Requirement of Nuclear Factor-B in Angiotensin II– and Isoproterenol-Induced Cardiac Hypertrophy In Vivo
http://www.100md.com
《循环学杂志》
the Franz-Volhard Clinic, Department of Cardiology, HELIOS Klinikum-Berlin, Charite Campus Berlin-Buch (C.F., S.D., W.S., R.D., M.W.B.)
Robert Koch Institut (P.L.)
Max Delbrück Center for Molecular Medicine (R.S.-U., A.B., A.E.-J., C.S.), Berlin, Germany.
Abstract
Background— In vitro experiments have proposed a role of nuclear factor-B (NF-B), a transcription factor, in cardiomyocyte hypertrophy and protection against apoptosis. Currently, the net effect on cardiac remodeling in vivo under common stress stimuli is unclear.
Methods and Results— We have generated mice with cardiomyocyte-restricted expression of the NF-B super-repressor IBN (NMHC) using the Cre/lox technique. NMHC mice displayed an attenuated hypertrophic response compared with control mice on infusion of angiotensin II (Ang II) or isoproterenol by micro-osmotic pumps, as determined by echocardiography (left ventricular wall dimensions: control plus Ang II, x1.5±0.1 versus sham; NMHC plus Ang II, x1.1±0.1 versus sham; P<0.05; n9), heart weight, and histological analysis. Real-time reverse-transcriptase polymerase chain reaction showed significantly reduced expression of hypertrophy markers ;-myosin heavy chain and atrial natriuretic peptide in Ang II–treated NMHC mice (P<0.05 versus control plus Ang II; n=4). Neither cardiomyocyte apoptosis nor left ventricular dilatation was observed. In cultured adult rat cardiomyocytes, NF-B DNA binding activity was increased by both Ang II– and interleukin-6–related cytokines. The latter are known to be released by cardiac fibroblasts on Ang II stimulation and thus could locally increase the NF-B response of cardiomyocytes. Finally, results from in vitro and in vivo experiments suggest a role for NF-B in the regulation of prohypertrophic interleukin-6 receptor gp130 on mRNA levels.
Conclusions— These results indicate that targeted inhibition of NF-B in cardiomyocytes in vivo is sufficient to impair Ang II– and isoproterenol-induced hypertrophy without increasing the susceptibility to apoptosis.
Key Words: angiotensin ; genes ; hypertrophy ; myocytes ; nuclear factor-B
Introduction
The adult heart responds to hemodynamic stress, various cytokines, and growth factors by hypertrophic growth of cardiomyocytes, which eventually may lead to heart failure resulting from excessive workload. Current heart failure treatment relies on potent cell surface blockade of the renin-angiotensin system combined with ;-adrenoceptor antagonism. Genetic evidence suggests that Ca2+-dependent transcription factors may be interesting new treatment targets. Knockout mice of NF-ATc and GATA-4, both downstream of the calcineurin pathway, consistently revealed impaired cardiac hypertrophy in various stress models, including G-protein–coupled receptor (GPCR) stimulation by angiotensin II (Ang II), endothelin I, and phenylephrine (PE).1,2 However, pharmacological calcineurin inhibition revealed variable results.3 Therefore, analysis of additional transcription factors might be interesting in the context of cardiac hypertrophy signaling.
Nuclear factor-B (NF-B), a ubiquitous transcription factor, is known for its role in immunity, inflammation, regulation of cell growth, apoptosis, and embryonal development.4–6 In most resting cells, NF-B is sequestered in the cytoplasm by interaction with its inhibitory proteins, the IBs, which are degraded on stimulation. Recent in vitro studies have suggested that NF-B is an attractive target for antagonizing GPCR-induced cardiac hypertrophy.7,8 However, NF-B was also found to regulate the expression of the hypertrophy inhibitory molecule IEX-1.9 Furthermore, NF-B has been implicated in regulation of cardiomyocyte survival genes. NF-B was shown to protect isolated neonatal cardiomyocytes from tumor necrosis factor- (TNF)–induced apoptosis and to limit cardiac infarct size in vivo.10,11
Because the evidence linking NF-B to cardiac hypertrophy was derived from in vitro experiments, we studied the effect of targeted NF-B inhibition in vivo using Ang II and the ;-adrenoceptor agonist isoproterenol (Iso) as hypertrophic stimuli. The results presented here firmly establish that activation of NF-B in cardiomyocytes is a requisite to the development of cardiac hypertrophy by Ang II and Iso in vivo. NF-B inhibition does not lead to detectable levels of cardiomyocyte apoptosis after Ang II or Iso treatment. Furthermore, we provide evidence that the interleukin (IL)-6 receptor gp130 mRNA is downregulated in hearts of NMHC mice.
Methods
Transgenic Mice
The cloxPIBN mice were described previously.6 Mice expressing Cre-recombinase under the control of cardiomyocyte-specific -myosin heavy chain (-MHC) promoter (-MHC-Cre mice) were a generous gift from Dr Michael D. Schneider (Baylor College of Medicine, Houston, Tex).12 All mice were bred on a C57Bl/6 background for 6 generations. All aspects of animal care and experimental protocols were approved by the Berlin animal review board (Reg. 0135/01).
Induction of Cardiac Hypertrophy and Echocardiography
Micro-osmotic pumps (model 1002, Alzet) releasing Ang II (Sigma; 1.4 μg · kg–1 · min–1; solvent, 0.9% NaCl/0.01N acetic acid) or Iso (Sigma; 60 mg · kg–1 · d–1 in 0.9% NaCl) were implanted subcutaneously into age-matched (12 to 16 weeks old) male mice after anesthesia with ketamine/xylazine (30 mg/10 mg per 1 kg IP). Hypertrophy was assessed by echocardiography with an Accuson Sequoia (Siemens) instrument equipped with a 13-MHz microprobe. Ventricular measurements in M mode were taken before implantation and 12 days (Ang II) or 7 days (Iso) after, with 3 readings per mouse. The observer was blinded to genotype and treatment of the mice.
Histological Analysis
Hearts were fixed in 10% formalin/PBS and embedded in paraffin. Heart sections (10 μm) were stained with PAS or Masson trichrome as indicated to evaluate morphology and cellular dimensions. TUNEL assays were performed with the Apop Tag Plus system from Intergene according to the manufacturer’s instructions.
RNA Quantification
Total RNA was isolated with the RNeasy kit (Qiagen), and cDNA was synthesized with Superscript II reverse transcriptase (RT) (Invitrogen). cDNA was subjected to real-time RT–polymerase chain reaction (PCR) by SYBR Green Analysis (Qiagen) performed on an iCycler instrument (Biorad). Primers for mouse gene analysis were as described previously.13 Primer sets for rat genes were as follows: IB: forward, 5'-CAGCATCTCCACTCCGTCCT-3'; reverse, 5'-GCGTTGACATCAGCACCCAA-3'; gp130: forward, 5'-CAAGCACCGTGCAGTACTCC-3'; reverse, 5'-TGTCCACACTATCCACCAGCT-3'. GAPDH (Clonetech) was used as control.
Cell Culture and Adenoviral Infection
Cardiomyocytes were isolated from 12- to 14-week-old male Wistar rats (Moellegard, Schoenwalde, Germany) as described before.14 Three hours after attachment, cardiomyocytes were infected with recombinant adenoviral vector (Ad) encoding IBN (Ad5IBN) or empty vector (Ad5control; 50 MOI) and stimulated after another 36 hours. Cardiac fibroblasts obtained in parallel were grown to 70% confluence and serum deprived for 20 hours before stimulation. Adult mouse cardiomyocytes were isolated and cultured following the instructions given at www.signaling-gateway.org.
Immunoblotting and Electrophoretic Mobility Shift Assay
Cardiomyocytes were stimulated with Ang II, leukemia inhibitory factor (LIF), PE (all Sigma), TNF- (Biomol), IL-6 (Roche), cardiothrophin (CT)-1 (Calbiochem), or FCS as indicated. Nuclear and cytosolic extracts were prepared as described.15 Electrophoretic mobility shift assay (EMSA) was performed with 5 to 10 μg nuclear protein as described previously.11 For immunoblotting, 10 to 20 μg cytosolic protein was used. IBN from heart was immunoprecipitated as described.16 Total lysates of adult mouse cardiomyocytes were prepared as described.16 The antibodies for immunoblotting or immunoprecipitation were from Santa Cruz (IB, gp130), Cell Signaling (p- or total ERK and CREB, p-Stat3 [Tyr 705]), or Advanced ImmunoChemical Inc (GAPDH).
Immunostaining
After stimulation with TNF- (10 ng/mL, 30 minutes), mouse cardiomyocytes were fixed (4% formaldehyde/PBS, 25 minutes, 4°C), permeabilized (0.5% Triton X-100/PBS, 10 minutes, 4°C), and incubated with an antibody against NF-B–p65 (1:100, Santa Cruz) according to the manufacturer’s instructions. The TRITC-labeled secondary antibody was from Dianova; DAPI (0.5 μg/mL) was from Sigma.
Statistical Analysis
Differences between experimental groups were analyzed by use of a Student t test or 1-way ANOVA, followed by Bonferroni posttest when multiple groups were compared. Data are reported as mean±SEM. Values of P<0.05 were considered significant. Real-time RT-PCR data from adult cardiomyocytes were analyzed by paired t test, comparing cells derived from each preparation separately.
Results
Generation of Mice With Cardiomyocyte-Specific NF-B Inhibition
Mice heterozygous for floxed IBN (cloxPIBN, NloxP) were bred with heterozygous, -MHC–Cre (c-MHC-Cre, CreMHC) transgenic mice to generate mice with cardiomyocyte-restricted IBN expression, called NMHC mice (Figure 1A).6,12 IBN lacks the destruction box and acts as a super-repressor of NF-B.6 Heart-specific recombination was analyzed by PCR demonstrating efficient removal of the stop codon (Figure 1B). Heart-restricted IBN expression was confirmed by immunoblot analysis of immunoprecipitated tissue extracts of NMHC and cIBN mice, which express IBN ubiquitously (referred to as Nubi mice; Figure 1C).6 Less IBN protein is detected in heart tissue extracts from NMHC compared with Nubi mice because of up to 70% nonmyocytes in whole-heart extracts (Figure 1C).6 In addition, CreMHC mice exhibit mosaicism of Cre recombinase expression, which is estimated to be present in 70% of cardiomyocytes (described elsewhere,17 data not shown). Furthermore, recombination was confirmed in isolated cardiomyocytes from NMHC mice (Figure 1D). Effective NF-B inhibition in NMHC mice was confirmed by immunostaining of NF-B–p65 in TNF-–stimulated cultured cardiomyocytes. Although control cells showed robust nuclear translocation of p65, cardiomyocytes genetically expressing IBN displayed reduced p65 translocation (Figure 1E). In conclusion, NMHC mice exhibit cardiac-restricted, impaired NF-B activation.
Attenuation of Ang II– and Iso-Induced Cardiac Hypertrophy in NMHC Mice
NMHC mice did not exhibit any significant changes at histological or echocardiographic examination when left untreated (data not shown).18,19 Wild-type, CreMHC, or NloxP mice (summarized as control mice) and age-matched NMHC mice were exposed to Ang II (1.4 μg · kg–1 · min–1) infusion for 14 days. Although control mice exhibited a significant increase in diastolic wall dimensions and ratio of heart weight to tibia length on Ang II treatment, NMHC mice did not mount a hypertrophic response (Figure 2A and 2B and Table 1). Histological examination confirmed these results (Figures 2C and 3A through 3C). Similarly, Iso-induced hypertrophy at 7 days was attenuated in NMHC mice (Table 2). Thus, suppression of NF-B activity has a protective effect on Ang II– and Iso-induced cardiac hypertrophy.
In addition to morphological changes, mRNA expression of hypertrophy markers was analyzed by real-time RT-PCR (Figure 2D). As expected, we found a significant induction of atrial natriuretic peptide (ANP; expression ratio versus sham, 2.6±0.3), brain natriuretic peptide (BNP; expression ratio versus sham, 1.3±0.28), ;-MHC (expression ratio versus sham, 2.2±0.4), and -skeletal actin (expression ratio versus sham, 2.6±0.3) gene expression in cardiac tissue from Ang II–treated control animals. -MHC and transforming growth factor (TGF)-; levels remained largely unchanged after 14 days of Ang II treatment. In contrast, NMHC mice showed a significant reduction in ;-MHC and ANP upregulation by Ang II (Figure 2D).
Cytokines and/or chemokines secreted from cardiomyocytes may contribute to Ang II–induced inflammatory responses. As expected, histological analysis of Ang II–infused control mice revealed perivascular infiltration of inflammatory cells (Figure 3E) compared with sham-operated controls (Figure 3D). Ang II–induced perivascular inflammation was not altered in NMHC mice (Figure 3F). Hence, NF-B activity in cardiomyocytes may not contribute significantly to the Ang II–induced inflammatory reaction.
Because NF-B has been hypothesized to regulate survival signals in cardiomyocytes, we evaluated apoptosis of cardiomyocytes in hearts of mice. In histological sections from control and NMHC mice challenged with Ang II, we did not detect apoptotic cells by TUNEL assay (Figure 3G and 3J, respectively) or fragmentation of DAPI-stained nuclei (Figure 3H and 3K, respectively). As a positive control, one section was treated with DNase I before the TUNEL assay (Figure 3I). Furthermore, dilatation of the left ventricle resulting from apoptotic loss of cardiomyocytes was not detectable by echocardiography (Tables 1 and 2). Expression of IBN does not induce cardiomyocyte apoptosis and/or impairment of LV function in this model.
Activation of NF-B in Adult Cardiomyocytes by Ang II and IL-6 Cytokines
We analyzed NF-B DNA binding in adult rat cardiomyocytes after Ang II stimulation. Moderate NF-B DNA binding activity was induced by physiological concentrations of Ang II as early as 15 minutes (1.45±0.12-fold) and was sustained for 1 hour (Figure 4A). Ang II induced less NF-B DNA binding than TNF- stimulation (2.46±0.3-fold). NF-B DNA complexes induced by Ang II contained p50 and p65 (Figure 4B).
Activation of cardiac fibroblasts by Ang II significantly contributes to cardiac hypertrophy.20 We therefore analyzed the effect of Ang II on NF-B activity in cardiac fibroblasts. Ang II activated both ERK and CREB but not NF-B binding activity (Figure 4C). These pathways were demonstrated to regulate the expression of IL-6 in neonatal cardiac fibroblasts. Paracrine secretion of IL-6–related cytokines augments cardiac hypertrophy.21 Therefore, we studied the effect of IL-6 cytokines on NF-B activation in adult rat cardiomyocytes. Indeed, IL-6, CT-1, and LIF, alone or in combination, induced NF-B DNA binding at 30 minutes (Figure 4D, top). Interestingly, PE did not induce NF-B in adult cardiomyocytes, in contrast to previously published data from neonatal cardiomyocytes.7
ERK1/2 is known to be a central kinase of cardiac hypertrophy signaling.22 Ang II and TNF- did not induce ERK1/2 phosphorylation in adult cardiomyocytes at any time points, whereas PE activated ERK1/2 most potently (Figure 4D, bottom). These data imply that PE-induced hypertrophy is regulated mainly by ERK, whereas IL-6 cytokine-induced hypertrophy involves NF-B in addition to a modest ERK activation. Ang II induced NF-B activation but not ERK phosphorylation in adult rat cardiomyocytes (Figure 4D).
NF-B–Dependent Gene Regulation
A DNA microarray analysis was performed to identify possible downstream targets of NF-B involved in hypertrophy (data not shown). Adult rat cardiac myocytes transfected with Ad5IBN were compared with Ad5control or untransfected cells. Downregulation of IB mRNA was used as a positive control for efficient NF-B inhibition because the IB promoter contains several regulatory NF-B binding sites.4 The microarray data were confirmed by real-time RT-PCR for gp130 mRNA showing significant downregulation in IBN-transfected cells (Figure 5A). A similar trend was observed in whole-heart extracts from NMHC mice compared with control animals at baseline (Figure 5B B, left bar; P=0.056; n=4). In addition, levels of gp130 mRNA were significantly reduced in hearts of Ang II–treated NMHC mice (Figure 5B, right bar; P<0.05; n=4). In cultured cardiomyocytes from control or NMHC mice, protein levels of gp130 were comparable under nonstimulated conditions (Figure 5C). However, after 30 minutes of Ang II and IL-6, gp130 protein expression was reduced in cardiomyocytes from NMHC mice compared with control mice. Phosphorylation of transcription factor STAT3, which is a typical downstream target of IL-6/gp130 signaling, remained unchanged in NMHC cardiomyocytes after stimulation with Ang II and IL-6.
Discussion
Ca2+-dependent signaling molecules have been shown to mediate cardiac hypertrophy in vivo. Here, we provide genetic evidence for a Ca2+- independent signaling pathway required for cardiac hypertrophy, namely the NF-B activation cascade. Our results establish that inhibition of transcription factor NF-B by cardiomyocyte-restricted expression of the NF-B superrepressor IBN is sufficient to attenuate Ang II– and Iso-induced cardiomyocyte hypertrophy in vivo without a detectable effect on cardiomyocyte apoptosis. Interestingly, NF-B exerts its effect on cardiac hypertrophy by affecting a different subset of hypertrophy-related genes compared with the calcineurin-dependent signaling molecules. In calcineurin-deficient mice, inhibition of Ang II–induced hypertrophy was associated with a reduction in -skeletal actin mRNA.23 Similarly, inhibition of hypertrophy in NF-Atc3–/– mice was accompanied by attenuation of -skeletal actin upregulation.24 In contrast, NMHC mice displayed impaired ;-MHC and ANP upregulation while leaving BNP and -skeletal actin induction intact. BNP exerts positive effects in cardiac remodeling by antagonizing TGF-;–induced deposition of extracellular matrix proteins.25
In vitro experiments using neonatal rat cardiomyocytes as a model system proposed various hypertrophic pathways to regulate NF-B activity. The GPCR agonists endothelin 1, Ang II, and PE were all found to upregulate NF-B–dependent reporter genes.7,8 However, the upregulation may be indirect because the induction of NF-B DNA binding in response to these stimuli was not shown in these studies. Using a different system, namely adult rat cardiomyocytes, we observed only a modest increase in NF-B DNA binding on Ang II stimulation. Because IL-6 family cytokines contribute significantly to Ang II–induced cardiac hypertrophy,21 we analyzed NF-B activation by IL-6, CT-1, and LIF. All IL-6 cytokines were found to activate NF-B in adult cardiomyocytes substantially. These data are in line with previous reports in which activation of NF-B downstream of the IL-6 receptor gp130 protected neonatal cardiomyocytes from hypoxia-induced apoptosis.26 We found that PE strongly stimulates ERK1/2 phosphorylation but not NF-B DNA binding in adult cardiomyocytes. This result is in contrast to published data on neonatal cardiomyocytes.7 In our view, PE signaling differs from Ang II– and IL-6-induced pathways in cardiac hypertrophy in the adult system.
NF-B has been shown to regulate the expression of numerous genes, including survival factors and cell growth regulatory molecules. More precisely, the expression of apoptosis inhibitory molecules iAP1, bcl-2, and bcl-xL was described to be NF-B dependent.27 In our mouse model, NF-B inhibition attenuated cardiac hypertrophy without any detectable effects on cardiomyocyte apoptosis. These results suggest an important cardiomyocyte-specific role for NF-B in regulating genes involved in cardiac hypertrophy, whereas expression of survival factors might be affected only by more stringent NF-B inhibition. Alternatively, other transcription factors might compensate for reduced NF-B activity in cardiomyocytes concerning its role in apoptosis but not hypertrophy. The latter hypothesis is supported by our previously published observation that even complete NF-B inhibition by Ad5IBN in isolated neonatal rat cardiomyocytes did not lead to a detectable regulation of iAP1+2, bcl-2, or bcl-xL protein.11
The IL-6 receptor gp130 is a central regulator of cardiac hypertrophy.28 Here, we found gp130 mRNA to be downregulated in rat cardiomyocytes with NF-B inhibition and tissue extracts from NMHC mice after Ang II stimulation. Further studies are needed to determine whether this effect on gp130 mRNA is crucial for the observed effect on cardiac hypertrophy. Promoter analysis of the human, rat, and mouse gp130 gene did not reveal a canonical NF-B binding site, suggesting an indirect regulatory mechanism (data not shown).
Taken together, our data suggest that the NF-B signaling cascade is a potential target for pharmacological intervention in cardiac hypertrophy (Figure 5D). Further experiments are warranted to assess the long-term effect of NF-B inhibition on cardiac remodeling under pressure overload and other models of cardiac failure. In addition, pharmacological inhibitors of the NF-B pathway already studied in the context of chronic inflammatory lung and bowel diseases should be tested in animal models of cardiac hypertrophy and failure.
Acknowledgments
This study was supported by an MDC clinical research grant to Drs Bergmann and Scheidereit, a European Union Marie Curie Host Development Fellowship to Drs Bergmann and Scheidereit, and a grant-in aid from Pinguin Stiftung, Germany to Dr Bergmann. This study was supported in part by a grant from BMBF to Dr Scheidereit. We thank Gabi Welsch, B;rbel Pohl, and Sarah Ugowski for expert technical assistance.
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Robert Koch Institut (P.L.)
Max Delbrück Center for Molecular Medicine (R.S.-U., A.B., A.E.-J., C.S.), Berlin, Germany.
Abstract
Background— In vitro experiments have proposed a role of nuclear factor-B (NF-B), a transcription factor, in cardiomyocyte hypertrophy and protection against apoptosis. Currently, the net effect on cardiac remodeling in vivo under common stress stimuli is unclear.
Methods and Results— We have generated mice with cardiomyocyte-restricted expression of the NF-B super-repressor IBN (NMHC) using the Cre/lox technique. NMHC mice displayed an attenuated hypertrophic response compared with control mice on infusion of angiotensin II (Ang II) or isoproterenol by micro-osmotic pumps, as determined by echocardiography (left ventricular wall dimensions: control plus Ang II, x1.5±0.1 versus sham; NMHC plus Ang II, x1.1±0.1 versus sham; P<0.05; n9), heart weight, and histological analysis. Real-time reverse-transcriptase polymerase chain reaction showed significantly reduced expression of hypertrophy markers ;-myosin heavy chain and atrial natriuretic peptide in Ang II–treated NMHC mice (P<0.05 versus control plus Ang II; n=4). Neither cardiomyocyte apoptosis nor left ventricular dilatation was observed. In cultured adult rat cardiomyocytes, NF-B DNA binding activity was increased by both Ang II– and interleukin-6–related cytokines. The latter are known to be released by cardiac fibroblasts on Ang II stimulation and thus could locally increase the NF-B response of cardiomyocytes. Finally, results from in vitro and in vivo experiments suggest a role for NF-B in the regulation of prohypertrophic interleukin-6 receptor gp130 on mRNA levels.
Conclusions— These results indicate that targeted inhibition of NF-B in cardiomyocytes in vivo is sufficient to impair Ang II– and isoproterenol-induced hypertrophy without increasing the susceptibility to apoptosis.
Key Words: angiotensin ; genes ; hypertrophy ; myocytes ; nuclear factor-B
Introduction
The adult heart responds to hemodynamic stress, various cytokines, and growth factors by hypertrophic growth of cardiomyocytes, which eventually may lead to heart failure resulting from excessive workload. Current heart failure treatment relies on potent cell surface blockade of the renin-angiotensin system combined with ;-adrenoceptor antagonism. Genetic evidence suggests that Ca2+-dependent transcription factors may be interesting new treatment targets. Knockout mice of NF-ATc and GATA-4, both downstream of the calcineurin pathway, consistently revealed impaired cardiac hypertrophy in various stress models, including G-protein–coupled receptor (GPCR) stimulation by angiotensin II (Ang II), endothelin I, and phenylephrine (PE).1,2 However, pharmacological calcineurin inhibition revealed variable results.3 Therefore, analysis of additional transcription factors might be interesting in the context of cardiac hypertrophy signaling.
Nuclear factor-B (NF-B), a ubiquitous transcription factor, is known for its role in immunity, inflammation, regulation of cell growth, apoptosis, and embryonal development.4–6 In most resting cells, NF-B is sequestered in the cytoplasm by interaction with its inhibitory proteins, the IBs, which are degraded on stimulation. Recent in vitro studies have suggested that NF-B is an attractive target for antagonizing GPCR-induced cardiac hypertrophy.7,8 However, NF-B was also found to regulate the expression of the hypertrophy inhibitory molecule IEX-1.9 Furthermore, NF-B has been implicated in regulation of cardiomyocyte survival genes. NF-B was shown to protect isolated neonatal cardiomyocytes from tumor necrosis factor- (TNF)–induced apoptosis and to limit cardiac infarct size in vivo.10,11
Because the evidence linking NF-B to cardiac hypertrophy was derived from in vitro experiments, we studied the effect of targeted NF-B inhibition in vivo using Ang II and the ;-adrenoceptor agonist isoproterenol (Iso) as hypertrophic stimuli. The results presented here firmly establish that activation of NF-B in cardiomyocytes is a requisite to the development of cardiac hypertrophy by Ang II and Iso in vivo. NF-B inhibition does not lead to detectable levels of cardiomyocyte apoptosis after Ang II or Iso treatment. Furthermore, we provide evidence that the interleukin (IL)-6 receptor gp130 mRNA is downregulated in hearts of NMHC mice.
Methods
Transgenic Mice
The cloxPIBN mice were described previously.6 Mice expressing Cre-recombinase under the control of cardiomyocyte-specific -myosin heavy chain (-MHC) promoter (-MHC-Cre mice) were a generous gift from Dr Michael D. Schneider (Baylor College of Medicine, Houston, Tex).12 All mice were bred on a C57Bl/6 background for 6 generations. All aspects of animal care and experimental protocols were approved by the Berlin animal review board (Reg. 0135/01).
Induction of Cardiac Hypertrophy and Echocardiography
Micro-osmotic pumps (model 1002, Alzet) releasing Ang II (Sigma; 1.4 μg · kg–1 · min–1; solvent, 0.9% NaCl/0.01N acetic acid) or Iso (Sigma; 60 mg · kg–1 · d–1 in 0.9% NaCl) were implanted subcutaneously into age-matched (12 to 16 weeks old) male mice after anesthesia with ketamine/xylazine (30 mg/10 mg per 1 kg IP). Hypertrophy was assessed by echocardiography with an Accuson Sequoia (Siemens) instrument equipped with a 13-MHz microprobe. Ventricular measurements in M mode were taken before implantation and 12 days (Ang II) or 7 days (Iso) after, with 3 readings per mouse. The observer was blinded to genotype and treatment of the mice.
Histological Analysis
Hearts were fixed in 10% formalin/PBS and embedded in paraffin. Heart sections (10 μm) were stained with PAS or Masson trichrome as indicated to evaluate morphology and cellular dimensions. TUNEL assays were performed with the Apop Tag Plus system from Intergene according to the manufacturer’s instructions.
RNA Quantification
Total RNA was isolated with the RNeasy kit (Qiagen), and cDNA was synthesized with Superscript II reverse transcriptase (RT) (Invitrogen). cDNA was subjected to real-time RT–polymerase chain reaction (PCR) by SYBR Green Analysis (Qiagen) performed on an iCycler instrument (Biorad). Primers for mouse gene analysis were as described previously.13 Primer sets for rat genes were as follows: IB: forward, 5'-CAGCATCTCCACTCCGTCCT-3'; reverse, 5'-GCGTTGACATCAGCACCCAA-3'; gp130: forward, 5'-CAAGCACCGTGCAGTACTCC-3'; reverse, 5'-TGTCCACACTATCCACCAGCT-3'. GAPDH (Clonetech) was used as control.
Cell Culture and Adenoviral Infection
Cardiomyocytes were isolated from 12- to 14-week-old male Wistar rats (Moellegard, Schoenwalde, Germany) as described before.14 Three hours after attachment, cardiomyocytes were infected with recombinant adenoviral vector (Ad) encoding IBN (Ad5IBN) or empty vector (Ad5control; 50 MOI) and stimulated after another 36 hours. Cardiac fibroblasts obtained in parallel were grown to 70% confluence and serum deprived for 20 hours before stimulation. Adult mouse cardiomyocytes were isolated and cultured following the instructions given at www.signaling-gateway.org.
Immunoblotting and Electrophoretic Mobility Shift Assay
Cardiomyocytes were stimulated with Ang II, leukemia inhibitory factor (LIF), PE (all Sigma), TNF- (Biomol), IL-6 (Roche), cardiothrophin (CT)-1 (Calbiochem), or FCS as indicated. Nuclear and cytosolic extracts were prepared as described.15 Electrophoretic mobility shift assay (EMSA) was performed with 5 to 10 μg nuclear protein as described previously.11 For immunoblotting, 10 to 20 μg cytosolic protein was used. IBN from heart was immunoprecipitated as described.16 Total lysates of adult mouse cardiomyocytes were prepared as described.16 The antibodies for immunoblotting or immunoprecipitation were from Santa Cruz (IB, gp130), Cell Signaling (p- or total ERK and CREB, p-Stat3 [Tyr 705]), or Advanced ImmunoChemical Inc (GAPDH).
Immunostaining
After stimulation with TNF- (10 ng/mL, 30 minutes), mouse cardiomyocytes were fixed (4% formaldehyde/PBS, 25 minutes, 4°C), permeabilized (0.5% Triton X-100/PBS, 10 minutes, 4°C), and incubated with an antibody against NF-B–p65 (1:100, Santa Cruz) according to the manufacturer’s instructions. The TRITC-labeled secondary antibody was from Dianova; DAPI (0.5 μg/mL) was from Sigma.
Statistical Analysis
Differences between experimental groups were analyzed by use of a Student t test or 1-way ANOVA, followed by Bonferroni posttest when multiple groups were compared. Data are reported as mean±SEM. Values of P<0.05 were considered significant. Real-time RT-PCR data from adult cardiomyocytes were analyzed by paired t test, comparing cells derived from each preparation separately.
Results
Generation of Mice With Cardiomyocyte-Specific NF-B Inhibition
Mice heterozygous for floxed IBN (cloxPIBN, NloxP) were bred with heterozygous, -MHC–Cre (c-MHC-Cre, CreMHC) transgenic mice to generate mice with cardiomyocyte-restricted IBN expression, called NMHC mice (Figure 1A).6,12 IBN lacks the destruction box and acts as a super-repressor of NF-B.6 Heart-specific recombination was analyzed by PCR demonstrating efficient removal of the stop codon (Figure 1B). Heart-restricted IBN expression was confirmed by immunoblot analysis of immunoprecipitated tissue extracts of NMHC and cIBN mice, which express IBN ubiquitously (referred to as Nubi mice; Figure 1C).6 Less IBN protein is detected in heart tissue extracts from NMHC compared with Nubi mice because of up to 70% nonmyocytes in whole-heart extracts (Figure 1C).6 In addition, CreMHC mice exhibit mosaicism of Cre recombinase expression, which is estimated to be present in 70% of cardiomyocytes (described elsewhere,17 data not shown). Furthermore, recombination was confirmed in isolated cardiomyocytes from NMHC mice (Figure 1D). Effective NF-B inhibition in NMHC mice was confirmed by immunostaining of NF-B–p65 in TNF-–stimulated cultured cardiomyocytes. Although control cells showed robust nuclear translocation of p65, cardiomyocytes genetically expressing IBN displayed reduced p65 translocation (Figure 1E). In conclusion, NMHC mice exhibit cardiac-restricted, impaired NF-B activation.
Attenuation of Ang II– and Iso-Induced Cardiac Hypertrophy in NMHC Mice
NMHC mice did not exhibit any significant changes at histological or echocardiographic examination when left untreated (data not shown).18,19 Wild-type, CreMHC, or NloxP mice (summarized as control mice) and age-matched NMHC mice were exposed to Ang II (1.4 μg · kg–1 · min–1) infusion for 14 days. Although control mice exhibited a significant increase in diastolic wall dimensions and ratio of heart weight to tibia length on Ang II treatment, NMHC mice did not mount a hypertrophic response (Figure 2A and 2B and Table 1). Histological examination confirmed these results (Figures 2C and 3A through 3C). Similarly, Iso-induced hypertrophy at 7 days was attenuated in NMHC mice (Table 2). Thus, suppression of NF-B activity has a protective effect on Ang II– and Iso-induced cardiac hypertrophy.
In addition to morphological changes, mRNA expression of hypertrophy markers was analyzed by real-time RT-PCR (Figure 2D). As expected, we found a significant induction of atrial natriuretic peptide (ANP; expression ratio versus sham, 2.6±0.3), brain natriuretic peptide (BNP; expression ratio versus sham, 1.3±0.28), ;-MHC (expression ratio versus sham, 2.2±0.4), and -skeletal actin (expression ratio versus sham, 2.6±0.3) gene expression in cardiac tissue from Ang II–treated control animals. -MHC and transforming growth factor (TGF)-; levels remained largely unchanged after 14 days of Ang II treatment. In contrast, NMHC mice showed a significant reduction in ;-MHC and ANP upregulation by Ang II (Figure 2D).
Cytokines and/or chemokines secreted from cardiomyocytes may contribute to Ang II–induced inflammatory responses. As expected, histological analysis of Ang II–infused control mice revealed perivascular infiltration of inflammatory cells (Figure 3E) compared with sham-operated controls (Figure 3D). Ang II–induced perivascular inflammation was not altered in NMHC mice (Figure 3F). Hence, NF-B activity in cardiomyocytes may not contribute significantly to the Ang II–induced inflammatory reaction.
Because NF-B has been hypothesized to regulate survival signals in cardiomyocytes, we evaluated apoptosis of cardiomyocytes in hearts of mice. In histological sections from control and NMHC mice challenged with Ang II, we did not detect apoptotic cells by TUNEL assay (Figure 3G and 3J, respectively) or fragmentation of DAPI-stained nuclei (Figure 3H and 3K, respectively). As a positive control, one section was treated with DNase I before the TUNEL assay (Figure 3I). Furthermore, dilatation of the left ventricle resulting from apoptotic loss of cardiomyocytes was not detectable by echocardiography (Tables 1 and 2). Expression of IBN does not induce cardiomyocyte apoptosis and/or impairment of LV function in this model.
Activation of NF-B in Adult Cardiomyocytes by Ang II and IL-6 Cytokines
We analyzed NF-B DNA binding in adult rat cardiomyocytes after Ang II stimulation. Moderate NF-B DNA binding activity was induced by physiological concentrations of Ang II as early as 15 minutes (1.45±0.12-fold) and was sustained for 1 hour (Figure 4A). Ang II induced less NF-B DNA binding than TNF- stimulation (2.46±0.3-fold). NF-B DNA complexes induced by Ang II contained p50 and p65 (Figure 4B).
Activation of cardiac fibroblasts by Ang II significantly contributes to cardiac hypertrophy.20 We therefore analyzed the effect of Ang II on NF-B activity in cardiac fibroblasts. Ang II activated both ERK and CREB but not NF-B binding activity (Figure 4C). These pathways were demonstrated to regulate the expression of IL-6 in neonatal cardiac fibroblasts. Paracrine secretion of IL-6–related cytokines augments cardiac hypertrophy.21 Therefore, we studied the effect of IL-6 cytokines on NF-B activation in adult rat cardiomyocytes. Indeed, IL-6, CT-1, and LIF, alone or in combination, induced NF-B DNA binding at 30 minutes (Figure 4D, top). Interestingly, PE did not induce NF-B in adult cardiomyocytes, in contrast to previously published data from neonatal cardiomyocytes.7
ERK1/2 is known to be a central kinase of cardiac hypertrophy signaling.22 Ang II and TNF- did not induce ERK1/2 phosphorylation in adult cardiomyocytes at any time points, whereas PE activated ERK1/2 most potently (Figure 4D, bottom). These data imply that PE-induced hypertrophy is regulated mainly by ERK, whereas IL-6 cytokine-induced hypertrophy involves NF-B in addition to a modest ERK activation. Ang II induced NF-B activation but not ERK phosphorylation in adult rat cardiomyocytes (Figure 4D).
NF-B–Dependent Gene Regulation
A DNA microarray analysis was performed to identify possible downstream targets of NF-B involved in hypertrophy (data not shown). Adult rat cardiac myocytes transfected with Ad5IBN were compared with Ad5control or untransfected cells. Downregulation of IB mRNA was used as a positive control for efficient NF-B inhibition because the IB promoter contains several regulatory NF-B binding sites.4 The microarray data were confirmed by real-time RT-PCR for gp130 mRNA showing significant downregulation in IBN-transfected cells (Figure 5A). A similar trend was observed in whole-heart extracts from NMHC mice compared with control animals at baseline (Figure 5B B, left bar; P=0.056; n=4). In addition, levels of gp130 mRNA were significantly reduced in hearts of Ang II–treated NMHC mice (Figure 5B, right bar; P<0.05; n=4). In cultured cardiomyocytes from control or NMHC mice, protein levels of gp130 were comparable under nonstimulated conditions (Figure 5C). However, after 30 minutes of Ang II and IL-6, gp130 protein expression was reduced in cardiomyocytes from NMHC mice compared with control mice. Phosphorylation of transcription factor STAT3, which is a typical downstream target of IL-6/gp130 signaling, remained unchanged in NMHC cardiomyocytes after stimulation with Ang II and IL-6.
Discussion
Ca2+-dependent signaling molecules have been shown to mediate cardiac hypertrophy in vivo. Here, we provide genetic evidence for a Ca2+- independent signaling pathway required for cardiac hypertrophy, namely the NF-B activation cascade. Our results establish that inhibition of transcription factor NF-B by cardiomyocyte-restricted expression of the NF-B superrepressor IBN is sufficient to attenuate Ang II– and Iso-induced cardiomyocyte hypertrophy in vivo without a detectable effect on cardiomyocyte apoptosis. Interestingly, NF-B exerts its effect on cardiac hypertrophy by affecting a different subset of hypertrophy-related genes compared with the calcineurin-dependent signaling molecules. In calcineurin-deficient mice, inhibition of Ang II–induced hypertrophy was associated with a reduction in -skeletal actin mRNA.23 Similarly, inhibition of hypertrophy in NF-Atc3–/– mice was accompanied by attenuation of -skeletal actin upregulation.24 In contrast, NMHC mice displayed impaired ;-MHC and ANP upregulation while leaving BNP and -skeletal actin induction intact. BNP exerts positive effects in cardiac remodeling by antagonizing TGF-;–induced deposition of extracellular matrix proteins.25
In vitro experiments using neonatal rat cardiomyocytes as a model system proposed various hypertrophic pathways to regulate NF-B activity. The GPCR agonists endothelin 1, Ang II, and PE were all found to upregulate NF-B–dependent reporter genes.7,8 However, the upregulation may be indirect because the induction of NF-B DNA binding in response to these stimuli was not shown in these studies. Using a different system, namely adult rat cardiomyocytes, we observed only a modest increase in NF-B DNA binding on Ang II stimulation. Because IL-6 family cytokines contribute significantly to Ang II–induced cardiac hypertrophy,21 we analyzed NF-B activation by IL-6, CT-1, and LIF. All IL-6 cytokines were found to activate NF-B in adult cardiomyocytes substantially. These data are in line with previous reports in which activation of NF-B downstream of the IL-6 receptor gp130 protected neonatal cardiomyocytes from hypoxia-induced apoptosis.26 We found that PE strongly stimulates ERK1/2 phosphorylation but not NF-B DNA binding in adult cardiomyocytes. This result is in contrast to published data on neonatal cardiomyocytes.7 In our view, PE signaling differs from Ang II– and IL-6-induced pathways in cardiac hypertrophy in the adult system.
NF-B has been shown to regulate the expression of numerous genes, including survival factors and cell growth regulatory molecules. More precisely, the expression of apoptosis inhibitory molecules iAP1, bcl-2, and bcl-xL was described to be NF-B dependent.27 In our mouse model, NF-B inhibition attenuated cardiac hypertrophy without any detectable effects on cardiomyocyte apoptosis. These results suggest an important cardiomyocyte-specific role for NF-B in regulating genes involved in cardiac hypertrophy, whereas expression of survival factors might be affected only by more stringent NF-B inhibition. Alternatively, other transcription factors might compensate for reduced NF-B activity in cardiomyocytes concerning its role in apoptosis but not hypertrophy. The latter hypothesis is supported by our previously published observation that even complete NF-B inhibition by Ad5IBN in isolated neonatal rat cardiomyocytes did not lead to a detectable regulation of iAP1+2, bcl-2, or bcl-xL protein.11
The IL-6 receptor gp130 is a central regulator of cardiac hypertrophy.28 Here, we found gp130 mRNA to be downregulated in rat cardiomyocytes with NF-B inhibition and tissue extracts from NMHC mice after Ang II stimulation. Further studies are needed to determine whether this effect on gp130 mRNA is crucial for the observed effect on cardiac hypertrophy. Promoter analysis of the human, rat, and mouse gp130 gene did not reveal a canonical NF-B binding site, suggesting an indirect regulatory mechanism (data not shown).
Taken together, our data suggest that the NF-B signaling cascade is a potential target for pharmacological intervention in cardiac hypertrophy (Figure 5D). Further experiments are warranted to assess the long-term effect of NF-B inhibition on cardiac remodeling under pressure overload and other models of cardiac failure. In addition, pharmacological inhibitors of the NF-B pathway already studied in the context of chronic inflammatory lung and bowel diseases should be tested in animal models of cardiac hypertrophy and failure.
Acknowledgments
This study was supported by an MDC clinical research grant to Drs Bergmann and Scheidereit, a European Union Marie Curie Host Development Fellowship to Drs Bergmann and Scheidereit, and a grant-in aid from Pinguin Stiftung, Germany to Dr Bergmann. This study was supported in part by a grant from BMBF to Dr Scheidereit. We thank Gabi Welsch, B;rbel Pohl, and Sarah Ugowski for expert technical assistance.
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