Abstract 1369: Bnip3 Mediates Mitochondrial Dysfunction via Two Distinct Pathways in Cardiac Cells
Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with mitochondrial dysfunction and cell death in the myocardium. We previously found that Bnip3 contributes to ischemia/reperfusion (I/R) injury and in this study we investigated the mechanism of Bnip3-mediated mitochondrial dysfunction during I/R. Using 3-D fluorescent imaging of HL-1 myocytes overexpressing GFP-Bax, we found that overexpression of the dominant negative Bnip3ΔTM reduced Bax translocation to the mitochondria by ~45% during simulated I/R (sI/R) (n=3, p<0.05). Moreover, Bnip3 overexpression alone resulted in increased activation of Bax by ~48% and Bak by ~56% (n=3, p<0.05) which correlated with loss of mitochondrial membrane potential (ΔΨ m) as measured by TMRM. Bnip3-mediated mitochondrial dysfunction was reduced by overexpression of Bcl-2 or treatment with Cyclosporin A (CsA), a permeability transition pore (PTP) inhibitor, at 24 h posttransfection (n=3, p<0.05). However, a time-course study revealed that Bcl-2 and CsA only delayed the onset of mitochondrial dysfunction and at 48 h posttransfection, >85% of Bnip3 expressing cells had lost their ΔΨm even in the presence of Bcl-2 or CsA. To further investigate the role of Bax and Bak in Bnip3-mediated mitochondrial dysfunction, we overexpressed Bnip3 in mouse embryonic fibroblasts (MEFs) derived from mice deficient in Bax and Bak. Bax/Bak deficient MEFs were initially resistant to Bnip3-mediated mitochondrial dysfunction, but by 72 h posttransfection >90% of cells displayed loss of ΔΨm (n=3, p<0.05). Interestingly, overexpression of Bcl-2, treatment with CsA or zVAD-fmk, a broad spectrum caspase inhibitor, did not protect against Bnip3-mediated loss of ΔΨ m in cell lacking Bax and Bak. These data suggest that Bnip3 mediates mitochondrial dysfunction through two distinct pathways: one early activated pathway through opening of PTP and activation of Bax/Bak and a second delayed Bax/Bak independent pathway. Both Bnip3 and Bcl-2 have been reported to be upregulated during heart failure. Thus, an anti-apoptotic response by Bcl-2 may be futile in protecting the heart against mitochondrial dysfunction by Bnip3 due to activation of the delayed Bax/Bak independent cell death pathway.