Abstract 209: Role Of Apoptosis Inducing Factor In Caspase-independent Apoptosis In Hypertrophic Cardiomyocytes
Caspases are thought to be central mediators of the apoptotic program, but recent data indicate that apoptosis can also be activated by caspase-independent mechanisms, such as via apoptosis inducing factor (AIF). We and others have shown that the hypertrophic heart may be more prone to apoptotic insult. Thus, we seek to investigate the characteristics of AIF-induced cardiac apoptosis in hypertrophic cardiomyocytes in vitro and describe its role.
Method: Hypertrophic (H-CMs) and non-hypertrophic cardiomyocytes (N-CMs) were isolated from Dahl salt sensitive rats fed a high salt or a normal diet for 6 weeks, respectively; exposed to apoptotic stimulation, such as hypoxia/reoxygenation (H/R) or staurosporine (ST); and analyzed for the activation of caspases, AIF and cytochrome c release from mitochondria, and the rate of apoptosis.
Results: Both H-CMs and N-CMs responded to H/R and ST with a significant release of AIF (evidence of AIF activation), cytochrome c (evidence of caspase-dependent apoptosis), and apoptosis. Apoptotic stimuli did not cause significant caspase activation in H-CMs, but the release of AIF from the mitochondria in these cells was a significant 2.5 times greater than in N-CMs. These results suggest that AIF-induced apoptosis is more prominent in H-CMs. We also found that in N-CMs, inhibiting caspase by adenoviral overexpression of CrmA (a known caspase inhibitor), or pre-treatment with zVAD-fmk (a pan-caspase inhibitor) significantly blocked apoptosis after H/R and ST (39% and 66%, p<0.05 for both). Caspase inhibition did not block the release of AIF from mitochondria in H-CMs, however, and failed to provide significant protection against apoptosis. Finally, pre-treatment with 4-amino-1,8-napthalimide (4-AN), a potent inhibitor of PARP (an important regulator of caspase-independent apoptosis), completely blocked the release of AIF (98%, p<0.01), and apoptosis in H-CMs (54%, p<0.05), but not in N-CMs (11%, p<NS).
Conclusion: Apoptotic mechanisms in hypertrophic cardiomyocytes appear to be biased towards caspase independent pathways, and therefore the inhibition of AIF-induced apoptosis may be an effective anti-apoptotic therapy in hypertrophic heart.