Investigating the impact of antioxidant supplementation on muscle antioxidant status and the skeletal muscle proteome in thoroughbred racehorses

Glutathione (GSH) and Coenzyme Q10 (CoQ10) are potent cellular antioxidants that work to mitigate oxidative stress arising from reactive oxygen species (ROS) production. GSH is not well absorbed because it is broken down into individual amino acids in the small intestines. Cysteine is the rate limiting amino acid in GSH synthesis but is not well absorbed and is instead largely produced from methionine intracellularly. N-acetyl cysteine (NAC) supplementation has been studied as an alternative to cysteine or GSH supplements and has been shown to increase the amount of circulating cysteine and increase transport activity for this reduced form in humans. GSH concentrations in skeletal muscle have been measured in only a few studies in other species. Neither GSH nor NAC have been previously studied in the skeletal muscle of horses.CoQ10 is an electron transporter in the mitochondrial electron transport chain (ETC) where it transfers electrons either from complex I or complex II to complex III. Within the ETC, complex I has been shown to be the primary source of ROS during exercise in comparison to the other complexes. Due to its location and function, CoQ10 can also function as a potent membrane bound antioxidant and mitigate ROS produced through the ETC. CoQ10's function as an antioxidant and electron transporter has not been studied in great depth in horses. Branched chain amino acid (BCAA) supplements are three essential amino acids that have been shown to have positive effects on protein synthesis through the mammalian target of rapamycin pathway (mTOR). BCAA can serve as an energy source in skeletal muscle where they are directly metabolized in the tricarboxylic acid cycle. There are a limited number of studies of the impact of BCAA supplementation on equine skeletal muscle and none have looked at BCAA combined with antioxidant supplements. The overarching objective of this dissertation was to evaluate the impact of antioxidant supplementation on fit, healthy Thoroughbred horses. The first study supplemented NAC and CoQ10 to maximally exercising Thoroughbreds to determine its effect on the redox equilibrium and skeletal muscle proteome. We determine that NACQ increases muscle GSH concentrations post exercise while increasing TCA cycle enzymes and enhancing capacity for cellular NADPH production. The second study measured the effect of a single dose of differing amounts and formulations of CoQ10 on plasma CoQ10 concentrations. We determined that individual horses have different absorption responses, with 50% showing no response. The third study analyzed chronic CoQ10 supplementation's effect on plasma CoQ10 concentrations, concentrations or activities of skeletal muscle antioxidants, mitochondrial respiration, and the skeletal muscle proteome. Results supported CoQ10's function as an antioxidant and ability to alter the contribution of complex I and complex II to electron transfer without increasing mitochondrial volume density. The final study of this dissertation analyzed the impact of NAC and BCAA supplementation on sub-maximally exercising Thoroughbreds by measuring antioxidant status and alterations to the skeletal muscle proteome before and after exercise. This study identified no changes in skeletal muscle GSH concentrations and ROS before or after exercise but did find differentially expressed proteins within the ETC, redox reactions, and glycolysis after submaximal exercise. All supplements warrant further investigation in horses with myopathies.