Cell division cycle protein 42-driven activation of the MKK3/6-p38 signaling pathway participates in cardiac remodeling in mice

Figures

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  Fig. 1. Cardiomyocyte-specific deletion of the Cdc42 gene inhibits AngII-induced cardiac hypertrophy in mice. Cardiomyocyte-specific Cdc42 knockout (Cdc42CKO) mice were generated by crossing Cdc42loxP/loxP mice with MLC2v-Cre mice. The expression of cardiac Cdc42 in heart tissues (C) was measured by semiquantitative qPCR (A), RT‒PCR (B), or western blotting. The data were analyzed with a t test to evaluate the statistical significance of differences between two groups. The left ventricle (LV) mass (mg), ejection fraction (EF) (%) and fraction shortening (FS) (%) were measured by echocardiography in Cdc42loxP/loxP and Cdc42CKO mice treated with or without AngII infusion (1500 ng/kg/min) for 7 days in three independent experiments (n = 4–6 per group per group, total n = 14–15 per group) (D-F). Representative images of mouse hearts (G), the ratio of heart weight (HW)/body weight (BW) (%) (H), and histological images of cardiac fibrosis by Masson’s trichome staining (I) and the relative fibrotic area comparison (J) are presented for Cdc42loxP/loxP and Cdc42CKO mice with or without AngII infusion for 7 days. The scale bars are indicated in the figures. Two-way ANOVA and an unpaired two-tailed…

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  Fig. 2. Cardiomyocyte-specific deletion of the Cdc42 gene inhibits AngII-induced activation of the MEKK3/6-p38 signaling pathway in mouse heart tissues. Phosphorylated MKK3/6 and p38 (A, B), GSK3β (C, D), PI3K and AKT (E, F), ERK1/2 (G, H), JNK (I, J), NF-κB/P65 (K, L), calcineurin, NFAT-C4 and CaMK II (M, N) were quantified by western blot analysis. GAPDH served as a control. Two-way ANOVA and an unpaired two-tailed Tukey test were used to evaluate the statistical significance of differences between the groups. The data are representative of three independent experiments. *P < 0.05, **P < 0.01

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  Fig. 3. Cdc42 deficiency inhibits AngII-induced activation of the MEKK3/6-p38 signaling pathway in cardiomyocytes. Cardiomyocytes were isolated from adult Cdc42LoxP/LoxP and Cdc42CKO mice and treated with or without 200 ng/ml AngII for 15 min. Phosphorylated protein levels of MKK3/6 and p38 (A, B), GSK3β (C, D), PI3K and AKT (E, F), ERK1/2 (G, H), JNK (I, J), NF-κB/P65 (K, L), calcineurin, NFAT-C4 and CaMK II (M, N) were quantified by western blot analysis. GAPDH served as a control. Two-way ANOVA and an unpaired two-tailed Tukey test were used to evaluate the statistical significance of differences between the groups. The data are representative of three independent experiments. *P < 0.05

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  Fig. 4. The overexpression of Cdc42 promoted cardiomyocytic hypertrophy by activating the p38 signaling pathway in vitro. H9c2 cells were transfected with the PCMV-Cdc42-myc vector (Cdc42-OV group), stimulated with AngII (200 nmol/L, AngII group) for 24 h, or pretreated with 2 µM ML141 (Cdc42 inhibitor) or 1 µM SB 203580 (p38 inhibitor) for 30 min in a 5% CO2 incubator at 37 °C. The cells were subsequently transfected with the PCMV-myc vector as a control (vehicle group). The efficiency of Cdc42 overexpression was confirmed by Western Blot and real-time PCR in H9c2 cell lines transfected with expressing vector of Cdc42 (A-C). Representative immunofluorescence images (red, phalloidine; blue, DAPI) of H9c2 cells with overexpression of Cdc42 (D) or AngII stimulation (F) and with or without pretreatment with a p38 inhibitor or a Cdc42 inhibitor (D, F) were acquired, and the cellular surface area was quantified (E, G). The mRNA (H) and protein (I) expression levels of reprogrammed hypertrophic genes (ANP and BNP) were quantified by RT‒PCR in Cdc42-OV cells treated with or without SB203580, and GAPDH was used as a reference gene. The total or phosphorylated p38 in H9c2 cells treated with or…

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  Fig. 5. Cardiac-specific deletion of Cdc42 protects cardiac functions from TAC-induced cardiac remodeling. Representative echocardiography images of a restricted mouse aorta (as indicated by the orange arrow) and increased aortic blood flow are presented as indicators of successful TAC modeling in mice (A). Echocardiograms were obtained at 2 or 8 weeks after TAC in Cdc42loxP/loxP and Cdc42CKO mice (n = 6–8 per group); representative images are shown (B). The systolic diameter (diameter; s) and volume (volume; s), diastolic diameter (diameter; d) and volume (volume; d), ejection fraction (EF) (%), fraction shortening (FS) (%) and LV mass were measured by echocardiography in Cdc42loxP/loxP and Cdc42CKO mice (C-I). Two-way ANOVA and an unpaired two-tailed Tukey test were used to evaluate the statistical significance of differences between the groups. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 6. Cardiac Cdc42 deficiency inhibited cardiac hypertrophy and fibrosis and p38 phosphorylation in TAC-induced cardiac remodeling. Representative images of mouse hearts were obtained at 8 weeks after TAC (A). The protein expression levels of Cdc42, ANP and BNP were quantitatively analyzed in TAC-8wk hearts (B-D). Histological images of cardiac fibrosis in Cdc42 loxP/loxP and Cdc42 CKO mice are shown in blue by Masson’s trichrome staining (E). The protein expression levels of Collagen I, TNF-α, p38 and p-p38 were quantitatively analyzed in heart tissues from TAC-2-week-old mice (F-I). The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 7. Cardiac Cdc42 deficiency suppresses phospho-p38 and apoptosis in TAC myocardium and reduces the secretion of the proinflammatory cytokines IL6 and TNFα in cardiac remodeling. The representative images of phospho-p38 and TUNEL signals in TAC 2 wk hearts (A). The serum levels of IL-6 and TNF-α were quantitatively analyzed at 2 and 8 weeks after TAC (B). H9c2 cells were plated on glass coverslips and stimulated with 200 nM AngII for 12 h with or without pretreatment with ML141 (Cdc42 inhibitor) or SB203580 (p38 inhibitor) for 30 min. IL6 expression or quantification was performed by immunofluorescence staining (C-D), and the immunofluorescence intensity was recorded and analyzed by ImageJ software (D). The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001