Although
ischemia-reperfusion (I/R) can initiate apoptosis, the timing and contribution of the mitochondrial/
cytochrome c apoptosis death pathway to I/R injury is unclear. We studied the timing of
cytochrome c release during I/R and whether subsequent
caspase activation contributes to
reperfusion injury in confluent chick cardiomyocytes. One-hour simulated
ischemia followed by 3-h reperfusion resulted in significant cell death, with most cell death evident during the reperfusion phase and demonstrating mitochondrial
cytochrome c release within 5 min after reperfusion. By contrast, cells exposed to prolonged
ischemia for 4 h had only marginally increased cell death and no detectable
cytochrome c release into the cytosol.
Caspase activation could not be detected after
ischemia only, but it significantly increased after reperfusion.
Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl
ketone, Ac-Asp-Gln-Thr-Asp-H, or benzyloxycarbonyl-Leu-Glu (Ome)-His-Asp-(Ome)-fluoromethyl
ketone given only at reperfusion significantly attenuated cell death and resulted in return of contraction. Antixoxidants decreased
cytochrome c release, nuclear condensation, and cell death. These results suggest that reperfusion
oxidants initiate
cytochrome c release within minutes, and apoptosis within hours, significant enough to increase cell death and contractile dysfunction.