TEA domain transcription factor 1(TEAD1) induces cardiac fibroblasts cells remodeling through BRD4/Wnt4 pathway

Abstract

Cardiac fibroblasts (CFs) are the primary cells tasked with depositing and remodeling collagen and significantly associated with heart failure (HF). TEAD1 has been shown to be essential for heart development and homeostasis. However, fibroblast endogenous TEAD1 in cardiac remodeling remains incompletely understood. Transcriptomic analyses revealed consistently upregulated cardiac TEAD1 expression in mice 4 weeks after transverse aortic constriction (TAC) and Ang-II infusion. Further investigation revealed that CFs were the primary cell type expressing elevated TEAD1 levels in response to pressure overload. Conditional TEAD1 knockout was achieved by crossing TEAD1-floxed mice with CFs- and myofibroblasts-specific Cre mice. Echocardiographic and histological analyses demonstrated that CFs- and myofibroblasts-specific TEAD1 deficiency and treatment with TEAD1 inhibitor, VT103, ameliorated TAC-induced cardiac remodeling. Mechanistically, RNA-seq and ChIP-seq analysis identified Wnt4 as a novel TEAD1 target. TEAD1 has been shown to promote the fibroblast-to-myofibroblast transition through the Wnt signalling pathway, and genetic Wnt4 knockdown inhibited the pro-transformation phenotype in CFs with TEAD1 overexpression. Furthermore, co-immunoprecipitation combined with mass spectrometry, chromatin immunoprecipitation, and luciferase assays demonstrated interaction between TEAD1 and BET protein BRD4, leading to the binding and activation of the Wnt4 promoter. In conclusion, TEAD1 is an essential regulator of the pro-fibrotic CFs phenotype associated with pathological cardiac remodeling via the BRD4/Wnt4 signalling pathway.

Fig. 1

TEAD1 expression is increased in human and mouse remodeling hearts. a Schematic illustration of the RNA-seq analysis strategy. the blue dots represent DEGs with foldchange < 1/1.5, adjust P value < 0.05, the red dots represent DEGs with foldchange >1.5, adjust P value < 0.05. Grey dots represent gene that the expression change had no significant difference. The number of genes upregulated in TAC and Ang-II samples were indicated in the Venn diagram. b GO term enrichment analysis of 118 genes upregulated in both TAC and Ang-II samples (foldchange >1.5, adjust P value < 0.05). c Heatmap of 32 genes upregulated in both TAC and Ang-II samples (foldchange >2, adjust P value < 0.05). d Quantitative real time polymerase chain reaction (qRT-PCR) analyses of TEAD1 mRNA levels in heart samples from WT mice at 4 weeks after Sham or TAC (n = 4 for Sham and n = 8 for TAC per group). e Western blot and quantification of TEAD1 and α‑SMA protein levels in heart samples from WT mice at 4 weeks after Sham or TAC (n = 4 per group). f qRT-PCR analyses of TEAD1 mRNA levels in heart samples from WT mice at 4 weeks after Saline or Ang-II infusion (n = 4 for Saline and n = 8 for Ang-II per group). g Western blot and quantification of TEAD1 and α‑SMA protein levels in heart samples from WT mice at 4 weeks after Saline or Ang-II infusion (n = 4 per group). h Western blot of TEAD1 in heart samples from non-HCM and HCM patients. For all statistical plots, the data are presented as mean ± SD i Western blot and quantification of TEAD1, α‑SMA and Vimentin protein levels in isolated CFs at 4 weeks after TAC or Sham (n = 4 per group). d-g and i by two-tailed unpaired Student’s t-test. TAC, transverse aortic constriction; α-SMA, α-smooth muscle actin

Fig. 2

Knockout of TEAD1 in cardiac fibroblasts attenuates TAC-induced cardiac remodeling. a Schematic for echocardiography and sample collection from 4 groups: TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ at 4 weeks after sham or TAC. b Left ventricular EF assessed by echocardiography in TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ mice after 4 weeks sham or TAC (n = 4–10 per group). c Heart sections from TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ mice after sham or TAC surgery were stained with picrosirius red to visualize collagen deposition (n = 4–10 mice per group; scale bar=50 μm). d. Heart sections from TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ mice after sham or TAC surgery were stained with WGA to demarcate the cell boundaries (n = 4-10 mice per group; scale bar=20 μm). e Heart sections from TEAD1^fl/fl and TEAD1^fl/flcol1a2+ mice after sham or TAC surgery were stained with hematoxylin and eosin to show whole-heart gross images (n = 4–10 mice per group; scale bar=5 mm). f Representative immunofluorescence images of Collagen I and Collagen III staining in the hearts from TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ mice after sham or TAC surgery (n = 4 per group; scale bar=20 μm). g Western blot and quantification of α‑SMA and Galectin-3 protein levels in the heart homogenates extracted from TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ mice after sham or TAC surgery (n = 4 per group). h qRT-PCR analyses of the mRNA levels of ANP, BNP and β-MHC in heart samples from TEAD1^fl/fl and TEAD1^fl/flcol1a2⁺ mice after sham or TAC surgery (n = 4 per group). For all statistical plots, the data are presented as mean ± SD. ns. indicates no significance between the 2 indicated groups. b–d and g by two-way ANOVA with Bonferroni multiple comparison test. h by two-way ANOVA with Dunnett’s T3 post hoc analysis. TAM, tamoxifen; EF, ejection fraction; WGA, wheat germ agglutinin; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain

Fig. 3

Myofibroblast-specific TEAD1 deficiency attenuates TAC-induced cardiac remodeling. a Schematic for echocardiography and sample collection from 4 groups: TEAD1^fl/fl and TEAD1^fl/flpostn⁺ 4 weeks after 4 weeks sham or TAC. b Left ventricular EF assessed by echocardiography of TEAD1^fl/fl and TEAD1^fl/flpostn⁺ mice after 4 weeks sham or TAC (n = 4-10 per group). c Heart sections from TEAD1^fl/fl and TEAD1^fl/flpostn⁺ mice after sham or TAC surgery were stained with picrosirius red to visualize collagen deposition (n = 4-10 mice per group; scale bar=50 μm). d Heart sections from TEAD1^fl/fl and TEAD1^fl/flpostn⁺ mice after sham or TAC surgery were stained with WGA to demarcate the cell boundaries (n = 4-10 mice per group; scale bar=20 μm). e Heart sections from TEAD1^fl/fl and TEAD1^fl/flpostn+ mice after sham or TAC surgery were stained with hematoxylin and eosin to show whole-heart gross images (n = 4-10 mice per group; scale bar=5 mm). f Representative immunofluorescence images of Collagen I and Collagen III staining in the hearts from TEAD1^fl/fl and TEAD1^fl/flpostn⁺ mice after sham or TAC surgery (n = 4 per group; scale bar=20 μm). g Western blot and quantification of α‑SMA and Galectin3 protein levels in the heart homogenates extracted from TEAD1^fl/fl and TEAD1^fl/flpostn⁺ mice after sham or TAC surgery (n = 4 per group). h. qRT-PCR analyses of the mRNA levels of ANP, BNP and β-MHC in heart samples from TEAD1^fl/fl and TEAD1^fl/flpostn⁺ mice after sham or TAC surgery (n = 4 per group). For all statistical plots, the data are presented as mean ± SD. ns. indicates no significance between the 2 indicated groups. b–d and g by two-way ANOVA with Bonferroni multiple comparison test. h by two-way ANOVA with Dunnett’s T3 post hoc analysis. TAM tamoxifen; EF ejection fraction; WGA wheat germ agglutinin; ANP atrial natriuretic peptide; BNP brain natriuretic peptide; β-MHC β-myosin heavy chain

Fig. 4

TEAD1 regulates Ang-II-induced fibroblast-to-myofibroblast transition in vitro. a qRT-PCR analyses of the mRNA levels of TEAD1 in CFs infected with adenovirus expressing TEAD1 or NC (n = 3 per group). b Western blot and quantification of TEAD1 protein levels in CFs infected with indicated adenovirus (n = 3 per group). c. Western blot and quantification of TEAD1, Collagen I and α‑SMA protein levels in CFs transfected with si-NC or si-TEAD1 and then treated with saline or Ang-II for 48 h (n = 3 per group) or infected with indicated adenovirus (n = 3 per group). d Representative images of immunofluorescence staining and quantification of α‑SMA in CFs transfected with si-NC or si-TEAD1 and then treated with saline or Ang-II for 48 h (n = 3 per group, scale bar=100 μm). e. Representative images of immunofluorescence staining against α-SMA and quantification of the CFs infected with indicated adenovirus (n = 3 per group, scale bar=100 μm). f Collagen gel contraction seeded CFs transfected with si-NC or si-TEAD1 and then treated with saline or Ang-II for 48 h (n = 3 per group). g Collagen gel contraction seeded CFs infected with indicated adenovirus (n = 3 per group). h. Migration of CFs transfected with si-NC or si-TEAD1 and then treated with saline or Ang-II (n = 3 per group; scale bar=100 μm). i. Migration of CFs infected with indicated adenovirus (n = 3 per group; scale bar=100 μm). For all statistical plots, the data are presented as mean ± SD. ns. indicates no significance between the 2 indicated groups. a by Welch’s t-test. b, c, e, g, and i by two-tailed unpaired Student’s t-test; c, d, f and h by two-way ANOVA with Bonferroni multiple comparison test

Fig. 5

TEAD1 regulates CFs differentiation via the bromodomain-containing protein 4 (BRD4)/Wnt4 signalling pathway. a Western blot and quantification of Wnt4 protein levels in CFs transfected with si-NC or si-TEAD1 and then treated with saline or Ang-II for 48 h (n = 4 per group); Western blot and quantification of Wnt4 protein levels in CFs infected with adenovirus expressing TEAD1 or NC (n = 4 per group). b Western blot and quantification of TEAD1 and β-catenin protein levels in cytoplasmic and nuclear fractions extracted from CFs transfected with si-NC or si-TEAD1 and then treated with saline or Ang-II (n = 4 per group); Western blot and quantification of TEAD1 and β-catenin protein levels in cytoplasmic and nuclear fractions extracted from CFs infected with adenovirus expressing TEAD1 or NC (n = 4 per group). c Western blot of TEAD1, Wnt4, α‑SMA and Galectin-3 protein levels in CFs infected with adenovirus expressing TEAD1 and transfected with si-Wnt4. d TEAD1 binding tracks at Wnt4 gene loci in CFs based on CHIP-seq dataset. e ChIP-qPCR analysis using a TEAD1-specific antibody to detect TEAD1 binding to the Wnt4 promoter in CFs (n = 4 per group). f–g Endogenous immunoprecipitation of TEAD1 and BRD4 in the presence (f) or absence (g) of JQ1. h Luciferase activity in HEK293T transfected with WT or mutating Wnt4 luciferase reporter plasmids (n = 4 per group). i. ChIP-qPCR analysis using an BRD4-specific antibody to detect BRD4 binding to the Wnt4 promoter in CFs (n = 4 per group). j. Luciferase activity in CFs transfected with a Wnt4 luciferase reporter plasmid, along with adenovirus expressing TEAD1 in the presence or absence of JQ1 (1 μM) (n = 4 per group). k Luciferase activity in CFs transfected with an Wnt4 luciferase reporter plasmid, along with adenovirus expressing indicated proteins (n = 4 per group). l Western blot of TEAD1, BRD4, Wnt4, α‑SMA and Galectin-3 protein levels in CFs infected with adenovirus expressing TEAD1 in the presence or absence of JQ1. For all statistical plots, the data are presented as mean ± SD. ns. indicates no significance between the 2 indicated groups. a–b by two-way ANOVA with Bonferroni multiple comparison test. e and i by two-tailed unpaired Student’s t-test. j–k by two-way ANOVA with Dunnett’s T3 multiple comparisons test. h by one-way ANOVA with Dunnett’s T3 multiple comparisons test. ChIP, chromatin immunoprecipitation

Fig. 6

VT103 attenuates TAC-induced cardiac remodeling by inhibiting TEAD1. a Schematic for echocardiography and sample collection from 4 groups: WT mice were subjected to sham or TAC subsequently treated with vehicle or VT103 (20 mg/kg/day) every 2 days via intraperitoneal injection for 28 days (n = 4–10 per group). b Representative echo image of M-mode after 4 weeks TAC or sham. c Left ventricular EF and FS assessed by echocardiography in WT mice after 4 weeks sham or TAC subsequently treated with vehicle or VT103 (n = 4–10 per group). d The ratios of HW to BW and HW to TL in WT mice after 4 weeks sham or TAC subsequently treated with vehicle or VT103 (n = 4–10 per group). e–g Heart sections were stained with hematoxylin and eosin, WGA or picrosirius red from WT mice subjected to sham or TAC surgery subsequently treated with vehicle or VT103 (n = 4–10 per group; for hematoxylin and eosin staining, scale bar=5 mm; for WGA staining, scale bar=20 μm; for picrosirius red staining, scale bar = 50 μm). h Western blot and quantification of α‑SMA and Galectin-3 protein levels in the heart homogenates extracted from WT mice after sham or TAC surgery treated with vehicle or VT103 (n = 4 per group). i qRT-PCR analyses of the mRNA levels of ANP, BNP and β-MHC in heart homogenates extracted from WT mice after sham or TAC surgery treated with vehicle or VT103 (n = 4 per group). For all statistical plots, the data are presented as mean ± SD. ns. indicates no significance between the 2 indicated groups. c–d and f–h by two-way ANOVA with Bonferroni multiple comparison test. i by two-way ANOVA with Dunnett’s T3 post hoc analysis

Fig. 7

TEAD1 promotes the fibroblast-to-myofibroblast transition during TAC or Ang-II-induced pathological cardiac remodelling through the Wnt signalling pathway. TAC or Ang II stimulation induces TEAD1 expression, which binds to the promoter of Wnt4 to promote its expression through interaction with BRD4. The overexpressed Wnt4 enhances the nuclear translocation of β-catenin, thus activating the canonical Wnt signalling pathway to promote the fibroblast-to-myofibroblast transition. This figure was drawn by using pictures from Servier Medical Art (https://smart.servier.com/). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)