A Novel Hypoxia-Inducible Spliced Variant of Mitochondrial Death Gene Bnip3 Promotes Survival of Ventricular Myocytes

Rationale: Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. Previously, we established hypoxia-inducible death factor Bnip3 as a critical component of the intrinsic death pathway. Objective: To investigate alternative splicing of Bnip3 pre-mRNA in postnatal ventricular myocytes during hypoxia. Methods and Results: We identify a novel previously unrecognized spliced variant of Bnip3 (Bnip3 Delta ex3) generated by alternative splicing of exon3 exclusively in cardiac myocytes subjected to hypoxia. Sequencing of Bnip3 Delta ex3 revealed a frame shift mutation that terminated transcription up-stream of exon5 and exon6 ablating translation of the BH3-like domain and critical carboxyl-terminal transmembrane domain crucial for mitochondrial localization and cell death. Notably, although the 26-kDa Bnip3 protein (Bnip3FL) encoded by full-length mRNA was localized to mitochondria and provoked cell death, the 8.2-kDa Bnip3 Delta ex3 protein encoded by the truncated spliced mRNA was defective for mitochondrial targeting but interacted with Bnip3FL resulting in less association of Bnip3FL with mitochondria and diminished apoptotic and necrotic cell death. Forced expression of Bnip3FL in cardiac myocytes or Bnip3(-/-) mouse embryonic fibroblasts triggered widespread cell death that was inhibited by coexpression of Bnip3 Delta ex3. Conversely, RNA interference targeted against sequences encompassing the unique exon2-exon4 junction of the Bnip3 Delta ex3 sensitized cardiac myocytes to mitochondrial perturbations and cell death induced by Bnip3FL. Conclusions: Given the otherwise lethal consequences of deregulated Bnip3FL expression in postmitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the mitochondrial defects and cell death of ventricular myocytes that is obligatorily linked and mutually dependent on alternative splicing of Bnip3FL during hypoxia or ischemic stress. (Circ Res. 2011;108:1084-1092.)