Published online before print July 11, 2002, doi: 10.1161/01.RES.0000029232.42227.16
© 2002 American Heart Association, Inc.
Cellular Biology |
Inducible Expression of BNIP3 Provokes Mitochondrial Defects and Hypoxia-Mediated Cell Death of Ventricular Myocytes
From The Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, and the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba.
Correspondence to Dr Lorrie A. Kirshenbaum, Institute of Cardiovascular Sciences, St Boniface General Hospital, Research Centre Rm 3016, 351 Taché Ave, Winnipeg, Manitoba, Canada R2H 2A6. E-mail Lorrie{at}sbrc.umanitoba.ca
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Abstract |
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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
TM), necessary for mitochondrial insertion. The pan-caspase inhibitor z-VAD-fmk (25 to 100 µmol/L) suppressed BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner. Bongkrekic acid (50 µmol/L), an inhibitor of the PT pore, prevented BNIP3-induced mitochondrial defects and cell death. Expression of BNIP3TM 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.
Key Words: ventricular myocytes • cell death • apoptosis • hypoxia • gene
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Introduction |
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Oxygen deprivation for prolonged periods leads to cardiac cell death and ventricular dysfunction. Although the molecular events that underlie hypoxia-induced cell death remain cryptic, there is evidence that a mitochondrial pathway may be centrally involved.1–3 Perturbations to mitochondria resulting in the loss of
m, from the opening of a large multiprotein conductance channel referred to as permeability transition (PT) pore, have been reported (reviewed in Reed4). The PT pore, which comprises in part the adenine nucleotide translocator, porin/voltage-activated anion channel (VDAC) as well as other mitochondrial membrane proteins presumably opens in response to pro-death signals, resulting in mitochondrial swelling, generation of reactive oxygen species, and dissipation of m.5 PT pore opening has been reported during hypoxia, increased intracellular calcium, as well as oxidative stress injury.2,6 However, the signaling factors that result in mitochondrial perturbations and PT pore opening by pro-death signals are poorly defined. BNIP3 is a novel subclass of death-inducing mitochondrial proteins with homology to the BH3 domain of the Bcl-2 gene family.7,8 BNIP3 was initially identified as an adenovirus E1B 19-kd interacting protein.8 Other members of this sub-group are known to include Nix and the Caenorhabditis elegans ortholog ceBNIP3.9 The mode by which BNIP3 provokes apoptosis is unknown but recent evidence suggests it may impinge on mitochondrial function.9,10 The carboxyl terminal transmembrane domain (TM) of BNIP3 was identified to be necessary for mitochondrial targeting.9,11
Using a yeast 2-hybrid approach, we have found that BNIP3 is expressed in human heart tissue (K.M. Regula and L.A. Kirshenbaum, unpublished data, 2002). However, the significance of this finding and the relation of BNIP3 to cardiac cell death is undetermined. Given that increased transcript levels of BNIP3 have been detected in immortalized cells during chronic hypoxia12,13 raises the interesting possibility that BNIP3 may be an inducible factor that initiates cell death during hypoxia. Whether BNIP3 provokes mitochondrial defects leading to PT pore changes and cell death of ventricular myocytes during hypoxia is unknown and has not been formally tested. Therefore, as a first step toward elucidating a role for BNIP3 in myocardial cell death, we ascertained whether BNIP3 is involved in mitochondrial defects and cell death of ventricular myocytes during hypoxia.
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Materials and Methods |
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Cell Culture and Hypoxia
Ventricular myocytes were isolated from 1- to 2-day-old Sprague-Dawley rats (University of Manitoba, Winnipeg, Canada) and submitted to primary culture as reported.14 Myocytes were subjected to hypoxia for 24 hours in an air-tight chamber continually gassed with 95% N2-5% CO2, as previously reported.15–17 Adult rat hearts (n=3) were excised from adult rats (250 to 300 g) and subjected to global hypoxia for 1 hour on a modified Langendorff perfusion system with buffer continually gassed with 95% N2-5% CO2, as previously reported.18 Heart failure was induced in rats by surgical ligation of the left anterior descending coronary artery, as previously reported.19 Non-failing sham control hearts and those with heart failure were excised from animals 8 weeks after myocardial infarction. Hearts (n=3) were dissected to remove the infarcted scar tissue, and the left ventricular free wall was homogenized and analyzed by Western blot for BNIP3 expression. Animals were handled according to guidelines established by the University of Manitoba and Canadian Council on Animal Care.
Recombinant Adenovirus
The cDNAs for BNIP3 full-length (BNIP3) and carboxyl terminal transmembrane domain deletion mutant (BNIP3TM) have been described previously.9 Recombinant adenoviruses encoding the full-length BNIP3 or transmembrane deletion mutant are designated AdBNIP3 or AdBNIP3TM, respectively, and generated as reported previously.20
Cell Viability
Cell viability was determined using the vital dyes 2 µmol/L calcein acetoxymethyl ester (green) to determine the number of living cells and 2 µmol/L ethidium homodimer-1 (red) (Molecular Probes) to identify the number of dead cells.14 The number of green cells versus red cells was used to determine changes in cell viability.15 Cells were analyzed from at least n=3 independent myocyte isolations counting 
200 cells for each condition tested. Data are expressed as mean±SE percent reduction from control.
Western Blot Analysis
For detection of BNIP3 protein, total cardiac cell lysate was subjected to Western blot analysis using a murine antibody directed toward BNIP3 (generously provided by Dr A.H. Greenberg, Cancer Care Manitoba, Winnipeg, Canada).9 To isolate mitochondria from ventricular myocytes, cells were washed in HIM buffer (200 mmol/L mannitol, 70 mmol/L sucrose, 10 mmol/L HEPES-KOH, 1 mmol/L EGTA, pH 7.5). The crude cell pellet was disrupted and resuspended in HIM buffer containing 2.0% wt/vol bovine serum albumin. The homogenate was ultracentrifuged to obtain the mitochondrial fraction and resuspended in 200 mmol/L sucrose, 10 mmol/L HEPES-KOH, 1 mmol/L ATP, 5 mmol/L sodium succinate, 0.08 mmol/L ADP, and 2 mmol/L K2HPO4. To assess whether BNIP3 integrates into mitochondrial membranes during hypoxia, mitochondria were subjected to alkali extraction using 0.01 mol/L Na2CO3 (pH 11.5); this will dissociate and solubilize proteins that are not membrane integrated.9,21 Antibodies directed toward the following Bcl-2 family members were obtained from respective suppliers: Bad (Transduction Laboratories Inc), Bax (Santa Cruz Inc), and Bak and Bcl-2 (Pharmingen). Bound proteins were visualized using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Inc).
Mitochondrial Permeability Transition Pore
To monitor mitochondrial permeability transition (PT), ventricular myocytes were loaded with 5 µmol/L calcein-acetoxymethylester (calcein-AM, Molecular Probes) in the presence of 2 to 5 mmol/L cobalt chloride as previously reported.2,22
Statistical Analysis
Multiple comparisons between groups were determined by 1-way ANOVA. Unpaired 2-tailed Student’s t test was used to compare mean differences between groups. Differences were considered to be statistically significant to a level of P<0.05. In all cases, the data were obtained from at least 3 to 4 independent myocyte isolations using 3 replicates for each condition tested.
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Results |
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Previous work from our laboratory has demonstrated that hypoxia-induced apoptosis of ventricular myocytes involves perturbations to the mitochondria including loss of
m and PT pore opening.2,15 Our data further indicated that interventions that retained PT pore in the closed configuration suppressed hypoxia-induced loss of m and cell death of ventricular myocytes, establishing a link between perturbations to mitochondria and hypoxia-induced apoptosis of ventricular myocytes. As a step toward identifying putative regulators of cardiac cell death, we ascertained whether hypoxia leads to the induction of BNIP3. As shown by Western blots analysis (Figure 1), there was no apparent change in the expression of BNIP3 after short durations of hypoxia compared with normoxic control cells. This finding is consistent with our previous work indicating that cell viability was not significantly different from normoxic control cells before 24 hours of chronic hypoxia. Interestingly, a significant 19.5-fold (P<0.05) induction of BNIP3 protein expression was observed in cells subjected to hypoxia for 24 hours. The induction of BNIP3 is concordant with our previous studies and coincident with the occurrence of hypoxia-induced mitochondrial defects and apoptosis of ventricular myocytes at this time point.2 Moreover, there was no apparent change in the expression of Bad, Bak, Bax, or Bcl-2 in cells subjected to hypoxia at this time point (Figure 2).
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Given that BNIP3 expression was increased during hypoxia relative to the other death-promoting factors studied suggested that it may be important for cardiac cell death. Therefore, we focused our efforts on the involvement of BNIP3 during hypoxia-induced apoptosis of ventricular myocytes. To verify the hypoxia-induced BNIP3 was not developmentally restricted or its expression confined to the neonatal myocardium, we ascertained whether BNIP3 induction occurs in the context of the adult heart under in vitro and in vivo conditions. Concordant with our findings was an observed increase in BNIP3 expression in isolated Langendorff perfused adult rat hearts subjected to global hypoxia (Figure 3A), verifying our BNIP3 data in neonatal ventricular myocytes. Furthermore, in contrast to normal, non-failing hearts, a significant increase in BNIP3 expression was detected in adult rat hearts with chronic heart failure after myocardial infarction in vivo (Figure 3B). These findings indicate that induction and expression of BNIP3 is not limited to the neonatal heart and that BNIP3 may be involved in promoting cell death during hypoxic/ischemic pathologies.
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To determine whether BNIP3 is cytotoxic and provokes cell death of ventricular myocytes, we generated a replication defective adenovirus encoding full-length BNIP3. For these experiments, myocytes were stained with the vital dyes calcein-AM and ethidium homodimer-1 to mark the number of live cells green and dead cells red, respectively.23 As shown in Figure 4A, a significant 8.3-fold increase (P<0.05) in myocyte death was observed in the presence of BNIP3 compared with cells infected with a control virus lacking the BNIP3 cDNA insert. Moreover, Hoechst 33258 staining for nuclear morphology revealed a significant increase in nucleosomal DNA fragmentation typical of apoptosis in the presence of BNIP3 (Figure 4B). Importantly, the pan caspase inhibitor z-VAD-fmk (25 to 100 µmol/L) prevented BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner (Figure 4C). These results corroborate the notion that BNIP3 provokes apoptosis of ventricular myocytes and suggest the involvement of a caspase-regulated pathway. Importantly, BNIP3 had no apparent effect on the expression levels of pro-death family members Bad, Bax, or Bcl-2 in ventricular myocytes (Figure 5).
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A recent model for the induction of apoptosis purports that mitochondrial defects follow the integration of death-inducing Bcl-2 family members.21 In this study, endogenous Bax protein was found loosely associated with mitochondria but readily integrated into mitochondrial membranes following pro-death signals. Similarly, we found that BNIP3 is associated with the mitochondria in normoxic cells (K.M. Regula and L.A. Kirshenbaum, unpublished data, 2002). To ascertain whether hypoxia provokes the integration of BNIP3 into mitochondrial membranes, we subjected mitochondria of normoxic and hypoxic cells to alkali extraction, which will dissociate and solubilize unintegrated membrane proteins.21 After alkali extraction of mitochondria, BNIP3 was undetectable in the mitochondrial membranes of normoxic cells, indicating it was alkali soluble and not membrane integrated. In contrast, BNIP3 was readily detectable in the mitochondrial fraction of hypoxic cells after alkali extraction, suggesting that it was tightly associated with mitochondrial membranes (Figure 6). These results indicate that hypoxia is sufficient to provoke the integration of BNIP3 protein into mitochondrial membranes. Because perturbations to mitochondria resulting from opening of the mitochondrial PT pore have been suggested to be an underlying feature of the apoptotic program, we next addressed whether BNIP3 promotes mitochondrial PT changes. For these experiments, ventricular myocytes were loaded with calcein-AM in the presence of cobalt chloride to quench the cytoplasmic signal.2 The loss of green fluorescence is a measure of mitochondrial membrane permeability and can be used as an index of PT pore opening.22 As shown in Figure 7A, control cells displayed punctate green-staining mitochondria, indicative of the PT pore in a closed configuration; however, a marked reduction in mitochondrial fluorescence was observed in the presence of BNIP3, consistent with PT pore opening. To verify that the BNIP3-induced loss of mitochondrial calcein staining was related to PT changes, we treated cells with Bongkrekic acid (50 µmol/L), a known inhibitor of the adenine nucleotide translocator (ANT), and the PT pore.24,25 As shown in Figure 7A, Bongkrekic acid suppressed BNIP3-induced loss of mitochondrial calcein staining, confirming the involvement of PT pore. Together our data suggest that BNIP3 induces perturbations to mitochondria in ventricular myocytes consistent with PT pore opening.
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To substantiate the conclusion that integration of BNIP3 into mitochondria is required for cell death of ventricular myocytes during hypoxia, we tested whether a carboxyl terminal transmembrane deletion mutant of BNIP3 (BNIP3TM), defective for mitochondrial integration could suppress hypoxia-induced cell death of ventricular myocytes. As shown in Figure 7B, cells expressing the BNIP3TM were not different from control cells with respect to viability, indicating that the BNIP3TM was not toxic to myocytes and did not provoke cell death. A 5.6-fold increase (P<0.05) in myocyte death was observed in cells subjected to hypoxia, a finding concordant with our previously reported data.2 In addition, hypoxia-induced cell death of ventricular myocytes was markedly blunted in the presence of the BNIP3TM (Figures 7B and 7C). Importantly, BNIP3TM had no apparent effect on the expression levels pro-death Bcl-2 family members or Bcl-2 expression itself (Figure 5). These results suggest BNIP3TM may act in a dominant-negative manner to block the cytotoxic actions of the endogenous BNIP3 protein. This notion is founded on the observation that hypoxia-induced mitochondrial integration of the endogenous BNIP3 protein was suppressed by the BNIP3TM (Figure 6). Collectively, the data verify the involvement of BNIP3 and the importance of mitochondrial integration during hypoxia-induced cell death of ventricular myocytes.
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Discussion |
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In the present report, we provide the first direct evidence for the involvement of BNIP3 as an inducible factor that provokes mitochondrial defects and cell death of ventricular myocytes during hypoxia. Furthermore, our data strongly suggest that BNIP3 induces cell death through a mechanism that impinges on mitochondrial PT pore. This notion is substantiated by several key observations. First, we demonstrated that hypoxia is sufficient to provoke the integration of BNIP3 into mitochondrial membranes. Next, we found that BNIP3 expression in ventricular myocytes resulted in mitochondrial defects consistent with PT opening that could be suppressed by Bongkrekic acid, an inhibitor of PT pore. Lastly, BNIP3
TM, which is defective for mitochondrial targeting, suppressed hypoxia-induced cell death of ventricular myocytes, suggesting that this property of BNIP3 may be crucial for induction of death of ventricular myocytes. Precedence for the regulation of mitochondrial PT by proapoptotic Bcl-2 family members has been reported.21 For example, BAX can reportedly influence PT pore changes by interacting with VDAC/porin and ANT.21,26 Whether BNIP3 impinges on these or other mitochondrial proteins to trigger PT changes is unknown and awaits further investigation.
Because deregulated expression of BNIP3 would otherwise provoke mitochondrial defects and cell death of ventricular myocytes, the question is raised as to how BNIP3 is regulated in the absence of a pro-death signal. Although the mechanism(s) responsible for retaining BNIP3 in a functionally inactive state during normoxia is unknown, it is tempting to speculate that BNIP3 requires some posttranslation modification, similar to that reported for other proapoptotic Bcl-2 family members. Examples include the Akt-dependent phosphorylation of Bad, which retains phosphorylated Bad in a functionally inactive state bound to the cytoplasmic 14-3-3 protein.27 Similarly, the caspase 8-dependent cleavage of Bid prompts the translocation of t-Bid to mitochondria, resulting in cytochrome c release and cell death.28 Alternatively, BNIP3 may require a chaperone or accessory protein that directly or indirectly influences its mitochondrial targeting. Whether these or other mechanisms regulate BNIP3 function during hypoxia awaits further investigation.
Given that BNIP3TM mutant, which is defective for homodimeration and mitochondrial targeting,29,30 had no apparent effect on the expression levels of other pro-death Bcl-2 family members or Bcl-2 itself, suggests it likely rescues hypoxia-induced cell death through an alternative mechanism. The fact that mitochondrial integration of the endogenous BNIP3 was impaired in the presence of the BNIP3TM supports the notion that BNIP3TM likely acts as a dominant-negative inhibitor of the endogenous BNIP3 by competing with some regulatory factor(s) required for mitochondrial targeting.
Although the mechanism by which BNIP3 triggers cell death of ventricular myocytes is unknown, its ability to provoke cell death may not be mutually dependent or obligatorily linked to an apoptotic pathway. Given its reported ability to provoke a "necrotic-like" cell death through a caspase-independent pathway,9 this feature of BNIP3 is less clear and warrants further explanation. In this regard, apoptosis and necrosis have been distinguished at least operationally by whether or not caspase processing occurs. The reported ability for apoptosis to occur through a caspase-independent pathway would argue that caspase activation alone may not be sufficient to distinguish between a given cell death pathway.9
However, the ability of BNIP3 to provoke apoptosis, necrosis, or both may instead reflect features of the same cell death pathway that converge on down targets in a cell type- and context-specific manner. In this regard, it was previously reported that BNIP3 induces cell death through a caspase-independent pathway and without cytochrome c release.9 In the present study, we demonstrate that the pan caspase inhibitor z-VAD-fmk suppressed BNIP3-induced cell death in ventricular myocytes in a dose-dependent manner, suggesting the involvement of caspase-regulated pathway. Further, expression of BNIP3 in ventricular myocytes provoked mitochondrial cytochrome c release (K.M. Regula and L.A. Kirshenbaum, unpublished data, 2002). Therefore, we believe that the apparent discrepancies between our study and the report by Vande Velde et al9 may be due to differences in the model system used. For example, we used primary cultured ventricular myocytes for our study in contrast to the immortalized transformed cell lines used by Vande Velde et al.9 Although transformed cell lines can provide important mechanistic insight into the signaling molecules involved in the cell death pathway, they may not reflect the pathways used in nontransformed cells. Consequently, BNIP3 may utilize an alternative signaling pathway (ie, caspase-dependent versus caspase-independent) in a cell type- and context-specific manner. Therefore, we believe our data in primary cultured ventricular myocytes is physiologically relevant and may reflect more closely the operation of BNIP3 in vivo. Further, the fact that z-VAD-fmk was sufficient to suppress BNIP3-induced cell death is concordant with our previously published work indicating that hypoxia-mediated cell death of ventricular myocytes is a caspase-mediated process.2 Therefore, we believe that the induction of BNIP3 during hypoxia may be centrally involved in the cell death process by its ability to impinge on at least 2 mutually related components of the cell pathway, namely mitochondria and caspases.
The fact that BNIP3 was detected in the hypoxic adult heart in vitro indicates that its expression is not confined or developmentally restricted to the neonatal myocardium. Given that BNIP3 was induced in an infarction model of chronic heart failure in vivo, it is tempting to speculate that BNIP3 may contribute to the reported apoptosis and ventricular remodeling associated with chronic hypoxia and/or ischemia, and awaits further investigation.
Although the molecular mechanisms that regulate BNIP3 expression during hypoxia are unknown, the fact that the BNIP3 appeared to be the only proapoptotic Bcl-2 family member to be induced during hypoxia is intriguing and suggests the possibility that BNIP3 is regulated at least transcriptionally, different from the other death factors. In this regard, BNIP3 promoter was identified to contain canonical binding sites for the hypoxia-inducible transcription factor HIF-1
,12,31 supporting our contention that BNIP3 is an inducible factor that initiates the cell death program during hypoxia. Whether HIF-1 or alternative cellular factors mechanistically account for the induction of BNIP3 during hypoxic injury of ventricular myocytes remains a point of active investigation.
Nevertheless, under the conditions tested, we provide the first direct evidence for BNIP3 as an inducible factor that promotes mitochondrial defects and cell death of ventricular myocytes during hypoxia. Our data further suggest that interventions designed to functionally modulate BNIP3 activity may prove beneficial in preventing cardiac cell death during conditions of chronic hypoxia or ischemia.
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Acknowledgments |
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This work was supported by grants from The Canadian Institutes for Health Research, and L.A.K. is a Canada Research Chair in Molecular Cardiology. We are grateful to Dr Arnold H. Greenberg for generous gift of reagents cited. Drs J. Davie and H. Weisman for critical comments on the manuscript. Expert technical assistance was provided by C. Robinson and J. Roth.
Received February 5, 2002; revision received June 21, 2002; accepted June 25, 2002.
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H. Liu and S. A. Fisher Hypoxia-Inducible Transcription Factor-1{alpha} Triggers an Autocrine Survival Pathway During Embryonic Cardiac Outflow Tract Remodeling Circ. Res., June 6, 2008; 102(11): 1331 - 1339. [Abstract] [Full Text] [PDF]
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K. Shivakumar, S. J. Sollott, M. Sangeetha, S. Sapna, B. Ziman, S. Wang, and E. G. Lakatta Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2653 - H2658. [Abstract] [Full Text] [PDF]
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 S.-D. Lee, W.-W. Kuo, D.-T. Bau, F.-Y. Ko, F.-L. Wu, C.-H. Kuo, F.-J. Tsai, P. S. Wang, M.-C. Lu, and C.-Y. Huang The coexistence of nocturnal sustained hypoxia and obesity additively increases cardiac apoptosis J Appl Physiol, April 1, 2008; 104(4): 1144 - 1153. [Abstract] [Full Text] [PDF]
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N. Yurkova, J. Shaw, K. Blackie, D. Weidman, R. Jayas, B. Flynn, and L. A. Kirshenbaum The Cell Cycle Factor E2F-1 Activates Bnip3 and the Intrinsic Death Pathway in Ventricular Myocytes Circ. Res., February 29, 2008; 102(4): 472 - 479. [Abstract] [Full Text] [PDF]
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 A. Diwan, J. Wansapura, F. M. Syed, S. J. Matkovich, J. N. Lorenz, and G. W. Dorn II Nix-Mediated Apoptosis Links Myocardial Fibrosis, Cardiac Remodeling, and Hypertrophy Decompensation Circulation, January 22, 2008; 117(3): 396 - 404. [Abstract] [Full Text] [PDF]
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A. B. Gustafsson and R. A. Gottlieb Heart mitochondria: gates of life and death Cardiovasc Res, January 15, 2008; 77(2): 334 - 343. [Abstract] [Full Text] [PDF]
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N. Bodyak, D. L. Rigor, Y.-S. Chen, Y. Han, E. Bisping, W. T. Pu, and P. M. Kang Uncoupling protein 2 modulates cell viability in adult rat cardiomyocytes Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H829 - H835. [Abstract] [Full Text] [PDF]
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 Z. Zhang, X. Yang, S. Zhang, X. Ma, and J. Kong BNIP3 Upregulation and EndoG Translocation in Delayed Neuronal Death in Stroke and in Hypoxia Stroke, May 1, 2007; 38(5): 1606 - 1613. [Abstract] [Full Text] [PDF]
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 A. B. Gustafsson and R. A. Gottlieb Bcl-2 family members and apoptosis, taken to heart Am J Physiol Cell Physiol, January 1, 2007; 292(1): C45 - C51. [Abstract] [Full Text] [PDF]
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J. Shaw, T. Zhang, M. Rzeszutek, N. Yurkova, D. Baetz, J. R. Davie, and L. A. Kirshenbaum Transcriptional Silencing of the Death Gene BNIP3 by Cooperative Action of NF-{kappa}B and Histone Deacetylase 1 in Ventricular Myocytes Circ. Res., December 8, 2006; 99(12): 1347 - 1354. [Abstract] [Full Text] [PDF]
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J. Shaw and L. A. Kirshenbaum HAX-1 Represses Postmitochondrial Caspase-9 Activation and Cell Death During Hypoxia-Reoxygenation Circ. Res., August 18, 2006; 99(4): 336 - 338. [Full Text] [PDF]
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 A. S. Galvez, E. W. Brunskill, Y. Marreez, B. J. Benner, K. M. Regula, L. A. Kirschenbaum, and G. W. Dorn II Distinct Pathways Regulate Proapoptotic Nix and BNip3 in Cardiac Stress J. Biol. Chem., January 20, 2006; 281(3): 1442 - 1448. [Abstract] [Full Text] [PDF]
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D. Baetz, K. M. Regula, K. Ens, J. Shaw, S. Kothari, N. Yurkova, and L. A. Kirshenbaum Nuclear Factor-{kappa}B-Mediated Cell Survival Involves Transcriptional Silencing of the Mitochondrial Death Gene BNIP3 in Ventricular Myocytes Circulation, December 13, 2005; 112(24): 3777 - 3785. [Abstract] [Full Text] [PDF]
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 M. P. Biju, Y. Akai, N. Shrimanker, and V. H. Haase Protection of HIF-1-deficient primary renal tubular epithelial cells from hypoxia-induced cell death is glucose dependent Am J Physiol Renal Physiol, December 1, 2005; 289(6): F1217 - F1226. [Abstract] [Full Text] [PDF]
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 M. Murai, M. Toyota, H. Suzuki, A. Satoh, Y. Sasaki, K. Akino, M. Ueno, F. Takahashi, M. Kusano, H. Mita, et al. Aberrant Methylation and Silencing of the BNIP3 Gene in Colorectal and Gastric Cancer Clin. Cancer Res., February 1, 2005; 11(3): 1021 - 1027. [Abstract] [Full Text] [PDF]
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K. M. Regula, D. Baetz, and L. A. Kirshenbaum Nuclear Factor-{kappa}B Represses Hypoxia-Induced Mitochondrial Defects and Cell Death of Ventricular Myocytes Circulation, December 21, 2004; 110(25): 3795 - 3802. [Abstract] [Full Text] [PDF]
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 A. Vengellur and J. J. LaPres The Role of Hypoxia Inducible Factor 1{alpha} in Cobalt Chloride Induced Cell Death in Mouse Embryonic Fibroblasts Toxicol. Sci., December 1, 2004; 82(2): 638 - 646. [Abstract] [Full Text] [PDF]
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K. M. Regula, M. J. Rzeszutek, D. Baetz, C. Seneviratne, and L. A. Kirshenbaum Therapeutic opportunities for cell cycle re-entry and cardiac regeneration Cardiovasc Res, December 1, 2004; 64(3): 395 - 401. [Abstract] [Full Text] [PDF]
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M. T. Crow, K. Mani, Y.-J. Nam, and R. N. Kitsis The Mitochondrial Death Pathway and Cardiac Myocyte Apoptosis Circ. Res., November 12, 2004; 95(10): 957 - 970. [Abstract] [Full Text] [PDF]
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 R. M. Graham, D. P. Frazier, J. W. Thompson, S. Haliko, H. Li, B. J. Wasserlauf, M.-G. Spiga, N. H. Bishopric, and K. A. Webster A unique pathway of cardiac myocyte death caused by hypoxia-acidosis J. Exp. Biol., August 15, 2004; 207(18): 3189 - 3200. [Abstract] [Full Text] [PDF]
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 J. Okami, D. M. Simeone, and C. D. Logsdon Silencing of the Hypoxia-Inducible Cell Death Protein BNIP3 in Pancreatic Cancer Cancer Res., August 1, 2004; 64(15): 5338 - 5346. [Abstract] [Full Text] [PDF]
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A. B. Gustafsson, J. G. Tsai, S. E. Logue, M. T. Crow, and R. A. Gottlieb Apoptosis Repressor with Caspase Recruitment Domain Protects against Cell Death by Interfering with Bax Activation J. Biol. Chem., May 14, 2004; 279(20): 21233 - 21238. [Abstract] [Full Text] [PDF]
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 M.-J. Lee, J.-Y. Kim, K. Suk, and J.-H. Park Identification of the Hypoxia-Inducible Factor 1{alpha}-Responsive HGTD-P Gene as a Mediator in the Mitochondrial Apoptotic Pathway Mol. Cell. Biol., May 1, 2004; 24(9): 3918 - 3927. [Abstract] [Full Text] [PDF]
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B. L. Copple, C. M. Rondelli, J. F. Maddox, N. C. Hoglen, P. E. Ganey, and R. A. Roth Modes of Cell Death in Rat Liver after Monocrotaline Exposure Toxicol. Sci., January 1, 2004; 77(1): 172 - 182. [Abstract] [Full Text] [PDF]
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M. T. Crow Hypoxia, BNip3 Proteins, and the Mitochondrial Death Pathway in Cardiomyocytes Circ. Res., August 9, 2002; 91(3): 183 - 185. [Full Text] [PDF]
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