M3 subtype of muscarinic acetylcholine receptor inhibits cardiac fibrosis via targeting microRNA-29b/beta-site app cleaving enzyme 1 axis.

Background:Previous studies have confirmed that choline exerts anti-fibrotic effect in the heart by activating the M3 subtype of muscarinic acetylcholine receptor (M3 receptor), but the mechanism remains to be clarified. MicroRNA-29b (miR-29b) plays an important role in the fibrotic process and can directly target collagen to resist myocardial fibrosis. This study investigated whether miR-29b is involved in the anti-fibrotic effect of activating M3 receptor. Methods:Proliferation of cardiac fibroblasts was induced by transforming growth factor (TGF)-β1 in vitro. The expression of miR-29b in cardiac fibroblasts was detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Protein levels of collagens I, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA) and beta-site app cleaving enzyme 1 (BACE1) were determined by Western blot analysis. Fibroblast-myofibroblast transition was identified by immunofluorescence staining. Proliferation and migration of cardiac fibroblasts as indicated by transwell and scratch assays. Results:The expression of miR-29b decreased when treated with TGF-β1 (P=0.0389) and increased after choline stimulated (P=0.0001). Overexpression of miR-29b could reverse the high expression of collagen I (P<0.0001), α-SMA (P=0.0007), and CTGF (P=0.0038) induced by TGF-β1, whereas inhibition of miR-29b had a tendency to even further increase the expression of fibrosis markers. Meanwhile, inhibition of miR-29b could reverse the anti-fibrotic effect of choline, increasing the expression of collagen I (P=0.0040), α-SMA (P=0.0001), and CTGF (P=0.0185), and promoting the fibroblast proliferation and migration. Moreover, BACE1 protein level, increased after TGF-β1 treatment (P=0.0037) and reversed by overexpression of miR-29b (P=0.0493). Choline could reduce the increase of BACE1 induced by TGF-β1 (P=0.0264), and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) increased the expression of BACE1 (P=0.0060). Furthermore, overexpression of BACE1 could reverse the protective effect of miR-29b in cardiac fibrosis, increasing the protein level of collagen I (P=0.0404). Conclusions:The results suggested that M3 receptor activation could exert cardioprotective effects in cardiac fibrosis by mediating miR-29b/BACE1 axis.