TEAD1 expression is elevated in response to pathological cardiac remodeling in humans and mice

To identify potential key regulators associated with cardiac remodeling, we performed RNA-sequencing (RNA-seq) analyses in myocardial samples from mice subjected to TAC or Ang-II infusion for 4 weeks (Fig. 1a). Overall, 423 and 909 genes were upregulated (false discovery rate q-value < 0.05; fold change > 1.5) in the TAC samples and Ang-II samples compared with the control samples, respectively (Fig. 1a). Of these, 118 genes were simultaneously upregulated in the TAC and Ang-II models (Fig. 1a). Gene Ontology (GO) analysis (false discovery rate q-value < 0.05) of 118 genes suggested that they were involved in ECM organisation, angiogenesis, and other cardiac remodeling process (Fig. 1b). Further analysis with fold change > 2 undergoing hierarchical clustering revealed 32 genes with elevated expression in both TAC and Ang-II models, of which we found that TEAD1 was particularly significantly elevated (Fig. 1c). To confirm the RNA-seq results, TEAD1 expression was further assessed in wild-type (WT) mice after TAC surgery or Ang-II infusion. Consistently, myocardial TEAD1 mRNA and protein levels increased 4 weeks post TAC and Ang-II infusion (Fig. 1d–g). Next, we determined whether these changes in TEAD1 expression also occurred in hypertrophic cardiomyopathy (HCM) patients. As expected, the expression of TEAD1 was upregulated in the HCM (Fig. 1h and Supplementary Fig. 1a). Altogether, these results suggest that the transcription factor TEAD1 may be associated with the development of pathological cardiac remodeling.

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

Next, we examined the potential cardiac cell populations responsible for elevated TEAD1 expression in response to cardiac remodeling. CMs and CFs were isolated from the hearts of adult mice subjected to TAC, and TEAD1 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Low TEAD1 expression was detected in both cardiac cell populations from the sham-operated mice. There was a marked elevation observed in both the mRNA and protein expression of TEAD1 within CFs after TAC (Fig. 1i). In contrast, TAC had no significant effect on the protein levels of TEAD1 in CMs (Supplementary Fig. 1b). Immunofluorescence staining also showed that TEAD1 was upregulated in tissue sections after TAC (Supplementary Fig. 1c). These results revealed that CFs-derived TEAD1 may play a key role in cardiac remodeling.

TEAD1 knockout in CFs ameliorates TAC- and Ang-II-induced cardiac remodeling and dysfunction

Next, we explored the in vivo effects of TEAD1 knockout in mice. Knockout of TEAD1 in CFs (TEAD1fl/flcol1a2+) was generated by crossing TEAD1-floxed (TEAD1fl/fl) mice with col1a2-cre/ERT mice, thus allowing tamoxifen (TAM)-inducible deletion of TEAD1 in CFs (Supplementary Fig. 2a). The genotype of TEAD1fl/fl and col1a2+ mice was determined through PCR analysis (Supplementary Fig. 2b). TAM administation for 5 days successfully reduced the expression of TEAD1 in CFs from TEAD1fl/flcol1a2+ mice, while TEAD1 expression remained normal in CFs from the control TEAD1fl/fl mice not expressing Cre recombinase (Supplementary Fig. 2c, d). This decrease was specific to CFs (Supplementary Fig. 2e, f). After continuous administration of TAM for 5 days, TEAD1fl/flcol1a2+ and TEAD1fl/fl mice had 7 days to acclimate the effects of TAM and then were performed TAC for 4 weeks (Fig. 2a). Although no remarkable changes were observed at the basal level, the TEAD1fl/flcol1a2+ mice exhibited significantly improved ejection fraction (EF) values after TAC compared with the TEAD1fl/fl mice (Fig. 2b). Specific cardiac ultrasound parameters were shown in Supplementary Table 3. Sirius red and immunofluorescence staining showed that TAC-induced fibrosis was significantly attenuated in the TEAD1fl/flcol1a2+ mice compared with the TEAD1fl/fl mice (Fig. 2c, f). Wheat germ agglutinin (WGA) staining revealed a significantly reduced CMs cross-sectional area (CSA) in the TEAD1fl/flcol1a2+ mice compared with that in the TEAD1fl/fl mice (Fig. 2d). Whole-heart gross images also showed that TEAD1 knockout in CFs reduced hypertrophy induced by TAC (Fig. 2e). Further investigation revealed the reduced levels of cardiac fibrotic markers α‑SMA and galectin-3 in the TEAD1fl/flcol1a2+ mice after TAC compared with the TEAD1fl/fl mice (Fig. 2g). The atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) expression was also dramatically inhibited in the TEAD1fl/flcol1a2+ mice (Fig. 2h).

Fig. 2

Knockout of TEAD1 in cardiac fibroblasts attenuates TAC-induced cardiac remodeling. a Schematic for echocardiography and sample collection from 4 groups: TEAD1fl/fl and TEAD1fl/flcol1a2+ at 4 weeks after sham or TAC. b Left ventricular EF assessed by echocardiography in TEAD1fl/fl and TEAD1fl/flcol1a2+ mice after 4 weeks sham or TAC (n = 4–10 per group). c Heart sections from TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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

We next investigated whether TEAD1 deficiency could elicit similar cardioprotective effects in response to neurohumoral cardiac stress. After continuous administration of TAM for 5 days, TEAD1fl/flcol1a2+ and TEAD1fl/fl mice had 7 days to acclimate the effects of TAM and then were infused with Ang-II for 4 weeks via an implantable minipump (Supplementary Fig. 3a). Echocardiography revealed no significant differences between the EF values of the TEAD1fl/flcol1a2+ and TEAD1fl/fl mice following Ang-II infusion (Supplementary Fig. 3b). Specific cardiac ultrasound parameters were shown in Supplementary Table 4. Furthermore, consistent with what was observed in response to TAC, the TEAD1fl/flcol1a2+ mice exhibited a lower cardiac fibrosis and CSA after Ang-II infusion (Supplementary Fig. 3c, d). Additionally, the Ang-II-induced increase in cardiac fibrotic markers α‑SMA and galectin-3 was also significantly attenuated in the TEAD1fl/flcol1a2+ mice (Supplementary Fig. 3e). The ANP, BNP and β-MHC expression was also dramatically inhibited in the TEAD1fl/flcol1a2+ mice (Supplementary Fig. 3f). These results demonstrated that TEAD1 deficiency in CFs effectively prevented cardiac remodeling in response to TAC and Ang-II infusion.

Myofibroblasts-specific TEAD1 deficiency attenuates stress-induced cardiac remodeling and dysfunction

To further explore the effects of TEAD1 deficiency in response to TAC, TEAD1fl/fl mice were crossed with postn-MerCreMer mice to knockout TEAD1 in myofibroblast (TEAD1fl/flpostn+) (Supplementary Fig. 4a). PCR analysis was used to determine the genotyping of TEAD1fl/fl and postn+ mice (Supplementary Fig. 4b). Postn has been characterized as a marker only responsive to injury in myofibroblasts from adult tissues.17 TEAD1fl/flpostn+ mice were subjected to TAC for 7 days to transform fibroblasts into myofibroblasts, TAM was then administrated at the 8th day post TAC for subsequent 5 days (Fig. 3a). Knockout specificity in heart and myofibroblasts was verified at 13th day after TAC by qRT-PCR and immunoblot analyses (Supplementary Fig. 4c–f). The TEAD1fl/flpostn+ mice exhibited significantly preserved EF values after TAC compared with the TEAD1fl/fl mice (Fig. 3b). Specific cardiac ultrasound parameters were shown in Supplementary Table 5. Sirius red and immunofluorescence staining showed that TAC-induced fibrosis was significantly attenuated in the TEAD1fl/flpostn+ mice after TAC compared with the TEAD1fl/fl mice (Fig. 3c, f). WGA staining revealed a significantly reduced CMs CSA in the TEAD1fl/flpostn+ mice compared with the TEAD1fl/fl mice (Fig. 3d). Likewise, whole-heart gross images also showed that TEAD1 knockout in myofibroblasts reduced hypertrophy induced by TAC (Fig. 3e). The TAC-induced upregulation of cardiac fibrotic markers α‑SMA and galectin-3 was also attenuated in the TEAD1fl/flpostn+ mice (Fig. 3g). A significant reduction of ANP, BNP, and β-MHC was also observed in the TEAD1fl/flpostn+ mice after pressure overload (Fig. 3h).

Fig. 3

Myofibroblast-specific TEAD1 deficiency attenuates TAC-induced cardiac remodeling. a Schematic for echocardiography and sample collection from 4 groups: TEAD1fl/fl and TEAD1fl/flpostn+ 4 weeks after 4 weeks sham or TAC. b Left ventricular EF assessed by echocardiography of TEAD1fl/fl and TEAD1fl/flpostn+ mice after 4 weeks sham or TAC (n = 4-10 per group). c Heart sections from TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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 TEAD1fl/fl and TEAD1fl/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

We further confirmed the role of TEAD1 in myofibroblast in Ang-II-induced cardiac remodeling model. This conditional knockout of TEAD1 was induced in mice subjected to Ang-II for 7 days, then followed by administration of TAM for subsequent 5 days (Supplementary Fig. 5a). Knockout specificity in heart and myofibroblasts was verified at 13th day after Ang-II by qRT-PCR and immunoblot analyses (Supplementary Fig. 4g–j). The mitigation of remodeling was similar with our previous TAC model. The difference in the EF was not observed between the TEAD1fl/flpostn+ and TEAD1fl/fl mice post Ang-II treatment (Supplementary Fig. 5b). Specific cardiac ultrasound parameters were shown in Supplementary Table 6. Cardiac fibrosis was significantly reduced in the TEAD1fl/flpostn+ mice compared with the TEAD1fl/fl mice after Ang-II stimulation (Supplementary Fig. 5c). The TEAD1fl/flpostn+ mice exhibited a significantly reduced CMs CSA after Ang-II stimulation (Supplementary Fig. 5d). The fibrotic markers α‑SMA and galectin-3 (Supplementary Fig. 5e) as well as ANP, BNP, and β-MHC (Supplementary Fig. 5f) were significantly reduced in the TEAD1fl/flpostn+ mice compared with the TEAD1fl/fl mice after Ang-II stimulation. These results suggested that TEAD1 knockout in myofibroblasts may be an effective strategy to reverse cardiac remodeling after cardiac injury.

TEAD1 inhibition and knockdown ameliorate CFs differentiation and collagen secretion in vitro

Our results prompted us to explore the role of TEAD1 in the pathological differentiation of CFs in vitro. We assessed whether the pharmacological inhibition of TEAD1 signalling via VT103, which is known to strongly inhibit the TEAD/Yes-associated protein (YAP) interaction, could recapitulate the beneficial effects of TEAD1 deficiency in CFs after stimulation with Ang-II. VT103 treatment did not affect the TEAD1 mRNA or protein expression levels in these cells (Supplementary Fig. 6a, b). However, as expected, co-immunoprecipitation assay revealed that VT103 treatment significantly inhibited the TEAD1/YAP interaction in vitro and in vivo (Supplementary Fig. 6c, d). RNA-seq analysis with VT103 revealed that 41 genes were upregulated in the NMCFs (Neonatal Mice Cardiac Fibroblasts) after Ang-II stimulation, but this upregulation was reversed by VT103 treatment (Supplementary Fig. 7a). Gene Ontology (GO) analysis (false discovery rate q-value < 0.05) revealed that genes downregulated in response to VT103 treatment were associated with ECM organisation and cell adhesion (Supplementary Fig. 7b). Kyoto Encyclopaedia of Genes and Genomes analysis (KEGG, false discovery rate q-value < 0.05) further revealed the Wnt signalling pathway as the most significantly enriched category (Supplementary Fig. 7c). Immunofluorescence staining confirmed that Ang-II stimulation significantly increased α-SMA expression and that this effect was reversed by VT103 treatment (Supplementary Fig. 7d). Next, Ang-II increased collagen gel contraction over a 48-hour period, but this effect was inhibited by VT103 treatment (Supplementary Fig. 7e). VT103 also ameliorated Ang-II-induced fibroblasts migration (Supplementary Fig. 7f). Ang-II-induced increased expression of genes associated with extracellular matrix deposition was abolished with VT103 treatment in NMCFs (Supplementary Fig. 7g).

Since VT103 only occupies the interface on TEAD1 and disrupts YAP-TEAD1 interaction in cells18,19 and thus cannot reduce the TEAD1 protein expression, we next determined whether specific knockdown of TEAD1 by siRNA would phenocopy the effects of VT103 on suppressing the CFs differentiation and whether adenovirus expressing TEAD1 would promote the activation of CFs. After adenovirus infection, significantly elevated TEAD1 mRNA and protein levels were confirmed in NMCFs (Fig. 4a, b). Transfection with siRNA successfully knocked down TEAD1 protein levels in NMCFs (Fig. 4c). Consistent with our findings in antagonist treatment, the increases of TEAD1, α-SMA and collagen I at protein level were reversed by TEAD1 knockdown in NMCFs (Fig. 4c). However, overexpression of TEAD1 alone can induce upregulation of α-SMA and collagen I protein levels in NMCFs (Fig. 4c). Moreover, TEAD1 knockdown significantly reduced α-SMA expression, collagen gel contraction, and cell migration following Ang-II stimulation (Fig. 4d, f and h), whereas TEAD1 overexpression enhanced these effects (Fig. 4e, g and i). These findings further supported an association between TEAD1 expression and Ang-II-induced CFs differentiation and that these effects could be effectively attenuated via TEAD1 knockdown.

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

TEAD1 regulates CFs differentiation via the bromodomain-containing protein 4 (BRD4)/Wnt4 signalling pathway

To fully understand the molecular mechanisms underlying TEAD1-mediated cardiac remodeling, we next performed genome-wide RNA-seq to identify the transcriptomic changes in response to TEAD1 knockdown and overexpression in NMCFs. Hierarchical clustering analysis of all identified genes revealed six clearly defined clusters that were associated with their respective experimental cohorts (si-NC, si-TEAD1, Ang-II, si-TEAD1+Ang-II, OE-NC, and OE-TEAD1); hence we performed additional differential gene expression analysis (Supplementary Fig. 8a). Overall, 77 genes were upregulated in the NMCFs following Ang-II stimulation, but this upregulation was reversed by si-TEAD1 (Supplementary Fig. 8c). Furthermore, 20 of these 77 differentially expressed genes were upregulated after TEAD1 overexpression (Supplementary Fig. 8b). GO analysis (false discovery rate q-value < 0.05) revealed genes that were downregulated in response to TEAD1 knockdown were associated with ECM organisation, extracellular structure organisation, and angiogenesis, all of which are known to be involved in cardiac remodeling (Supplementary Fig. 8d). KEGG analysis (false discovery rate q-value < 0.05) further revealed the Wnt signalling pathway as the most significantly enriched category (Supplementary Fig. 8e). Notably, gene set enrichment analysis (GSEA) (false discovery rate q-value < 0.05) revealed that the Wnt signalling pathway was significantly downregulated in response to TEAD1 knockdown and significantly upregulated in response to TEAD1 overexpression (Supplementary Fig. 8f, g). Among the differential genes regulating the Wnt signaling pathway, we found that Wnt4 was significantly down-regulated in si-TEAD1 and up-regulated in TEAD1-OE (Fig. 5a). Interestingly, the Wnt4 expression was also upregulated in the HCM patients (Supplementary Fig. 9a, b). We found that under no matter pathological stimulation or TEAD1 overexpression, TEAD1 was almost absent in the cytoplasm (Fig. 5b). Furthermore, we found that si-TEAD1 alone did not significantly affect the nuclear translocation of β-catenin but prevented beta-catenin nuclear import in the presence of Ang-II stimulation, suggesting that si-TEAD1 might sensitively function in response to neurohumoral stimulation instead of under physiological homeostasis (Fig. 5b). Interestingly, overexpression of TEAD1 alone could induce the nuclear import of β-catenin (Fig. 5b), which was connected with the previous results that overexpression of TEAD1 alone could induce the differentiation of CFs. There were no notable alterations detected in the protein levels of c-Jun N-terminal kinase (JNK) or calcium/calmodulin-dependent protein kinase II (CaMKII), indicating that the non-canonical Wnt signalling pathway remained inactivated (Supplementary Fig. 10a, b). In vivo, decreased Wnt4 expression was observed in mice with knocking out TEAD1 in CFs or myofibroblasts following TAC or Ang-II infusion (Supplementary Fig. 11a–d).

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

Next, Wnt4 was knocked down in NMCFs using siRNA to confirm the requirement of Wnt signalling in TEAD1-induced CFs differentiation. Indeed, Wnt4 deficiency reduced the expression of α-SMA and Galectin-3 induced by TEAD1 overexpression (Fig. 5c). Furthermore, we used existing CFs-specific TEAD1 knockout mice and injected AAV9-Tcf21-Wnt4 to observe whether Wnt4 overexpression in CFs would block the protection provided by TEAD1 knockout after TAC (Supplementary Fig. 12a). Wnt4 expression was significantly upregulated in CFs after AAV9 injection (Supplementary Fig. 12b). Wnt4 overexpression blocked the protective effect of TEAD1 knockout after TAC, mainly reflected by EF, FS (Supplementary Fig. 12d, e), HE staining (Supplementary Fig. 12c), sirius red staining (Supplementary Fig. 12f) and fibrotic markers (Supplementary Fig. 12g). These results illustrated the importance of Wnt4 activity as an essential downstream signaling pathway in TEAD1-mediated CFs transformation. TEAD1NC and TEAD1OE were subjected to whole-genome chromatin immunoprecipitation-sequencing (ChIP-seq) and the gene regulatory elements bound by TEAD1 were mapped. TEAD1 was enriched at the promoters, with 15.56% of the bound regions located within 2 kb of transcription start site (TSS). Moreover, TEAD1 overexpression increased TEAD1 chromatin occupancy within the promoter of Wnt4 (Fig. 5d). Chromatin immunoprecipitation (ChIP)-qPCR confirmed high enrichment efficiency of the Wnt4 promoter with the TEAD1 antibody than anti-IgG antibody (Fig. 5e), indicating TEAD1 serves as a transcriptional regulator of Wnt4. The ChIP-seq predicted TEAD1 binding sites within the Wnt4 promoter located at -526-516. To further confirm the above binding site, the mutating plasmid within the TEAD1 binding sites in Wnt4 promoter was transfected into HEK293T cells. Significantly decreased Wnt4 promoter activity was detected in missing mutants compared with the WT fragment (Fig. 5h).

We found that knocking down YAP could reduce the expression of Wnt4 (Supplementary Fig. 13a), confirming that both TEAD1 and YAP could regulate the expression of Wnt4. Our research is focused on identifying additional proteins or co-factors that interact with TEAD1 and YAP to influence their transcriptional activity. To further investigate the mechanisms underlying TEAD1-induced CFs differentiation through Wnt signalling pathway, MS was performed to identify proteins that specifically interacted with TEAD1. Among the co-purified proteins, BRD4, an important regulator of CFs differentiation, was found to be enriched among the purified protein complexes. Co-immunoprecipitation assays were performed to confirm this direct interaction between TEAD1 and BRD4 in CFs (Fig. 5f). However, after treatment with VT103, TEAD1 cannot bind to YAP or BRD4 (Supplementary Fig. 13b, c). In addition, we found that VT103 did not affect the activation status of YAP induced by Ang-II (Supplementary Fig. 13d). We speculate that VT103 mainly affects the binding of TEAD1 to YAP or BRD4 in the nucleus, but has no effect on phosphorylated YAP in the cytoplasm. After VT103 treatment, TEAD1 was inactivated and could not bind to the promoter of Wnt4, thus losing the cooperation with BRD4. Additionally, blocking bromodomain (BD) of BRD4 through the inhibitor JQ1 affected the binding of BRD4 and TEAD1 (Fig. 5g). Next, we tested whether BRD4 directly regulates the expression of Wnt4 and thus affects the downstream Wnt signaling pathway and fibroblast transformation. ChIP-qPCR assay results indicated that there was no BRD4 binding site in the promoter region of Wnt4 (Fig. 5i). Furthermore, TEAD1-activated luciferase reporter activity driven by Wnt4 promoter was reduced in response to JQ1 treatment (Fig. 5j), while co-transfection of both TEAD1 and BRD4 resulted in additive activation of this reporter (Fig. 5k). Next, qRT-PCR and Western blot results showed that the BRD4 inhibitor JQ1 could indeed inhibit the increase of Wnt4 and α-SMA expression caused by TEAD1 overexpression (Supplementary Fig. 14a and Fig. 5l). Moreover, JQ1 treatment significantly reduced α-SMA expression, collagen gel contraction, and cell migration following TEAD1 overexpression (Supplementary Fig. 14d–f). More significant upregulation of Wnt4 and ACTA2 expression was also evidenced upon co-transfection of both TEAD1 and BRD4 compared with transfection of TEAD1 alone (Supplementary Fig. 14b, c). Taken together, TEAD1 and BRD4 interact in a bromodomain-dependent manner to control the differentiation of CFs, suggesting BRD4 can be preferentially targeted to specific genomic loci by combination with TEAD1.

TEAD1 inhibitor VT103 exhibits promising pharmacological effects for preventing stress-induced cardiac remodeling

To investigate the potential therapeutic possibility of TEAD1 inhibition in cardiac remodeling, we evaluated the effects of the TEAD1 inhibitor VT103 on TAC-induced cardiac remodeling. To test the in vivo effects of VT103, we intraperitoneally administered VT103 (20 mg/kg every other day for 28 days) to mice 3 days after TAC or sham surgery (Fig. 6a). The safety of TEAD1 inhibitors VT103 was evaluated during the implementation of the experiment. We found that the use of VT103 did not affect the body weight and heart rate as well as physiological functions of major organs (liver and kidney) in mice through histological (HE staining) and biochemical analyses (creatine kinase, LDH, AST, ALT and creatinine) (Supplementary Fig. 15a–g). VT103 treatment prevented TAC-induced cardiac hypertrophy and dysfunction in WT mice, as demonstrated by preserved EF and FS values and lower HW/BW and HW/TL ratios in these mice compared with the vehicle control (Fig. 6b–d). Specific cardiac ultrasound parameters were shown in Supplementary Table 7. Consistently, histological analyses revealed that VT103 reduced the cardiac hypertrophy, CMs CSA and fibrotic area following TAC in these mice (Fig. 6f, g). Additionally, VT103 treatment reduced the expression of cardiac fibrotic markers α‑SMA and galectin-3 as well as ANP, BNP, and β-MHC, which were all upregulated in the vehicle control (Fig. 6h, i). These data reinforced a predominant role of TEAD1 in cardiac remodeling and suggested the clinical translation of TEAD1 inhibitor in treating cardiac diseases associated with pathological remodeling.

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/)