Kelly M. Regula

Inducible Expression of BNIP3 Provokes Mitochondrial Defects and Hypoxia-Mediated Cell Death of Ventricular Myocytes

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 (P <0.05) 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 (P <0.05) 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&Dgr;TM), necessary for mitochondrial insertion. The pan-caspase inhibitor z-VAD-fmk (25 to 100 &mgr;mol/L) suppressed BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner. Bongkrekic acid (50 &mgr;mol/L), an inhibitor of the PT pore, prevented BNIP3-induced mitochondrial defects and cell death. Expression of BNIP3&Dgr;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.

Distinct pathways regulate proapoptotic Nix and BNip3 in cardiac stress

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 μm), angiotensin II (100 nm), or isoproterenol (10 μm). 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 PKCα, but not PKCϵ or dominant negative PKCα, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKCα-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 Gq overexpression model of hypertrophy. Luciferase activity increased in Gαq-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 hypoxia-inducible, whereas Nix expression was induced by Gαq-mediated hypertrophic stimuli. PKCα, a Gq effector, transduced Nix transcriptional induction via Sp1.

Nuclear Factor-κB–Mediated Cell Survival Involves Transcriptional Silencing of the Mitochondrial Death Gene BNIP3 in Ventricular Myocytes

BACKGROUND A survival role for the transcription factor nuclear factor-kappaB (NF-kappaB) 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-kappaB impinge on the hypoxia-inducible death factor BNIP3. METHODS AND RESULTS Activation of the NF-kappaB signaling pathway by IKKbeta in ventricular myocytes suppressed mitochondrial permeability transition pore (PTP) opening and cell death provoked by BNIP3. Expression of IKKbeta or p65 NF-kappaB suppressed basal and hypoxia-inducible BNIP3 gene activity. Deletion analysis of the BNIP3 promoter revealed the NF-kappaB elements to be crucial for inhibiting basal and inducible BNIP3 gene activity. Cells derived from p65(-/-)-deficient mice or ventricular myocytes rendered defective for NF-kappaB signaling with a nonphosphorylative IkappaB exhibited increased basal BNIP3 gene expression, mitochondrial PTP, and cell death. Genetic or functional ablation of the BNIP3 gene in NF-kappaB-defective myocytes rescued them from mitochondrial defects and cell death. CONCLUSIONS The data provide new compelling evidence that NF-kappaB suppresses mitochondrial defects and cell death of ventricular myocytes through a mechanism that transcriptionally silences the death gene BNIP3. Collectively, our data provide new mechanistic insight into the mode by which NF-kappaB suppresses cell death and identify BNIP3 as a key transcriptional target for NF-kappaB-regulated expression in ventricular myocytes.

Nuclear Factor-κB Represses Hypoxia-Induced Mitochondrial Defects and Cell Death of Ventricular Myocytes

Background—Oxygen deprivation for prolonged periods of time provokes cardiac cell death and ventricular dysfunction. Preventing inappropriate cardiac cell death in patients with ischemic heart disease would be of significant therapeutic value as a means to improve ventricular performance. In the present study, we wished to ascertain whether activation of the cellular factor nuclear factor (NF)-&kgr;B suppresses mitochondrial defects and cell death of ventricular myocytes during hypoxic injury. Methods and Results—In contrast to normoxic control cells, ventricular myocytes subjected to hypoxia displayed a 9.1-fold increase (P<0.05) in cell death, as determined by Hoechst 33258 nuclear staining and vital dyes. Mitochondrial defects consistent with permeability transition pore opening, loss of mitochondrial membrane potential (&Dgr;&PSgr;m), and Smac release were observed in cells subjected to hypoxia. An increase in postmitochondrial caspase 9 and caspase 3 activity was observed in hypoxic myocytes. Adenovirus-mediated delivery of wild-type IKK&bgr; (IKK&bgr;wt) resulted in a significant increase in NF-&kgr;B–dependent DNA binding and gene transcription in ventricular myocytes. Interestingly, subcellular fractionation of myocytes revealed that the p65 subunit of NF-&kgr;B was localized to mitochondria. Hypoxia-induced mitochondrial defects and cell death were suppressed in cells expressing IKK&bgr;wt but not in cells expressing the kinase-defective IKK&bgr; mutant. Conclusions—To the best of our knowledge, the data provide the first direct evidence that activation of the NF-&kgr;B signaling pathways is sufficient to suppress cell death of ventricular myocytes during hypoxia. Moreover, our data further suggest that NF-&kgr;B averts cell death through a mechanism that prevents perturbations to the mitochondrion during hypoxic injury.

Serpin Protein CrmA Suppresses Hypoxia-Mediated Apoptosis of Ventricular Myocytes

BackgroundIn this study, we ascertain whether caspase 8 activation and mitochondrial defects underlie apoptosis of ventricular myocytes during hypoxia. As an approach to circumvent the potential shortcomings surrounding the limited permeability and short half-life of the synthetic peptide inhibitors designed to block caspase activation, we constructed a replication-defective adenovirus encoding the serpin caspase inhibitor protein CrmA to ensure efficient and continual inhibition of caspase 8 activity during chronic hypoxia. Methods and ResultsIn contrast to normoxic cells, oxygen deprivation of postnatal ventricular myocytes for 24 hours resulted in a 9-fold increase (P <0.05) in apoptosis as determined by Hoechst 33258 staining and nucleosomal DNA laddering. Moreover, hypoxia provoked a 1.5-fold increase (P <0.01) in caspase 8–like activity. Furthermore, hypoxia provoked perturbations to mitochondria consistent with the mitochondrial death pathway, including permeability transition pore (PT) opening, loss of mitochondrial membrane potential (&Dgr;&PSgr;m), and cytochrome c release. Importantly, CrmA suppressed caspase 8 activity, PT pore changes, loss of &Dgr;&PSgr;m, and apoptosis but had no effect on hypoxia-mediated cytochrome c release. Furthermore, Bongkrekic acid, an inhibitor of PT pore, prevented hypoxia-induced PT pore changes, loss of &Dgr;&PSgr;m, and apoptosis but had no effect on hypoxia-mediated cytochrome c release. ConclusionsTo our knowledge, we provide the first direct evidence for the operation of CrmA as an antiapoptotic factor in ventricular myocytes during prolonged durations of hypoxia. Furthermore, our data suggest that perturbations to mitochondria including PT pore changes and &Dgr;&PSgr;m loss are caspase-regulated events that appear to be separable from cytochrome c release.

Dissecting apoptosis and intrinsic death pathways in the heart.

The limited regenerative capacity of postnatal ventricular myocytes coupled with their meager ability for genetic manipulation has presented a major technical obstacle for deciphering apoptosis initiation and execution signals in the heart. In this report, we describe the technical approaches used to study the intrinsic death pathways in postnatal ventricular myocytes during acute hypoxic injury. Discussed are methods for hypoxia, recombinant adenovirus-mediated gene transfer, cellular viability assays using the vital dyes calcein acetomethoxyester and ethidium homodimer-1, analysis of nuclear morphology by use of Hoechst dye 33258, and assessment of the state of the mitochondrial permeability transition pore. Our work has established that hypoxia triggers perturbations to mitochondria consistent with loss of mitochondrial membrane potential, permeability transition pore opening, and apoptotic cell death by the intrinsic pathway.