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

Recurrent exertional rhabdomyolysis (RER) is a chronic muscle disorder of unknown etiology in racehorses. A potential role of intramuscular calcium (Ca 2+ ) 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 Ca 2+ 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 Ca 2+ 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 Ca 2+- 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 Ca 2+ release, showed no DEG compared to control horses. We conclude that RER-susceptibility is associated with alterations in proteins, genes and pathways impacting myoplasmic Ca 2+ 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 Ca 2+ release and subsequent mitochondrial buffering of excessive myoplasmic Ca 2+ .