Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation

Abstract

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 (ΔΨ m) was assessed with tetramethylrhodamine methyl ester. During ischemia, ΔΨ 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 ΔΨ 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 ΔΨ m during ischemia. Finally, 143B-ρ 0 osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of ΔΨ 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.