophy, or Enlargement primary statement categories or “Suggests䡠 䡠 䡠” secondary statement category

406

Change in interpretation without significant change in waveform

Used when a primary or secondary statement is added or removed despite no real change in the tracing; ie, an interpretive disagreement exists between the readers of the first and second ECGs

QTc indicates corrected QT interval; HR, heart rate; bpm, beats per minute; AV, atrioventricular; and IV, intraventricular.

Mason et al TABLE 5.

Standardization and Interpretation of the ECG, Part II

General Use Rules

TABLE 6.

1331

Secondary–Primary Statement Pairing Rules

1

Secondary statements must be accompanied by a primary statement

Secondary Code

2

Modifiers must be accompanied by a primary statement

200

145–147

May Accompany These Primary Codes

3

A primary statement may be accompanied by nothing, by ⱖ1 modifiers, by ⱖ1 secondary statements, or by both.

201

21, 105, 109, 120, 131, 141, 145–147

202

105, 106, 145–146

4

Each secondary statement can accompany only certain primary statements (see Table 6)

203

109, 120, 125, 128, 131, 141, 143

5

Each general modifier can accompany only certain primary statements (see Table 7)

204

147

205

145–147

6

Each specific modifier can accompany only primary statements within its category

206

145–147

207

149

208

147

209

142

210

148

211

147–148, 150

212

14, 154

213

82, 105–106, 121

214

124

215

42, 131, 145–147

220

145–147, 151

221

55, 56

222

55, 56

223

148, 149

224

128

225

22, 24–26, 37, 38

226

145–147

227

145–147

228

80, 105, 128, 155

229

109, 120, 122–123, 125, 128, 131, 141, 143

230

128, 131

231

128

e316

Circulation

March 13, 2007

Figure 1. Chest radiograph obtained on admission showing localized bulge of the left cardiac contour (arrows).

Zeina et al

Pericardial Hemangioma Evaluated With MDCT

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Figure 2. A, Transesophageal echocardiography demonstrating an epicardial mass (M). The color Doppler short-axis view shows the left coronary artery system traversing the mass (M). B and C, Cardiac computed tomography angiography. B, Maximum-intensity projection reconstruction shows a large hypodense solid mass (M), located in the epicardium posteromedially to the right ventricle outflow tract (RVOT) and ascending aorta (A), causing compression and displacement of the left atrium (LA) and the left superior pulmonary vein (PV). Small areas of enhancement are noted within the mass. C, Multiplanar reformatted image demonstrating the left main coronary artery (LM), left anterior descending artery (LAD), and first diagonal (D1) completely surrounded by the tumor and slightly narrowed. D, Sagittal ECG-gated breath-hold cine magnetic resonance (FIESTA) image shows a soft tissue mass (arrows) compressing the anterolateral wall of the left ventricle (LV), with no evidence of myocardial involvement. Note a moderate pericardial effusion (open arrows). E, Histological examination of the tumor (hematoxylin and eosin, magnification ⫻2.5) showing irregular cavernous vascular spaces filled with blood and separated by fibrous stroma. Some spaces contain thrombi. The cavernous vascular spaces are lined by bland, flattened endothelium. The walls of the blood vessels contain fibrous and smooth muscular tissue.

Images in Cardiovascular Medicine Cardiovocal Syndrome Associated With Huge Left Atrium Okan Gulel, MD; Diyar Koprulu, MD; Zafer Kucuksu, MD; Mustafa Yazici, MD; Senem Cengel, MD

A

attributable to compression of the left recurrent laryngeal nerve by the huge left atrium. That hypothesis was proven by the flexible fiberoptic laryngoscopy, which showed paralytic left vocal cord (Figure 2, Movie). Because of these findings, the patient underwent operation for mitral valve replacement and atrial reconstruction. No complications occurred in the postoperative period. The patient’s voice quality improved somewhat, but it did not become totally normal. Cardiovocal syndrome or Ortner’s syndrome is a clinical condition with hoarseness attributable to left recurrent laryngeal nerve palsy in cardiovascular diseases. In our patient, nerve palsy developed because of compression of a very big left atrium.

46-year-old woman was admitted to our cardiology department because of dyspnea with exertion and hoarseness lasting for a long time. She had irregular medical follow-ups because of rheumatic mitral valve disease. Atrial fibrillation with a ventricular rate of 90/min was present in her electrocardiography. Chest teleradiography showed an increased cardiothoracic ratio. Transthoracic echocardiography revealed normal left ventricle systolic function (ejection fraction⫽60%) and mildly enlarged left ventricular dimensions (end-diastolic diameter⫽56 mm, end-systolic diameter⫽36 mm). Although systolic pulmonary artery pressure was 50 mm Hg, dimensions of the right heart chambers were normal. Important findings of the transthoracic echocardiography were found to be present at the mitral valve and left atrium. Mitral valve stenosis (planimetric mitral valve area⫽1.5 cm2), severe mitral regurgitation (3⫹), and huge left atrium with dimensions of 13⫻13 cm were determined (Figure 1). It was thought that the patient had hoarseness

Disclosures None.

From the Departments of Cardiology (O.G., D.K., Z.K., M.Y.) and Otorhinolaryngology (S.C.), Faculty of Medicine, Ondokuz Mayis University, Samsun, Turkey. The online-only Data Supplement, consisting of a movie, is available with this article at http://circ.ahajournals.org/cgi/content/full/115/10/ e318/DC1. Correspondence to Okan Gulel, MD, Universite Lojmanlari, A-Blok No:7, 55139 Kurupelit/Samsun/Turkey. E-mail [email protected] (Circulation. 2007;115:e318-e319.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.649814

e318

Gulel et al

Figure 1. Appearance of the huge left atrium during transthoracic echocardiography. LV indicates left ventricle; RV, right ventricle; LA, left atrium; and RA, right atrium.

Cardiovocal Syndrome

e319

Figure 2. Appearance of the paralytic left vocal cord during flexible fiberoptic laryngoscopy. The asterisk shows the left vocal cord.

Images in Cardiovascular Medicine Infarction-Like Electrocardiographic Changes Due to a Myocardial Metastasis From a Primary Lung Cancer Panagiotis Samaras, MD*; Frank Stenner-Liewen, MD*; Stefan Bauer, MD; Gerhard W. Goerres, MD; Lotta von Boehmer, MD; Nina Kotrubczik, MD; Rolf Jenni, MD; Christoph Renner, MD; Alexander Knuth, MD

A

By transbronchial biopsy, a squamous cell carcinoma was diagnosed. The respiratory symptoms resolved after an Ultraflex stent had been placed in the right main bronchus. Three months after onset of chemotherapy with carboplatin and gemcitabine, the patient’s cancer progressed, with growth of the myocardial metastasis. In ECG, more pronounced ST-segment elevations and new elevations in leads V1 and V6 were detected (Figure 1B). Chest pain and infarction-like ECG changes were associated with tumor in this case. Usually, myocardial metastases remain clinically unapparent and are only discovered at autopsy. Although acute myocardial infarction is the most frequent cause of ST-segment elevations, the possibility of a myocardial metastasis should be considered when electrocardiographic changes are seen in patients with malignancies.

69-year-old man (a heavy smoker) presented with chest pain and dyspnea that he had experienced for several days. The initial ECG revealed ST-segment elevations in the leads V2 through V5, suggesting an acute myocardial infarction (Figure 1A). Laboratory tests showed normal levels of creatine kinase and troponin T, but an elevated pro-brain natriuretic peptide (1895 ng/L; normal value for adult males is ⬍227 ng/L). Chest radiography revealed a tumorous mass in the right upper lobe. A computed tomography scan demonstrated a lung tumor occluding the right main bronchus. In addition, a 3.7-cm, hypodense lesion was seen in the apical near-right ventricular myocardium, consistent with a metastasis (Figure 1A). Echocardiography confirmed a right ventricular mass filling the apex and one third of the right ventricle (Figure 2). Left ventricular function was unimpaired. The biplane ejection fraction was 65% (fractional shortening: 47%; left ventricular end-diastolic volume: 132 mL; left ventricular end-systolic volume: 29 mL; interventricular septum: 1.2 cm, akinetic; left ventricular posterior wall: 1.1 cm, contraction normal).

Disclosures None.

From the Departments of Oncology (P.S., F.S.-L., S.B., L.v.B., N.K., C.R., A.K.), Radiology (G.W.G.), and Cardiology (R.J.), University Hospital Zurich, Zurich, Switzerland. *The first 2 authors contributed equally to this work. The online-only Data Supplement, consisting of a movie, is available with this article at http://circ.ahajournals.org/cgi/content/full/115/10/ e320/DC1. Correspondence to Panagiotis Samaras, MD, University Hospital Zurich, Department of Oncology, Ramistrasse 100, 8091 Zurich, Switzerland. E-mail [email protected] (Circulation. 2007;115:e320-e321.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.650762

e320

Samaras et al

ECG Changes in Lung Cancer

e321

Figure 1. Metastatic growth and ECG changes. A, At diagnosis in January 2006, the myocardial metastasis had a diameter of 3.7 cm (arrow). ST-segment elevations were seen in chest leads V2 to V5. B, Three months later, in April 2006, the metastasis was 7 cm (arrow). The ST-segment elevations in leads V2 to V4 were more pronounced than in the initial ECG, and new elevations in V1 and V6 indicated growth of the metastasis.

Figure 2. The apical 4-chamber plane showed a right ventricular mass that was 5⫻3 cm in size (arrow). Left ventricular function was unimpaired, with a biplane ejection fraction of 65%.

Correspondence Letter by Pischon et al Regarding Article, “Adiponectin and Coronary Heart Disease: A Prospective Study and Meta-Analysis”

relative risk of 0.56 (95% CI: 0.32 to 0.99) for quintiles2 was reduced to a relative risk of 0.65 (0.44 to 0.98) in the meta-analysis by the use of tertiles.1 Interestingly, this estimate of effect is very similar to the results these authors have reported in their previously published study among women (relative risk of 0.63; 0.36 to 1.08).5 Further, although not documented in the meta-analysis, the studies that were included had different degrees of adjustment. Clearly, much more data are needed before meta-analyses can clarify the potential and important inverse association between adiponectin and clinically diagnosed coronary heart disease.

To the Editor: We read with interest the report by Sattar and colleagues1 that shows no significant relationship of plasma adiponectin levels with risk of coronary heart disease in the British Regional Heart Study. In a summary of published studies, Sattar et al1 also report only a modest inverse association. However, we have concerns about their pooling of only a few studies when several different methodologies, populations, and analysis methods were used. For example, the Health Professionals Follow-Up Study was the only study that had blood specimens stored in liquid nitrogen at temperatures ⬍⫺130°C.2 Adiponectin was significantly inversely associated with coronary heart disease risk in the Health Professionals Follow-Up Study.2 In the British Regional Heart Study, in which serum was stored for approximately 16 years at ⫺20°C, reported adiponectin levels were considerably lower, and correlations of adiponectin with high-density lipoprotein cholesterol (r⫽0.33) and triglycerides (r⫽⫺0.25) were substantially weaker as compared with the Health Professionals Follow-Up Study (r⫽0.44; r⫽⫺0.39). The body mass index levels between the 2 studies were similar, but the correlations with body mass index were also weaker, even though body mass index was measured in the British Regional Heart Study (r⫽⫺0.21) but self-reported in the Health Professionals Follow-Up Study (r⫽⫺0.27). Therefore, additional data are required to better understand the impact of blood storage methods and characteristics on levels of adiponectin. We were surprised to read that Wolk et al3 and Zoccali et al4 had measured adiponectin using a radioimmunoassay by Linco (St Charles, Mo) in fresh samples at baseline in 1997 to 1998, although—to our knowledge—this assay had not been introduced to the market at this time. Further, the publication by Wolk et al3 does not contain information about adiponectin. It would be informative to know how the authors identified these unpublished data. Meta-analyses should be used to provide greater insight across the quantitative distribution of biomarkers instead of ignoring these differences across studies. It is also unclear to us why the authors have chosen tertiles for comparison, because the size of their study would have allowed more informative comparisons. Previous studies, including their own prior analysis, have reported relative risks for quartiles or quintiles.2,5 Our observed

Disclosures None. Tobias Pischon, MD, MPH Matthias B. Schulze, DrPH Department of Epidemiology German Institute of Human Nutrition Potsdam-Rehbruecke, Germany Eric B. Rimm, ScD Departments of Nutrition and Epidemiology Harvard School of Public Health Boston, Mass 1. Sattar N, Wannamethee G, Sarwar N, Tchernova J, Cherry L, Wallace AM, Danesh J, Whincup PH. Adiponectin and coronary heart disease: a prospective study and meta-analysis. Circulation. 2006;114:623– 629. 2. Pischon T, Girman CJ, Hotamisligil GS, Rifai N, Hu FB, Rimm EB. Plasma adiponectin levels and risk of myocardial infarction in men. JAMA. 2004;291:1730 –1737. 3. Wolk R, Berger P, Lennon RJ, Brilakis ES, Johnson BD, Somers VK. Plasma leptin and prognosis in patients with established coronary atherosclerosis. J Am Coll Cardiol. 2004;44:1819 –1824. 4. Zoccali C, Mallamaci F, Tripepi G, Benedetto FA, Cutrupi S, Parlongo S, Malatino LS, Bonanno G, Seminara G, Rapisarda F, Fatuzzo P, Buemi M, Nicocia G, Tanaka S, Ouchi N, Kihara S, Funahashi T, Matsuzawa Y. Adiponectin, metabolic risk factors, and cardiovascular events among patients with end-stage renal disease. J Am Soc Nephrol. 2002;13: 134 –141. 5. Lawlor DA, Davey Smith G, Ebrahim S, Thompson C, Sattar N. Plasma adiponectin levels are associated with insulin resistance, but do not predict future risk of coronary heart disease in women. J Clin Endocrinol Metab. 2005;90:5677–5683.

(Circulation. 2007;115:e322.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/10.1161/CIRCULATIONAHA.106.666677

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Correspondence Response to Letter Regarding Article, “Adiponectin and Coronary Heart Disease: A Prospective Study and Meta-Analysis”

Disclosures None. Naveed Sattar, MD Lynne Cherry, PhD A. Michael Wallace, PhD University Department of Vascular Biochemistry Glasgow, Scotland

We thank Dr Pischon and colleagues for their interest in our work.1 We have reported the largest single prospective populationbased study of adiponectin levels and coronary heart disease thus far, reinforced by a meta-analysis of 6 previous studies totaling 1313 incident cases.1 Overall, the adjusted risk ratio for coronary heart disease was 0.84 (95% CI: 0.70 to 1.01) in a comparison of individuals in the top third with those in the bottom third of baseline adiponectin concentrations, and there was no evidence of heterogeneity among the 7 reports (␹2⫽8.4; P⫽0.21; I2⫽29% [95% CI: 0% to 69%]). We analyzed the new data using different cut points, such as comparisons of extreme fifths (adjusted odds ratio: 1.12; 95% CI: 0.75 to 1.66) and per 50% increase in loge adiponectin levels (adjusted odds ratio: 1.02; 95% CI: 0.93 to 1.12). Although the available evidence suggests that adiponectin levels are less strongly associated with coronary heart disease risk than was previously suspected, we have stated that further data are needed. Pischon et al draw attention to potential underestimation of effects due to prolonged storage of serum samples at ⫺20°C. We have acknowledged this possibility, but we also have noted that it was unlikely to be a major factor because (1) adiponectin concentrations (and correlations with several risk markers) in our study were comparable with those previously reported,2–4 and (2) adiponectin concentrations were reasonably stable for a 4-year duration in paired measurements (self-correlation coefficient: 0.58; 95% CI: 0.49 to 0.66), reflecting our study’s reasonably robust sample storage and assay methods. There are no data suggesting that adiponectin is influenced by delayed measurement, prolonged storage, or repeated freeze–thaw cycles, but we agree that such considerations merit further study. The assay method attributed to the study of Zoccali et al5 in Table 4 of our report was incorrectly stated as the assay by Linco (St Charles, Mo); it was an in-house ELISA (leptin levels in this study were measured with the radioimmunoassay adiponectin assay kit described). The study of Wolk et al6 did use the radioimmunoassay adiponectin assay kit by Linco, but on frozen rather than fresh blood samples. Five of the 6 principal investigators of previous studies (including Wolk,6 who have published on leptin levels) have provided standard comparisons of odds ratios for coronary heart disease adjusted for at least age, sex, smoking, blood pressure, and lipids (the majority involving further adjustment for markers of adiposity). This information enabled reasonable consistency of data analysis in our literature-based review, but, as implied by Pischon et al3, more detailed pooling of available data (such as on the basis of individual-participant meta-analysis) would be required to achieve even greater consistency.

Goya Wannamethee, PhD Julia Tchernova, BSc Department of Primary Care and Population Sciences Royal Free UCL Medical School London, England Nadeem Sarwar, MPhil John Danesh, DPhil Department of Public Health and Primary Care University of Cambridge Cambridge, England Peter H. Whincup, FRCP Division of Community Health Sciences St George’s, University of London London, England 1. Sattar N, Wannamethee G, Sarwar N, Tchernova J, Cherry L, Wallace AM, Danesh J, Whincup PH. Adiponectin and coronary heart disease: a prospective study and meta-analysis. Circulation. 2006;114:623– 629. 2. Lawlor DA, Davey Smith G, Ebrahim S, Thompson C, Sattar N. Plasma adiponectin levels are associated with insulin resistance, but do not predict future risk of coronary heart disease in women. J Clin Endocrinol Metab. 2005;90:5677–5683. 3. Pischon T, Girman CJ, Hotamisligil GS, Rifai N, Hu FB, Rimm EB. Plasma adiponectin levels and risk of myocardial infarction in men. JAMA. 2004;291:1730 –1737. 4. Lindsay RS, Resnick HE, Zhu J, Tun ML, Howard BV, Zhang Y, Yeh J, Best LG. Adiponectin and coronary heart disease: the Strong Heart Study. Arterioscler Thromb Vasc Biol. 2005;25:e15– e16. 5. Zoccali C, Mallamaci F, Tripepi G, Benedetto FA, Cutrupi S, Parlongo S, Malatino LS, Bonanno G, Seminara G, Rapisarda F, Fatuzzo P, Buemi M, Nicocia G, Tanaka S, Ouchi N, Kihara S, Funahashi T, Matsuzawa Y. Adiponectin, metabolic risk factors, and cardiovascular events among patients with end-stage renal disease. J Am Soc Nephrol. 2002;13: 134 –141. 6. Wolk R, Berger P, Lennon RJ, Brilakis ES, Johnson BD, Somers VK. Plasma leptin and prognosis in patients with established coronary atherosclerosis. J Am Coll Cardiol. 2004;44:1819 –1824.

(Circulation. 2007;115:e323.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

DOI: 10.1161/CIRCULATIONAHA.106.671289

e323

Correction The article, “Carotid Intima-Media Thickness Is Associated With Premature Parental Coronary Heart Disease: The Framingham Heart Study” by Wang et al that appeared in the August 5, 2003, issue (Circulation. 2003;108:572-576) contained an error. Because of a programming error, the age cutoff used to define premature coronary heart disease was 70 years, rather than 60 years as stated in the Methods (fourth paragraph) and the title of Table 2. The corrected title for Table 2 should read, “Table 2: Mean Internal Carotid IMT in mm (Standard Error), by Parental Premature (Age ⬍70) CHD Status.” If age 60 years is used rather than 70 years, the results do not change in men (age-adjusted internal carotid intima-media thickness [IMT] in those with versus without premature parental coronary heart disease [CHD], 1.17 vs. 1.05 mm, P⫽0.003) but are slightly attenuated in women (age-adjusted internal carotid IMT in those with versus without premature parental CHD, 0.92 vs. 0.86 mm, P⫽0.13). The authors regret this error and any confusion it may have caused. DOI: 10.1161/CIRCULATIONAHA.107.181837

(Circulation. 2007;115:e324.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

e324

Correction In the article, “Adiponectin and Coronary Heart Disease: A Prospective Study and Meta-Analysis” by Sattar et al that published in the August 15, 2006, issue (Circulation. 2006;114:623– 629), the assay method attributed to the study of Zoccali et al in Table 4 was incorrectly stated as the assay by Linco; it was an in-house ELISA and done on frozen rather than fresh samples. In addition, with respect to the study of Wolk et al in the same table, frozen rather than fresh blood samples were used. The authors regret these errors. DOI: 10.1161/CIRCULATIONAHA.107.181838

(Circulation. 2007;115:e325.) © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org

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March 13, 2007

European Perspectives in Cardiology

Spotlight: Michal Tendera, MD, FESC Dr Michal Tendera stepped down from the presidency of the European Society of Cardiology in September 2006. He shares his thoughts about his tenure with Sarah Ramsay, MA.

hen Dr Tendera stepped into the spotlight as president of the European Society of Cardiology (ESC) in 2004 it was a time of great change both in Europe and in cardiology. The past 2 years in this office have been a whirlwind of activity for Dr Tendera, who is a past president of the Polish Cardiac Society and professor and chair of cardiology at the Upper-Silesian Cardiac Center, Silesian School of Medicine, Katowice, Poland. “The ESC is in essence a federation of 50 national societies, 5 subspecialty associations, 18 working groups, and 3 councils,” Dr Tendera explains. “We also have affiliated members from outside Europe and individual members — the fellows. The total number of individuals who belong to the ESC is now in excess of 50 000.” Dr Tendera feels that his tenure came at the end of a decade of progress that saw the ESC develop into an increasingly active and professionally relevant organisation. With a mission to reduce the burden of cardiovascular disease in Europe, the ESC has had to broaden and strengthen its portfolio of projects to accommodate changes within and outside the cardiology profession. This portfolio is extensive and includes a collection of congresses and meetings, an active publishing arm with 7 journals, and the development of guidelines, education, and auditing processes. “Although the list of ESC projects and

W

initiatives is long,” says Dr Tendera, “if I were to name some of the main achievements of this board and this presidency, a significant one has been the integration of cardiovascular medicine.” He continues, “I think that cardiology is at a very interesting point in time when there are 2 forces acting on this speciality. The first is attracting other subspecialities of medicine and is developing into cardiovascular medicine, which includes aspects of cardiovascular surgery, molecular biology, nephrology, and other specialities.” Dr Tendera says that the second force resembles that which led to the fragmentation of internal medicine as a discipline around 20 years ago, with the advent of subspecialities such as cardiology, nephrology and gastroenterology. “This is starting to happen in cardiology. We at the ESC think that talking together and staying under one umbrella is something that is extremely important,” says Dr Tendera. “So far, we have been quite successful because we have created new branches representing subspecialities within the ESC. We started with 4 such branches: the European Heart Rhythm Association, the European Association of Echocardiography, the Heart Failure Association of the ESC, and the European Association for Cardiovascular Prevention and Rehabilitation. They are all independent organisations that act within the ESC.” To add to this list, in the past year

On other pages... Cardiology Careers: The Netherlands

Viewpoint: Michele Brignole, MD

Cardiology fellows training in the Netherlands will soon have to choose a subspeciality. Freek W.A. Verheugt, MD, PhD, FESC, talks about this and other training issues facing young cardiologists. Page f39

Dr Michele Brignole, chief of an arrhythmologic centre in Lavagna, Italy, believes syncope is often unexplained or misdiagnosed. He outlines how he thinks management can be improved. Page f41

More Pages for Circulation: European Perspectives in Cardiology. This successful addition to Circulation now has a new look and has also increased in size, with 2 more pages in every issue.

Circulation: European Perspectives

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an agreement was struck to merge EuroPCR with the ESC Working Group on Interventional Cardiology to form the European Association of Percutaneous Cardiovascular Interventions. Dr Tendera emphasises that these activities marked an important step in maintaining and strengthening the cohesion of European cardiovascular medicine. Another area in which the ESC has been particularly industrious during Dr Tendera’s presidency revolves around its stronger relationships with both national cardiology societies and with the European Union (EU). There have been several initiatives, but the main focus of collaboration in recent years has been prevention of cardiovascular disease. Dr Tendera says, “This will be summarised in a document that is likely to be launched in a few months’ time under the German presidency of the European Union —the European Heart Health Charter.” Dr Tendera points out that the decision to Dr Tendera (left) is presented with the gold medal of the ESC by incoming president Kim Fox, take this initiative was made at a conference MD, FRCP, FESC at the ESC’s general assembly in Barcelona, Spain, September 2006. organised jointly by the European Commission, the ESC, the European Heart Network, and been done in the field of guidelines is quite impressive, and World Health Organisation Europe. “So what we have now,” what is important is that those guidelines are now adopted by he says, “is a common view of how cardiovascular health most of the national societies. Thus, they do not produce promotion should be approached in all EU countries. Of their own guidelines any longer but endorse those of the course, development of such projects would have been ESC. They provide translations and comment on specifics to impossible without a major contribution from different do with their own geographical, epidemiological and finannational societies. There were proposals from everyone and cial situations. So, this activity has been harmonised to a large several meetings which included, perhaps for the first time in extent.” Importantly, all the guidelines are accompanied by history, representatives of ministries of health and presidents derivative educational tools that are available in different of national cardiac societies of different countries, sitting languages, and education is another major area where Dr together at one table and discussing cardiovascular disease Tendera feels that improvement has been made. prevention. So it was a joint project that really involved all One example of this improvement is the emphasis that the national members.” ESC has put on the development of educational material in With regard to the more mainstream activities of the ESC, print and especially electronic form, rather than on meetings. Dr Tendera notes, “There is a need to audit whatever we do Dr Tendera explains, “Another aspect of education is the clinically. And this is what we are doing with the developdevelopment of the core curriculum, and this is a very ment of surveys and registries. In the last 2 years the structure detailed outline of what kind of training should be required of the ESC survey program has been changed, and, most of all cardiologists throughout Europe.” This harmonisation importantly, the participating countries and individual centres is a critical development, especially in light of the increasing receive feedback on their performance, which has allowed mobility of the medical workforce in Europe. The draft docbenchmarking of the results between different program ument outlining these requirements has been agreed to by participants.” most national societies, and Dr Tendera is confident that it Dr Tendera is keen to see the ESC audit flourish and will soon be implemented. inform future clinical practice throughout Europe. “I think Dr Tendera mentions a third example where there has been this is very valuable because we sometimes don't know the progress in education. “For the first time, we have produced optimal use of high technology, for example,” he says. a textbook of cardiovascular medicine that is based on guide“There are no good reasons why in one country the rate of lines. It will be a key tool for cardiologists in training and is implantation of implantable cardioverter defibrillators is being translated into several languages.” Perhaps it is a fitting high, while in another the rate of use of drug-eluting stents is tribute to Dr Tendera’s energetic 2 years at the head of the high. And we don't know whether this is reflected in patient ESC that the first non-English version of the ESC Textbook of outcomes. Now we have an improved tool to see how differCardiovascular Medicine will be in Polish. ences in practice have an impact on clinical results.” Sarah Ramsay is a freelance medical writer. In addition, Professor Tendera notes, “I think what has © World Congress of Cardiology 2006

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Climbing the Cardiology Career Ladder: The Netherlands Starting in 2007, cardiology fellows training in the Netherlands will have to choose a subspeciality. Freek W.A. Verheugt, MD, PhD, FESC, chairman of the Committee of Cardiology Training Directors of the Netherlands, calls for more women and more mobility. Barry Shurlock, MA, PhD, talks to him about this and other training issues.

he Netherlands has about 120 hospitals, of which 80 have by newly qualified doctors in the Netherlands, where they are catheter laboratories, 20 practice primary coronary interattracted by the charisma associated with the speciality and vention, and 13 have facilities for the full range of cardiology by the fact that it is a hands-on discipline using sophisticated interventions, including cardiothoracic surgery. Each year, technology, with results that are often quickly and unamabout 60 new cardiology trainees (termed fellows), who biguously apparent. already have had 6 years of basic medical training for their Training in the country is highly developed and well MD, are admitted to the 13 major centres. Here, they train for organised, according to Dr Verheugt, but he points to 2 areas 2 years in internal medicine and for 4 years in cardiology. where he would like to see progress. “First, it is difficult in From the middle of this year, all fellows will have to decide the Netherlands, as in other countries, to persuade young in their final year which of 6 subspecialities they wish to women to train, though the reasons are not clear.” He sugpursue (Figure 1). These are interventional cardiology, electrogests, “Perhaps it is the need for intense work at weekends physiology, congenital cardiology, cardiac intensive care, and during night shifts. We are trying to tackle the problem cardiac imaging, or general cardiology. by encouraging more women to be involved in cardiac Dr Freek W.A. Verheugt, professor and chairman of the research, in the hope that they will later take up the speciality.” Department of Cardiology at the Heart Lung Center, Dr Verheugt also would like doctors to be much more University Medical Center in Nijmegen, the Netherlands, mobile in the early years of their careers in the Netherlands, comments, “It will be a big change, but the need for this is where it is normal to do both basic medical training and spegenerally accepted. Plans are already in place for the 1-year ciality training in the same hospital and/or medical school subspeciality courses, although there is some talk that and then to stay on in a career post. “It is a weakness, not only for electrophysiology and interventional cardiology it should in the Netherlands, but in continental Europe as a whole,” he be 2 years. Not every cardiology fellow will opt for a named says. “People tend to stay in the hospital or the university subspeciality, and that’s why we will have the category of where they train. Even moving from, say, Amsterdam to general cardiology.” Rotterdam, which is only a distance of 65 kilometres, is rarely Cardiology in Dutch hospitals is completely separate from done. I try to encourage my own fellows to move about, but internal medicine, explains Dr Verheugt. “I regard this as a it’s not easy to get the jobs or the funds. Also, they don’t great strength in terms of raising money for research, though for some complex clinical cases, specialists from other disciplines may need to be called in.” Much of the financial support for cardiology comes from the Netherlands Heart Foundation, a national charity. A major interest at the Heart Lung Centre in Nijmegen is congenital heart disease and paediatric cardiology. Dr Verheugt’s personal interest is primary coronary intervention for ST-elevation myocardial infarction and related conditions. At Nijmegen, patients with infarcts are accompanied in the ambulance by specialist nurses who perform electrocardiography, proceed to make a preliminary diagnosis, and fax or e-mail ECG readings to the hospital in advance of admission. Clinical audits show that, on average, patients in Nijmegen are in the catheter laboraFigure 1. Cardiology fellows in training in the Heart Lung Centre in Nijmegen. tory within an hour of an emergency call.1 Cardiology is much sought after as a speciality They will have to select a subspeciality to pursue for l year as from mid 2007. Dr Freek Verheugt

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move because of family, friends, roots—it’s a cultural thing. Yet, it is always good to see how things are done in another hospital, where the axioms are probably different.” Because the teaching programmes at the 8 medical schools in the Netherlands are not well coordinated, it is sometimes difficult to shift from 1 programme to the other midcourse. As a young doctor, Dr Verheugt took his own advice: After doing his MD and PhD at Amsterdam and his cardiology training at Rotterdam, he and his family spent a year in the United States, where he worked as an assistant professor of medicine in the Cardiology Division of the University of Colorado Health Sciences Center, Denver, Colo. After training for their MD, Dutch doctors who wish to specialise in cardiology are free to apply to any of the 13 hospitals Figure 2. The University Medical Centre, Nijmegen. Two-thirds of the cardiologists that that have training programmes. “Many work there are hired for their research skills, and most will have taken a PhD. apply to all 13 to maximise their chances of acquiring a place. Applications by letter are screened, and a tion) before being eligible for licensing. Licensing is the task short list of candidates for interview is prepared,” explains Dr of the Medical Specialist Registration Committee of the Verheugt, who says that a key requirement he looks for is the Royal Dutch Medical Society. Dr Verheugt strongly recomability to make quick decisions in stressful circumstances. mends that cardiologists who wish to reach the top of the proHe says, “Every 3 months, a colleague and I interview 5 fession study for a PhD before undertaking speciality trainor 6 applicants during the course of an afternoon. The appliing. Although this route is only taken by about 20% of Dutch cation letter is very important. I look for good reasons why cardiologists, 2 out of 3 cardiologists at the Heart Lung they have chosen cardiology. Did they opt for cardiology durCenter in Nijmegen are hired for their research skills, and ing their 3-month preferred internship? What did they do durmost will have taken a PhD between their MD and specialist ing the obligatory research project for their MD? If it was cartraining. He comments, “Other cardiologists are employed diology, that’s obviously important. Why have they chosen for specific technical skills, as dedicated teachers, or because this hospital? I expect them to have some knowledge of our they are able to handle complex clinical cases.” research and training programmes. I’m not impressed if their Asked about the future, Dr Verheugt says, “Cardiology father was a cardiologist, though I’m interested!” training will be split up into the subspecialities, but only after Explaining how the interview progresses, Dr Verheugt at least 2 years of medicine and 3 years of general cardiology. says, “For me, the first 10 seconds are the most important— Society demands subspecialists, but general cardiology will how did they come across, how did they look, how did they always be necessary.” shake hands? It sounds trivial, but it’s important in a specialBarry Shurlock is a freelance medical writer ity where close contact with patients in stressful situations is Reference the norm.” 1. Verheugt FWA. Reperfusion therapy starts in the ambulance. Once accepted for training, fellows have their progress Circulation. 2006;113:2377–2379. assessed informally with tutors 4 times a year. Until they have completed 3 years of specialist training, they can be taken off the course, but this only occurs in about 5% of cases, according to Dr Verheugt. The training programmes themselves are assessed every 5 years through visitations; each visitation is conducted by 2 programme directors, accompanied by a fellow, all from elsewhere. Shortcomings may require a repeat assessment after 2 years, but this is uncommon. Once a fellow has obtained a diploma as a cardiologist, he or she is free to work throughout Europe, subject to language restrictions, but in practice, almost all of them stay in the Netherlands. Doctors from outside the Netherlands who wish to practise in the country have to be able to speak Dutch (nonspeakers usually take a 6-month course with a final examinaUniversity Medical Centre, Nijmegen

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Viewpoint: Michele Brignole, MD, FESC Some Thoughts on Unexplained Syncope Syncope is often unexplained or misdiagnosed. Dr Michele Brignole, chief of the Arrhythmologic Centre, Department of Cardiology, Ospedali del Tigullio, Lavagna, Italy, outlines to Ingrid Torjesen, BSc, how he believes management of syncope in Europe can be improved. ccurate diagnosis of syncope is essential because treatfor managing syncope.2 Dr Brignole chaired the task force ment needs to be tailored to its cause, says Dr Michele and says their objective was to set a standard for management Brignole, and he hopes this better management will be and promote both the development of syncope specialists and achieved for patients in Europe in the near future. the availability of specialist facilities. Their rationale was that “A syncopal episode is experienced by 30% to 40% of all doctors are likely to see a patient with syncope so should young adults and by 50% of people during their lifetime,” Dr be trained to perform an initial evaluation. This would enable Brignole states. “It is one of the most frequent causes of them to select those patients who require referral to arrhythemergency room visits and hospitalisations.” mologic facilities. Diagnosing the cause of syncope is difficult because it can In the 1980s, the cause of syncope remained unexplained be related to several diseases and patients are usually asympin around 30% to 40% of patients. This percentage has protomatic at evaluation. An accurate diagnosis is essential for gressively decreased and now stands at around 20%. determining the most effective treatment strategy and the However, a recent study showed that strict adherence to reclikely prognosis, but, at present, according to Dr Brignole, the ommended guidelines led to a diagnosis for virtually all diagnostic process at many European centres involves “costly patients, with the cause unexplained in only 2%.2 and often useless diagnostic procedures.” Around two thirds of patients are diagnosed with neurally He explains: “As syncope is so common, virtually all mediated reflex syncope, and Dr Brignole says that the incitypes of doctor come across it, including general practitiondence of this type is actually higher because some patients ers, emergency doctors, cardiologists, neurologists, internal will not seek medical attention for their episodes. Another medicine specialists, and geriatricians. This has made it 10% to 15% of patients are diagnosed with cardiac syncope difficult to improve the standardisation and organisation of (see Figure), which has a poor prognosis unless there is effecsyncope.” tive treatment. “One of the most important goals of syncope Expressing his frustration, Dr Brignole continues, “We evaluation is to identify cardiac syncope and to differentiate it tried for 20 years to teach the best methodology, to teach a from other causes, especially the most frequent, benign neurally standard, but it is impossible. If there is a relatively small mediated,” Dr Brignole says. group of homogeneous doctors, you select a few who are In addition to failure to attribute a cause, misdiagnosis is very keen on that selected topic. It is their job, so it is easy to also a problem with syncope. According to Dr Brignole, obtain a quality standard. But, if you have to teach thousands physicians frequently confound the prognostic significance of doctors because syncope is only a minor part of their time, of syncope with that of underlying heart disease and treat it is almost impossible.” accordingly. “An implantable cardioverter defibrillator (ICD) The fact that strategies for assessing 66 bpm syncope vary widely among physicians 40 bpm P P P P and hospitals creates enormous difficulties, says Dr Brignole. “The result is a wide variation in the diagnostic tests 60 bpm P applied, the proportion and types of attributable diagnoses, and the proportion of Asystole = 43 s syncope patients in whom the diagnosis 60 bpm remains unexplained.” The lack of a uniform strategy for the management of syncope in everyday practice was confirmed by 50 bpm a prospective study of patients attending 28 general hospitals in Italy.1 In 2004, the European Society of Cardiology Syncope Task Force tried to An example of cardiac syncope,which has a poor prognosis unless there is effective treatment. address this issue by publishing guidelines An important goal is to identify cardiac syncope and differentiate it from other causes. Dr Michele Brignole

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is currently recommended for patients with syncope who have structural heart disease and an ejection fraction