Electroacupuncture pretreatment mediates sympathetic nerves to alleviate myocardial ischemia-reperfusion injury via CRH neurons in the paraventricular nucleus of the hypothalamus

Abstract

Background: Myocardial ischemia-reperfusion can further exacerbate myocardial injury and increase the risk of death. Our previous research found that the paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in the improvement of myocardial ischemia-reperfusion injury (MIRI) by electroacupuncture (EA) pretreatment, but its mechanism of action is still unclear. CRH neurons exhibit periodic concentrated expression in PVN, but further research is needed to determine whether they are involved in the improvement of MIRI by EA pretreatment. Meanwhile, numerous studies have shown that changes in sympathetic nervous system innervation and activity are associated with many heart diseases. This study aims to investigate whether EA pretreatment improves MIRI through sympathetic nervous system mediated by PVN^CRH neurons.

Methods: Integrated use of fiber-optic recording, chemical genetics and other methods to detect relevant indicators: ECG signals were acquired through Powerlab standard II leads, and LabChart 8 calculated heart rate, ST-segment offset, and heart rate variability (HRV); Left ventricular ejection fraction (LVEF), left ventricular short-axis shortening (LVFS), left ventricular end-systolic internal diameter (LVIDs) and interventricular septal thickness (IVSs) were measured by echocardiography; Myocardial infarct area (IA) and area at risk (AAR) were calculated by Evans-TTC staining. Pathological changes in cardiomyocytes were observed by HE staining; Changes in PVN^CRH neuronal activity were recorded by fiber-optic photometry; Sympathetic nerve discharges were recorded for in vivo electrophysiology; NE and TH protein expression was assayed by Western blot.

Results: Our data indicated that EA pretreatment can effectively alleviate MIRI. Meanwhile, we found that in the MIRI model, the number and activity of CRH neurons co labeled with c-Fos in the PVN area of the rat brain increased, and the frequency of sympathetic nerve discharge increased. EA pretreatment could reverse this change. In addition, the results of chemical genetics indicated that inhibiting PVN^CRH neurons has a similar protective effect on MIRI as EA pretreatment, and the activation of PVN^CRH neurons can counteract this protective effect.

Conclusion: EA pretreatment can inhibit PVN^CRH neurons and improve MIRI by inhibiting sympathetic nerve, which offers fresh perspectives on the application of acupuncture in the management of cardiovascular disease.

Keywords: CRH neurons; Electroacupuncture pretreatment; Myocardial ischemia- reperfusion injury; Neural mechanism; Paraventricular nucleus of hypothalamus.

Fig.1

Effects of EA pretreatment on cardiac function in MIRI rats. A ECG recordings of the EA group. B The statistical analysis of ST deviations in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. C The statistical analysis of LF/HF ratio in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, *p < 0.05, n = 6 rats/group. D Comparison of frequency domain analysis in each group of rats. E Comparison of left ventricular echocardiography in each group of rats. F The statistical analysis of LVEF, LVFS, LVIDs, and IVSs values in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group

Fig.2

Effects of EA pretreatment on myocardial tissue and sympathetic nerves in MIRI rats. A Comparison of TTC-Evans blue double staining results of rat heart tissues in various groups. B The statistical analysis of area of myocardial infarction and area at risk in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. C Comparison of HE staining results of rat heart tissues in each group (magnification, × 20; scale bar, 100 µm). D The statistical analysis of NE and CK-MB values in myocardial tissue homogenates in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. E Comparison of original trajectory maps of sympathetic nerve activity in rats of various groups. F The statistical analysis of the frequency of sympathetic nerve discharges in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, **p < 0.01, n = 6 rats/group. G Comparison of NE and TH protein expression in each group of rats. H The statistical analysis of NE and TH protein expression in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, **p < 0.01, *p < 0.05, n = 3 rats/group

Fig.3

Results of immunofluorescence staining in the PVN. A Location of PVN (magnification, × 5; scale bar, 200 µm). B Comparison of c-Fos-positive neuronal expression in the PVN of rats in each group (magnification, × 20; c-Fos, red; DAPI, blue; scale bar, 50 µm). C The statistical analysis of the number of c-Fos + neurons in the PVN in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. D Comparison of CRH neurons and GABA neurons co-labeled with c-Fos in the PVN of MIRI rats (magnification, × 40; scale bar, 20 µm). E The statistical analysis of the number of CRH neurons and GABA neurons co-labeled with c-Fos in the PVN of MIRI rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group

Fig.4

Results of fiber-optic recording of CRH neurons in the PVN. A Fiber optic burial diagram. B Fiber optic embedding sites and virus injection sites (magnification, × 10; scale bar, 100 µm; magnification, × 40; scale bar, 20 µm). C Comparison of the original trajectories of calcium activity in CRH neurons in 300 s in each group of rats. D Comparison of ΔF/F with time trajectory at 470 nm for each group of rats (n = 6). E Comparison of thermograms of ΔF/F over time trajectories in groups of rats (n = 6). F The statistical analysis of ΔF/F for 1–300 s and calcium events occurring in PVN^CRH neurons in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group

Fig.5

Results of chemogenetic regulation of PVN^CRH neurons. A Chemical genetic virus injection site (magnification, × 10; scale bar, 100 µm). B Experimental protocol for ECG recording under physiologic conditions in rats. C Experimental design diagram. D Comparison of various indices of ECG in rats injected with different kinds of viruses. Data are expressed as the mean ± SD. ***p < 0.001; **p < 0.01; ns, means no significance, n = 6 rats/group

Fig.6

Effects of inhibition of PVN^CRH neurons on cardiac function in MIRI rats. A ECG recordings of the hM4Di + Model group. B The statistical analysis of ST deviations in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. C The statistical analysis of LF/HF ratio in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, **p < 0.01, n = 6 rats/group. D Comparison of frequency domain analysis in each group of rats. E Comparison of left ventricular echocardiography in each group of rats. F The statistical analysis of LVEF, LVFS, LVIDs, and IVSs values in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, **p < 0.01, n = 6 rats/group

Fig.7

Effects of inhibition of PVN^CRH neurons on myocardial tissue and sympathetic nerves in MIRI rats. A Comparison of TTC-Evans blue double staining results of rat heart tissues in various groups. B The statistical analysis of area of myocardial infarction and area at risk in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, *p < 0.05, n = 6 rats/group. C Comparison of HE staining results of rat heart tissues in each group (magnification, × 20; scale bar, 100 µm). D The statistical analysis of NE and CK-MB values in myocardial tissue homogenates from various groups of rats. Data are expressed as the mean ± SD. ***p < 0.001, **p < 0.01, n = 6 rats/group. E Comparison of original trajectory maps of sympathetic nerve activity in rats of various groups. F The statistical analysis of the frequency of sympathetic nerve discharges in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group

Fig.8

Effect of activation of PVN^CRH neurons on cardiac function in MIRI rats. A ECG recordings of the hM3Dq + EA + Model group. B The statistical analysis of ST deviations in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats / group. C The statistical analysis of LF/HF ratio in each group of rats. Data are expressed as the mean ± SD. **p < 0.01, *p < 0.05, n = 6 rats/group. D Comparison of frequency domain analysis in each group of rats. E Comparison of left ventricular echocardiography in each group of rats. F The statistical analysis of LVEF, LVFS, LVIDs, and IVSs values in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, **p < 0.01, *p < 0.05, n = 6 rats/group

Fig.9

Effects of activation of PVN^CRH neurons on myocardial tissue and sympathetic nerves in MIRI rats. A Comparison of TTC-Evans blue double staining results of rat heart tissues in various groups. B The statistical analysis of area of myocardial infarction and area at risk in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. C Comparison of HE staining results of rat heart tissues in each group (magnification, × 20; scale bar, 100 µm). D The statistical analysis of NE and CK-MB values in myocardial tissue homogenates from various groups of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group. E Comparison of original trajectory maps of sympathetic nerve activity in rats of various groups. F The statistical analysis of the frequency of sympathetic nerve discharges in each group of rats. Data are expressed as the mean ± SD. ***p < 0.001, n = 6 rats/group