Calcineurin/NFATc3 pathway mediates myocardial fibrosis in diabetes by impairing enhancer of zeste homolog 2 of cardiac fibroblasts

Figures

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  Fig. 1. The cardiac function in diabetic rats. (A) The body weight over 8 weeks in Con and DM groups. (B) The blood glucose over 8 weeks in different groups. (C-G) The analysis of left ventricular EF, FS, LVIDd and LVIDs in different groups. Con: control group; DM: diabetes mellitus group; FS: left ventricular fractional shortening; EF: ejection fraction; LVIDd: left ventricular internal diameter at end-diastole; LVIDs: left ventricular end systolic diameter. n = 5 per group, *P < 0.05

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  Fig. 2. Hyperglycemia promoted myocardial fibrosis. (A) Representative images of Masson and hematoxylin and eosin (HE) staining for myocardial tissue in the Con and DM groups (n = 3 per group). (B, D, E) The protein expressions of Collagen I, Collagen III and TGF-β1 and in rat hearts of the two groups (n = 6 per group). (C, F, G) The protein expressions of TGF-β1 and collagen I in NG and HG groups (n = 6 per group). The blots in Fig. 2B were from different parts of the same gel. The blots in Fig. 2C were from different gels. Uncropped protein blots are provided in the supplementary file. Con: control group; DM: diabetes mellitus group; HG: high glucose group. *P < 0.05

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  Fig. 3. Hyperglycemia promoted the expression of CaN/NFATc3 pathway and EZH2 protein. (A-E) The protein expressions of CaN, NFATc3, EZH2 and H3K27me3 in rat hearts of the two groups (n = 6 per group). (F-J) The protein expressions of CaN, NFATc3, EZH2 and H3K27me3 in NRCFs of the two groups (n = 6 per group). The blots in Fig. 3 were from different gels. Con: control group; DM: diabetes mellitus group; HG: high glucose group. *P < 0.05

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  Fig. 4. Role of CaN/NFATc3 pathway in hyperglycemia-induced myocardial fibrosis. (A) Representative western blotting bands of Collagen I, Collagen III and TGF-β1; (B-D) Analysis of the expression of Collagen I, Collagen III and TGF-β1 among different groups. The blots for Collagen III and TGF-β1 in Fig. 4A were from different parts in the same gels; The blots for Collagen I were from different gels. Con: normal glucose group; Con + CsA: normal glucose group with CsA; DM: high glucose group; DM + CsA: high glucose group with CsA. n = 5 per group, *P < 0.05

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  Fig. 5. Role of CaN/NFATc3 on EZH2 in hyperglycemia. (A-B) the expression of CaN protein levels after use of CsA (n = 5 per group); (C) the expression of NFATc3 protein levels after the use of CaN inhibitors (n = 5 per group); (D) the expression of EZH2 protein expression after using of CsA (n = 5 per group); (E) the expression of H3K27me3 protein expression after using of CsA (n = 5 per group); (F) the nuclear translocation of NFATc3. The NRCFs were labeled with anti-NFATc3 (green) and DAPI (blue) (n = 3 per group). The blots in Fig. 5A were from different gels. Con: normal glucose group; Con + CsA: normal glucose group with CsA; DM: high glucose group; DM + CsA: high glucose group with CsA. *P < 0.05

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  Fig. 6. Role of EZH2 in hyperglycemia-mediated myocardial fibrosis in vitro. (A) Representative Western blotting bands of Collagen I, Collagen III and TGF-β1 after using GSK126; (B-D) Analysis of the expression of Collagen I, Collagen III and TGF-β1 among different groups. (E) Schematic depicting the role of CaN/NFATc3/EZH2 pathway in diabetes-induced myocardial fibrosis. The blots in Fig. 6A were from different gels. Con: normal glucose group; Con + EZH2: normal glucose group with EZH2; DM: high glucose group; DM + EZH2: high glucose group with EZH2. n = 3–5 per group, *P < 0.05