Furosemide does not enhance mitochondrial capacities and ATP production in mature Thoroughbred horses

Furosemide, a loop diuretic widely used to mitigate exercise-induced pulmonary hemorrhage in performance horses, remains controversial due to uncertainty over whether it serves purely as a welfare measure or also enhances performance. The objective of this study was to test the hypothesis that repeated furosemide administration would impair mitochondrial respiratory capacities and adenosine triphosphate (ATP) production in mature Thoroughbred horses. Horses were matched by body weight and randomly assigned to receive either 250 mg furosemide (FUR; 5 females, 4 castrated males; mean ± SD 4.1 ± 0.8 yr) or an equal volume of saline (CON; 3 females, 3 castrated males; 3.8 ± 0.4 yr) intravenously once weekly for 14 wk. All horses completed standardized treadmill exercise training three times weekly to develop and maintain fitness. At wk 14, gluteus medius muscle samples were collected. Samples were flash frozen for determination of citrate synthase (CS) activity as a proxy for mitochondrial volume density by kinetic colorimetry. Additional tissue was maintained in biopsy preservation solution at 4C and divided to prepare (1) mechanically teased, chemically permeabilized fibers and (2) isolated mitochondria. High-resolution respirometry (permeabilized fibers) and fluororespirometry (isolated mitochondria) were performed using an Oroboros O2k within 24 h of collection. Data were analyzed by one-way ANOVA. Mitochondrial volume density (CS activity) did not differ among groups. In permeabilized fibers, nonphosphorylating leak respiration tended to be greater in FUR than CON horses (P < 0.01) only when normalized to CS activity and expressed relative to maximal noncoupled electron transfer capacity (EN+S). This difference was not present when leak was normalized to tissue wet weight. No other respiratory capacity, including NADH-linked oxidative phosphorylation supporting complex I (PN), maximal P with substrates for complexes I and II (PN+S), maximal noncoupled EN+S, or E with complex II substrate (succinate) alone (ES), was impacted by furosemide treatment. In isolated mitochondria, ATP production relative to oxygen consumption during maximal PN+S tended to be lower in FUR than CON horses (P = 0.10), suggesting a possible reduction in energy-production efficiency. However, given the absence of effects on respiratory capacities or other ATP production metrics, the biological relevance of this trend is unclear and warrants further investigation. Overall, these findings support that repeated furosemide administration does not enhance skeletal muscle mitochondrial function or ATP-generating capacity in mature Thoroughbred horses and therefore does not appear to act as a performance-enhancing agent through this mechanism. Funding: Supported in part by the Horseracing Integrity and Safety Authority (HISA). The funders had no role in study design, data analysis, or interpretation. This abstract was presented at the American Physiology Summit 2026 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.