Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses

Abstract

Recurrent exertional rhabdomyolysis (RER) is a chronic muscle disorder of unknown etiology in racehorses. A potential role of intramuscular calcium (Ca2+) dysregulation in RER has led to the use of dantrolene to prevent episodes of rhabdomyolysis. We examined differentially expressed proteins (DEP) and gene transcripts (DEG) in gluteal muscle of Thoroughbred race-trained mares after exercise among three groups of 5 horses each; 1) horses susceptible to, but not currently experiencing rhabdomyolysis, 2) healthy horses with no history of RER (control), 3) RER-susceptible horses treated with dantrolene pre-exercise (RER-D). Tandem mass tag LC/MS/MS quantitative proteomics and RNA-seq analysis (FDR <0.05) was followed by gene ontology (GO) and semantic similarity of enrichment terms. Of the 375 proteins expressed, 125 were DEP in RER-susceptible versus control, with 52 ↑DEP mainly involving Ca2+ regulation (N = 11) (e.g. RYR1, calmodulin, calsequestrin, calpain), protein degradation (N = 6), antioxidants (N = 4), plasma membranes (N = 3), glyco(geno)lysis (N = 3) and 21 DEP being blood-borne. ↓DEP (N = 73) were largely mitochondrial (N = 45) impacting the electron transport system (28), enzymes (6), heat shock proteins (4), and contractile proteins (12) including Ca2+ binding proteins. There were 812 DEG in RER-susceptible versus control involving the electron transfer system, the mitochondrial transcription/translational response and notably the pro-apoptotic Ca2+-activated mitochondrial membrane transition pore (SLC25A27, BAX, ATP5 subunits). Upregulated mitochondrial DEG frequently had downregulation of their encoded DEP with semantic similarities highlighting signaling mechanisms regulating mitochondrial protein translation. RER-susceptible horses treated with dantrolene, which slows sarcoplasmic reticulum Ca2+ release, showed no DEG compared to control horses. We conclude that RER-susceptibility is associated with alterations in proteins, genes and pathways impacting myoplasmic Ca2+ regulation, the mitochondrion and protein degradation with opposing effects on mitochondrial transcriptional/translational responses and mitochondrial protein content. RER could potentially arise from excessive sarcoplasmic reticulum Ca2+ release and subsequent mitochondrial buffering of excessive myoplasmic Ca2+.

Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis

“…STIM1 regulates store operated calcium entry into the muscle cell when sarcoplasmic reticulum stores are depleted [ 50 ]. CASQ1 is a high affinity calcium storage protein in the sarcoplasmic reticulum that was a ↑DEP and ↓DEG in RER-susceptible Thoroughbred mares and a ↓DEG in RER-susceptible Thoroughbred geldings between episodes of rhabdomyolysis in previous studies [ 7 , 51 ]. Enhanced sarcoplasmic reticulum calcium stores and post-translational modulation of RYR1 calcium release by oxidation (S-nitrosylation) or beta-adrenergic-induced phosphorylation are potential mechanisms that could enhance RYR1 calcium release and trigger an episode of rhabdomyolysis.…”

“…In further agreement with the study of acute rhabdomyolysis in French Trotters, numerous mitochondrial genes were downregulated in RER-susceptible horses in our study [ 9 ]. In the acute rhabdomyolysis study, this was interpreted to indicate a decrease in mitochondrial protein expression arising from mitochondrial buffering of calcium as was found in Thoroughbreds susceptible to RER [ 7 , 9 ]. Our proteomic analysis, however, found that 89% (17/19) of the DEP mitochondrial proteins were upregulated and the genes encoding 11 of these proteins had significantly downregulated gene expression.…”

“…The expression of RER is impacted by a genetic predisposition coupled with environmental factors that complicate the identification of susceptibility loci in both breeds [ 4 , 5 , 6 ]. A lack of a defined genetic predisposition could be due to a polygenic mode of inheritance with small effect size for RER susceptibility, allele specific expression, transcriptional or translational modifications, alternate splicing, or selective isoform expression induced in a race training environment [ 4 , 7 ].…”

“…This type of transcriptomic and proteomic analysis was recently performed in Thoroughbred racehorses susceptible to, but not experiencing clinical signs of rhabdomyolysis. Results of that study identified increased protein and decreased gene expression in pathways of calcium regulation and mitochondrial function between episodes of rhabdomyolysis [ 7 ]. An underlying cause for RER was hypothesized to be environmental stressors causing post-translational modification of the calcium release channel (RYR1) that triggered excessive release of sarcoplasmic reticulum calcium stores, muscle contracture, mitochondrial calcium buffering and damage [ 7 ].…”

The Impact of N-Acetyl Cysteine and Coenzyme Q10 Supplementation on Skeletal Muscle Antioxidants and Proteome in Fit Thoroughbred Horses

“…Skeletal muscle glutathione concentrations were measured previously in several species with wide-ranging results depending on the method of analysis ( Table S1 ) [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ]. When predominantly fast-twitch muscle such as the human vastus lateralis or “quadriceps” (~60% fast-twitch muscle fiber; glutathione 1.2–1.4 nmol/mg wet weight) [ 42 ] were compared to Thoroughbred gluteal muscle (~80% fast-twitch muscle fibers 0.85 nmol/kg wet weight) [ 43 ] measured by HPLC, horses appeared to have approximately 40% lower glutathione concentrations than humans. This could be in part be due to the higher fast-twitch fiber type composition of horse gluteal muscle compared to human vastus lateralis or, intriguingly, equine muscle could have a lesser capacity to synthesize glutathione or an enhanced turnover of glutathione with exercise.…”