Abstract P281: Bcl-2 Limits Ischemia-Reperfusion Injury of the Naive Myocardium by Preserving Mitochondrial Integrity and Respiratory Complex Functionality and Is Modulated by PKCε

Over-expression of Bcl-2 protects against myocardial ischemia/reperfusion (I/R) injury. Nevertheless, the participation of Bcl-2 in basal myocardium, and its subcellular targets under such conditions, remains elusive. Using a mouse line with an ablation of the Bcl-2 gene, we found that myocardial infarct size (IS) was exacerbated vs. wild type (WT) mice, demonstrating that Bcl-2 limit IS in basal I/R injury. The exacerbated IS in Bcl-2 KO was abolished by in vivo treatment with the selective Mitochondrial Permeability Transition (MPT) inhibitor cyclosporine A (10 mg/kg, iv) , while isolated cardiac mitochondria from Bcl-2 null mice exhibited increased matrix swelling in response to CaCl 2 , showing an increased susceptibility to MPT. However, recombinant Bcl-2 or PKCε were both sufficient to attenuate the increased susceptibility to MPT. Interestingly, spectrophotometric analysis of baseline activities of Mitochondrial Electron Transport Chain Complexes (ETC) I and V (but not of ETC II, III and IV), were increased in cardiac mitochondria from Bcl-2 null mice as compared to mitochondria from normal WT mice, demonstrating an altered mitochondrial respiratory complex functionality. In addition, immunoprecipitation with PKCε in AE-PKCε mouse hearts (i.e. mice with an increased activity of PKCε) followed by immunoblotting for Bcl-2, showed an 2-fold increased interaction between PKCε and Bcl-2. Similarly, immunoprecipitation with Phospho-Serine followed by immunoblotting for Bcl-2 indicated a 2-fold increased Serine residue phosphorylation of Bcl-2. Mass spectrometry analysis further showed that PKCε can phosporylate Bcl-2 at its Serine24 residue site in vitro, indicating that PKCε can directly interact and phosphorylate Bcl-2. These data suggest that Bcl-2 is pivotal in limiting IS in basal I/R injury by counteracting MPT and preserving mitochondrial respiratory complex functionality, and implicate a direct interaction and phosphorylation of Bcl-2 by PKCε in this process.