Abstract
Caspase inhibition is thought to be an effective strategy for inhibiting apoptosis in heart. Recent data, however, suggest that caspase-independent apoptosis might be activated when caspases are inhibited. Here, we examined whether caspase-independent apoptosis involving apoptosis inducing factor (AIF) is important in the development of heart failure in the setting of caspase inhibition in vivo. Method: We created transgenic mice with cardiac specific overexpression of cytokine response modifier A (CrmA), a known inhibitor of caspases, using αMHC promoter (CrmA Tg mice). A single dose of doxorubicin (DOX; 20 mg/kg) was injected i.p. into male mice to generate acute cardiomyopathy. A quantitative analysis of cardiac apoptosis was performed using TUNEL assay. Results: Caspase activity was completely inhibited in CrmA Tg mice hearts 6 days after DOX injection, whereas the WT hearts showed significant caspase activation. We also observed significantly improved survival in CrmA Tg compared to WT mice (81% vs 38%, p<0.05) 6 days after DOX injection. However, at 12 days, CrmA Tg and WT mice had a similar rate of cardiac apoptosis, and the survival benefit did not persist. Further analysis revealed that CrmA Tg but not WT mice hearts were characterized by a significant release of AIF from the mitochondria at 12 days. This evidence of AIF-induced apoptosis suggests a late activation of AIF in the setting of caspase inhibition. CrmA Tg mice were also pre-treated with 4-AN (4-amino-1,8-napthalimide, 3 mg/kg), a potent inhibitor of PARP, and examined 12 days after DOX injection. PARP is an important regulator of AIF-induced apoptosis, and PARP inhibition significantly improved DOX-impaired cardiac function (+91%, p<0.01) and survival compared to CrmA Tg mice without 4-AN (93% vs. 13%, p<0.05). In addition 4-AN pretreatment resulted in a significant inhibition of PARP activity, blockade of AIF released from mitochondria, and less apoptosis compared to CrmA Tg without 4-AN. Conclusion: In the setting of caspase inhibition, apoptosis can be induced through caspase-independent mechanisms, predominately via AIF, in a model of heart failure in vivo . The concurrent inhibition of AIF activation provides a more effective and complete inhibition of apoptosis.
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