Conserved skeletal muscle transcriptomic responses to pacing strategies in Thoroughbred horses

This study investigated whether different pacing patterns during high-intensity exercise elicit distinct transcriptional responses in equine skeletal muscle. Eight Thoroughbred horses completed two treadmill exercise sessions in a randomized crossover design. In the positive-pacing condition, horses exercised at 110% maximal O₂ uptake (V˙O₂max) for 1 min followed by 90% V˙O₂max for 1 min, whereas in the negative-pacing condition, the order was reversed. At 4 h after exercise, the positive-pacing protocol resulted in upregulation of 1,989 genes and downregulation of 840 genes, whereas the negative-pacing protocol resulted in upregulation of 1,710 genes and downregulation of 593 genes (false discovery rate <0.05; fold change ≥1.5). Despite these differences, most exercise-responsive genes and pathways related to hypoxia signaling, extracellular matrix remodeling, and metabolic regulation were shared between protocols. A direct comparison of gene expression between the two protocols identified four genes with higher expression after positive pacing, including RP1, MORN5, and two unannotated equine transcripts (ENSECAG00000060378 and ENSECAG00000057614), whereas five genes (OLFML2B, POSTN, ANGPTL1, MAP1A, and ZNF554) showed higher expression after negative pacing. These findings indicate that the skeletal muscle transcriptomic response to workload-matched high-intensity exercise is largely conserved between pacing strategies in Thoroughbred horses, with only limited pacing-dependent differences.