Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion

Abstract

Oxidants such as reactive oxygen species (ROS) have been shown to participate in myocardial ischemia/reperfusion injury. While many studies report a burst of ROS at reperfusion, few reports have presented evidence of significant ROS generation during ischemia. Our previous studies of cultured cardiomyocytes indicated that antioxidants are most effective when given prior to reperfusion during ischemia. Therefore, we hypothesized that significant ROS generation may occur during ischemia prior to reperfusion. We tested this in a perfused isolated cardiomyocyte system (i.e. without neutrophils, endothelial cells, or xanthine/xanthine oxidase) during simulated ischemia/reperfusion while measuring oxidant generation using intracellular fluorescent probes. During ischemia, the ROS probes dihydroethidium and 2',7'-dichlorofluorescin were significantly oxidized, suggesting superoxide and H2O2 generation. At reperfusion following 1 h ischemia, these probes suggested a further burst of H2O2 and hydroxyl radicals. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline used during ischemia attenuated oxidant generation, increased cell viability, and improved return of contraction after ischemia. To further evaluate the relationship between residual O2 and ROS generation, we administered O2 scavengers during ischemia and measured corresponding changes in oxidant generation, cell viability and contraction during reperfusion. Enzymatic scavenging of residual O2 during ischemia (reducing PO2 from 3.5 to 2.5 tau) paradoxically improved subsequent viability and contraction. These results indicate that cultured cardiomyocytes generate significant ROS during ischemia. This ROS generation is related to residual O2 present during ischemia and contributes significantly to the cellular injury seen at reperfusion.