Abstract 2572: Doxorubicin Induces Caspase-independent Apoptosis in Transgenic Mice Overexpressing CrmA
Apoptosis is implicated in the development of heart failure, and caspases have been the central mediators that cause apoptosis. However, recent evidences suggest that caspase-independent apoptosis may be activated in setting of caspase inhibition, and the contribution of caspase-independent apoptosis to the development of heart failure is not known. In this study, we examined the doxorubicin-induced cardiac apoptosis in transgenic (Tg) mice expressing a cowpox viral serpin protein, cytokine response modifier A (CrmA) that is known to inhibit caspases. Male wild-type (WT) and CrmA Tg mice (~3-mo of age) were i.p. injected with 20 mg/kg doxorubicin. We observed significant mortality benefit at 6 days after the injection in CrmA Tg mice (p<0.05), but after 12 days there was no mortality benefit compared to the WT mice. The quantitative analysis of cardiac apoptosis using terminal deoxynucleotidyl trans-ferase (TdT)-mediated dUTP nick end labeling (TUNEL) showed significant but similar amount of increase in cardiac apoptosis in both WT and CrmA Tg mice in response to doxorubicin. We hypothesize that the initial benefit was due to caspase inhibition in CrmA Tg mice, but there was activation of caspase-independent apoptosis after 12 days in CrmA Tg mice. Indeed, caspase activity assays showed that caspase-3 and caspase-8 were activated only in WT but not in CrmA Tg mice heart after doxorubicin treatment. To determine if caspase-independent apoptosis was activated, we measured the cytosolic release of caspase-independent apoptotic factors, which normally reside in mitochondria but are released to cytosol in response to apoptotic stimulation. We found that there was significant release of AIF and Omi/HtrA2 from mitochondria to cytosol suggesting activation of caspase-independent apoptosis in CrmA Tg mice, but not in WT mice. These findings suggest that apoptosis induced by doxorubicin occurs even in the heart that is lacking of caspase activation possibly through activation of caspase-independent apoptotic mechanism. We speculate that inhibition of both caspase-dependent and -independent apoptosis are needed for complete inhibition of apoptosis in heart during apoptotic stimulation.