BMAL1 attenuates myocardial infarction-induced fibrosis via suppressing p-SMAD3/SMAD3 in TGF-β1 pathway

Figures

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  Fig. 1. BMAL1 is downregulated in MI mice and TGF-β1-treated-mCFs. (A) Western blot analysis of Col1, BMAL1 and α-SMA protein levels in two different mice groups. (B) Statistical analysis of relative protein expressions in (A) (n = 3). (C) Immunohistochemical staining of BMAL1 from the heart of mice in Sham and MI group. (D) The relative IOD/area of BMAL1 in (C) (n = 6). (E) Western blot analysis of fibrosis-related protein levels in two different mCFs groups. (F) mRNA levels of Col1, BMAL1 and α-SMA in TGF-β1–stimulated mCFs (n = 3). (G) Bar charts of relative expressions of Col1, BMAL1 and α-SMA in (E) (n = 3). Col1: COL1A1. Data are presented as means ± SD. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005).

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  Fig. 2. BMAL1 suppressed differentiation, proliferation and migration in TGF-β1–stimulated mCFs. (A) Fn, Col1A1 and α-SMA proteins detected in four different mCFs groups (n = 3). (B) Statistical analysis of Fn, Col1A1 and α-SMA in (A). (C) Quantification of Fn, Col1A1 and α-SMA mRNA levels in four different mCFs groups (n = 3). (D) Representative images of the 0-h and 24-h wound healing assay (n = 3). (E) Statistical graphs of wound closure in (D). (F) Transwell assay in four different groups (n = 3). (G) Count of migrated cells in (F). (H) EdU proliferation assay (Red fluorescence indicates proliferating cells, blue fluorescence indicates cell nuclei) (n = 3). (I) Statistical graphs of EdU assay in (H). (J) CCK-8 assay (n = 3). Fn: fibronectin, Col1: COL1A1. Group1: NC, Group2: oe-BMAL1, Group3: TGF-β1, Group4: oe-BMAL1 +TGF-β1. Data are presented as means ± SD. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001).

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  Fig. 3. Silenced expression of BMAL1 demoted the differentiation of mCFs. (A) Protein expression levels of si#BMAL1 compared with si#NC (n = 3). (B) Western blot of Fn, Col1 and α-SMA in three different groups (n = 3). (C) Wound healing assay (n = 3). (D) Statistical graphs of wound healing assay in (C). (E) Transwell assay (n = 3). (F) Statistical graphs of Transwell assay in (E). (G) The relative expressions of BMAL1 in (A). (H) The relative expressions of Fn, Col1 and α-SMA in (B). (I) mRNA expression levels of BMAL1 in three different mice groups (n = 3). (J) mRNA levels of Fn, Col1 and α-SMA in three different mice groups (n = 3). (K) EdU assay (n = 3). (L) Statistical graphs of EdU assay in (K). (M) CCK-8 assay (n = 3). Fn: fibronectin, Col1: COL1A1. Group 1: TGF-β1-si#NC, Group 2: TGF-β1-si#1, Group 3: TGF-β1-si#2. Data are presented as means ± SD. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗P < 0.0001).

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  Fig. 4. BMAL1 confers a protective role for MI by improving survival and preserving cardiac function. (A) Survival curves following four different interventions (n = 6). (B) Heart Rate, Bone Length and Body Weight of mice following different interventions (n = 6). (C) Representative image of echocardiography following different interventions (n = 6) (D) Cardiac function analysis following different interventions (n = 6). AAV means AAV-BMAL1. Data are presented as means ± SD. (ns, not significant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001).

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  Fig. 5. BMAL1 overexpression attenuates myocardial fibrosis in vivo. (A) H&E and MASSON staining in cardiac tissue. (B) Statistical charts of fibrosis and left wall thickness in (A). (C) Protein expression levels of Fn, Col1, BMAL1 and α-SMA (n = 3). (D) The relative expressions of Fn, Col1, BMAL1 and α-SMA in (C). Immunohistochemical staining of Fn, COL1 and α-SMA from the heart of following AAV treatment. (F) The relative IOD/area of Fn, COL1 and α-SMA in (E) (n = 6). Fn: fibronectin, Col1: COL1A1. Data are presented as means ± SD. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001).

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  Fig. 6. BMAL1 overexpression attenuate the classical TGF-β pathway by blocking the expression in SMAD3. (A) SMAD3 and p-SMAD3 protein expressions under BMAL1 overexpression and TGF-β1 stimulation. (B) AKT and p-AKT protein expressions under different interventions. (C) SMAD3 and p-SMAD3 protein expressions in mouse cardiac tissue following AAV-mediated BMAL1 overexpression. (D) AKT and p-AKT protein expressions in mouse cardiac tissue under different interventions. (E) Western blot of SMAD3 and p-SMAD3 under activin A treatment. (F) Ratios of p-SMAD3/SMAD3 in (A) (n = 3). (G) Ratios of p-AKT/AKT in (B) (n = 3). (H) Ratios of p-SMAD3/SMAD3 in (C) (n = 3). (I) Ratios of p-AKT/AKT in (D) (n = 3). (J) Western blot analysis of Fn, Col1 and α-SMA under activin A treatment. (K) Ratios of p-SMAD3/SMAD3 in (E) (n = 3). (L) Ratios of Fn, COL1 and α-SMA in (I) (n = 3). Fn: fibronectin, Col1: COL1A1. Data are presented as means ± SD. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001).

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  Fig. 7. The schematical illustration of BMAL1 involving in the mechanism of myocardial infarction. Following myocardial infarction, BMAL1 expression in cardiac fibroblasts is significantly reduced. This reduction diminishes the suppression of SMAD3 phosphorylation by inhibiting SMAD7 expression, resulting in elevated Fibronectin, Col1A1 and α-SMA levels, which ultimately drives the differentiation of fibroblasts into myofibroblasts. Image was created with BioRender.