Establishment and functional studies of a model of cardiomyopathy with cardiomyocyte-specific conditional knockout of Arhgef18

Figures

• 

  Fig. 1.. The Arhgef18 conditional knockout (cKO) mouse model was successfully constructed. (A) Pie chart of four genotypes of F3 generation mice, approximately 1/4 each. (B) Pie chart of the genders of F3 generation mice, approximately 1/2 each. (C) Identification of mouse genotypes by PCR and agarose gel electrophoresis. Line M is a molecular size ladder for electrophoresis. The LoxP band was 432 bp, the wild-type band was 375 bp, and the Cre band was 603 bp in length. (D) Arhgef18 mRNA expression in heart (n=6 per group), liver (n=4 per group), lung (n=4 per group) and kidney (n=5 per group) tissues from Arhgef18fl/fl, Arhgef18fl/+ cKO and Arhgef18fl/fl cKO mice at 4 weeks of age by quantitative real-time PCR (q-PCR). (E,F) Expression levels of ARHGEF18 in heart (n=6 per group), liver (n=4 per group), lung (n=4 per group) and kidney (n=4 per group) tissues from three groups of mice at 4 weeks of age, detected using western blotting (E) and quantitatively analysed using HSP90 for normalization (F). The data are presented as the mean±s.d.. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus respective control group (one-way ANOVA).

• 

  Fig. 2.. Arhgef18 cKO mice develop a cardiomyopathy phenotype. (A) Body weight of Arhgef18fl/fl, Arhgef18fl/+ cKO and Arhgef18fl/fl cKO mice from the same litter at 1, 2, 3, 4, 8, and 12 weeks of age (n=40 per group for 1-4 weeks, n=20 per group for 8 weeks, n=15 per group for 12 weeks). Arhgef18fl/fl cKO mice presented pronounced deceleration of body weight growth beginning at 3 weeks of age. (B) Representative images of M-mode echocardiography of the heart in three groups of mice at 2 and 4 weeks of age. (C) Echocardiographic parameters of left-ventricular (LV) ejection fraction (LVEF), LV fraction shortening (LVFS), LV anterior wall thickness at end systole (LVAWs), LV inner diameter at end systole (LVIDs) and LV volume at end systole [LV volume (s)] were analysed in three groups of mice (n=5-8 mice per time point in each group) at 2, 4, 12 and 24 weeks of age, suggesting reduced systolic function, cardiac chamber enlargement and ventricular wall thinning in Arhgef18fl/fl cKO mice. (D) Nppa and Nppb mRNA expression in three groups of mice (n=5-6 per group) at 4 weeks of age by q-PCR, suggesting reduced cardiac function in Arhgef18fl/fl cKO mice. (E) Representative images of gross…

• 

  Fig. 3.. Cytoskeletal rearrangement of myocardium in Arhgef18 cKO mice. (A) mRNA expression of cytoskeleton-related genes, including vinculin, αβ-tubulin, cTNT and α-actinin, in myocardial tissue from three groups of mice (n=4-6 per group) at 4 weeks of age by q-PCR, showing reduced expression in Arhgef18 cKO mice. (B,C) The expression levels of cytoskeletal proteins, including vinculin, αβ-tubulin, cTNT and α-actinin, in myocardial tissue from three groups of mice (n=4-6 per group) at 4 weeks of age, detected using western blotting (B) and quantitatively analysed using HSP90 for normalization (C), suggesting that the expression of these proteins is lower in Arhgef18 cKO mice than in Arhgef18fl/fl mice. (D,E) Fluorescence intensity of cytoskeletal proteins (E), including vinculin, αβ-tubulin, cTNT and α-actinin, in cardiomyocytes from three groups of mice (n=3 mice per group; >20 cardiomyocytes per mouse) at 4 weeks of age, detected using immunofluorescence (D), showing that the fluorescence intensity of these proteins in Arhgef18 cKO mice was weaker than that in Arhgef18fl/fl mice. Scale bars: 10 μm. Blue, DAPI; purple, vinculin; dark green, αβ-tubulin; orange, cTNT; light green,…

• 

  Fig. 4.. Disruption of myocardial tissue polarity in Arhgef18 cKO mice. (A) The mRNA expression of polarity-related genes, including Pard3 and Scrib in the myocardial tissue from three groups of mice (n=4-6 per group) at 4 weeks of age by q-PCR, showing reduced mRNA expression of polarity-related genes in Arhgef18 cKO mice. (B,C) The expression levels of polarity proteins, including SCRIB and CRB2, in myocardial tissue from three groups of mice (n=5-6 per group) at 4 weeks of age, detected using western blotting (B) and quantitatively analysed using HSP90 for normalization (C), suggesting reduced expression of polarity proteins in Arhgef18 cKO mice. (D,E) Fluorescence intensity of the polarity protein PARD3 (E) in cardiomyocytes from three groups of mice (n=3 mice per group; >20 cardiomyocytes per mouse) at 4 weeks of age, detected using immunofluorescence (D), showing that the fluorescence intensity of PARD3 in Arhgef18 cKO mice was weaker than that in Arhgef18fl/fl mice. Scale bars: 10 μm. Blue, DAPI; red, PARD3. The data are presented as the mean±s.d. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus respective control group (one-way ANOVA).