Yes-associated protein (YAP) mediates adaptive cardiac hypertrophy in response to pressure overload

Abstract

Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, Yap^+/flox;Cre^α-MHC (YAP-CHKO) and Yap^+/flox mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO.

Keywords: YAP; cardiac hypertrophy; cardiomyocyte; cardiovascular disease; heart failure; signal transduction.

Figure 1.

YAP is activated after acute PO. Wildtype (WT) mice were subjected to TAC. The hearts were harvested at indicated time points (6 h to 7 days (d)). A, immunoblot analyses were performed to determine the phosphorylation status of YAP and Last2 in ventricular lysates. n = 5. B, mRNA expression of YAP in the mouse heart was measured by quantitative real-time PCR assays. n = 3. C, quantification of Ser-127–phosphorylated YAP and total YAP. D, immunoblot analyses of Ser-397–phosphorylated YAP and total YAP. E, quantification of phosphorylated Lats2 and total Lats2. F, immunostaining for YAP 7 days after TAC in the WT mouse heart. DAPI was used for nuclear staining. n = 4. G, subcellular localization of endogenous YAP in mouse ventricular extracts 7 days after TAC. Rho-GDIα and lamin A/C served as markers of cytosol- and nucleus-enriched fractions, respectively. n = 4. H, quantification of the data shown in G. The data are expressed as ratios relative to the mean value of the sham group. Data in graphs represent mean ± S.D.; *, p < 0.05; **, p < 0.01, compared with Sham. Statistical analyses were conducted with ANOVA or Student's t test. Post hoc analysis was conducted with Tukey's test.

Figure 2.

PO-induced up-regulation of YAP was inhibited in YAP-CHKO mice. TAC or sham operation was applied to WT or YAP-CHKO mice for 7 days. A and B, whole-heart lysates were subjected to immunoblotting with anti-YAP antibody. GAPDH antibody was used as loading control. Data are expressed as a ratio compared with the mean value of the WT-sham group. n = 6 per group. C and D, total RNA was extracted from the heart and subjected to quantitative RT-PCR analyses. Relative mRNA expression of Ctgf (C) and Ankrd (D) is shown. n = 5–6. The data are expressed as ratios relative to the mean value of the WT-sham group. Data in graphs represent mean ± S.D.; **, p < 0.01; ***, p < 0.005 compared with Shams; $, p < 0.05 in comparison with WT-sham and YAP-CHKO-Sham; #, p < 0.05; ##, p < 0.01, in comparison with WT-TAC and YAP-CHKO-TAC. Statistical analyses were conducted with ANOVA. Post hoc analysis was conducted with Tukey's test.

Figure 3.

Heterozygous down-regulation of YAP significantly attenuates PO-induced cardiac hypertrophy. TAC was applied to WT or YAP-CHKO mice for 1 week. A, LVW/TL. n = 12–14. B, lung W/TL. n = 12–14. C, cardiomyocyte cross-sectional area (CSA) was evaluated with wheat germ agglutinin staining. Values are relative to the WT-sham group. Representative images from each group are shown in the upper panel. Scale bars, 200 μm. n = 5 per group. D, mRNA expression of Anf was evaluated with quantitative RT-PCR. n = 6. Values are mean ± S.D. relative to the WT-sham group. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; versus shams. #, p < 0.05; ###, p < 0.001 in comparison with WT-TAC and YAP-CHKO-TAC. Statistical analyses were conducted with ANOVA. Post hoc analysis was conducted with Tukey's test.

Figure 4.

TAC-induced cardiac dysfunction was exacerbated in YAP-CHKO mice. WT and YAP-CHKO mice were subjected to either TAC or sham operation for 7 days. Echocardiography and hemodynamic analyses were performed before euthanasia 7 days after TAC or sham operation. A, representative images of M mode echocardiography. B, % fractional shortening. C, left ventricular end-diastolic diameter (LVEDd, mm). D, wall stress calculated using the data set obtained by echocardiography and hemodynamic measurement. E, left ventricular end-diastolic pressure (LVEDP, mm Hg) evaluated with a Millar catheter. n = 12–14 per group. B–E, values are mean ± S.D. **, p < 0.01; ***, p < 0.001 versus shams; #, p < 0.05 in comparison with WT-TAC and YAP-CHKO-TAC. Values are means ± S.D. Statistical analyses were conducted with ANOVA. Post hoc analysis was conducted with Tukey's test.

Figure 5.

TAC-induced apoptosis and fibrosis were significantly increased in YAP-CHKO mice. WT and YAP-CHKO mice were subjected to either TAC or sham operation for 7 days. A, left, representative images of TUNEL staining and DAPI staining from each group. TUNEL-positive CMs are indicated by white arrows. Scale bars, 100 μm. A, right, number of TUNEL-positive CMs/the total number of nuclei. Values are mean ± S.D. n = 5. B, left, Masson's trichrome staining showing interstitial fibrosis. Representative images from each group are shown. Scale bars, 100 μm (right). The quantitative analysis of the fibrotic areas. n = 6. Values are the means ± S.D. ***, p < 0.0005; ****, p < 0.0001 versus shams. ##, p < 0.01; ###, p < 0.0005 in comparison with WT-TAC and YAP-CHKO-TAC. Statistical analyses were conducted with ANOVA. Post hoc analysis was conducted with Tukey's test.

Figure 6.

Heterozygous down-regulation of YAP inhibits CM cell cycle re-entry 7 days after TAC. WT and YAP-CHKO mice were subjected to either TAC or sham operation for 7 days. A, heart sections were stained with anti-Ki-67 (green), anti-troponin T (red), and DAPI (blue). The arrow indicates a Ki-67–positive CM. B, quantification of Ki-67–positive CMs (%) detected in WT and YAP-CHKO mouse hearts. n = 4. Values are mean ± S.D. *, p < 0.05 versus WT-sham; #, p < 0.05, comparison with WT-TAC and YAP-CHKO-TAC. Statistical analyses were conducted with ANOVA. Post hoc analysis was conducted with Tukey's test.

Figure 7.

RhoA mediates activation of YAP in response to mechanical stress. A, cultured neonatal rat CMs were pre-incubated with either C3 toxin (1 mg/ml), an inhibitor of RhoA, or vehicle (veh) for 4 h before stretch stress. CMs were then cyclically stretched by 20% for 1 h. Cytosolic (cyto)- and nucleus (nuc)-enriched fractions were prepared by subcellular fractionation. Representative immunoblots are shown in A. Rho-GDI and lamin A/C served as markers of cytosolic- and nucleus-enriched fractions, respectively. The experiments were repeated four times. *, p < 0.05 in comparison with vehicle (Veh) and vehicle + stretch; #, p < 0.05 in comparison with vehicle + stretch and C3 + stretch. B, mice were subjected to either sham or TAC for 1 or 7 days, and then heart homogenates were prepared. The RhoA-binding domain of rhotekin was used to selectively pull down activated GTP-bound RhoA. Samples were analyzed by SDS-PAGE. The relative binding of GTP-loaded RhoA to rhotekin is shown. n = 6. *, p < 0.05, versus sham. C and D, neonatal rat CMs were transduced with either GFP or active RhoA (RhoA (Q63L), aRhoA) adenovirus. C, cytosolic- and nucleus-enriched fractions were prepared by subcellular fractionations and then subjected to immunoblot analyses with anti-YAP antibody. Immunoblots with anti-RhoGDI and anti-lamin A/C antibodies were conducted to assess the purity of each fraction. D, quantification of the data shown in C. n = 4. Data are mean ± S.D. *, p < 0.05; **, p < 0.01, comparison with aRhoA and GFP. Statistical analyses were conducted with ANOVA. Post hoc was conducted with Tukey's test.

Figure 8.

Heterozygous down-regulation of YAP inhibits Akt phosphorylation after 1 week of TAC. WT and YAP-CHKO mice were subjected to either TAC or sham operation for 7 days. A, whole-heart lysates were subjected to immunoblotting for Thr-308–phosphorylated and total Akt or Ser-9–phosphorylated and total GSK-3β antibody. GAPDH antibody was used as loading control. Representative immunoblots are shown. B, quantification of relative pAkt/Akt shown in A. C, quantification of relative pGSK-3β/GSK-3β shown in A. B and C, data are normalized by the mean value in the WT-sham group. Values are mean ± S.D. n = 6 animals per group. *, p < 0.05; **, p < 0.01 versus sham; ##, p < 0.01; ###, p < 0.0005, compared with WT-TAC and YAP-CHKO-TAC. Statistical analyses were conducted with ANOVA. Post hoc analysis was conducted with Tukey's test.

Figure 9.

YAP negatively regulates PTEN in CMs. A, hearts were harvested from Yap^+/F, Yap^+/F;Cre^αMHC, and Yap^F/F;Cre^αMHC mice under baseline conditions. Whole-heart lysates were subjected to immunoblotting for PTEN and β-actinin. The data are normalized by the mean value in Yap^+/F group. In the presence of decreased of YAP expression, PTEN expression was increased. Values are the means ± S.D. *, p < 0.05 versus Yap^+/F. n = 3. B, neonatal rat CMs were transduced with LacZ control (Ad-LacZ) or YAP adenovirus (Ad-YAP). Cell lysates were subjected to immunoblotting for PTEN and β-actinin. The data are normalized by the mean value of the LacZ-transduced CMs. The experiments were repeated four times. Values are mean ± S.D. *, p < 0.05 versus the LacZ-transduced CMs. Statistical analyses were conducted with ANOVA or Student's t test. Post hoc analysis was conducted with Tukey's test.