Doxorubicin Cytotoxicity in Differentiated H9c2 Cardiomyocytes: Evidence for Acute Mitochondrial Superoxide Generation.

Although a mitochondrial redox-cycling superoxide-generating mechanism for the cardiotoxicity of doxorubicin was suggested from experiments with isolated mitochondria, its occurrence and contribution to cytotoxicity in intact cardiomyocytes is not fully established. Therefore, we determined the immediate and delayed effects of doxorubicin on the generation of reactive oxygen species (ROS) and cytotoxicity in differentiated H9c2 cardiomyocytes. Although relatively short incubations (3 or 6 h) with 1 or 5 µM doxorubicin did not acutely decrease cell survival, exposure to 5 µM doxorubicin for 3 h was sufficient to cause a significant delayed decrease in cell survival after an additional 24 h without doxorubicin. Mitochondrial superoxide generation was observed to increase within 30 min of incubation with 5 µM doxorubicin. Increased intracellular ROS generation, decreased mitochondrial metabolic activity, and decreased mitochondrial membrane potential (MMP) were observed after more extended periods (6-12 h). Overall, these observations support that the toxicity of doxorubicin to differentiated cardiomyocytes involves acute mitochondrial superoxide generation with subsequent intracellular ROS generation, mitochondrial dysfunction, and cell death.