Pro‐Apoptotic Bnip3 Functions as a Mitochondrial Sensor of Oxidative Stress in Myocardial Ischemia/Reperfusion

Abstract

Bnip3 is a member of the BH3‐only subfamily of pro‐apoptotic Bcl‐2 proteins and is associated with mitochondrial dysfunction and cell death. We have investigated potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT‐sensitive complex in isolated myocytes treated with H2O2 and in hearts subjected to ischemia/reperfusion (I/R). Overexpression of Bnip3 in HL‐1 myocytes revealed that most of Bnip3 existed in the 48‐kDa complex which correlated with increased cell death. Bnip3 contains a single Cys at position 64 and mutation of the Cys or deletion of the N‐term (aa 1–64) caused a reduction in the cell death activity of Bnip3. Co‐immunoprecipitation studies and separation of purified Bnip3 on SDS‐PAGE showed the presence of the same DTT sensitive complex, suggesting that the complex is a Bnip3 homodimer. Mutation of a His residue in the C‐terminal transmembrane domain resulted in complete loss of homodimerization and cell death activity of Bnip3 compared to wild type. 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. This works was supported by grants from TRDRP (14KT‐0109) and NIH (HL087023).