Improving vagal activity ameliorates cardiac fibrosis induced by angiotensin II: in vivo and in vitro

Abstract

Cardiac remodeling is characterized by overactivity of the renin-angiotensin system (RAS) and withdrawal of vagal activity. We hypothesized that improving vagal activity could attenuate cardiac fibrosis induced by angiotensin II (Ang II) in vivo and in vitro. Rats were subjected to abdominal aorta constriction (AAC) with or without pyridostigmine (PYR) (31 mg/kg/d). After 8 weeks, PYR significantly decreased Ang II level, AT1 protein expression, and collagen deposition in cardiac tissue and improved heart rate variability, baroreflex sensitivity and cardiac function, which were abolished by atropine. In vitro, treatment of cardiac fibroblasts (CFs) with Ang II (10(-7) M) increased cell proliferation, migration, transformation, and secretory properties, which were significantly diminished by acetylcholine (ACh, 10(-6) M). Subsequently, Ang II significantly increased collagen type I expression as well as metalloproteinase (MMP)-2 expression and activity. Transforming growth factor (TGF)-β1 expression and Smad3 phosphorylation presented a similar trend. Notably, the knockdown of the acetylcholine M2 receptor by siRNA could abolish ACh anti-fibrotic action. These data implicated cholinesterase inhibitor can increase vagal activity and reduce local Ang II level, and ACh inhibit Ang II pro-fibrotic effects. Our findings suggested that the parasympathetic nervous system can serve as a promising target for cardiac remodeling treatment.

Figures

Figure 1. PYR inhibits cardiac fibrosis and improves cardiac function.

(a) The image shows Masson’s trichrome stained collagen in the cardiac interstitium. Blue parts represent collagen. Bar = 50 μm. (b) Representative LV sections stained with picrosirius red for collagen type I (red) and III (orange yellow) from all groups as indicated. Bar = 25 μm. (c,d) LV tissues were analyzed for collagen type I and III by western blot. (e) MAP, mean arterial pressure. (e) HR, heart rate. (f) LVSP, left ventricular systolic pressure. (h) LVEDP, left ventricular end-diastolic pressure. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control group; #P < 0.05 vs. AAC group; &P < 0.05 vs. PYR group.

Figure 2. PYR decreases Ang II level and improves vagal activity.

(a) Radioimmunoassay was performed to detect Ang II in serum and (b) left ventricular tissues. (c) Western blot and quantitative evaluation of AT1 expression. (d) Baroreflex sensitivity (BRS). (e) Standard deviation of RR Interval (SDNN). (f) Normalized low frequency power (LF power; %). (g) Normalized high frequency power (HF power, %). (h) Absolute values of low/high frequency power ratio (LF/HF). Data are presented as means ± SEM. **P < 0.01 and ***P < 0.001 vs. control group; #P < 0.05 and # #P < 0.01 vs. AAC group; &P < 0.05 and &&P < 0.01 vs. PYR group.

Figure 3. ACh attenuates Ang II-induced collagen production.

(a) Fluorescence micrographs of vimentin, Von Willebrand Factor and desmin. Bar = 25 μm. (b,c) For collagen type I assay, CFs were cultured in the indicated experimental conditions and immunostained with an antibody against collagen I (green), and the histograms showed the corresponding densitometric analyses of the intensity of collagen I fluorescence signal. Bar = 50 μm. (d) Western blot analysis of collagen I expression in CFs. The densitometric analysis of the bands normalized to GAPDH is reported in the histograms. (e) The content of hydroxyproline was measured by using a bioluminescent assay. (f) MMP-2 activity was measured by gelatin zymography. (g) Western blot and quantitative evaluation of MMP-2 expression. Data are presented as means ± SEM. **P < 0.01 vs. control group; #P < 0.05 and # #P < 0.01 vs. Ang II group; &P < 0.05 and &&P < 0.01vs. ACh group.

Figure 4. ACh suppresses Ang II-induced proliferation and viability.

(a) Representative photomicrographs of 5-ethynil-2-deoxyuridine (EdU) staining (left panel) and corresponding total cell photomicrographs (middle panel). Proliferative cell nuclei were labeled with EdU (green). Cell nuclei were labeled by using PI (Red). (b) Effect of ACh on cell proliferation as determined by EdU staining. Quantitative data showing the percentage of EdU-positive cells (number of green vs. red nuclei). Bar = 50 μm. Data are presented as means ± SEM. *P < 0.05 and **P < 0.01 vs. control group; ##P < 0.01 vs. Ang II group; &P < 0.05 vs. ACh group.

Figure 5. ACh inhibits CFs migration and transformation induced by Ang II.

(a,b) Fluorescence immunohistochemistry using a specific α-SMA first antibody following by a second antibody conjugated to FITC was performed to demonstrate fibroblast transformation induced by Ang II. Nuclei were stained with PI. Bar = 25 μm. (c,d) Representative pictures of cell migration detected by transwell migration assay. Bar = 50 μm. Data are presented as means ± SEM. **P < 0.01 and ***P < 0.001 vs. control; ##P < 0.001 vs. Ang II; &P < 0.05 and &&P < 0.01 vs. ACh.

Figure 6. ACh inhibits the Ang II-induced activation of the TGF-β1/Smad3 pathway.

(a) Immunofluroescence images show that ACh blocks Smad3 nuclear translocation induced by Ang II. (b,c) Western blot evaluated expression of TGF-β1 and P-Smad3. Bar = 25 μm. Data are presented as means ± SEM. *P < 0.05 and **P < 0.01 vs. control; #P < 0.05 and ##P < 0.01 vs. Ang II; &P < 0.05 and &&P < 0.01 vs. ACh.

Figure 7. M2 AChR knockdown abrogates ACh anti-fibrotic effect.

(a) Western blot was performed to assess M2 AChR knockdown. (b) Hydroxyproline content was measured by using a bioluminescent assay. (c) M2 AChR siRNA transfection increases collagen I contents in the presence of ACh in Ang II treated cells. Bar = 50 μm. (d) M2 AChR siRNA transfection increases fibroblasts migration. Bar = 50 μm. (e) Cell proliferation was measured as the percentage of EdU-positive cells. Bar = 50 μm. (f) M2 AChR siRNA transfection increases fibroblast transformation. Bar = 25 μm. (g) Expression of TGF-β1, P-Smad3, and collagen I. Data are presented as means ± SEM. *P < 0.05 and **P < 0.01 vs. control siRNA group.