Published online before print March 1, 2007, doi: 10.1161/01.RES.0000261693.13269.bf
© 2007 American Heart Association, Inc.
Molecular Medicine |
CHF1/Hey2 Plays a Pivotal Role in Left Ventricular Maturation Through Suppression of Ectopic Atrial Gene Expression
From the Vascular Medicine Research, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Center for Cardiovascular Biology, University of Washington, 815 Mercer St, Rm 353, Seattle, WA 98109. E-mail mtchin{at}u.washington.edu
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Abstract |
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We previously reported that mice lacking the hairy-related basic helix–loop–helix (bHLH) transcription factor CHF1/Hey2 develop a thin-walled left ventricle. To explore the basis for this phenotype, we examined regional gene expression patterns in the developing myocardium. We found that atrial natriuretic factor (ANF), which is normally expressed in the atria and trabeculae and is restricted from the developing compact myocardium beginning at embryonic day 13.5, is persistently expressed in the left ventricular compact myocardium of the knockout animals. We also examined the expression pattern of the T-box transcription factor Tbx5, a known regulator of ANF, and an additional Tbx5-dependent gene, connexin 40 (Cx40), both of which share a similar expression pattern to ANF during development. Tbx5 and Cx40 were similarly expressed ectopically in the compact myocardium of the CHF1/Hey2 knockout mouse. The atrial contractile genes mlc1a and mlc2a were also expressed ectopically in the left ventricular compact myocardium, providing evidence for a general dysregulation of atrial gene expression. Crossing of a myocardial-specific CHF1/Hey2 transgenic mouse with the knockouts led to rescue of the thin-walled myocardial phenotype and restoration of the normal patterns of gene expression. Myocardial cell proliferation, which has been shown previously to be suppressed by Tbx5, was also decreased in the knockout mice and rescued by the transgene. Our findings suggest that CHF1/Hey2 suppresses atrial identity in the left ventricular compact myocardium, facilitates myocardial proliferation by suppressing Tbx5, and thereby promotes proper ventricular myocardial maturation.
Key Words: CHF1/Hey2 • Tbx5 • cardiac development • knockout mouse
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Introduction |
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We and others have previously identified a family of cardiovascular restricted, basic helix–loop–helix (bHLH) transcription factors related to hairy, a drosophila bHLH gene involved in Notch signaling and patterning of the peripheral nervous system.1–6 This family has received various names, including Hey, Hesr, HRT, CHF, gridlock, and HERP. In zebrafish, gridlock has been found to play an important role in development of the aorta and in arterial versus venous cell fate decisions.5,7 Numerous studies have postulated roles for these genes in Notch signaling,6,8,9 endothelial differentiation,10,11 skeletal muscle differentiation,12 transcriptional repression,2,12,13 smooth muscle function,9,14–16 morphogenesis of the heart,17–20 and cardiac hypertrophy.21
CHF1/Hey2 (also called Hesr-2, HRT2, gridlock, and HERP1) was originally described as a bHLH protein expressed primarily in the developing ventricular myocardium and vasculature.2–4 Loss-of-function studies in zebrafish initially suggested that the primary effect of CHF1/Hey2 is on vascular development.5,7 Loss-of-function studies in mice, however, revealed primarily cardiac abnormalities.17–20,22 The spectrum of defects varies, with reports of cardiomyopathy,17,19 ventricular septal defects,18,19 valvular abnormalities,18,20,22 vascular abnormalities,16,20 and a thin-walled myocardium.20 The basis for this variation is primarily genetic, as the phenotype varies significantly by genetic background, although there is some variation even when the background is inbred.20
Our previous work on the phenotype of the CHF1/Hey2 knockout mice inbred to the C57BL/6 background has revealed the importance of CHF1/Hey2 in the developing myocardium. In these mice, the myocardium is thin walled, because of a marked decrease in the thickness of the compact myocardium,20 and this phenotype is completely penetrant. The importance of CHF1/Hey2 in the myocardium is further underscored by our finding that a transgenic mouse expressing CHF1/Hey2 in the myocardium can not only rescue the myocardial phenotype in the knockout mice but can also partially rescue the septal and valvular phenotypes.20
To identify potential mechanisms by which CHF1/Hey2 affects myocardial development, we performed a systematic analysis of regional myocardial gene expression, using markers of the atria, trabeculae, and ventricles, and also assessed for an alteration in myocardial cell proliferation. We have found that loss of CHF1/Hey2 leads to ectopic expression of atrial genes in the left ventricular compact myocardium, including the transcription factor Tbx5 and its target genes ANF and Cx40, and a profound decrease in myocardial cell proliferation. Our findings suggest an important mechanism by which CHF1/Hey2 regulates ventricular maturation.
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Materials and Methods |
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Animals
The mice lacking CHF1/Hey2 and expressing CHF1/Hey2 under the control of mlc2v promoter on a C57BL/6 background were generated and bred as described previously.20 All animals were managed according to guidelines published in the Use and Care of Laboratory Animals and in accordance with IACUC approved experimental protocols.
In Situ Hybridization and Immunohistochemistry
In situ hybridization was performed as described previously,20,23,24 with slight modifications. The embryos were fixed (4% formaldehyde, 0.1 mol/L 4-morpholinepropanesulfonic acid, 2 mmol/L EGTA, 1 mmol/L MgSO4) for 4 hours at room temperature and stored at –20°C in methanol. The embryos were bleached with 30% H2O2 in methanol at 4°C for 1 hour and embedded in paraplast (Fisher HealthCare). Deparaffinized 15-µm-thick sections were briefly rinsed in PBS, followed by 10-minute treatment with 5 µL/mL acetic anhydride in 0.1 mol/L triethanolamine and 15-minute treatment with 2 mg/mL glycine in PBS/1% Tween 20. The sections were prehybridized with hybridization solution24 at 70°C for 1 hour and hybridized with hybridization solution23 containing 0.5 µg/mL digoxigenin-labeled riboprobe at 70°C for 16 hours. The sections were washed with 50% formamide, 2x SSC at 70°C for 2 hours and rinsed in PBS at room temperature. After blocking and reaction with alkaline phosphatase-conjugated anti-Dig antibody (1:2000 Boehringer) at 4°C for 16 hours, color was developed using BM purple (Boehringer).
The cDNAs used for generation of Digoxigenin-labeled mouse riboprobes were ANF (nucleotides 173 to 744; GenBank no. NM_ 008725), ß-MHC (nucleotides 7 to 237; GenBank no. NM_080728), BMP10 (nucleotides 86 to 1222; GenBank no. NM_009756), CHF1/Hey2,20 Cx40 (nucleotides 144 to 2127; GenBank no. NM_008121), GATA4 (nucleotides 1413 to 3272; GenBank no. NM_008092), mlc1a (nucleotides 46 to 339; GenBank no. NM_010858), mlc2a (nucleotides 65 to 529; GenBank no. NM_022879), mlc2v,25 Nkx2.5 (nucleotides 231 to 1181; GenBank no. NM_008700), N-myc (nucleotides 525 to 1762; GenBank no. NM_008709), and Tbx5 (nucleotides 360 to 1950; GenBank no. NM_011537). At least 3 embryos were examined for each gene.
Cell Proliferation
Sections (5-µm thick) were stained immunohistochemically using rabbit anti–phospho-histone H3 (Cell Signaling Technology) at 1:50 dilution. Alkaline phosphatase-conjugated anti-rabbit IgG (Chemicon) was used at 1:1000 dilution for the second antibody. The total number of positive cells in the left ventricular compact myocardium was recorded, and then sections were counterstained with hematoxylin to count total cell number, to derive the percentage of positive cells. We examined hearts from 2 embryos of each genotype and sampled 7 sections from each heart. Statistical comparison was done by an unpaired Student’s t test.
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Results |
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CHF1/Hey2 Is Expressed in the Compact but Not Trabecular Myocardium
Previous reports have demonstrated that CHF1/Hey2 is expressed primarily in the developing ventricle through whole-mount and radioactive in situ hybridization.2–4,26 To examine the expression pattern in pure C57BL/6 background mice in detail, we performed nonradioactive section in situ hybridization for CHF1/Hey2 as previously described,2,20 with slight modifications. As shown in Figure 1, CHF1/Hey2 is expressed predominantly in the compact myocardium and interventricular septum at all time points examined, from embryonic day 11.5 (E11.5) through E15.5. Expression is also seen in the epithelial cells lining the endocardial cushions, but is not seen in the trabecular myocardium.
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ANF Is Expressed Ectopically in the Left Ventricular Compact Myocardium of CHF1/Hey2 Knockout Hearts
We and others have previously shown that CHF1/Hey2 can inhibit expression of the ANF promoter in vitro,27 during development,26 and during development of cardiac hypertrophy,21 through an interaction with the transcription factor GATA4. To assess for alteration in ANF expression in our C57BL/6 CHF1/Hey2 knockout mice, we performed in situ hybridization for ANF expression at E12.5 through E15.5. As shown in Figure 2, ANF is normally expressed in the atria, trabeculae, and left ventricular compact myocardium at E12.5, but becomes restricted from the left ventricular compact myocardium beginning at E13.5, while persisting in the atrial and trabecular myocardium. In the CHF1/Hey2 knockout embryos, however, ANF is expressed continuously in the left ventricular compact myocardium after E13.5 through E15.5. This pattern of ectopic expression is consistent with a previous study,26 occurs in a domain where CHF1/Hey2 is normally expressed, and is suggestive of derepression of ANF after deletion of CHF1/Hey2. Derepression of ANF through increased GATA4 activity alone, however, cannot explain the ectopic expression of ANF limited to the left ventricular compact myocardium, as GATA4 and CHF1/Hey2 are both expressed in the left and the right ventricle (Figure 1 and Figure I in the online data supplement, available at http://circres.ahajournals.org).
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Tbx5, an Upstream Regulator of ANF, and Its Target Gene, Cx40, Are Also Expressed Ectopically in the Left Ventricular Compact Myocardium of the Knockout Hearts
The ANF promoter has been studied extensively in vitro and is positively regulated by the cardiac transcription factors GATA4, Nkx2.5, and Tbx5.28–30 To determine the potential contribution of these factors, we investigated their expression patterns by in situ hybridization. As shown in supplemental Figures I and II, the expression patterns of GATA4 and Nkx2.5 are indistinguishable in wild-type and knockout embryos. Tbx5 has previously been reported to be expressed weakly in the developing left ventricle at E13.5.31 As shown in Figure 3, we observe weak expression of Tbx5 in the developing ventricle at E13.5; however, this expression is decreased by E15.5 in wild-type hearts. The expression pattern for Tbx5 in the knockout embryos, however, is similar to that observed for ANF, in that it is persistently expressed in the left ventricular compact myocardium through E15.5 (Figure 3). These findings suggest that ectopic expression of ANF results from persistent expression of Tbx5.
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To verify that persistent expression of Tbx5 is sufficient to promote expression of downstream target genes, we assessed expression of another Tbx5-dependent gene, connexin40 (Cx40).29 Cx40 is normally expressed in the atrial and trabecular myocardium and is also expressed in the vasculature at later stages in development. As shown in Figure 4, Cx40 is expressed normally in both wild-type and knockout embryos at E12.5 but is expressed ectopically in the left ventricular compact myocardium of the CHF1/Hey2 knockout embryos beginning at E13.5. The punctate pattern seen in both the wild-type and knockout heart compact myocardium beginning at E14.5 reflects expression in the vasculature, as Cx40 is known to be expressed in both the myocardium and the vasculature at later stages of development.32 The myocardial expression pattern of Cx40 is similar to that of Tbx5 and ANF in the knockout embryos. These findings indicate that Tbx5 is a downstream target of CHF1/Hey2 and that CHF1/Hey2 normally suppresses Tbx5 expression in the left ventricular compact myocardium, resulting in suppression of ANF and Cx40.
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Atrial Contractile Markers mlc1a and mlc2a Are Also Expressed Ectopically in the Left Ventricular Compact Myocardium, Suggesting Perturbation in Ventricular Maturation
ANF, Tbx5, and Cx40 are normally expressed in the atria and trabeculae. Dysregulation of ANF, Tbx5, and Cx40 with ectopic expression in the left ventricular compact myocardium suggested that the left ventricular compact myocardial cells might have atrial or trabecular cell characteristics instead of or in addition to ventricular characteristics. To explore these possibilities, we examined the expression pattern of atrial, ventricular and trabecular markers. We first examined the expression of the ventricular markers mlc2v, ß-MHC, and N-myc, to determine whether ventricular cell identity was altered in the compact myocardium. As shown in supplemental Figure III, there is no difference in regional expression of these markers in the knockout embryos, demonstrating that the left ventricular compact myocardial cells have ventricular cell characteristics. Overall N-myc expression may be decreased in the knockout ventricles, however, which may contribute in part to decreased myocardial proliferation, as described below.
We next examined the expression of the atrial and trabecular markers mlc1a and mlc2a. Neither is known to be dependent on Tbx5. As shown in Figure 5, both mlc1a and mlc2a show ectopic expression in the left ventricular compact myocardium of the CHF1/Hey2 knockout embryos after E13.5, providing further evidence that ventricular maturation is dysregulated and that the left ventricular myocardium has characteristics not only of ventricular cells but also of atrial or trabecular cells.
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Because ANF, Tbx5, Cx40, mlc1a, and mlc2a are also expressed in the trabeculae, we considered the possibility that the cells ectopically expressing these genes may have trabecular rather than atrial characteristics. To assess this possibility, we examined expression of the trabecular marker BMP10. BMP10 is expressed predominantly in the trabecular myocardium, and mice lacking BMP10 demonstrate decreased trabeculation and a thin-walled myocardium associated with decreased myocyte proliferation.33 As shown in Figure 6, BMP10 is expressed normally in both wild-type and knockout embryos, suggesting that the knockout left ventricular compact myocardial cells are more atrial in character than trabecular. Overall, these findings suggest that the compact myocardial cells have both atrial and ventricular characteristics, which is reminiscent of undifferentiated cardiac myocytes. These findings also suggest a role for CHF1/Hey2 in promoting proper myocardial maturation beyond E13.5.
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A CHF1/Hey2 Transgene Expressed Diffusely in the Myocardium Suppresses Ectopic, but Not Normal, Expression of Atrial Genes
We have previously reported that an mlc2v-driven CHF1/Hey2 transgene expressed diffusely in all 4 cardiac chambers can rescue the myocardial and partially rescue the valvular and septation phenotypes in the knockout mice and improve survival.20 To assess whether this diffusely expressed transgene can suppress ectopic gene expression, we performed in situ hybridization on CHF1/Hey2 knockout embryos carrying this transgene at E12.5 and E15.5. As shown in Figure 7 and supplemental Figure IV, the transgene is expressed diffusely in all 4 chambers and in both the trabecular and compact myocardium of the ventricles. Knockout embryos carrying the transgene did not express ANF, Tbx5, Cx40, mlc1a, or mlc2a in the compact myocardium at E15.5, demonstrating that CHF1/Hey2 does indeed rescue gene expression patterns by suppressing ectopic expression. Interestingly, expression of the CHF1/Hey2 transgene in the trabecular myocardium and atria was not sufficient to suppress the normal expression of these genes in the atria and trabeculae, or suppress expression at E12.5. Our findings suggest that CHF1/Hey2 is likely a facilitator of normal ventricular maturation, but is not sufficient to suppress atrial gene expression and act as a determination gene.
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Myocyte Proliferation Is Decreased in CHF1/Hey2-Null Embryos
To investigate the mechanistic basis for the observed thin-walled myocardium, we hypothesized that the thin myocardium may result from either increased myocyte apoptosis or from decreased myocyte proliferation. We analyzed apoptosis at various developmental stages by both TUNEL and activated caspase 3 immunostaining and found no significant differences between wild-type and CHF1/Hey2 knockout embryos (data not shown). Our analysis of myocyte cell proliferation by immunostaining for phospho-histone H3, however, revealed a marked decrease in the number of proliferating cells at E13.5 in the left ventricular compact myocardium of the CHF1/Hey2 knockout embryos (Figure 8B and 8D). Expression of a myocardial CHF1/Hey2 transgene rescued the proliferation defect (Figure 8C and 8D). These findings suggest that absence of CHF1/Hey2 in the myocardium diminishes the capacity for cellular growth and are consistent with our previous findings that absence of CHF1/Hey2 in vascular smooth muscle inhibits proliferation16 and can lead to thin-walled arterial vessels.20
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Discussion |
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Regional myocardial gene expression patterns change drastically during cardiac development and are linked to the local differentiation of working myocardium.34–36 Under normal conditions, expression of atrial genes (ANF, Cx40, Tbx5, mlc1a, and mlc2a) decreases after E13.5 in the left ventricular compact myocardium. Suppression of atrial genes after E13.5 is likely to be important for proper differentiation of the left ventricular compact myocardium and, consequently, will be important for myocardial maturation and function.
We and others have previously reported that CHF1/Hey2 knockout mice have multiple developmental defects; however, the transcriptional mechanisms and effects on regional myocardial differentiation are poorly understood. Here we report that absence of CHF1/Hey2 leads to severe perturbation in regional myocardial gene expression, with dysregulation of Tbx5-dependent genes and ectopic expression of atrial genes in the compact myocardium. This "atrialization" of the compact myocardium may contribute to the development of the thin-walled myocardial phenotype. One possible mechanistic link would be that the increased expression of Tbx5 has been associated with decreased cellular proliferation and is postulated to provide a growth arrest signal.37 Accordingly, ectopic expression of Tbx5 may contribute to the decreased myocardial cell proliferation observed in our study. In addition, ectopic expression of the contractile genes mlc1a and mlc2a may contribute to the cardiomyopathy that has previously been noted in CHF1/Hey2 knockout mice.17,19 The link among CHF1/Hey2, Tbx5-dependent transcriptional programs, and ectopic atrial gene expression is a novel finding that may provide a general mechanism by which a thin-walled myocardium arises.
One curious aspect of our study is that the ectopic expression of Tbx5, ANF, and Cx40 is limited to the left ventricular myocardium, even though CHF1/Hey2 is expressed in the compact myocardium of both ventricles. These findings demonstrate that CHF1/Hey2 normally suppresses Tbx5 expression only in the left ventricle and that the left and right ventricular transcriptional programs are distinct, as has been noted in other studies.35,38 Another interesting aspect is that transgenic expression of CHF1/Hey2 diffusely in the myocardium, although sufficient to suppress ectopic atrial gene expression at later stages, is insufficient to suppress normal atrial gene expression in the atria and trabecula. CHF1/Hey2 is therefore a facilitator of myocardial maturation, rather than a determination gene for the ventricular myocardium.
A general theme in the biology of CHF1/Hey2 is that it functions as a regulator of cardiovascular cell responses to external stimuli, such as growth factors16 and hypertrophic agents,21 and has been shown to respond to cellular signaling.27 We have found that CHF1/Hey2 strongly affects regional myocardial gene expression and maturation. It is tempting to speculate that CHF1/Hey2 affects the response of the developing myocyte to developmental signaling cues, although the exact nature of this effect will be the subject of future study.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants HL076232 and HL081088 (to M.T.C.) and a Japan Heart Foundation fellowship (to N.K.).
Disclosures
None.
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Footnotes |
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Original received October 13, 2006; revision received January 17, 2007; accepted February 19, 2007.
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