Previously, work has identified genetic predictors of exercise performance or racing success, but the functional effect of these variants has been less well explored. In this study we sought to test the use of a commercial ELISA for detection of myostatin in equine serum and thereafter to examine the effects of a MSTN gene promoter SINE insertion in vivo. Crucially, this study has revealed the biological consequences of the SINE mutation at the protein level in Thoroughbred racehorses. The results corroborate those found in previous in vitro cell culture reporter studies 17,18 and help explain in vivo effects on performance.
Morrison et al. described use of an ELISA for detection of serum myostatin in mixed breed horses and ponies of unknown MSTN genotype revealing that circulating myostatin concentration is significantly higher in obese compared to lean animals 23. All the Thoroughbreds used in the current trial were all from the same racing yard, and were of similar lean body condition; however, given the variation of myostatin expression even within genotyped groups, it would be of interest to see if body condition score is also associated with myostatin serum concentration in racehorses.
There is debate as to whether sex affects serum myostatin concentration in humans. Tanaka et al. revealed that obese men had higher circulating myostatin concentration than obese women 24. Similarly, it has also been suggested that only men have an association between muscle mass and serum myostatin 25. However, Yano et al. found no difference in serum myostatin between sexes26, as we report in the current work. Circulating myostatin concentrations decline with age in mammals, including horses 27 and there is much interest in the hormone’s role in development of age-associated sarcopaenia in older mammals 28. An association with age was not seen in this study; however, the majority of horses in this investigation were between 2 and 3 years old (i.e. active racehorses), with the oldest being 8. As such, serum myostatin concentrations might decline in older horses than those in this study.
Physically fit humans have higher serum myostatin concentrations than unfit humans 29. In the current work all samples were collected from racehorses in training, and likely therefore with similar levels of fitness. However, other work has found that musculoskeletal condition has little effect on serum myostatin concentrations in humans 3031,32 including those in high-velocity resistance training 33. Recent studies suggest that myostatin expression changes in equine skeletal muscle in response to exercise and training 34: in future it would be of interest to examine the effects of exercise and training on serum myostatin concentrations within MSTN-genotyped horse groups.
MSTN genotype is associated with variation in muscle mass 35: WT horses have the lowest muscle mass, and those with SINE mutations have the highest. Similarly, a study in a child with marked muscle hypertrophy detected exceedingly low myostatin concentrations in the individual’s serum 36. This is comparable to the effect seen in homozygous MSTN-null, double-muscled meat-producing animals that have significantly lower circulating myostatin than wild type counterparts. Homozygous SINE horses also have a greater body weight to wither height ratio 35. We found no difference in the height of homozygous and wild type horses but since horses were not weighed, the mass to height ratio could not be calculated; our data does though suggest that the SINE mutation in horses is not directly linked to stature. Myostatin has an indirect effect on bone formation due to decreased muscle growth and thereby a reduction in mechanical loading. The protein is also expressed around fracture sites and reduces callus formation 37-39. Wu et al. discovered that circulating myostatin correlates with lower bone density 30. Consequently, it would be of interest to examine whether horses with the SINE insertion are more at risk of musculoskeletal injury.
In this study, myostatin was detectable in the serum of homozygous SINE animals. Unlike Belgian Blue cattle 19, mice 20 and dogs 22 with null mutations that prevent the expression of myostatin entirely, these horses produce the hormone, albeit at a lower concentration than the heterozygous and WT animals. This may be the reason that these animals are outstanding athletes whereas those with myostatin mutations in other species are not. Similarly, perhaps, heterozygous “bully” (myostatin haploinsufficient) Whippets are faster than wild type dogs 22 and notably, the mother of a human myostatin null infant (who was therefore presumably a heterozygote) was an Olympic athlete 36.
Whilst the range of myostatin concentrations in homozygous horses is clearly defined, there is a marked overlap between heterozygous and WT horses. It seems likely therefore that there are other genetic or environmental factors that influence serum myostatin concentration in these animals. It will be of interest to examine daily and seasonal variability within horses and to determine whether heterozygote horses that have consistently lower serum myostatin concentrations excel over shorter distances, similar to homozygous types. Determining the reasons for this variation might reveal other performance-associated gene variants in Thoroughbreds.
To conclude, within Thoroughbreds the circulating myostatin concentration differs, and is dependent on the number of copies of a SINE mutation within the MSTN gene promoter. Determining the reason why myostatin expression varies between horses of the same genotype might reveal additional performance-associated genes. Finally, the underlying mechanism that links altered myostatin expression with the fibre type proportions of skeletal muscles in mammals remains unknown and is worthy of further study.
Acknowledgments: We are grateful to Mr Mark Johnston for access to samples. This study was approved by the Royal Veterinary College’s research office and assigned the following unique research number (URN): 1442543.