ANO1 relieves pressure overload-induced myocardial fibrosis in mice by inhibiting TGF-β/Smad3 signaling pathway.

Abstract

The aim of this study was to measure the expression of anoctamin 1 (ANO1) in myocardial tissues of mice with pressure overload-induced myocardial fibrosis, and to further investigate the effect of ANO1 on myocardial fibrosis in mice and its mechanism. A total of 40 male C57/B6 mice aged 6-8 weeks old were divided into 2 groups using a random number table, namely sham operation group (Sham group, n=20) and thoracic aortic constriction group (TAC group, n=20). Meanwhile, 20 ANO1 transgenic (TG) mice aged 6-8 weeks old were enrolled for TAC as TAC + ANO1 TG group. At 8 weeks after TAC, ejection fraction (EF%) and fraction shortening (FS%) in each group of mice were detected via echocardiography. Western blotting and immunofluorescence staining assays were conducted to measure the protein expression of ANO1 in myocardial tissues of mice in each group. The pathological changes in myocardial tissues of mice were evaluated through hematoxylin-eosin (H&E) staining. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay was performed to measure the messenger ribonucleic acid (mRNA) expression levels of hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in myocardial tissues of mice in each group. The deposition of collagen fibers in heart tissues was determined by Masson staining assay. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect the apoptosis of myocardial cells and fibroblasts in heart tissues. Additionally, the protein expressions of oxidative stress markers superoxide dismutase 1 (SOD1) and 4-hydroxynonenal (4-HNE) in myocardial tissues were detected as well. Finally, Western blotting was employed to detect the effect of ANO1 overexpression on the expression of transforming growth factor-β (TGF-β)/Smad3 signaling pathway-related proteins in myocardial tissues of mice. At 8 weeks after TAC, ANO1 expression was overtly reduced in myocardial tissues of mice (p<0.05). Echocardiographic results showed that ANO1 overexpression significantly alleviated TAC-induced cardiac function deterioration in mice (p<0.05). The mRNA expression levels of ANP and BNP in myocardial tissues of TAC + ANO1 TG group were evidently lower than those in TAC group (p<0.05). Meanwhile, myocardial interstitial collagen deposition was significantly ameliorated in TAC + ANO1 TG group compared with TAC group (p<0.05). ANO1 overexpression notably mitigated the apoptosis of myocardial cells and oxidative stress in mice with cardiac pressure overload (p<0.05). Western blotting results further indicated that after overexpression of ANO1, the protein levels of TGF-β and phosphorylated Smad3 (p-Smad3) were significantly inhibited in mice undergoing TAC (p<0.05). In the case of cardiac pressure overload in mice, ANO1 is lowly expressed in myocardial tissues. Meanwhile, its overexpression is able to attenuate pressure overload-induced myocardial fibrosis in mice by repressing the TGF-β/smad3 signaling pathway. All our findings indicate that ANO1 can serve as a potential gene target for the treatment of myocardial fibrosis in the future.