Over-Expression of Inhibitor of Differentiation 2 Attenuates Post-Infarct Cardiac Fibrosis Through Inhibition of TGF-β1/Smad3/HIF-1α/IL-11 Signaling Pathway

Figures

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  Figure 1. Myocardial Fibrosis and Id2 expression in rat hearts post-MI. (A) Masson’s trichrome staining post-MI after 2 weeks (n = 6). Blue staining represents fiber tissue. Scale bars represent 100 um. (B) HE staining post-MI after 2 weeks (n = 6). Scale bars represent 100 um. (C) Immunofluorescence images of myofibroblasts derived from CFs (n = 6). Green, vimentin; red, α-SMA; blue, nuclei. Scale bars represent 100 um (n = 6). (D) Immunofluorescence images show Id2 expressions in MI rats and sham rats. Red, Id2; blue, nuclei. Scale bars represent 100 um. (E, F) Id2 protein levels detected by western blot analysis after MI. (G, H) Rats were intramyocardially injected with Ad-GFP-Id2 or Ad-GFP for 3 days, 7 days, 2 weeks and 4 weeks. The fluorescence of Ad-GFP and Ad-GFP-Id2 in injected area of rat heart after transfected 2 weeks (G). The protein levels of Id2 in left ventricular myocardium were examined by Western Blot (H). h, hours; d, days; w, weeks. β-actin was used as the loading control. Data represent means ± SEM (n = 3). *,P < 0.05 VS Sham group, 0 h or GFP group.

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  Figure 2. Id2 improved cardiac structure and function. (A–E) Four groups’ comparison represented Id2 can ameliorate cardiac structure by reducing the HW/BW or HW/LW ratio index. (F–L) Densitometric analysis of the data demonstrated a significantly improvement of cardiac function in Id2 groups. BW body weight, HW heart weight, Lung weight, FS fractional shortening, LVEF left ventricular ejection fraction, LVESD left ventricular end-systolic diameter, LVEDD left ventricular end-diastolic diameter, LVESV left ventricular end-systolic volume, LVEDV left ventricular end-diastolic volume, HR heart rate, Bp blood pressure. Data represent means ± SEM. Heart weight index (n = 10), Transthoracic echocardiography measurements (n = 7). *P <0.05, vs sham group; #P <0.05, vs MI group and GFP group.

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  Figure 3. Adenoviral Id2 delivery ameliorated cardiac remodeling post-MI. (A, B) Representative Masson’s trichrome staining (A) and histogram of connective tissue percentage (B) of hearts. Blue staining indicates connective tissue (n = 6). Scale bars represent 100 um. (C) Representative HE staining images (n = 6). Scale bars indicate 100 um. (D) immunofluorescence images of myofibroblasts derived from CFs (n = 6). Green, vimentin; red, α-SMA; blue, nuclei. Scale bars represent 100 um. (E, F) Western blot analysis of protein expressions in rat hearts among the four groups (n = 3). β-actin was used as the loading control. Data represent means ± SEM. *,P < 0.05, vs sham group; # ,P < 0.05, vs MI group and GFP group; in panel (E) including α-SMA, Collagen I, Collagen III in both MI and GFP group; in panel (E) including MMP2, TIMP-1 in both MI and GFP group.

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  Figure 4. NRVMs or CFs treated with hypoxia or TGF-β1. (A–C) Comparison of Id2 in NRVMs and in CFs (n = 3). (D) The proliferation rate of CFs in different hypoxia time (n = 5). (E, F) The mRNA levels of α-SMA (E) and rat Id2 (F) in CFs treated with the multi-hypoxia time (n = 3). (G–J) qRT-PCR and Western blot were performed to detect the mRNA levels of α-SMA, Collagen I, Collagen III in CFs treated with TGF-β1(10 ng/ml) for 24 (G) or hypoxia environment for 12 h (H) and the protein expressions of α-SMA, Collagen I, Collagen III in CFs treated with TGF-β1(10 ng/ml) for 24 h (I) or hypoxia environment for 12 h (J) (n = 3). (K, L) immunofluorescence images of myofibroblasts derived from CFs, which were treated with TGF-β1 (G) and put under hypoxia environment (H), respectively (n = 3). Green, vimentin; red, α-SMA; blue, nuclei. Scale bars represent 100 um. β-actin was used as the loading control. Data represent means ± SEM. *P < 0.05 VS control group or 0 h ; #P < 0.05 VS TGF-β1 group, TGF-β1+GFP group, hypoxia group, or hypoxia+GFP group; in panels (G, I) including α-SMA, Collagen I, Collagen III in both TGF-β1 group and TGF-β1+GFP group; in panels (H, J) including α-SMA, Collagen I,…

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  Figure 5. Id2 inhibited myofibroblast differentiation via TGF-β1/smad3/HIF-1α/IL-11 pathways. (A) The protein levels of TGF-β1, smad3, HIF-1α, IL-11 in rat hearts. (B, C) qRT-PCR and Western blot were performed to detect the mRNA levels (C) or the protein expressions (B) of smad3, HIF-1α, IL-11 in CFs treated with TGF-β1 (10 ng/ml) for 24 h. (D) The mRNA levels of TGF-β1, smad3, HIF-1α, IL-11 in CFs treated with different hypoxia time. (E–F) qRT-PCR and Western blot were performed to detect the mRNA levels (F) or the protein expressions (E) of TGF-β1, smad3, HIF-1α, IL-11 in CFs treated with hypoxia for 12 h. (G) The protein levels of HIF-1α in six groups (n = 3). (H, I) immunofluorescence images of CFs among six groups in vitro (n = 6). Red, HIF-1α; blue, nuclei. Scale bars represent 100 um. β-actin was used as the loading control. Data represent means ± SEM (n = 3). *P < 0.05, vs sham group or control group. #P <0.05, vs MI group, GFP group, TGF-β1 group, TGF-β1+GFP group, hypoxia group, or hypoxia+GFP group; in panel (A) including TGF-β1, smad3, HIF-1α, IL-11 in both MI group and GFP group; in panels (B, C) including smad3, HIF-1α, IL-11 in both TGF-β1 group and TGF-β1+GFP group; in…

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  Figure 6. Id2 reduced carmyocyte apoptosis post-MI. (A, B) Representative TUNEL staining. The apoptotic cells show yellow-brown nuclei, while the normal cells exhibit light blue nuclei. (n = 6). Scale bars represent 100 um. (C) The protein levels of c-caspase3, c-caspase9, bax and bcl-2 in rat hearts (n = 3). (D) The apoptotic cell rate in four groups. (E) Immunofluorescence images of cardiomyocytes among four groups in vivo (n = 6). Red, c-caspase3; blue, nuclei. Scale bars represent 100 um. β-actin was used as the loading control. Data represent means ± SEM. *P <0.05, vs control group. #P <0.05, vs MI group and GFP group; in panel (C) including c-caspase3, c-caspase9, bax and bcl-2 in both MI group and GFP group; in panel (C) including c-caspase3, c-caspase9, and bcl-2 in Id2 group.

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  Figure 7. NRVMs treated with hypoxia. (A–E) Flow cytometry of NRVMs’ apoptosis rate after treated with different hypoxia time (n = 3). (A) 0 h, (B) 4 h, (C) 6 h, (D) 8 h, (E) 12 h. (F) The mRNA levels of rat Id2 in NRVMs along with different hypoxia time. (G) The mRNA levels of bax/bcl, c-caspase3 and c-caspase9 in NRVMs with different hypoxia time. (H. I) qRT-PCR and Western blot were performed to detect the mRNA levels (H) or the protein expressions (I) of bax/bcl, c-caspase3 and c-caspase9 in NRVMs treated with hypoxia for 6 h. (J) The protein level of HIF-1α in NRVMs in four groups. β-actin was used as the loading control. Data represent means ± SEM (n = 3). *P <0.05, vs control groups or 0 h; #P <0.05, vs H groups and H+G groups; in panel (G) including c-caspase3, c-caspase9, bax/bcl-2 in both 6 h and 8 h; in panel (H) including c-caspase3, c-caspase9, bax/bcl-2 in both H group and H+G group; in panel (H) including c-caspase3, c-caspase9, and bax/bcl-2 in Id2 group; in panel (I) including c-caspase3, c-caspase9, bax and bcl-2 in both H group and H+G group; in panel (I) including c-caspase3, c-caspase9, bax, and bcl-2 in Id2 group.