Cholesterol metabolic enzyme Ggpps regulates epicardium development and ventricular wall architecture integrity in mice

Figures

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  Figure 1. Ggpps deletion in cardiomyocytes results in epicardial cell hyperproliferation and fibroblast incline in the ventricular wall. (A) Immunofluorescence staining of WT1, cTnT, and DAPI in control and Nkx2.5KO hearts at E10.5, E11.5, and E12.5. Scale bar, 100 μm. (B) Immunofluorescence staining of Ki67 and WT1 indicates epicardial cell proliferation in control and Nkx2.5KO hearts at E10.5, E11.5, and E12.5. Scale bar, 100 μm. (C) Immunofluorescence staining of vimentin detects fibroblasts in control and Nkx2.5KO hearts at E10.5, E11.5, and E12.5. Scale bar, 100 μm.

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  Figure 2. Ggpps deletion in epicardial cells results in the damage to ventricular wall architecture integrity. (A) Survival rate of Wt1KO mice when tamoxifen injected at E10.5. (B) Survival rate of Wt1KO mice when tamoxifen injected at E13.5. (C) Histology analysis of the epicardium in control and Wt1KO embryos at E13.5. Scale bar, 250 μm (left) and 50 μm (right). (D) Immunofluorescence staining of cTNT in the embryonic heart myocardium of Wt1KO and control mice. Scale bar, 50 μm. (E) Immunofluorescence staining of WT1, cTnT, and DAPI in the embryonic heart myocardium of Wt1KO and control mice. Scale bar, 50 μm. (F) Immu nofluorescence staining of vimentin and DAPI in the embryonic hearts of Wt1KO and control mice. Scale bar, 50 μm. (G) Immunofluorescence staining of PECAM and DAPI in the embryonic hearts of Wt1KO and control mice. Scale bar, 50 μm.

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  Figure 3. Ggpps deficiency disrupts cell‒cell contact of epicardial cells. (A) Epicardial myocytes isolated from E13.5 hearts of floxed Ggpps mice were treated with a vehicle or Cre adenovirus. (B) Immunofluorescence staining of phalloidin and DAPI of the epicardial cells isolated from E13.5 hearts of floxed Ggpps mice. Scale bar, 50 μm. (C) Immunofluorescence staining of β-catenin and DAPI of the epicardial cells from E13.5 hearts of floxed Ggpps mice. Scale bar, 50 μm. (D) Immunofluorescence staining of WT1, β-catenin, and DAPI in the epicardial cells from Wt1KO embryonic hearts with vehicle or GGPP (20 μM) treatment. Scale bar, 50 μm. (E) Immunofluorescence staining of N-cadherin, β-catenin, and DAPI in the embryonic hearts of Wt1KO and control mice at E13.5. Scale bar, 50 μm.

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  Figure 4. Ggpps deletion results in YAP inactivation. (A) Immunohistochemistry staining of YAP in the control (n = 5 for E13.5) and Wt1KO (n = 6 for E13.5) mice. Scale bar, 250 μm. (B) The protein expression of YAP in control and Wt1KO hearts was determined by western blotting analysis. n = 6. (C) Immunofluorescence staining of YAP in isolated epicardial cells from E13.5 control and Wt1KO mice. Scale bar, 50 μm.

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  Figure 5. Ggpps deletion is beneficial to MI recovery in mice. (A) Cardiac function was examined with echocardiography of Wt1KO and control mice (left) and after 24 h with MI (right). (B) LVIDD in sham and MI groups of Wt1KO and control mice. (C) LVIDS in sham and MI groups of Wt1KO and control mice. During systolic stage, Wt1KO mice after MI show no difference with sham (P = 0.011). (D) Representative histological sections through multiple levels of the heart (apex toward base) with Masson’s trichrome staining show the scar tissue in green and the healthy myocardium in red in wild-type and Wt1KO mouse hearts after MI. (E) The quantification of scar size in wild-type and Wt1KO mouse hearts after MI (n = 6, ***P < 0.001). Figures are representative of three independent experiments. Values are presented as mean ± SEM, with P-values by unpaired two-tailed Student’s t-test.