Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy

Loss of cardiomyocytes through programmed cell death is a key event in the development of heart failure, but the inciting molecular mechanisms are largely unknown. We used microarray analysis to identify a genetic program for myocardial apoptosis in Gq-mediated and pressure-overload cardiac hypertrophy. A critical component of this apoptotic program was Nix/Bnip3L. Nix localized to mitochondria and caused release of cytochrome c, activation of caspase-3 and apoptotic cell death, when expressed in HEK293 fibroblasts. A previously undescribed truncated Nix isoform, termed sNix, was not targeted to mitochondria but heterodimerized with Nix and protected against Nix-mediated apoptosis. Forced in vivo myocardial expression of Nix resulted in apoptotic cardiomyopathy and rapid death. Conversely, sNix protected against apoptotic peripartum cardiomyopathy in Gq-overexpressors. Thus, Nix/Bnip3L is upregulated in myocardial hypertrophy, and is both necessary and sufficient for Gq-mediated apoptosis of cardiomyocytes and resulting hypertrophy decompensation.

The mechanisms for apoptosis induction in hypertrophied myocardium are unknown, and identifying genetic modifiers of apoptosis is a major investigative goal in cardiovascular biology^{1, 8, 9}. In the heart, as in all tissues, caspases are essential downstream effectors of apoptotic signals^{1, 10}, but multiple upstream pathways activate caspases, including death-ligand receptors and the primary mitochondrial pathway¹. One approach for delineating molecular determinants of apoptosis in cardiac hypertrophy might be transcriptome analysis of a relevant experimental model of apoptotic hypertrophy decompensation^{11, 12}, such as Gq-dependent cardiac hypertrophy progressing to apoptosis in the cardiac-specific Gq overexpressing mouse^{5, 13}. Gq is the heterotrimeric G protein that couples membrane receptors for angiotensin II, endothelin-1 and epinephrine to the cardiac hypertrophy response. In vivo inhibition or gene ablation has demonstrated that Gq signaling is necessary for normal embryonic cardiac development and for pressure overload hypertrophy in the adult heart^{14, 15, 16}. Indeed, enhanced receptor-mediated or autonomous cardiac Gq signaling is sufficient to recapitulate cellular, molecular and functional characteristics of pressure-overload hypertrophy^{5, 13, 17}, and can progress to apoptotic cardiac failure^{5, 18}.

Figure 1. Identification and cloning of mouse Nix and sNix. a, DNA microarray analysis showing 153 regulated genes in Gq transgenic mice (red +), of which 4 of 88 were apoptosis genes (black +). IAP, baculoviral IAP repeat; FAF-1; Fas-associated factor-1. b, Northern-blot analysis of IMAGE:656946/Nix showing increased expression in Gq transgenic (left) and transverse aortic coarcted (TAC; middle) mice, as well as human hypertensive left ventricular hypertrophy (LVH; right; representative of 3 individual experiments). Transcripts seen in mouse (black arrow) and human (red arrows) hearts are consistent with their respective known mRNA species35, 36. c, RT-PCR of Nix ORF from mouse hearts generated 2 products, identified by sequencing as: Nix and splice variant, sNix, which contains a 42-bp insert encoding a premature termination codon that truncates the protein by ten amino acids. Full Figure and legend (52K)

To identify the regulated transcript encoded by IMAGE:656945, we probed a custom Gq-transgenic mouse-heart cDNA library. Seven independent cDNA clones were isolated, the largest of which contained an incomplete open reading frame (ORF) identifying the gene as a recently described Bcl-2 family member, Nix/Bnip3L (ref. 20). The full-length mouse cardiac Nix ORF, encoding a 218-amino-acid protein, was obtained by reverse transcriptase (RT)-PCR (Fig. 1c and d). Also identified was a novel Nix splice variant, sNix, with a 42-base insertion that encodes a premature termination codon, truncating the C terminus by 10 amino acids (Fig. 1d). Because Nix is a pro-apoptotic mitochondria-targeted protein²⁰ and mitochondrial degeneration is characteristic of Gq-induced cardiomyocyte apoptosis²¹, we postulated that increased Nix expression could contribute to apoptotic degeneration.

Figure 2. Confocal analysis of Nix-expressing cells. a−d, Left column is FLAG−Nix (green) with light micrographs showing transfected and non-transfected cells; middle column is mitochondrial HSP-60 (a), cytochrome c (b), activated caspase-3 (c), and TUNEL (d). Right column is overlay. Full Figure and legend (85K)

The above results and recent published data²⁰ indicate that Nix can be a potent cell-suicide factor in vitro, but its in vivo function in myocardium or any other tissue is not known. Therefore, to determine if increased myocardial Nix expression is sufficient to cause cardiomyopathy, FLAG−Nix and -sNix were transgenically expressed in mouse hearts using the -myosin heavy-chain promoter (-MHC), chosen because its activity is minimal in the fetal ventricle, but induced shortly after birth²³. sNix-expressing mice from two independent lines were viable for nine months, and were normal in histological appearance, ventricular function, and cardiac gene expression (data not shown). Four Nix founders (F0) were identified (from 35 possible) (Fig. 3a), but only two mosaic founders, with 60 and 13 copies of the transgene, produced transgenic progeny. Myocardial Nix expression of first generation Nix mice correlated with transgene copy number (Fig. 3b). Immunoblot analysis demonstrated detectable myocardial transgene expression on postnatal day three, with expression that was near maximal on day five (Fig. 3b), and assays of cardiac gene expression revealed significantly increased atrial natriuretic peptide (ANF) (267 29% of nontransgenic siblings (NTG); P < 0.01), and decreased sarcoplasmic reticulum calcium (SERCA) (30 2% of NTG; P < 0.01) and phospholamban (25 1% of NTG; P < 0.01) gene expression, suggesting a molecular heart-failure phenotype²⁴ (Fig. 3c). At birth, Nix transgenic pups were indistinguishable from nontransgenic, but by six days old were strikingly smaller than their non-transgenic littermates (Nix body weight: 2.21 0.45 g, n = 13; nontransgenic littermates: 3.55 0.49 g, n = 25; P < 0.001) (Fig. 3d). All high-expressing Nix mouse pups died on day 6 or 7, whereas low-expressing Nix mice died between days 11 and 14. Although hearts of Nix mice appeared grossly normal, they occupied a substantially greater proportion of the thoracic cavity than those of their nontransgenic littermates (Fig. 3d) and echocardiography showed relative ventricular dilation, bradycardia, and depressed contractile function (Fig. 3d, inset).

Figure 3. Phenotypic analysis of Nix-expressing mice. a, Southern-blot analysis of 4 Nix founders, showing numbers of incorporated transgenes; top is quantitative standards. b, Southern (top) and western (middle) blots of Nix expression in neonatal F1 Nix-high (60) and Nix-low (13) mice. Bottom shows transgene protein expression as a function of mouse age. c, RNA dot blot analysis of cardiac gene expression in 6-day-old Nix mice and NTG. GAPDH, control; SERCA, sarcoplasmic reticulum calcium ATPase; bMHC, b myosin heavy chain; ANF, atrial natriuretic peptide; SKACT, skeletal actin; PLB, phospholamban; aMHC, a myosin heavy chain. d, Pathological analysis revealed stunted growth of 6-day-old mouse pups (left) and, on cross-section of mid thorax (right), relative cardiac enlargement (cardiac myosin light chains stained brown for visualization). Insets are m-mode echocardiograms; quantitative data are on top. ESD, end-systolic dimension; EDD, end-diastolic dimension; FS, fractional shortening; HR, heart rate. Full Figure and legend (90K)

Immuno-confocal microscopy demonstrated transgenic FLAG−Nix protein expression specifically in myocardial tissue (Fig. 4a). Positive signals from the terminal transferase−mediated dUTP-biotin nick end labeling (TUNEL) assay were widespread in Nix expressors, with apoptotic indices (positive nuclei/total nuclei) of 20 3% in Nix expressors versus 2 1% in controls (n = 5 each; P < 0.01). Apoptosis was specific for cardiomyocytes, as shown by costaining with -sarcomeric actin (Fig. 4b, left). Nuclear morphology was characteristic of apoptotic cells, with fragmentation and margination of DNA at the nuclear envelope (Fig. 4b, right). sNix-expressing mice showed none of the abnormalities seen in Nix mice (data not shown).

Figure 4. Cellular effects of myocardial Nix expression. a, Anti-FLAG fluorescence microscopy of Nix expression in ntg (left) and Nix (right) mice. LV, left ventricle; RV, right ventricle. Hearts are outlined and gain was increased to show non-staining tissues for clarity. b, Left panel, Confocal analysis of Nix ventricular myocardium. TUNEL-positive myocyte nuclei are green; normal nuclei are blue and cardiomyocytes are labeled red with -sarcomeric actin. Magnification, 60. Right panel, cardiomyocyte apoptotic bodies (arrow) visualized by TUNEL labeling. Magnification, 200. Full Figure and legend (83K)

Figure 5. Expression of recombinant Nix and sNix in HEK 293 cells. a, Immunoblot analysis demonstrated faster migration of FLAG−sNix, localized primarily in cytosol, compared to membrane-associated FLAG−Nix. b, Confocal microscopy of Nix/sNix (green) and mitochondrial HSP-60 (red) demonstrates localization of Nix, but not sNix in mitochondria (yellow). c, Immunoblot analyses of Nix homodimeric (column 2) and Nix/sNix heterodimeric (column 5) complexes. +, transfection or (IP) immunoprecipitation reagent. Columns 1 and 3, positive control; 4, negative control. d, Enhanced viability of HEK293 cells co-expressing Nix/sNix, relative to Nix alone. *, P < 0.001 compared with control; #, P < 0.001 compated with Nix. Full Figure and legend (56K)

The apparent dominant inhibitory activity of sNix for Nix was used to determine if Nix was essential for apoptosis in Gq-mediated peripartum cardiomyopathy⁵. Double transgenic sNix/Gq females were mated and followed for development of peripartum cardiomyopathy, including cardiac dilation, deterioration in contractile function, and death. At baseline, sNix did not affect Gq expression or the characteristic features of cardiac hypertrophy and contractile depression in this transgenic model (Fig. 6). However, co-expression of sNix with Gq delayed or prevented the early death seen in the peripartum period (Fig. 6a), strikingly diminished cardiomyocyte apoptosis (Fig. 6b), and largely prevented the characteristic ventricular postpartum dilation (Fig. 6c). However, cardiac mass (Gq = 220 10 mg; sNix/Gq = 200 20 mg) of Gq mice was not significantly changed by co-expression of Nix.

Figure 6. sNix effects on Gq-peripartum cardiomyopathy. a, Kaplan Meyer curve of peripartum survival. , Gq (n = 8); , sNix/Gq (n = 9). b, Apoptosis, assayed as apoptotic index. , Gq; , sNix/Gq; , NTG (*, P < 0.001). c, Serial echocardiography demonstrating progressive postpartum ventricular dilation and failure in Gq (right column), but not sNix/Gq (left column) mice. Top, prepartum; middle, 3 d postpartum; bottom, 7 d postpartum. Full Figure and legend (53K)

Myocardial hypertrophy, defined as increased cardiac mass and cardiomyocyte volume associated with characteristic changes in gene and protein expression, is the universal response to hemodynamic stress. Enhanced myocardial force-generating capacity and favorable geometry in the hypertrophied heart compensate for increased load, and thus permit increased cardiac work²⁷. However, in the face of unremitting hemodynamic overload, compensated hypertrophy inexorably and inevitably fails, leading to dilated cardiomyopathy through a poorly understood process termed decompensation²⁸. Loss of cardiomyocytes contributes to hypertrophy decompensation, and studies over the past decade have increasingly suggested an important pathophysiological role for cardiomyocyte apoptosis in progression to heart failure^{1, 2, 3, 4, 5, 6, 29}. To modify or abort decompensation, intrinsic determinants of myocardial apoptosis must be identified. Toward this end, an increase in pro-apoptotic Bcl-2 family members has been reported after in vitro cardiomyocyte oxidative stress³⁰ or mechanical stretch³¹, and pro-apoptotic Bax protein is increased after acute myocardial infarction in humans³². Particular importance as potential targets for therapeutic intervention would be assigned to apoptosis effectors that are induced in non-failing hypertrophy, thus preceding (and therefore possibly promoting) cardiomyocyte apoptosis and the heart-failure phenotype. The present studies identify Nix/Bnip3L as a hypertrophy-induced molecular trigger for cardiomyocyte suicide in Gq overexpression, experimental murine pressure overload and non-failing human cardiac hypertrophy.

Both pro- and anti-apoptotic Bcl-2 proteins are highly expressed in embryonic and neonatal hearts, in which apoptosis is required for normal development. Pro-apoptotic family members are, however, barely detectable in terminally differentiated adult myocardium³⁰, thus assuring a balance of Bcl-2-related proteins in the normal adult heart that is heavily shifted toward cell survival. We detected increased Nix expression as part of an upregulated four-member apoptosis gene program in cardiac hypertrophy secondary to Gq-overexpression, and murine and human pressure overload, all of which represent Gq-dependent processes^{5, 13, 14, 15, 16}. In vitro expression delineated the primary mitochondrial effects of Nix and revealed that its naturally occurring C-terminal truncated isoform, sNix, had no apoptotic activity, and was indeed a dominant inhibitor of Nix-mediated apoptosis. In vivo expression showed that forced Nix expression was sufficient to cause cardiomyocyte apoptosis and lethal dilated cardiomyopathy. Protection by sNix against apoptosis in the Gq-peripartum model further demonstrated that Nix function/mitochondrial targeting is necessary for this form of apoptotic heart failure.

The existence of a genetic program for apoptosis in cardiomyocytes may at first seem surprising, but is less so in the context of a hypertrophy-associated 'fetal gene program', that is, re-expression of genes that are abundant in the embryonic heart (including ANF and noncardiac isoforms of myosin and actin)²⁴. Because apoptosis is necessary for normal embryonic cardiac development, re-induction of apoptosis genes may represent another facet of this fetal program. However, only 4 of 88 assayed apoptosis genes were regulated in Gq-dependent myocardial hypertrophy; a generalized increase in apoptotic mediators did not occur. Generalized anti-apoptotic approaches for prevention of hypertrophy decompensation are therefore not likely to be as effective as specific targeting of a single critical regulated component, such as Nix. It must also be considered that apoptosis is not the only relevant mechanism for cardiomyocyte death in decompensating hypertrophy. A phenotype of neonatal heart failure from myocyte necrosis has been reported in the -MHC Arg403Gly mutation mouse model of hypertrophic cardiomyopathy^{33, 34} suggesting that an effective approach to prevent cardiomyocyte death in decompensating hypertrophy must target both processes.

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The cardiac-specific Gq transgenic mouse model of myocardial hypertrophy, and its proclivity for apoptotic decompensation under defined conditions, have been described in detail^{5, 13, 17, 18}. Gq ventricular poly(A)⁺ RNA for DNA microarray and northern-blot analysis was prepared from 8-wk-old male mice. Transgenic mice expressing murine Nix, or its truncated variant sNix, were created using the full-length mouse -MHC promoter²³ and were identified by genomic Southern-blot analysis of tail clip DNA using the Nix or sNix cDNAs as radioactive probes. Transverse aortic banding to induce pressure overload, was performed as described¹⁷. Transaortic gradients averaged 93 3 mmHg in 3-day banded mice. Studies were performed in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Mouse and human ventricular mRNA was prepared using Triazol (Gibco-BRL, Grand Island, New York) and the Oligotex mRNA isolation kit (Qiagen, Valencia, California) according to manufacturer's instructions. Total RNA was passed twice through oligo d(T) columns and mRNA was stored as ethanolic precipitates at -70 °C. Experiments were performed with RNA pooled from 2 (northern blotting, RT-PCR) or 8 (microarray analyses) individual mouse ventricles. DNA-microarray hybridizations were performed in duplicate on different samples of Gq mRNA using Incyte mouse gene-expression microarrays (GEM), version 1.12 (Genome Systems, St. Louis, Missouri). Poly(A)⁺ mRNA from Gq transgenic hearts and their corresponding non-transgenic controls were used to prepare Cy3 and Cy5 derivatized cDNAs, which were competitively hybridized to the DNA chips. Data were examined and analyzed as described¹⁹. Northern-blot analysis used standard methodology and formaldehyde/agarose gels in which 2 g (mouse) or 7 g (human) of poly(A)⁺ mRNA was loaded per lane. Mouse blots were probed with ³²P-labeled (random priming) IMAGE EST:656945 obtained from Incyte, and human northern blots were probed with a 2.6-kb fragment of human Nix 3' UTR (Genbank Accession #AL132665).

A custom Gq mouse-heart cDNA library was screened using IMAGE:656945 as radioactive template. The longest cDNA clone was 2.7 kb with an incomplete ORF of 642 base pairs (bp), which permitted identification of the gene product as mouse Nix²⁰ (Genbank Accession #AF067395). The full mouse Nix coding region was obtained by RT-PCR of Gq ventricular mRNA using, as forward primer (5'-CTCGAGAGCCGGATACTGTC-3') and as reverse primer (5'-CGACTGAGCACACCTTCT-3'). On multiple occasions, from both Gq and non-transgenic mouse hearts, two products were obtained, subcloned into PCR2 vector, and verified by DNA sequencing. Amino terminal epitope tags were added using PCR mutagenesis and the modified cDNAs were subcloned into pcDNA3 for transient expression in HEK293 fibroblasts. Expression was quantified by immunoblotting or fluorescence immunohistochemistry. Cellular viability was assessed with Trypan blue 0.4% (Sigma).

Paraffin-embedded heart sections fixed in 10% neutral buffered formalin underwent antigen unmasking by heating to 95 °C in 10 mM sodium citrate buffer, pH = 6.0, for 20 minutes. Confocal imaging was performed on a dual laser Nikon PCM2000 system and a Nikon Eclipse E800 microscope with emission wavelengths of 515 30 nm (fluorescein), 605 32 nm (Texas red), and 650 nm long-pass (Cy5). Light micrographs used differential interference contrast optics. Antibodies to HSP60 (Santa Cruz Biotech, #SC-1052), cytochrome-c (Santa Cruz Biotech, #SC-7159), active caspase-3 (R&D Systems, #AF835) and -sarcomeric actin (Zymed #18-0177) were identified with Texas Red, fluorescein, or Cy5-linked secondary antibodies (Vector Laboratories, Burlingame, California, and Amersham Pharmacia, Piscataway, New Jersey). Epitope tags were labeled using anti-FLAG M2 monoclonal antibody (Sigma #F3165) or anti-HA (12CA5) (Roche #1583816). Apoptosis was determined using the TUNEL assay and Promega fluorescein or horseradish peroxidase apoptosis detection systems. Immunoprecipitations were performed in 0.2% CHAPS and 150 mM NaCl buffer.

Data are expressed as mean SEM. Comparisons used Student's t test or ANOVA as appropriate. Survival of peripartum Gq and sNix/Gq mice was compared by Wilcoxon test. P value of < 0.05 was assigned statistical significance.

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1. MacLellan, W.R. & Schneider, M.D. Death by design. Programmed cell death in cardiovascular biology and disease. Circ. Res. 81, 137–144 (1997). | PubMed  | ISI | ChemPort |
2. Narula, J. et al. Apoptosis in myocytes in end-stage heart failure. N. Engl. J. Med. 335, 1182–1189 (1996). | Article | PubMed  | ISI | ChemPort |
3. Olivetti, G. et al. Apoptosis in the failing human heart. N. Engl. J. Med. 336, 1131–1141 (1997). | Article | PubMed  | ISI | ChemPort |
4. Mallat, Z. et al. Evidence of apoptosis in arrhythmogenic right ventricular dysplasia. N. Engl. J. Med. 335, 1190–1196 (1996). | Article | PubMed  | ISI | ChemPort |
5. Adams, J.W. et al. Enhanced Gq signaling: A common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proc. Natl. Acad. Sci. USA 95, 10140–10145 (1998). | Article | PubMed  | ChemPort |
6. Hirota, H. et al. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress. Cell 97, 189–198 (1999). | Article | PubMed  | ISI | ChemPort |
7. Levy, D. et al. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N. Engl. J. Med. 322, 1561–1566 (1990). | PubMed  | ISI | ChemPort |
8. Chien, K.R. Molecular advances in cardiovascular biology. Science 260, 916–917 (1993). | PubMed  | ISI | ChemPort |
9. Williams, R.S. Apoptosis and heart failure. N. Engl. J. Med. 341, 759–760 (1999). | Article | PubMed  | ISI | ChemPort |
10. Thornberry, N.A. & Lazebnik, Y. Caspases: Enemies within. Science 281, 1312–1316 (1998). | Article | PubMed  | ISI | ChemPort |
11. MacLellan, W.R. & Schneider, M.D. Genetic dissection of cardiac growth control pathways. Annu. Rev. Physiol. 62, 289–319 (2000). | Article | PubMed  | ISI | ChemPort |
12. Schneider, M.D. & Schwartz, R.J. Chips ahoy: Gene expression in failing hearts surveyed by high-density microarrays. Circulation 102, 3026–3027 (2000). | PubMed  | ISI | ChemPort |
13. D'Angelo, D.D. et al. Transgenic Gq overexpression induces cardiac contractile failure in mice. Proc. Natl. Acad. Sci. USA 94, 8121–8126 (1997). | Article | PubMed  | ChemPort |
14. Offermanns, S. et al. Embryonic cardiomyocyte hypoplasia and craniofacial defects in Gq/G11-mutant mice. EMBO J. 17, 4304–4312 (1998). | Article | PubMed  | ISI | ChemPort |
15. Akhter, S.A. et al. Targeting the receptor–Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science 280, 574–577 (1998). | Article | PubMed  | ISI | ChemPort |
16. Wettschureck, N. et al. Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Gq/G11 in cardiomyocytes. Nat. Med. 7, 1236–1240 (2001). | Article | PubMed  | ISI | ChemPort |
17. Sakata, Y. et al. Decompensation of pressure-overload hypertrophy in Gq-overexpressing mice. Circulation 97, 1488–1495 (1998). | PubMed  | ISI | ChemPort |
18. De Windt, L.J. et al. Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: An apoptosis-independent model of dilated heart failure. Circ. Res. 86, 255–263 (2000). | PubMed  | ISI | ChemPort |
19. Aronow, B.J. et al. Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy. Physiol. Genomics 6, 19–28 (2001). | PubMed  | ISI | ChemPort |
20. Chen, G. et al. Nix and Nip3 form a subfamily of pro-apoptotic mitochondrial proteins. J. Biol. Chem. 274, 7–10 (1999). | Article | PubMed  | ISI | ChemPort |
21. Adams, J.W. et al. Cardiomyocyte apoptosis induced by Gq signaling is mediated by permeability transition pore formation and activation of the mitochondrial death pathway. Circ.Res. 87, 1180–1187 (2000). | ChemPort |
22. Shi, Y. A structural view of mitochondria-mediated apoptosis. Nat. Struct. Biol. 8, 394–401 (2001). | Article | PubMed  | ISI | ChemPort |
23. Subramaniam, A. et al. Tissue-specific regulation of the -myosin heavy chain gene promoter in transgenic mice. J. Biol. Chem. 266, 24613–24620 (1991). | PubMed  | ISI | ChemPort |
24. Chien, K.R., Knowlton, K.U., Zhu, H. & Chien, S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: Molecular studies of an adaptive physiologic response. FASEB J. 5, 3037–3046 (1991). | PubMed  | ISI | ChemPort |
25. Ray, R. et al. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J. Biol. Chem. 275, 1439–1448 (2000). | Article | PubMed  | ISI | ChemPort |
26. Chen, G. et al. The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J. Exp. Med. 186, 1975–1983 (1997). | Article | PubMed  | ISI | ChemPort |
27. Mirsky, I. Left ventricular stresses in the intact human heart. Biophys. J. 9, 189–208 (1969). | PubMed  | ISI | ChemPort |
28. Katz, A.M. Cardiomyopathy of overload. A major determinant of prognosis in congestive heart failure. N. Engl. J. Med. 322, 100–110 (1990). | PubMed  | ISI | ChemPort |
29. Zhang, D. et al. TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice. Nat. Med. 6, 556–563 (2000). | Article | PubMed  | ISI | ChemPort |
30. Cook, S.A., Sugden, P.H. & Clerk, A. Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: Association with changes in mitochondrial membrane potential. Circ. Res. 85, 940–949 (1999). | PubMed  | ISI | ChemPort |
31. Leri, A. et al. Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin–angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell. J. Clin. Invest. 101, 1326–1342 (1998). | PubMed  | ISI | ChemPort |
32. Misao, J. et al. Expression of bcl-2 protein, an inhibitor of apoptosis, and Bax, an accelerator of apoptosis, in ventricular myocytes of human hearts with myocardial infarction. Circulation 94, 1506–1512 (1996). | PubMed  | ISI | ChemPort |
33. Geisterfer-Lowrance, A.A. et al. A mouse model of familial hypertrophic cardiomyopathy. Science 272, 731–734 (1996). | PubMed  | ChemPort |
34. Fatkin, D. et al. Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the cardiac myosin heavy chain gene. J. Clin. Invest. 103, 147–153 (1999). | PubMed  | ISI | ChemPort |
35. Bruick, R.K. Expression of the gene encoding the pro-apoptotic Nip3 protein is induced by hypoxia. Proc. Natl. Acad. Sci. USA 97, 9082–9087 (2000). | Article | PubMed  | ChemPort |
36. Yasuda, M. et al. BNIP3: A human homolog of mitochondrial proapoptotic protein BNIP3. Cancer Res. 59, 533–537 (1999). | PubMed  | ISI | ChemPort |

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Competing interests statement:  The authors declare that they have no competing financial interests.