Mitochondrial ROS Production at Complexes I and III in Human Myocardium and Skeletal Muscle: A Distinct Pattern Compared with Rat Tissue

Mitochondrial reactive oxygen species (ROS) play a central role in cardiac ischemia/reperfusion injury, heart failure, and arrhythmogenesis, while also serving essential signaling functions under physiological conditions. Among the eleven identified mitochondrial ROS-producing sites, complexes I and III are considered the major contributors, particularly under conditions of impaired electron flow. However, much of the existing knowledge comes from rodent models or cultured cells and is often assumed to apply to humans. Here, ROS production from complexes I and III was measured directly in human myocardial and skeletal muscle biopsies and compared with corresponding rat tissues under identical experimental conditions. Hydrogen peroxide generation was quantified using Amplex UltraRed, with simultaneous monitoring of mitochondrial respiration using a Clark-type oxygen electrode. Across all examined mechanisms—reverse and forward electron transport at complex I and the ubiquinol oxidation site of complex III, rat tissues produced more ROS than human tissues, consistent with their higher respiratory rates. However, the dominant ROS-producing sites differed: in rats, complex III was the primary source, whereas in human tissues the highest ROS production occurred during reverse electron transport at complex I. When normalized to respiration, human tissues showed relatively greater ROS generation at complex I but markedly lower production at complex III. These direct measurements of mitochondrial ROS production in human myocardium provide new insight into cardiac redox physiology and may explain the limited clinical translation of cardioprotective strategies targeting mitochondrial ROS production, such as interventions aimed at modulating reperfusion injury or preconditioning.