Mitochondrial content and intrinsic coupling‐related differences shape bioenergetic output and redox responses across striated muscles in rats

Abstract

Striated muscles differ markedly in energetic demand, but whether mitochondrial respiration and reactive oxygen species emission differ intrinsically across muscle types remains unclear. This study compared mitochondrial respiration, hydrogen peroxide (H ₂ O ₂ ) emission and antioxidant enzyme expression in heart, diaphragm and hindlimb skeletal muscle from healthy rats. Mitochondrial O ₂ flux and H ₂ O ₂ emission were measured simultaneously using high‐resolution respirometry coupled with fluorometry in tissue homogenates subjected to substrate‐uncoupler‐inhibitor titration protocols. Mitochondrial content was estimated by citrate synthase activity, and antioxidant enzyme expression was measured by immunoblotting. Heart tissue displayed the highest mitochondrial respiration and H ₂ O ₂ emission when expressed per tissue mass, followed by diaphragm and skeletal muscle. However, normalisation by citrate synthase activity eliminated differences in respiratory flux between muscles, indicating that variations in tissue‐level respiration were primarily driven by mitochondrial content. Net H ₂ O ₂ emission was greatest during leak respiration and decreased during oxidative phosphorylation and uncoupled states in all muscle types. Cardiac mitochondria exhibited a higher H ₂ O ₂ /O ₂ ratio during leak respiration and higher coupling‐related ratios than those of diaphragm and skeletal muscle. In association, cardiac muscle showed greater expression of the mitochondrial antioxidant enzymes superoxide dismutase 2 and catalase in a separate cohort. These findings indicate that absolute oxidative metabolism across striated muscles is largely determined by mitochondrial content, whereas respiratory capacity per mitochondrial unit was conserved. Cardiac muscle exhibits elevated leak‐associated reactive oxygen species emission but compensates by enhancing antioxidant defences, highlighting coordination between bioenergetic demand and redox regulation in continuously active muscles.