Oxygen deprivation for prolonged periods leads to cardiac cell death and
ventricular dysfunction. The ability to prevent myocardial cell death would be of significant therapeutic value in maintaining cardiac function after injury. While
caspases have been suggested to play a critical role in apoptosis, their involvement during hypoxic injury has not been formally determined. In this report, we show that adult ventricular myocytes subjected to
hypoxia for 1 h undergo a three-fold increase (P<0.05) in the incidence of apoptosis as determined by TUNEL analysis and
Hoechst 33258 nuclear staining. Western blot analysis of hypoxic myocytes revealed a 10-fold increase in the proteolytic processing of
caspase 3 to p17 with a concomitant cleavage of the
caspase 3 substrate PARP from 116 kd to p85 kd compared to normoxic controls. Defects in mitochondrial membrane integrity were also observed as evidenced by the translocation of
cytochrome c from the mitochondrial to cytosolic compartment of hypoxic cells. Pretreatment of ventricular myocytes with the
peptide-
caspase inhibitor known to block
caspases related to
caspase 1 (
Ac-YVAD-CHO) attenuated
cytochrome c release, processing of
caspase 3, and apoptosis. While the
caspase inhibitor (
Ac-DEVD-CHO) which blocks
caspases related to
caspase 3, suppressed the cleavage of PARP and apoptosis, it had no effect on
cytochrome c release by mitochondria. The data provide direct evidence for the proteolytic activation of
caspases during
hypoxia-mediated apoptosis of adult ventricular myocytes. Furthermore, the data suggest a hierarchical scheme for
caspase activation with mitochondrial
cytochrome c release occurring proximally to
DEVD-CHO-inhibitable
caspases.