Irisin inhibits high glucose‐induced endothelial‐to‐mesenchymal transition and exerts a dose‐dependent bidirectional effect on diabetic cardiomyopathy

Figures

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  Figure 1. Effect of r‐irisin on myocardial remodelling in mice with STZ‐induced diabetes. A1: Heart size comparison (scale bar: 3 mm). A2: Representative histologic cross‐sectional anatomy at the papillary muscle level (scale bar: 3 mm). A3: Representative haematoxylin and eosin staining (H&E) of left ventricular (LV) longitudinal sections (scale bar: 20 μm). A4: Representative H&E staining of LV transverse sections (scale bar: 20 μm). (B) Lung weight/tibial length (LW/TL) ratios. (C) Heart weight/tibial length (HW/TL) ratios. (D) Cross‐sectional area of cardiomyocytes quantified from H&E‐stained sections. Control: normal mice treated with PBS. DM: diabetic mice treated with PBS. Low‐dose Irisin: diabetic mice treated with low dose of r‐irisin. High‐dose Irisin: diabetic mice treated with high dose of r‐irisin. Results shown are means ± S.D.; *P < 0.05.

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  Figure 2. Effect of r‐irisin on diabetes‐induced myocardial dysfunction. A1: Representative 2D echocardiograms. A2: Representative M‐mode echocardiograms. A3: Representative pulsed‐wave Doppler echocardiograms of mitral inflow. A4: Representative tissue Doppler echocardiograms. (B) LV ejection fraction (LVEF). (C) Fractional shortening (FS). (D) Early to late mitral flow (E/A). (E): E′/A′. (F): LV end‐diastolic dimension (LVEDd). (G) LV posterior wall thickness at diastole (LVPWd). Control: normal mice treated with PBS. DM: diabetic mice treated with PBS. Low‐dose Irisin: diabetic mice treated with low dose of r‐irisin. High‐dose Irisin: diabetic mice treated with high dose of r‐irisin. Results are shown as means ± S.D.; *P < 0.05.

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  Figure 3. Effect of r‐irisin on diabetes‐induced myocardial fibrosis. A1‐A2: Representative Masson's trichrome staining of the myocardium (scale bar: 30 μm). A3‐A6: Representative Picrosirius red staining of the myocardium (scale bar: 50 μm). (B) Quantified intramyocardial fibrosis area. (C) Quantified perivascular fibrosis area. Control: normal mice treated with PBS. DM: diabetic mice treated with PBS. Low‐dose Irisin: diabetic mice treated with low dose of r‐irisin. High‐dose Irisin: diabetic mice treated with high dose of r‐irisin. Results are means ± S.D.; *P < 0.05.

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  Figure 4. Effect of r‐irisin on collagen I and III levels in mice with STZ‐induced diabetes. (A) Representative immunohistochemical staining of collagens I (A1‐A2) and III (A3‐A4) (scale bar: 30 μm). (B–C) Quantification of intramyocardial (B) and perivascular (C) areas positive for collagen I. (D–E) Quantification of intramyocardial (D) and perivascular (E) areas positive for collagen III. Control: normal mice treated with PBS. DM: diabetic mice treated with PBS. Low‐dose Irisin: diabetic mice treated with low dose of r‐irisin. High‐dose Irisin: diabetic mice treated with high dose of r‐irisin. Results shown are means ± S.D.; *P < 0.05.

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  Figure 5. Effect of r‐irisin on high glucose (HG) induced EndMT in vivo and in vitro. (A) Double immunofluorescence staining of antibodies to CD31 (Red) with antibodies to α‐SMA (green) in coronary arterioles of all groups mouse. Colocalization of CD31 with α‐SMA expression in coronary arterioles is shown in yellow. DAPI (Blue) was used to stain nucleus. Scale bars = 30 μm. (B) Morphological change in HUVECs after HG exposure with or without r‐irisin treatment, which results in a fibroblast‐like spindle‐shaped form (scale bar: 100 μm). (C) Double immunofluorescence staining with antibodies against α‐SMA (green) and VE‐cadherin (red) (scale bar: 40 μm). (D) Double immunofluorescence staining with antibodies against CD31 (green) and S100A4/FSP‐1 (red) (scale bar: 40 μm). Nuclei were counterstained with DAPI (blue in A, C and D). E‐K: Western blotting analysis (E) and quantification of the protein expression of S100A4/FSP‐1(F), α‐SMA(G), vimentin(H), CD31(I), VE‐cadherin(J) and collagen I(K). NG (normal glucose): 5 mmol/l glucose. HG: 33 mmol/l glucose. HG+Irisin (2 nmol/l): 33 mmol/l glucose + 2 nmol/l r‐irisin. HG+Irisin (20 nmol/l): 33 mmol/l glucose + 20 nmol/l r‐irisin. HG+Irisin (40…

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  Figure 6. Effect of r‐irisin on cardiac fibroblast (CF) proliferation and migration. (A) EDU assay of HG/r‐irisin induced CF proliferation (scale bar: 40 μm). (B) Percentages of EDU‐positive cells. (C) Flow cytometry performed with propidium iodide (PI) staining to analyse CF proliferation. (D) Analysis of cell cycle distribution. (E) CFs were cultured in Transwells (pore size, 8 μm) for 12 hrs, and then, the CFs on the external surface of the Transwells were stained with crystal violet and photographed under a microscope. (F) Quantification of CF chemotaxis and migration towards HG/r‐irisin. (G) Western blotting analysis of the expression of collagens I and III in cells treated with different doses of irisin. NG: 5 mmol/l glucose. NG+Irisin (20 nmol/l): 5 mmol/l glucose + 20 nmol/l r‐irisin. NG+Irisin (40 nmol/l): 5 mmol/l glucose + 40 nmol/l r‐irisin. HG: 33 mmol/l glucose. HG+Irisin (20 nmol/l): 33 mmol/l glucose + 20 nmol/l r‐irisin. HG+Irisin (40 nmol/l): 33 mmol/l glucose + 40 nmol/l r‐irisin. Results are shown as means ± S.D.; *P < 0.05.

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  Figure 7. Effect of r‐irisin on Smad expression/phosphorylation in HUVECs and matrix metalloproteinase (MMP) expression in CFs. (A–B) Western blotting analysis (A) and quantification (B) of Smad7, p‐Smad2/t‐Smad2 and p‐Smad3/t‐Smad3 levels in HUVECs after HG exposure with or without r‐irisin treatment. (C) CFs were stimulated with 0, 2, 20, 40 or 80 nmol/l r‐irisin in NG medium, and then, MMP‐2 and MMP‐9 expression was analysed through Western blotting. (D) Quantification of MMP‐2 and MMP‐9 expression. (E–F) Western blotting analysis (E) and quantification (F) of MMP‐2 and MMP‐9 expression in CFs after NG exposure or after HG exposure with or without r‐irisin at 0, 40 or 80 nmol/l. NG: 5 mmol/l glucose. HG: 33 mmol/l glucose. HG+Irisin (2 nmol/l): 33 mmol/l glucose + 2 nmol/l r‐irisin. HG+Irisin (20 nmol/l): 33 mmol/l glucose + 20 nmol/l r‐irisin. HG+Irisin (40 nmol/l): 33 mmol/l glucose + 40 nmol/l r‐irisin. HG+Irisin (80 nmol/l): 33 mmol/l glucose + 80 nmol/l r‐irisin. Results are mean ± S.D.; *P < 0.05.

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  Figure 8. R‐irisin intensifies HG‐induced activation of p38MAPK and ERK in CFs. (A‐B) Western blotting analysis (A) and quanti fication (B) of p‐p38/t‐p38 and p‐ERK/t‐ERK levels in CFs at the indicated time‐points. (C–D) Western blotting analysis (C) and quantification (D) of p‐p38/t‐p38 and p‐ERK/t‐ERK levels in CFs after treatment with r‐irisin at 0, 2, 20, 40 nmol/l in NG medium. (E–F) Western blotting analy sis (E) and quantification (F) of p‐p38/t‐p38 and p‐ERK/t‐ERK levels in CFs after NG exposure or after HG exposure with r‐irisin treatment at 0, 20 or 40 nmol/l. G‐H: Western blotting analysis (G) and quantification (H) of MMP‐2 and MMP‐9 expression in CFs after NG or HG/r‐irisin treatment with PD or SB. NG: 5 mmol/l glucose. HG: 33 mmol/l glucose. HG+Irisin (20 nmol/l): 33 mmol/l glucose + 20 nmol/l r‐irisin.HG+Irisin (40 nmol/l): 33 mmol/l glucose + 40 nmol/l r‐irisin. Irisin+PD: 40 nmol/l r‐irisin with ERK inhi bitor PD98059. Irisin+SB: 40 nmol/l r‐irisin with p38 inhibitor SB203580. Results are shown as means ± S.D.; *P < 0.05.