Electroacupuncture Improves Cardiac Function via Inhibiting Sympathetic Remodeling Mediated by Promoting Macrophage M2 Polarization in Myocardial Infarction Mice

Figures

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  Figure 1. Experimental protocol.

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  Figure 2. EA treatment improved post-MI cardiac function and reduced fibrosis. (a) Representative M-mode of echocardiographic images of mice at 28 days after MI (n = 11 per group) are shown. (b–d) Quantitative group data for EF (b), FS (c), and HR (d) are shown (n = 11 per group). (e) Representative images of Sirius red staining are shown at 28 days after MI (n = 3 per group). Scale bar =100 μm. (f) Quantitative analysis of Sirius red staining in all groups (n = 3 per group). EF, ejection fraction; FS, fractional shortening; HR, heartbeats per minute. Data are shown as mean ± SEM. ns, no significance, ∗∗∗∗P < 0.0001.

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  Figure 3. EA treatment inhibited post-MI systemic sympathetic activity and reduced macrophage infiltration: (a) serum NE levels at 28 days after MI (n = 6 per group); (b) heart tissue NE levels 28 days after MI (n = 3 per group); (c–e) spectral analysis of heart rate variability reflecting sympathetic activity: ratios of LF (c), HF (d), and LF/HF (e) (n = 6 per group); (f) Representative flow cytometric analysis of macrophage infiltration in the heart 28 days after MI (n = 4 per group). (g–i) Representative flow cytometric analysis of M1 and M2 macrophages in the heart at 28 days after MI (n = 4 per group). NE, noradrenaline; LF, low frequency (0.20–0.75 Hz); HF, high frequency (0.75–0.2 Hz); LF/HF, low-frequency/high-frequency ratio. Data are shown as mean ± SEM. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

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  Figure 4. EA treatment improved post-MI cardiac function, reduced cardiac fibrosis and decreased sympathetic activation. (a) Representative M-mode echocardiographic images of mice at 7 days after MI are shown (n = 10 per group). (b–d) Quantitative groups data for EF (b), FS (c), and HR (d) are shown (n = 10 per group). (e) Representative images of Sirius red staining are shown at 7 days after MI (n = 3 per group). Scale bar = 100 μm. (f) Quantitative analysis of Sirius red staining in all groups (n = 3 per group). (g–i) Spectral analysis of heart rate variability reflecting sympathetic activity: ratios of LF (g), HF (h) and LF/HF (i) (n = 5 per group). EF, ejection fraction; FS, fractional shortening; HR, heartbeats per minute. Data are shown as mean ± SEM. ns, no significance, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

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  Figure 5. Effect of EA treatment on post-MI macrophage infiltration, macrophage polarization, and inflammatory factor: (a and b) representative flow cytometric analysis of macrophage infiltration in the heart at 1, 3, and 7 days after MI (n = 5 per group); (c–e) representative flow cytometric analysis of M1 and M2 macrophages in the heart at 1, 3 and 7 days after MI (n = 5 per group); (f–h) expression of inflammatory cytokines TNF-α (n = 5 per group), IL-1β (n = 5 per group) and IL-6 (n = 3 per group) in myocardium at 7 days after MI was assessed by RT-qPCR. Data are expressed as mean ± SEM. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

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  Figure 6. Effect of EA treatment on post-MI cardiac sympathetic nervous system remodeling in mice post-MI. (a, c) Representative TH immunohistochemical staining and analysis of heart tissues in 28 days after MI (n = 3 per group), scale bar = 50 μm. (b, d) Representative GAP43 immunohistochemical staining and analysis of heart tissues at 28 days after MI (n = 3 per group). Scale bar = 50 μm. (e) The relative mRNA expression of TH in heart (n = 4 per group). (f) The relative mRNA expression of GAP43 in the heart (n = 4 per group). Data are expressed as mean ± SEM. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

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  Figure 7. Effects of EA treatment on the MI-induced NGF production and the expression of GAP43 and TH post-MI. (a, c) Representative Western blots and quantification of NGF expression (n = 3 per group). (b, d) Representative Western blots and quantification of TH expression (n = 3 per group). (e) TH mRNA levels in cardiac tissue at 7 days (n = 5 per group). (f) Representative images of immunofluorescence staining for NGF/F480+ cell expression in the heart at 7 days (n = 3 per group). Positive staining for F4/80 is red and NGF is green. Cell nuclei were counterstained with DAPI (blue). Scale bar = 200 μm.(g) Immunostaining quantification of staining for NGF/F4/80+ cells at 7 days after (n = 3 per group). (h) GAP43 mRNA levels in cardiac tissue 7 days after (n = 5 per group). Data are shown as mean ± SEM. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.