CCAAT/enhancer‐binding protein β overexpression alleviates myocardial remodelling by regulating angiotensin‐converting enzyme‐2 expression in diabetes

Figure 1. Functional and pathological features of hearts. (A1 and A2) Representative two‐dimensional echocardiograms and M‐mode echocardiograms. (A3) Representative pulsed‐wave Doppler echocardiograms of mitral inflow. (B–F) Sequential statistics results: LVEF, FS, E/A, LVPWd and LVEDd. (G) Quantitative analysis of cardiomyocyte cell diameter. (H) Heart size (scale bar: 3 mm). (I) Representative cross‐sectional image of histological staining illustrating the anatomy at the papillary muscle level (scale bar: 3 mm). (J) Representative images of H&E‐stained sections (scale bar: 20 μm). Control: normal mice. DM + sh‐N.C.: negative shRNA control in diabetic mice. DM + C/EBPβ: lentivirus‐mediated delivery of C/EBPβ in diabetic mice. DM + sh‐C/EBPβ: lentivirus‐mediated delivery of sh‐C/EBPβ in diabetic mice. DM + valsartan: valsartan treatment in diabetic mice. Data are presented as the means ± SD. *P < 0.05 versus control, #P < 0.05 versus DM + sh‐N.C., &P < 0.05 versus DM + C/EBPβ.

Figure 2. Histological and immunohistochemical analyses of myocardial fibrosis. (A) Masson's trichrome staining (first row; scale bar: 20 μm). (B–D) Immunohistochemical staining and quantitative analysis of collagen III (B, second row; scale bar: 20 μm), collagen I (C, third row; scale bar: 20 μm) and TGF‐β1 expression (D, fourth row; scale bar: 20 μm). Data are presented as the means ± SD. *P < 0.05 versus control; #P < 0.05 versus DM + sh‐N.C.; &P < 0.05 versus DM + C/EBPβ.

Figure 3. Effects of C/EBPβ on collagen deposition and TGF‐β1, MMP‐2 and MMP‐9 expression in vivo and in vitro. Serum MMP‐2 and MMP‐9 levels were measured using enzyme‐linked immunosorbent assay (ELISA). (A–E) Sequential Western blot evaluations of collagen III (A), collagen I (B), MMP‐9 (C), TGF‐β1 (D) and MMP‐2 (E) levels in the myocardium. (F–G) ELISA analysis of serum MMP‐9 and MMP‐2 levels. (H–L) Sequential Western blot evaluations of collagen III (H), collagen I (I), TGF‐β1 (J), MMP‐9 (K) and MMP‐2 (L) levels in primary neonatal mouse CFs. (M and N) MMP‐9 and MMP‐2 activities in cell culture supernatants were assessed using zymography. Control: 5.5 mM glucose. oc: osmotic control, 5.5 mM glucose + 27.5 mM mannose. HG + sh‐N.C.: high glucose (33.3 mM glucose) + negative shRNA control. HG + C/EBPβ: high glucose + lentivirus vector containing C/EBPβ. HG + sh‐C/EBPβ: high glucose + lentivirus vector containing shRNA‐C/EBPβ. HG + valsartan: high glucose + valsartan (10−6 mol/l). Data are presented as the means ± SD. *P < 0.05 versus control; #P < 0.05 versus DM + sh‐N.C. or HG + sh‐N.C.; &P < 0.05 versus DM + C/EBPβ or HG + C/EBPβ.

Figure 4. Effects of C/EBPβ on apoptosis and inflammation. (A) Western blot analysis of Bax and Bcl‐2 expression in the myocardium. (B) Western blot analysis of AT2R expression in vivo. (C) Bax and Bcl‐2 protein expression in H9C2 cells. (D) Western blot analysis of AT2R expression in H9C2 cells. (E–F) Serum interleukin‐6 (IL‐6) and monocyte chemoattractant protein‐1 (MCP‐1) levels were measured using ELISAs. Data are presented as the means ± SD. *P < 0.05 versus control; #P < 0.05 versus DM + sh‐N.C. or HG + sh‐N.C.; &P < 0.05 versus DM + C/EBPβ or HG + C/EBPβ.

Figure 5. C/EBPβ and ACE2 protein expression levels in vivo and in vitro. ACE protein expression level in the myocardium. Western blot analysis of the levels of the C/EBPβ (A) and ACE2 (B) ACE (C) proteins in mouse heart tissue. Levels of C/EBPβ (D) and ACE2 (E) proteins in CFs. Immunohistochemical analysis of myocardial C/EBPβ (F) and ACE2 (G) expression. Data are presented as the means ± SD. *P < 0.05 versus control; #P < 0.05 versus DM + sh‐N.C. or HG + sh‐N.C.; &P < 0.05 versus DM + C/EBPβ or HG + C/EBPβ.

Figure 6. Ang II and Ang(1–7) levels in the myocardium and serum were measured using ELISAs. Western blots of the levels of the ACE, AT1R and MasR proteins in vivo and in vitro. Immunohistochemical analysis of myocardial ACE expression. (A–D) Ang II and Ang(1–7) levels in the myocardium (A and B) and serum (C and D) were measured using ELISAs. (E and F) Western blot analysis of the levels of the AT1R and MasR proteins in mouse heart tissue. (G and H) Levels of the AT1R and MasR proteins in CFs. (I and J) Western blots and immunohistochemical staining for ACE in CFs and in the myocardium (scale bar: 20 μm). Data are presented as the means ± SD. *P < 0.05 versus control; #P < 0.05 versus DM + sh‐N.C.; &P < 0.05 versus DM + C/EBPβ.

Figure 7. Mechanisms by which C/EBPβ and ACE2 respond to the HG treatment in CFs. Cells were divided into sh‐N.C. + si‐N.C., si‐N.C. + C/EBPβ and C/EBPβ + si‐ACE2 groups. (A–D) Western blot analysis of C/EBPβ (A), ACE2 (B), collagen III (C) and TGF‐β1 (D) levels. HG: high glucose, 33.3 mM glucose. sh‐N.C. + si‐N.C.: shRNA negative control + siRNA negative control. si‐N.C. + C/EBPβ: siRNA negative control + lentivirus‐mediated delivery of C/EBPβ. C/EBPβ + si‐ACE2: lentivirus‐mediated delivery of C/EBPβ + siRNA‐ACE2. (E) Chromatin immunoprecipitation (ChIP) analysis of the relationship between C/EBPβ and ACE2. IgG: negative control. H3: positive control. C/EBPβ: C/EBPβ antibody. HG + IgG: high glucose + negative control. HG + C/EBPβ: high glucose + C/EBPβ antibody. Data are presented as the means ± SD. *P < 0.05 versus sh‐N.C. + si‐N.C.; #P < 0.05 versus si‐N.C. + C/EBPβ. δ < 0.05 versus C/EBPβ.