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.