Abstract 1326: Hypoxia-Induced Disruption of Rb/E2F-1 Inhibitory Complexes Provokes Mitochondrial Perturbations and Cell Death of Ventricular Myocytes Through De-regulated Expression of the Death Protein BNIP3
The cellular factor E2F-1 is known to regulate a variety of processes including G1 exit and apoptosis. Previously we have shown that E2F-1 provokes cell death of post-mitotic adult and neonatal ventricular myocytes, however, the underlying mechanism was undetermined. In this report we show that E2F-1 triggers cell death of ventricular myocytes through a mechanism that directly impinges upon the intrinsic apoptotic pathway and involves the activation of the death factor Bnip3. Overexpression of E2F-1 in cells caused a 4.9 fold increase (p<0.01) in myocyte cell death compared to control cells. E2F-1 provoked mitochondrial perturbations consistent with hypoxic injury and the intrinsic death pathway including permeability transition pore opening, Smac/Dibalo release and caspase-3 activation. Bnip3 gene transcription was increased by 2.1 fold in cells expressing wild type E2F-1 but not in cells expressing a transactivation defective mutant. During hypoxia, Rb was proteolytically cleaved and inactivated in ventricular myocytes. In contrast to normoxic control cells, ChIP analysis verified increased binding of E2F-1 to the Bnip3 promoter during hypoxia- a finding consistent with the induction of Bnip3 transcription. The Bnip3 homologue, Nix/Bnip3L was unaffected in ventricular myocytes by either E2F-1 or hypoxia. Genetic ablation of E2F-1 or expression of a caspase resistant form of Rb suppressed basal and hypoxia-inducible Bnip3 gene transcription. Furthermore, loss of function mutations of Bnip3 abrogated mitochondrial defects and cell death elicited by E2F-1. To our knowledge the data provide the first direct evidence that de-regulated E2F-1 activity during hypoxia impinges upon the intrinsic death pathway through the transcriptional activation of Bnip3 in the heart. Further we show mechanistically that basal and hypoxia-inducible activation of the Bnip3 promoter is continent upon the presence of the cellular factor E2F-1.