In 1997, scientists McFerron and Lee uncovered a “secret” to the public of an anomaly that breeders had benefited from since the late 1800s: a gene responsible for the development of large, meaty cows. (1) Over the course of a century ago, European herders noticed that some of their cattle were more muscular than others. As genetics buffs, they began to selectively cross these robes in order to increase the lineage with this trait. Thus, two breeds of cattle (Blu Belga and Piemontese) have been developed that generally show an increase in muscle mass compared to other breeds. These breeders never imagined that Mighty Mouse would become more than just a cartoon after many years.
A group of scientists led by McFerron and Lee from Johns Hopkins University studied a group of proteins that regulate cell growth and differentiation. During their research, they discovered a gene that may be responsible for the muscle gain phenomenon, also called “double muscle”. (1) (2) . Myostatin, a protein encoded by a gene, is part of a superfamily of related molecules called beta transforming growth factors (TGF-b). It is also called growth and differentiation factor 8 (GDF-8). By blocking the myostatin gene in guinea pigs, the researchers were able to show that transgenic guinea pigs develop two to three times more muscle mass than guinea pigs with the same intact gene. Lee claimed that the guinea pigs with the blocked myostatin gene “were guinea pigs like Schwarzenegger.” (3)
Further studies of the genes present in the skeletal muscle of two cattle breeds revealed mutations in the gene encoding myostatin. The dual muscle trait of myostatin gene-blocked guinea pigs and dual-muscle cattle demonstrates that myostatin performs the same biological function in the two species. Myostatin appears to inhibit skeletal muscle growth factor. Gene blocking in transgenic guinea pigs or gene mutations such as in double-muscled livestock leads to increased muscle mass. This discovery paved the way for a myriad of futuristic implications, from the creation of supermuscular livestock to the treatment of muscle-wasting ailments in humans.
Researchers are developing methods that influence the expression and function of myostatin and its gene to produce livestock with more muscle mass and less fat. Myostatin inhibitors can be developed to treat muscle wasting diseases such as muscular dystrophy in humans. However, many media outlets immediately raised the issue of the abuse of myostatin inhibitors by athletes. In addition, it has been hypothesized that a genetic propensity for high levels of myostatin is responsible for the lack of muscle gain in some athletes who train with resistance. Therefore, this article provides an overview of the science of myostatin and its role in sport.
What is myostatin
Before we can understand the consequences of interfering with myostatin and its genome, we need to understand what myostatin is and what it does. Upper organisms are composed of many different types of cells, the growth, development and functions of which must be coordinated for the proper functioning of individual tissues and the whole organism. This is possible due to specific intercellular signals that control tissue growth, development and function. These molecular signals stimulate a cascade of events in cells called cellular signaling that triggers a final response in or through the cell.
Classic hormones are widespread signaling molecules (called endocrines). These substances are produced and secreted by cells or tissues and enter the bloodstream through blood and other body fluids to affect the activity of other cells or other tissues in the body. However, growth factors are usually synthesized by cells and affect the cellular functions of the same cell (autocrine) or another neighboring cell (paracrini). These molecules are determinants of differentiation, growth, motility, and cellular gene expression, as well as how a group of cells acts as a tissue or organ.
Growth factors (GFs) are usually effective at very low concentrations and have a strong affinity for their corresponding receptors on affected cells. For each type of GF, there is a specific receptor in the cell membrane or in the nucleus. Once bound, the receptor-ligand complex produces an intracellular signal within the cell (i.e. in the nucleus) and alters the function of the cell.
GF can have different biological effects depending on the type of cell it interacts with. The cell’s response is highly dependent on the type of receptors present on the cell. Some GFs, such as insulin-like growth factor 1 (IGF-1), have broad specificity and affect many cell categories. Others act on only one type of cell and stimulate a specific response.
Many growth factors promote or inhibit cellular function and can be multifactorial. In other words, two or more substances may be required to elicit a specific cellular response. The proliferation, growth and development of most cells requires a specific combination of several GFs rather than one GF. Agents that inhibit growth can counterbalance those that promote growth (and vice versa), as in a metabolic system. The point at which many of these substances coincide to elicit a specific response depends on other regulatory factors, environmental and not.
Growth Factor Conversion
Some GFs stimulate cell proliferation, while others inhibit it, others can be stimulants when present at one concentration and inhibitors at a different concentration. Based on their biological function, GFs are a large array of proteins. They are usually grouped by amino acid sequence and tertiary structure. The large GF group consists of the beta-transforming growth factor (TGFb) superfamily, of which many subtypes exist. They have multiple effects on cellular function and are expressed in large quantities.
A common feature of TGFb is that they are secreted by cells in an inactive complex form. As a result, they show little or no biological activity until the hidden complex is destroyed. The exact mechanisms involved in the activation of these latent complexes are not fully understood, but may involve specific enzymes. This further explains how growth factors are involved in complex interactions.
Another common feature of TGFb is that their biological activity is often expressed in the presence of other growth factors. Therefore, we can understand that the biological activity of TGFb is complex, since they depend on the physiological state of the affected cell and on the presence of other growth factors.
There are many TGFb subtypes, classified according to their structure. One of these subtypes consists of growth and differentiation factors (GDFs), which, in particular, regulate growth and differentiation. GDF-8, also called myostatin, is a skeletal muscle protein associated with double muscle in guinea pigs and livestock.
McPherron et al. identified the expression of myostatin in the late stages of guinea pig embryo development and in many developing skeletal muscles. (1) Myostatin has also been identified in adult animals. Although myostatin mRNA was found almost exclusively in skeletal muscle, lower concentrations were also found in adipose tissue.
To determine the biological role of myostatin in skeletal muscle, McPherron and colleagues blocked a gene that codes for the myostatin protein in guinea pigs, causing a decrease in its function. The transgenic animals thus obtained had a gene incapable of producing myostatin. Reproduction of these transgenic guinea pigs resulted in offspring:
- or homozygous with both mutated genes (i.e. carrier of both mutated genes)
- or homozygous with both natural genes (i.e. carrier of both genes with normal function)
- or heterozygous carrier of the mutant gene and the normal gene.
The main difference between the various phenotypes was in muscle mass. For everything else, they were apparently healthy. They all grew to maturity and were fertile.
Homozygous mutants in guinea pigs (often called gene-blocked guinea pigs) were 30% larger than heterozygotes and the normal type of the same litter, regardless of gender and age. The adult guinea pig mutants had an abnormal body shape with very large hips and shoulders and body fat similar to those of their natural counterparts. The individual muscles of the mutant guinea pigs weighed 2-3 times more than that of natural guinea pigs. Histological analysis showed that the large muscle mass of the mutant guinea pigs was the result of both hyperplasia (more muscle fibers) and hypertrophy (larger size of individual muscle fibers).
Following this discovery, McFerron and other researchers examined the presence of myostatin and possible genetic mutations in other animal species. Scientists have identified myostatin sequences in 9 other vertebrates, including pigs, chickens and humans. (2) (4) A group of researchers found separately two independent mutations of the myostatin gene in two breeds of cattle: the Belgian blue and the Piedmontese. (2) (5) a deletion in the myostatin gene Belgian blue removes the entire active region of the molecule, rendering it inactive, this mutation causing hypertrophy and muscle gain. The coding sequence for myostatin in Piedmont contains a meaning mutation. This means that a point in the sequence encodes a different amino acid. This mutation is likely to result in complete or near complete loss of myostatin function.
McPherron et al. analyzed the DNA of other pure cattle breeds (16 breeds), which are not usually considered gable, and found only a similar mutation in the myostatin gene. (2) . the mutation was found in an allele of one non-double-muscled animal. Other mutations have been identified, but they did not affect protein function.
Previous studies have found high levels of myostatin in the skeletal muscle of cattle and rodents. (2) (7) In addition, mRNA expression varied between individual muscles. As a result, it was believed that myostatin is assigned to skeletal muscle and that the gene’s role is limited to skeletal muscle development. However, a team of New Zealand researchers recently identified mRNA and myostatin protein in heart muscle. (8)
Members of the TGF-b superfamily are found in many different types of cells, including adult and developing heart muscle cells. The three known TGF-b isoforms (TGF-b 1, -b 2 and -b 3) are expressed differently at both the mRNA and protein levels during cardiac development. (9) This indicates that these isoforms play different roles in regulating tissue development and growth. Thus, Sharma and colleagues examined the distribution of the myostatin gene in other tissues using more sensitive detection methods than those used by previous researchers. (8)
The researchers found a DNA sequence in the heart tissue of sheep and cows that was identical to the corresponding myostatin sequence in skeletal muscle, indicating the presence of the myostatin gene in these tissues. A deletion of the myostatin gene, which is present in muscle tissue, was found in the heart tissue of a Belgian blue fetus. The researchers identified the untreated precursor and transformed the myostatin protein in skeletal muscle in sheep and normal livestock, but not in Belgian blue muscle. In addition, only the protein of untreated myostatin has been found in adult heart tissue.
Animals with induced myocardial infarction (leading to cell death in heart tissue) showed high levels of myostatin protein even 30 days after the infarction in cells immediately surrounding the fatal injury. However, the intact cells surrounding the infarcted area contained very low levels of myostatin protein, similar to that in control tissue. Given the increased TGF-b levels in artificially infarcted heart tissue (10) , these growth factors may be involved in accelerating tissue healing.
At Purdue University, Shaoquan and colleagues identified myostatin mRNA in pig mammary glands, which may have a regulatory role in piglets. (12) The researchers also identified similar mRNAs in pig skeletal tissue, but not in connective tissue. In addition, most studies confirm that high levels of prenatal myostatin mRNA and reduced levels of postanatal in animals reflect the regulatory role of myostatin in the growth, differentiation and fusion of the myoblast (the precursor of muscle cells).
Mutation of the myostatin gene in two breeds of cattle is not as beneficial as in guinea pigs. In cattle, the increase in muscle mass is negligible compared to myostatin-blocked guinea pigs (20-25% in Belgian blue and 200-300% in null guinea pigs). In addition, cattle with mutated myostatin have smaller internal organs, lower fertility in women, delayed puberty and lower offspring survival. (6) Although no abnormalities have been found in myostatin-treated guinea pigs, the Belgian Blue has a smaller heart. (11) Although the decrease in organ mass is attributable to an increase in skeletal muscle mass, this remains to be confirmed. Since there is evidence that the effects of myostatin mutation on cardiac tissue differ from species to species, there may be other variations in tissue. In addition, studies have identified myostatin mRNA in tissues other than skeletal muscle, showing that its expression is not transferred to muscle tissue as originally thought. Only future research will clarify these possibilities.
Although many members of the TGF-b superfamily are found in heart and skeletal muscle tissues, their exact role in development is still unclear. Apparently, based on previous studies, the myostatin protein may play different roles in developing and adult tissues. Sharma et al. Argue that “myostatin performs different functions at different times in the development of the heart.” (8) As we will see, the same concept can be applied to skeletal muscle.
Myostatin and skeletal muscle regulation
While many studies show that myostatin is involved in prenatal muscle growth, we know little about its association with muscle regeneration. The regeneration of damaged skeletal muscle tissue is a complex system and the ability to regenerate changes during the life of an animal. The effect of various growth factors on tissues varies throughout life. In the embryo and in young animals, hormones and growth factors promote muscle growth. However, many of these factors are attenuated in adults. Changes in growth factors inside and outside muscle cells can reduce their ability to maintain protein expression. Although mRNA protein is found in the cell, there are many protein regulation sites beyond the mRNA level. As mentioned above, myostatin protein is presented in unchanged (inactive) and modified (active) forms. Therefore, it is possible to regulate the biological activity of myostatin at any moment of its synthesis and secretion.
Remember that almost all of the body’s regulatory systems are subject to positive and negative control. This also applies to cardiac tissue and skeletal muscle tissue. Myoblasts in animal embryos respond to various signals that control cell proliferation and migration. Instead, differentiated muscle cells respond to a different set of different signals. Different combinations of signals regulate the transition from undefined cells to differentiated cells and ensure regular formation and differentiation in cellular tissues. However, many of the factors that regulate various developmental pathways in muscle tissue are still poorly understood.
Products of MOD, IGF-1 and myogenin (growth promoters in muscle cells) are associated with muscle cell differentiation and activation of muscle-specific gene expression. (14) Expression of mRNA of muscle regulating factor (MRF-4) increases after birth and is the dominant factor in adult muscle. This growth factor is believed to play an important role in maintaining muscle cells. Besides myostatin, there are other inhibitory gene products such as Id (DNA binding inhibitor). Although in vitro experiments reveal the mechanisms of these specific proteins, we know little about their role.
While we know that lack of myostatin protein is associated with skeletal muscle hypertrophy in gene-blocked McPherron guinea pigs and dioecious cattle, we know little about the physiological expression of myostatin in normal skeletal muscle. Recent studies in human and animal models point to a paradox in the role of myostatin in muscle growth.
For example, some results indicate that myostatin may be fiber type specific. Small piglets with lower birth weight than normal piglets had lower proportions of type I skeletal muscle fibers in certain muscles. (12) Similar observations were made in guinea pigs with undetectable myostatin mRNA levels in the atrophied sole (type I fibers). (13) A temporary increase in myostatin mRNA was found in muscles in rapidly twitching atrophied, but not in slowly twitching muscles. Hence, myostatin can modulate the expression of genes that control muscle fiber type.
Research also shows no metabolic effects on myostatin expression in piglets and guinea pigs. (12) (13) Reducing food intake in both pigs, both in guinea pigs did not affect skeletal muscle myostatin mRNA levels. In piglets, neither dietary polyunsaturated fatty acids nor the administration of exogenous growth hormone altered the expression of myostatin. (12) These and other studies conclusively show that the physiological role of myostatin is mainly related to prenatal muscle growth, when myoblasts proliferate, differentiate, and fuse to form muscle fibers.
Although the authors speculate that myostatin exerts its influence in an autocrine / paracrine way, myostatin has also been found in the blood, showing that it is also secreted in the blood. (8) (4) A protein found in human blood is considered modified myostatin (active form). High levels of this protein have been associated with muscle loss in men with HIV 4. However, this association does not necessarily demonstrate that myostatin directly contributes to muscle loss. We don’t know if myostatin acts directly on muscles or other regulatory systems that regulate muscle growth. Although many authors have suggested that myostatin may play an important role in muscle regeneration after injury, this has yet to be confirmed.
Myostatin and Athletes
Further complicating the question of the role of myostatin in the regulation of muscle growth is the report by a group of scientists, according to which mutations in the human myostatin gene have little effect on the response of muscle mass to strength training (15) , unpublished data. Based on the report that muscle mass is an inherited trait in humans (16) , Ferrell and colleagues studied changes in the myostatin gene sequence in humans. The researchers also looked at the effect of changes in myostatin on muscle mass in response to strength training.
The subjects came from different ethnic groups and were categorized based on the increase in muscle mass that was observed after strength training. Bodybuilders were present in the world’s top 10 and lowest categories. There were also soccer players, powerlifters and previously untrained people. Quadriceps muscle volume in all subjects was measured by magnetic resonance imaging before and after nine weeks of hard training with knee extensors. The subjects were grouped and matched based on the degree of response and ethnicity.
In the DNA samples taken from the subjects, there were many variations in the genetic coding sequence. One subject had two variants and two others had two others. The subjects were heterozygous for the natural allele, which means that they had the mutated allele and the other allele was normal. Other differences were present in the generality of the subjects and certain municipalities were identified. One variation was common in a mixed group of Caucasian and African American subjects. However, the less common allele has been reported more frequently in African Americans. Although the authors comment that “these variable sites (in the gene sequence) have the potential to modify the function of the myostatin gene product and alter nutrient distribution in heterozygous individuals for the allele variant,” the data are taken so far from this and other studies suggest that this may not happen. … This study showed no significant difference between genotypes and response to strength training. There were also no significant differences between African American respondents and non-respondents in strength training, or between Caucasian respondents and non-respondents.
Further research is needed to determine if myostatin plays an active role in muscle growth after birth and in adult tissue. To figure out the health benefits of humans, we also need to figure out its role in muscle atrophy and regeneration after injury. Only in-depth research will show the possible existence of these benefits.
The future of myostatin
Now that we have covered some of the biology of biostatin, its gene and related research, what are the implications of its use?
Many authors of myostatin studies suggest that interfering with myostatin activity in humans may reverse muscle loss associated with muscular dystrophy, AIDS, and cancer. Some speculate that manipulating this gene could lead to very muscular animals. Indeed, ongoing research is trying to explore and develop these possibilities. Of course, a large pharmaceutical company recently applied for a patent for vaccination with an antibody to the myostatin protein.
A physician who has written articles on resistance training argues that myostatin overexpression is the cause of the problems that weightlifters face with increasing muscle mass. A spokesperson for the R&D Supplement Laboratory erroneously claimed that a “rarer” form of myostatin gene mutation is responsible for the massive gains in muscle mass among elite agonist bodybuilders, not counting the performance-enhancing substances these bodybuilders might suggest. The media apparently predicted that athletes who are already “steroid users” would use myostatin inhibitors to gain a competitive advantage. (3)
Many of these claims are unfounded or misinterpret scientific research. There is, of course, the possibility that manipulation of the myostatin gene in humans could be the key to reversing diseases that cause muscle loss. However, little is known about the role of myostatin in regulating muscle growth. It is important that research shows that decreased myostatin activity in adults can induce muscle growth. Likewise, studies should also demonstrate that overexpression and administration of myostatin causes a decrease in muscle mass. It is also important to know if manipulating myostatin will interfere with other growth systems, especially in other tissues, which will lead to pathological pathologies. Although guinea pigs with a blocked Macferron gene have not experienced serious disruption, guinea pigs are not human.
We do not fully understand the role of myostatin in exercise-induced muscle hypertrophy or regeneration after muscle injury. Until then, it may be premature to blame the overexpression of myostatin in the absence of hypertrophy in weightlifters. The study does not even support the claim that the increase in muscle mass in elite bodybuilders is the result of a mutation in the myostatin gene. Research simply does not point to genetic variations in myostatin as a source of significant differences in human phenotypes.
Given the history of athletes’ propensity to abuse performance-enhancing substances in the public eye, the media hypothesis of myostatin inhibitors may or may not be justified. We all know that in today’s competitive arena, you need to gain a competitive edge to stay on top of the competition. For many athletes, this is achieved by integrating hard training with substances that increase growth or performance. It is difficult to predict whether myostatin inhibitors will be added to the arsenal. As long as science uncovers the true nature of this growth factor and its role in the complex regulation of muscle tissue, and researchers determine its therapeutic effects, we can only speculate. Despite attempts to tightly control any pharmaceutical use of manipulating myostatin protein, it will sooner or later appear on the black bodybuilding market. We hope that then science identified side effects and beneficial ones.