Abstract 628: Pro-Apoptotic Bnip3 Functions as a Mitochondrial Sensor of Oxidative Stress in Myocardial Ischemia/Reperfusion
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, but it is not known how I/R causes activation of Bnip3. We have investigated potential mechanism(s) by which Bnip3 activity is regulated. Western blot analysis of heart lysates revealed that Bnip3 forms a ∼48 kDa complex after I/R that is sensitive to reduction by DTT. Complex formation was reduced when hearts were perfused with the reactive oxygen species (ROS) scavenger N-acetyl cysteine prior to I/R. The complex also increased in isolated myocytes treated with hydrogen peroxide, suggesting that the DTT-sensitive complex is formed by increased oxidative stress. To further investigate the function of this complex, we overexpressed Bnip3 in HL-1 myocytes and found that most of Bnip3 existed in the 48-kDa complex which correlated with increased cell death. In contrast, endogenous Bnip3 in the heart exists mainly as a monomer under normal conditions. Scanning of the Bnip3 protein sequence revealed a single conserved Cys residue at position 64, which may be susceptible to oxidation. Mutation of the Cys to an Ala or deletion of the N-term (aa 1–64) resulted in reduced cell death activity of Bnip3 compared to wild type when overexpressed in HL-1 myocytes. Separation of purified Bnip3 on SDS-PAGE showed the presence of the same DTT sensitive complex, suggesting that the complex is a Bnip3 homodimer. The transmembrane (TM) domain of Bnip3 has previously been identified to be important for homodimerization, and contains a His residue at position 173 that is essential for homodimerization. Mutation of the His to an Ala also resulted in reduced cell death activity of Bnip3 compared to wild type when overexpressed in HL-1 myocytes, suggesting that homodimerization is important for cell death activity of Bnip3. A consequence of I/R is the production of ROS and oxidation of proteins, which promotes formation of Cys disulfide bonds between proteins. Thus, these studies suggest that Bnip3 functions as a redox sensor in cells where increased oxidative stress induces homodimerization of Bnip3 via N-terminal Cys residue and the C-terminal TM domain.