Kelly M. Regula

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 (BNIP3DeltaTM), necessary for mitochondrial insertion. The pan-caspase inhibitor z-VAD-fmk (25 to 100 micromol/L) suppressed BNIP3-induced cell death of ventricular myocytes in a dose-dependent manner. Bongkrekic acid (50 micromol/L), an inhibitor of the PT pore, prevented BNIP3-induced mitochondrial defects and cell death. Expression of BNIP3DeltaTM 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.

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. 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. Cardiomyocyte apoptosis contributes to functional deterioration in ischemic, hypertrophic, and dilated cardiomyopathies (1Narula J. Haider N. Virmani R. DiSalvo T.G. Kolodgie F.D. Hajjar R.J. Schmidt U. Semigran M.J. Dec G.W. Khaw B.A. N. Engl. J. Med. 1996; 335: 1182-1189Crossref PubMed Scopus (1246) Google Scholar, 2Olivetti G. Abbi R. Quaini F. Kajstura J. Cheng W. Nitahara J.A. Quaini E. Di Loreto C. Beltrami C.A. Krajewski S. Reed J.C. Anversa P. N. Engl. J. Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1483) Google Scholar, 3Bialik S. Geenen D.L. Sasson I.E. Cheng R. Horner J.W. Evans S.M. Lord E.M. Koch C.J. Kitsis R.N. J. Clin. Investig. 1997; 100: 1363-1372Crossref PubMed Scopus (348) Google Scholar, 4Condorelli G. Morisco C. Stassi G. Notte A. Farina F. Sgaramella G. de Rienzo A. Roncarati R. Trimarco B. Lembo G. Circulation. 1999; 99: 3071-3078Crossref PubMed Scopus (250) Google Scholar, 5Hirota H. Chen J. Betz U.A. Rajewsky K. Gu Y. Ross Jr., J. Muller W. Chien K.R. Cell. 1999; 97: 189-198Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). 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 (4Condorelli G. Morisco C. Stassi G. Notte A. Farina F. Sgaramella G. de Rienzo A. Roncarati R. Trimarco B. Lembo G. Circulation. 1999; 99: 3071-3078Crossref PubMed Scopus (250) Google Scholar, 6Misao J. Hayakawa Y. Ohno M. Kato S. Fujiwara T. Fujiwara H. Circulation. 1996; 94: 1506-1512Crossref PubMed Scopus (327) Google Scholar). 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 (7Guo K. Searfoss G. Krolikowski D. Pagnoni M. Franks C. Clark K. Yu K.T. Jaye M. Ivashchenko Y. Cell Death. Differ. 2001; 8: 367-376Crossref PubMed Scopus (252) Google Scholar, 8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar, 9Kubasiak L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12825-12830Crossref PubMed Scopus (391) Google Scholar, 10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar). These two factors are each expressed in the heart, localized to mitochondria, and sufficient to induce apoptosis via the intrinsic, or mitochondrial, pathway (8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar, 10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar, 11Zhang H.M. Cheung P. Yanagawa B. McManus B.M. Yang D.C. Apoptosis. 2003; 8: 229-236Crossref PubMed Scopus (74) Google Scholar). The potential for BNip3 or Nix, alone or in association with other Bcl2 family proteins (12Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (723) Google Scholar, 13Zong W.X. Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: 1481-1486Crossref PubMed Scopus (715) Google Scholar), 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 (14Graham R.M. Frazier D.P. Thompson J.W. Haliko S. Li H. Wasserlauf B.J. Spiga M.G. Bishopric N.H. Webster K.A. J. Exp. Biol. 2004; 207: 3189-3200Crossref PubMed Scopus (159) Google Scholar). Indeed, mitochondrial disruption may have especially profound consequences for the heart as myocardium is enriched in mitochondria and has a high rate of energy utilization (15Marin-Garcia J. Goldenthal M.J. J. Card. Fail. 2002; 8: 347-361Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We examined the hypothesis that cardiac regulation of these two closely related mitochondrial death proteins would, because of their functional similarities, be distinct, i.e. that each was the apoptotic effector of a different cell stress pathway. Previous reports have defined in detail the physiological mechanisms and molecular determinants of BNip3 gene regulation; BNip3 expression varies between tissue types (16Yasuda M. Han J.W. Dionne C.A. Boyd J.M. Chinnadurai G. Cancer Res. 1999; 59: 533-537PubMed Google Scholar), and in the heart, is strikingly increased in response to in vitro cardiomyocyte hypoxia/acidosis or in vivo transient myocardial ischemia (8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar, 9Kubasiak L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12825-12830Crossref PubMed Scopus (391) Google Scholar, 14Graham R.M. Frazier D.P. Thompson J.W. Haliko S. Li H. Wasserlauf B.J. Spiga M.G. Bishopric N.H. Webster K.A. J. Exp. Biol. 2004; 207: 3189-3200Crossref PubMed Scopus (159) Google Scholar). In cultured carcinoma cells, hypoxic induction of BNip3 was reported to be hypoxia-inducible factor-1α-(HIF-1α) 3The abbreviations used are: HIF-1hypoxia-inducible factor-1HREhypoxia-response elementsPKCprotein kinase CPMAphorbol myristate acetateNixPNix promoterluciferaseTACtransverse aortic coarctationPP2aprotein phosphatase 2aNRCMneonatal rat cardiac myocyteslucluciferase. dependent, consistent with the presence of multiple hypoxia-response elements (HRE) that are canonical HIF-1α-binding sites in the 5′ promoter region of the BNip3 gene (17Sowter H.M. Ratcliffe P.J. Watson P. Greenberg A.H. Harris A.L. Cancer Res. 2001; 61: 6669-6673PubMed Google Scholar). Nix, on the other hand, is constitutively expressed in many tissues, including the heart (18Matsushima M. Fujiwara T. Takahashi E. Minaguchi T. Eguchi Y. Tsujimoto Y. Suzumori K. Nakamura Y. Genes Chromosomes Cancer. 1998; 21: 230-235Crossref PubMed Scopus (94) Google Scholar, 19Chen G. Cizeau J. Vande V.C. Park J.H. Bozek G. Bolton J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: 7-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), and has not been reported to increase with myocardial hypoxia/ischemia (14Graham R.M. Frazier D.P. Thompson J.W. Haliko S. Li H. Wasserlauf B.J. Spiga M.G. Bishopric N.H. Webster K.A. J. Exp. Biol. 2004; 207: 3189-3200Crossref PubMed Scopus (159) Google Scholar). Instead, Nix expression is strikingly increased in Gq-dependent cardiac hypertrophies (10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar). hypoxia-inducible factor-1 hypoxia-response elements protein kinase C phorbol myristate acetate Nix promoterluciferase transverse aortic coarctation protein phosphatase 2a neonatal rat cardiac myocytes luciferase. Notwithstanding the absence of positive data for Nix transcriptional regulation in ischemic cardiac tissue, the presence of three putative HREs in the human Nix promoter (20Aerbajinai W. Giattina M. Lee Y.T. Raffeld M. Miller J.L. Blood. 2003; 102: 712-717Crossref PubMed Scopus (91) Google Scholar) and a report of hypoxic induction of Nix in cultured tumor cell lines (17Sowter H.M. Ratcliffe P.J. Watson P. Greenberg A.H. Harris A.L. Cancer Res. 2001; 61: 6669-6673PubMed Google Scholar) have led to general acceptance of the that cardiac Nix and BNip3 are hypoxia-inducible Circ. Res. 2002; 91: PubMed Scopus Google Scholar) and are in a by H.M. Cheung P. Yanagawa B. McManus B.M. Yang D.C. Apoptosis. 2003; 8: 229-236Crossref PubMed Scopus (74) Google Scholar). The of a high of functional and is not In contrast, distinct mechanisms for Nix and BNip3 apoptotic to defined be for cell death in a as the have the and in vitro and in vivo functional of the Nix with the basal of Nix expression between tissue and in the heart, Nix is induced by different physiological stimuli. cardiac Nix expression was by protein kinase C activation and of the transcription factor Sp1. These hypoxic induction of cardiac Nix and that Nix and BNip3 the of distinct stress-mediated cardiomyocyte apoptosis for hypertrophy and hypoxia. of the Nix and BNip3 and of a high to the region between Nix and protein phosphatase 2a the Nix A was the and To the were with and sites respectively. The were with and and the and sites of The Nix promoter region was by The Nix promoter was with and to a of 5′ that were to Nix promoter-luciferase reporter A was used to a luciferase reporter the human BNip3 Cardiomyocyte and myocytes were and cultured as (8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar). of was 24 cardiomyocyte were with PKC of of hypoxic with by or with (10 nm phorbol myristate acetate in 10 10 isoproterenol, or nm or angiotensin of Nix Nix promoter-luciferase and was the of cell were by of and were was in of the transgenic by transverse aortic coarctation for and of by 24 were as (10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar, Odley A. Y. Lynch R.A. D.L. R. Dorn G.W. Circ. Res. PubMed Scopus Google Scholar). were in with of and used of with for Nix or BNip3. Nix, were to a of the the molecular Nix (10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar). A multiple tissue was Luciferase were in cell and by and the was for luciferase and other were and in of cell and for and were for luciferase activity in a the Luciferase luciferase activity was to protein are reported as induction to for myocytes or of protein for tissue were as M.G. Dorn G.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of was with of human protein in the presence or absence of a of for were by and by were as of the GC is and underwent by in 10 and were with or used with or with were examined on a are reported as ± were in were or of A was used for with In Nix and BNip3 Nix is on of We used to of the Nix promoter revealed the absence of or transcription elements but the presence of several GC boxes 5′ to the transcription is of The presence of several HREs in the human Nix promoter has been (20Aerbajinai W. Giattina M. Lee Y.T. Raffeld M. Miller J.L. Blood. 2003; 102: 712-717Crossref PubMed Scopus (91) Google Scholar) and a for the Nix is in cardiac hypoxia Circ. Res. 2002; 91: PubMed Scopus Google Scholar). Indeed, the Nix promoter and human BNip3 promoter are in that multiple putative and GC To determine Nix and BNip3 are for these two different Nix expression with in whereas BNip3 between different in heart, and These distinct of gene expression suggested in to distinct basal expression the Nix and BNip3 be In of Nix and BNip3 identify for regulation of Nix and BNip3 in the heart, of each gene were to a luciferase reporter and expressed in cultured neonatal rat cardiac myocytes of in a hypoxic increased but not Nix, activity The transcriptional response to Nix promoter activity was induced by a hypertrophic in N.H. J. Clin. Investig. PubMed Scopus Google Scholar, J. Clin. Investig. PubMed Scopus Google Scholar), but not isoproterenol or angiotensin In contrast, BNip3 promoter activity was not induced by of these and transcription in response to phenylephrine and isoproterenol was the in vitro distinct regulation of Nix and BNip3 to and BNip3 is induced by whereas Nix is induced by the hypertrophic Since the mechanisms for hypoxic BNip3 induction have been in detail L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12825-12830Crossref PubMed Scopus (391) Google Scholar), and for hypertrophic induction of Nix are studies on The functional of in the Nix promoter was in of basal and activity of 5′ Nix promoter-luciferase reporter and by in basal Nix activity in to the of the to A Nix the region a of basal transcriptional to elements the gene not and was not of GC boxes with of basal promoter reports have of GC in several with of the transcription to GC elements M.G. Dorn G.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. S.M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. Y. Y. R. Biol. PubMed Scopus Google Scholar, S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Chen J. 2003; PubMed Scopus Google Scholar), used to determine the be for Nix and with a was with a the to region the to region and the to region of the Nix promoter but not A with a GC with the and the of in a These that to in the Nix The for regulation of has been reported as induction of factor by PKC M.G. Dorn G.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. S.M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. Y. Y. R. Biol. PubMed Scopus Google Scholar, S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Chen J. 2003; PubMed Scopus Google Scholar). studies that is to induce hypertrophy in by PKC K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. J. Biol. Chem. Full Text PDF PubMed Google Scholar), increased Nix promoter activity the consequences on Nix transcription of PKC with phorbol myristate acetate (PMA, 10 nm for 24 in PMA increased Nix transcriptional activity in on the of Nix promoter activity and elements in the of Nix and the that some of these elements bound the transcription factor M.G. Dorn G.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), that inhibition of Nix transcription. Indeed, to GC boxes with mithramycin A E. S.M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) basal activity and PMA for each of the Nix In the phorbol of Nix promoter activity was as a for activation of To determine PKC was of Nix transcription and to activity of PKC in with NixP were with or the two and PKC Y. Odley A. A. M.G. A. Dorn G.W. Circ. Res. 2003; PubMed Scopus Google Scholar). of with induced cell death by apoptosis and not be in the in PKCα, but not increased Nix promoter activity in cardiac myocytes to with with consistent with a major for negative was effects on Nix transcription these studies a for Nix transcriptional activation by in In of the Nix in vivo determinants of Nix transcriptional regulation were evaluated in three transgenic lines the with expression on the basal luciferase activity was in was in hearts in other with cardiac activity was increased in hearts ± for and in mice the transgenic Y. Lorenz J.N. R.A. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) but not in hearts 24 of ischemia of the a of to be sufficient for induction of BNip3 (8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar). Nix transcriptional with the luciferase was proportional to the of cardiac hypertrophy as heart for To determine the for Nix induction in and cardiac hypertrophy was as suggested by the activity was in of mice for with mithramycin in in vivo of Nix induction by mithramycin that Nix transcriptional regulation is Sp1. with these expression was increased in with Nix transcriptional activity as luciferase protein in mice with The the that Nix and the two major cardiac BH3-only proteins, are by mechanisms H.M. Cheung P. Yanagawa B. McManus B.M. Yang D.C. Apoptosis. 2003; 8: 229-236Crossref PubMed Scopus (74) Google Scholar, Circ. Res. 2002; 91: PubMed Scopus Google Scholar). Instead, each of these two mitochondrial death proteins to be induced by a different of physiological and stimuli. Nix is the apoptotic effector for myocardial whereas BNip3 is the apoptotic effector for cardiac hypoxia. These not of for cardiomyocyte but defined for apoptosis in ischemic myocardial cardiac hypertrophy apoptosis is to have in heart and myocardial are the to the Nix and BNip3 are In ischemic and dilated human the of apoptotic is increased (1Narula J. Haider N. Virmani R. DiSalvo T.G. Kolodgie F.D. Hajjar R.J. Schmidt U. Semigran M.J. Dec G.W. Khaw B.A. N. Engl. J. Med. 1996; 335: 1182-1189Crossref PubMed Scopus (1246) Google Scholar, 2Olivetti G. Abbi R. Quaini F. Kajstura J. Cheng W. Nitahara J.A. Quaini E. Di Loreto C. Beltrami C.A. Krajewski S. Reed J.C. Anversa P. N. Engl. J. Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1483) Google Scholar). has not been to determine apoptosis is a factor in human heart decompensation is a of functional have the potential for D. M. K. W. S. S.M. J. Kitsis R.N. J. Clin. Investig. 2003; PubMed Scopus Google Scholar) or H. Chen J. Betz U.A. Rajewsky K. Gu Y. Ross Jr., J. Muller W. Chien K.R. Cell. 1999; 97: 189-198Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar) myocardial apoptosis to heart Indeed, human cardiac hypertrophy is associated with Nix induction (10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar), and inhibition of Nix with a dominant negative in mitochondrial is apoptotic heart in the transgenic model (10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar). These data the that cell death in and apoptosis in contributes to hypertrophy decompensation and the of heart In contrast, BNip3 is not in cardiac hypertrophy or by hypertrophic but is increased in expression by hypoxia or ischemia (8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar, 9Kubasiak L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12825-12830Crossref PubMed Scopus (391) Google Scholar). The distinct for Nix and BNip3 are of particular because the two proteins have are BH3-only members of the of apoptotic proteins, are to mitochondria by a and with other proteins, as or to apoptosis by and activation of the pathway H.M. Cheung P. Yanagawa B. McManus B.M. Yang D.C. Apoptosis. 2003; 8: 229-236Crossref PubMed Scopus (74) Google Scholar, 14Graham R.M. Frazier D.P. Thompson J.W. Haliko S. Li H. Wasserlauf B.J. Spiga M.G. Bishopric N.H. Webster K.A. J. Exp. Biol. 2004; 207: 3189-3200Crossref PubMed Scopus (159) Google Scholar, Circ. Res. 2002; 91: PubMed Scopus Google Scholar). Indeed, has been some as to Nix and BNip3 were of the or different to by the for Nix as (18Matsushima M. Fujiwara T. Takahashi E. Minaguchi T. Eguchi Y. Tsujimoto Y. Suzumori K. Nakamura Y. Genes Chromosomes Cancer. 1998; 21: 230-235Crossref PubMed Scopus (94) Google Scholar, 19Chen G. Cizeau J. Vande V.C. Park J.H. Bozek G. Bolton J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: 7-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The studies that these related apoptotic factors are the of different that are by distinct physiological in the is that Nix and BNip3 be in other as by hypoxia in human tumor (17Sowter H.M. Ratcliffe P.J. Watson P. Greenberg A.H. Harris A.L. Cancer Res. 2001; 61: 6669-6673PubMed Google Scholar), the in cardiac regulation functional the protein be to the of Nix and BNip3 in the in vivo The molecular mechanisms of cardiac BNip3 induction have been elucidated in detail (8Regula K.M. Ens K. Kirshenbaum L.A. Circ. Res. 2002; 91: 226-231Crossref PubMed Scopus (279) Google Scholar, 9Kubasiak L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12825-12830Crossref PubMed Scopus (391) Google Scholar). is a for BNip3 by the in the promoter In contrast, Nix transcription was not increased by in vitro hypoxia or angiotensin II Instead, was increased by that are associated with of myocardial or cardiac i.e. phenylephrine and (10Yussman M.G. Toyokawa T. Odley A. Lynch R.A. Wu G. Colbert M.C. Aronow B.J. Lorenz J.N. Dorn G.W. Nat. Med. 2002; 8: 725-730Crossref PubMed Scopus (265) Google Scholar, N.H. J. Clin. Investig. PubMed Scopus Google Scholar, J. Clin. Investig. PubMed Scopus Google Scholar, Y. Lorenz J.N. R.A. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). A of each of these K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. Lorenz J.N. R.A. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, G.W. Wu G. A. Google Scholar), was sufficient to induce Nix promoter activity in cultured and may be the of in vivo Nix induction as the studies be in is that is increased and in Gq-dependent hypertrophy Y. Lorenz J.N. R.A. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, G.W. Wu G. A. Google Scholar) and that has been to with Gq to and heart in Y. Odley A. A. M.G. A. Dorn G.W. Circ. Res. 2003; PubMed Scopus Google Scholar). transcriptional of Nix is of many effects of activation in myocardial hypertrophy. In the of the Nix transcriptional induction was via to elements the of transcriptional regulation has been with phorbol of to other including for the M.G. Dorn G.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), T. M. Y. Y. R. Biol. PubMed Scopus Google Scholar), S. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), E. S.M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and II Chen J. 2003; PubMed Scopus Google Scholar). particular to the and K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) a transcriptional for cardiomyocyte PKCα, and that the promoter in vitro hypertrophy. of in vivo Nix induction by mithramycin the of pathway to the These studies the cardiac Nix induction is by the to increased Nix expression activation of by hypertrophic induction of and to GC in the In overexpression and pathway. The distinct and mechanisms Nix and BNip3 in the heart a and for of cell death in the In the the of for apoptosis proteins a gene expression the and the of

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.