Abstract P21: Deletion of Caspase-2 Inhibits Ischemia/Reperfusion-induced Oxidant Burst and Apoptosis in Cardiomyocytes
Introduction: Caspase-2 is one of the most conserved caspases across animal species and shares structural and functional features of both the initiator caspases such as caspases (casp)-8 or -9 and the more downstream effector caspases such as casp-3. We have demonstrated that ischemia/reperfusion (I/R) in cardiomyocytes induces a mitochondrial pathway of apoptosis, demonstrating rapid casp-2 activation and cytochrome (cyt) c release, which can be blocked by inhibition of casp-2 activity. Here, we report the effects of specific inhibition via siRNA “knock-down” of casp-2 and casp-8 in chick cardiomyocytes and casp-2 knockout (C2−/−) in murine cardiomyocytes on I/R-induced mitochondrial oxidant generation and cell death.
Methods: Cardiomyocytes, isolated from chick embryos or 1–2-day old B6129 mice, were exposed to 60 or 90 min simulated ischemia and 1 to 3 h reperfusion. Inhibitors were added at the beginning of reperfusion. Cell viability was evaluated by propidium iodide, and oxidant production was measured using 6-carboxy-2′, 7′-dichloro-dihydrofluorescein diacetate (6-carboxy-H2DCFDA). Mitochondrial integrity was assessed using MitoTracker Red CMXH2Ros.
Results: Specific depletion of casp-2 mRNA (as confirmed by RT-PCR) inhibited I/R-induced cell death (23.1±9.07% vs. 52.4±7.39%) in chick cells, while casp-8 mRNA depletion did not. Similarly, I/R-induced death in C2−/− cells was reduced from 49.2±4.25% in wild-type (wt) to 35.4±2.95% (p<0.05). Analyses of caspase activity suggested that the residual death in C2−/− cells was due to compensatory actions of casp-3. In chick cells, casp-2 inhibition also blocked the mitochondrial reperfusion ROS burst by more than 80%, as measured by DCF fluorescence, but inhibition of casp-8 or casp-3 had no effect. In murine cardiomyocytes, C2−/− cells also exhibited significantly reduced oxidant generation compared to wt cells (14.66±1.10 a.u. to 7.69±1.72 a.u., p<0.05). Blockade of casp-2, but not of casp-8, also preserved mitochondrial integrity after reperfusion.
Conclusion: Together, these data support a general role for casp-2 upstream of the mitochondria, with effects on critical I/R-induced apoptotic events such as mitochondrial ROS generation, mitochondrial integrity and cyt c release.