Ischemia-reperfusion injury induces cell death, but the responsible mechanisms are not understood. This study examined mitochondrial depolarization and cell death during
ischemia and reperfusion. Contracting cardiomyocytes were subjected to 60-min
ischemia followed by 3-h reperfusion. Mitochondrial membrane potential (DeltaPsi(m)) was assessed with
tetramethylrhodamine methyl ester. During
ischemia, DeltaPsi(m) decreased to 24 +/- 5.5% of baseline, but no recovery was evident during reperfusion. Cell death assessed by
Sytox Green was minimal during
ischemia but averaged 66 +/- 7% after 3-h reperfusion.
Cyclosporin A, an inhibitor of mitochondrial permeability transition, was not protective. However, pharmacological
antioxidants attenuated the fall in DeltaPsi(m) during
ischemia and cell death after reperfusion and decreased lipid peroxidation as assessed with C11-BODIPY. Cell death was also attenuated when residual O(2) was scavenged from the perfusate, creating anoxic
ischemia. These results suggested that
reactive oxygen species (ROS) were important for the decrease in DeltaPsi(m) during
ischemia. Finally, 143B-rho(0)
osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of DeltaPsi(m) during
ischemia and were significantly protected against cell death during reperfusion. Collectively, these studies identify a central role for mitochondrial ROS generation during
ischemia in the mitochondrial depolarization and subsequent cell death induced by
ischemia and reperfusion in this model.