Impacts of repeated fatiguing exercise on mitochondria in Thoroughbreds

Abstract

Racehorses are exceptional athletes but may be prone to fatigue-induced injury. Quantification of fatigue remains elusive, posing a significant hurdle to predict injury susceptibility. We hypothesized that gluteus medius mitochondrial metrics would decrease and production of H₂O₂, a reactive oxygen species, would increase as horses performed repeated fatiguing exercise. Before (wk 0) and after (wk 9) 8 wk of moderate intensity training, 12 mature, unfit Thoroughbred geldings (mean ± SD 8 ± 2 yr; 510 ± 35 kg) performed standardized exercise-to-fatigue tests (SET) on d 1, 3, and 5. Isolated mitochondrial oxidative phosphorylation capacities (P) and production of adenosine triphosphate (ATP) and H₂O₂ were quantified in muscle samples collected on d 0, 2, 4, and 6 of each SET week. Data were analyzed by PROC MIXED with repeated measures in SAS v9.4; fixed effects were day, week, and day × week. At wk 0, LEAK and maximal P (P_CI+II) decreased by d 6 (P = 0.02) while P with complex I (P_CI) increased on d 2 (P = 0.04) but dropped below d 0 at d 6 (P = 0.002); P with complex II (P_CII) also increased on d 2 (P = 0.05) but returned to d 0 levels by d 4. At wk 9, LEAK was unchanged while P_CI, P_CI+II and P_CII were greater on d 4 than 2 (P < 0.04) but no day differed from d 0. LEAK on d 2 was greater at wk 0 than 9 (P = 0.01). On d 0 and 2, P_CI, P_CI+II, and P_CII were greater at wk 0 than 9 (P ≤ 0.008). Maximal (CI+II) ATP flux at wk 0 increased from d 0 to 2 (P = 0.0007), decreased to d 0 levels at d 4 (P = 0.03), and was similar to d 2 and 4 but greater than d 0 (P = 0.01) at d 6. ATP flux through CI and CII individually was stable through wk 0. At wk 9, CI ATP flux was greater at d 2 than 4 and 6 (P < 0.007) but none differed from d 0. Maximal ATP flux at wk 9 decreased at d 2 (P = 0.0007) but returned to d 0 levels by d 4, and ATP flux through CII was unchanged. Complex I ATP flux on d 2 was greater at wk 9 than 0 (P = 0.005) and maximal ATP flux on d 2 and 6 were lower at wk 9 than 0 (P < 0.03). Overall, H₂O₂ flux was greater during P_CI+II (P = 0.04) and tended to be greater during P_CI (P = 0.07) at wk 9 than 0. Across wk 0 and 9, H₂O₂ flux relative to O₂ flux (H₂O₂/O₂) increased on d 4 during LEAK and P_CI+II (P < 0.05) and was greater on d 6 during LEAK, P_CI, and P_CI+II (P < 0.05). H₂O₂/O₂ during P_CII increased on d 4 at wk 0 (P = 0.01) but returned to d 0 levels on d 6, while decreasing on d 2 at wk 9 (P = 0.04) but returning to d 0 levels by d 4. In partial support of our hypothesis, repeated SETs impaired mitochondrial respiratory capacities in an untrained state, which appeared to be mitigated by training, but did not align with ATP production. Regardless of fitness, repeated SETs increased mitochondrial ROS production, which may be related to development of fatigue.