Abstract 1322: Bnip3 Mediates Mitochondrial Fragmentation and Cell Death via Sarco/Endoplasmic Reticulum
Myocardial ischemia and reperfusion (I/R) is associated with increased mitochondrial fragmentation and cell death. We previously found that the mitochondrial death protein Bnip3 contributes to I/R injury by perturbing mitochondrial function. In this study, we investigated the mechanism(s) by which Bnip3 causes mitochondrial dysfunction. We utilized transfections of HL-1 myocytes coupled with fluorescence deconvolution microscopy for the analysis of Bnip3 function. Imaging of cells transfected with Mito-DsRed to label mitochondria allowed us to monitor the dynamics of mitochondrial morphology. Cell death was measured by assessing plasma membrane permeability to fluorescent dyes. Under normal conditions, cells contain a tubular connected network of mitochondria. We found that overexpression of Bnip3 dramatically altered mitochondrial morphology resulting in small round fragments of mitochondria which correlated with increased cell death. Bcl-2 is an anti-apoptotic protein which localizes to mitochondria, endoplasmic reticulum and nuclear envelope in cells and has been shown to protect against Bnip3-mediated cell death. Interestingly, we found that targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER) by replacing the Bcl-2 transmembrane domain with the C-terminal targeting sequence from cyt b5 also protected against cell death by Bnip3. Examination of mitochondrial morphology in HL-1 myocytes overexpressing S/ER-Bcl-2 and Bnip3 revealed that targeting Bcl-2 to the S/ER also reduced Bnip3-mediated mitochondrial fragmentation. Subcellular fractionation of heart lysates coupled with Western blotting for endogenous Bnip3 and co-localization studies by immunocytochemistry of HL-1 cells overexpressing myc-Bnip3 and FLAG-S/ER-Bcl-2 confirmed that Bnip3 and S/ER-Bcl-2 did not co-localize. Bnip3 localized to the mitochondria whereas S/ER-Bcl-2 localized only in the S/ER. Targeting Bcl-2 to the S/ER has been shown to protect against cell death by depletion of S/ER Ca2+ stores. Since Ca2+ has been shown to induce mitochondrial fragmentation and cell death, these studies suggest that activation of Bnip3 in the mitochondria may trigger the release of Ca2+ from the S/ER which leads to mitochondrial fragmentation and cell death.