Cardiovascular Research Advance Access published March 8, 2011

Establishment of the mouse ventricular conduction system

Lucile Miquerol, Sabrina Beyer and Robert G. Kelly

Developmental Biology Institute of Marseilles – Luminy (IBDML) CNRS UMR6216 Université de la Méditerranée Campus de Luminy, case 907 13288 Marseille cedex 9 France

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Fax: + 33 4 91 26 97 26 Correspondance to Lucile Miquerol. E-mail [email protected]

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2011. For permissions please email: [email protected]

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Phone: +33 4 91 26 97 34

2 Abstract The ventricular conduction system represents the electrical wiring responsible for the coordination of cardiac contraction. Defects in the circuit produce a delay or conduction block and induce cardiac arrhythmias. Understanding how this circuit forms and identification of the factors important for its development thus provide insights into the origin of cardiac arrhythmias. Recent advances, using genetically modified mouse models, have contributed to understanding how the ventricular conduction system is established during heart development. Transgenic mice carrying different reporter genes have highlighted the conservation of the anatomy and development of the ventricular conduction system between mouse and man. Lineage tracing and retrospective clonal analysis have established the myogenic origin of the ventricular conduction system and determined properties of

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defects, have led to the identification of transcription factors important for the development of the ventricular conduction system. These transcription factors operate at the levels of both conduction system morphogenesis and differentiation by controlling the expression of genes responsible of the electrical activity of the heart. In summary, defects in the ventricular conduction system are a major cause of arrhythmias and deciphering the molecular pathways responsible for conduction system morphogenesis and the differentiation of conductive myocytes furthers our understanding of the mechanisms underlying heart disease.

This article is part of the Spotlight Issue on: Cardiac Development

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conductive progenitor cells. Finally, gene knockout models reproducing human cardiac

3 Introduction

The primary role of the heart, to pump blood around the body, relies on two main functions, conduction and contraction. Active contraction of cardiac muscle is ensured by the working myocardium, upon electrical stimulation. Efficient expulsion of blood depends on tightly coordinated sequential contraction of the atria and ventricles. The dual role of the cardiac conduction system is to produce this electrical activity and to propagate it in a coordinated manner. The electrical activity of the heart is generated by pacemaker cells localized in the sino-atrial node (SAN) in the dorsal region of the right atrium. From the SAN, electrical activity spreads rapidly to both atria provoking simultaneous atrial contraction. The atria and ventricles are

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reach the ventricles, the electrical impulse follows a specialized route termed the atrioventricular conduction system (AVCS). Electrical activity from the atria converges on the atrioventricular node (AVN) where its conduction velocity is reduced to delay the spread of electrical activity and allow atrial contraction before ventricular activation. Electrical activity is then rapidly propagated to the ventricular apex through a specialized fast conducting conduction system or ventricular conduction system (VCS), including the atrio-ventricular bundle (AVB) also known as the His bundle, the right and left bundle branches (RBB, LBB) on either side of the interventricular septum, and a complex network of Purkinje fibers (PF) ramifying over the subendocardial ventricular surface. The VCS plays a critical role in coordinating the heartbeat by rapidly conducting the electrical impulse to the ventricular apex in order to activate contraction from the apex and maximize efficiency of expulsion of the blood through the great arteries at the base of the heart.

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electrically isolated by the annulus fibrosis composed of non-myocardial cells1. In order to

4 The first anatomical description of the human VCS was made by Tawara at the beginning of the 20th century2. Conductive cells can be distinguished from working myocardium by the presence of a poor contractile apparatus with few sarcomeres, enriched glycogen content and reduced numbers of T tubules3. The function of conductive cells has been investigated by electrophysiological experiments demonstrating specific electrical properties, including rapid conduction4. However it is the global anatomy of the VCS that orchestrates coordination of the heartbeats and disturbances in VCS anatomy result in arrhythmias5. The availability of genetically modified models makes the mouse a highly attractive species in which to study the anatomy and development of the conduction system. This review will focus on the murine VCS with emphasis on transgenic mouse models that have provided insight into the origin

1- Mouse models for studying the ventricular conduction system

a. The asymmetrical anatomy of the VCS In order to visualize the anatomy of the entire murine VCS, we generated a transgenic mouse line in which a GFP (Green Fluorescent Protein) reporter gene was inserted at the Cx40 locus, encoding a connexin expressed throughout the VCS6. Cx40 defines the adult VCS both by its pattern of expression and its role in conduction. Cx40 forms gap junction channels with a high conductance and is expressed in all compartments of the fast conduction system including the AVB, left and right BB, and PF network but not in working myocardium or slow conducting compartments of the conduction system such as the SAN and AVN7. Cx40GFP expression delineates the adult ventricular conduction system revealing a network extremely similar to the drawings of Tawara for the human heart (Figure 1) and consistent with classical histological images of the mouse VCS8. 3D imaging of Cx40GFP hearts reveals that the VCS

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and development of the VCS.

5 represents only one percent of the volume of the ventricles9. A precise 3D image of the AV conduction axis was recently published by Aahnaanen and collaborators, by analyzing gene expression patterns in the central conduction system10. These data reveal the existence of specific subregions of the AV axis delineated by distinct molecular signatures. From the atria, two successive rings of cells form figure of eight-like structures around the AV junctions described as the transitional AV ring and nodal AV ring that appear to play a role in slowing down atrial activation. The AV or central conduction axis is composed of a distinct compact AVN and an AVB including lower nodal cells (LNC) anatomically similar to that described using the Cx40GFP reporter line (Figure 2A, B). One striking feature of the anatomy of the VCS is the asymmetrical morphology of the BB and PF network between right and left ventricles (Figure 2). The proximal RBB is formed by

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IVS has shown that the electrical pathways in the proximal part of the VCS are also asymmetrical6. The PF network is localized at the surface of the right ventricular free wall while in the left ventricle the PF covers the entire septal surface (Figure 2D-F). Modeling of the ventricular conduction system demonstrates a precise alignment of activation maps and PF anatomy11. This consistency between anatomy and physiology illustrates how morphology underlies function in the VCS. In human patients, RBB block is more frequent than left block, which may be explained by the fact that a single branch is likely to be more sensitive to damage than multiple branches12. In general, the pattern of electrical activation follows the direction of blood flow13. The asymmetry of the peripheral VCS may thus be explained by the necessity of coupling differential patterns of activation in each ventricle with maximal haemodynamic efficiency.

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a single fascicle while the LBB is formed by numerous fascicles. Optical mapping of the adult

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b. Markers of the developing VCS The first peristaltic contractions of the mouse heart appear by embryonic day (E) 8.5 and are rapidly replaced as the cardiac tube loops by sequential contraction of the atria and ventricles detected by a regular ECG14, 15. Based on histological criteria, Viragh and Challice carried out exhaustive analysis of the development of the mouse conduction system and did not find specialized conductive cells at these early stages3. Instead, sequential contraction of the primary heart tube is thought to be ensured by alternating regions of fast conducting (future atria and ventricles) and slow conducting myocytes (atrioventricular canal, AVC, and outflow tract, OFT)16-18. By E9-E10, the primordium of the AV conduction pathway appears in the inner dorsal wall of the AVC and becomes increasingly compact as development proceeds to

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extending from the primordium of the AVN to the middle portion of the rim of the interventricular foramen where it divides into right and left branches3. More recently, a number of transgenic mice carrying reporter genes have allowed visualization of these cells15, 19, 20

. In Mink-LacZ or CCS-LacZ embryos, the primordium of the AVN can be seen in the

dorsal wall of the outer curvature of the heart, while reporter gene expression is observed in rings at the crest of the IVS and around the atrioventricular canal21-23. A similar ring pattern is also observed for the T-box transcription factor, Tbx3, which identifies the developing central conduction system24. The peripheral conduction system is not yet delineated at this stage of development. At E9.5, Cx40 positive trabeculae form at the subendocardial surface of both ventricles and are thought to play the role of a fast conducting network in the embryonic heart14, 25. Optical mapping of embryonic hearts has shown that ventricular activation switches from an apex to base pattern at E10.5, to biventricular activation points by E12.523. A

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form the AVN3. At the same time, a group of cells differentiates along the ridge of the IVS

7 functional embryonic route equivalent to the His-Purkinje network is thus present prior to completion of ventricular septation. More recently, Christoffels and Moorman have published a recapitulative table of the expression pattern of known markers of conduction system development15. Many of these molecular markers are not selective for the definitive conduction system and are also expressed in surrounding tissues that share a common developmental pathway. Tbx3, for example, is expressed in the AVC and in AVC derived cells including valve leaflets as well as the central conduction system, and broad expression of the CCS-LacZ transgene is observed in the IVS and valves21, 24. Cx40, which as we have discussed above is a definitive marker of the mature VCS, is expressed in ventricular myocardium and but not in the AVN and AVB before E14.57. In contrast, the CCS-LacZ transgene is present in the AVC and IVS at E10.5,

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subsequently convergent expression pattern throughout the definitive VCS (Figure 3). These results reveal the complexity underlying the development of the VCS and suggest that the central and peripheral components of the conduction system develop independently prior to integration in a single conductive pathway. More recently, a new marker of the VCS, contactin 2 (cntn-2), encoding a cell adhesion molecule critical for neuronal patterning and ion channel clustering, has been identified by a transcriptome analysis of PFs purified from CCS-LacZ mice26 . Immunohistochemistry and electrophysiological recordings showed that cntn-2-GFP+ cells have a conductive phenotype. The expression pattern of contactin2 during embryonic development remains to be described and no cardiac phenotype has been reported in mutant mice27, 28. The absence of transgenic mice specifically delineating the entire VCS during development may stem from the multiplicity of VCS cell lineages.

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thus differing in expression from the Cx40eGFP allele in early development, despite a

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2- The developmental origin and establishment of the VCS a. The origin of conductive cells A long-standing debate in the cardiac field concerns the origin of conductive cells. This debate reflected observations that conductive cells express certain neuronal genes and the heterogeneity of cardiac cell populations potentially contributing to the VCS, i.e. cardiomyocytes, neural crest cells (NCC) and epicardially derived cells (EPDC)29. Cardiomyocytes originate from two contiguous populations of mesodermal progenitor cells, the first and second heart fields30. The first heart field constitutes the cardiac crescent at E7.5 and gives rise to linear heart tube. The heart tube subsequently elongates by addition of second heart field cells in pharyngeal mesoderm to the venous and arterial poles. The first

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the second heart field contributes to the right ventricle and outflow tract, in addition to part of the atria. The outflow tract is also colonized by cardiac neural crest cells that drive septation and participate in valve formation29. Epicardial derived cells give rise to coronary vessels and cardiac fibroblasts31. In order to address the cellular origin of the VCS, Mikawa and coworkers carried out pioneering clonal analysis experiments in the avian primary heart tube using replication-defective retroviral infection17. They discovered that conductive cells are always found in clones together with adjacent working myocytes32,

33

. This was the first

evidence of the myogenic origin of the CS using an experimental procedure independent of the expression of molecular markers. Recently, we have taken a similar approach in the mouse heart using the α-cardiac-actin-nlaacZ mouse line that permits retrospective clonal analysis 9. This approach is based on a defective reporter gene carrying a duplication with a stop codon in the β-galactosidase coding sequence34. Low frequency spontaneous intragenic recombination leads to loss of the duplication and restores the nlacZ open reading frame. The

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heart field participates predominantly in the development of the left ventricle and atria while

9 cell in which this event occurs thus carries a functional nlacZ gene that will be stably transmitted to all daughter cells, allowing visualization of clusters of clonally related myocytes. In order to define the lineage relationship between cells of the murine VCS and the surrounding working myocardium, clusters of clonally related myocytes were screened for conductive cells using Connexin40 driven eGFP expression. The presence of mixed clusters, composed of conductive and working myocytes, revealed that both cell types develop from common progenitor cells. Mixed clusters were observed in all compartments of the VCS including the AVB, right and left BB and PF network, demonstrating that the entire VCS shares a common cellular origin with surrounding working myocytes9. However, these results do not exclude a minor participation of other cell lineages in the VCS. Such a possibility is suggested by a genetic lineage analysis identifying a small proportion of the VCS that

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allele, suggested that NCC may contribute to the central conduction system36, although the participation of NCC in the VCS was not clearly demonstrated. The contribution of epicardial-derived cells to the VCS was evaluated by lineage tracing analysis using a Tbx18Cre allele. The T-box transcription factor encoding gene Tbx18 is specifically expressed in the sinus horn and epicardium during cardiac development10. Tbx18 derived cells were shown to contribute to the sheath insulation of the AV axis but not to cardiomyocytes of the AV conduction system. To conclude, both retroviral and retrospective clonal analysis clearly demonstrate the participation of myogenic progenitors in all compartments of the VCS. As yet there is no convincing evidence that EPDC or NCC make cellular contributions to the VCS. Other Cre lines have been used to follow the progeny of different myocardial progenitor cells populations during cardiac development. Observations with cGATA-6-enhancer-Cre mice suggested that the AVC contributes to the AVN and AVB37; however, as this enhancer is also active in these structures later in development these mice cannot be reliably used for lineage

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originates from Mesp1-negative cells35. A genetic lineage analysis of NCC using a Wnt1-Cre

10 analysis of the AVC. Expression of the T-box transcription factor Tbx2 is restricted to the primary myocardium of the AVC and outflow tract38. Using a Tbx2Cre allele, Aanhaanen et al. showed that AVC primary myocardium participates in formation of the AVN but not of the AVB and BB. These results indicate that the AVN and AVB do not develop from a single progenitor cell population but that central conduction system components segregate early in development. Indeed, the AVN originates from an Isl1 positive progenitor population, suggestive of a second heart field origin, although Isl1 is also expressed in a subset of cells in the AVN39. The Mef2c-AHF-Cre line has been used to label the anterior component of the second heart field, which provides the precursors of the outflow tract and right ventricle40. Observations using this line have revealed a large contribution of labeled cells to the right ventricle and IVS including the AVB. However, during cardiac development this transgene is

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SHF cells to the ventricles10. However, analysis of the Mef2c-Cre lineage revealed the absence of a ventricular contribution to the AVN, thus confirming the Tbx2-Cre result. More recently, we have generated an inducible Cre allele expressed under transcriptional control of Cx40 regulatory sequences41. Cx40Cre mice allow precise temporal control of Cre recombination in the Cx40 expression domain. As mentioned earlier, Cx40 expression initiates at the onset of trabeculation and persists in trabeculae throughout cardiac development42. Recombination at E10.5 demonstrates that Cx40 is expressed in cells giving rise to both conductive and working myocytes in each ventricle. In contrast, recombination at E16.5 reveals that Cx40 expressing cells at this stage contribute only to the VCS including the AVB, right and left BB and PF network9. These data illustrate the lineage relationship between Cx40+ embryonic trabeculae and the peripheral PF network.

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continuously expressed in the IVS and so, again, it is not suitable to follow the contribution of

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b. Biphasic development of the VCS Two models have been proposed to explain the mode of development of the VCS. The “outgrowth” model is based on the expression pattern of specific markers describing populations of conductive progenitors organized in rings along the heart tube15, 19. However, as mentioned above, none of these markers are exclusive to the conduction system and this model depends on approximating histological, immunological and functional criteria. The alternate “in-growth” or recruitment model was postulated based on lineage analysis experiments in the avian system43. Using replication defective retroviral labeling Mikawa and colleagues demonstrated that conductive cells share common progenitors with working myocytes and that the VCS develops by a process of induction and recruitment of myocytes

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proliferative and subsequent growth of the conduction system occurs by further myocyte recruitment. The nlaacZ clonal analysis has provided insights into the mode of development of the murine VCS9. Retrospective clonal analysis consists of studying the properties of clonally related cells such as clone size, cell identity and cell dispersion to provide information on the events that take place during development of a particular structure44. In double transgenic α-cardiacactin-nlaacZ/Cx40GFP hearts, two classes of conductive clusters were identified, containing only conductive cells (unmixed conductive clusters) or both conductive and working cells (mixed clusters) (Figure 4A). While the presence of mixed clusters demonstrates that conductive and working myocytes share a common progenitor, as discussed above, unmixed conductive clusters indicate that cells retain their proliferative potential after restriction to a conductive fate9. Analysis of mixed and unmixed cluster size reveals that development of the mammalian VCS is biphasic: conductive myocytes develop from common progenitors with

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through endothelial-derived signals17. According to this model conductive myocytes are non-

12 working myocytes followed by limited proliferative outgrowth. This conclusion is supported by genetic fate mapping using an inducible Cre allele at the Cx40 locus9. Pre-existing controversies can therefore be reconciled by a sequential model of development according to which “outgrowth” follows “ingrowth” during development of the mammalian VCS (Figure 4B). The recruitment model is based on the arrest of proliferation of differentiating conductive cardiomyocytes32. However, our clonal analysis suggests that conductive cardiomyocytes continue to proliferate, albeit limited to 4-5 cell divisions. This discrepancy may originate in the time of label induction in the mouse and avian experiments. Labeling is restricted to the cardiac tube stage in the chick model whereas it can occur at any stage prior to observation in the nlaacZ experiment. Another discordant point concerns the nature of the progenitor cell from which VCS differentiates, for example, whether it was a

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. The nlaacZ experiment reveals the existence of

common progenitors but does not address their phenotype. These cells are likely to be embryonic myocytes that are distinct from mature contractile and conductive cardiomyocytes. Analyses of mixed nlaacZ labeled clusters provides information as to the populations of progenitor cells in different compartments of the VCS (Figure 5). The percentage of conductive cells per cluster tend to be small in the PF network and large in the AVB, suggesting that progenitors of the central components of the VCS give rise to limited numbers of working myocytes9. This conclusion is consistent with the broad expression pattern of reporter genes from the different mouse models described above15. These data are also consistent with birthdating studies showing that differentiation of the central conduction system precedes that of the peripheral conduction system32. Moreover, this is also consistent with the early specification model recently proposed by Moorman and Christoffels according to which a pool of Tbx2/Tbx3 positive cells maintain an undifferentiated phenotype and low proliferation rates compared to chamber or ventricular myocardium15. In the peripheral

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contractile or conductive myocyte14,

13 conduction system, the nlaacZ clonal analysis revealed a striking difference in the potential of these progenitor cells between right and left ventricles. While the relative number of conductive cells is equivalent in both ventricles, the number of working cardiomyocytes is greater in mixed clusters in the left compared to the right ventricle. Using the Cx40Cre allele to genetically label trabecular myocardium, we observed a more extensive contribution to the left than to the right ventricular myocardium in the same proportion as nlaacZ clusters (Figure 5). These data suggest that PF progenitor cells in both ventricles are Cx40 positive and that the mode of VCS development differs in each ventricle. This difference may underline the origin of these progenitor cell populations, which derive from the first heart field for the left ventricle and second heart field for the right ventricle29. Moreover, the T-box transcription factor, Tbx5, both by its pattern of expression45, 46, and by its role of transcriptional activator

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progenitors. By E16.5, Cx40 expressing cells are restricted to ventricular Purkinje fibers9. The fast conducting phenotype is thus broadly present in the embryonic ventricle prior to establishment of the specialized VCS and the mass of working chamber myocytes. Trabeculated hearts therefore do not require a fully specialized Purkinje fiber network for coordinated contraction. This conclusion has implications for both the ontogeny and evolution of the VCS14.

3- Molecular mechanisms regulating VCS development a. Transcription factors A panel of different transcription factors have been implicated in the differentiation and development of the VCS15,

47, 48

. Concerning central components of the VCS, AVN

development is highly dependent on the transcription factor Nkx2.549. In Nkx2.5 null embryos, the lack of Mink-LacZ expressing cells in the AVC at E9.0 suggests the absence of

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of the Cx40 gene, may be one of the major gene involved in the development of these

14 an AVN primordium. In Nkx2.5 heterozygous adult hearts, the AVN is significantly reduced in size, being comprised only of a core of Cx40+/Cx45+ cells with no Cx40-/Cx45+ cells49. Using the nomenclature of Aanhaanen, this corresponds to the loss of the compact AVN and maintenance of lower nodal cells (LNC)10. Nkx2.5+/- mice display AV conduction defects as shown by a prolonged PR interval on ECG recordings, consistent with absence of the AVN49, 50

. Conditional loss of Nkx2.5 in the ventricles or at perinatal stages induces the development

of a hypoplastic AVN and progressive AV block51, 52. The progression of the phenotype is attributed to the selective degeneration of the AVN after birth52. These data demonstrate that Nkx2.5 plays a role in the maintenance of the AVN in the postnatal heart. In man, mutations in NKX2.5 cause a variety of cardiac anomalies frequently associated with conduction blocks53. Histological examination of postmortem hearts has shown that progressive AV

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tissues52. Several transcription factors have been implicated in the specification, development and maturation of the AVB and BB. Tbx3, encoding a T-box containing transcriptional repressor, plays a role in the specification of the AVB and BB. Tbx3 null embryos die between E12.5 and E15.5 and present cardiac malformations including ventricular septal defect (VSD) with a short and blunted septum54. In the absence of Tbx3, a number of working myocardial markers are expressed at the crest of the septum and Nkx2.5, normally overexpressed in the developing AVB, is present at the same level throughout the septum. These molecular changes are accompanied by the absence of cell cycle exit of cells at the crest of the septum. Tbx3 thus appears to induce specification of AV conduction system progenitors by repressing expression of working myocardial genes. The early death of Tbx3 null embryos has prevented investigation of Tbx3 function in later steps of VCS development, nevertheless heterozygous mice display a normal cardiac phenotype structurally and functionally 54. This is not the case

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block is associated with a degeneration of the AVN and replacement by fibrosis and adipose

15 for two other transcription factors, Nkx2.5 and Tbx5, for which conduction defects are observed when only one copy of the gene is removed46, 49. As mentioned above, a hypoplastic AVB is observed in adult Nkx2.5 heterozygote hearts or ventricular restricted Nkx2.5 knockout mice49,

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. These mice display specific conduction defects in the AVB as shown by

intracardiac electrophysiological recordings. Tbx5 is also expressed in the developing AVB and BB at the crest of the IVS46. In Tbx5 heterozygous mice, the RBB is absent or severely abnormal correlating with AV conduction defects. The AV conduction system develops in a ring structure that is maintained after birth in Tbx5+/- but not wildtype hearts. Tbx5 is thus required for the morphological maturation of the AVB and LBB and essential for patterning and function of the RBB. These observations underscore the link between defective patterning of the developing conduction system and functional abnormalities of the mature conduction

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heart defects and conduction system anomalies including variable degrees of AV block55. Moskowitz and colleagues have shown that Tbx5 and Nkx2.5 cooperate during development of the VCS56. This cooperation is mediated by the expression of Id2, encoding a helix-loophelix transcriptional repressor. In Id2-/- mice, the AVN develops normally but the development of the AVB and BB is impaired, correlating with conduction defects such as long QRS and LBB block. In Tbx5+/-/Nkx2.5+/- compound heterozygotes mice, Id2 is not expressed and cells at the crest of the IVS fail to exit the cell cycle. These data suggest that Id2, like Tbx3, acts through the repression of myogenic genes and promotes VCS differentiation15. However, in Tbx3-/- embryos, the expression of Tbx5 and Id2 are unchanged indicating that these factors are not sufficient to promote VCS differentiation54. Together, it appears that differentiation and maturation of the AVB and BB requires a combinatorial effect of different transcription factors including Tbx3, Tbx5, Id2 and Nkx2.5.

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system. Mutations in human TBX5 cause Holt-Oram syndrome characterised by congenital

16 To date, Nkx2.5 is the only transcription factor implicated in the differentiation and maturation of the PF network. Ventricular restricted inactivation of Nkx2.5 provokes a massive trabecular overgrowth and a prolonged QRS on ECG52. Transcriptional profiling revealed upregulation of BMP10 and conduction specific genes in mutant hearts suggesting a fundamental defect in ventricular cell lineage maturation. In addition, the peripheral PF network is severely hypoplastic in Nkx2.5 haploinsufficient mice49, 57. Using Cx40GFP mice, we have shown that while development of trabeculae in Nkx2.5+/- fetuses is normal, postnatal development of the PF network fails (Figure 6). Furthermore, chimeric analyses demonstrated that Nkx2.5 plays a cell autonomous role during the perinatal period in the normal formation of the PF network57. In addition to these transcription factors involved in the differentiation and maturation of the

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gap junction protein encoding genes without overtly affecting VCS morphoplogy. In Hf1b-/or Hop-/- mice, conduction defects were found to be secondary to disregulation of the gap junction gene Cx40 in the absence of structural abnormalities of the conduction system58, 59. Ventricular restricted knock-out of Hf-1b causes electrophysiological abnormalities including fatal ventricular arrhythmias60. This phenotype is correlated with abnormalities in Cx43 level, myocyte size, activation spread and coronary arterial structure and function. The Iroquois homeobox gene Irx5 is detected in the endocardial myocardium of the ventricles and Irx5-/mice are susceptible to tachyarryhtmias61. This phenotype is associated with the loss of transmural potassium current Ito important for cardiac repolarisation. Two categories of transcription factors can thus be distinguished based on their roles in the cardiac conduction system. Cardiac conduction disturbances may originate from defects in either the function and/or the structure of the conduction system. In the first category, several transcription factors regulate expression of genes encoding ion channel or gap junction

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VCS, several other factors have been implicated in the expression of specific ion channel and

17 proteins. Ion channels are essential for the acquisition of electrophysiological properties of conductive cardiomyocytes and gap junctions for the conduction of electrical activity. The second category contains transcription factors that specify cell lineages destined to become part of the cardiac conduction system. Defects in specification entail abnormal morphological development of the conduction system. Nkx2.5 can be classified in both categories and represents the major transcription factor playing a role in differentiation and development of the VCS, as the development of all compartments of the VCS requires a dose and time dependent expression of Nkx2.5.

b. Extrinsic signaling pathways VCS development also depends on extrinsic parameters which in turn induce the

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cells. As described in section 2, the heart is comprised of diverse cell populations: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. These cells originate from different progenitor populations including the first and second heart fields, NCC and EPDC. As discussed previously, while NCC and EPDC may not contribute directly to the VCS, these cells play a role in VCS development. After NCC ablation in chick embryos, the electrical activation switch from apex to base does not occur but is maintained from base to apex in the IVS62. This phenotype results from the absence of compaction and electrical isolation of conduction system bundles. NCC migrate to the crest of the IVS in close vicinity to the AV conduction system36. NCC restricted deletion of Hf1b led to atrial and atrioventricular dysfunction resulting from deficiencies in the neurotrophin receptor trkC63. Moreover, the inhibition of EPDC in chick embryos disturbs the development of the PF network64. Together, these results suggest that NCC and EPDC are required for normal VCS development65.

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transcriptional pathways directly involved in the differentiation and maturation of conductive

18 Crosstalk between endothelial and myocardial cells plays a major role during cardiac development. Endothelin-1 and neuregulin are two factors secreted by endothelial cells that play important roles in the development of ventricular trabeculae. Endothelin receptor and neuregulin null embryos die early during embryogenesis due to cardiac defects and absence of trabeculae66,

67

. Gourdie et al have demonstrated that an endothelial signal induces

perivascular PF differentiation in chick hearts68,

69

. Avian and murine embryonic

cardiomyocytes express conductive markers when cultured in the presence of ET-1. In contrast, ET-1 does not upregulate the expression of the CCS-LacZ transgene during in vitro culture of embryonic mouse hearts, but neuregulin is able to do so during a restricted time window70. The modification of CCS-lacZ expression under neuregulin treatment is associated with conduction disturbances detected by optical mapping. ET-1 and neuregulin have been

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cardiomyocytes71. Together, these data identify an early role of the endothelium mediated by neuregulin or endothelin in trabeculae formation and consequently in VCS development. ET-1 signaling acts through the activity of the endothelin-converting enzyme ECE-1 which is responsible for cleavage of the precursor Big-ET into active ET-172. ECE-1 is expressed in the heart in close apposition with the developing VCS in both chick and mouse73. Overexpression of ECE-1 in chick hearts induces development of ectopic peripheral PF72. ECE-1 thus plays a key role in defining the time and the location of PF differentiation within the embryonic myocardium. The expression of ECE-1 depends on hemodynamic parameters74. Pressure overload induces the expression of ECE-1 associated with precocious emergence of the mature apex-first activation pattern. Sedmera and collaborators have shown that mechanical loading is required for normal differentiation of the conduction system as well as for acquisition of the apex to base activation pattern underlying the functional HisPurkinje system75,

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. These findings identify hemodynamics as a key epigenetic factor in

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shown to direct the differentiation of embryonic murine cardiomyocytes into conductive

19 development of the cardiac conduction system. However, these studies were all performed in chick with emphasis on periarterial PFs and there is as yet no in vivo evidence that ET signaling is necessary for differentiation of the VCS in the mouse. In addition to the murine and avian models, new insights into the genetic and epigenetic control of conduction system development stem from mutagenesis approaches in the zebrafish. Indeed the power of the zebrafish experimental model is revealed in a recent study using optogenetics to delimit and control the electrical activity of the heart in vivo77. This work opens up the possibility for developmental biologists to investigate the onset of cardiac function more precisely. In addition, analysis of the effect of disruption of cardiac conduction in the absence of contraction in the zebrafish has recently demonstrated that electrical activation per se influences cardiomyocyte architecture and overall cardiac morphogenesis78.

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heart development.

Concluding remarks The VCS is responsible for the synchronization of the heartbeat and physiologists have extensively studied the automatic and rhythmic activity of these specialized cardiomyocytes. In this review, we have seen that, despite important discrepancies between human and mouse cardiac physiology, murine VCS anatomy is close to that of the human heart making the mouse an attractive model for developmental biologists. Indeed murine genetics, including the use of VCS reporter lines, together with experimental biology in avian systems has addressed the cellular origin of the VCS and defined a number of the genes and signaling molecules regulating establishment of the VCS. Moreover, mouse models of congenital heart defects emphasize the role of VCS morphology in cardiac function. These data show that transcription factors play roles in differentiation of conductive cells as well as in the

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Feedback between form and function is thus essential for development of the VCS and normal

20 development and maturation of the network. Convergent genetic and physiological studies in different model systems will further define the relationship between structure and function in the VCS and cardiac rhythm and contribute to new therapeutic approaches for cardiac arrhythmia.

Funding: Supported by the European Community’s FP6 contract Heart Repair (LSHM-C7-2005018630) and FP contract CardioGeNet (Health-2007-B-223463) and the Association Française contre les Myopathies.

Acknowledgements:

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Conflict of interest: none declared.

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We are grateful to Vincent Christoffels for discussion and comments on the manuscript.

21 References: [1]

Anderson RH, Ho SY. The morphology of the cardiac conduction system. Novartis

Found Symp 2003;250:6-17; discussion 18-24, 276-279. [2]

Tawara S. Das Reisleitungssystem des Saugetierherzens. Gustav Fisher 1906;Jena.

[3]

Viragh S, Challice CE. The development of the conduction system in the mouse

embryo heart. II. Histogenesis of the atrioventricular node and bundle. Dev Biol 1977;56:397411. [4]

Anumonwo JM, Tallini YN, Vetter FJ, Jalife J. Action potential characteristics and

arrhythmogenic properties of the cardiac conduction system of the murine heart. Circ Res 2001;89:329-335. [5]

Haissaguerre M, Shah DC, Jais P, Shoda M, Kautzner J, Arentz T et al. Role of

Accepted Manuscript

2002;359:677-678. [6]

Miquerol L, Meysen S, Mangoni M, Bois P, van Rijen HV, Abran P et al.

Architectural and functional asymmetry of the His-Purkinje system of the murine heart. Cardiovasc Res 2004;63:77-86. [7]

Gros D, Dupays L, Alcolea S, Meysen S, Miquerol L, Theveniau-Ruissy M.

Genetically modified mice: tools to decode the functions of connexins in the heart-new models for cardiovascular research. Cardiovasc Res 2004;62:299-308. [8]

Lev M, Thaemert JC. The conduction system of the mouse heart. Acta Anat (Basel)

1973;85:342-352. [9]

Miquerol L, Moreno-Rascon N, Beyer S, Dupays L, Meilhac SM, Buckingham ME et

al. Biphasic development of the mammalian ventricular conduction system. Circ Res 2010;107:153-161.

Downloaded from by guest on January 20, 2017

Purkinje conducting system in triggering of idiopathic ventricular fibrillation. Lancet

22 [10]

Aanhaanen WT, Mommersteeg MT, Norden J, Wakker V, de Gier-de Vries C,

Anderson RH et al. Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart. Circ Res 2010;107:728-736. [11]

Tusscher KH, Panfilov AV. Modelling of the ventricular conduction system. Prog

Biophys Mol Biol 2008;96:152-170. [12]

Bashore TM. Adult congenital heart disease: right ventricular outflow tract lesions.

Circulation 2007;115:1933-1947. [13]

Sedmera D, Reckova M, Bigelow MR, Dealmeida A, Stanley CP, Mikawa T et al.

Developmental transitions in electrical activation patterns in chick embryonic heart. Anat Rec 2004;280A:1001-1009. [14]

Moorman AF, Christoffels VM. Cardiac chamber formation: development, genes, and

Accepted Manuscript

[15]

Christoffels VM, Moorman AF. Development of the cardiac conduction system: why

are some regions of the heart more arrhythmogenic than others? Circ Arrhythm Electrophysiol 2009;2:195-207. [16]

de Jong F, Opthof T, Wilde AA, Janse MJ, Charles R, Lamers WH et al. Persisting

zones of slow impulse conduction in developing chicken hearts. Circ Res 1992;71:240-250. [17]

Gourdie RG, Harris BS, Bond J, Justus C, Hewett KW, O'Brien TX et al.

Development of the cardiac pacemaking and conduction system. Birth Defects Res C Embryo Today 2003;69:46-57. [18]

Moorman AF, Christoffels VM. Development of the cardiac conduction system: a

matter of chamber development. Novartis Found Symp 2003;250:25-34; discussion 34-43, 276-279.

Downloaded from by guest on January 20, 2017

evolution. Physiol Rev 2003;83:1223-1267.

23 [19]

Jongbloed MR, Mahtab EA, Blom NA, Schalij MJ, Gittenberger-de Groot AC.

Development of the cardiac conduction system and the possible relation to predilection sites of arrhythmogenesis. ScientificWorldJournal 2008;8:239-269. [20]

Myers DC, Fishman GI. Toward an understanding of the genetics of murine cardiac

pacemaking and conduction system development. Anat Rec 2004;280A:1018-1021. [21]

Jongbloed MR, Wijffels MC, Schalij MJ, Blom NA, Poelmann RE, van der Laarse A

et al. Development of the right ventricular inflow tract and moderator band: a possible morphological and functional explanation for Mahaim tachycardia. Circ Res 2005;96:776783. [22]

Kupershmidt S, Yang T, Anderson ME, Wessels A, Niswender KD, Magnuson MA et

al. Replacement by homologous recombination of the minK gene with lacZ reveals restriction

Accepted Manuscript

[23]

Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE et al.

Visualization and functional characterization of the developing murine cardiac conduction system. Development 2001;128:1785-1792. [24]

Hoogaars WM, Tessari A, Moorman AF, de Boer PA, Hagoort J, Soufan AT et al. The

transcriptional repressor Tbx3 delineates the developing central conduction system of the heart. Cardiovasc Res 2004;62:489-499. [25]

Viragh S, Challice CE. The development of the conduction system in the mouse

embryo heart. Dev Biol 1982;89:25-40. [26]

Pallante BA, Giovannone S, Fang-Yu L, Zhang J, Liu N, Kang G et al. Contactin-2

expression in the cardiac Purkinje fiber network. Circ Arrhythm Electrophysiol 2010;3:186194.

Downloaded from by guest on January 20, 2017

of minK expression to the mouse cardiac conduction system. Circ Res 1999;84:146-152.

24 [27]

Fukamauchi F, Aihara O, Wang YJ, Akasaka K, Takeda Y, Horie M et al. TAG-1-

deficient mice have marked elevation of adenosine A1 receptors in the hippocampus. Biochem Biophys Res Commun 2001;281:220-226. [28]

Law CO, Kirby RJ, Aghamohammadzadeh S, Furley AJ. The neural adhesion

molecule TAG-1 modulates responses of sensory axons to diffusible guidance signals. Development 2008;135:2361-2371. [29]

Vincent SD, Buckingham ME. How to make a heart: the origin and regulation of

cardiac progenitor cells. Curr Top Dev Biol 2010;90:1-41. [30]

Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two

sources of myocardial cells. Nat Rev Genet 2005;6:826-835. [31]

Carmona R, Guadix JA, Cano E, Ruiz-Villalba A, Portillo-Sanchez V, Perez-Pomares

Accepted Manuscript

Med 2010;14:2066-2072. [32]

Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, Gourdie RG.

Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development 1999;126:5041-5049. [33]

Gourdie RG, Mima T, Thompson RP, Mikawa T. Terminal diversification of the

myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development 1995;121:1423-1431. [34]

Meilhac SM, Kelly RG, Rocancourt D, Eloy-Trinquet S, Nicolas JF, Buckingham ME.

A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart. Development 2003;130:3877-3889. [35]

Kitajima S, Miyagawa-Tomita S, Inoue T, Kanno J, Saga Y. Mesp1-nonexpressing

cells contribute to the ventricular cardiac conduction system. Dev Dyn 2006;235:395-402.

Downloaded from by guest on January 20, 2017

JM et al. The embryonic epicardium: an essential element of cardiac development. J Cell Mol

25 [36]

Nakamura T, Colbert MC, Robbins J. Neural crest cells retain multipotential

characteristics in the developing valves and label the cardiac conduction system. Circ Res 2006;98:1547-1554. [37]

Davis DL, Edwards AV, Juraszek AL, Phelps A, Wessels A, Burch JB. A GATA-6

gene heart-region-specific enhancer provides a novel means to mark and probe a discrete component of the mouse cardiac conduction system. Mech Dev 2001;108:105-119. [38]

Aanhaanen WT, Brons JF, Dominguez JN, Rana MS, Norden J, Airik R et al. The

Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. Circ Res 2009;104:1267-1274. [39]

Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J et al. Isl1 identifies a cardiac

progenitor population that proliferates prior to differentiation and contributes a majority of

Accepted Manuscript

[40]

Verzi MP, McCulley DJ, De Val S, Dodou E, Black BL. The right ventricle, outflow

tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field. Dev Biol 2005;287:134-145. [41]

Beyer S, Kelly RG, Miquerol L. Inducible Cx40-Cre expression in the cardiac

conduction system and arterial endothelial cells. Genesis 2010. [42]

Delorme B, Dahl E, Jarry-Guichard T, Briand JP, Willecke K, Gros D et al.

Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system. Circ Res 1997;81:423-437. [43]

Mikawa T, Hurtado R. Development of the cardiac conduction system. Semin Cell

Dev Biol 2007;18:90-100. [44]

Miquerol L, Kelly RG. Monitoring Clonal Growth in the Developing Ventricle.

Pediatr Cardiol 2009;30:603-608.

Downloaded from by guest on January 20, 2017

cells to the heart. Dev Cell 2003;5:877-889.

26 [45]

Bruneau BG, Logan M, Davis N, Levi T, Tabin CJ, Seidman JG et al. Chamber-

specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev Biol 1999;211:100-108. [46]

Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S et al. The T-

Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development 2004;131:4107-4116. [47]

Harris BS, Jay PY, Rackley MS, Izumo S, O'Brien T X, Gourdie RG. Transcriptional

regulation of cardiac conduction system development: 2004 FASEB cardiac conduction system minimeeting, Washington, DC. Anat Rec 2004;280A:1036-1045. [48]

Hatcher CJ, Basson CT. Specification of the cardiac conduction system by

transcription factors. Circ Res 2009;105:620-630.

Accepted Manuscript

Jay PY, Harris BS, Maguire CT, Buerger A, Wakimoto H, Tanaka M et al. Nkx2-5

mutation causes anatomic hypoplasia of the cardiac conduction system. J Clin Invest 2004;113:1130-1137. [50]

Kasahara H, Wakimoto H, Liu M, Maguire CT, Converso KL, Shioi T et al.

Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein. J Clin Invest 2001;108:189-201. [51]

Briggs LE, Takeda M, Cuadra AE, Wakimoto H, Marks MH, Walker AJ et al.

Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects. Circ Res 2008;103:580-590. [52]

Pashmforoush M, Lu JT, Chen H, Amand TS, Kondo R, Pradervand S et al. Nkx2-5

pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block. Cell 2004;117:373-386.

Downloaded from by guest on January 20, 2017

[49]

27 [53]

Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP et al.

Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 1998;281:108-111. [54]

Bakker ML, Boukens BJ, Mommersteeg MT, Brons JF, Wakker V, Moorman AF et

al. Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system. Circ Res 2008;102:1340-1349. [55]

Basson CT, Huang T, Lin RC, Bachinsky DR, Weremowicz S, Vaglio A et al.

Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc Natl Acad Sci U S A 1999;96:2919-2924. [56]

Moskowitz IP, Kim JB, Moore ML, Wolf CM, Peterson MA, Shendure J et al. A

molecular pathway including Id2, Tbx5, and Nkx2-5 required for cardiac conduction system

Accepted Manuscript

[57]

Meysen S, Marger L, Hewett KW, Jarry-Guichard T, Agarkova I, Chauvin JP et al.

Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system. Dev Biol 2007;303:740-753. [58]

Ismat FA, Zhang M, Kook H, Huang B, Zhou R, Ferrari VA et al. Homeobox protein

Hop functions in the adult cardiac conduction system. Circ Res 2005;96:898-903. [59]

Nguyen-Tran VT, Kubalak SW, Minamisawa S, Fiset C, Wollert KC, Brown AB et al.

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages. Cell 2000;102:671-682. [60]

Hewett KW, Norman LW, Sedmera D, Barker RJ, Justus C, Zhang J et al. Knockout

of the neural and heart expressed gene HF-1b results in apical deficits of ventricular structure and activation. Cardiovasc Res 2005;67:548-560.

Downloaded from by guest on January 20, 2017

development. Cell 2007;129:1365-1376.

28 [61]

Costantini DL, Arruda EP, Agarwal P, Kim KH, Zhu Y, Zhu W et al. The

homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. Cell 2005;123:347-358. [62]

Gurjarpadhye A, Hewett K, Justus C, Wen X, Stadt H, Kirby M et al. Cardiac neural

crest ablation inhibits compaction and electrical function of conduction system bundles. Am J Physiol Heart Circ Physiol 2007;292:H1291-1300. [63]

St-Amand T, Lu J, Zamora M, Gu Y, Stricker J, Hoshijima M et al. Distinct roles of

HF-1b/Sp4 in ventricular and neural crest cells lineages affect cardiac conduction system development. Dev Biol 2006;291:208-217. [64]

Eralp I, Lie-Venema H, Bax N, Wijffels M, Laarse AVD, Deruiter M et al.

Epicardium-derived cells are important for correct development of the Purkinje fibers in the

Accepted Manuscript

[65]

Poelmann RE, Jongbloed MR, Molin DG, Fekkes ML, Wang Z, Fishman GI et al. The

neural crest is contiguous with the cardiac conduction system in the mouse embryo: a role in induction? Anat Embryol (Berl) 2004;208:389-393. [66]

Clouthier DE, Hosoda K, Richardson JA, Williams SC, Yanagisawa H, Kuwaki T et

al. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development 1998;125:813-824. [67]

Meyer D, Birchmeier C. Multiple essential functions of neuregulin in development.

Nature 1995;378:386-390. [68]

Gourdie RG, Wei Y, Kim D, Klatt SC, Mikawa T. Endothelin-induced conversion of

embryonic heart muscle cells into impulse-conducting Purkinje fibers. Proc Natl Acad Sci U S A 1998;95:6815-6818.

Downloaded from by guest on January 20, 2017

avian heart. Anat Rec A Discov Mol Cell Evol Biol 2006;288:1272-1280.

29 [69]

Hyer J, Johansen M, Prasad A, Wessels A, Kirby ML, Gourdie RG et al. Induction of

Purkinje fiber differentiation by coronary arterialization. Proc Natl Acad Sci U S A 1999;96:13214-13218. [70]

Rentschler S, Zander J, Meyers K, France D, Levine R, Porter G et al. Neuregulin-1

promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci U S A 2002;99:10464-10469. [71]

Patel R, Kos L. Endothelin-1 and Neuregulin-1 convert embryonic cardiomyocytes

into cells of the conduction system in the mouse. Dev Dyn 2005;233:20-28. [72]

Takebayashi-Suzuki K, Yanagisawa M, Gourdie RG, Kanzawa N, Mikawa T. In vivo

induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1. Development 2000;127:3523-3532.

Accepted Manuscript

Sedmera D, Harris BS, Grant E, Zhang N, Jourdan J, Kurkova D et al. Cardiac

expression patterns of endothelin-converting enzyme (ECE): implications for conduction system development. Dev Dyn 2008;237:1746-1753. [74]

Hall CE, Hurtado R, Hewett KW, Shulimovich M, Poma CP, Reckova M et al.

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart. Development 2004;131:581-592. [75]

Reckova M, Rosengarten C, deAlmeida A, Stanley CP, Wessels A, Gourdie RG et al.

Hemodynamics is a key epigenetic factor in development of the cardiac conduction system. Circ Res 2003;93:77-85. [76]

Sankova B, Machalek J, Sedmera D. Effects of mechanical loading on early

conduction system differentiation in the chick. Am J Physiol Heart Circ Physiol 2010;298:H1571-1576.

Downloaded from by guest on January 20, 2017

[73]

30 [77]

Arrenberg AB, Stainier DY, Baier H, Huisken J. Optogenetic control of cardiac

function. Science 2010;330:971-974. [78]

Chi NC, Bussen M, Brand-Arzamendi K, Ding C, Olgin JE, Shaw RM et al. Cardiac

conduction is required to preserve cardiac chamber morphology. Proc Natl Acad Sci U S A

Downloaded from by guest on January 20, 2017

Accepted Manuscript

2010;107:14662-14667.

31 Legend to figures Figure 1: Anatomy of the ventricular condution system in mouse and man. A: GFP expression in the left ventricular conduction system of a Cx40GFP mouse heart. AVB: Atrioventricular bundle; LBB: Left bundle branch; LPF: Left Purkinje fibers. B: Drawing of the ventricular conduction system in the human left ventricle by Tawara (1906)2.

Figure 2: Right-left asymmetry of the mouse ventricular conduction system. A-B: 3D reconstruction of the atrioventricular bundle (AVB) of an adult Cx40GFP heart showing a small right bundle branch (RBB) (A) and large left bundle branch (LBB) (B). The IVS is colored yellow. C-E: Right-Left asymmetry of the peripheral conduction system. C: Transverse section of an adult Cx40GFP heart showing the large number of Purkinje fibers

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(D). In contrast, in the left ventricle PFs are mainly present on the septal surface (E) and absent between the two papillary muscles (stars) in the left ventricular wall (E’). Left and right ventricular lumens are colored grey. 3D reconstruction of the right (D-D’) and left (EE’) Purkinje fiber network of a Cx40GFP adult heart. RV: Right ventricle; LV: Left ventricle; RS: Right septum; LS: Left septum.

Figure 3: Comparison of the expression pattern of CCS-LacZ and Cx40GFP VCS reporter lines during heart development. A-B: The expression of CCS-LacZ reproduced with permission from Jongbloed et al. in Circ. Res. (2005)21 (A) and Cx40GFP (B) differs at E10.5. RV: Right ventricle; LV: Left ventricle; AVC: Atrioventricular canal; RA: Right atrium; LA: Left atrium. C-D: Expression patterns of CCS-LacZ reproduced with permission from Rentschler et al. in Development (2001)23 (C) and Cx40GFP (D) converge at neonatal stages (P0). See text

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(PF) on the right ventricular free wall (D’ arrowhead) compared to the right septal surface

32 for details. H: His bundle; LBB: Left bundle branch; RBB: Right bundle branch; LPF: Left Purkinje fibers.

Figure 4: Biphasic development of the ventricular conduction system. A: Retrospective nlaacZ clonal analysis of the ventricular conduction system demonstrating two types of clusters: mixed clusters composed of conductive and working myocytes and unmixed clusters composed of either conductive or working myocytes. B: Biphasic model showing differentiation from a common myogenic progenitor followed by limited proliferation of conductive myocytes.

Figure 5: Model of development of the VCS from two populations of progenitor cells. A: The

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ventricle. This right/left discordance is also observed after Cx40-Cre induction at E10.5, suggesting that VCS progenitor cells are Cx40 positive. By E16.5, Cx40-derived cells give rise only to conductive cardiomyocytes. B-C: At E10.5, the central conduction system derives from AV progenitors (red) that proliferate at low rates and express markers such as Tbx3 or CCS-LacZ. nlaacZ clusters in the central conduction system contain a high percentage of conductive cells per mixed cluster. The peripheral conduction system is derived from a distinct population of Cx40-positive cells that is more extensive in the left than right ventricle. C: At E16.5, the results from Cx40-cre lineage analysis suggest that differentiation step has definitively occurred and that both the central and peripheral VCS are derived from Cx40derived cells.

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percentage of working myocytes in nlaacZ mixed clusters is higher in the left than right

33 Figure 6: Hypoplasia of the Purkinje fiber network in Nkx2.5 haploinsufficient mice. GFP expression of the left VCS show a PF network organized of dense ellipses in Cx40GFP mice while the PF network is reduced and fails to organized in elliptic structures in Nkx2.5+/- mice.

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AVB: Atrioventricular bundle; LBB: Left bundle branch; LPF: Left Purkinje network.

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