Comparison of Respiratory Capacities of Permeabilized Muscle Fibers and Isolated Mitochondria in Exercising Horses

Consensus within the research community suggests that permeabilized skeletal muscle fibers (pfi) better maintain complex cellular environments allowing for greater confidence in respiration measures, by comparison to isolated mitochondria. Isolated mitochondria (Mt), however, offer the advantage of greater reproducibility between technical replicates compared to pfi. Further, to date, high-resolution fluororespirometry analyses in an Oroboros O2k ( eg., adenosine triphosphate and H 2 O 2 production) must be performed in Mt due to interference of pfi membranes with fluorophores. Obtaining reliable, accurate respiration measures is especially important when comparing subjects of varying degrees of fitness for diagnostic purposes or in disease conditions where mitochondrial damage might be expected. We hypothesized that gluteus medius mitochondrial oxidative phosphorylation capacities (P) would be comparable between pfi and isolated mitochondria (Mt) both before and after fatiguing exercise. Adult castrated male Thoroughbred horses (n = 12; mean ± SD 8 ± 2 yr; 510 ± 35 kg) performed repeated standardized exercise-to-exhaustion tests (SET) on d 1, 3, and 5 before (wk0) and after (wk9) 8 wk of moderate intensity conditioning. Gluteus medius biopsies were retrieved on d 0, 2, and 4 of each SET wk for pfi and Mt P measurements by high-resolution respirometry. Outputs for pfi were integrative (relative to mg tissue) and intrinsic (relative to CS activity) leak, P with complex I (P CI ), and maximal P with CI and complex II (P CI+II ); for Mt, outputs were integrative (relative to volume) and intrinsic (relative to protein in the suspension) leak, P CI , and P CI+II . Using SET times, horses were classified as fatigue responsive (RESP, n = 6 at wk0, n = 4 at wk9) with declining sequential SET times, or non-responsive (NON, n = 6 at wk0, n = 7 at wk9), no change in SET times. Pearsons’s correlation coefficients were calculated for the respiration measures using SASv9.4. There were no relationships between pfi and Mt in RESP horses at wk0. In NON horses, pfi and Mt integrative leak were positively correlated on d 0 (r = 0.97; P = 0.03), and pfi integrative and Mt intrinsic P CI (r = 0.88) and pfi and Mt phosphorylation control of CI (r = -0.88) showed relationships on d 4 ( P = 0.02) of wk0. At wk9, pfi intrinsic and Mt integrative leak on d 0 (r = -0.91), pfi integrative and Mt intrinsic P CI+II on d 2 (r = -0.93), and pfi intrinsic and Mt integrative P CI+II on d 4 (r = -0.91) tended to be negatively correlated in RESP horses ( P < 0.1). Finally, on d 2 of wk9, pfi and Mt integrative P CI were positively correlated (r = 0.81; P = 0.03) and pfi and Mt intrinsic P CI (r = 0.73), pfi integrative and Mt intrinsic P CI+II (r = 0.74), and pfi and Mt intrinsic P CI+II (r = 0.72) tended to be correlated in NON horses ( P < 0.1). Overall, there was poor agreement between mitochondrial respiratory capacities measured in pfi and Mt from the same biopsy. Greater sample type agreement was observed in NON compared to RESP horses, however, possibly due to mitochondrial damage that compromises healthy organelle isolation in RESP horses. United States Department of Agriculture Award 2024-67015-42246, the Texas A&M AgriLife Institute for Equine Sciences, and the Link Endowed Chair in Equine Science This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.