Abstract 252: Doxorubicin-induced H9c2 Cell Death is Mediated by Excessive Mitochondrial Fission and Mitophagy

Abstract

Doxorubicin (DOX) is a widely used antineoplastic agent that can cause heart failure. DOX cardiotoxicity is closely associated with mitochondrial damage. Mitochondrial fission and mitophagy are quality control mechanisms that help maintain a pool of healthy mitochondria. However, too much mitochondrial fission and/or mitophagy may compromise cell viability. Indeed, Mdivi-1, an inhibitor of the fission protein Drp1, can attenuate DOX-induced cardiac injury, suggesting that mitochondrial fragmentation may play a role in DOX cardiotoxicity. Using genetic gain- and loss-of function approaches, we determined whether mitochondrial fragmentation and/or mitophagy contribute to DOX-induced cardiomyocyte death. H9c2 cardiac myoblast cells were transfected with siRNA targeting Drp-1 before DOX administration. Mitochondrial morphology was examined with confocal microscopy after infection of the cells with the adenovirus encoding mitochondria-targeted fluorescent protein MitoDsRed. Morphometric analysis demonstrated that Drp-1 knockdown markedly diminished DOX-induced mitochondrial fragmentation as shown by form factor, aspect ratio, and mean mitochondrial size. This led to reduced cardiomyocyte death as revealed by the percentage of propidium iodide (PI)-positive cells and the cleavage of caspase-3 and Poly ADP ribose polymerase (PARP). Not surprisingly, Drp-1 knockdown also attenuated DOX-induced mitophagy flux as assessed by the dual fluorescent mitophagy reporter mt-Rosella. Further, knockdown of Parkin, a key regulator of mitophagy, dramatically diminished DOX-induced H9c2 cell death. Although Drp1 overexpression did not markedly increase DOX-induced cell death, Parkin overexpression predisposed H9c2 cells to DOX toxicity. Together, these results suggest that DOX-induced cardiotoxicity may be due to excessive mitochondrial fragmentation and accelerated mitochondrial degradation through autophagy. Strategies that limit mitochondrial fission and mitophagy within the physiological range may help reduce DOX cardiotoxicity. However, further studies are clearly warranted to make sure that these strategies will not compromise the antitumor efficacy of DOX.