Erythropoietin is a protein that stimulates the development of red blood cells, which carry oxygen. The synthetic form of this protein, a drug called Epoietin, or EPO for short, was developed to treat anemia but was widely abused by athletes. The best known case was that of cyclists in the "Tour de France" of 1998. An entire team was suspended from the race when it was discovered that they had used EPO, but despite this the use of EPO in the sport continued.
By H. Lee Sweeney, Scientific American
On the same subject: The dilemma of drugs in sports
Gene therapy to restore muscles, which have degenerated due to aging or disease, is about to enter clinical use and top athletes desire it to improve their achievements.
Won't the day be far away and drugs derived from changes in genes will change the face of sports?
In August 2004, many athletes flocked to Athens to take part in a tradition that began in Greece more than 2,000 years ago. While the world's best athletes were testing the limits of human strength, speed and agility, some were apparently testing a newer, less noble Olympic tradition: performance-enhancing drugs. Despite repeated scandals, many athletes cannot resist the temptation of drugs, if only to not fall behind the competitors who do. As long as winning is paramount, there will always be athletes who will jump at any opportunity to gain a few fractions of a second in speed or a slight improvement in endurance.
Sports authorities fear a new method of drug use that will be impossible to detect, and certainly not prevent. Treatments that regenerate the muscle, strengthen it and protect it from degeneration will soon enter clinical trials in humans, for the treatment of muscle wasting diseases. In some methods, patients receive a synthetic gene, which can last for years and produce large amounts of chemicals that naturally build muscle.
This type of gene therapy could change the lives of the elderly and those with muscular dystrophy. Unfortunately, it's also a dream come true for the doping athlete. The chemicals are only produced locally in the muscle tissue, and there is no difference between them and their natural counterparts. Nothing enters the bloodstream, so the authorities will have nothing to discover in blood and urine tests. The World Anti-Doping Agency (WADA) has already appealed to scientists to find ways to prevent gene therapy from becoming the new means of doping the body (gene doping). But with the entry of these treatments into clinical trials, and eventually into widespread use, it will not be possible to prevent athletes from accessing them.
Is gene therapy about to lay the groundwork for sophisticated fraud in athletics? This is absolutely possible. Will a day come when gene therapy will be so common for the treatment of diseases, that the manipulation of genes to improve achievements will be accepted by all? Maybe. In any case, the world may have watched one of the last Olympics in which genetically enhanced athletes did not participate.
From loss comes profit
The research on improving muscle size and strength did not begin to serve elite athletes. I started my own work by watching my family members, many of whom lived to be more than 80 and 90 years old. Although their health was generally good, their quality of life was poor due to the frailty associated with aging. Muscle mass and strength may decrease by a third between the ages of 30 and 80.
There are three types of muscles in the body: smooth muscles, which surround internal spaces such as the digestive tract, skeletal muscles, which most of us envision when we think of a muscle and the heart muscle. The skeletal muscles together make up the largest organ in the body, and they are the ones that deteriorate with age. Deterioration occurs especially in a certain type of skeletal muscle known as "fast-twitch fibers", which is the strongest type. When the muscle weakens, the tendency to lose balance increases and it is more difficult to stabilize and prevent a fall. If, as a result of a fall, the pelvis is cracked or another serious injury is caused, the person may completely lose his mobility.
Skeletal muscle loss occurs with age in all mammals, and most likely results from a cumulative failure to repair damage caused by normal use. It is interesting to note that there is a similarity between the changes in the skeletal muscles resulting from aging and the physical and functional changes observed in the group of diseases known as muscular dystrophy, although the rate in aging is much lower.
In the most common and severe version of muscular dystrophy, Duchenne muscular dystrophy, an inherited genetic mutation causes the absence of the protein dystrophin, which protects the muscle fibers from the damage caused by the force they exert in their normal movement. In muscular dystrophy, the muscles are good at repairing themselves, but their normal recovery mechanism cannot keep up with the accelerated rate of damage. In aging muscles, the rate of damage may be normal, but the repair mechanisms no longer respond as they did before. Because of this, both in aging and in manure-type muscle atrophy, muscle fibers die and are replaced by infiltrating fibrous tissue and fat.
In contrast, the severe muscle loss experienced by astronauts in subgravity and immobile patients is probably caused by a complete shutdown of the muscle's repair and growth mechanisms, along with an acceleration of apoptosis, the programmed cell death mechanism. This phenomenon, called atrophy of disuse, is still not fully understood, but it makes evolutionary sense. Skeletal muscle is a tissue whose metabolic cost of maintenance is high, and maintaining a close relationship between the size of the muscle and its activity therefore saves energy. Skeletal muscle is incredibly responsive to changes in functional requirements. Just as it depletes due to disuse, so it increases in response to repeated efforts (hypertrophy). The increased load activates several signaling pathways that lead to the addition of new cellular components within the muscle fibers, to changes in the type of fiber, and in extreme situations, also to the addition of new muscle fibers.
Scientists seeking to learn how to influence muscle growth piece together the molecular details of the muscle's natural building and depletion processes. Unlike a typical cell, whose membrane encloses a liquid cytoplasm and a single nucleus, a muscle cell is an elongated cylinder with many nuclei, and its cytoplasm consists of tiny long fibers called myofibrils. The myofibrils are made up of contractile units, arranged side by side, called sarcomeres. The coordinated shortening of the sarcomeres generates the muscle contractions, but the force they exert, if not channeled out, may damage the muscle fibers. The protein dystrophin, which is missing in patients with muscular dystrophy of the dung type, conducts this energy through the membrane of the muscle cell and protects the fiber.
Still, muscle fibers are damaged during normal activity, even with the moderating effect of dystrophin. In fact, physical training probably does, building muscle mass and strength. Microscopic tears in the fiber, which are created due to the effort, activate a chemical alarm that triggers regeneration of the tissues. In muscle, the regeneration is not expressed in the production of new muscle fibers but in the repair of the outer membrane of existing fibers and their growth in new myofibrils. For the production of this new protein, the relevant genes are activated within the muscle cell nuclei, and when the demand for myofibrils is great, additional nuclei are needed to increase the production capacity of the muscle cells.
Local satellite cells residing outside the muscle fibers respond to this call. The muscle-specific Al[EK1] stem cells first multiply through normal cell division, then some of their progeny fuse with the muscle fiber and donate their nuclei to the cell. Both growth-promoting and growth-inhibiting factors are involved in the regulation of this process. Satellite cells respond to the insulin-like growth factor called IGF-I by increasing the number of divisions, while another growth-regulating factor, myostatin, inhibits their proliferation.
Knowing these mechanisms, we began seven years ago, my group at the University of Pennsylvania in collaboration with Nadia Rosenthal and her colleagues at Harvard University, to investigate the possibility of using IGF-I to change muscle function. We knew that if you inject IGF-I protein alone, it dissipates within a few hours. But if we insert a gene into a cell, the gene will continue to function as long as the cell lives, and muscle fibers live for a very long time. A single dose of the IFG-I gene given to an elderly person may last for the rest of their life. We therefore set out to find a way to inject the IGF-I gene directly into the muscle tissue.
wearing new genes
Then as now, a major obstacle on the way to successful gene healing was the difficulty of inserting a selected gene into the desired tissue. Like many other researchers, we chose a virus as a means of introduction, called a vector, because viruses are adept at sneaking genes into cells. They survive and spread by tricks that trick the host cells into letting them in, like a biological Trojan horse. A virus that enters the nucleus of a host cell, uses the cell's mechanisms to copy its genes and produce proteins. The gene cure takes advantage of this ability by implanting a synthetic gene in the virus and removing any gene that the virus might use to cause disease or to copy itself. We chose a tiny virus called AAV (adeno-associated virus) as our vector, partly because it easily infects human muscles, but is not known to cause any disease.
We modified it using a synthetic gene that produces IGF-I only in skeletal muscle, and started trying it in normal mice. After we injected the processed virus (AAV-IGF-I) into young mice, we saw that the overall size of the muscles and their growth rate were 15 to 30 percent higher than normal, even though the mice were inactive. Moreover, when we injected the gene into adult mice and let them reach old age, their muscles did not weaken.
To supplement and test the approach and its degree of safety, Rosenthal developed genetically modified mice whose production of IGF-I in their skeletal muscles is increased. Happily, their development was normal except that their skeletal muscles were 20 to 50 percent larger than those of normal mice. As the transgenic mice aged, their muscles retained the regenerative capacity characteristic of younger animals. And just as important, their IGF-I levels were high only in the muscles, not in the blood; High levels of IGF-I in the blood can cause heart problems and increase the risk of cancer. Further experiments showed that overproduction of IGF-I accelerates muscle repair, even in mice with a particularly severe form of muscular dystrophy.
Increasing the local production of IGF-I allows us to achieve one of the main goals of gene therapy in its fight against muscle degenerative diseases: to break the close connection between the use of the muscle and its size. Such an imitation of the results of muscle exercises is a magician, of course, for top athletes. The rate of muscle growth in young, inactive animals suggests that the treatment also has the power to improve genetic enhancement of the ability of a healthy muscle. Recently, my lab worked with a group of exercise physiology researchers led by Dr. P. Ferrar from the University of Texas at Austin to test this theory.
We injected AAV-IGF-I into a muscle in just one leg of each of our lab rats, then put the animals through eight weeks of a weight-bearing muscle development protocol. By the end of the training period, the AAV-IGF-I-injected muscles had strengthened almost twice as much as the non-injected leg muscles in the same animals. After the training was stopped, the injected muscles lost their strength much more slowly than the non-enhanced muscles. Even in inactive rats, AAV-IGF-I resulted in a 15 percent increase in muscle strength, similar to what we saw in the previous experiments in mice.
We intend to continue researching IGF-I gene therapy in dogs, because Golden Retriever dogs tend to develop a particularly severe form of muscular dystrophy. At the same time, we will also conduct the study in healthy dogs to add and test the effect and safety of inducing IGF-I overproduction. It is a powerful growth and signaling factor that tumors also respond to.
Considering the safety issue and the fact that the best way to introduce AAV into humans has not yet been decided, whether through the bloodstream or by direct injection into the muscle, it seems that it will take at least another ten years until IGF-I gene therapy treatment is approved. In the short term, experiments to insert genes to replace the dystrophin gene in humans are already in the planning stages, and the Muscular Dystrophy Association will soon begin a clinical trial of injecting IGF-I to treat myotonic dystrophy, a condition of continuous muscle contraction that destroys the muscle.
An even more immediate approach to encouraging muscle hypertrophy (growth) may come from drugs to block the growth inhibitor myostatin. It is still not clear exactly how inhibiting myostatin builds muscle, but the protein probably limits muscle growth during embryonic development and adult life. It acts as a brake on the normal growth of the muscle and possibly as an atrophy promoter when the functional requirements of the muscle decrease. Experiments with genetically modified mice show that when this anti-growth factor is absent, the muscles grow considerably both due to hypertrophy of the existing muscle fibers and due to hyperplasia, which is an increase in the number of muscle fibers.
make muscle and more
Pharmaceutical and biotechnology companies are working on a variety of myostatin inhibitors. At first, the possibility of producing meatier animals sparked commercial interest. Nature has already provided examples of the effect of blocking myostatin in the "Belgian Blue" and "Piedmont" cattle breeds, both of which have an inherited genetic mutation that produces a truncated and ineffective version of myostatin. The appearance of these muscular beasts is even more impressive, as the absence of myostatin also impairs fat layering, giving them a sculpted, fat-free appearance.
The first drugs based on blocking myostatin are antibodies to myostatin, and one of these drugs may soon enter clinical trials in muscular dystrophy patients. A different approach mimics the cattle mutation by creating a shorter version of myostatin, which lacks the signaling ability of a normal myostatin molecule but retains the structures that allow it to bind to nearby satellite cells. The reduced protein, or peptide, actually blocks the anchoring sites and prevents normal myostatin molecules from binding to them. Injecting the peptide into mice causes the skeletal muscles to grow, and my colleague and I will try to create a similar effect in dogs by introducing a synthetic gene for the production of the peptide.
Even healthy people who desire rapid muscle development are interested in blocking myostatin. Although systemic drugs do not target a specific muscle like gene insertion, the advantage of the drugs is the ease of taking them, which means that you can immediately stop using them if a problem arises. However, sports authorities will be able to detect such drugs relatively easily in blood tests.
But what if athletes used gene therapy in an approach similar to our AAV-IGF-I strategy? The gene product will only be found in muscle, not in blood or urine, and will be identical to its natural counterpart. Only a biopsy of the muscle can test the presence of a specific synthetic gene or vector. But in the case of AAV, it is possible that many people are naturally infected with this innocent virus, so the test will not conclusively prove drug use. Furthermore, since most athletes will refuse to undergo an invasive biopsy before competition, this type of genetic enhancement will remain hidden for all intents and purposes.
And what about the safety of rapidly increasing muscle mass by 20 to 40 percent? Is an athlete exercising his genetically enlarged muscles likely to exert a force strong enough to crack his bones or tendons? Apparently not, we are more concerned about building muscle in the elderly whose bones have weakened due to osteoporosis. In a young, healthy person, muscle growth over weeks or months will give skeletal support factors time to grow to meet the new demands.
This safety question is just one of the many questions that we must continue to investigate in animals before we can even consider using such treatments on healthy people just for the purpose of improving their performance. However, with gene healing about to become a routine medical treatment, it is clear that the use of gene doping will not be far behind, and muscle augmentation is just one of those uses. In sports such as sprinting, there will be a demand for gene manipulation to convert normal muscle fibers into fast-twitch fibers. For marathon runners, the most important thing is to increase endurance.
Muscle tissue will most likely be the first to undergo genetic improvements, but others may follow. For example, endurance is affected by the amount of oxygen reaching the muscles. Erythropoietin is a protein that stimulates the development of red blood cells, which carry oxygen. The synthetic form of this protein, a drug called Epoietin, or EPO for short, was developed to treat anemia but was widely abused by athletes. The best known case was that of cyclists in the "Tour de France" of 1998. An entire team was suspended from the race when it was discovered that they had used EPO, but despite this the use of EPO in the sport continued.
Inserting genes to increase the production of erythropoietin has already been tried in animals, and the results clearly demonstrate the dangers inherent in trying to apply such treatments to humans prematurely. In 1997 and 1998, scientists tested synthetic genes for erythropoietin on monkeys and beavers. In both experiments, the animals' red blood cell counts nearly doubled within 10 weeks, and their blood became so thick that it had to be regularly thinned to prevent heart failure.
The technology necessary for the unethical use of gene insertion is certainly not within the reach of the average athlete. But the sports community fears that, just as "steroids to order" are now manufactured and sold, the day will not be far away when genetic improvements will also be offered for sale on the free market. The use of these substances will be much more difficult to monitor than to monitor drugs, because it is difficult to detect their presence in the body.
There is also a good chance that in the coming decades some of these protective treatments will be proven safe for use and offered to the general public. If a day comes and genetic enhancement methods are widely used to improve the quality of life, society's ethical position regarding gene manipulation will probably be very different than it is today. Sports authorities already recognize that muscle rehabilitation therapies may help in recovery from sports injuries.
Will we one day find ourselves engineering a super athlete, or improving the health of the entire population through the insertion of genes? Even in its infancy, this technology has tremendous potential to change both the face of sports and the face of our society. The ethical issues involved in genetic enhancement are many and complex. But this time at least we have time to discuss and debate them before we have the ability to use this power.
[EK1] The comment may be true, but we want to be clear first, the term stem cells has penetrated the academic world as well. And for reference, this issue has a whole article on the subject, and the translator, a biologist by training, and the scientific editor, also a biologist, chose this term. This is what Prof. Michel Revel did in his lecture at Hamda - Eitan
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Overview \ Molecular muscle building
The control of muscle growth and repair is done by chemical signals, and these, in turn, are controlled by genes. Stimulating or blocking these signals using a synthetic gene makes it possible to regenerate muscles lost due to old age or disease.
Athletes can use the same technique to increase their muscles and strength, and the traces of the treatment will not be detected.
When gene therapy becomes a routine medical treatment, it will be very difficult to prevent its misuse. However, the attitudes regarding genetic improvement of the body may also change.
The body's powerhouse
Skeletal muscles make up more than a third of the body mass of an average healthy thirty-year-old person. These muscle cells are different from the rest of the body cells. The muscle cells are actually long cylindrical fibers, up to 30 cm long, containing many nuclei. Bundles of smaller fibers within each muscle cell contract and thus provide the stable support we need to sit up straight in the movie theater or the burst of power needed to jump off the line and run a mile in 2.5 minutes.
In order to meet the constant and changing demands of all kinds, the muscles have different types of fibers adapted to continuous efforts or quick bursts of power, as well as cellular structures that prevent the force of the contractions of the fibers from damaging the fibers themselves.
Bundles of muscle fibers, self-bundled into bundles, surrounded by connective tissue and fat (left). There are two main types of muscle fibers. The dark, "slow" fibers burn energy more slowly; These tire less but also react less when a burst of power is needed. The light, "fast" fibers are faster and stronger, but certain subtypes of them tire easily. The fibers can adapt to changing strength and endurance needs by changing their type.
Myofibrils fill each muscle cell. Each myofibril is made of units called sarcomeres (below), arranged side by side. A sarcomere is a lattice of the proteins actin and myosin (above). These protein fibers slide past each other to contract the sarcomere. As a result of the coordinated contractions of the sarcomeres, the entire muscle fiber contracts.
The force exerted by the contracting sarcomeres is channeled out of the fibers by proteins that cross the cell membrane and connect to the tissue of the extracellular material. Among these, dystrophin also acts as a shock absorber and protects the cell from damage.
About the author
H. Lee Sweeney is Professor and Head of the Department of Physiology at the University of Pennsylvania School of Medicine. He is a member of the scientific advisory board of the National Institute of Arthritis and Musculoskeletal Diseases, scientific director of the parents' project to fight muscular dystrophy and a member of the protein translation research advisory board of the Muscular Dystrophy Association. His work encompasses both basic research of structures that allow cells to move and generate force, in particular the family of molecular motors of the myosin protein, and the translation of insights into the structure and behavior of muscle cells for treatment through gene therapy in various diseases, including muscular dystrophy. He participated in the 2002 Symposium on the Future of Gene Therapy organized by the World Agency on Drug Abuse.
A natural advantage
In June 2004, the New England Journal of Medicine published the first documented description of a person carrying a genetic mutation that prevents the production of myostatin. Such cases were discussed in scientific circles, but were never published because the people and their families did not want to risk exposure. Rumor has it that a European champion in weightlifting is a member of one of these families, which would not be surprising if it turned out to be true, considering the huge advantage that a natural myostatin-blocking mutation gives when it comes to muscle development and strengthening.
But would this be an unfair advantage for the athlete, and would this justify other competitors using myostatin inhibitor drugs or simple gene therapy to put everyone on the same starting line? These questions will no doubt be raised in the ongoing debate about the possibility of athletes using new genetic therapies to improve their performance.
Natural "mutants" among athletes have been documented in the past, including an Olympic gold medal winner. The Finnish ski sprinter, Aro Montirante, won two gold medals at the 1964 Winter Olympics. Only a few decades later, Finnish scientists located a genetic mutation in Montirante's entire family, a mutation that causes an overreaction to erythropoietin, as a result of which the number of red blood cells, which carry oxygen, greatly increases. It turned out that many of Montirante's family members were also champions in sports that require high endurance.
Besides mutations that have a dramatic effect, researchers have also begun to discover different versions of natural genes that confer an advantage in different types of sports, although in a less pronounced way. For example, last year Australian researchers tested a gene called ACTN3 in a group of elite runners of both sexes. Almost 20% of humans lack a functional version of this gene, which produces a protein specific to fast-twitch muscle fibers. Usually, another, less effective protein compensates for its lack. The scientists found an abnormally high prevalence of the active gene ACTN3 among male and female runners. In particular, among the female runners, two copies of the gene were found at a higher frequency than expected if the group had been selected at random.
Many research groups are trying to identify variants of other genes that give athletes an advantage by increasing oxygen uptake, cardiac efficiency, power output, endurance and other traits. So far, more than 90 genes or chromosomal sites have been linked to athletic performance, and this research is already raising its ethical controversies. Critics fear that children will be recruited into certain sports based on their genetic makeup or, if not born with the right gene mix, will not be given any opportunity to reach the highest level of sports training. Some even predicted selective hybridization to produce a super-athlete.
A more certain result of scanning the genomes of athletes will be the discovery that some of them, like Montirante's, contain genetic mutations that actually equate to genetic enhancement. Such discoveries will add even greater complexity to ethical debates about the future of doping in sports.
And more on the subject
Viral Mediated Expression of Insulin-like Growth Factor I Blocks the Aging-Related Loss of Skeletal Muscle Function. Elisabeth R. Barton-Davis et al. in Proceedings of the National Academy of Sciences USA, Vol. 95, no. 26, pages 15,603–15,607; December 22, 1998.
Muscle, Genes and Athletic Performance. Jasper L. Anderson, Peter Schjerling and Bengt Saltin in Scientific American, Vol. 283, no. 3, pages 48–55; September 2000.
Towards Molecular Talent Scouting. Gary Taubes in Scientific American Presents: Building the Elite Athlete, Vol. 11, no. 3, pages 26–31; Fall 2000.
Viral Expression of Insulin-like Growth Factor-I Enhances Muscle Hypertrophy in Resistance-Trained Rats. Sukho Lee et al. in Journal of Applied Physiology, Vol. 96, no. 3, pages 1097–1104; March 2004.
Epoetin Wikipedia
This article originally appeared in Scientific American in May 2004
