The Biochemical and Bioenergetic Consequences of the Myostatin SNP g.66493737C>T and SINE insertion 227 bp polymorphism in Skeletal Muscle of Thoroughbred Horses

Abstract

The Thoroughbred horse is a tremendous athletic breed, which can be attributed to years of genetic selection for speed and stamina. This selection has led to the Thoroughbred having a very high aerobic capacity relative to muscle mass. Skeletal muscle is the location of most of the oxygen consumption that occurs during exercise and the density of mitochondria in skeletal muscle has been found to be proportional to the maximum aerobic rate and hence the standard metabolic rate. It is known that Thoroughbred skeletal muscle has a remarkable ability to adapt to physical training. Although the physiological adaption to training has been well described in the Thoroughbred, little is known about mitochondrial adaption in skeletal muscle of Thoroughbreds. Myostatin is a negative regulator of skeletal muscle growth known to have pronounced effects on skeletal muscle mass. Multiple polymorphisms in the equine myostatin (MSTN) gene have been identified and two have become of considerable interest: SNP g.66493737C>T in the first intron and SINE insertion 227 bp polymorphism in the promoter region. Both variants have been found to have highly significant associations with best race distance in Thoroughbred racehorses. Associations of these polymorphisms with muscle fibre proportion and body composition in the Thoroughbred have also been reported. Although the correlation between the variants and optimum race distance is substantial, the exact mechanism by which the variants affect activity of the gene is unknown. The present work characterises the influence of the MSTN polymorphisms on myostatin activity in the Thoroughbred horse, elucidating the mechanism by which the polymorphisms affect the MSTN gene. Molecular biology techniques were employed to create myostatin constructs of various genotypes within a luciferase expression vector in order to establish the activity of the MSTN gene in the presence and absence of the SINE insertion 227 bp polymorphism and SNP g.66493737C>T, enabling us to determine the effect of both polymorphisms in combination and alone on the MSTN gene. The study tested the hypothesis that mitochondrial function in Thoroughbreds is influenced by MSTN SINE insertion 227 bp polymorphism and SNP g.66493737C>T, by employing enzyme activity assays, qPCR and immunoblotting. We firstly determined that the MSTN SNP g.66493737C>T and SINE insertion 227 bp polymorphism are in full concordance in the Thoroughbred horse. We also observed a significant association between MSTN SNP g.66493737C>T and SINE insertion 227 bp polymorphism genotype with MSTN gene expression in untrained Thoroughbred horses. Importantly, we demonstrated that the SINE insertion 227 bp polymorphism had a significant effect on myostatin protein production and gene expression. We found that the SNP g.66493737C>T has no effect on myostatin activity, providing strong evidence that the SINE insertion 227 bp polymorphism acts as the direct functional effector of myostatin expression. For the first time we have described the mechanism by which the SINE insertion 227 bp polymorphism influences the MSTN gene; an alternative transcription start site is created by the presence of the SINE insertion 227 bp polymorphism, resulting in a lengthened transcript and subsequent decreased gene expression. We observed significant associations between MSTN genotype and mitochondrial abundance and muscle fibre proportion in untrained Thoroughbred horses. Additionally, higher combined mitochondrial complex I+III and II+III activities were observed in skeletal muscle tissue from SINE insertion 227 bp polymorphism/SNP g.66493737 Callele horses when compared to no SINE insertion 227 bp polymorphism/SNP g.66493737 T-allele, apparently due to increased coenzyme Q. Subsequent transcript analysis suggests decreased coenzyme Q biosynthesis in these horses. The information contained within this thesis provides convincing evidence for the influence of the SINE insertion 227 bp polymorphism on myostatin expression in the Thoroughbred. Furthermore, the research elucidated a number of additional associations with these MSTN polymorphisms which may be relevant for future enhancement of Thoroughbred horse training strategies.