Abstract-In this study, we provide evidence for the operation of BNIP3 as a key regulator of mitochondrial function and cell death of ventricular myocytes during hypoxia. In contrast to normoxic cells, a 5.6-fold increase (PϽ0.05) in myocyte death was observed in cells subjected to hypoxia. Moreover, a significant increase in BNIP3 expression was detected in postnatal ventricular myocytes and adult rat hearts subjected to hypoxia. An increase in BNIP3 expression was detected in adult rat hearts in vivo with chronic heart failure. Subcellular fractionation experiments indicated that endogenous BNIP3 was integrated into the mitochondrial membranes during hypoxia. Adenovirus-mediated delivery of full-length BNIP3 to myocytes was toxic and provoked an 8.3-fold increase (PϽ0.05) in myocyte death with features typical of apoptosis. Mitochondrial defects consistent with opening of the permeability transition pore (PT pore) were observed in cells expressing BNIP3 but not in cells expressing BNIP3 missing the carboxyl-terminal transmembrane domain (BNIP3⌬TM), necessary for mitochondrial insertion. The pan-caspase inhibitor z-VAD-fmk (25 to 100 mol/L) suppressed BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner. Bongkrekic acid (50 mol/L), an inhibitor of the PT pore, prevented BNIP3-induced mitochondrial defects and cell death. Expression of BNIP3⌬TM suppressed the hypoxia-induced integration of the endogenous BNIP3 protein and cell death of ventricular myocytes. To our knowledge, the data provide the first evidence for the involvement of BNIP3 as an inducible factor that provokes mitochondrial defects and cell death of ventricular myocytes during hypoxia.

Background-A survival role for the transcription factor nuclear factor-B (NF-B) in ventricular myocytes has been reported; however, the underlying mechanism is undefined. In this report we provide new mechanistic evidence that survival signals conferred by NF-B impinge on the hypoxia-inducible death factor BNIP3.

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 M), angiotensin II (100 nM), or isoproterenol (10 M). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC␣, but not PKC⑀ or dominant negative PKC␣, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA-or PKC␣-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G q overexpression model of hypertrophy. Luciferase activity increased in G␣ q -NixP hearts 3.2 ؎ 0.4-fold and in TAC hearts 2.8 ؎ 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxiainducible, whereas Nix expression was induced by G␣ q -mediated hypertrophic stimuli. PKC␣, a G q effector, transduced Nix transcriptional induction via Sp1.Cardiomyocyte apoptosis contributes to functional deterioration in ischemic, hypertrophic, and dilated cardiomyopathies (1-5). A critical but poorly understood feature of the cardiomyocyte cell death program is stress-mediated induction of gene expression for several pro-apoptotic factors belonging to the Bcl2 family of apoptosis-regulating proteins (4, 6). Recent studies of apoptosis gene induction in cardiac hypoxia and hypertrophy decompensation have assigned particular importance to two members of the BH3-only subgroup of Bcl2-like proteins, BNip3 and Nix (7-10). These two factors are each expressed in the heart, localized to mitochondria, and sufficient to induce apoptosis via the intrinsic, or mitochondrial, pathway (8, 10, 11). The potential for BNip3 or Nix, alone or in association with other Bcl2 family proteins (12, 13), to disrupt mitochondrial integrity by communicating with the mitochondrial permeability transition pore has suggested to some that a major function of the BH3-only proteins is to determine the on/off state of the mitochondrial permeability transition pore (14). Indeed, mitochondrial disruption may have especially profound consequences for the heart as myocardium is enriched in mit...