Small ubiquitin-related modifier (SUMO)ylation of SIRT1 mediates (-)-epicatechin inhibited- differentiation of cardiac fibroblasts into myofibroblasts

Figures

• 

  Figure 1.. The effect of EPI on cardiac function and myocardial structure in mice. (A) Representative cardiac echocardiography of the indicated groups. (B-I) LVEF: left ventricular ejection fraction, LVFS: left ventricular fractional shortening, LVIDs: left ventricular systolic internal diameter, LVIDd: left ventricular diastolic internal diameter, LVPWs: Left ventricular posterior wall thickness in systole, LVPWd: left ventricular posterior wall thickness in diastole, IVSs: interventricular septal thickness at end systole, IVSd: interventricular septal thickness at end-diastole. Statistical analysis of LVEF, LVFS, LVIDs, LVIDd, LVPWs, LVPWd, IVSs, IVSd (n = 5). Representative graph of H&E (J) and Masson (K). The magnification is ×200, scale bar = 100 μm. Statistical analysis of collagen deposition (L) and HW/BW index (M). Data are shown as mean ± SEM (n = 6). *p < 0.05, **p < 0.01 compared with control group; #p < 0.05, ##p < 0.01 compared with TAC group.

• 

  Figure 2.. EPI blocks Ang II-induced myofibroblast transformation. (A) CFs stained with α-SMA (red) and DAPI (blue) antibody in each group. (B) Statistical analysis of α-SMA. The magnification is ×200, scale bar = 50 μm. (C) Representative western blot bands of α-SMA, COL I, COL III, TGF-β1 and P-SMAD3 after incubation with EPI or-/and Ang II in CFs. (D-H) Statistical analysis of α-SMA, COL I/COL III, TGF-β1 and P-SMAD3. Data are shown as mean ± SEM (n = 3–6). *p < 0.05, **p < 0.01 compared with the control group; #p < 0.05, ##p < 0.01 compared with the Ang II group.

• 

  Figure 3.. EPI activates SIRT1-SUMO1. Representative images of IHC staining in mice heart with SIRT1 (A) and SUMO1 (C) antibody. The magnification is ×200, scale bar = 50 μm. Statistical analysis of SIRT1 (B) and SUMO1 (D) (n = 5). (E) Representative immunoblots of SIRT1-SUMOylation in CFs (n = 3). (F-G) Representative immunoblots of SIRT1-SUMOylation after incubation with EPI or /and Ang II in CFs (n = 3). (H) Representative western blot bands of SIRT1-SUMO1 after incubation with EPI or /and Ang II in H9C2, CMs and CFs. (I-N) Statistics of SIRT1-SUMO1 expression in H9C2, CMs and CFs. Data are shown as mean ± SEM (n = 6). *p < 0.05, **p < 0.01 compared with the control group; #p < 0.05, ##p < 0.01 compared with the Ang II/TAC group.

• 

  Figure 4.. The effect of SIRT1-SUMO1 on EPI-induced protein levels of P-AKT and P-GSK3β. (A) Representative western blot bands of P-AKT and P-GSK3β. (B-C) Statistics of P-AKT/P-GSK3β expression in CFs. (D) Representative western blot bands of SIRT1-SUMO1, P-AKT, and P-GSK3β after incubation with EPI, Si-SIRT1, and NC in CFs. (E-H) Statistics of SIRT1, SUMO1, P-AKT, and P-GSK3β protein expression levels. Data are shown as mean ± SEM (n = 6). *p < 0.05, **p < 0.01 compared with control group; #p < 0.05, ##p < 0.01 compared with EPI group; &p < 0.05, &&p < 0.01 compared with EPI + Si-SIRT1 group.

• 

  Figure 5.. The effect of SIRT1-SUMO1 on myofibroblast transformation and protein levels of P-AKT/P-GSK3β. (A) Western blot bands of TGF-β1, P-SMAD3, SIRT1, SUMO1, P-AKT and P-GSK3β after incubation with Ang II, EPI, Si-SIRT1, and Si-N in CFs. (B-G) Statistics of TGF-β1, P-SMAD3, SIRT1, SUMO1, P-AKT, and P-GSK3β expression in each group. (H) CFs stained with α-SMA (red) and DAPI (blue) antibody in each group. (I) Statistical analysis of α-SMA. The magnification is ×200, scale bar = 50 μm. Data are shown as mean ± SEM (n = 3–6). ** p< 0.01 compared with control group; #p < 0.05, ##p < 0.01 compared with Ang II group; && p< 0.01 compared with Ang II + EPI group; △ p< 0.05, △△p < 0.01 compared with Ang II + EPI + Si-SIRT1 group.

• 

  Figure 6.. The effect of SP1 on protein levels of SIRT1-SUMO1 /AKT/GSK3β pathway. (A) Representative western blot bands of SP1 in H9C2, CMs, and CFs. (B-D) Statistics of SP1 expression in H9C2, CMs, and CFs. (E) Representative western blot bands of SP1, SIRT1, SUMO1, P-AKT, and P-GSK3β after incubation with Ang II, EPI, and MTM in CFs. (F-J) Statistics of SP1, SIRT1-SUMO1, P-AKT, P-GSK3β expression in CFs. Data are shown as mean ± SEM (n = 6). *p < 0.05, **p < 0.01 compared with control group; #p < 0.05, ##p < 0.01 compared with Ang II group; &p < 0.05, &&p < 0.01 compared with Ang II + EPI group.

• 

  Figure 7.. The effect of SP1 on Ang II-induced myofibroblast transformation. (A) CFs stained with α-SMA (red) and DAPI (blue) antibody in each group. (B) Statistical analysis of α-SMA. The magnification is ×200, scale bar = 50 μm. (C) Western blot bands of α-SMA, COLI/COLIII. (D-F) Statistical analysis of α-SMA, COL I/COL III. Data are shown as mean ± SEM (n = 6). *p < 0.05 compared with control group; #p < 0.05, ##p < 0.01 compared with Ang II group; &p < 0.05, &&p < 0.01 compared with Ang II + EPI group.