History of Discovery
In addition to sports training, environmental exposure, nutrition, and the professional activity of a person, genetic factors also have a great influence on the strength indicators of an athletes’ skeletal muscles . The study of genetic foundations, including gene polymorphisms and their connections with a body’s resistance to physical load as a whole, and their contribution to an athletes’ strength abilities and development should reasonably be considered as one of the most important and significant areas of modern sports science .
Myostatin (MSTN) protein was discovered in 1997 and was encoded by MSTN gene, located on chromosome 2 2q32.2; it encodes 375 amino acids in three exons and occupies a site of approximately 8 kb . This discovery was considered a significant success in the study of genetic factors for increasing muscle mass and developing strength abilities.
This gene was named myostatin because of its ability to inhibit muscle differentiation and growth , whereas the overexpression of myostatin is associated with muscle atrophy . However, these studies have confirmed the central and critical role of MSTN in suppressing muscle growth [6, 7].
Special attention is given to MSTN because the very first publications on this factor concluded that its absence affects the increase in muscle mass due to hypertrophy and hyperplasia of the muscle fibers . The increase in detailed scientific studies of MSTN and the possibilities of using the published data for various biomedical and sports purposes, including gene doping  has increased the interest in the subject.
The ability of MSTN to limit the growth of muscle mass immediately attracted the attention of researchers as it can be used in sports and sports medicine.
MSTN, also known as the growth differentiation factor-8 (GDF-8), is a protein-based hormone that acts as a negative regulator of muscle growth. This was first mentioned by McPherron et al. . The authors found that a mutation in MSTN leads to an increase in the size of muscle tissue. During the initial stages, these were primarily conducted on animals followed by on humans. MSTN is particularly of interest in sports, wherein one can monitor its correlation with the performance, especially in sports that require muscle strength and mass .
Mutations in MSTN lead to a significant increase in muscle mass . It is an important gene that affects myogenesis as its role is to regulate the growth and differentiation of muscle cells . In particular, the genetic predisposition to gain muscle mass is due to the low expression of MSTN, which is advantageous in the improvement of strength .
As MSTN is the most common type of skeletal muscle, it is of interest in studies related to sports science . However, its expression is also noted in the heart and adipose tissues [15, 16].
The growing interest in MSTN has led to the publication of a large number of scientific papers that can be found in the Web of Science and PubMed database (Fig. 1). A review of the publications confirms that MSTN is an endogenous negative regulator of skeletal muscle mass, which acts as an antianabolic agent that suppresses the activation, replication, DNA and protein synthesis of muscle satellite cells affecting myogenic differentiation .
Researchers from Taiwan found that MSTN was negatively correlated with age and the percentage of fat mass in healthy young men . The results of the experiments proved that the reference value of MSTN concentration in blood serum in healthy young men is 12.3 ± 3.6 ng/mL and that it negatively correlates with age .
Considerable attention should be paid to the factors contributing to the inhibition of MSTN expression. According to scientific sources, such factors include hypodynamia, various origin diseases, state of weightlessness, and aging [9, 20]. The level of MSTN in skeletal muscles is also influenced by power-oriented physical exercise .
There are a number of factors that act as inhibitors of MSTN synthesis, including myocyte 2 enhancing factor (MEF2); gamma-receptors activated by peroxisome proliferator (PPARγ); MyoD; and hormones, such as insulin-like growth factor (IGF-1), angiotensin II, thyroid hormone, erythropoietin , sex steroids, follistatin, and estradiol .
One of the main factors in sports that significantly affect the level of MSTN secreted is power-oriented physical activity, hypoxia, and dietary supplements. Moreover, the production of MSTN is influenced by essential amino acids, which are often consumed by athletes after intensive training .
Currently, the study of antibodies against MSTN, e.g., MYO‑029 and BYM338, are attracting much attention, but their effectiveness is still poorly studied [25, 26]. In addition to antibodies, other MSTN inhibitors, such as the hormone follistatin, can also suppress its activity [27–29].
Recent studies have shown that essential amino acids suppress MSTN expression in human skeletal muscles [30, 31].
In high-performance sports, myostatin inhibition is prohibited by WADA (https://www.wada-ama.org/en/prohibited-list/prohibitedat-all-times/hormone-and-metabolic-modulators).
MSTN is a potential genetic marker of the athletic abilities in strength sports because of the involvement of a large number of skeletal muscles and the functions of myokines. Some research related to the study of MSTN and its role in hypertrophy and skeletal muscle strength seemed contradictory [20, 32, 33]. Therefore, in this study, attention was given to the influence of the K153R rs1805086 genotype on the manifestation of skeletal muscle strength in athletes.
Mechanism of Effect of MSTN on Skeletal Muscle Mass and Strength
Physical activity causes muscle hypertrophy, and performing physical power-oriented exercises clearly demonstrated this. This type of exercise causes mechanical damage to sarcomeres and sarcolemmas. After a certain period of time, the balance shifts toward protein synthesis and, as a result, phenotypic changes increase the volume and strength of skeletal muscles. These processes release active MSTN, which affects satellite cells and fibroblasts located near the damaged area. MSTN can cause protein degradation in myofibrils, which are important for the normal functioning of muscle fibers as they removes unnecessary, wasted proteins from the muscle cells .
MSTN is one of the main factors associated with muscle atrophy. In studies involving humans, it was found that by the 25th day of the sedentary regime, the level of myostatin increased by 12% . Myostatin can regulate the function of muscle fibers and nearby cells, which include fibroblasts and satellite cells or satellites. Mature muscle fibers are the products of final differentiation .
An increase in muscle size is achieved by the fusion of satellite proliferating cells with fibers. Primarily, microtrauma in a single muscle fiber act as a stimulus for the proliferation of satellite cells in adult organisms. When these cells are activated and emerge from a dormant state, genes characteristic of myoblasts are also activated. Therefore, satellite cells become myoblasts that migrate to the damaged areas of muscle tissue and depending on the degree of damage, either merge with the damaged muscle fiber (hypertrophy) or merge with each other, thus creating new fibers (hyperplasia). Therefore, satellite cells regulate the functional state of skeletal muscles in the adult body. They are necessary for the restoration of damaged muscle fibers and are a source of additional nuclei in case of muscle hypertrophy after training sessions. MSTN negatively affects the proliferation of satellite cells . Power-oriented training sessions result in mechanical stretching of the muscle and lead to microdamage. There is also evidence that MSTN negatively regulates the activation of resting satellite cells, hindering their development. Such inhibitory effects are necessary for normal muscle regeneration as premature fusion of satellite cells with myofibrils can impair muscle fiber functions (Fig. 2).
In general, the mechanism by which MSTN controls the number of muscle fibers is not well studied. It is synthesized as an inactive protein and undergoes changes to turn into mature active form in two stages . It enters the bloodstream as a latent precursor protein and undergoes a proteolytic process, turning into a mature peptide that binds to the extracellular type II receptor (ActRIIB) activin. Binding of MSTN to ActRIIB induces the intracellular activation of proteins, by which MSTN modulates the proliferation and differentiation of myoblasts, and ultimately, the muscle mass [30, 37, 38].
Effect of Myostatin on Tendons and Bones
Tendons are an important component in the manifestation of maximum strength of the skeletal muscle. Weightlifting and speed-strength sports athletes with high indicators of skeletal muscle strength often have tendon injuries as their muscle strength exceeds their endurance. During strength training, fibroblasts proliferate, collagen synthesis increases, and the cross-sectional area of the tendons increases to make them stiffer. This allows the tendons to withstand high-intensity physical loads and reduce the risk of damage to them .
MSTN can change the mechanical properties of tendons by impairing their ability to stretch, increasing the risk of damage. Such data cast doubt on the feasibility of inhibiting MSTN expression for sports purposes . The exact mechanisms of the effect of myostatin on tendons and ligaments are still unknown, and further studies are needed to assess its regulatory role in these processes . When studying the regeneration of muscles and tendon fibroblasts, it is assumed that myostatin affects the expression of type 1 collagen. Recent studies have reported that local injections of exogenous myostatin during tendon healing increase the cross-sectional area of the tendon .
In both human and animal studies, there is evidence that myostatin is an important regulator of muscle mass as well as bone density. The mechanisms by which myostatin regulates bone formation are not completely understood, but it is clear that it has a direct effect on the proliferation and differentiation of stem cells [42, 43]. Since myostatin and its receptor are expressed during bone regeneration, it affects bone density . It is likely that myostatin directly affects bones, increasing bone mineral density. Some features of different phenotypes may be associated with increased biomechanical load, e.g., in weightlifters or under the influence of other factors, such as mechanical growth factors or growth hormones. These issues have yet to be studied in more detail, but if a number of studies prove that myostatin does have an effect on bones, then it can be assumed that myostatin inhibitors will be useful not only for increasing muscle mass, but also for bone density. This assumption is supported by recent data showing that myostatin significantly increases bone volume during fibular healing .
In previous studies, it was found that a number of missense substitutions in exons 1 and 2 of MSTN are of great interest to researchers to confirm MSTN connection with athletes’ strength abilities, muscle hypertrophy  and recovery after intensive strength exercises. The polymorphisms K153R, A55T, E164K P198A, I225T and c.373 + 5 [20, 46, 47] are of particular interest to the gene as well.
MSTN Mutation (rs397515373, c. 373 + 5 G>A)
This mutation is very rare, with an average prevalence of 0.0004% in the population. It was necessary to obtain 500,000 samples to detect a mutation once. In 2004, a paper describing a case of MSTN mutation in a child was published . In both the allelic copies of MSTN, the newborn boy had mutations that suppressed the synthesis of the functioning myostatin protein. The child was observed to have enlarged muscles of the thighs and upper extremities at birth. Ultrasonography of this child showed that the cross-section of the quadriceps femoris muscle was 7.2 SD, which was higher than the average (± standard deviation) value for 10 persons matched for age and gender. Moreover, the thickness of his subcutaneous fat was 2.88 SD below the average value of that of his peers. All reflexes of the child were normal, except for those associated with tendons. Interestingly, this mutation was also present in other members of this family. One of the relatives was extraordinarily strong, and the 24-year-old mother of the child was a professional athlete and had developed muscles, although to a lesser extent than her son. This study showed for the first time that the MSTN rs397515373 mutation (c.373 + 5 G>A) leads to an increase in muscle mass and strength [49, 50].
MSTN A55T Mutation (rs180565, 163 G>A)
A55T is important for the stability of the inhibitory activity of myostatin and affects mature myostatin .
A study devoted to physical exercise reported that subjects with AT and TT genotypes of the A55T polymorphism had greater muscle hypertrophy than those with AA genotype after 8 weeks of exercise with weights . Studies have shown that myostatin polymorphisms can affect the skeletal muscle phenotype after exercise with weights. However, previous studies of myostatin SNPs associated with muscle hypertrophy in response to prolonged power-oriented strength exercises have not confirmed pronounced muscle hypertrophy after strength physical load .
Studies on an Asian sample set (n = 500) showed that the A55T polymorphism can affect the activity of myostatin, mass of skeletal muscles, and the amount of fat in the body. The results have shown that the A55T polymorphism determines the genetic predisposition to the development of excessive obesity and low muscle mass in Asians .
Chinese scientists found that people with the genotype AT + TT of the A55T polymorphism of MSTN showed a significant increase in the thickness of biceps (0.292 ± 0.210 cm, P = 0.03) but not quadriceps (0.254 ± 0.198 cm, P = 0.07) compared to those of the carriers of the AA genotype. Thus, the obtained results suggest a possible association between polymorphism A55T and muscle hypertrophy caused by strength training in Chinese individuals .
Korean researchers have found that the A55T polymorphism is associated with skeletal muscle recovery after strength training. The study sample included 48 young healthy college students (age 24.8 ± 2.2 years, height 176.7 ± 5.3 cm, weight 73.7 ± 8.3 kg) who performed 50 repetitions in strength exercises. The A55T polymorphism was classified into the homozygous allele of MSTN A55T (AA, n = 34.72%), heterozygous allele of MSTN A55T (AT, n = 13.26%), and homozygous mutant carriers (TT, n = 1.2%). After strength exercises, subjects with heterozygous AT showed significantly faster muscle recovery than those in the AA group (P = 0.042). These results prove that the AT genotype of the A55T polymorphism is associated with a faster recovery of skeletal muscle strength after intense strength exercise .
Turkish scientists failed to identify the relationship between the A55T polymorphism and the morphological data of arm wrestlers [52, 55, 56]. Moreover, no statistically significant relationships have been found in highly qualified athletes in endurance sports [57, 58].
Mutation of MSTN E164K rs35781413 (с.490G>A, p.Glu164Lus)
In a number of studies related to the influence of this genotype on the phenotype of athletes and people not engaged in sports, the results of experiments showed no statistically significant differences . This is also due to the very low frequency of this genotype in humans. According to the website http://www.ensembl.org, the average frequency of a rare allele was 1%. Such a low allele frequency obviously limits the possibility of studying large groups of people with minor alleles .
There are only indirect assumptions that this mutation can affect the manifestation of muscle mass and strength in humans. These assumptions are based on the fact that this polymorphism can make a significant contribution to the biochemical variability of mature myostatin, and accordingly, affect the state of the vertebrate muscular system. However, this assumption requires further study .
Mutation of MSTN K153R (rs1805086, p.Lys153Arg, c.458A>G)
The RR genotype of the MSTN rs1805086 gene is more common in top-class athletes of weightlifting . Some researchers found a positive association between the K153R rs1805086 allele and the manifestation of strength abilities and muscle hypertrophy [13, 46, 52, 61], whereas other researchers did not find any significant connection [33, 46, 62]. Some studies have proven a connection with high performance in high jumps (P < 0,05) . Studies on the relationship between K153R and skeletal muscle phenotypes in elderly Caucasian women have shown that the heterozygote MSTN rs1805086 KR is a favorable genotype for the increased muscle mass in the biceps of the shoulder .
In studies conducted on 16 women and 34 men of the Caucasus native and African–American and Afro–European ethnicities that participated in the European Championships and the Olympic games in sports, such as football (n = 4), basketball (n = 10), tennis (n = 6), volleyball (n = 6), canoeing (n = 2), rugby (n = 10), baseball (n = 6), and track-and-fields (sprint, javelin, and shot put) (n = 6), who were compared with a control group of 100 people, including 40 women and 60 men that not involved in sports, the authors failed to find statistically significant differences between the elite athletes and people in the control group in terms of the allele K153 occurrence frequency and the success in competitions .
Studies on the relationship between myostatin and muscle pathologies in healthy elderly people are contradictory . The association between low myostatin levels and low skeletal muscle mass was observed only in men and not in women. The authors point to the need for further research on myostatin as a biomarker of muscle mass and strength .
Allele Frequency of MSTN K153R (rs1805086)
According to the Ensembl database, the frequency of the rare allele 153R is on an average 7% (3% in Caucasians and 22% in Africans), and large sample sizes are necessary to reliably identify the association of this polymorphism with strength abilities and muscle hypertrophy (Figure 3).
The conducted studies could not always prove the connection between the athletes’ skeletal muscle strength, muscle mass, and competitive performance [32, 62]. Due to the low frequency of K153R polymorphism in Caucasian athletes of cyclic sports, the authors point out the possibility of evaluating MSTN K153R polymorphism during sports selection and that this mutation needs further study.
The problem of studying mutations in MSTN is the low frequency of some alleles. To obtain the required number of subjects and statistically significant data, we would need the subjects to be very specific, for example, involving highly qualified athletes of weightlifting sports or people with an exceptional proportion of skeletal muscles . Such subjects, for instance, can also include some ethnicities, considering their residence .
Problems with Sample Collection
Because the occurrence of the MSTN K153R rs1805086 allele in populations is 7% on an average, it creates problems in identifying people with the rare genotype . Two of the factors that significantly affect the association of myostatin genotypes with muscle mass and skeletal muscle strength are sex and age. Experiments to identify the effect of myostatin on muscle mass and strength showed different results depending on these two factors [65, 66].
Contradictory data were also obtained in cases where the subjects were representative of different sports . The impact of myostatin on skeletal muscle strength depends on the sport. In the type of sport requiring the ability to maintain a given physical load for a long time, no statistically significant data were found on the association of MSTN polymorphisms with muscle mass and strength .
Some studies have reported that female estrogens affect the change in the expression of myostatin caused by power-oriented physical exercises . In addition, differences in ethnicity, sample size, body weight, and level of physical activity may be potential reasons for the different results in studies related to myostatin . The authors pointed out that nutritional factors should be considered when assessing the level of myostatin. Sex is also an important factor in the reduction of muscle strength and age-related decrease in muscle mass. Men usually begin to lose muscle mass after 40 years of age, when the level of testosterone in serum drops. Women can gradually lose 10–15% of their muscle mass from the age over 25 until the onset of menopause, after which it increases at a rate of 2% annually. Therefore, the amount of muscle mass is also affected by sex and diet.
In addition, studies often consider a specific human muscle as an object, such as the biceps or quadriceps, and this is also a limiting factor for the full assessment of the relationship between myostatin and the muscle mass and strength of the entire body as a whole.
The absence of a control group in some studies does not allow us to solve the problem of statistical validity of the obtained data . To obtain the most reliable data, larger sample sizes are required.
Finally, after testing for strength exercises, researchers considered only some indicators of muscle fatigue; therefore, they were limited to confirming the connection mechanism between specific strength indicators, myostatin genotype, and muscle strength. It should be noted that most scientific publications are primarily based on previous research [67–69].
There are studies in which the authors concluded that the K153R MSTN allele does not affect muscle phenotypes in women, wherein their sample of subjects was 33 people aged between 90–97-years-old. Considering that this allele is very rare, the results of such studies seem very doubtful .
Thus, in this manuscript, the systematic review of publications related to the influence of the K153R allele of MSTN helped conclude that the data obtained can be regarded as contradictory. With such a discrepancy, the question arises whether the allelic variants K and R of the MSTN rs1805086 gene are genetic factors that can affect human strength abilities and skeletal muscle hypertrophy.
Meta-analysis overcomes the limitation of a small sample size by combining the results of a number of individual studies to obtain a single best estimate.