Calcium‑sensing receptor promotes high glucose‑induced myocardial fibrosis via upregulation of the TGF‑β1/Smads pathway in cardiac fibroblasts

Figures

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  Figure 1.. Successful modeling of type 1 diabetic cardiomyopathy. (A) Random blood glucose was assessed at weeks 2, 4, 8 and 12. Other indicators were analyzed at week 12 after successful modeling. Blood glucose, TG, TC and insulin levels in the serum. (B) HW/BW. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the Control; #P<0.05 vs. T1D rats (n=8). TG, triacylglycerol; TC, total cholesterol; HW/BW, heart weight to body weight ratio; T1D, type 1 diabetes.

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  Figure 2.. (A) Effects of CaSR on collagens and morphological changes of myocardial tissue in rats. Masson's trichrome staining (collagen deposition displayed in blue) and Sirius red staining (collagen fibers displayed in red). Representative images of H&E staining examined by transmission electron microscopy. (B) Representative western blots for the detection of CaSR, Col-I, Col-III and TGF-β1 in comparison with β-actin expression in rats. *P<0.05, **P<0.01, ****P<0.0001 vs. Controls; #P<0.05, ##P<0.01, ###P<0.001 vs. T1D rats (n=8). TGF, transforming growth factor; T1D, type 1 diabetes; CaSR, calcium-sensing receptor; Col, collagen.

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  Figure 3.. Proliferation and migration of diabetic CFs. CFs were cultured for 24 h in a control group (5.5 mM), HG group (40 mM), HG+R568 (5 µM) group and HG+Calhex231 (3 µM) group. CF migration and proliferation (cell viability) were detected by (A) scratch wound repair assays, as well as (B) a CCK-8 assay, respectively. ***P<0.001, ****P<0.0001 vs. the Control; #P<0.05, ##P<0.01, ###P<0.001 vs. HG (n≥16). HG, high glucose; CFs, cardiac fibroblasts.

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  Figure 4.. Measurement of intracellular Ca2+ in CFs, the level of p-PKC/p38 and the expression of TGF-β1 in the culture medium. CFs were cultured for 24 h at 37°C in a control group (5.5 mM), HG group (40 mM), HG+R568 (5 µM) group and HG+Calhex231 (3 µM) group. (A) Cytosolic Ca2+ was stained with Fluo-3/AM and assessed by measuring the fluorescence intensity. (B) The protein levels of p-PKC and p-p38 were evaluated by western blot analysis. (C) The expression of TGF-β1 in the culture supernatants of the CFs was determined using ELISA. *P<0.05, **P<0.01, ***P<0.001 vs. the Control; #P<0.05, ##P<0.01, ###P<0.001 vs. HG (n≥16). TGF, transforming growth factor; p-PKC, phosphorylated protein kinase C; HG, high glucose; CFs, cardiac fibroblasts.

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  Figure 5.. Effects of TβRI/II kinase inhibitor (LY2109761) on TGF-β1/Smads pathway in CFs. CFs were cultured for 48 h at 37°C in an HG group (40 mM), R568 (5 µM), HG+Calhex231 (3 µM) and HG+R568+LY2109761 (20 µM) group. Representative western blots of TGF-β1, TβRI, TβRII in comparison with β-actin expression and p-Smad2 in comparison with t-Smad2 expression in CFs are provided. **P<0.01, ***P<0.001, ****P<0.0001 vs. the Control; #P<0.05, ##P<0.01, ###P<0.001 vs. HG; @@P<0.01 vs. R568 (n≥3). HG, high glucose; p-/t-Smad, phosphorylated/total Smad; TGF, transforming growth factor; TβRI, TGF-β-type I receptor; CFs, cardiac fibroblasts.

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  Figure 6.. Effects of TGF-β1 in culture medium on the changes of extracellular matrix components. Cardiac fibroblasts were treated with HG (40 mM) in the presence or absence of TGF-β1 (5, 10, 20 ng/ml) for 48 h at 37°C. (A) After different treatments, the protein expression of Col-I, Col-III, MMP-2 and MMP-9 was evaluated by western blot analysis. (B) The changes in the mRNA levels of Col-I, Col-III, MMP-2 and MMP-9 were measured by RT-qPCR, and the mRNA levels were normalized to β-actin, which was used as the housekeeping gene. (C) The cell supernatants were collected for the determination of Col-I, Col-III, MMP2 and MMP9 expression using ELISA. *P<0.05, **P<0.01 vs. the Control; #P<0.05, ##P<0.01, ###P<0.001 vs. HG (n≥8). Col, collagen; SMA, smooth muscle actin; MMP, matrix metallopeptidase; TGF, transforming growth factor; HG, high glucose.

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  Figure 7.. Schematic diagram illustrating the role of CaSR in the activation of high glucose-treated cardiac fibroblasts. CaSR, calcium-sensing receptor.