Lack of JunD Promotes Pressure Overload–Induced Apoptosis, Hypertrophic Growth, and Angiogenesis in the Heart
http://www.100md.com
《循环学杂志》
the Departments of Cardiology and Angiology (D.H.-K., K.K., A.S., K.M., A.Q., H.D.), Cardiovascular Surgery (A.H.), and Nephrology (J.P.), Hannover Medical School, Hannover, Germany
Unit of Gene Expression and Disease, Pasteur Institute, Paris, France (M.Y., J.B.W.).
Abstract
Background— The Jun family of activator protein 1 (AP-1) transcription factors (c-Jun, JunB, and JunD) is involved in fundamental biological processes such as proliferation, apoptosis, tumor angiogenesis, and hypertrophy. The role of individual AP-1 transcription factors in the stressed heart is not clear. In the present study we analyzed the role of JunD in survival, hypertrophy, and angiogenesis in the pressure-overloaded mouse heart after thoracic aortic constriction.
Methods and Results— Mice lacking JunD (knockout [KO]) showed increased mortality and enhanced cardiomyocyte apoptosis and fibrosis associated with increased levels of hypoxia-induced factor-1, vascular endothelial growth factor (VEGF), p53, and Bax protein and reduced levels of Bcl-2 protein after 7 days of severe pressure overload compared with wild-type (WT) siblings. Cardiomyocyte hypertrophy in surviving KO mice was enhanced compared with that in WT mice. Chronic moderate pressure overload for 12 weeks caused enhanced left ventricular hypertrophy in KO mice, and survival and interstitial fibrosis were comparable with WT mice. Cardiac function, 12 weeks after operation, was comparable among shams and pressure-overloaded mice of both genotypes. In addition, KO mice exposed to chronic pressure overload showed higher cardiac capillary density associated with increased protein levels of VEGF.
Conclusions— Thus, JunD limits cardiomyocyte hypertrophy and protects the pressure-overloaded heart from cardiac apoptosis. These beneficial effects of JunD, however, are associated with antiangiogenic properties.
Key Words: angiogenesis apoptosis hypertrophy
Introduction
Arterial hypertension imposes biomechanical stress on the left ventricle (LV), forcing the heart to work harder. Under these conditions, cardiomyocytes undergo hypertrophic growth. Although cardiac hypertrophy is initially compensatory for increased workload, prolongation of this process leads to congestive heart failure, arrhythmias, and sudden death.1–3 Insufficient oxygenation due to abnormalities in the microvascularization is involved in the pathogenesis of heart failure. In this respect, cardiomyocyte hypertrophy increases the diffusion distance, and failure of angiogenesis to compensate for this, together with the increased oxygen demand in the work-overloaded heart, has been shown to induce cardiac apoptosis and is associated with heart failure.4 For instance, therapeutic enhancement of angiogenesis has been shown to improve cardiac function.5
One of the earliest responses of the heart to stress (eg, acute infarction, ischemic preconditioning, pressure overload, or neurohumoral stimulation) is the reexpression of silenced activator protein 1 (AP-1) transcription factors and the formation of AP-1 transcriptional complexes.6–11 In this respect, AP-1 transcription factors have been implicated in cardiomyocyte hypertrophy, apoptosis, and cardiac fibrosis.9,12–19
AP-1 transcriptional complexes are composed of Jun proteins (c-Jun, JunB, and JunD) that can form homodimers or heterodimers with Fos and ATF proteins.20 Despite the observation that Jun proteins share a high degree of sequence homology, they often exert opposing effects on cell growth, division, and survival. Analysis of AP-1 complex function in vitro has demonstrated distinct roles for each Jun protein and has suggested that various homodimer and heterodimer combinations determine the nature of the AP-1–mediated cellular responses. In this respect, c-Jun has been associated with cell proliferation, angiogenesis, and hypertrophic growth.13,21–25 Activation of c-Jun NH2-terminal kinases (JNK), which phosphorylates c-Jun and activates AP-1 complexes, has been associated with different forms of cardiac pathology (reviewed by Petrich and Wang26). More specifically, JNK via c-Jun regulates cardiomyocyte hypertrophy and apoptosis and is involved in contractile function and extracellular matrix remodeling of the heart.26–28 In contrast to c-Jun, JunD has no JNK docking site and seems only to be efficiently phosphorylated by JNK when it dimerizes with c-Jun.29 Furthermore, JunD has been associated with the antiproliferative state and cell differentiation.22 A recent report showed that JunD protects cells from oxidative stress but attenuates tumor angiogenesis by promoting proteasomal degradation of hypoxia-induced factor-1 (HIF1), thereby reducing expression of proangiogenic vascular endothelial growth factor (VEGF).30 The precise role of JunD in the heart, however, is not known.
During embryogenesis, c-Jun and JunB are necessary for the development of the cardiovascular system but are downregulated in the adult heart and rapidly reinduced in the adult myocardium under stress.13,24,31,32 In contrast, JunD protein is dispensable for development of the embryonic cardiovascular system, even though it is constitutively expressed throughout cardiac development and remains present at substantial levels in cardiomyocytes and nonmyocytes of the adult heart.32,33 Surprisingly, JunD protein levels are decreased in the failing myocardium of transgenic mice with cardiac-specific overexpression of tumor necrosis factor- (TNF-).34 Furthermore, reduced levels of JunD mRNA have been reported in the myocardium of patients with end-stage heart failure due to dilated cardiomyopathy.35
To elucidate the role of JunD under pathophysiological stress in the heart, we explored the effects of pressure overload in mice lacking JunD by thoracic aortic constriction (TAC) and analyzed the role of JunD in survival, cardiac hypertrophy, and apoptosis as well as in angiogenesis.
Methods
The targeted disruption of the junD gene (junD–) in mice (129/SvJ background) has been previously described.33 For this study, homozygous mutant (junD–/–; knockout [KO]), heterozygous mutant (junD–/+), and wild-type (WT) (junD+/+) mice were raised from heterozygous mutant (junD–/+) breeding pairs. Primers used for genotyping were as follows: endogenous junD gene: 5'-CACTG-CACCTTCTGCGCTGCG-3'; 5'-CGCTTGCGGCATTTGGAGGC-3'; for the inserted LacZ transgene: 5'-CATCGGAGCCGGTCTGTA-3'; 5'-GCGATTAAGTTGGGTAACGC-3'.
Animal Experiments
Male mice (aged 12 weeks) were sedated with propofol (10 mg/kg IV), anesthetized with 3% enflurane, and connected to a rodent ventilator (Harvard). Transverse TAC was performed as described36 with the use of wires with diameter of 300 μm to generate severe pressure overload (sTAC) or wires with diameter of 400 μm to generate moderate pressure overload (mTAC). Sham-operated mice, one group analyzed 7 days and a second group analyzed 12 weeks after sham operation, served as controls. The surgeon was blinded to the genotype of the mice. After the mice were euthanized, hearts were rapidly excised; LVs were dissected and snap-frozen in liquid nitrogen. For histological analyses, hearts were embedded in OCT after retrograde perfusion with PBS (pH 7.4) containing 50 mmol/L KCl and 200 U/mL heparin or embedded in paraffin after paraformaldehyde in situ fixation as described previously.37
Hemodynamic Measurement and Transthoracic Echocardiography
Hemodynamic measurements were performed in mice under anesthesia with the use of a 1.4F micromanometer conductance catheter (SPR-719; Millar Instruments) as described.37,38 In brief, mice were anesthetized and mechanically ventilated with enflurane (3%), a bilateral vagotomy was performed, and the catheter was inserted in the LV cavity via the right carotid artery. Echocardiography was performed in sedated mice (ketamine 100 mg/kg and xylazine 1.25 mg/kg IP) as described.39 In brief, short-axis M-mode images were recorded at the papillary muscle level in sedated mice with the use of a linear 15-MHz transducer (ATL HDI 5000 CV). Fractional shortening (FS) was calculated as follows: FS(%)=[(LVEDD–LVESD)/LVEDD]x100, where LVEDD is LV end-diastolic diameter and LVESD is LV end-systolic diameter.
All animal studies were in compliance with the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health and were approved by the local animal care committees.
Patient Population
LV myocardium was obtained from patients undergoing heart transplantation due to end-stage dilated cardiomyopathy (n=10; mean age, 44±13 years; NYHA functional class III or IV; LV ejection fraction, 16±7%; end-diastolic diameter, 65±14 mm). Seven patients had been treated with ACE inhibitors, 6 patients with diuretics, 7 patients with digoxin, and 5 patients with -blockers. For comparison, we studied LV tissue samples from donor hearts that could not be transplanted for technical reasons (nonfailing donors) (n=7; mean age, 43±2 years). Tissue samples were snap-frozen in liquid nitrogen no later than 2 hours after explantation.
Tissue Morphometry and Fibrosis
Tissue morphometry was performed in a blinded fashion in in situ fixed LV tissue slices stained with picrosirius red or hematoxylin and eosin (H&E) with the use of the Quantimet 500MC digital image analyzer as described.37 Mean cardiomyocyte cross-sectional area (CSA) was determined in in situ fixed LV sections stained with H&E or stained with antibodies recognizing wheat germ agglutinin (WGA) (Vector) as described.40 For nuclear staining, hematoxylin or Hoechst 33258 (Sigma) was used. Apoptotic nuclei were determined by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) (QBIOGENE)/Hoechst 33258 (Sigma) staining, and cardiomyocytes were identified by sarcomeric -actinin staining (Sigma) as described previously.40
Capillary Density and Number of Coronary Resistance Vessels
Capillary density was determined in LV sections with transversely sectioned cardiomyocytes immunostained against isolectin B4 (Vector) or platelet–endothelial cell adhesion molecule–1 (PECAM-1) (Santa Cruz) and counterstained with WGA and Hoechst 33258 (Sigma) as described previously.40 In brief, the ratio of isolectin B4– or PECAM-1–positive cells to total number of nuclei (Hoechst stain) per field was calculated. High-power fields (x400; 200x200 μm; 8 fields per section of LV basis, middle part, and apex; n=4 per genotype) with transversely sectioned cardiomyocytes were digitally recorded (Quantimet 500MC). Capillary-to-cardiomyocyte ratio was determined in LV sections double stained for isolectin B4 and WGA. For each mouse, high-power fields (x400; from LV basis, middle part, and apex with transversely sectioned myocytes) were digitally recorded to calculate the number of capillaries per cardiomyocyte in 50 to 100 cardiomyocytes.
Arterioles and coronary resistance vessels were counted in -smooth muscle actin– (Sigma) and WGA-stained transverse LV sections (3 sections per heart: basis, middle part, and apex; n=4 for each genotype) as described.40 We distinguished arterioles with inner diameters <25 μm and 25 to 50 μm and vessels with diameters >50 μm and counted them separately.
Investigators were blinded to the genotype of the material for capillary and coronary density quantification.
Polymerase Chain Reaction and Immunoblot Analyses
Reverse transcriptase–polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting were performed according to standard procedures.41 Real-time measurements of PCR amplification were performed with the Stratagene MX4000 multiplex QPCR System with the SYBR green dye method (Brilliant SYBR Green Mastermix Kit, Stratagene). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal standard. Primers used for real-time PCR were as follows: ANP: 5'-GCCGGTAGAA-GATGAGGTCA-3'; 5'-GGGCTCCAATCCTGTCAATC-3'; skeletal muscle -actin: 5'-ATCTCACGTTCAGCTGTGGTCA-3'; 5'-ACCA-CCGGCATCGTGTTGGAT-3'; MHC: 5'-GGAAGAGCGAGCGG-CGCATCAAGG-3'; 5'-CTGCTGGACAGGTTATTCCTCA-3'; G3PDH: 5'-AACGACCCCTTCATTGAC-3'; 5'-TCCACGACAT-ACTCAGCAC-3'; VEGF: 5'-TTACTGCTGTACCTCCACC-3'; 5'-ACAGGACGGCTTGAAGATG-3'. Antibodies against JunD, c-Jun, c-Fos, p53, Bax, Bcl-2, HIF1, VEGF, phospho-JNK1/2, JNK, phospho-ERK1/2, ERK1/2, actin (Santa Cruz), and active caspase-3 (BD Pharmingen) were used for immunoblots.
Statistical Analysis
All data are given as mean±SD. Differences between groups were evaluated by Student t test or ANOVA followed by Bonferroni test as appropriate. Statistical significance was defined as P<0.05.
Results
Cardiac Phenotype of JunD KO Mice
Examination of mice lacking JunD (KO) revealed that they had normal life spans and no gross morphological abnormalities of the heart, but they had reduced body weights (BW),33 LV weights, and LV/BW ratios (Table). Morphometric analysis of in situ fixed and H&E-stained myocardial cross sections showed that cardiac dimensions (Table) and CSA were reduced in KO mice (Figure 1). Heterozygous mutant mice (junD–/+) did not differ significantly from WT animals with respect to survival, size, LV/BW ratio, cardiac dimensions, and cardiac function under basal conditions and after severe (7 days) or moderate (12 weeks) pressure overload (data not shown); therefore, in this study only data from WT and KO mice are presented.
Morphometric, Hemodynamic, and Echocardiographic Analyses of WT and KO Mice After 12 Weeks of Sham Operation or Moderate Pressure Overload (mTAC)
Severe Pressure Overload Resulted in High Mortality, Enhanced Myocardial Apoptosis, and Elevated Expression of p53 in KO Mice
We examined the response to severe pressure overload (sTAC) in WT (WT-sTAC) and KO (KO-sTAC) mice. sTAC resulted in a higher mortality in KO-sTAC mice (Figure 1A); 7 of 11 KO-sTAC mice died within the first 7 days of sTAC, whereas only 3 of 14 WT-sTAC mice died within the same period. All WT-sham (n=8) and KO-sham (n=8) mice survived 7 days (Figure 1A). After 7 days, the surviving WT-sTAC and KO-sTAC mice showed a similar rise in prestenotic systolic blood pressure: WT-sham, 122±7 mm Hg versus WT-sTAC, 179±20 mm Hg, P<0.05; KO-sham, 123±10 mm Hg versus KO-sTAC, 168±15 mm Hg, P<0.05; WT-sTAC versus KO-sTAC, P=NS. After 7 days of sTAC, apoptosis in cardiomyocytes and nonmyocytes, assessed by TUNEL assay, was more pronounced and protein levels of active caspase-3 were augmented in LVs of KO-sTAC mice compared with WT-sTAC mice (Figure 1B and 1C). In addition, compared with WT-sTAC mice, KO-sTAC mice showed higher increases in protein levels of p53 (WT-sTAC, 130%; KO-sTAC, 243%; P<0.05) and of the proapoptotic Bcl-2 family member Bax (WT-sTAC, 240%; KO-sTAC, 349%; P<0.05), whereas the increase in protein levels of the antiapoptotic protein Bcl-2 (WT-sTAC, 325%; KO-sTAC, 189%; P<0.05) was attenuated (Figure 1C). Protein levels of JunD were not significantly altered in WT-sTAC mice 7 days after sTAC (data not shown). The expression pattern of c-Jun and c-Fos and the phosphorylation of JNK were similar in both genotypes after sTAC (Figure 1C).
Seven days after sTAC, surviving KO-sTAC mice showed a larger increase in CSA compared with WT-sTAC mice (Figure 1D and 1E). Echocardiography in KO-sTAC mice was not possible because these mice did not survive anesthesia.
Chronic Moderate Pressure Overload Triggered an Enhanced Hypertrophic Response in KO Mice
Moderate pressure overload (mTAC) for 7 days or for 12 weeks did not result in increased mortality in KO or WT mice. mTAC for 7 days resulted in a similar rise in systolic prestenotic blood pressure in both genotypes (WT-mTAC, 150±17 mm Hg versus KO-mTAC, 147±11 mm Hg; P=NS) and in a similar increase in the LV/BW ratio (WT [n=5], 38±22% versus KO [n=5], 34±9%; P=NS) in KO-mTAC and WT-mTAC mice. The prevalence of TUNEL-positive cells tended to be higher in KO-mTAC mice than in WT-mTAC mice (data not shown); however, no increase in activated caspase-3 protein could be detected in WT-mTAC or KO-mTAC LVs after 7 days of mTAC (data not shown).
After 12 weeks of mTAC, KO-mTAC mice showed a significantly stronger hypertrophic response in LVs compared with WT-mTAC mice, which was also reflected by a larger increase in the LV/BW ratio (33% in WT-mTAC versus 85% in KO-mTAC; P<0.01; Table, Figure 2A) and a higher increase in CSA (Table, Figure 2A). There was a similar rise in the prestenotic blood pressure (Table), and there were no differences in cardiac function (determined by echocardiography; Table) or interstitial fibrosis (determined by picrosirius red [not shown] and H&E staining [Figure 2B]) between WT-mTAC and KO-mTAC mice after 12 weeks of mTAC. Real-time PCR analyses showed enhanced increases in mRNA levels of ANP and skeletal muscle -actin in KO-mTAC LVs, whereas the reduction in MHC was similar in LVs of both genotypes 12 weeks after mTAC (Figure 2C).
Chronic Moderate Pressure Overload Reduces Protein Levels of JunD
Previous work demonstrated substantial expression of JunD in the adult mouse heart, mainly in blood vessels and cardiomyocytes.33,34 After mTAC for 12 weeks, protein levels of JunD in WT-mTAC LVs were significantly decreased (–43±25%; P<0.05) compared with WT-shams (Figure 2D). No JunD protein was detected in KO LVs (Figure 2D). Protein levels of c-Fos and c-Jun tended to be higher (P=NS) in KO-mTAC LVs compared with WT-mTAC LVs, whereas activation of JNK and Erk1/2 proteins was not significantly different between the 2 genotypes 12 weeks after mTAC (Figure 2D).
Increased VEGF Expression and Higher Capillary Density in KO Mice After Pressure Overload
It has recently been shown that JunD attenuates tumor angiogenesis by protecting cells from oxidative stress, which in turn promotes proteasomal degradation of HIF1 and results in lower expression of the proangiogenic factor VEGF.30 We observed a higher increase in the expression of HIF1 protein and VEGF mRNA and protein after sTAC for 7 days in KO-sTAC mice compared with WT-sTAC mice (Figure 3A).
After mTAC for 12 weeks, KO-mTAC mice showed markedly increased VEGF protein levels compared with WT-mTAC mice (Figure 3B). However, this increase in the amount of VEGF proteins was not paralleled by substantially increased levels of VEGF mRNA or HIF1 protein (Figure 3B).
We performed immunostaining of LV sections from WT-sham, KO-sham, WT-mTAC, and KO-mTAC mice 12 weeks after operation with PECAM-1 or isolectin B4 to mark capillary endothelial cells. The increases in the capillary density and in the capillary-to-cardiomyocyte ratio were higher in KO-mTAC mice than in WT-mTAC mice (Figure 4A and 4C). In addition, antibody staining to smooth muscle cells revealed that KO-mTAC LVs showed significantly more arterioles than WT-mTAC or sham LVs (Figure 4B and 4C).
JunD Protein Levels Are Decreased in Patients With End-Stage Heart Failure
In patients with end-stage heart failure due to dilated cardiomyopathy, JunD protein levels in LVs were severely reduced compared with nonfailing LVs (Figure 5).
Discussion
The present study shows that in the absence of JunD, there is increased apoptosis and hypertrophy in the myocardium in response to pressure overload. Moreover, JunD reduces pressure overload–induced cardiac angiogenesis. These data extend the biological function of JunD as regulator of cell proliferation and tumor progression by providing evidence that JunD plays a critical role in cardioprotection and hypertrophy and that it acts as a negative modulator of angiogenesis in response to pathophysiological stress in the heart.
Mice lacking JunD show a high rate of mortality in response to severe pressure overload, associated with an increase in cardiac apoptosis, indicating that JunD protects against cardiomyocyte loss in response to increased workload. It has been shown that lack of JunD promotes p53-dependent apoptosis in fibroblasts in vitro.16 Furthermore, KO mice show enhanced hepatocyte apoptosis on injection with bacterial lipopolysaccharide, indicating that JunD plays a role in antiapoptotic pathways.16 A recent study demonstrated that JunD protects cells from oxidative stress by regulating genes involved in antioxidant defense.30 Oxidative stress, in turn, has been associated with increased expression of p53,42,43 HIF1, and VEGF.30,44,45 Moreover, p53 reduces the expression of genes opposing cell death, such as Bcl-2, and upregulates genes promoting apoptosis, such as Bax.46 Pressure overload is associated with oxidative stress and with increased production of reactive oxygen species.47 Indeed, KO mice exposed to sTAC showed enhanced cardiac expression of p53 and HIF1, which may be related to oxidative stress. The increased activation of proapoptotic p53-dependent pathways in ventricles of KO-sTAC mice, eg, upregulation of Bax and downregulation of Bcl-2, supports the notion that the high mortality rate in KO-sTAC mice could result, at least in part, from increased cardiomyocyte apoptosis due to enhanced oxidative stress. These findings suggest that JunD may protect the heart from pressure overload by attenuating proapoptotic pathways.
The inability to increase cardiac perfusion by limited cardiac neoangiogenesis is considered to represent a key element in the transition of compensated to decompensated hypertrophy.4 KO mice exposed to mTAC for 12 weeks show a higher cardiac capillary and arteriole density and a higher capillary-to-cardiomyocyte ratio. The KO-mTAC mice therefore developed more severe LV hypertrophy, and this growth response of the myocardium was associated with increased angiogenesis, which represents a physiologically adequate vascular adaptation to increased oxygen demand of hypertrophied cardiomyocytes. This could, at least in part, explain why no significant alterations in cardiac function, fibrosis, or apoptosis occurred in KO-mTAC mice after chronic pressure overload despite profound cardiomyocyte hypertrophy. Previous studies have shown that regulation of the proangiogenic factor VEGF is critical for myocardial angiogenesis.48 Remarkably, KO-sTAC mice express higher levels of VEGF mRNA and protein and of HIF1 protein, a transcription factor known to promote VEGF expression, after 7 days of severe pressure overload. Biomechanical and oxidative stress induce HIF1 expression, mainly by attenuating its proteasomal degradation.30 In this respect, JunD has been shown to promote HIF1 protein degradation in tumors,30 and thus the lack of JunD in the heart might promote HIF1 protein stabilization and VEGF transcription, leading to enhanced stress-induced angiogenesis. Interestingly, after mTAC for 12 weeks, elevated VEGF protein levels in KO-mTAC mice were not associated with a significant increase in HIF1 protein accumulation or paralleled by significantly increased VEGF mRNA levels. This finding suggests that JunD, in addition to the transcriptional regulation, may be involved in the posttranscriptional regulation of VEGF, probably by indirect regulation of proteasomal degradation. Alternatively, the half-life of VEGF mRNA could be shorter than the VEGF protein half-life in KO-mTAC mice. Future studies will be needed to investigate this observation.
It has previously been reported that human and mouse adult myocardium constitutively expresses high levels of JunD protein in cardiomyocytes and nonmyocytes.33–35 Pollack and coworkers35 showed that JunD mRNA levels are reduced in human failing myocardium. Consistent with these observations, we found decreased JunD protein levels in the LVs of patients with end-stage heart failure (Figure 5). In the present study we show that loss of JunD results in increased mortality and cardiomyocyte apoptosis after acute pressure overload but enhances angiogenesis in the chronic phase of the disease. These results suggest that lower levels of JunD in failing human hearts may lead to cardiomyocyte loss in response to acute pathophysiological stress but may allow a higher degree of cardiac angiogenesis.
One limitation of this study is that loss of JunD is associated with a modestly smaller body and heart size. These features indicate that KO mice may differ from WT animals in their metabolism and growth. In addition, the lack of JunD may lead to the activation of compensatory mechanisms, which in turn may influence the cardiac phenotype independently of JunD. Our findings concerning apoptosis and angiogenesis, however, are consistent with biological functions of JunD described in cell culture and tumorigenesis models.16,30
In conclusion, the absence of JunD causes enhanced cardiomyocyte hypertrophy and cardiac apoptosis in response to pressure overload, possibly because JunD attenuates oxidative stress. These beneficial effects of JunD, however, are associated with antiangiogenic properties.
Acknowledgments
This work was supported by the DAAD and by grants of the association for International Cancer Research and the European Community. We are grateful to Birgit Brandt and Silvia Gutzke for excellent technical assistance.
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Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard JC, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, D’Amore PA, Shima DT. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat Med. 1999; 5: 495–502.(Denise Hilfiker-Kleiner, )
Unit of Gene Expression and Disease, Pasteur Institute, Paris, France (M.Y., J.B.W.).
Abstract
Background— The Jun family of activator protein 1 (AP-1) transcription factors (c-Jun, JunB, and JunD) is involved in fundamental biological processes such as proliferation, apoptosis, tumor angiogenesis, and hypertrophy. The role of individual AP-1 transcription factors in the stressed heart is not clear. In the present study we analyzed the role of JunD in survival, hypertrophy, and angiogenesis in the pressure-overloaded mouse heart after thoracic aortic constriction.
Methods and Results— Mice lacking JunD (knockout [KO]) showed increased mortality and enhanced cardiomyocyte apoptosis and fibrosis associated with increased levels of hypoxia-induced factor-1, vascular endothelial growth factor (VEGF), p53, and Bax protein and reduced levels of Bcl-2 protein after 7 days of severe pressure overload compared with wild-type (WT) siblings. Cardiomyocyte hypertrophy in surviving KO mice was enhanced compared with that in WT mice. Chronic moderate pressure overload for 12 weeks caused enhanced left ventricular hypertrophy in KO mice, and survival and interstitial fibrosis were comparable with WT mice. Cardiac function, 12 weeks after operation, was comparable among shams and pressure-overloaded mice of both genotypes. In addition, KO mice exposed to chronic pressure overload showed higher cardiac capillary density associated with increased protein levels of VEGF.
Conclusions— Thus, JunD limits cardiomyocyte hypertrophy and protects the pressure-overloaded heart from cardiac apoptosis. These beneficial effects of JunD, however, are associated with antiangiogenic properties.
Key Words: angiogenesis apoptosis hypertrophy
Introduction
Arterial hypertension imposes biomechanical stress on the left ventricle (LV), forcing the heart to work harder. Under these conditions, cardiomyocytes undergo hypertrophic growth. Although cardiac hypertrophy is initially compensatory for increased workload, prolongation of this process leads to congestive heart failure, arrhythmias, and sudden death.1–3 Insufficient oxygenation due to abnormalities in the microvascularization is involved in the pathogenesis of heart failure. In this respect, cardiomyocyte hypertrophy increases the diffusion distance, and failure of angiogenesis to compensate for this, together with the increased oxygen demand in the work-overloaded heart, has been shown to induce cardiac apoptosis and is associated with heart failure.4 For instance, therapeutic enhancement of angiogenesis has been shown to improve cardiac function.5
One of the earliest responses of the heart to stress (eg, acute infarction, ischemic preconditioning, pressure overload, or neurohumoral stimulation) is the reexpression of silenced activator protein 1 (AP-1) transcription factors and the formation of AP-1 transcriptional complexes.6–11 In this respect, AP-1 transcription factors have been implicated in cardiomyocyte hypertrophy, apoptosis, and cardiac fibrosis.9,12–19
AP-1 transcriptional complexes are composed of Jun proteins (c-Jun, JunB, and JunD) that can form homodimers or heterodimers with Fos and ATF proteins.20 Despite the observation that Jun proteins share a high degree of sequence homology, they often exert opposing effects on cell growth, division, and survival. Analysis of AP-1 complex function in vitro has demonstrated distinct roles for each Jun protein and has suggested that various homodimer and heterodimer combinations determine the nature of the AP-1–mediated cellular responses. In this respect, c-Jun has been associated with cell proliferation, angiogenesis, and hypertrophic growth.13,21–25 Activation of c-Jun NH2-terminal kinases (JNK), which phosphorylates c-Jun and activates AP-1 complexes, has been associated with different forms of cardiac pathology (reviewed by Petrich and Wang26). More specifically, JNK via c-Jun regulates cardiomyocyte hypertrophy and apoptosis and is involved in contractile function and extracellular matrix remodeling of the heart.26–28 In contrast to c-Jun, JunD has no JNK docking site and seems only to be efficiently phosphorylated by JNK when it dimerizes with c-Jun.29 Furthermore, JunD has been associated with the antiproliferative state and cell differentiation.22 A recent report showed that JunD protects cells from oxidative stress but attenuates tumor angiogenesis by promoting proteasomal degradation of hypoxia-induced factor-1 (HIF1), thereby reducing expression of proangiogenic vascular endothelial growth factor (VEGF).30 The precise role of JunD in the heart, however, is not known.
During embryogenesis, c-Jun and JunB are necessary for the development of the cardiovascular system but are downregulated in the adult heart and rapidly reinduced in the adult myocardium under stress.13,24,31,32 In contrast, JunD protein is dispensable for development of the embryonic cardiovascular system, even though it is constitutively expressed throughout cardiac development and remains present at substantial levels in cardiomyocytes and nonmyocytes of the adult heart.32,33 Surprisingly, JunD protein levels are decreased in the failing myocardium of transgenic mice with cardiac-specific overexpression of tumor necrosis factor- (TNF-).34 Furthermore, reduced levels of JunD mRNA have been reported in the myocardium of patients with end-stage heart failure due to dilated cardiomyopathy.35
To elucidate the role of JunD under pathophysiological stress in the heart, we explored the effects of pressure overload in mice lacking JunD by thoracic aortic constriction (TAC) and analyzed the role of JunD in survival, cardiac hypertrophy, and apoptosis as well as in angiogenesis.
Methods
The targeted disruption of the junD gene (junD–) in mice (129/SvJ background) has been previously described.33 For this study, homozygous mutant (junD–/–; knockout [KO]), heterozygous mutant (junD–/+), and wild-type (WT) (junD+/+) mice were raised from heterozygous mutant (junD–/+) breeding pairs. Primers used for genotyping were as follows: endogenous junD gene: 5'-CACTG-CACCTTCTGCGCTGCG-3'; 5'-CGCTTGCGGCATTTGGAGGC-3'; for the inserted LacZ transgene: 5'-CATCGGAGCCGGTCTGTA-3'; 5'-GCGATTAAGTTGGGTAACGC-3'.
Animal Experiments
Male mice (aged 12 weeks) were sedated with propofol (10 mg/kg IV), anesthetized with 3% enflurane, and connected to a rodent ventilator (Harvard). Transverse TAC was performed as described36 with the use of wires with diameter of 300 μm to generate severe pressure overload (sTAC) or wires with diameter of 400 μm to generate moderate pressure overload (mTAC). Sham-operated mice, one group analyzed 7 days and a second group analyzed 12 weeks after sham operation, served as controls. The surgeon was blinded to the genotype of the mice. After the mice were euthanized, hearts were rapidly excised; LVs were dissected and snap-frozen in liquid nitrogen. For histological analyses, hearts were embedded in OCT after retrograde perfusion with PBS (pH 7.4) containing 50 mmol/L KCl and 200 U/mL heparin or embedded in paraffin after paraformaldehyde in situ fixation as described previously.37
Hemodynamic Measurement and Transthoracic Echocardiography
Hemodynamic measurements were performed in mice under anesthesia with the use of a 1.4F micromanometer conductance catheter (SPR-719; Millar Instruments) as described.37,38 In brief, mice were anesthetized and mechanically ventilated with enflurane (3%), a bilateral vagotomy was performed, and the catheter was inserted in the LV cavity via the right carotid artery. Echocardiography was performed in sedated mice (ketamine 100 mg/kg and xylazine 1.25 mg/kg IP) as described.39 In brief, short-axis M-mode images were recorded at the papillary muscle level in sedated mice with the use of a linear 15-MHz transducer (ATL HDI 5000 CV). Fractional shortening (FS) was calculated as follows: FS(%)=[(LVEDD–LVESD)/LVEDD]x100, where LVEDD is LV end-diastolic diameter and LVESD is LV end-systolic diameter.
All animal studies were in compliance with the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health and were approved by the local animal care committees.
Patient Population
LV myocardium was obtained from patients undergoing heart transplantation due to end-stage dilated cardiomyopathy (n=10; mean age, 44±13 years; NYHA functional class III or IV; LV ejection fraction, 16±7%; end-diastolic diameter, 65±14 mm). Seven patients had been treated with ACE inhibitors, 6 patients with diuretics, 7 patients with digoxin, and 5 patients with -blockers. For comparison, we studied LV tissue samples from donor hearts that could not be transplanted for technical reasons (nonfailing donors) (n=7; mean age, 43±2 years). Tissue samples were snap-frozen in liquid nitrogen no later than 2 hours after explantation.
Tissue Morphometry and Fibrosis
Tissue morphometry was performed in a blinded fashion in in situ fixed LV tissue slices stained with picrosirius red or hematoxylin and eosin (H&E) with the use of the Quantimet 500MC digital image analyzer as described.37 Mean cardiomyocyte cross-sectional area (CSA) was determined in in situ fixed LV sections stained with H&E or stained with antibodies recognizing wheat germ agglutinin (WGA) (Vector) as described.40 For nuclear staining, hematoxylin or Hoechst 33258 (Sigma) was used. Apoptotic nuclei were determined by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) (QBIOGENE)/Hoechst 33258 (Sigma) staining, and cardiomyocytes were identified by sarcomeric -actinin staining (Sigma) as described previously.40
Capillary Density and Number of Coronary Resistance Vessels
Capillary density was determined in LV sections with transversely sectioned cardiomyocytes immunostained against isolectin B4 (Vector) or platelet–endothelial cell adhesion molecule–1 (PECAM-1) (Santa Cruz) and counterstained with WGA and Hoechst 33258 (Sigma) as described previously.40 In brief, the ratio of isolectin B4– or PECAM-1–positive cells to total number of nuclei (Hoechst stain) per field was calculated. High-power fields (x400; 200x200 μm; 8 fields per section of LV basis, middle part, and apex; n=4 per genotype) with transversely sectioned cardiomyocytes were digitally recorded (Quantimet 500MC). Capillary-to-cardiomyocyte ratio was determined in LV sections double stained for isolectin B4 and WGA. For each mouse, high-power fields (x400; from LV basis, middle part, and apex with transversely sectioned myocytes) were digitally recorded to calculate the number of capillaries per cardiomyocyte in 50 to 100 cardiomyocytes.
Arterioles and coronary resistance vessels were counted in -smooth muscle actin– (Sigma) and WGA-stained transverse LV sections (3 sections per heart: basis, middle part, and apex; n=4 for each genotype) as described.40 We distinguished arterioles with inner diameters <25 μm and 25 to 50 μm and vessels with diameters >50 μm and counted them separately.
Investigators were blinded to the genotype of the material for capillary and coronary density quantification.
Polymerase Chain Reaction and Immunoblot Analyses
Reverse transcriptase–polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting were performed according to standard procedures.41 Real-time measurements of PCR amplification were performed with the Stratagene MX4000 multiplex QPCR System with the SYBR green dye method (Brilliant SYBR Green Mastermix Kit, Stratagene). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal standard. Primers used for real-time PCR were as follows: ANP: 5'-GCCGGTAGAA-GATGAGGTCA-3'; 5'-GGGCTCCAATCCTGTCAATC-3'; skeletal muscle -actin: 5'-ATCTCACGTTCAGCTGTGGTCA-3'; 5'-ACCA-CCGGCATCGTGTTGGAT-3'; MHC: 5'-GGAAGAGCGAGCGG-CGCATCAAGG-3'; 5'-CTGCTGGACAGGTTATTCCTCA-3'; G3PDH: 5'-AACGACCCCTTCATTGAC-3'; 5'-TCCACGACAT-ACTCAGCAC-3'; VEGF: 5'-TTACTGCTGTACCTCCACC-3'; 5'-ACAGGACGGCTTGAAGATG-3'. Antibodies against JunD, c-Jun, c-Fos, p53, Bax, Bcl-2, HIF1, VEGF, phospho-JNK1/2, JNK, phospho-ERK1/2, ERK1/2, actin (Santa Cruz), and active caspase-3 (BD Pharmingen) were used for immunoblots.
Statistical Analysis
All data are given as mean±SD. Differences between groups were evaluated by Student t test or ANOVA followed by Bonferroni test as appropriate. Statistical significance was defined as P<0.05.
Results
Cardiac Phenotype of JunD KO Mice
Examination of mice lacking JunD (KO) revealed that they had normal life spans and no gross morphological abnormalities of the heart, but they had reduced body weights (BW),33 LV weights, and LV/BW ratios (Table). Morphometric analysis of in situ fixed and H&E-stained myocardial cross sections showed that cardiac dimensions (Table) and CSA were reduced in KO mice (Figure 1). Heterozygous mutant mice (junD–/+) did not differ significantly from WT animals with respect to survival, size, LV/BW ratio, cardiac dimensions, and cardiac function under basal conditions and after severe (7 days) or moderate (12 weeks) pressure overload (data not shown); therefore, in this study only data from WT and KO mice are presented.
Morphometric, Hemodynamic, and Echocardiographic Analyses of WT and KO Mice After 12 Weeks of Sham Operation or Moderate Pressure Overload (mTAC)
Severe Pressure Overload Resulted in High Mortality, Enhanced Myocardial Apoptosis, and Elevated Expression of p53 in KO Mice
We examined the response to severe pressure overload (sTAC) in WT (WT-sTAC) and KO (KO-sTAC) mice. sTAC resulted in a higher mortality in KO-sTAC mice (Figure 1A); 7 of 11 KO-sTAC mice died within the first 7 days of sTAC, whereas only 3 of 14 WT-sTAC mice died within the same period. All WT-sham (n=8) and KO-sham (n=8) mice survived 7 days (Figure 1A). After 7 days, the surviving WT-sTAC and KO-sTAC mice showed a similar rise in prestenotic systolic blood pressure: WT-sham, 122±7 mm Hg versus WT-sTAC, 179±20 mm Hg, P<0.05; KO-sham, 123±10 mm Hg versus KO-sTAC, 168±15 mm Hg, P<0.05; WT-sTAC versus KO-sTAC, P=NS. After 7 days of sTAC, apoptosis in cardiomyocytes and nonmyocytes, assessed by TUNEL assay, was more pronounced and protein levels of active caspase-3 were augmented in LVs of KO-sTAC mice compared with WT-sTAC mice (Figure 1B and 1C). In addition, compared with WT-sTAC mice, KO-sTAC mice showed higher increases in protein levels of p53 (WT-sTAC, 130%; KO-sTAC, 243%; P<0.05) and of the proapoptotic Bcl-2 family member Bax (WT-sTAC, 240%; KO-sTAC, 349%; P<0.05), whereas the increase in protein levels of the antiapoptotic protein Bcl-2 (WT-sTAC, 325%; KO-sTAC, 189%; P<0.05) was attenuated (Figure 1C). Protein levels of JunD were not significantly altered in WT-sTAC mice 7 days after sTAC (data not shown). The expression pattern of c-Jun and c-Fos and the phosphorylation of JNK were similar in both genotypes after sTAC (Figure 1C).
Seven days after sTAC, surviving KO-sTAC mice showed a larger increase in CSA compared with WT-sTAC mice (Figure 1D and 1E). Echocardiography in KO-sTAC mice was not possible because these mice did not survive anesthesia.
Chronic Moderate Pressure Overload Triggered an Enhanced Hypertrophic Response in KO Mice
Moderate pressure overload (mTAC) for 7 days or for 12 weeks did not result in increased mortality in KO or WT mice. mTAC for 7 days resulted in a similar rise in systolic prestenotic blood pressure in both genotypes (WT-mTAC, 150±17 mm Hg versus KO-mTAC, 147±11 mm Hg; P=NS) and in a similar increase in the LV/BW ratio (WT [n=5], 38±22% versus KO [n=5], 34±9%; P=NS) in KO-mTAC and WT-mTAC mice. The prevalence of TUNEL-positive cells tended to be higher in KO-mTAC mice than in WT-mTAC mice (data not shown); however, no increase in activated caspase-3 protein could be detected in WT-mTAC or KO-mTAC LVs after 7 days of mTAC (data not shown).
After 12 weeks of mTAC, KO-mTAC mice showed a significantly stronger hypertrophic response in LVs compared with WT-mTAC mice, which was also reflected by a larger increase in the LV/BW ratio (33% in WT-mTAC versus 85% in KO-mTAC; P<0.01; Table, Figure 2A) and a higher increase in CSA (Table, Figure 2A). There was a similar rise in the prestenotic blood pressure (Table), and there were no differences in cardiac function (determined by echocardiography; Table) or interstitial fibrosis (determined by picrosirius red [not shown] and H&E staining [Figure 2B]) between WT-mTAC and KO-mTAC mice after 12 weeks of mTAC. Real-time PCR analyses showed enhanced increases in mRNA levels of ANP and skeletal muscle -actin in KO-mTAC LVs, whereas the reduction in MHC was similar in LVs of both genotypes 12 weeks after mTAC (Figure 2C).
Chronic Moderate Pressure Overload Reduces Protein Levels of JunD
Previous work demonstrated substantial expression of JunD in the adult mouse heart, mainly in blood vessels and cardiomyocytes.33,34 After mTAC for 12 weeks, protein levels of JunD in WT-mTAC LVs were significantly decreased (–43±25%; P<0.05) compared with WT-shams (Figure 2D). No JunD protein was detected in KO LVs (Figure 2D). Protein levels of c-Fos and c-Jun tended to be higher (P=NS) in KO-mTAC LVs compared with WT-mTAC LVs, whereas activation of JNK and Erk1/2 proteins was not significantly different between the 2 genotypes 12 weeks after mTAC (Figure 2D).
Increased VEGF Expression and Higher Capillary Density in KO Mice After Pressure Overload
It has recently been shown that JunD attenuates tumor angiogenesis by protecting cells from oxidative stress, which in turn promotes proteasomal degradation of HIF1 and results in lower expression of the proangiogenic factor VEGF.30 We observed a higher increase in the expression of HIF1 protein and VEGF mRNA and protein after sTAC for 7 days in KO-sTAC mice compared with WT-sTAC mice (Figure 3A).
After mTAC for 12 weeks, KO-mTAC mice showed markedly increased VEGF protein levels compared with WT-mTAC mice (Figure 3B). However, this increase in the amount of VEGF proteins was not paralleled by substantially increased levels of VEGF mRNA or HIF1 protein (Figure 3B).
We performed immunostaining of LV sections from WT-sham, KO-sham, WT-mTAC, and KO-mTAC mice 12 weeks after operation with PECAM-1 or isolectin B4 to mark capillary endothelial cells. The increases in the capillary density and in the capillary-to-cardiomyocyte ratio were higher in KO-mTAC mice than in WT-mTAC mice (Figure 4A and 4C). In addition, antibody staining to smooth muscle cells revealed that KO-mTAC LVs showed significantly more arterioles than WT-mTAC or sham LVs (Figure 4B and 4C).
JunD Protein Levels Are Decreased in Patients With End-Stage Heart Failure
In patients with end-stage heart failure due to dilated cardiomyopathy, JunD protein levels in LVs were severely reduced compared with nonfailing LVs (Figure 5).
Discussion
The present study shows that in the absence of JunD, there is increased apoptosis and hypertrophy in the myocardium in response to pressure overload. Moreover, JunD reduces pressure overload–induced cardiac angiogenesis. These data extend the biological function of JunD as regulator of cell proliferation and tumor progression by providing evidence that JunD plays a critical role in cardioprotection and hypertrophy and that it acts as a negative modulator of angiogenesis in response to pathophysiological stress in the heart.
Mice lacking JunD show a high rate of mortality in response to severe pressure overload, associated with an increase in cardiac apoptosis, indicating that JunD protects against cardiomyocyte loss in response to increased workload. It has been shown that lack of JunD promotes p53-dependent apoptosis in fibroblasts in vitro.16 Furthermore, KO mice show enhanced hepatocyte apoptosis on injection with bacterial lipopolysaccharide, indicating that JunD plays a role in antiapoptotic pathways.16 A recent study demonstrated that JunD protects cells from oxidative stress by regulating genes involved in antioxidant defense.30 Oxidative stress, in turn, has been associated with increased expression of p53,42,43 HIF1, and VEGF.30,44,45 Moreover, p53 reduces the expression of genes opposing cell death, such as Bcl-2, and upregulates genes promoting apoptosis, such as Bax.46 Pressure overload is associated with oxidative stress and with increased production of reactive oxygen species.47 Indeed, KO mice exposed to sTAC showed enhanced cardiac expression of p53 and HIF1, which may be related to oxidative stress. The increased activation of proapoptotic p53-dependent pathways in ventricles of KO-sTAC mice, eg, upregulation of Bax and downregulation of Bcl-2, supports the notion that the high mortality rate in KO-sTAC mice could result, at least in part, from increased cardiomyocyte apoptosis due to enhanced oxidative stress. These findings suggest that JunD may protect the heart from pressure overload by attenuating proapoptotic pathways.
The inability to increase cardiac perfusion by limited cardiac neoangiogenesis is considered to represent a key element in the transition of compensated to decompensated hypertrophy.4 KO mice exposed to mTAC for 12 weeks show a higher cardiac capillary and arteriole density and a higher capillary-to-cardiomyocyte ratio. The KO-mTAC mice therefore developed more severe LV hypertrophy, and this growth response of the myocardium was associated with increased angiogenesis, which represents a physiologically adequate vascular adaptation to increased oxygen demand of hypertrophied cardiomyocytes. This could, at least in part, explain why no significant alterations in cardiac function, fibrosis, or apoptosis occurred in KO-mTAC mice after chronic pressure overload despite profound cardiomyocyte hypertrophy. Previous studies have shown that regulation of the proangiogenic factor VEGF is critical for myocardial angiogenesis.48 Remarkably, KO-sTAC mice express higher levels of VEGF mRNA and protein and of HIF1 protein, a transcription factor known to promote VEGF expression, after 7 days of severe pressure overload. Biomechanical and oxidative stress induce HIF1 expression, mainly by attenuating its proteasomal degradation.30 In this respect, JunD has been shown to promote HIF1 protein degradation in tumors,30 and thus the lack of JunD in the heart might promote HIF1 protein stabilization and VEGF transcription, leading to enhanced stress-induced angiogenesis. Interestingly, after mTAC for 12 weeks, elevated VEGF protein levels in KO-mTAC mice were not associated with a significant increase in HIF1 protein accumulation or paralleled by significantly increased VEGF mRNA levels. This finding suggests that JunD, in addition to the transcriptional regulation, may be involved in the posttranscriptional regulation of VEGF, probably by indirect regulation of proteasomal degradation. Alternatively, the half-life of VEGF mRNA could be shorter than the VEGF protein half-life in KO-mTAC mice. Future studies will be needed to investigate this observation.
It has previously been reported that human and mouse adult myocardium constitutively expresses high levels of JunD protein in cardiomyocytes and nonmyocytes.33–35 Pollack and coworkers35 showed that JunD mRNA levels are reduced in human failing myocardium. Consistent with these observations, we found decreased JunD protein levels in the LVs of patients with end-stage heart failure (Figure 5). In the present study we show that loss of JunD results in increased mortality and cardiomyocyte apoptosis after acute pressure overload but enhances angiogenesis in the chronic phase of the disease. These results suggest that lower levels of JunD in failing human hearts may lead to cardiomyocyte loss in response to acute pathophysiological stress but may allow a higher degree of cardiac angiogenesis.
One limitation of this study is that loss of JunD is associated with a modestly smaller body and heart size. These features indicate that KO mice may differ from WT animals in their metabolism and growth. In addition, the lack of JunD may lead to the activation of compensatory mechanisms, which in turn may influence the cardiac phenotype independently of JunD. Our findings concerning apoptosis and angiogenesis, however, are consistent with biological functions of JunD described in cell culture and tumorigenesis models.16,30
In conclusion, the absence of JunD causes enhanced cardiomyocyte hypertrophy and cardiac apoptosis in response to pressure overload, possibly because JunD attenuates oxidative stress. These beneficial effects of JunD, however, are associated with antiangiogenic properties.
Acknowledgments
This work was supported by the DAAD and by grants of the association for International Cancer Research and the European Community. We are grateful to Birgit Brandt and Silvia Gutzke for excellent technical assistance.
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