CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signaling pathway

Figures

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  Figure 1. The Expression of CD9 in TAC Myocardial Tissue (A) The mRNA-seq data (GSE101977, GSE180720, and GSE89714) displayed increased CD9 expression in a stress-induced cardiac hypertrophy mouse model and hypertrophic cardiomyopathy patients' heart tissues (p < 0.05 for CD9). (B) Western blot analysis of CD9 protein expression in heart tissue from mice 4 weeks post-TAC surgery and the Sham group, with quantification in the right panel (n = 6). (C) RT-PCR detected CD9 mRNA expression in heart tissue from mice 4 weeks post-TAC surgery and Sham group (n = 6). (D) CD9 protein expression in cardiomyocytes and fibroblasts analyzed by Western blot, with quantification below (n = 3). (E) CD9 protein expression in cardiomyocytes under Ang II stimulation examined by Western blot, with quantification below (n = 3). (F) CD9 mRNA expression in cardiomyocytes under Ang II stimulation detected through RT-PCR. (G) Representative immunofluorescence image depicting CD9 (Green) co-stained with cTNT (Red) and DAPI in Sham and TAC groups, Scale bar: 100 μm, with quantification in the right panel (n = 3). See also Figure S1A. (H) Western blot analysis of HIF1α and CD9 protein expression in cardiomyocytes,…

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  Figure 2. Knockdown of CD9 Ameliorates TAC-Induced Pathological Cardiac Hypertrophy C57 mice injected with AAV CD9-shRNA two weeks pre-TAC surgery, sacrificed 4 weeks post-surgery. (A and B) Ultrasound assessed cardiac hypertrophy degree pre-sacrifice (n = 7). (C and D) Ultrasound evaluated cardiac function pre-sacrifice (n = 7). (E–G) Stats on heart weight/body weight, lung weight/body weight, and heart weight/tibia length (n = 7). (H and I) HE staining of cardiac tissue, with cardiomyocyte cross-sectional area statistics (n = 7). Scale bar: 1 mm in upper panel, 20 μm in lower panel. (J and K) Sirius Red staining and cardiac tissue statistics (n = 7). Scale bar: 100 μm. (L) RT-PCR measured Anp, Bnp, and Myh7 expression in cardiac tissue (n = 3). (M) RT-PCR assessed cardiac tissue fibrosis marker expression (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S1B–S1F.

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  Figure 3. Overexpression of CD9 Aggravates TAC-Induced Pathological Cardiac Hypertrophy C57 mice injected with AAV CD9 two weeks pre-TAC surgery, sacrificed 4 weeks post-surgery. (A and B) Ultrasound assessed cardiac hypertrophy degree pre-sacrifice (n = 7). (C and D) Ultrasound evaluated cardiac function pre-sacrifice (n = 7). (E–G) Stats on heart weight/body weight, lung weight/body weight, and heart weight/tibia length (n = 7). (H and I) HE staining of cardiac tissue, with cardiomyocyte cross-sectional area statistics (n = 7). Scale bar: 1 mm in upper panel, 20 μm in lower panel. (J and K) Sirius Red staining and cardiac tissue statistics (n = 7). Scale bar: 100 μm. (L) RT-PCR measured Anp, Bnp, and Myh7 expression in cardiac tissue (n = 3). (M) RT-PCR assessed cardiac tissue fibrosis marker expression (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S2.

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  Figure 4. CD9 Promotes Cardiac Hypertrophy by Regulating GP130 to Activate STAT3 (A and B) Immunoprecipitation and Western blot assays used anti-CD9 and anti-GP130 antibodies to detect GP130 binding with CD9 in cardiomyocytes, with IgG as control (n = 1). (C) Cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA underwent cycloheximide treatment (CHX). GP130 protein abundance analyzed through Western blot over time (0, 0.5, 1, and 2 h), quantified below (n = 3). (D) Effects of lysosome inhibitor CQ and proteasome inhibitor MG132 on GP130 expression in cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA examined (n = 3). (E) Cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA underwent co-IP of GP130, followed by immunoblotting of precipitates for ubiquitin (n = 3). (F) Cardiomyocytes transfected with Ad CD9 or Ad Control co-IP’d for GP130, followed by immunoblotting of precipitates for ubiquitin (n = 3). (G and H) Western blot assays performed on cardiomyocytes with CD9 knockdown or overexpression under Ang II stimulation, assessing CD9, GP130, p-STAT3, and STAT3 expression. GAPDH was loading control, with quantification below (n = 3). (I) Western blot nuclear…

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  Figure 5. Knockdown of GP130 Reverses CD9-Induced Pathological Cardiac Hypertrophy C57 mice injected with AAV CD9 and AAV GP130-shRNA two weeks pre-TAC surgery, sacrificed 4 weeks post-surgery. (A and B) Ultrasound assessed cardiac hypertrophy degree pre-sacrifice (n = 7). (C and D) Ultrasound evaluated cardiac function pre-sacrifice (n = 7). (E–G) Stats on heart weight/body weight, lung weight/body weight, and heart weight/tibia length (n = 7). (H–I) HE staining of cardiac tissue, with cardiomyocyte cross-sectional area statistics (n = 7). Scale bar: 1 mm in upper panel, 20 μm in lower panel. (J and K) Sirius Red staining and cardiac tissue statistics (n = 7). Scale bar: 100 μm. (L) RT-PCR measured Anp, Bnp, and Myh7 expression in cardiac tissue (n = 3). (M) RT-PCR assessed cardiac tissue fibrosis marker expression (n = 3). (N) Western blot detected the phosphorylation level of STAT3, with quantification below (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S3.