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

Abstract

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.

Keywords: Cardiac fibrosis; M3 subtype of muscarinic acetylcholine receptor (M3 receptor); beta-site app cleaving enzyme 1 (BACE1); microRNA-29b (miR-29b).

Figure 1

The anti-fibrotic effects of miR-29b and it participates in the role of activating M₃ receptor in cardiac fibroblasts. (A) qRT-PCR was performed to detect the relative expression of miR-29b in TGF-β1-induced cardiac fibroblasts when choline and 4-DAMP stimulation (n=6). (B-G) Relative protein level of collagen I (B, n=6; E, n=6), α-SMA (C, n=6; F, n=4), and CTGF (D, n=7; G, n=6) in cardiac fibroblasts detected by western blot. ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05. GAPDH was included as a control. The data are expressed as the mean ± SEM. miR-29b, microRNA-29b; TGF-β1, transforming growth factor beta 1; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; AMO-miR-29b, miR-29b inhibitor; M3 receptor, M3 subtype of muscarinic acetylcholine receptor; qRTPCR, quantitative real-time reverse transcription polymerase chain reaction; SEM, standard error of mean.

Figure 2

Inhibition of miR-29b after M₃ receptor activation increases the protein level of cardiac fibrosis markers. Relative protein level of collagen I (A, n=6), α-SMA (B, n=4), and CTGF (C, n=7) in cardiac fibroblasts detected by western blot. ***, P<0.001; **, P<0.01; *, P<0.05. GAPDH was included as a control. The data are expressed as the mean ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TGF-β1, transforming growth factor beta 1; AMO-miR-29b, miR-29b inhibitor; NC, negative control; miR-29b, microRNA-29b; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; M₃ receptor, M₃ subtype of muscarinic acetylcholine receptor; SEM, standard error of mean.

Figure 3

Inhibition of miR-29b reverses the anti-fibrotic effects of activating M₃ receptor and promotes the expression of collagen I and α-SMA. (A,E) Relative collagen I protein level in cardiac fibroblasts detected by immunofluorescence. Scale bar =50 µm. (B,F) Statistical results of immunofluorescence of collagen I (B, n=6; F, n=3). (C,G) Relative α-SMA protein level in cardiac fibroblasts detected by immunofluorescence. Scale bar =50 µm. (D,H) Statistical results of immunofluorescence of α-SMA (D, n=6; H, n=3). ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05. The data are expressed as the mean ± SEM. TGF-β1, transforming growth factor beta 1; AMO-miR-29b, miR-29b inhibitor; miR-29b, microRNA-29b; NC, negative control; α-SMA, α-smooth muscle actin; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; M₃ receptor, M₃ subtype of muscarinic acetylcholine receptor; SEM, standard error of mean.

Figure 4

Inhibition of miR-29b after M₃ receptor activation promotes the migration ability of cardiac fibroblasts. (A) The migration rate of cardiac fibroblasts detected by scratch test. Scale bar =500 µm. (B) Statistical results about the migration rate of cardiac fibroblasts, n=4. (C) The migration ability of cardiac fibroblasts was detected by transwell (crystal violet staining). Scale bar =150 µm. (D) Statistical results about migration ability of cardiac fibroblasts, n=4. ****, P<0.0001; ***, P<0.001; **, P<0.01. The data are expressed as the mean ± SEM. TGF-β1, transforming growth factor beta 1; AMO-miR-29b, miR-29b inhibitor; miR-29b, microRNA-29b; NC, negative control; M₃ receptor, M₃ subtype of muscarinic acetylcholine receptor; SEM, standard error of mean.

Figure 5

MiR-29b participates in the anti-fibrotic effects after M₃ receptor activation through targeting BACE1. (A) Screening target genes of miR-29b by online prediction websites: miRDB, miRTarBase, TargetScan, and PicTar. (B) The binding sites of miR-29b and BACE1 in different species. (C-F) Relative protein level of BACE1 in cardiac fibroblasts detected by western blot (C, n=4; D, n=4; E, n=5; F, n=4) (G) qRT-PCR assay was used to examine the expression of miR-29b in the presence of BACE1 overexpression, n=8. (H) Relative protein level of collagen I in cardiac fibroblasts detected by western blot, n=6. ***, P<0.001; **, P<0.01; *, P<0.05. The data are expressed as the mean ± SEM. Student’s t-test was used for two-group comparisons. BACE1, beta-site app cleaving enzyme 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; TGF-β1, transforming growth factor beta 1; miR-29b, microRNA-29b; AMO-miR-29b, miR-29b inhibitor; 4-DAMP, 4-diphenylacetoxy-Nmethylpiperidine methiodide; M₃ receptor, M₃ subtype of muscarinic acetylcholine receptor; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.