N. Bachl, MD Prof. Center of Sports Science University of Vienna Department of Sports- and Exercise Physiology

Tempus Sport, final event 01.12. 2011 Tirana

• 15 billion euros are spent by illegal trade with doping worldwide. The main winners: producers and dealers. • More than 15,5 millions are consuming regularly doping substances. 70 percent of them are bodybuilder and hobby sportsmen. • Over six trade ways 60 percent of doping substances are dealed. Big Player in the doping-business: the Russian mafia and the Olymic host country China. 32/ 08 Format

For each individual, there is a limit to the capacity to perform exercise. The limitation, however, depends on the nature of the task and is also influenced by a number of other factors. Maughan 2005

LIMITS OF SPORTS PERFORMANCE

To become an Olympic athlete choose your parents well! Per-Olaf Astrand „or made a gene-scan“

IT IS MORE IMPORTANT PARTICIPATIN G THAN WINNING

„or try to manipulate your genes ..“

AND TRAIN WHAT EVER YOUR ORGANISM WILL TOLERATE.

Gene Doping Direct approach: • gene therapy in vivo and in vitro • germ line gene therapy Indirect approach: • manipulation of the gene-expression • Stemcell-therapy + manipulation of the gene-expression

Gerlinger u.a.: Gendoping, 2008

Gene therapy Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development.

Types of gene therapy Gene therapy may be classified into the two following types: Germ line gene therapy In the case of germ line gene therapy, Germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are integrated into their genomes. Somatic gene therapy In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient.

Animal Studies: It’s All in the Genes Walsh et al. J. Appl. P hysiol. 2006

(meters run to exhaustion)

Intrinsic edurance capacity

1200

High-capacity running

900 800 700

347%

600

171%

500

Low-capacity running

400 300 200

1

2

3

4

5

6

Generation of selective breeding

11

Heritability of these phenotypes is generally low (about 25 % or less) and rarely exceeds 50 %.

Individuals with the same genotype respond more similarly to training than those with different genotypes. The search for genetic markers of trainability status will likely be more productive than the investigation of molecular markers of the performance phenotype in the untrained state. C.Bouchard et al, 1997

ACTN3 is the first structural skeletal-muscle gene for which an association concerning speed/strength or endurance has been demonstrated. Thus, α-actinin-3 may promote the formation of fast-twitch fibers or alter glucose metabolism in response to training. In addition, α-actinin-3 may be evolutionarily optimized for the minimization of damage caused by eccentric muscle contraction.

ACTN3 GENOTYPES FREQUENCY

N. Yang, 2003

Scoring the genotypes Assigning a genotype score of 2 for the preferable (optimal) genotype and of 0 for the less optimal genotype Summing up the genotype scores Transforming to a scale of 0-100  total genotype score (TGS) • None of the studied world class athletes hat the optimal genotype score. • One top three finischer of the Tour de France had only 3 endurance related genotypes. Ruiz, JR., et al., J Physiol 2009

Populationsgröße zur Auffindung eines “optimalen” Ausdauerathleten

Williams et al., J Physiol 586.1 (2008)

MANIPULATION OF GENE-EXPRESSION Control of the RNA processing (splicing etc.) Gene therapy with Neutralisation of RNA with viruses

si/antisense RNA Manipulation of the RNA-stability

Control of the DNA-structure (histones, DNAmethylisation etc.

Manipulation of the stability and activity of transcription factors Control of the transcription Gene therapy with plasmides

Gene Therapies that could be abused (IOC-MC)

- Systemic protein: EPO, Growth Hormone, Growth Factor - Wound or injury healing: PDGF, KGF, bone repair factors - Increase muscle mass: angiogenic factors to skeletal and heart muscle, MGF, GDF’s Myostatin; - Blood vessel growth: FGF-1, 2, 4 or 5; vascular endothelial growth factors, VEGF

Gene Therapies that could be abused

- Pain relief: (endorphins, enkephalins, other analgesic peptides) - Neurological: hormones/growth factors Pituitary/hypothalamic i.e. GHRF, cognition/memory enhancers; mood altering

Polymorphism and Muscle • Myostatin (Growth differentiation factor 8 - gdf8) – inhibitor of muscle hypertrophy – many knock-out and knock-in animal-studies are available – in humans, 6 different polymorphisms have been described (Seibert et al, J Am Geriatr Soc 49, 2001) – the R153 polymorphism is reported to affect muscle phenotypes (Ferrell et al, Genomics 62, 1999) – Strength training is associated with decreased myostatin levels in both old and young (Roth et al, Exp Biol Med 228, 2003)

Myostatin- Blockade Genetic approach: • Forbidden as germ-line – gene-therapy • Inhibiting RNA • Enzymatic blockade of the proteins produced after translation. Non genetic approach: • Blockade of the myostatin receptor with competing proteins • Blockade of myostatin with antibodies

Approaches for Gene-Doping: 

Myostatin-Gene * If this gene is switched off in mice (knock out mices) –> abnormal muscle hypertrophy

T.Schultz et al, F.I.T. 1/98

T. Hertrampf et al, FIT 1/2004

Polymorphism and Muscle – inhibitor of muscle hypertrophy – Myostatin deficiency can also occur in humans naturally as seen in a German boy

– 2008/ 4,5 y – 2-3 kg Barbels – M. quadriceps Cross sectional area 6,72 cm (double from normal)

Geoff Goldspink Royal Free & University College Medical School, London, UK

MGF and the regulation of muscle strength.

Alternative Splicing of the Human IGF-I Gene 3

Promoter 1

6

IGF – IEa (systemic) 5 4 3

Hormones effect on the liver 1

4

2

4 - IEb 5 IGF

3

6

Promoter 2

Human IGF–I gene Mechanical signals including local cell damage

3

4

MGF (autocrine) reading frame shift

5

6

* 49 base insert

Mechanical Growth Factor – (MGF) This growth factor appears to be involved in protecting heart as well as skeletal muscle by inducing local repair (increased damage limitation) and preventing apoptosis. * Increasing muscle mass and strength adaptation * There is also evidence that MGF involved in maintaining nervous tissue, as IGF-1 is known to be transported within neurons.

Approaches for Gene-Doping: Mechanical Growth Factor – (MGF) • When liver type IGF-1 is introduced into muscles with a similar approach, the increase of muscle mass is also 20 %, but only after 4 months! • If MGF is placed in an engineered gene and injected into muscles of a laboratory mouse: 20 % increase in muscle mass in 3 weeks.

The effect of treating the muscles of mdx dystrophic mice with MGF in a plasmid vector after only 3 weeks on muscle strength

Percentage of treated muscle tetanic force compared to their contralateral untreated muscle (%)

G. Goldspink

40

***

35 30 25 20 15 10 5 0 -5 -10 MGF

G. Goldspink, 2005

Vector

PPARGC1 – peroxisome proliferative activated receptor gene coactivator1 The protein encoded by the gene PPAR-gamma is a regulator of adipocyte differentiation. Additionally, PPAR-gamma has been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer. Multiple transcript variants that use alternate promoters and splicing have been identified for this gene. At least three of these variants encode the same isoform. The PPARGC1 gene is preferentially expressed in ST fibers, and its activation leads to mitochondrial biogenesis, up-regulation of mitochondrial enzymes involved in fatty acid metabolism (assimilation of lipids via anabolic pathways) and electron transport, and reduced susceptibility to fatigue during repetitive contractions PPARG

PPARG

chromosome: 3; Location: 3p25

Ferre P. (2004), Diabetes 53(1); 42-50

AICAR (Exercise in a pill)

Gene insertion Transgenic overexpression

GW1516 



GW1516, in oral application, which stimulates activation of PPARd in skeletal muscle, sedentary mice showed no benefit by just applying GW1516,

• only in combination with exercise an improvement of running time and distance by 68% and 70% respectively occurred compared to the exercising cohort,

• combined GW1516 treatment and exercise established a unique gene expression pattern that was neither found in individual interventions.

AICAR-AMPK Agonist • Application: – oral – 500mg/kg/day – daily over 4 weeks • Result: – inactive mice fed with AICAR ran 44% farther on a treadmill than the exercisetrained-mice – AMP-mimetic AICAR can increase endurance in sedentary mice by genetically reprogramming muscle metabolism in a PPARd-dependent manner. – establishment of a unique muscle endurance gene signature

Hypoxia Inducible Factor • HIF mediates protective responses to hypoxia HIF is found in virtually all cells and is normally inactive. Under conditions of hypoxia, however, HIF moves to the nucleus of the cell where it functions as a transcription factor, causing the expression (i.e., "turning on") of certain genes. The expression of HIF-mediated genes results in the production of proteins that play key roles in bringing about compensatory changes to oxygen deprivation on both cellular and physiological levels.

HIF – Prolyl Hydroxylases •

Using different HIF-PH inhibitors, FibroGen seeks to selectively harness and direct HIF-mediated protective mechanisms, such as erythropoiesis and cytoprotection, for the treatment of anemia and conditions associated with tissue damage or injury, respectively. In addition, FibroGen believes that the ability to pharmacologically control certain aspects of metabolism mediated by HIF will prove useful in treating metabolic disorders, such as obesity, and that directing certain aspects of HIFmediated vascular biology will find application in wound healing.

•

Guided by this new insight on HIF-PH and possessing a diverse collection of PH inhibitors, FibroGen was uniquely positioned to advance HIF Stabilization technology and soon demonstrated proof-of-concept in preclinical models of anemia and tissue injury. FG-2216 is the Company's lead erythropoietic agent in clinical development for the treatment of anemia (300fold increase in EPO-plasma concentration in primates).

S107 What Slows Down Marathoners May Also Tire Heart Failure Patients

Membrane excitation small amount of calcium enters the cell (red dots), binds to the RyR release channel and calcium is released (dots in the bubble stream out) 

muscle contraction occurs

Calcium is pumped back into the sarcoplasmatic reticulum, which causes muscle relaxation

This cycle repeats every time a muscle contracts.

S107 What Slows Down Marathoners May Also Tire Heart Failure Patients

Depletion of calstabin-1 (stabilizing subunit of RyR1 channels) cause “leaky” RyR1 channels (major CA2+ release channel), which leads to defective Ca2+ signaling, muscle damage, and impaired exercise capacity in mice and humans.

If this channel is leaking, it can cause severe problems, for example: – heart failure – defects in muscle function

S107 What Slows Down Marathoners May Also Tire Heart Failure Patients To assess whether remodeling of the RyR1-channel-macromolecularcomplex observed in exercised mice occurs in humans, thigh muscle biopsies were obtained from trained athletes before and after near-maximum aerobic exercise. The results suggest that similar remodeling of the RyR1 channel complex occurs in humans. The leak was present in the muscle of mice after an intense three-week daily swimming regimen and in human athletes after three days of daily intense cycling. The same leak was previously discovered by Marks and colleagues in the muscles of animals with heart failure.

With S107, the mice were still energetic, had lost less exercise capacity after 3 weeks, and their muscles showed fewer signs of calcium leakage, atrophy, and less muscle damage and showed up to 30% less muscle fatigue.

Bellinger et al: Remodeling of ryanodine receptor complex causes ‘‘leaky’’ channels: A molecular mechanism for decreased exercise capacity. PNAS 2008; 105: 2198-2202 Bellinger et al. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle; Nature Medicine 2009

Doping potential

Substance S107 Sport any sport with increased muscle performance Prohibited not yet Used probably Detection easy – non natural compound Application osmotic pump (mice)

PEPCK

PEPCK – Phosphoenolpyruvate caboxykinase •PEPCK-C has a key role in glucose and lipid metabolism •usually found in liver, adipose tissue and kidneys –liver: gluconeogenesis –adipose tissue: generation of triglycerides •for examining the role in muscle Hanson and Hakimi (2008) created transgenic PEPCK-C mice overexpressing the enzyme

PEPCK – Phosphoenolpyruvate caboxykinase Transgenic mice overexpressing PEPCK-C –ate 60% more than control, but –had half the body weight and –10% the body fat of controls –extended life span relative to controls –increased VO2max –increased running distance –old mice ran twice as fast as young controls

Conclusion: overexpression of PEPCK-C repatterns energy metabolism and leads to greater longevity (Hakimi et al, J Biol Chem 45, 2008)

PEPCK – Phosphoenolpyruvate caboxykinase “Mighty Mice”: "At the beginning of exercise, the concentration of lactate was similar in two groups of mice, but by the end of the exercise period, the –control group had elevated levels of blood lactate with – little change in the levels in the PEPCK-Cmus mice.“

Case Western Reserve researchers breed a “Lance Armstrong” mouse “They are metabolically similar to Lance Arnstrong biking up the Pyrenees: They utilize mainly fatty acids for energy and produce very little lactic acid (Richard W. Hanson, Journal of Biological Chemistry, 2007). "Armstrong's most unusual attribute may be his low lactate levels. During intense training, the levels of –most racers range from 12 μL/kg to as much as 20 μL/kg; –Armstrong is below 6 μL/kg." Lactate is believed to be a useful fuel for the body.

Doping potential

Substance PEPCK S107 Sport any sport with increased muscle performance endurance sports Prohibited not yet Used probably ?? Detection easy – non natural compound Application osmotic pump (mice) PEPCK mice had been created by introducing the cDNA for the enzyme into their germ line. “One thing is for sure, if Mother Nature wanted 9 units/g of PEPCK-C in our skeletal muscle, instead of 0.08 units/g, she would have put it there!”, so the authors and creators of the PEPCK-mice thinking that doping might not play a role yet.

SCN9A – No Pain, No Gain The gene SCN9A encodes for a subunit of the voltage-gated sodium channel NaV1.7, which is strongly expressed in nociceptive neurons. A mutation in this gene causes loss of function of the channel, which leads to the inability to sense pain.

Cox J., Reinmann F. An SCN9A channelopathy causes congenital inability to experience pain. Nature 2006

SCN9A – No Pain, No Gain These findings stimulate the search for novel analgesics selective targeting of the sodium channel subunit less side effects than current pain medications Developing such a new generation of drugs that can reduce the pain intensity also opens the gate for misuse and a new generation of pain killers, which are popular in any kind of sports.

Waxman SG (December 2006). "Neurobiology: a channel sets the gain on pain". Nature 444 (7121): 831–2. Dib-Hajj SD, Cummins TR, Black JA, Waxman SG (November 2007). "From genes to pain: Nav1.7 and human pain disorders". Trends in Neurosciences 30 (11): 555–63.

SCN9A – No Pain, No Gain Some studies already deal with different compounds for blocking the NaV1.7 channel.

So did for example Desaphy et al (2009): „Low, clinically relevant orphenadrine concentration produces a significant block of Nav1.7 channels, which are critical for experiencing pain...“ Desaphy JF et al (2009). Involvement of voltage-gated sodium channels blockade in the analgesic effects of orphenadrine. Pain;142(3):225-35.

Like the Lords of Genesis?! Gene technology and ist consequences are the challenge of the 21st century. How we will deal with this challenge will be more important than allour measure against NUCLEAR POWER, AIDS and XENOPHOBIA together. Michael Weight.