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.