This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/9/1353    most recent 01.RES.0000266605.63681.5av1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baurand, A. Articles by Bergmann, M. W. Search for Related Content PubMed PubMed Citation Articles by Baurand, A. Articles by Bergmann, M. W. Related Collections Contractile function Biochemistry and metabolism Other myocardial biology Other heart failure Apoptosis Physiological and pathological control of gene expression

(Circulation Research. 2007;100:1353.)
© 2007 American Heart Association, Inc.

Integrative Physiology

ß-Catenin Downregulation Is Required for Adaptive Cardiac Remodeling

Anthony Baurand^*, Laura Zelarayan^*, Russell Betney, Christina Gehrke, Sandra Dunger, Claudia Noack, Andreas Busjahn, Joerg Huelsken, Makoto Mark Taketo, Walter Birchmeier, Rainer Dietz, Martin W. Bergmann

From the Max Delbrück Center for Molecular Medicine (A. Baurand, L.Z., C.N., W.B., M.W.B.), Berlin, Germany; Department of Cardiology (R.B., C.G., S.D., R.D., M.W.B.), Campus Buch and Campus Virchow–Klinikum, Charité–Universitätsmedizin Berlin, Franz Volhard Klinik, HELIOS Klinikum Berlin–Buch, Germany; HealthTwiSt GmbH (A. Busjahn), Berlin, Germany; Institut Suisse de Recherche Expérimentale sur le Cancer (J.H.), Epalinges, Switzerland; and Department of Pharmacology (M.M.T.), Graduate School of Medicine, Kyoto University, Japan.

Correspondence to Martin W. Bergmann, MD, Franz Volhard Clinic, Wiltbergstrasse 50, 13125 Berlin, Germany. E-mail martin.bergmann{at}charite.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The armadillo-related protein ß-catenin has multiple functions in cardiac tissue homeostasis: stabilization of ß-catenin has been implicated in adult cardiac hypertrophy, and downregulation initiates heart formation in embryogenesis. The protein is also part of the cadherin/catenin complex at the cell membrane, where depletion might result in disturbed cell–cell interaction similar to N-cadherin knockout models. Here, we analyzed the in vivo role of ß-catenin in adult cardiac hypertrophy initiated by angiotensin II (Ang II). The cardiac-specific mifepristone-inducible MHC-CrePR1 transgene was used to induce ß-catenin depletion (loxP-flanked exons 3 to 6, ß-cat^ex3–6 mice) or stabilization (loxP-flanked exon 3, ß-cat^ex3 mice). Levels of ß-catenin were altered both in membrane and nuclear extracts. Analysis of the ß-catenin target genes Axin2 and Tcf-4 confirmed increased ß-catenin–dependent transcription in ß-catenin stabilized mice. In both models, transgenic mice were viable and healthy at age 6 months. ß-Catenin appeared dispensable for cell membrane function. Ang II infusion induced cardiac hypertrophy both in wild-type mice and in mice with ß-catenin depletion. In contrast, mice with stabilized ß-catenin had decreased cross-sectional area at baseline and an abrogated hypertrophic response to Ang II infusion. Stabilizing ß-catenin led to impaired fractional shortening compared with control littermates after Ang II stimulation. This functional deterioration was associated with altered expression of the T-box proteins Tbx5 and Tbx20 at baseline and after Ang II stimulation. In addition, atrophy-related protein IGFBP5 was upregulated in ß-catenin–stabilized mice. These data suggest that ß-catenin downregulation is required for adaptive cardiac hypertrophy.

Key Words: transcription factor • signaling • ß-catenin • hypertrophy • development

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ß-catenin is a plakophilin protein family member. The plakophilins belong to the armadillo-related proteins, which are essential components of the desmosomal plaque. In addition to adhesive function, the plakophilin ß-catenin has been ascribed an important signaling function. For instance, ß-catenin is a transcriptional coactivator of the T-cell factor/lymphoid enhancer factor (TCF/LEF) complex, which regulates embryonic, postnatal, and oncogenic growth in many tissues, including the heart.¹

Cardiomyocyte growth occurs during left ventricular (LV) remodeling following chronic pressure overload and/or ischemic heart disease. Increased ß-catenin levels were detected in the intercalated disc in heart specimens from patients with inherited cardiac hypertrophy. The ß-catenin increase at the cell membrane was accompanied by reduced nuclear ß-catenin levels.² Recently, reduced ß-catenin–dependent transcription via Tcf/Lef1 transcription factors was suggested to be the molecular mechanism behind the fibroadipocytic replacement in arrhythmogenic right ventricular cardiomyopathy caused by the loss of desmoplakin.³

LV remodeling includes reactivation of the fetal gene program and possibly the embryonic gene program as well.⁴ WNT/ß-catenin regulation is among the earliest events in cardiac development. Specifically, ß-catenin downregulation following inhibitory Dickkopf signaling in the embryonic endoderm precedes cardiac development from the embryonic mesoderm.⁵ Because universal ß-catenin deletion is lethal, inducible tissue-specific modulation of ß-catenin has been used to analyze the role of this factor in adult heart LV remodeling.

Two recent publications have analyzed the effect of ß-catenin depletion in the adult heart. Zhou et al⁶ found no phenotype concerning membrane function attributable to the compensatory upregulation of plakoglobin in the intercalated disc as previously described in other tissues.⁷ Furthermore, Chen et al confirmed their previous in vitro observations concerning ß-catenin in adult cardiac hypertrophy: the increase in heart weight/body weight after transaortic constriction was attenuated in ß-catenin–depleted transgenic mice. Here, we extended these observations by generating 2 complementary mouse models to understand the role of ß-catenin in adult cardiac remodeling in vivo on G protein–coupled receptor activation. One model exhibits conditional cardiomyocyte ß-catenin depletion (ß-cat^ex3–6), whereas the other exhibits ß-catenin stabilization (ß-cat^ex3). In contrast to earlier studies, these data suggest that ß-catenin downregulation is required for adaptive cardiac remodeling preserving LV systolic function under increased stress.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Mice
Mice with cardiac-restricted ß-catenin depletion (ß-cat^ex3–6) (Figure 1) as well as enhanced ß-catenin expression (ß-cat^ex3) (Figure 2) were generated through mating of MHC-CrePR1 mice with either ß-catenin^{ex3–6,flox/flox} or ß-catenin^{ex3,flox/flox}; each individual strain has been described previously.^6,8,9 Whereas excision of exons 3 to 6 (ß-cat^ex3–6) generates a functional inactive ß-catenin mutant, excision of exon 3 results in a nondegradable mutant of ß-catenin, thereby increasing cellular ß-catenin levels (ß-cat^ex3). Mice were bred in a mixed C57BL/6+FVB background (ß-cat^ex3–6) or FVB background (ß-cat^ex3).

View larger version (21K): [in this window] [in a new window]   Figure 1. Generation of mice with inducible, heart-specific ß-catenin depletion (ß-catex3–6). A, Representative mating scheme between mice carrying ß-catenin floxed exons 3 to 6 and MHC-CrePR1 mice. Mice have been described previously. For heart-specific, progesterone-inducible Cre recombinase expression MHC-CrePR1 mice were used. In ß-catex3–6,flox/floxxMHC-CrePR1, administration of mifepristone leads to loxP site recombination with the appearance of a 600-bp band. Hybridization sites of the primers are indicated (red arrows). B, Western blot (WB) from whole heart lysate of both control and ß-catex3–6 mice (upper left). A significant reduction of ß-catenin protein expression in ß-catex3–6 is detected by a C-terminal binding antibody. A second Western blot (lower left) using an N-terminal binding antibody indicates diminished expression of ß-catenin in all cellular compartments (cytosol, nucleus, and membrane). ß-Actin was used as control for cytosol and nucleus and E-cadherin for membrane. Also at the mRNA level, ß-catenin quantification by real-time PCR normalized to GAPDH revealed decreased expression of full-length ß-catenin cDNA (right). C, ß-Catenin and N-cadherin immunofluorescence (IF) (green) of heart sections of control (CT) or ß-catex3–6 mice. Nuclei were stained with DAPI (blue). ß-Catenin staining was clearly reduced in the mutants, whereas N-cadherin was not affected. The right panels show an enlargement of an area containing a gap junction, which shows the apparent depletion of ß-catenin expression. Scale bar=20 µm.

View larger version (14K): [in this window] [in a new window]   Figure 2. Generation of mice with inducible, heart-specific stabilization of ß-catenin (ß-catex3). A, The scheme depicts the mating between MHC-CrePR1 mice and ß-catex3,flox/flox, which, on recombination, lack the glycogen synthase kinase 3ß phosphorylation site, resulting in organ-specific stabilization of ß-catenin. Genomic PCR analysis shows the truncated ß-catenin product (700 bp) following specific recombination in the heart of in ß-catex3 mice; hybridization sites of the primers are indicated (red arrows). B, Western blot depicting expression of the truncated form of ß-catenin protein in ß-catex3 mice in different cellular compartments (cytosol, nucleus, and membrane). C, ß-Catenin target genes Axin2, Tcf-4, and Lef-1 are upregulated in ß-catex3 total heart RNA extracts, as measured by real-time PCR. Fold expression is relative to GAPDH calculated as copy numbers.

In MHC-CrePR1 mice, heart-specific expression of the Cre fusion protein is activated by intraperitoneal administration of the estrogen derivate mifepristone (RU486, 30 µg per gram of body weight; Sigma-Aldrich) once every day for 5 days in 3-month-old mice.⁸ Throughout the study, littermates negative for either Cre or loxP sites were used as controls (for primers, see the online data supplement, available at http://circres.ahajournals.org). The Berlin Animal Review Board approved the study (regulation no. 0135/01).

Induction of Cardiac Hypertrophy and Echocardiography
Ten days after Cre induction, microosmotic pumps (model 1002, Alzet) releasing angiotensin II (Ang II) (Sigma; 1.4 µg·kg^–1·min^–1, solvent 0.9% NaCl/0.01N acetic acid) were implanted subcutaneously into age-matched mice after anesthesia with ketamine/xylazine (30 mg/10 mg·kg^–1 IP) as described.¹⁰ Hypertrophy was assessed by echocardiography with either an Accuson Sequoia (Siemens) instrument equipped with a 15-MHz microprobe (ß-cat^ex3–6) or a VisualSonics Vevo 770 High-Resolution Imaging System equipped with a 30-MHz RMV-707B scanning head (ß-cat^ex3). For echocardiography, mice were anesthetized by isoflurane inhalation (2% isoflurane). Ventricular measurements in M mode were taken before implantation and 12 days after with 3 readings per mouse. The observer was unaware of the genotypes and treatments.

RNA Quantification, Reporter Assays, Protein Analyses, and Immunohistochemistry
Total RNA was isolated from mouse heart tissue using the RNeasy kit (Qiagen). cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen). cDNA was subjected to real-time PCR by SYBR Green Analysis (Qiagen) performed on an iCycler instrument (Bio-Rad). All real-time PCR sample reactions were performed in triplicate and normalized to GAPDH mRNA expression (for primers, see Table II in the online data supplement). A standard curve was run with dilution series of the amplified fragment allowing for mRNA copy number calculations.

Protein was isolated from heart tissue using a mortar and pestle with liquid nitrogen and then with a compartmental protein extraction kit (Chemicon). The method isolates proteins specifically from nuclear, cytoplasmic, and membrane compartments. Antibodies for the ß-catenin C terminus (BD Bioscience), ß-catenin N terminus (nanoTools), Tbx5 (Abcam), N-cadherin (1:100, Zymed), and GAPDH (Advanced ImmunoChemicals) were used. Horse radish peroxidase–conjugated secondary antibodies were from DAKO. For wheat germ agglutin/5'-fluorescein isothiocyanate (WGA-FITC) staining, heart sections were deparaffinized and stained overnight with 100 microL of lectin WGA-FITC (20 mg/mL) per slide. The cell area was calculated by measuring at least 300 cells per animal using ImageJ software. Detection of apoptosis was performed by indirect TUNEL with the ApopTag Fluorescein in situ apoptosis detection kit (Chemicon) according to the instructions of the manufacturer. The specimens were costained with antibody to cardiac troponin T (Santa Cruz Biotechnology) for visualization of cardiomyocytes.

Statistical Analysis
Differences between experimental groups were analyzed using SPSS 14.0. Within and between effects for repeated measures were analyzed by general linear models with post hoc paired t tests. Data are reported as mean±SEM. P<0.05 values were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ß-catenin Heart-Specific Mouse Models
We used an inducible Cre/loxP system restricting ß-catenin depletion to -myosin heavy chain (MHC)-positive cells after hormone induction. Recombination efficiency was tested through mating of MHC-CrePR1 mice to ROSA 26 reporter mice expressing the lacZ expression cassette under the control of a floxed stop codon.⁸ Recombination efficiency after mifepristone injection was determined to be approximately 70% of cardiomyocytes as described before (see supplemental Figure I).⁸ Induction of Cre recombinase excised exons 3 to 6 in hearts from ß-cat^{ex3–6,flox/flox}xMHC-CrePR1, resulting in animals that had a heart-specific ß-catenin depletion (ß-cat^{ex 3–6}, Figure 1A). Successful heart-specific recombination was confirmed by PCR using primers for the recombined DNA that result in a 600-bp fragment (Figure 1A). The downregulation of ß-catenin was detected both at the mRNA and protein level: active Cre recombinase led to dramatically reduced ß-catenin mRNA and protein levels in total heart extracts from several mice (Figure 1B). Biochemical methods showed depletion of ß-catenin in all cellular compartments including the nucleus. Using antibodies against both the N and C terminus, expression of a truncated ß-catenin isoform was excluded as described before (Figure 1B).⁶ In tissue, ß-catenin was readily detectable in gap junctions between cardiomyocytes in control hearts but not in sections from ß-catenin heart-depleted mice. Staining of N-cadherin complexing with ß-catenin at the cell junction showed no apparent phenotype in ß-cat^ex3–6 mice with this method (Figure 1C). Electrocardiograms showed no rhythm disturbances in the mice (data not shown).

Deletion of exon 3 renders ß-catenin to be resistant against glycogen synthase kinase 3ß–induced phosphorylation and consequently blocks proteasome-mediated degradation. Mifepristone administration to ß-cat^{ex3,flox/flox} mice mated to MHC-CrePR1 mice generated ß-cat^ex3 animals (Figure 2A).⁹ Heart-specific Cre recombinase activation excised exon 3 and resulted in mice with heart-restricted, stabilized ß-catenin (Figure 2A). PCR using primers designed to give a 700-bp fragment after successful recombination in contrast to 900 bp beforehand confirmed the genotype (Figure 2A). Stabilized ß-catenin was detectable in cytosolic, membrane, and nuclear fractions of whole heart lysates as a smaller band than endogenous ß-catenin (Figure 2B). Staining with N-cadherin, which is complexing with ß-catenin at the cell junction, did not reveal an apparent phenotype at the membrane compared with control mice (data not shown). Next, genes regulated transcriptionally by ß-catenin were analyzed by real-time PCR with extracts from ß-cat^ex3 mutants compared with control littermates¹: Axin2 (7.03±0.7-fold, P<0.001), Tcf-4 (2.92±0.16-fold, P<0.05), and Lef-1 (2.91±0.4-fold, P<0.05) expression was significantly increased (Figure 2C). These data prove a functional increase in ß-catenin–dependent transcription in ß-cat^ex3 mice as described before concerning other tissues.^9,11

ß-Catenin Depletion Leads to Mild Cardiac Hypertrophy
Fourteen days after the final mifepristone injection, the cardiac transverse ventricular cross-sections appeared thicker in ß-catenin–depleted mice compared with wild-type mice (Figure 3A). Cardiac hypertrophy was confirmed by cross sectional area analysis in vivo: the average cell size from ß-catenin depleted mice (9.4x10⁷±0.14x10⁷ arbitrary units) increased significantly over control cell size (7.9x10⁷±0.15x10⁷ arbitrary units; P<0.001) (Figure 3A). Echocardiographic examination demonstrated increased diameters of the diastolic interventricular septum as well as the LV posterior wall (Figure 3B). No significant changes were seen regarding cavity size or fractional shortening. A summary of all echocardiographic measurements can be found in supplemental Table I. RNA quantification revealed increased expression of hypertrophy gene markers atrial (ANP) and brain (BNP) natriuretic peptides, ßMHC, and -sarcomeric actin (Figure 3C). In summary, depletion of ß-catenin leads to moderate cardiac hypertrophy in the adult heart. This initial phenotype did not exaggerate and was not accompanied by a loss of cardiac function or any other cardiac deterioration up to the age of 6 months (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 3. Baseline characterization reveals modest hypertrophy in ß-catex3–6 and diminished growth in ß-catex3. A, Transverse sections (upper panels) of control (CT), ß-catex3–6, and ß-catex3 hearts. Cross-sectional area analysis by WGA-FITC staining demonstrates modest cardiac hypertrophy in mice with cardiac-specific ß-catenin depletion (ß-catex3–6) as well as diminished growth after ß-catenin stabilization (ß-catex3); quantification is shown (bottom); n=8 to 11 mice per group. B, Echocardiographic diastolic diameters of interventricular septum (IVSd) and LV posterior wall (LVPWd) demonstrate cardiac hypertrophy after ß-catenin depletion. No significant change in wall dimensions after ß-catenin stabilization was observed (n15 for each group). C, RNA quantification by real-time RT-PCR analysis of hypertrophic markers after ß-catenin depletion and stabilization. *P<0.05, **P<0.01; expression fold is relative to GAPDH control.

ß-Catenin Stabilization Leads to Impaired Cardiac Growth
Deletion of the endogenous exon 3 results in a single-copy gene of stabilized ß-catenin under the control of the endogenous reporter. In contrast to our initial hypothesis based on previously published in vitro and in vivo data,^12,13 ß-catenin stabilization (ß-cat^ex3) did not cause hypertrophy (Figure 3). In fact, when cross-sectional area was calculated 14 days after Cre recombinase induction by mifepristone, a significantly decreased cell size was observed (7.8x10⁷±1.1x10⁶ [control] versus 6.4x10⁷±1.4x10⁶ [ß-cat^ex3], P<0.001; Figure 3A). Cells from stabilized ß-catenin mice were approximately 82% the size of control cells. Hypertrophy gene marker expression was the inverse of the ß-catenin–depletion experiment. Hypertrophic markers were either stable (ßMHC) or downregulated in the ß-cat^ex3 samples compared with controls (Figure 3C). Over 6 months, the animals developed and behaved normally. Taken together, the above data suggest that depleting ß-catenin in the adult heart results in adaptive cardiac hypertrophy, whereas expression of a nondegradable mutant results in a phenotype of smaller cardiomyocytes indicating impaired cardiac growth or cardiac muscular atrophy.

Responses to Ang II in the ß-Catenin Mouse Models
After 12 days of Ang II infusion at pressure doses (1.4 µg·kg^–1·min^–1), control animals developed hypertrophy, as determined by echocardiography, gene regulation, in vivo cross sectional area, and heart/body weight ratio as described before (Figure 4).¹⁰ From the M-mode echocardiography imaging, a thickened interventricular septum and LV posterior wall after Ang II was identified in control animals (Figure 4A and supplemental Table I). Even if at baseline ß-catenin–depleted mice exhibited an increase of the cardiomyocyte area (Figure 3A), treatment with Ang II further enhanced cellular cross-sectional area (12.1x10⁷±0.24x10⁷ arbitrary units) to absolute values similar to control mice (12.3x10⁷±0.19x10⁷ arbitrary units) (Figure 4B). LV wall thickening was identical in ß-catenin heart-depleted animals and control littermates after Ang II (Figure 4A). Heart weights normalized to body weight showed a significant increase for both control and ß-catenin–depletion mice (Figure 4A and supplemental Table I). Moreover, Ang II increased gene expression of several hypertrophy gene markers in both control and ß-catenin heart-depleted animals, with the exception of ßMHC (Figure 4C). A lack of ßMHC upregulation in response to stress stimuli might be indicative of adaptive cardiac remodeling preserving LV function, as described before.¹⁴

View larger version (35K): [in this window] [in a new window]   Figure 4. Ang II stimulation induces hypertrophy in both control (CT) and ß-catenin–depleted mice. A, Both control and ß-catenin–depleted ß-catex3–6 mice exhibit cardiac hypertrophy after 14 days of microosmotic minipump infusion of Ang II (1.4 µg·kg–1·min–1). Representative examples of M-mode echocardiograms, quantification of echocardiographic parameters, and heart/body weight from control and ß-catex3–6 mice with and without Ang II infusion are shown (n6 for each group). B, Myocyte cross-sectional area increases in both control and ß-catenin–depleted ß-catex3–6 mice, as measured by WGA-FITC staining of cardiac tissue sections and quantification from control or ß-catex3–6 mice with Ang II infusion. More than 500 cells were measured for each animal; n4 mice per group. Scale bar=20 µm. C, Significant upregulation of ANP, BNP, and -sarcomeric actin normalized to GAPDH after Ang II in control and ß-catenin–depleted ß-catex3–6 mice. Quantification by real-time PCR of whole heart RNA samples from control (white) or ß-catex3–6 (gray) mice without (open bars) and with (shaded bars) Ang II infusion. *P<0.05, **P<0.01, ***P<0.001.

The effects of 12 days of Ang II infusion in mice with cardiac stabilized ß-catenin (ß-cat^ex3) were opposite in kind to those of mice with ß-catenin heart depletion. Echocardiography showed no significant change in the wall thickness following Ang II treatment in ß-catenin–stabilized mice in contrast to control littermates, in which wall thickness increased significantly (Figure 5A and also see supplemental Table I). ANP (ß-cat^ex3, 0.4±0.15; ß-cat^ex3+Ang II, 1.03±0.15; P<0.01) and BNP (ß-cat^ex3, 0.43±0.24; ß-cat^ex3+Ang II, 1.4±0.27; P<0.01) expression was induced by Ang II, but final values were generally lower than in control mice (control: ANP, Ang II, 2.3±0.8; BNP, Ang II, 2.6±0.6). In vivo, cardiomyocyte size following Ang II treatment significantly increased over baseline in both models but was significantly smaller in ß-cat^ex3 compared with control animals treated with Ang II (9.07x10⁷±0.14x10⁷ [control] versus 8.14x10⁷±0.14[x10⁷ [ß-cat^ex3], P<0.001; Figure 5B).

View larger version (34K): [in this window] [in a new window]   Figure 5. ß-Catenin stabilization attenuates Ang II–induced cardiac hypertrophy but leads to decreased cardiac function. A, Attenuation of cardiac hypertrophy after Ang II in ß-catenin–stabilized mice is accompanied by significant deterioration of systolic function, as measured by fractional shortening (right bar graph). Representative examples of M-mode echocardiograms and quantification from control (CT) or ß-catex3 mice with and without Ang II infusion. Quantification of echocardiographic wall size and fractional shortening from control or ß-catex3 mice with and without Ang II infusion. *P<0.05, **P<0.01; n3. B, Increase of myocyte cross-sectional area after Ang II is attenuated in ß-catenin–stabilized ß-catex3 mice. More than 400 cells counted per animal; n3 mice per group. ***P<0.001. C, Rates of TUNEL/-sarcomeric actin/DAPI-positive cardiomyocytes after Ang II infusion decrease after ß-catenin stabilization in ß-catex3. In addition to the TUNEL assay (red fluorescence), slides were costained with -sarcomeric actin (green fluorescence) and DAPI (blue stain) in control mice and ß-catex3 mice before and after Ang II stimulation.

As expected, fractional shortening increased in control mice after Ang II treatment (31.1±1.6% to 36.2±1.6%). Instead, ß-catenin heart stabilization led to reduced fractional shortening after Ang II infusion (34.7±2.9% to 22.4±4.5%, P<0.05; Figure 5A and supplemental Table I). These absolute values in animals aged 10 to 14 weeks are identical to a serial analysis of the age-dependent changes using the same high-resolution echo techniques as used for these studies (see also supplemental Figure II).¹⁵ The reduced fractional shortening in ß-catenin–stabilized mice was not caused by increased cardiomyocyte apoptosis as ß-catenin–stabilized mice demonstrated rather fewer TUNEL/-sarcomeric actin/2',6'-diamidino-2-phenylindole (DAPI)-positive cells after Ang II treatment compared with controls (ß-cat^ex3, 0.058±0.027% at baseline to 0.120±0.049% after Ang II; P<0.001; compared with control+Ang II, 0.297±0.025%; Figure 5C). In contrast, analysis of mice with ß-catenin depletion showed a nonsignificant increase of TUNEL-positive cells at baseline with similar results after Ang II treatment (control, 0.004±0.004; ß-cat^{ex 3–6}, 0.112±0.044%; after Ang II: control, 0.09±0.02%; ß-cat^ex3–6, 0.2±0.13%). These absolute values are similar to other reports, ie, rates of apoptotic cardiomyocytes after transaortic constriction.¹⁶ Moreover, no significant differences concerning fibrosis occurring focally throughout the LV wall was observed when comparing both transgenic models to their respective controls. In summary, Ang II–induced hypertrophy is not affected by ß-catenin depletion, whereas mice with stabilized ß-catenin exhibit an impaired hypertrophic response to Ang II. Apoptosis-related mechanisms do not explain these findings.

Analysis of Cardiac ß-Catenin Target Genes
Next, we analyzed several target genes previously described to be regulated in a ß-catenin–dependent fashion either in embryonic development (GATA-4, Nkx2.5, MEF2a, Tbx5, and Tbx20) or have been described in the context of chronic phosphatidylinositol 3-kinase (PI3K)/AKT signaling (atrogin 1, insulin-like growth factor–binding protein 5 [IGFBP5]).¹⁷ Analysis was performed in whole heart RNA extracts. GATA-4, Nkx2.5, MEF2a, and atrogin 1 expression was not significantly altered by Ang II treatment or MHC-dependent changes in ß-catenin levels. Axin2 levels were increased by Ang II in both control and ß-catenin–stabilized mice, although the baseline levels were already significantly increased in ß-cat^ex3 mice (Figures 6A and 2C).

View larger version (21K): [in this window] [in a new window]   Figure 6. Gene regulation in ß-catex3 mice. RNA normalized to GAPDH levels in whole heart extracts as measured by real-time PCR in ß-catenin–stabilized mice (ß-catex3). A, Upregulation of ß-catenin target gene Axin2 by Ang II treatment in ß-catex3 compared with control (CT) animals. B, Upregulation of insulin-like growth factor binding protein 5 (IGFBP5) was found by RT-PCR at baseline in the ß-catex3 mice in comparison to controls, whereas no significant change was observed by Ang II stimulation. C, T-box proteins 5 (Tbx5) and 20 (Tbx20) mRNA levels are differentially regulated in ß-catex3 mice on Ang II stimulation. D, Tbx5 Western blot showing the downregulation of its expression in control mice after Ang II treatment (top) as well as in ß-catex3 mice at baseline (bottom). Western blot quantification was performed by densitometry normalized to GAPDH (right).

Interestingly, ß-catenin levels affected gene expression of IGFBP5, a protein inhibitory for the IGF-signaling cascade. IGFBP5 was found upregulated in hearts with genetically mediated chronic AKT activation.¹⁷ Functionally, IGFBP5 is implicated in muscle atrophy. Here, IGFBP5 was upregulated in ß-cat^ex3 mice compared with control littermates (9.02±3.2-fold). No significant change of expression levels were observed on Ang II stimulation.

At baseline, ß-catenin stabilization led to reduced Tbx5 mRNA and protein levels, whereas Tbx20 mRNA was found to be upregulated (Figure 6C and 6D; Tbx20 versus control: 3.3±0.31-fold; P<0.05). Ang II treatment significantly downregulated Tbx5 gene expression in control mice; this effect was lost in the ß-catenin–stabilized mice (Figure 6C). No significant effect of Ang II on Tbx20 gene expression was observed (Figure 6C). These results suggest differential regulation of T-box proteins downstream of ß-catenin in association with a phenotype of impaired cardiac growth and performance.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here we studied the role of ß-catenin in adult heart homeostasis and LV remodeling after chronic G protein–coupled receptor stimulation. We found that decreased ß-catenin expression caused modest cardiac hypertrophy at baseline, compared with controls. Ang II infusion induced cardiac hypertrophy both in control and in ß-catenin–depleted mice but not in ß-catenin–stabilized mice. This decreased hypertrophic response was accompanied by reduced fractional shortening. The phenotype of deteriorating heart function on ß-catenin stabilization was associated with altered regulation of the T-box proteins Tbx5 and Tbx20 as well as the AKT-regulated, atrophy-related protein IGF-binding protein 5 (IGFBP5). We conclude that ß-catenin downregulation is required for adaptive cardiac hypertrophy preserving LV function under chronic Ang II stimulation.

The ß-Catenin Mouse Models
The 2 complementary mouse models analyzed here were generated using previously described ß-catenin floxed transgenic mice. Depletion or stabilization of ß-catenin both at the membrane as well as concerning its nuclear function has been achieved in various tissues before.^6,9,11 No apparent phenotype linked to the membrane function of ß-catenin in complex with N-cadherin was observed. The compensatory upregulation of plakoglobin (-catenin) demonstrated by Zhou et al in heart tissue might account for this phenomenon.⁷ This hypothesis is also supported by data from other tissues using this very same floxed mouse strain.^6,7

Similar considerations concerning the relevance of membrane-associated ß-catenin versus nuclear ß-catenin apply to the mouse model with ß-catenin stabilization: whereas no apparent phenotype concerning rhythm disturbances or membrane dysfunction was observed, the classic ß-catenin target genes Axin2, Tcf-4, and Lef-1 were found upregulated in cardiac tissue extracts. The nuclear localization of the stabilized truncated ß-catenin mutant in ß-cat^{exon3,flox/flox} also accounts for other phenotypes including the recently described block in hematopoietic stem cell differentiation.^9,11

ß-Catenin and Cardiac Hypertrophy
We were surprised to find that by stabilizing ß-catenin in the adult heart, the hypertrophic response after Ang II stimulation was abrogated, although at the expense of deteriorating heart function. This observation contradicts earlier findings, where ß-catenin depletion blocked cardiac hypertrophy after transaortic constriction (TAC).^12,13 Several differences between the studies may explain the discrepant findings. The Ang II stimulus used here activates signaling cascades different from pressure overload as in TAC. Discrepant results concerning the same signaling molecule have been described before, ie, concerning the role of MEKK1 in cardiac hypertrophy.^18,19 In ß-catenin–depleted mice, Chen et al describe an increase in heart weight and upregulation of hypertrophy markers ANP and ßMHC when compared with baseline levels, indicating intact hypertrophy in ß-catenin–depleted mice. The definition of hypertrophy was based on heart weight and cardiomyocyte width of isolated cardiomyocytes; neither diastolic wall size nor cell surface area in tissue sections was reported.¹³ Intriguingly, concerning the point of adaptive versus maladaptive cardiac remodeling, ß-catenin–depleted mice preserved LV function, despite ongoing stress by TAC, supporting a concept of adaptive hypertrophy mediated by ß-catenin downregulation.

Another major difference between the studies is the time course of the experiments: here, mice were stimulated with Ang II 10 days after mifepristone-induced Cre recombinase activation and analyzed 2 weeks later; Chen et al exposed their mice to TAC 6 weeks after induction of Cre recombinase and analyzed them 2 weeks later. The initial delay after ß-catenin depletion might have induced secondary effects.¹³

Our complimentary studies using mice with ß-catenin stabilization seemingly confirmed a beneficial role of ß-catenin downregulation in cardiac hypertrophy: after Ang II stimulation, the ß-catenin-stabilized mice exhibited a deterioration of cardiac function. To the best of our knowledge, in vivo studies describing the phenotype of mice with genetic stabilization of ß-catenin have not been described previously.

ß-Catenin and LV Remodeling
First, we tested whether cardiac ß-catenin manipulation might affect cardiomyocyte apoptosis. Whereas we observed an increase of TUNEL/-sarcomeric actin/DAPI-positive nuclei after Ang II treatment in control and ß-catenin–depleted mice to levels reported before, this was not observed in the mice with ß-catenin stabilization.¹⁶ Although determination of absolute values of apoptosis would require the combination of several techniques, these data clearly exclude increased apoptosis to account for the observed phenotype. This observation is in line with previous findings: several studies analyzing tissue-specific alterations of ß-catenin levels found that ß-catenin regulated expression of survival genes in brain, thymocytes, and vascular smooth muscle cells.²⁰ We previously described the association of stabilized ß-catenin and cell survival of isolated rat cardiomyocytes after activation of the PI3K/AKT pathway.²¹

Next, we analyzed previously described target genes of ß-catenin and PI3K/AKT. The PI3K/AKT pathway was previously shown to control ß-catenin levels in cardiomyocytes.¹² As ß-catenin downregulation initiates heart formation in the embryo, the gene expression levels of different transcription factors related to cardiomyocyte differentiation were investigated. Interestingly, differential regulation of the T-box proteins Tbx5 and Tbx20 was found: Ang II treatment downregulated Tbx5 mRNA and protein expression in control mice, whereas this effect was attenuated in ß-cat^ex3 mice. Tbx20 was upregulated in mice with ß-catenin stabilization. Differential roles have been identified for these T-box proteins: Tbx5 was previously shown to promote cardiomyocyte differentiation in association with Nkx2.5.²² In addition, heterozygote Tbx 5 deletion is a model for the Holt–Oran syndrome; interestingly, by 8 weeks after birth, such mice develop a phenotype of diastolic dysfunction known to precede systolic dysfunction.¹⁵ In contrast, Tbx20 is known to exert multiple transcriptional repressive functions including inhibition of ANP expression.^23,24 Increased expression of Tbx20 might therefore inhibit cardiac growth in ß-cat^ex3 mice.

In addition, regulation of the IGFBP5 was observed. In general, IGFBP5 inhibits cell proliferation and differentiation but increases cell survival rates.²⁵ Upregulation of IGFBP5 has been detected in mouse models with chronic activation of AKT.²⁶ Prolonged activation of this pathway was recently found to induce a phenotype of dilated cardiomyopathy.^27,28 The known functions of IGFBP5 and its expression levels observed here make this protein another candidate for explaining the effect of ß-catenin stabilization on cardiac LV remodeling.

In summary, we found that downregulation of ß-catenin initiates adaptive cardiomyocyte hypertrophy in the adult heart necessary for preservation of LV function under chronic stress. Stabilizing ß-catenin in the adult heart blocks signaling pathways required for protective hypertrophy in the presence of chronic Ang II-induced stress. This effect was not associated with detectable changes in intercellular adhesion but with altered gene expression concerning Tbx5 and Tbx20, which belong to the cardiac embryonic gene program. This finding suggests that on hypertrophic stress, the adult heart reverts to regulation of ß-catenin in a fashion similar to ß-catenin involvement in embryonic cardiac development.

	Acknowledgments

 
We thank Baerbel Pohl and Martin Taube for expert technical assistance; Friedrich C. Luft and Jeff Molkentin for critical reading of the manuscript and helpful comments; and Michael D. Schneider for providing the MHC-CrePR1 mice and for critical reading of the manuscript.

Sources of Funding

This study was supported by Marie Curie Host Development funding (to M.W.B.), a grant-in-aid by the Deutsche Forschungsgemeinschaft, and a grant from The Jürgen Manchot Foundation (to M.W.B.).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this work.

Original received September 12, 2006; resubmission received January 3, 2007; revised resubmission received March 23, 2007; accepted March 27, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Brembeck FH, Rosario M, Birchmeier W. Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr Opin Genet Dev. 2006; 16: 51–59.[CrossRef][Medline] [Order article via Infotrieve]

2. Masuelli L, Bei R, Sacchetti P, Scappaticci I, Francalanci P, Albonici L, Coletti A, Palumbo C, Minieri M, Fiaccavento R, Carotenuto F, Fantini C, Carosella L, Modesti A, Di Nardo P. Beta-catenin accumulates in intercalated disks of hypertrophic cardiomyopathic hearts. Cardiovasc Res. 2003; 60: 376–387.[Abstract/Free Full Text]

3. Garcia-Gras E, Lombardi R, Giocondo MJ, Willerson JT, Schneider MD, Khoury DS, Marian AJ. Suppression of canonical Wnt/beta-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy. J Clin Invest. 2006; 116: 2012–2021.[CrossRef][Medline] [Order article via Infotrieve]

4. Liang Q, Molkentin JD. Divergent signaling pathways converge on GATA4 to regulate cardiac hypertrophic gene expression. J Mol Cell Cardiol. 2002; 34: 611–616.[CrossRef][Medline] [Order article via Infotrieve]

5. Lickert H, Kutsch S, Kanzler B, Tamai Y, Taketo MM, Kemler R. Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev Cell. 2002; 3: 171–181.[CrossRef][Medline] [Order article via Infotrieve]

6. Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell. 2001; 105: 533–545.[CrossRef][Medline] [Order article via Infotrieve]

7. Zhou J, Qu J, Yi XP, Graber K, Huber L, Wang X, Gerdes AM, Li F. Upregulation of gamma-catenin compensates for the loss of beta-catenin in adult cardiomyocytes. Am J Physiol Heart Circ Physiol. 2007; 292: H270–H276.[Abstract/Free Full Text]

8. Minamino T, Gaussin V, DeMayo FJ, Schneider MD. Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein. Circ Res. 2001; 88: 587–592.[Abstract/Free Full Text]

9. Harada N, Tamai Y, Ishikawa T-o, Sauer B, Takaku K, Oshima M, Taketo MM. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J. 1999; 18: 5931–5942.[CrossRef][Medline] [Order article via Infotrieve]

10. Freund C, Schmidt-Ullrich R, Baurand A, Dunger S, Schneider W, Loser P, El-Jamali A, Dietz R, Scheidereit C, Bergmann MW. Requirement of nuclear factor-kappaB in angiotensin II- and isoproterenol-induced cardiac hypertrophy in vivo. Circulation. 2005; 111: 2319–2325.[Abstract/Free Full Text]

11. Scheller M, Huelsken J, Rosenbauer F, Taketo MM, Birchmeier W, Tenen DG, Leutz A. Hematopoietic stem cell and multilineage defects generated by constitutive ß-catenin activation. Nat Immunol. 2006; 7: 1037–1047.[CrossRef][Medline] [Order article via Infotrieve]

12. Haq S, Michael A, Andreucci M, Bhattacharya K, Dotto P, Walters B, Woodgett J, Kilter H, Force T. Stabilization of beta-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth. Proc Natl Acad Sci U S A. 2003; 100: 4610–4615.[Abstract/Free Full Text]

13. Chen X, Shevtsov SP, Hsich E, Cui L, Haq S, Aronovitz M, Kerkela R, Molkentin JD, Liao R, Salomon RN, Patten R, Force T. The {beta}-catenin/T-cell factor/lymphocyte enhancer factor signaling pathway is required for normal and stress-induced cardiac hypertrophy. Mol Cell Biol. 2006; 26: 4462–4473.[Abstract/Free Full Text]

14. van Rooij E, Doevendans PA, Crijns HJGM, Heeneman S, Lips DJ, van Bilsen M, Williams RS, Olson EN, Bassel-Duby R, Rothermel BA, De Windt LJ. MCIP1 overexpression suppresses left ventricular remodeling and sustains cardiac function after myocardial infarction. Circ Res. 2004; 94: e18–e26.[CrossRef][Medline] [Order article via Infotrieve]

15. Zhou Y-Q, Zhu Y, Bishop J, Davidson L, Henkelman RM, Bruneau BG, Foster FS. Abnormal cardiac inflow patterns during postnatal development in a mouse model of Holt-Oram syndrome. Am J Physiol Heart Circ Physiol. 2005; 289: H992–H1001.[Abstract/Free Full Text]

16. van Empel VP, Bertrand AT, van der Nagel R, Kostin S, Doevendans PA, Crijns HJ, de Wit E, Sluiter W, Ackerman SL, De Windt LJ. Downregulation of apoptosis-inducing factor in harlequin mutant mice sensitizes the myocardium to oxidative stress-related cell death and pressure overload-induced decompensation. Circ Res. 2005; 96: e92–e101.[Abstract/Free Full Text]

17. Skurk C, Izumiya Y, Maatz H, Razeghi P, Shiojima I, Sandri M, Sato K, Zeng L, Schiekofer S, Pimentel D, Lecker S, Taegtmeyer H, Goldberg AL, Walsh K. The FOXO3a transcription factor regulates cardiac myocyte size downstream of AKT signaling. J Biol Chem. 2005; 280: 20814–20823.[Abstract/Free Full Text]

18. Minamino T, Yujiri T, Terada N, Taffet GE, Michael LH, Johnson GL, Schneider MD. MEKK1 is essential for cardiac hypertrophy and dysfunction induced by Gq. Proc Natl Acad Sci U S A. 2002; 99: 3866–3871.[Abstract/Free Full Text]

19. Sadoshima J, Montagne O, Wang Q, Yang G, Warden J, Liu J, Takagi G, Karoor V, Hong C, Johnson GL, Vatner DE, Vatner SF. The MEKK1-JNK pathway plays a protective role in pressure overload but does not mediate cardiac hypertrophy. J Clin Invest. 2002; 110: 271–279.[CrossRef][Medline] [Order article via Infotrieve]

20. Wang X, Xiao Y, Mou Y, Zhao Y, Blankesteijn WM, Hall JL. A Role for the {beta}-catenin/T-cell factor signaling cascade in vascular remodeling. Circ Res. 2002; 90: 340–347.[Abstract/Free Full Text]

21. Bergmann MW, Rechner C, Freund C, Baurand A, El Jamali A, Dietz R. Statins inhibit reoxygenation-induced cardiomyocyte apoptosis: role for glycogen synthase kinase 3ß and transcription factor ß-catenin. J Mol Cell Cardiol. 2004; 37: 681–690.[CrossRef][Medline] [Order article via Infotrieve]

22. Hiroi Y, Kudoh S, Monzen K, Ikeda Y, Yazaki Y, Nagai R, Komuro I. Tbx5 associates with Nkx2–5 and synergistically promotes cardiomyocyte differentiation. Nat Genet. 2001; 28: 276–280.[CrossRef][Medline] [Order article via Infotrieve]

23. Stennard FA, Costa MW, Lai D, Biben C, Furtado MB, Solloway MJ, McCulley DJ, Leimena C, Preis JI, Dunwoodie SL, Elliott DE, Prall OWJ, Black BL, Fatkin D, Harvey RP. Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development. 2005; 132: 2451–2462.[Abstract/Free Full Text]

24. Plageman TF Jr, Yutzey KE. Differential expression and function of Tbx5 and Tbx20 in cardiac development. J Biol Chem. 2004; 279: 19026–19034.[Abstract/Free Full Text]

25. Schneider MR, Wolf E, Hoeflich A, Lahm H. IGF-binding protein-5: flexible player in the IGF system and effector on its own. J Endocrinol. 2002; 172: 423–440.[Abstract]

26. Matsui T, Li L, Wu JC, Cook SA, Nagoshi T, Picard MH, Liao R, Rosenzweig A. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem. 2002; 277: 22896–22901.[Abstract/Free Full Text]

27. Shiojima I, Sato K, Izumiya Y, Schiekofer S, Ito M, Liao R, Colucci WS, Walsh K. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J Clin Invest. 2005; 115: 2108–2118.[CrossRef][Medline] [Order article via Infotrieve]

28. Nagoshi T, Matsui T, Aoyama T, Leri A, Anversa P, Li L, Ogawa W, del Monte F, Gwathmey JK, Grazette L, Hemmings B, Kass DA, Champion HC, Rosenzweig A. PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury. J Clin Invest. 2005; 115: 2128–2138.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				L. Zelarayan, A. Renger, C. Noack, M.-P. Zafiriou, C. Gehrke, R. van der Nagel, R. Dietz, L. de Windt, and M. W. Bergmann NF-{kappa}B activation is required for adaptive cardiac hypertrophy Cardiovasc Res, December 1, 2009; 84(3): 416 - 424. [Abstract] [Full Text] [PDF]

				L. C. Zelarayan, C. Noack, B. Sekkali, J. Kmecova, C. Gehrke, A. Renger, M.-P. Zafiriou, R. van der Nagel, R. Dietz, L. J. de Windt, et al. {beta}-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation PNAS, December 16, 2008; 105(50): 19762 - 19767. [Abstract] [Full Text] [PDF]

				T. Grigoryan, P. Wend, A. Klaus, and W. Birchmeier Deciphering the function of canonical Wnt signals in development and disease: conditional loss- and gain-of-function mutations of {beta}-catenin in mice Genes & Dev., September 1, 2008; 22(17): 2308 - 2341. [Abstract] [Full Text] [PDF]

				A. E. Zemljic-Harpf, J. C. Miller, S. A. Henderson, A. T. Wright, A. M. Manso, L. Elsherif, N. D. Dalton, A. K. Thor, G. A. Perkins, A. D. McCulloch, et al. Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy Mol. Cell. Biol., November 1, 2007; 27(21): 7522 - 7537. [Abstract] [Full Text] [PDF]

				T. Force, K. Woulfe, W. J. Koch, and R. Kerkela Molecular Scaffolds Regulate Bidirectional Crosstalk Between Wnt and Classical Seven-Transmembrane Domain Receptor Signaling Pathways Sci. Signal., July 31, 2007; 2007(397): pe41 - pe41. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/9/1353    most recent 01.RES.0000266605.63681.5av1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baurand, A. Articles by Bergmann, M. W. Search for Related Content PubMed PubMed Citation Articles by Baurand, A. Articles by Bergmann, M. W. Related Collections Contractile function Biochemistry and metabolism Other myocardial biology Other heart failure Apoptosis Physiological and pathological control of gene expression