ERK1/2 communicates GPCR and EGFR signaling pathways to promote CTGF-mediated hypertrophic cardiomyopathy upon Ang-II stimulation

Figures

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  Fig. 1. Ang-II Stimulation Promotes Cardiomyocyte Fibrosis and Hypertrophy. a Western blot detection for ANP and BNP expression in H9c2 cardiomyocytes with different concentrations or (b) time of Ang-II treatment. (c) 24 h after seeding H9c2 cardiomyocytes in 10-cm culture plates, 10− 8 M AngII was added first. And, the β-actin of H9c2 cardiomyocytes was immunofluorescent stain. d Western blot detection for α-SMA, Paxillin and Vinculin expression in H9c2 cardiomyocytes with or without Ang-II treatment. All experiments were repeated at least three times. Data represent mean ± SD; * P < 0.05

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  Fig. 2. Ang-II stimulation promotes CTGF expression and reduces EGFR expression. a The mRNA expression of CTGF and EGFR in H9c2 cardiomyocytes with or without Ang-II treatment. b The expression of CTGF, EGFR, p-ERK1/2 and ERK1/2 were detected by western blot in H9c2 cardiomyocytes with or without Ang-II treatment. c The secretion of CTGF was detected by ELISA in medium of H9c2 cardiomyocytes with or without Ang-II treatment. All experiments were repeated at least three times. Data represent mean ± SD; * P < 0.05

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  Fig. 3. The expression of CTGF and EGFR under the stimulation of Ang-II in vivo. a The hematoxylin and eosin (HE)-staining (scale bar, 30 μm) of histological sections of the left ventricle in the indicated groups (n = 4 mice per experimental group). b The hematoxylin and eosin (HE)-staining (scale bar, 30 μm) of histological sections of the left ventricle in the indicated groups (n = 4 mice per experimental group). The immunohistochemistry assay shows expressions of CTGF and EGFR (brown) in the cytoplasm of cardiomyocytes (n = 4 mice per experimental group), scale bar, 30 μm. All experiments were repeated at least three times. Data represent mean ± SD; * P < 0.05

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  Fig. 4. Ang-II stimulates activation of ERK1/2 signal and is mediated by GPCR. a The expression of ANP, BNP, p-ERK1/2 and ERK1/2 were detected by Western blot in H9c2 cardiomyocytes with or without Ang-II or U0126 treatment. b 24 h after seeding H9c2 cardiomyocytes in 10-cm culture plates, 10− 8 M AngII was added first with or without U0126 or ARBs treatment. And, the β-actin of H9c2 cardiomyocytes was immunofluorescent stain. c The expression of ANP, BNP, CTGF, p-ERK1/2 and ERK1/2 were detected by western blot in H9c2 cardiomyocytes with or without Ang-II or ARBs treatment. d The cell viability of H9c2 cardiomyocytes was detected using CCK-8 assay after treatment with Ang-II, ARBs or U0126. All experiments were repeated at least three times. Data represent mean ± SD; * P < 0.05

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  Fig. 5. Reduced interaction between CTGF and EGFR stimulated by Ang-II. a The interaction of CTGF and EGFR was detected by co-IP in H9c2 cardiomyocytes with or without Ang-II treatment. b H9c2 cardiomyocytes were transfected with siCTGF for 24 h and then treated with or without Ang-II to detect the expression of ANP, BNP and CTGF by western blot. c H9c2 cardiomyocytes were transfected with siCTGF for 24 h and then treated with or without Ang-II to detect the expression of α-SMA, Paxillin and Vinculin by western blot. d H9c2 cardiomyocytes were treated with or without CTGF and then detected the expression of ANP and BNP by western blot. e H9c2 cardiomyocytes were treated with or without CTGF and then detected the expression of α-SMA, Paxillin and Vinculin by western blot. All experiments were repeated at least three times

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  Fig. 6. The expression of CTGF and EGFR under the stimulation of Ang-II is affected by the activity of ERK1/2. a The mRNA expression of CTGF or (b) EGFR were detected by RT-PCR in H9c2 cardiomyocytes with or without Ang-II or U0126 treatment. c The expression of CTGF, EGFR, p-ERK1/2 and ERK1/2 were detected by western blot in H9c2 cardiomyocytes with or without Ang-II or U0126 treatment. d The secretion of CTGF was detected by ELISA in medium of H9c2 cardiomyocytes with or without Ang-II or U0126 treatment. All experiments were repeated at least three times. Data represent mean ± SD; * P < 0.05