Mitochondrial Ca 2+flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H 2O 2

Abstract

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H 2O 2 leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H 2O 2 treatment. At times ranging from 10 to 90 min after H 2O 2 treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca 2+ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H 2O 2 exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H 2O 2 exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca 2+ ([Ca 2+] m) using rhodamine 2 detected an early accumulation of [Ca 2+] m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H 2O 2-induced decline in complexes II and IV activities, indicating that [Ca 2+] m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca 2+] m flux represents an early pivotal event in H 2O 2-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.