Published online before print December 13, 2004, doi: 10.1161/01.CIR.0000150537.59754.55
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(Circulation. 2004;110:3795-3802.)
© 2004 American Heart Association, Inc.
Heart Failure |
Nuclear Factor-
B Represses Hypoxia-Induced Mitochondrial Defects and Cell Death of Ventricular Myocytes
From the Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, Department of Physiology, Faculty of Medicine University of Manitoba, Winnipeg, Manitoba, Canada.
Correspondence to Dr Lorrie A. Kirshenbaum, Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, Room 3016, 351 Taché Ave, Winnipeg, Manitoba, Canada, R2H 2A6. E-mail lorrie{at}sbrc.ca
Received June 1, 2004; revision received September 14, 2004; accepted September 27, 2004.
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Abstract |
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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)-
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 (
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 IKKß (IKKßwt) resulted in a significant increase in NF-B-dependent DNA binding and gene transcription in ventricular myocytes. Interestingly, subcellular fractionation of myocytes revealed that the p65 subunit of NF-B was localized to mitochondria. Hypoxia-induced mitochondrial defects and cell death were suppressed in cells expressing IKKßwt but not in cells expressing the kinase-defective IKKß mutant.
Conclusions— To the best of our knowledge, the data provide the first direct evidence that activation of the NF-B signaling pathways is sufficient to suppress cell death of ventricular myocytes during hypoxia. Moreover, our data further suggest that NF-B averts cell death through a mechanism that prevents perturbations to the mitochondrion during hypoxic injury.
Key Words: myocytes • mitochondria • cell death • apoptosis • hypoxia
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Introduction |
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Agrowing body of evidence suggests that mitochondria play a central role in the apoptotic process.1,2 Perturbations to mitochondria resulting in the release of cytochrome c, second mitochondrial activator of caspases (Smac), Apoptosis Inducing Factor (AIF), and others are key components of the mitochondrial death pathway (reviewed by Reed).3 We have reported previously that hypoxia provokes mitochondrial defects consistent with permeability transition pore (PTP) opening, loss of mitochondrial membrane potential (
m), and cytochrome c release,4–8 identifying the mitochondrion as a key regulator of myocyte death during hypoxia.
The cellular NF-B activity is regulated by phosphorylation and degradation of the cytoplasmic inhibitor protein IB
.9–11 The IB kinases, notably IKKß, phosphorylate IB at serine residues S32 and S36, resulting in its proteasomal degradation. Recently, we and others have shown that NF-B activation is needed to suppress tumor necrosis factor (TNF)-–induced cell death of ventricular myocytes12–15; however, the underlying mechanism by which NF-B averts cell death is unknown. Therefore, as a first step toward elucidating the mode by which NF-B suppresses cell death of ventricular myocytes, we ascertained in the present study whether NF-B activation is sufficient to suppress mitochondrial defects and cell death of ventricular myocytes subjected to hypoxia. In this report, we provide new compelling evidence to suggest that NF-B suppresses hypoxia-induced cell death of ventricular myocytes through a mechanism that impinges on mitochondrial death pathway activation.
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Methods |
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Cell Culture and Hypoxia
Ventricular myocytes were isolated from 1- to 2-day-old Sprague-Dawley rats and submitted to primary culture as reported previously.16 Twenty-four hours after plating, myocytes were subjected to hypoxia in an air-tight chamber continually gassed with 95% N2–5% CO2, PO2
5 mm Hg, for 24 hours, after which cells were harvested and analyzed for mitochondrial perturbations and cell death as detailed below. This time point was chosen because earlier reports from our laboratory and others established 24 hours of hypoxia to be required to provoke apoptotic changes in neonatal ventricular myocytes.17–19 In all cases, data were obtained from at least n=3 to 4 independent myocyte cultures using replicates of n=3 for each condition tested.
Recombinant Adenovirus
The cDNAs for IB kinase IKK ß wild-type (IKKßwt) or kinase-defective IKKßK-M (IKKßmt) were kindly provided by W. Greene.20 We generated recombinant adenoviruses encoding wild-type IKKßwt (Ad IKKßwt), a kinase-defective IKKß (Ad IKKßmt), and nonphosphorylatable IB (Ad IB SA) by methodologies we reported previously.21 Normoxic control myocytes were infected with an "empty" control adenovirus, which contains the adenovirus backbone and cytomegalovirus (CMV) promoter (Ad CMV) and was previously determined to be nontoxic to myocytes.16,17 Myocytes were infected with a multiplicity of infection of 10, which achieves >90% of gene delivery to ventricular myocytes.16,17
Cell Viability
Cell viability was determined by use of the vital dyes calcein acetoxymethylester (2 µmol/L) to determine the number of living cells (green fluorescence) and ethidium homodimer-1 (2 µmol/L) to determine the number of dead cells (red fluorescence) (Molecular Probes) as reported previously.16,17 Cells were analyzed from at least n=3 to 4 independent myocyte cultures, counting 
200 cells for each condition tested. Data are expressed as mean±SEM percent reduction from control.
Western Blot Analysis
For detection of mitochondrial Smac release, the cytoplasmic S-100 fraction of cardiac lysate was prepared from myocytes as reported previously1,2 and subjected to Western blot analysis using a murine antibody directed toward the 23-kDa Smac/Diablo (generously provided by Dr David L. Vaux, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia).22 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) as reported previously.5 The crude cell pellet was disrupted and resuspended in HIM buffer containing 2.0% wt/vol BSA. 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 K2HPO4.23 Murine antibodies directed toward -sarcomeric actin (1 µg/mL, Sigma) and cytochrome oxidase subunit I (1 µg/mL, Molecular Probes) were used as reported previously.24 Bound proteins were visualized by use of enhanced chemiluminescence reagents (Amersham Pharmacia Inc).
Detection of Caspases
The proteolytic activation of caspase 9 and caspase 3 activity was determined by fluorogenic assay using the substrate DEVD-7-amino-4-trifluoromethyl coumarin for caspase 3 and LEHD-7-amino-4-trifluoromethyl coumarin for caspase 9, using 40 µg of cardiac lysate protein as reported previously.1 Hydrolysis of the DEVD or LEHD substrate was followed in a monitored using an excitation filter of 400 nm for caspase 3 and 380 nm/460 nm excitation/emission filter set for caspase 9 as suggested by the manufacturer (Clontech). Data are expressed as mean±SEM. Caspase activity is expressed as nmol AFC · µg–1 · min–1.
Mitochondrial PTP and m
Mitochondrial PTP opening was determined by loading ventricular myocytes with 5 µmol/L calcein-acetoxymethylester (calcein-AM, Molecular Probes) in the presence of 2 to 5 mmol/L cobalt chloride as reported previously.1,25 To monitor mitochondrial m, myocytes were incubated with 50 nmol/L tetramethylrhodamine methyl ester perchlorate (TMRM), (Molecular Probes).1,4 Cells were visualized with an Olympus AX-70 Research fluorescence microscope (Carsen Group). Data are expressed as mean±SEM integrated optical density.5,26
Electrophoretic Mobility Gel Shift Assay
Nuclear extracts from myocytes were prepared as described previously by McKinsey et al.27 DNA-binding reactions (20 µg) were performed using a double-stranded oligonucleotide DNA template for NF-B as reported previously.28 NF-B supershift experiments were performed with a rabbit antibody directed toward the p65 subunit of NF-B clone C20 (1 µg/mL; Santa Cruz) to verify the migrating complex contained the p65 subunit of NF-B as reported previously.28 Nuclear-protein complexes were resolved on a native 5% polyacrylamide gel in 1x Tris-Borate EDTA, pH 8.0, and detected by autoradiography.
Statistical Analysis
Multiple comparisons between groups were determined by 2-way ANOVA. Unpaired 2-tailed Student’s t test was used to compare mean differences between groups. Differences were considered to be statistically significant at a level of P<0.05. In all cases, the data were obtained from at least n=3 to 4 independent myocyte isolations using n=3 replicates for each condition tested.
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Results |
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Because cellular NF-
B activity is regulated by IKKß-mediated phosphorylation and degradation of IB,13,29 we ascertained whether signaling pathways mediated by IKKß are functionally intact and operational for NF-B activation in ventricular myocytes. For these experiments, adenoviruses encoding wild-type and catalytically inactive versions of IKKß were generated. Cardiac myocytes were subsequently transfected with a luciferase reporter plasmid containing tandem binding elements for NF-B and infected with replication-defective adenovirus encoding either wild-type IKKßwt or kinase-inactive mutant IKKßmt.20,30 As shown in Figure 1A, cells expressing IKKß resulted in a 3.8-fold increase (P<0.05) in NF-B-dependent gene transcription compared with controls cells. Importantly, no apparent increase in NF-B-dependent gene transcription was observed with the IKKßmt, verifying that this mutation of IKKß was catalytically inactive and defective for activating NF-B in ventricular myocytes. Furthermore, IKKßwt-mediated NF-B gene transcription was inhibited with a nonphosphorylatable form of IB (IBSA), verifying that IKKßwt-mediated gene activation was contingent on the activation of the NF-B signaling pathway. Moreover, electromobility shift analysis revealed an increase in NF-B-dependent DNA binding in cells expressing the IKKßwt compared with vector control cells. Importantly, IKKßwt-mediated NF-B DNA binding was inhibited with the IBSA (Figure 1B), a finding consistent with our transcription data. The data verify that the signaling pathways mediated by IKKßwt are functionally intact and operational for NF-B activation in ventricular myocytes.14,21 Therefore, for subsequent experiments, we used IKKßwt as a surrogate for activating the NF-B signaling pathway in ventricular myocytes.
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To establish whether NF-B activation is sufficient to suppress hypoxia-induced apoptosis of ventricular myocytes, we subjected postnatal ventricular cells to hypoxia in the absence and presence of IKKß proteins. For these experiments, normoxic and hypoxic myocytes were infected with adenoviruses encoding either IKKßwt or IKKßmt and stained with the vital dyes calcein-AM and ethidium homodimer-1 to mark the number of live cells (green) and dead cells (red), respectively.13 As shown in Figure 2, A and B, myocytes expressing IKKßwt were indistinguishable from control cells (control versus IKKßwt, P=0.21) with respect to cell viability, indicating that IKKßwt was not cytotoxic to myocytes. Interestingly, a 3.3-fold increase (P<0.05) in myocyte death was observed in myocytes expressing the dominant-negative IKKßmt compared with control cells or cells expressing the IKKßwt, indicating that basal NF-B activity is required for cell survival. In contrast to normoxic control cells, a significant 9.1-fold increase (P<0.05) in myocyte death was observed in cells subjected to hypoxia. Moreover, a significant increase in nucleosomal DNA fragmentation by Hoechst 33258 nuclear staining was observed in cells subjected to hypoxia compared with normoxic control cells, (P<0.01) (Figure 2B), a finding concordant with our previous data verifying that hypoxia provokes apoptosis of ventricular myocytes.1,5 Importantly, hypoxia-induced apoptosis was suppressed in myocytes expressing IKKßwt (P<0.01) but not in cells expressing the IKKßmt and defective for NF-B activation (Figure 2B). These data strongly suggest that NF-B may be important for suppressing apoptosis in cardiac myocytes during hypoxia and are consistent with earlier work from our laboratory demonstrating a critical role for NF-B for suppression of apoptosis by the antiapoptotic factor Bcl-2.12,21 Because mitochondria are known targets of hypoxic injury, we reasoned that NF-B may avert cell death by operating at the level of the mitochondria. This prompted us to examine whether NF-B is associated with mitochondria. For these experiments, S100 cytoplasmic and mitochondrial fractions were prepared from ventricular myocytes and subjected to Western blot analysis. As shown in Figure 3, the p65 subunit of NF-B was readily detectable in the S100 cytoplasmic fraction. Interestingly, however, NF-B p65 was also detectable in the mitochondrial fraction under basal conditions (Figure 3). To verify that these novel and unanticipated findings were not a result of incomplete cytoplasmic and mitochondrial fractionation, we probed the Western blot with antibodies directed toward specific cytoplasmic and mitochondrial markers. As shown in Figure 3, the cytoplasmic marker -sarcomeric actin was detected exclusively in the S100 cytoplasmic but not in the mitochondrial fraction, whereas cytochrome oxidase subunit I was detected preferentially in the mitochondrial but not in the cytoplasmic fraction. This substantiates the integrity of our cellular fractions and verifies that detection of NF-B in the mitochondrial fraction of ventricular myocytes was not factitiously introduced by incomplete separation of cytoplasmic and mitochondrial compartments. Moreover, our data are concordant with a recent report documenting the presence of NF-B in mitochondria.31 The significance of the presence of NF-B in the mitochondria is unknown but raises the interesting possibility that the cytoprotective actions of NF-B may reside in its ability to suppress mitochondrial defects during hypoxic injury.
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statistically different from HYPX.
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Because perturbations to mitochondria resulting from the opening of the mitochondrial PTP have been suggested to be an underlying feature of the mitochondrial death pathway during hypoxia, we next ascertained whether IKKß-mediated NF-B activation influences mitochondrial function and hypoxia-induced PTP changes. For these experiments, ventricular myocytes were loaded with calcein-AM in the presence of cobalt chloride to quench the cytoplasmic signal.1 The loss of green fluorescence by mitochondria is a measure of mitochondrial membrane permeability and can be used as an index of PTP opening.17,25 As shown in Figure 4A, normoxic control cells displayed punctate green-staining mitochondria, indicative of the PTP in a closed configuration. In contrast, however, a significant 4.2-fold reduction (P<0.01) in mitochondrial green fluorescence was observed in cells subjected to hypoxia compared with normoxic control cells, indicative of the PTP in the open configuration. Importantly, hypoxia-induced mitochondrial PTP opening was suppressed in ventricular myocytes expressing IKKßwt but not in cells expressing the kinase-defective IKKßmt (Figure 4). Consistent with the PTP data were an observed 4.9-fold reduction (P<0.01) in mitochondrial m in cells subjected to hypoxia compared with normoxic control cells (Figure 5). Importantly, the hypoxia-induced loss of m was significantly less (1.3-fold) in cells expressing IKKßwt compared with normoxic controls (P<0.01). These findings confirm that hypoxia triggers perturbations to mitochondria consistent with PTP opening and loss of m through a pathway that is inhibitable by NF-B.
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Because earlier work postulated that mitochondrial defects resulting in PTP opening and loss of m were requisite events for the release of proapoptotic factors by mitochondria important for caspase activation and cell death, we next assessed whether hypoxia triggers the mitochondrial release of proapoptotic factors such as Smac, a key factor involved in postmitochondrial caspase activation. As shown by Western blot analysis (Figure 6A), Smac was absent from the S100 cytoplasmic fraction of normoxic cells but was readily detectable in the S100 fraction of cells subjected to hypoxia. Importantly, hypoxia-induced Smac release was suppressed in cells expressing IKKßwt but not in cells expressing the IKKßmt. Because mitochondrial defects leading to Smac release are considered to be important events for caspase activation, we next assessed whether caspase 9 and the death effector caspase 3 were activated in hypoxic cells. As shown in Figure 6B, a significant 4.8- and 4.4-fold increase (P<0.01) in caspase 9 and caspase 3 activities were observed in hypoxic cells compared with normoxic control cells. Importantly, hypoxia-induced caspase activation was suppressed in cells expressing the IKKßwt. Collectively, the data strongly suggest that IKKß-mediated NF-B activation suppresses cell death of ventricular myocytes through a mechanism that prevents hypoxia-induced mitochondrial perturbations.
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Discussion |
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In this report, we provide the first direct evidence that IKKß-mediated NF-
B activation activates a survival pathway that is sufficient to suppress hypoxia-induced apoptosis of ventricular myocytes. Moreover, our data strongly suggest that NF-B may suppress apoptosis through a mechanism that impinges on the mitochondrial death pathway. This is substantiated by the finding that hypoxia-induced mitochondrial defects, including PTP opening, loss of m, and Smac release, were suppressed in NF-B-activated cells. The mode by which hypoxia provokes apoptosis of ventricular myocytes is poorly defined; however, recent work by our laboratory and others has demonstrated that mitochondrial perturbations consistent with PTP opening, loss of m, and caspase activation are the predominant underlying cause.2,32,33 Furthermore, pharmacological interventions that suppressed hypoxia-induced PTP opening were also sufficient to suppress hypoxia-induced apoptosis of ventricular myocytes,1,17 establishing a strong association between hypoxia-induced mitochondrial perturbations and death of ventricular myocytes. The findings of the present study are intriguing and suggest that NF-B activation may suppress apoptosis of ventricular myocytes during hypoxia by preventing the mitochondrial defects that underlie PTP opening. The PTP, which is compromised in part of the VDAC, ANT, and cyclophilin D, presumably opens in response to death signals.34,35 PTP opening results in loss of m, large-amplitude swelling, and the release of proapoptotic factors by mitochondria, including Smac, procaspases, and others, ostensibly amplifying the death signal and caspase activation.34,36
Although the mode by which NF-B suppresses apoptosis is unknown, the fact that it can reportedly suppress cell death by different death signals37–39 implies that it most likely impinges on 1 or more components of the cell death pathway. This suggests that the survival properties conferred by NF-B may not be mutually dependent or obligatorily linked to its ascribed nuclear action, given a recent report documenting the localization of NF-B to mitochondria.31,40 The significance of this observation and its relation to cell death is unknown but raises the interesting possibility that NF-B may avert apoptosis in part by directly modulating mitochondrial function. This view is supported by the findings of the present study, which indicate that hypoxia-induced mitochondrial defects, including PTP opening, were suppressed by NF-B.
The underlying mechanism by which NF-B protects against mitochondrial perturbations during hypoxia is unknown. However, we provide 3 tenable explanations that may account for the observed findings. First, because PTP opening can reportedly be influenced by the proapoptotic factors Bak, Bax, and others, it is possible that NF-B may directly or indirectly impair the ability of these factors to open the PTP. Second, given that several antiapoptotic factors, including cellular inhibitors of apoptosis (c-IAP) cIAP1, c-IAP2, and Bcl-2, are known to be transcriptional targets of NF-B, we cannot exclude the possibility that NF-B may suppress cell death by activating 1 or more of these survival genes. However, the fact that hypoxia-induced PTP opening and Smac release were markedly suppressed in NF-B-activated cells would argue that NF-B suppresses mitochondrial events upstream of Smac release. Furthermore, no apparent changes in the expression levels of the c-IAP1, cIAP2, or Bcl-2 were observed in cells expressing IKKß (K.M. Regula, K. Ens, and L.A. Kirshenbaum, unpublished data). This raises the third and perhaps the most compelling possibility, that NF-B may influence PTP opening by directly influencing the activity of 1 or more components of the PTP itself. This notion is supported by a recent finding documenting the interaction between IB and the PTP component ANT.40 The significance of this finding and its relation to mitochondrial PTP opening and cell death remains to be elucidated but raises the possibility that NF-B-IB complexes at the level of the mitochondria influence PTP components during hypoxia. That transcription factors other than NF-B may have sites of action outside the nucleus is supported by a recent report documenting the localization of the nuclear protein p53 to mitochondria after a death signal.41 Importantly, the ability of p53 to provoke mitochondrial defects and cell death was independent of p53-mediated transcription. Whether or not NF-B is sufficient to avert apoptosis independently of de novo gene transcription, its ability to localize to mitochondria or both is unknown and is an active area of investigation.
Although our data substantiate a cytoprotective role for NF-B in ventricular myocytes, it must be stated that the ability of NF-B to suppress apoptosis may not be a universally conserved feature, because under certain instances, as in the case of Sindbis virus infection or in the case of proinflammatory cytokines, NF-B may trigger rather than prevent apoptosis.42,43 Thus, whether NF-B operates as a proapoptotic or antiapoptotic factor may instead depend on the cell type and the activating pathway. Furthermore, whether biological signals that activate NF-B outside the canonical IKK signaling complex are equivalently sufficient to avert hypoxia-induced mitochondrial defects and apoptosis in ventricular myocytes is unknown and has not been formally tested.
Nevertheless, under the conditions tested, our data provide new compelling evidence to suggest that IKKß-mediated NF-B activation activates a survival pathway that suppresses hypoxia-induced mitochondrial defects and apoptosis of ventricular myocytes. The fact that myocytes defective for NF-B activation with the IKKßmt exhibited an increased incidence of cell death strongly supports the contention that NF-B may be crucial for cell survival. This notion is concordant with previously published work by our laboratory demonstrating that functional inactivation of NF-B rendered cells susceptible to TNF-–induced apoptosis12,13 and is further corroborated by a recent report by Misra et al,44 who demonstrated an increased susceptibility of hearts defective for NF-B activation to ischemic injury. Although mitochondrial function was not assessed by Misra et al,44 the fact that Bcl-2 expression was reportedly reduced in NF-B-defective hearts strongly supports our data and the argument that NF-B suppresses cell death during ischemic/hypoxic injury through a mechanism that involves components of the cell death pathway that impinge on mitochondrial function. Therefore, interventions designed to selectively activate the NF-B signaling pathway may prove beneficial in suppressing mitochondrial death pathway activation and cardiac cell death in patients with ischemic heart disease.
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Acknowledgments |
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This work was supported by grants to Dr Kirshenbaum from the Canadian Institutes for Health Research (CIHR) and an Interdisciplinary Heart Research program grant from CIHR (CHFNET). Dr Regula holds a studentship from the CIHR, and Dr Kirshenbaum holds a Canada Research Chair in Molecular Cardiology. We are grateful to Drs W. Greene and D. Vaux for generous gifts of reagents cited and Dr H. Weisman for critical comments on the manuscript. Expert technical assistance by C. Robinson, J. Roth, and F. Aguilar is appreciated.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Gurevich RM, Regula KM, Kirshenbaum LA. Serpin protein CrmA suppresses hypoxia-mediated apoptosis of ventricular myocytes. Circulation. 2001; 103: 1984–1991.
2. Bialik S, Cryns VL, Drincic A, et al. The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ Res. 1999; 85: 403–414.
3. Reed JC. Cytochrome c: can’t live with it—can’t live without it [comment]. Cell. 1997; 91: 559–562.[CrossRef][Medline] [Order article via Infotrieve]
4. Regula KM, Kirshenbaum LA. p53 activates the mitochondrial death pathway and apoptosis of ventricular myocytes independent of de novo gene transcription. J Mol Cell Cardiol. 2001; 33: 1435–1445.[CrossRef][Medline] [Order article via Infotrieve]
5. Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res. 2002; 91: 226–231.
6. Kubasiak LA, Hernandez OM, Bishopric NH, et al. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3.
7. Gottlieb RA. Mitochondria: execution central. FEBS Lett. 2000; 482: 6–12.[CrossRef][Medline] [Order article via Infotrieve]
8. Yussman MG, Toyokawa T, Odley A, et al. Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy. Nat Med. 2002; 8: 725–730.[Medline] [Order article via Infotrieve]
9. Baeuerle PA, Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988; 242: 540–546.
10. Romashkova JA, Makarov SS. NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling [see comments]. Nature. 1999; 401: 86–90.[CrossRef][Medline] [Order article via Infotrieve]
11. Li S, Sedivy JM. Raf-1 protein kinase activates the NF-kappa B transcription factor by dissociating the cytoplasmic NF-kappa B-I kappa B complex. Proc Natl Acad Sci U S A. 1993; 90: 9247–9251.
12. de Moissac D, Zheng H, Kirshenbaum LA. Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis. J Biol Chem. 1999; 274: 29505–29509.
13. Mustapha S, Kirshner A, de Moissac D, et al. A direct requirement of nuclear factor-kappa B for suppression of apoptosis in ventricular myocytes. Am J Physiol. 2000; 279: H939–H945.
14. Cook SA, Novikov MS, Ahn Y, et al. A20 is dynamically regulated in the heart and inhibits the hypertrophic response. Circulation. 2003; 108: 664–667.
15. Kurrelmeyer KM, Michael LH, Baumgarten G, et al. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc Natl Acad Sci U S A. 2000; 97: 5456–5461.
16. Kirshenbaum LA, Schneider MD. Adenovirus E1A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains. J Biol Chem. 1995; 270: 7791–7794.
17. Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res. 2002; 91: 226–231.
18. Long X, Boluyt MO, Hipolito ML, et al. p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invest. 1997; 99: 2635–2643.[Medline] [Order article via Infotrieve]
19. Tanaka M, Ito H, Adachi S, et al. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res. 1994; 75: 426–433.
20. O’Mahony A, Lin X, Geleziunas R, et al. Activation of the heterodimeric IkappaB kinase alpha (IKKalpha)-IKKbeta complex is directional: IKKalpha regulates IKKbeta under both basal and stimulated conditions. Mol Cell Biol. 2000; 20: 1170–1178.
21. Regula KM, Ens K, Kirshenbaum LA. IKKbeta is required for Bcl-2 mediated NFkappa B activation in ventricular myocytes. J Biol Chem. 2002; 277: 38676–38682.
22. Tikoo A, O’Reilly L, Day CL, et al. Tissue distribution of Diablo/Smac revealed by monoclonal antibodies. Cell Death Differ. 2002; 9: 710–716.[CrossRef][Medline] [Order article via Infotrieve]
23. Vande VC, Cizeau J, Dubik D, et al. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol. 2000; 20: 5454–5468.
24. Moissac D, Gurevich RM, Zheng H, et al. Caspase activation and mitochondrial cytochrome c release during hypoxia-mediated apoptosis of adult ventricular myocytes. J Mol Cell Cardiol. 2000; 32: 53–63.[CrossRef][Medline] [Order article via Infotrieve]
25. Petronilli V, Miotto G, Canton M, et al. Imaging the mitochondrial permeability transition pore in intact cells. Biofactors. 1998; 8: 263–272.[Medline] [Order article via Infotrieve]
26. Regula KM, Kirshenbaum LA. p53 activates the mitochondrial death pathway and apoptosis of ventricular myocytes independent of de novo gene transcription. J Mol Cell Cardiol. 2000; 33: 1435–1445.[CrossRef]
27. McKinsey TA, Brockman JA, Scherer DC, et al. Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB. Mol Cell Biol. 1996; 16: 2083–2090.[Abstract]
28. de Moissac D, Mustapha S, Greenberg AH, et al. Bcl-2 activates the transcription factor NFkappaB through the degradation of the cytoplasmic inhibitor IkappaBalpha. J Biol Chem. 1998; 273: 23946–23951.
29. Beg AA, Baltimore D. An essential role for NF-kappaB in preventing TNF-alpha-induced cell death [see comments]. Science. 1996; 274: 782–784.
30. Devin A, Lin Y, Yamaoka S, et al. The alpha and beta subunits of IkappaB kinase (IKK) mediate TRAF2- dependent IKK recruitment to tumor necrosis factor (TNF) receptor 1 in response to TNF. Mol Cell Biol. 2001; 21: 3986–3994.
31. Cogswell PC, Kashatus DF, Keifer JA, et al. NF-kappa B and I kappa B alpha are found in the mitochondria: evidence for regulation of mitochondrial gene expression by NF-kappa B. J Biol Chem. 2003; 278: 2963–2968.
32. Ekhterae D, Lin Z, Lundberg MS, et al. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circ Res. 1999; 85: e70–e77.[Medline] [Order article via Infotrieve]
33. Ricci JE, Gottlieb RA, Green DR. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J Cell Biol. 2003; 160: 65–75.
34. Crompton M, Virji S, Doyle V, et al. The mitochondrial permeability transition pore. Biochem Soc Symp. 1999; 66: 167–179.[Medline] [Order article via Infotrieve]
35. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC [see comments]. Nature. 1999; 399: 483–487.[CrossRef][Medline] [Order article via Infotrieve]
36. Di Lisa F, Bernardi P. Mitochondrial function as a determinant of recovery or death in cell response to injury. Mol Cell Biochem. 1998; 184: 379–391.[CrossRef][Medline] [Order article via Infotrieve]
37. Wang CY, Mayo MW, Korneluk RG, et al. NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science. 1998; 281: 1680–1683.
38. Beg AA, Sha WC, Bronson RT, et al. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature. 1995; 376: 167–170.[CrossRef][Medline] [Order article via Infotrieve]
39. Barkett M, Gilmore TD. Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene. 1999; 18: 6910–6924.[CrossRef][Medline] [Order article via Infotrieve]
40. Bottero V, Rossi F, Samson M, et al. Ikappa B-alpha, the NF-kappa B inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator. J Biol Chem. 2001; 276: 21317–21324.
41. Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria: a potential role in apoptotic signaling. J Biol Chem. 2000; 275: 16202–16212.
42. Griffin DE, Levine B, Ubol S, et al. The effects of alphavirus infection on neurons. Ann Neurol. 1994; 35 (suppl): S23–S27.[CrossRef][Medline] [Order article via Infotrieve]
43. Matsushita H, Morishita R, Nata T, et al. Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)–mediated bcl-2 suppression: in vivo evidence of the importance of NF-kappaB in endothelial cell regulation. Circ Res. 2000; 86: 974–981.
44. Misra A, Haudek SB, Knuefermann P, et al. Nuclear factor-B protects the adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myocardial infarction. Circulation. 2003; 108: 3075–3078.
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  X. Lu, H. Liu, L. Wang, and S. Schaefer Activation of NF-{kappa}B is a critical element in the antiapoptotic effect of anesthetic preconditioning Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1296 - H1304. [Abstract] [Full Text] [PDF]
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 J. Shaw, N. Yurkova, T. Zhang, H. Gang, F. Aguilar, D. Weidman, C. Scramstad, H. Weisman, and L. A. Kirshenbaum Antagonism of E2F-1 regulated Bnip3 transcription by NF-{kappa}B is essential for basal cell survival PNAS, December 30, 2008; 105(52): 20734 - 20739. [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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N. C. Moss, W. E. Stansfield, M. S. Willis, R.-H. Tang, and C. H. Selzman IKKbeta inhibition attenuates myocardial injury and dysfunction following acute ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2248 - H2253. [Abstract] [Full Text] [PDF]
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 W. Li, N. Ma, L.-L. Ong, C. Nesselmann, C. Klopsch, Y. Ladilov, D. Furlani, C. Piechaczek, J. M. Moebius, K. Lutzow, et al. Bcl-2 Engineered MSCs Inhibited Apoptosis and Improved Heart Function Stem Cells, August 1, 2007; 25(8): 2118 - 2127. [Abstract] [Full Text] [PDF]
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 J. Shaw, I. A. Eydelnant, and L. A. Kirshenbaum Transgenic Expression of A20 Prevents Cardiac Cell Death and Myocardial Dysfunction After Myocardial Infarction Circulation, April 10, 2007; 115(14): 1827 - 1829. [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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 S. Javadov, D. Baetz, V. Rajapurohitam, A. Zeidan, L. A. Kirshenbaum, and M. Karmazyn Antihypertrophic Effect of Na+/H+ Exchanger Isoform 1 Inhibition Is Mediated by Reduced Mitogen-Activated Protein Kinase Activation Secondary to Improved Mitochondrial Integrity and Decreased Generation of Mitochondrial-Derived Reactive Oxygen Species J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1036 - 1043. [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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