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, 26 (12), 4462-73

The beta-catenin/T-cell Factor/Lymphocyte Enhancer Factor Signaling Pathway Is Required for Normal and Stress-Induced Cardiac Hypertrophy

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The beta-catenin/T-cell Factor/Lymphocyte Enhancer Factor Signaling Pathway Is Required for Normal and Stress-Induced Cardiac Hypertrophy

Xin Chen et al. Mol Cell Biol.

Abstract

In cells capable of entering the cell cycle, including cancer cells, beta-catenin has been termed a master switch, driving proliferation over differentiation. However, its role as a transcriptional activator in terminally differentiated cells is relatively unknown. Herein we utilize conditional, cardiac-specific deletion of the beta-catenin gene and cardiac-specific expression of a dominant inhibitory mutant of Lef-1 (Lef-1Delta20), one of the members of the T-cell factor/lymphocyte enhancer factor (Tcf/Lef) family of transcription factors that functions as a coactivator with beta-catenin, to demonstrate that beta-catenin/Tcf/Lef-dependent gene expression regulates both physiologic and pathological growth (hypertrophy) of the heart. Indeed, the profound nature of the growth impairment of the heart in the Lef-1Delta20 mouse, which leads to very early development of heart failure and premature death, suggests beta-catenin/Tcf/Lef targets are dominant regulators of cardiomyocyte growth. Thus, our studies, employing complementary models in vivo, implicate beta-catenin/Tcf/Lef signaling as an essential growth-regulatory pathway in terminally differentiated cells.

Figures

FIG. 1.
FIG. 1.
Deletion of β-catenin in tamoxifen-treated β-cateninfl/fl/Cre mice. Cardiomyocytes were isolated from hearts of β-cateninfl/fl/Cre mice that had been treated with tamoxifen or vehicle (−Tamoxifen) for 5 days and then were sacrificed 10 days (top panels) or 6 weeks (A, bottom panel, and B) later. Myocytes were immunostained (A) or immunoblotted (B) for expression of β-catenin. (A, top left panel) The arrow identifies residual β-catenin remaining in the adherens junction. (A, bottom panel) The arrows identify cardiomyocytes in which β-catenin has been deleted by Cre-mediated recombination. (B) GAPDH serves as a loading control.
FIG. 2.
FIG. 2.
Expression of β-catenin targets in the hearts of β-cateninfl/fl/Cre (β-catfl/fl/Cre) mice. Immunoblot for c-Myc (top panel) and c-Fos (middle panel) in lysates from hearts of β-cateninfl/fl/Cre mice treated with tamoxifen or vehicle (−Tamoxifen) and subjected to TAC versus sham TAC (−TAC). (Bottom panel) Quantification of c-Myc expression by densitometry, normalized to vinculin as a loading control. Data are from groups of 3 to 5 mice. *, P < 0.05.
FIG. 3.
FIG. 3.
Characterization of the hypertrophic response in hearts of mice deleted for β-catenin. A. Five-week-old female β-cateninfl/fl/Cre (β-catfl/fl/Cre) mice were injected with tamoxifen or vehicle (placebo), and β-cateninfl/fl/WT mice were injected with tamoxifen. Six weeks later, mice underwent echocardiographic examination (top panel, baseline) and then were subjected to TAC or sham TAC. Two weeks later, echocardiography was performed (top panel, TAC), and then animals were sacrificed for determination of morphometric measures (bottom panel). LV mass is normalized to tibial length. B. Relationship between LVSP and LV mass/tibial length (LVM/TL) in β-cateninfl/fl/Cre mice, treated with tamoxifen or placebo, and subjected to TAC or sham TAC. Mice underwent the protocol described for panel A. Prior to sacrifice, they underwent invasive hemodynamic assessment with a micromanometer-tipped catheter followed by determination of the ratio of LV mass to tibial length (LVM/TL). Correlation coefficients for the two groups (r = 0.76 and 0.92) were significant at P < 0.001. The slope of the regression line for β-cateninfl/fl/Cre mice treated with tamoxifen, identified with an arrow, is significantly less than that for the β-cateninfl/fl/Cre mice treated with placebo. n = 8 for each TAC group, and n = 5 for each sham group. C. Direct determination of cardiomyocyte width. Cardiomyocytes were isolated from β-cateninfl/fl/Cre mice treated with tamoxifen or placebo (Tamoxifen−) or from WT/Cre mice (Cre) treated with tamoxifen that had been subjected to TAC or sham TAC. Cardiomyocyte width was determined after staining for β-catenin. *, P < 0.05; #, P < 0.01. n = 3 to 4 in TAC groups, and n = 2 in sham groups. A minimum of 100 cardiomyocytes was measured per animal. Statistical significance was determined based on comparing the mean value of myocyte width for each animal, not total myocytes (i.e., n = 3 to 4). D. Analysis of TAC-induced gene expression by RT-PCR. Expression of β-MHC, α-skeletal actin, and ANF in hearts of β-cateninfl/fl/Cre mice treated with tamoxifen or placebo and subjected to TAC or sham TAC. n = 3 for TAC groups, and n = 1 to 2 for sham groups.
FIG.4.
FIG.4.
Characterization of the Lef-1Δ20 transgenic line. A. Expression of HA-Lef-1Δ20. Anti-HA immunoblot of lysates from transgenic and wild-type hearts demonstrating expression of the Lef-1Δ20 transgene. B. Neonatal hearts expressing Lef-1Δ20 have reduced cardiac growth. Shown are hearts from a 5-day-old Lef-1Δ20 transgenic mouse (TG) from line 1 and a wild-type (WT) littermate. Note the foreshortened long axis of the ventricle in the TG. C. Cardiomyocyte size is reduced in the Lef-1Δ20 transgenic neonate. Sections of hearts from 5-day-old Lef-1Δ20 transgenic and wild-type mice were stained with FITC-conjugated wheat germ agglutinin to delineate the cardiomyocyte sarcolemma. The transgenic sample is notable for significantly decreased cross-sectional areas of the cardiomyocytes. Cardiomyocyte cross-sectional area is quantified in the lower panel. D. Expression of Lef-1Δ20 and Tcf4 by RT-PCR in TG and WT hearts. E. M-mode echocardiograms of 5-week-old wild-type and Lef-1Δ20 transgenic hearts, in which the transgene has inserted into the X chromosone, showing markedly decreased posterior wall (PW) and anterior wall (AW) thicknesses as well as a dilated ventricle (as evidenced by an increased end-diastolic dimension [EDD]) and reduced contractile function (as evidenced by an increased end-systolic dimension [ESD]). F. Hematoxylin- and eosin (H and E)-stained sagittal section of an X chromosome-inserted Lef-1Δ20 transgenic heart. Note the markedly reduced wall thicknesses and the dilated ventricle. G. Cardiomyocyte size in the Lef-1Δ20 transgenic. H- and E-stained sections of the heart of a mouse from the X chromosome-inserted Lef-1Δ20 transgenic line. Notable are two vastly different populations of cardiomyocytes, one characterized by extremely small myocytes and the other by normal-sized myocytes. Also note the mosaic pattern with areas of hypotrophic myocytes adjacent to areas of normal-sized myocytes. H. Cardiomyocyte size in Lef-1Δ20 transgenic mice. Myocytes isolated from the X-inserted Lef-1Δ20 transgenic mouse versus wild-type littermates were stained with anti-α-actinin antibody. Again, two populations are readily apparent in the transgenic. The lower right panel is a DAPI stain of the cells shown in the upper right panel, identifying nuclei. This demonstrates that the smaller myocyte is not a fragment of a larger normal myocyte. These grossly hypotrophic myocytes were never seen in the wild-type mice. I. Hypotrophic cardiomyocytes express Lef-1Δ20. Cardiomyocytes were stained with anti-Lef-1 antibody (upper panels) or DAPI (lower panels). Arrows identify nuclei of cells expressing Lef-1Δ20. All cells staining positive for Lef-1Δ20 expression were markedly hypotrophic. J. Quantification of cardiomyocyte width and length in the Lef-1Δ20-expressing cardiomyocytes versus those not expressing Lef-1Δ20. Also shown are values for myocytes isolated from wild-type mice. *, P < 0.01 versus cells not expressing Lef-1Δ20 and versus wild type. #, P < 0.05 versus the wild type.
FIG.4.
FIG.4.
Characterization of the Lef-1Δ20 transgenic line. A. Expression of HA-Lef-1Δ20. Anti-HA immunoblot of lysates from transgenic and wild-type hearts demonstrating expression of the Lef-1Δ20 transgene. B. Neonatal hearts expressing Lef-1Δ20 have reduced cardiac growth. Shown are hearts from a 5-day-old Lef-1Δ20 transgenic mouse (TG) from line 1 and a wild-type (WT) littermate. Note the foreshortened long axis of the ventricle in the TG. C. Cardiomyocyte size is reduced in the Lef-1Δ20 transgenic neonate. Sections of hearts from 5-day-old Lef-1Δ20 transgenic and wild-type mice were stained with FITC-conjugated wheat germ agglutinin to delineate the cardiomyocyte sarcolemma. The transgenic sample is notable for significantly decreased cross-sectional areas of the cardiomyocytes. Cardiomyocyte cross-sectional area is quantified in the lower panel. D. Expression of Lef-1Δ20 and Tcf4 by RT-PCR in TG and WT hearts. E. M-mode echocardiograms of 5-week-old wild-type and Lef-1Δ20 transgenic hearts, in which the transgene has inserted into the X chromosone, showing markedly decreased posterior wall (PW) and anterior wall (AW) thicknesses as well as a dilated ventricle (as evidenced by an increased end-diastolic dimension [EDD]) and reduced contractile function (as evidenced by an increased end-systolic dimension [ESD]). F. Hematoxylin- and eosin (H and E)-stained sagittal section of an X chromosome-inserted Lef-1Δ20 transgenic heart. Note the markedly reduced wall thicknesses and the dilated ventricle. G. Cardiomyocyte size in the Lef-1Δ20 transgenic. H- and E-stained sections of the heart of a mouse from the X chromosome-inserted Lef-1Δ20 transgenic line. Notable are two vastly different populations of cardiomyocytes, one characterized by extremely small myocytes and the other by normal-sized myocytes. Also note the mosaic pattern with areas of hypotrophic myocytes adjacent to areas of normal-sized myocytes. H. Cardiomyocyte size in Lef-1Δ20 transgenic mice. Myocytes isolated from the X-inserted Lef-1Δ20 transgenic mouse versus wild-type littermates were stained with anti-α-actinin antibody. Again, two populations are readily apparent in the transgenic. The lower right panel is a DAPI stain of the cells shown in the upper right panel, identifying nuclei. This demonstrates that the smaller myocyte is not a fragment of a larger normal myocyte. These grossly hypotrophic myocytes were never seen in the wild-type mice. I. Hypotrophic cardiomyocytes express Lef-1Δ20. Cardiomyocytes were stained with anti-Lef-1 antibody (upper panels) or DAPI (lower panels). Arrows identify nuclei of cells expressing Lef-1Δ20. All cells staining positive for Lef-1Δ20 expression were markedly hypotrophic. J. Quantification of cardiomyocyte width and length in the Lef-1Δ20-expressing cardiomyocytes versus those not expressing Lef-1Δ20. Also shown are values for myocytes isolated from wild-type mice. *, P < 0.01 versus cells not expressing Lef-1Δ20 and versus wild type. #, P < 0.05 versus the wild type.

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