Endurance exercise induces distinct skeletal and cardiac mitochondrial adaptations in racehorses

Mitochondrial respiration sustains the high energy demands of endurance exercise, yet the extent to which atrial, ventricular, and skeletal muscle mitochondria adapt remains uncertain. At the same time, endurance athletes face an increased risk of atrial fibrillation (AF), but the role of cardiac metabolism in arrhythmia susceptibility is poorly understood. Here, we compared mitochondrial respiration in skeletal muscle and across all four cardiac chambers between trained and untrained racehorses (<i>n</i> = 34) to investigate adaptations associated with long-term endurance exercise. We further examined whether cardiac metabolism was linked to AF propensity. All horses underwent treadmill performance testing, and mitochondrial respiration was assessed in permeabilized skeletal and cardiac muscle fibers. Cardiac RNA-sequencing and in vivo AF inducibility testing were performed in a subset of horses. Mitochondrial function varied by region: the left ventricle showed the greatest oxidative capacity, and the ventricles exceeded the atria in mitochondrial content. Trained horses showed improved skeletal complex I- and II-linked respiration, and skeletal muscle respiration correlated with aerobic performance. In contrast, cardiac mitochondrial content and mass-specific respiration were unchanged by endurance exercise, despite enrichment of mitochondrial complex I pathways on transcriptomic analysis. A greater cardiac capacity for fatty acid oxidation, but not mitochondrial respiration, was associated with protection against AF induction. These findings reveal tissue-specific mitochondrial adaptations to endurance exercise and implicate cardiac substrate preference, rather than respiratory capacity, as a potential determinant of AF vulnerability. This raises new questions about how different tissues adapt metabolically to exercise and the potential role of cardiac energetics in arrhythmogenesis.<b>NEW & NOTEWORTHY</b> This study reveals how endurance training shapes mitochondrial function in the heart and skeletal muscle. Using racehorses as a natural large-animal model, we compared mitochondrial respiration across all four cardiac chambers and skeletal muscle. Skeletal muscle mitochondria from trained horses showed greater respiratory capacity, whereas cardiac mitochondria did not. These findings uncover tissue-specific metabolic adaptations to exercise and highlight how cardiac energetics may influence susceptibility to atrial fibrillation in endurance athletes.