Choline Attenuates Cardiac Fibrosis by Inhibiting p38MAPK Signaling Possibly by Acting on M3 Muscarinic Acetylcholine Receptor

Figures

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  Figure 1. Protein level of M3 muscarinic acetylcholine receptor (M3R) in transforming growth factor beta 1 (TGF-β1)-induced cardiac fibroblasts (CF). (A) Protein level of M3R in CF and cardiomyocytes (CM). n = 3. (B) Effect of TGF-β1 (20 ng/ml, 48 h) on messenger RNA level of TGF-β1. **p < 0.01 vs. Ctrl, n = 3. (C) Effect of TGF-β1 (20 ng/ml, 48 h) on mRNA level of collagen I. ***p < 0.001 vs. Ctrl, n = 3. (D) Effect of TGF-β1 (20 ng/ml, 48 h) on protein level of M3R in CF. *p < 0.05 vs. Ctrl, n = 4.

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  Figure 2. Effects of choline and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) on transforming growth factor beta 1 (TGF-β1)-induced collagen secretion. (A) Effects of different concentrations of choline (1, 5, 10 mM) on protein level of collagen I. ***p < 0.001 vs. Ctrl, ## p < 0.01 vs. TGF-β1, n = 5. (B) Immunofluorescence for collagen I in cardiac fibroblasts (CF). Images were obtained using fluorescence microscopy. Blue fluorescence indicates 4′,6-diamidino-2-phenylindole, green fluorescence indicates collagen I, scale bars: 50 μm. *p < 0.05 vs. Ctrl, # p < 0.05 vs. TGF-β1, n = 3. (C) Effect of choline on collagen I protein level in the different experimental groups. *p < 0.05 vs. Ctrl, ## p < 0.01 vs. TGF-β1, && p < 0.01 vs. choline, n = 7. (D) Effect of choline on collagen III protein level in the different experimental groups. *p < 0.05 vs. Ctrl, # p < 0.05 vs. TGF-β1, & p < 0.05 vs. choline, n = 6. (E) Effects of choline (1 mM) and 4-DAMP (3 nM) on protein level of M3 muscarinic acetylcholine receptor (M3R) in TGF-β1-treated CF. *p < 0.05 vs. Ctrl, # p < 0.05 vs. TGF-β1, n = 5. (F) Protein level of M3R after transfecting with three fragments of M3R-siRNA. *p < 0.05 vs.…

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  Figure 3. Effects of choline on cardiac function of transverse aortic constriction (TAC) mice model. (A) Representative hearts from sham, TAC, and the mice induced by TAC treated with choline (14 mg/kg per day) and 4-DAMP (0.7 μg/kg/day) for 8 weeks. (B) The ratio of heart weight/body weight (BW). (C) The ratio of left ventricular weight (LVW)/BW. (D) The ratio of lung weight/body weight. (E) Representative echocardiographic images of mouse hearts in each group. (F) Left ventricular ejection fraction (LVEF). (G) Systolic left ventricular internal diameter (LVID, s). (H) Diastolic left ventricular internal diameter (LVID, d). *p < 0.05, **p< 0.01 vs. sham, # p < 0.05, ## p < 0.01 vs. TAC, & p < 0.05, && p < 0.01 vs. choline, n = 6.

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  Figure 4. Effects of choline on transverse aortic constriction (TAC)-induced myocardial fibrosis. (A) Representative heart section with Masson staining in the experimental groups, scale bar: 100 μm. **p < 0.01 vs. sham, ## p < 0.01 vs. TAC, && p < 0.01 vs. choline, n = 5. (B) Effects of choline and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) on the protein level of collagen I. *p < 0.05 vs. sham, # p < 0.05 vs. TAC, & p < 0.05 vs. choline, n = 8. (C) Effects of choline and 4-DAMP on the protein level of collagen III. **p < 0.01 vs. sham, ## p < 0.01 vs. TAC, n = 8. (D) Cardiac M3 muscarinic acetylcholine receptor protein level after TAC surgery and choline or 4-DAMP treatment. *p < 0.05 vs. sham, ## p < 0.01 vs.TAC, && p < 0.01 vs. choline, n = 8.

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  Figure 5. Effects of choline and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) on protein levels of transforming growth factor beta 1 (TGF-β1)/Smad2/3. (A) Effect of choline and 4-DAMP on TGF-β1 protein level in TGF-β1-induced cardiac fibroblasts (CF). *p < 0.01 vs. Ctrl, ### p < 0.001 vs. TGF-β1, n = 5. (B) Effect of choline and 4-DAMP on p-Smad2/3 protein level in TGF-β1-induced CF. ***p < 0.001 vs. Ctrl, # p < 0.05 vs. TGF-β1, & p < 0.05 vs. choline, n = 6. (C) Effects of choline and 4-DAMP on the protein level of TGF-β1 in transverse aortic constriction (TAC) mice hearts. *p < 0.05 vs. sham, ## p < 0.01 vs. TAC, & p < 0.05 vs. choline, n = 6. (D) Effects of choline and 4-DAMP on the protein level of p-smad2/3 in TAC mice hearts. *p < 0.5 vs. sham, # p < 0.05 vs. TAC, n = 4.

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  Figure 6. Effect of choline and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) on protein levels of p38MAPK and ERK1/2. (A) Effect of choline and 4-DAMP on p-p38 protein level in transforming growth factor beta 1 (TGF-β1)-induced cardiac fibroblasts (CF). **p < 0.01 vs. Ctrl, ## p < 0.01 vs. TGF-β1, && p < 0.01 vs. choline, n = 5. (B) Effect of choline and 4-DAMP on p-ERK1/2 protein level in TGF-β1-induced CF. **p < 0.01 vs. Ctrl, ## p < 0.01 vs. TGF-β1, n = 6. (C) Effects of choline and 4-DAMP on the protein level of p-p38 in transverse aortic constriction (TAC) mice hearts. *p < 0.05 vs. sham, # p < 0.05 vs. TAC, & p < 0.05 vs. choline, n = 5. (D) Effects of choline and 4-DAMP on the protein level of p-ERK in TAC mice hearts. *p < 0.05 vs. sham, # P< 0.05 vs. TAC, n = 4.