The bHLH transcription factor CHF1/Hey2 regulates susceptibility to apoptosis and heart failure after pressure overload

Abstract

Cardiac hypertrophy is a common response to hemodynamic stress in the heart and can progress to heart failure. To investigate whether the transcription factor cardiovascular basic helix-loop-helix factor 1/hairy/enhancer of split related with YRPW motif 2 (CHF1/Hey2) influences the development of cardiac hypertrophy and progression to heart failure under conditions of pressure overload, we performed aortic constriction on 12-wk-old male wild-type (WT) and heterozygous (HET) mice globally underexpressing CHF1/Hey2. After aortic banding, WT and HET mice showed increased cardiac hypertrophy as measured by gravimetric analysis, as expected. CHF1/Hey2 HET mice, however, demonstrated a greater increase in the ventricular weight-to-body weight ratio compared with WT mice (P < 0.05). Echocardiographic measurements showed a significantly decreased ejection fraction compared with WT mice (P < 0.05). Histological examination of Masson trichrome-stained heart tissue demonstrated extensive fibrosis in HET mice compared with WT mice. TUNEL staining demonstrated increased apoptosis in HET hearts (P < 0.05). Exposure of cultured neonatal myocytes from WT and HET mice to H₂O₂ and tunicamycin, known inducers of apoptosis that work through different mechanisms, demonstrated significantly increased apoptosis in HET cells compared with WT cells (P < 0.05). Expression of Bid, a downstream activator of the mitochondrial death pathway, was expressed in HET hearts at increased levels after aortic banding. Expression of GATA4, a transcriptional activator of cardiac hypertrophy, was also increased in HET hearts, as was phosphorylation of GATA4 at Ser¹⁰⁵. Our findings demonstrate that CHF1/Hey2 expression levels influence hypertrophy and the progression to heart failure in response to pressure overload through modulation of apoptosis and GATA4 activity.

cardiac hypertrophy includes both physiological hypertrophy and pathological hypertrophy and is a common response to hemodynamic stress in the heart (7). Physiological hypertrophy is an adaptive response to overloading of the heart and allows the heart to compensate for increased load. Excessive overloading in diseases such as hypertension can cause a maladaptive response of the heart, referred to as pathological hypertrophy, a common precursor to heart failure, cardiac arrhythmia, and sudden death (16, 28).

Cardiovascular basic helix-loop-helix (bHLH) factor 1/hairy/enhancer of split related with YRPW motif 2 (CHF1/Hey2) is a member of the hairy family of bHLH transcription factors and is a downstream target of the Notch signaling pathway (2, 9, 14, 22). CHF1/Hey2 is important in the development of the cardiovascular system and has been associated with cardiomyopathy, septal defects, and valvular anomalies (3, 5, 15, 23, 24). Our previously published work has shown that CHF1/Hey2 can regulate aryl hydrocarbon receptor nuclear translocator/endothelial PAS domain protein 1-dependent transcription of the VEGF promoter (2) and plays critical roles in cardiovascular development (2, 13, 23, 24), cardiac hypertrophy (29, 33), and vascular occlusion (25).

A complete knockout of CHF1/Hey2 leads to cardiomyopathy in adults (23). Massive postnatal cardiac hypertrophy with neonatal lethality has also been reported (5), although it is not clear whether this hypertrophy is secondary to septation defects (23) or valvular regurgitation (15). In vitro experiments using embryonic stem cells lacking both CHF1/Hey2 and CHF2/Hey1 showed that loss of both genes increased the expression of GATA4, GATA6, and atrial natriuretic factor (ANF) in embryoid bodies, and forced expression of CHF/Hey genes strongly repressed the expression of GATA4 and GATA6 promoters (4). The GATA4 target gene ANF was also inhibited by Hey1, CHF1/Hey2, and HeyL. The repression was due to direct binding of Hey proteins to GATA4 and GATA6, blocking their transcriptional activity. In vitro studies (11, 29) in neonatal cardiac myocytes also showed that CHF1/Hey2 can inhibit GATA4-dependent cardiac genes such as ANF, specifically through the suppression of transcription driven by GATA4. An in vivo study (13) in mice showed that deletion of the CHF1/Hey2 gene resulted in persistent ANF expression during embryonic development. Overexpression of CHF1/Hey2 in the myocardium has also been shown to attenuate phenylephrine-induced cardiac hypertrophy in vivo (29) and promote physiological over pathological hypertrophy after aortic banding (33). In the present study, we report that heterozygosity for CHF1/Hey2 predisposes to cardiac hypertrophy and progression to heart failure in a mouse model of pressure overload, through enhancement of apoptosis and activity of GATA4.

MATERIALS AND METHODS

CHF1/Hey2 heterozygous mice.

Global knockout mice lacking CHF1/Hey2 have been previously described (23, 24). Most knockout mice die perinatally due to congenital heart defects (23, 24) and are limited in availability for adult models of cardiovascular disease. Consequently, we studied CHF1/Hey2 heterozygous (HET) mice on a C57BL/6 genetic background. These mice have no baseline phenotype. All animal experiments were approved by the University of Washington Institutional Animal Care and Use Committee.

Aortic banding surgery.

Male wild-type (WT) or CHF1/Hey2 HET mice on a C57BL/6 background were used in this study. Mice (age: 12 wk, body weight: 20–30 g) were anesthetized with ketamine (130 mg/kg ip) and xylazine (8.8 mg/kg ip) and then subjected to transverse aortic banding surgery as previously described (12, 27, 34).

Echocardiography.

For the evaluation of heart function, echocardiographic experiments were performed 1 wk before surgery and 1 wk after surgery to measure left ventricular (LV) wall thickness, LV end-diastolic dimension (LVEDD), and ejection fraction (EF) using a Visual Sonics VEVO 770 system equipped with a 707B scan head, as previously described (19). Mice were lightly anesthetized with 1% isoflurane. Data were measured in M mode from the short axis. LVEF (in %) was calculated as follows: LVEF = [(LVIDd)3 − (LVIDs)3]/(LVIDd)3 × 100, where LVIDd and LVIDs are LV internal diameters at diastole and systole, respectively (30).

Gravimetry, histology, and apoptosis detection.

One week after surgery, mice were euthanized by CO₂ inhalation followed by weighing, heart removal, and exsanguination. Hearts were rinsed in cold PBS, trimmed of atrial and vascular tissue, dried briefly, and weighed. For histology, hearts were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned by standard methods or embedded in OCT compound and frozen. Sections were deparaffinized, rehydrated, and stained with Masson trichrome by standard methods. To detect apoptotic cells, frozen sections were fixed in 4% paraformaldehyde, stained with TUNEL reagent, and counterstained with methyl green with a commercially available kit according to the manufacturer's instructions (Roche Molecular Diagnostics). The number of apoptotic cells was normalized to the total number of nuclei to derive the percentage of apoptotic cells.

To identify cell types undergoing apoptosis in the heart, TUNEL staining was combined with MF-20 immunostaining, which detects sarcomeric myosin heavy chain. Fixed frozen sections were stained with the TUNEL reaction mixture (Roche) containing fluorescein-dUTP for 1 h at 37°C in the dark and then incubated with MF-20 monoclonal antibody (Developmental Studies Hybridoma Bank) overnight at 4°C. Alexa fluor 546-labeled goat anti-mouse secondary antibody (Invitrogen) was used to visualize MF-20 antibody bound to sarcomeric myosin heavy chain. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Stained sections were visualized and photographed using confocal microscopy (Nikon A1R).

Measurements of cardiac myocyte cross-sectional area and cardiac fibrosis were assessed by Masson trichrome staining as previously described (12). At least 100–150 randomly chosen cardiomyocytes from each heart were analyzed to measure cross-sectional cardiomyocyte area using Image J software (National Institutes of Health). The quantification of fibrosis was analyzed as a percentage of fibrotic area to total area using Image J software (National Institutes of Health).

Neonatal mouse myocyte culture and in vitro assessment of hypertrophy and apoptosis.

Neonatal mouse cardiac myocytes were harvested and cultured as previously described (26, 29). To induce apoptosis, neonatal myocytes were exposed to H₂O₂ (100 μM) for 24 h. To induce endoplasmic reticulum stress, cells were exposed to tunicamycin (100 ng/ml) for 48 h. Apoptotic cells were detected by TUNEL staining with a commercially available kit (Roche Molecular Diagnostics), whereas the remaining cells were counterstained with methyl green by standard methods. The percentage of apoptotic cells was calculated by dividing the number of apoptotic cells by the total number of cells and multiplying by 100. Apoptotic cells were also detected by annexin V staining using a commercially available kit (BD Biosciences). Through this method, early apoptotic cells were detected by FITC-conjugated annexin V and necrotic cells were excluded by propidium iodide staining. Cell sorting and measurements of annexin V- and propidium iodide-positive cell populations were performed by flow cytometry.

Myocyte hypertrophy was induced in vitro by serum stimulation (20% FBS). Isolated neonatal myocytes were cultured with serum-free DMEM for 24 h and then changed to DMEM with 20% FBS with [³H]leucine (1 μCi/ml). After 24 h, protein was extracted from cells for liquid scintillation counting of [³H]leucine, and the results were normalized to [³H]leucine uptake from cells not treated with serum. To assess whether hypertrophy leads to apoptosis in cultured myocytes, serum-treated cells were processed for TUNEL staining after 48 h of serum stimulation.

RNA isolation, quantitative RT-PCR, protein isolation, and Western blot analysis.

Total RNA was isolated from mouse LVs by homogenization and TRIzol reagent (Invitrogen) extraction according to the manufacturer's instructions. RNA was subsequently reverse transcribed into cDNA using Superscript III (Invitrogen) and oligo (dT)₂₀ primers (Invitrogen). Quantitative real-time PCR to assess CHF1/Hey2 and Bid expression was performed on cDNA samples using SYBR green fluorescent reagent and an Applied Biosystems 7500 according to standard protocols. GAPDH was used as a control. The following primers were used for the real-time PCR: CHF1/Hey2, 5′-TGA AGA TGC TCC AGG CTA CA-3′ and 5′-CAC TCT CGG AAT CCA ATG CT-3′; Bid, 5′-CGT GAT GTC TTC CAC ACG AC-3′ and 5′-GTT CCT CTG GAG GCA GTG TC-3′; and GAPDH, 5′-CCT TCA TTG ACC TCA ACT AC-3′ and 5′-GGA AGG CCA TGC CAG TGA GC-3′. Data analyses were performed with the $2^{-\Delta \Delta {\mathrm{C}}_{\mathrm{T}}}$ method, where C_T is cycle threshold, as previously described (20).

Total protein was isolated from mouse LV tissue by homogenization and lysis in 50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% IGEPAL, and 0.5% sodium deoxycholate with complete protease inhibitor (Roche) as previously described (8, 21). Protein concentration was determined using the Bradford method according to the kit manufacturer's instructions (Bio-Rad). Total proteins were loaded onto 12% SDS-PAGE gels and transferred to membranes for Western blot analysis as previously described (8). The following antibodies were used: anti-GATA-4 (phospho-Ser¹⁰⁵, Invitrogen) for the detection of phosphorylated GATA4, anti-GATA-4 (Santa Cruz Biotechnology) for the detection of total GATA4, anti-Bid (Cell Signaling) for the detection of Bid, and GAPDH (Cell Signaling) as a control.

Statistical analysis.

All data are reported as means ± SE. Comparisons between groups were made using an unpaired Student's t-test (two groups). All analyses were performed using commercially available software (StatView, SAS Institute). P values of <0.05 were taken as the minimal level of significance.

RESULTS

Mice underexpressing CHF1/Hey2 develop increased hypertrophy and reduced ventricular function after aortic banding.

To investigate the effect of CHF1/Hey2 underexpression on pressure overload-induced cardiac hypertrophy, we performed transverse aortic constriction on 12-wk-old male WT and CHF1/Hey2 HET mice (C57BL/6 strain) lacking one functional CHF1/Hey2 allele. We also characterized CHF1/Hey2 expression in the hearts of these animals by quantitative RT-PCR and found that HET mice express at ∼50% of WT levels and that expression does not significantly change after aortic banding (Fig. 1A). After transverse aortic banding, both WT and HET mice showed hypertrophy (Fig. 1B). Gravimetric analysis demonstrated a greater increase in the ventricular weight-to-body weight ratio in HET mice compared with WT mice (7.132 vs. 6.045 mg/g, P = 0.025; Fig. 1C). Both WT and HET mice showed increased LV wall thickness compared with sham controls (1.10 vs. 0.75 mm in WT mice, P < 0.001, and 1.15 vs. 0.74 mm in HET mice, P < 0.001; Fig. 1D). HET mice, however, developed increased LVEDD compared with WT mice (4.05 vs. 3.62 mm, P = 0.012; Fig. 1E), which suggested eccentric hypertrophy in HET mice compared with concentric hypertrophy in WT mice. HET mice also demonstrated increased susceptibility to heart failure. One week after aortic banding, WT mice showed no decrement in EF (62% vs. 66% in sham-operated mice, P = 0.45). HET mice showed significantly decreased EF (46 vs. 66% in sham-operated mice, P = 0.003, and vs. 62% in WT mice, P = 0.008; Fig. 1F). These findings demonstrate that CHF1/Hey2 underexpression predisposes to increased cardiac hypertrophy and decreased heart function under conditions induced by pressure overload.

Fig. 1.

Cardiovascular basic helix-loop-helix factor 1/hairy/enhancer of split related with YRPW motif 2 (CHF1/Hey2) heterozygous (HET) mice demonstrate increased ventricular mass, wall thickness, and ventricular chamber size along with decreased ejection fraction (EF) after transverse aortic constriction. A: quantitative RT-PCR of mRNA for CHF1/Hey2 after aortic banding. WT, wild type; NS, not significant. B: long-axis view of the whole heart and hematoxylin/eosin staining of representative left ventricular (LV) sections 1 wk after banding shows LV chamber enlargement. C: comparison of ventricular weight-to-body weight ratios (VW/BW) in WT and HET mice after aortic banding. D: comparison of LV wall thickness in WT and HET mice after aortic banding. E: comparison of LV end-diastolic dimension (LVEDD) in WT and HET mice after aortic banding. F: comparison of LVEF in WT and HET mice after aortic banding. n = 7 WT sham-operated mice, 12 WT banded mice, 5 HET sham-operated mice, and 10 HET banded mice.

To investigate whether the greater cardiac hypertrophy in HET hearts is due to myocyte enlargement, we measured myocyte cross-sectional area as previously described (12). Histological analysis demonstrated greater cardiac myocyte cross-sectional areas in HET hearts (359 μm² in HET hearts vs. 295 μm² in WT hearts, P < 0.001; Fig. 2). We also assessed the effect of CHF1/Hey2 underexpression on cardiac myocyte hypertrophy in vitro. Cultured neonatal myocytes were treated with 20% serum to induce hypertrophy as previously described (33). [³H]leucine uptake demonstrated an increased hypertrophic response in HET cardiac myocytes compared with WT cardiac myocytes (Fig. 3A). To investigate whether hypertrophic stimuli promote apoptosis in vitro, we performed TUNEL staining on cultured WT and HET cells after serum treatment. TUNEL staining showed no increased apoptosis after serum stimulation in HET cardiac myocytes compared with WT cardiac myocytes (Fig. 3B). These findings suggest that the macroscopic cardiac hypertrophy observed in CHF1/Hey2 HET hearts originates from the enlargement of individual cardiac myocytes and that apoptosis is not a direct consequence of increased hypertrophy and may be more a consequence of injury after sustained pressure overload.

Fig. 2.

CHF1/Hey2 HET mice demonstrate increased cardiac myocyte cross-sectional areas after aortic banding. WT and HET mice were euthanized 1 wk after transverse aortic banding. A: hearts were fixed, embedded, sectioned, and stained with Masson trichrome. Representative images are shown. B: myocyte cross-sectional areas of 150 consecutive myocytes were measured with ImageJ software in both WT and HET hearts. n = 7 WT sham-operated mice, 7 WT banded mice, 5 HET sham-operated mice, and 7 HET banded mice.

Fig. 3.

Cultured CHF1/Hey2 HET cardiac myocytes demonstrate increased hypertrophy but no significant apoptosis after serum stimulation. A: hypertrophic responses were measured by [³H]leucine uptake after serum stimulation and normalized to uptake in untreated cells. Results are presented as fold changes after serum stimulation. B: apoptosis after serum stimulation was determined by TUNEL staining. The number of TUNEL-positive cells was quantified numerically as a percentage of the total cell number.

Underexpression of CHF1/Hey2 predisposes to myocardial fibrosis and apoptosis after aortic banding.

HET mice developed extensive fibrosis after transaortic banding compared with WT mice, as shown in Fig. 4. Quantification of the fibrotic area by analysis with ImageJ software demonstrated that the amount of fibrosis significantly increased in HET hearts compared with WT hearts. TUNEL staining demonstrated increased numbers of apoptotic nuclei in HET hearts compared with WT hearts after aortic banding (Fig. 5A). Quantification of TUNEL-positive cells as a percentage of total nuclei demonstrated a significant increase of apoptosis in HET knockout hearts compared with WT hearts after aortic banding (Fig. 5B). To identify the cell types undergoing apoptosis in the heart, we combined TUNEL staining with antibody staining against sarcomeric myosin heavy chain and performed confocal microscopy. As shown in Fig. 5C, cells that were both TUNEL positive and sarcomeric myosin heavy chain positive were detectable, demonstrating that apoptosis of cardiac myocytes was observed after banding. These findings suggest that CHF1/Hey2 influences the progression of hypertrophy to heart failure through the regulation of apoptotic pathways and fibrosis.

Fig. 4.

CHF1/Hey2 HET mice demonstrate increased cardiac fibrosis after aortic banding. WT and HET mice were euthanized 1 wk after transverse aortic banding. Hearts were fixed, embedded, sectioned, and stained with Masson trichrome. A: stained sections showed increased fibrosis after transverse aortic banding in HET mice compared with WT mice (indicated by arrow). B: quantification of the fibrotic area as a percentage of the total area was performed by digital image tracing of the fibrotic and total areas using ImageJ software.

Fig. 5.

CHF1/Hey2 HET mice demonstrate increased cardiac apoptosis after transverse aortic banding. WT and HET mice were euthanized 1 wk after transverse aortic banding. A,1–4: hearts were fixed, embedded, sectioned, and stained with TUNEL reagent and counterstained with methyl green. Arrows indicate representative apoptotic cells. Scale bars are shown. B: the number of TUNEL-positive cells was tabulated in multiple sections and quantified as a percentage of the total cell number. n = 3 WT sham-operated mice, 3 WT banded mice, 3 HET sham-operated mice, and 4 HET banded mice. C: confocal microscopy of TUNEL (green) and MF-20 (red) double-stained heart sections after aortic banding detected cells that were both TUNEL positive and sarcomeric myosin heavy chain positive (arrow), demonstrating that apoptosis of cardiac myocytes was observed after banding.

Isolated neonatal cardiac myocytes that underexpress CHF1/Hey2 are prone to apoptosis.

Based on our observation that HET mice developed increased cardiac apoptosis after aortic banding, we hypothesized that individual myocytes underexpressing CHF1/Hey2 may be more susceptible to apoptosis when stressed. To test this hypothesis, we cultured neonatal myocytes from WT and HET mice as previously described (29) and treated them with H₂O₂, a potent inducer of oxidative stress and subsequent apoptosis, using an established protocol (1). As shown in Fig. 6A, WT and HET myocytes show comparable rates of apoptosis in culture in the absence of H₂O₂. After treatment with H₂O₂, both WT and HET cells demonstrated significantly increased apoptosis compared with untreated cells (Fig. 6B). HET cells also demonstrated significantly more apoptosis than WT cells after treatment. Treatment with tunicamycin, which promotes endoplasmic reticulum stress and apoptosis, also resulted in increased apoptosis in HET cells compared with WT cells, adding further evidence that decreased expression of CHF1/Hey2 predisposes cells to apoptosis (Fig. 6, C and D). To confirm that the cells were truly undergoing apoptosis and not necrosis, we performed annexin V and propidium iodide staining of cultured myocytes treated with H₂O₂ and analyzed cell populations by flow cytometry (Fig. 7). The number of annexin V-positive cells increased in both WT and HET cells after H₂O₂ treatment, and the increase was significantly greater in treated HET cells compared with treated WT cells, confirming that HET cells are more susceptible to apoptosis.

Fig. 6.

Cultured CHF1/Hey2 HET cardiac myocytes demonstrate increased apoptosis in vitro after treatment with H₂O₂ or tunicamycin. A: cultured neonatal cardiac myocytes (WT and HET) were treated with H₂O₂ for 24 h, and apoptotic cells were detected by TUNEL staining followed by methyl green counterstaining. B: the number of TUNEL-positive cells was tabulated in multiple fields for both WT and HET cells treated with either H₂O₂ or vehicle and quantified as a percentage of the total cell number. C: WT and HET myocytes were treated with the indicated concentration of tunicamycin for 48 h followed by TUNEL staining and methyl green counterstaining. D: the number of TUNEL-positive cells was tabulated in multiple fields for both WT and HET cells treated with either tunicamycin or vehicle and quantified as a percentage of the total cell number.

Fig. 7.

Cultured CHF1/Hey2 HET cardiac myocytes demonstrate increased annexin V staining after treatment with H₂O₂ as measured by flow cytometry. Cultured neonatal cardiac myocytes (WT and HET) were treated with H₂O₂ for 24 h. A: apoptotic cells were detected by annexin V staining, and necrotic cells were excluded by propidium iodide counterstaining. Cells staining for annexin V, propidium iodide, both, or neither were sorted and quantified in both WT and HET cells treated with either H₂O₂ or vehicle. B: the number of apoptotic cells was quantified as the percentage of annexin V positive, propidium iodide negative cells relative to total cells in each group using flow cytometry analysis.

Underexpression of CHF1/Hey2 in the myocardium is associated with alterations in the expression of the apoptotic regulator Bid after aortic banding.

To determine the potential basis for the increased susceptibility to apoptosis, we examined the expression of numerous candidate genes associated with apoptosis by quantitative RT-PCR of RNA isolated from WT and HET hearts before and after aortic banding. As shown in Fig. 8, Bid, an integrator of multiple apoptotic signaling pathways that activates the common mitochondrial death pathway (32), was expressed at increased levels. This increase in expression was confirmed at the protein level by Western blot analysis. The alteration in Bid expression in conjunction with the observation that myocytes underexpressing CHF1/Hey2 are susceptible to unrelated apoptotic stimuli suggests that CHF1/Hey2 is a regulator of a distal common apoptotic pathway that influences the progression to heart failure.

Fig. 8.

Bid expression is increased in HET hearts after aortic banding. A: quantitative RT-PCR of mRNA for Bid after aortic banding. RNA was isolated from WT or HET hearts after either sham operation or aortic constriction by standard methods. Expression was normalized to GAPDH and compared between samples by the $2^{-\Delta \Delta {\mathrm{C}}_{\mathrm{T}}}$ method. B: evaluation of Bid protein expression after aortic banding. Total protein was isolated from WT or HET hearts after either sham operation or aortic banding. Protein lysates were analyzed for protein expression by Western blot analysis using antibodies for Bid. Adjacent lanes represent biological replicates in the banded groups.

Underexpression of CHF1/Hey2 is associated with increased expression and phosphorylation of GATA4.

We and others (4, 11, 29) have previously reported that CHF1/Hey2 can interact with GATA4 to suppress GATA4-dependent activation of hypertrophy-associated genes such as ANF. CHF1/Hey2 has also been reported to suppress activity of the GATA4 promoter (4), and GATA4 has also been shown to promote hypertrophy (17). To assess whether CHF1/Hey2 loss of function affects the expression of GATA4, we performed Western blot analysis on protein extracts from WT and HET hearts after aortic banding. As shown in Fig. 9, GATA4 expression increased after aortic banding in both WT and HET hearts, but the increase was larger in HET hearts. GATA4 DNA binding activity is reportedly activated by phosphorylation at Ser¹⁰⁵ by ERKs (18), which are activated by hypertrophic stimuli. To assess changes in GATA4 phosphorylation at Ser¹⁰⁵, we performed additional Western blot analysis with an antibody specific for phosphorylated Ser¹⁰⁵ in GATA4. As shown in Fig. 9, phosphorylation of GATA4 at Ser¹⁰⁵ increased after banding, but this increase was proportional to the increase in total GATA4. These findings suggest that increased GATA4 expression results from CHF1/Hey2 underexpression and that this increased expression is not accompanied by a significant increase in GATA4 phosphorylation.

Fig. 9.

Evaluation of GATA4 expression and phosphorylation after aortic banding. Total protein was isolated from WT or HET hearts after either sham operation or aortic banding. Protein lysates were analyzed for protein expression by Western blot analysis using antibodies for phosphorylated GATA4 [phospho-Ser¹⁰⁵ (pS¹⁰⁵)], total GATA4, and GAPDH as indicated. Adjacent lanes represent biological replicates in the banded groups.

DISCUSSION

We found that underexpression of CHF/Hey2 facilitates the development of cardiac hypertrophy, the progression from hypertrophy to heart failure, and predisposes to apoptosis and fibrosis. While previous studies have suggested that CHF1/Hey2 may attenuate hypertrophy, to the best of our knowledge, our study is the first to show that CHF1/Hey2 underexpression facilitates the development of heart failure in the absence of congenital abnormalities. Our data also demonstrate that CHF1/Hey2 regulates susceptibility to apoptosis in cultured cardiac myocytes, most likely through the regulation of distal pathways that are activated by diverse apoptotic stimuli. As LV dysfunction, fibrosis, and apoptosis are indicators of pathological rather than physiological hypertrophy, our findings also suggest that CHF1/Hey2 is an important suppressor of a pathological hypertrophic response. Our finding that CHF1/Hey2 underexpression is associated with increased induction of GATA4 is consistent with previous data showing that CHF1/Hey2 affects the development of hypertrophy through the attenuation of GATA4 activity (11, 29). These findings may consequently have important implications for future therapy.

As discussed above, the distinguishing features of pathological hypertrophy are the development of apoptosis, fibrosis, and LV dysfunction. Limited progress has been made in distinguishing the molecular processes that distinguish the pathological versus physiological response. The understanding of the progression to heart failure is also understood only at the most rudimentary level. Although many of the molecular events that lead to LV dysfunction are poorly understood, apoptosis has been postulated to play an important role (for a review, see Ref. 10). Our findings that the CHF1/Hey2 expression level affects apoptosis observed in vivo after aortic banding and myocyte apoptosis induced by H₂O₂ or tunicamycin exposure in vitro imply that the mechanism by which CHF1/Hey2 affects the progression to heart failure under conditions of pressure overload is through modulation of the apoptotic process. Interestingly, H₂O₂ and tunicamycin induce apoptosis by different mechanisms. H₂O₂ induces oxidative stress, and oxidative stress has been implicated in the development of cardiac hypertrophy (31). Tunicamycin, on the other hand, is an inducer of the endoplasmic reticulum stress pathway, which is also activated in ischemia, hypertrophy, and heart failure (for a review, see Ref. 6). These observations suggest that the effects of CHF1/Hey2 on apoptosis are probably mediated through effects on distal components of the apoptotic machinery that are common to multiple apoptotic signaling pathways. Our finding that Bid expression is altered is consistent with this hypothesis. Bid interacts with multiple Bcl-2 family members and is cleaved by multiple apoptotic proteases such as caspases, calpains, and cathepsins. Cleaved Bid induces mitochondrial dysfunction and thereby links peripheral death pathways with the central mitochondrial death pathway (for a review, see Ref. 32). Increased expression of Bid is likely to increase sensitivity to apoptotic stimuli. Further study is needed to determine whether CHF1/Hey2 functions as a direct regulator of Bid. Given its putative role as a transcriptional repressor, it is certainly plausible that it functions as a repressor of Bid.

GATA4 has previously been shown to promote cardiac hypertrophy (17) through the activation of downstream target genes. The DNA binding activity of GATA4 is enhanced by phosphorylation of Ser¹⁰⁵ through the action of ERK1 and ERK2. We and others (11, 29) have previously shown that CHF1/Hey2 can inhibit hypertrophy through direct binding of GATA4 and inhibition of GATA4-dependent gene expression. CHF1/Hey2 has also been shown to directly repress the GATA4 promoter during embryonic development (4). The potential role of CHF1/Hey2 in regulating the phosphorylation of GATA4 or in regulating the GATA4 promoter in the adult during hypertrophy has not been previously addressed. Our data indicate that heterozygosity for CHF1/Hey2 leads to increased expression of GATA4 after aortic banding and correlates with increased hypertrophy and the progression to heart failure. Although phosphorylation of Ser¹⁰⁵ also increased in HET hearts, the increase in phosphorylation was proportional to the increase in total GATA4. These findings suggest that another mechanism by which CHF1/Hey2 regulates the development of hypertrophy is through the attenuation of GATA4 transcription. Even though CHF1/Hey2 interacts directly with GATA4, it does not appear to affect serine phosphorylation at residue 105 by ERKs.

Although we observe both increased hypertrophy and apoptosis in hearts that underexpress CHF1/Hey2, our in vitro experiments on cultured myocytes demonstrate that treatment with hypertrophic stimuli does not result in apoptosis, demonstrating that the hypertrophic and apoptotic responses are mediated by different pathways. In vivo, we propose that myocyte hypertrophy occurs in response to hemodynamic stress and that myocyte apoptosis occurs when the myocytes can no longer compensate for prolonged hemodynamic stress. Our findings suggest that CHF1/Hey2 HET myocytes are more sensitive to hemodynamic stress and, therefore, demonstrate an increased degree of hypertrophy compared with WT cells. This increased sensitivity is also manifested as an increased susceptibility to apoptosis. Not all myocytes will die, however, because within the banded hearts, hemodynamic stress is not evenly distributed among all myocytes. All will undergo some degree of hypertrophy, whereas those experiencing greater degrees of hemodynamic stress will undergo apoptosis. The greater degree of hypertrophy and apoptosis in HET hearts is reflective of a decreased ability to compensate for the hemodynamic stress, leading to heart failure.

Possible limitations of our study include the potential effects of CHF1/Hey2 on valvular pathology and function as well as effects on the aorta that may contribute to the differential responses to aortic banding observed in WT and HET animals. Mice completely lacking CHF1/Hey2 have previously been shown to have valvular abnormalities and subtle aortic defects (24). Such defects have not been observed in HET mice, however, either before or after aortic constriction (unpublished observations). In addition, our echocardiographic data showed no evidence of valvular insufficiency after aortic banding (data not shown). To date, however, we have not ruled out functional defects in the aorta that may affect blood pressure gradients induced by aortic banding, although there were no differences in blood pressure at baseline (data not shown). To rule out a functional defect will require directly measuring the blood pressure gradient across the site of constriction, which we hope to do in future studies.

Overall, our findings demonstrate that CHF1/Hey2 is an important regulator of pathological hypertrophy and the progression to heart failure. Given that CHF1/Hey2 is a transcription factor, it is likely that many additional transcriptional target genes exist that influence many aspects of cardiac myocyte biology. Studies to identify additional relevant target genes are ongoing. The identification of additional downstream targets of CHF1/Hey2 in the myocardium that control apoptosis and the progression to heart failure will likely lead to important therapies for cardiovascular disease.

GRANTS

This work was supported by National Institutes of Health Grants HL-081088 and ES-015915 (to M. T. Chin) and by an American Heart Association Postdoctoral Fellowship (to L. Wu).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).