| Backgroud and ObjectiveIschemic heart disease (IHD) is characterized by high morbidity and mortality, which threatens human health seriously. The pharmacology and operation treatment have significant effect on IHD. However, these treatments still presence many shortcomings. As a convenient,cost-effective, and non-obvious-side-effect complementary therapy, acupuncture plays an important role in the treatment of IHD. It has been reported that acupuncture treatment can reduce the frequency of angina pectoris, decrease the mortality, and improve the life quality in patients treated by drugs and revascularization.The heart is regulated by both the sympathetic and parasympathetic nervous systems and it functions best when the two systems are in balance. The beta-1 adrenergic receptor (β1AR)and the M2 muscarinic cholinergic receptor (M2AChR) are the predominant receptor subtypes in cardiomyocyte. Imbalance of the autonomic nervous system and abnormal changes of the expression and function of these receptors play an important role in cardiac pathologic changes. It has been confirmed that acupuncture at Neiguan (PC6) acupoint can regulate the excitability of the autonomic nervous system. Nevertheless, there is scarcely of the systematic study on the mechanism underlying the protective effect of acupuncture mediated by the signal transduction pathway of β1 AR and M2AChR.In this study, exhaustive swimming (ES) without loading was adopted to establish an acute myocardial ischemia (MI) model. We aimed to observe whether electroacupuncture (EA) at PC6 could relieve MI injury. The β1 AR and M2AchR gene knockout mice(β1AR-/- and M2AchR-/- mice) were conducted into this study to verify whether β1AR and M2AchR mediation was involved in this effect. Cardiac electrophysiology technology, ELISA, Western blot were applied in this project to detect the effect of EA on the expression and function ofβ1 AR, M2AchR,as well as the posterior-receptor signal components. The changes and the response patterns of Pi AR and M2AchR signal transduction pathways is systematically discussed in this study.MethodsA total of 42 male C57BL6 mice and 12 male P,R-/- and M2AChR-/- mice were used in the study. Of which 18 C57BL6 mice were randomly devided into control (n=6), model (n=6) and PC6 groups (n=6). Animals in the model and PC6 groups were subject to the ES sessions on experimental days 1, 3, 5, and 7. A 30 min EA treatment session was administrated in the PC6 but not the model group. The control group was subject to a 30 min session of grasping and fixing daily without any other treatment. The Electrocardiograph (ECG) of the control group was recorded at the baseline (day 0), and experimental day 1 and 7. The ECG of the model and PC6 groups was recorded at the baseline, immediately following ES on day 1 and 7. Each group of mice were executed, and the hearts were harvested and stored in -80℃. Cyclic adenosine monophosphate (cAMP) and cyclic guanosinc monophosphate (cGMP) in the myocardial tissue were detected by ELISA, and β1AR, M2AchR, Gs protein and Gi protein were detected by Western blot. The β1AR-/- and M2AChR-/- mice were both randomly divided into the model (n=6) and PC6 groups (n=6), and treated the same way corresponding to the groups of C57BL6 mice. The ECG of the β1AR-/- and M2AChR-/- mice was recorded at baseline, and immediately following ES session on experimental days 1 and 7.Another 24 C57BL6 mice were divided into control (n=8), model (n=8) and PC6 group (n=8).The mice in each group were subject to the treatment sessions described above. The mice in control group were anaesthetized and the hearts were harvested on day 7. The model and PC6 groups were anaesthetized and the hearts were harvested immediately following ES. The hearts of 4 mice in each group were put on the Langendorff perfusion device and separated into single cardiomyocytes, and the blood was collected to detect the concentration of serous cTnT. The Ca2+ transient amplitude of single cardiomyocytes was tested under the action of dobutamine (β1 AR agonist), forskolin (adenylate cyclase agonist), 8-Br-cAMP (cAMP analogue), methacholine (MCh, M2AchR agonists) and 8-Br-cGMP (cGMP analogue) to observe the activity of β1 AR and M2AchR,adenylate cyclase (AC), protein kinase A (PKA)and phosphodiesterase (PDE). The hearts of the other 4 mice in each group were placed into the 4% paraformaldehyde solution for histological detection.Results(1) As demonstrated by ECG, ES was able to lead to significant deviation of J point (In the model group,P<0.05; and in the PC6 group,P<0.01,compared with the control group),decreased the heart rate (HR) (in the model group,P<0.05; and in the PC6 group,P<0.05,compared with the baseline of the same group) and increased serous cTnT (P<0.05).Histological examination showed that there were ischemic areas in the left ventricular myocardium and showed typical pathological changes of myocardial ischemia. The deviation of J point (P<0.01, compared with the same group on day 1; P<0.05, compared with the model group on day 7) and the concentration of serous cTnT (P<0.01) decreased significantly after 7 days EA. Histological examination showed that the pathological changes of myocardial ischemia were significantly improved, and the ischemic area and the degree of ischemia was significantly reduced (P<0.01). However, EA had no effect on the bradycardia induced by ES, HR was still much lower than the baseline (P<0.05).(2) The protective effect of EA was dispelled when the β1AR and M2AchR gene was knocked out.(3) The role of β1 AR signal transduction pathway in the prevention against myocardial ischemic injury: The expression of β1AR in the myocardium significantly decreased after ES (P<0.01), and the response to dobutamine decreased significantly as compared to that in the control group (P<0.05). The expression of Gs protein did not change significantly,and the content of cAMP decreased significantly (P<0.01). The response to forskolin in single cardiomyocytes did not change significantly as compared to that in the control group. While after EA, the expression of β1AR in the myocardial tissue increased significantly (P<0.05), and the response to dobutamine was much higher than that of the model group (P<0.05). The content of Gs protein and cAMP did not change significantly as compared to that in model group. The amplitude of calcium transients in single cardiomyocytes under the action of forskolin was significantly lower than that in the model group (P<0.05). There was no significant change in cardiomyocyte response to 8-Br-cAMP as compared to the model group.(4) The role of M2AchR signal transduction pathway in the prevention against myocardial ischemic injury: The expression of M2AchR and the concentration of cGMP in the myocardium decreased significantly in the model group (P<0.01), and there was no significant change in the expression of Gi protein. The amplitude of calcium transient in single cardiomyocytes was significantly increased as compared to that in the control group under the action of MCh (P<0.01) and 8-Br-cGMP (P<0.01). The expression of M2AchR in myocardium increased significantly after EA (P<0.01). The expression of Gi protein was increased, but there was no significant statistic difference as compared to that in model group. The amplitude of calcium transient in single cardiomyocytes of the PC6 group was significantly lower than that in model group under the action of MCh (P<0.01)and 8-Br-cGMP (P<0.05).ConclusionThe results in the present study suggested that EA treatment at acupoint PC6 was able to reduce the myocatdial ischemic injury induced by ES session and that β1 AR and M2AChR are both involved in the mechanism of protection effect. Additionally, EA treatment at PC6 could increase the expression of β1 AR in myocardium of the mice subject to ES and increase the sensitivity of β1AR. Meanwhile, EA at PC6 also could increase the expression of M2AchR in myocardium of the exhaustive mice thus would inhibit the over activation of AC and the cAMP dependent response in the downstream further.We propose that this effect would maintain the moderate excitation of β1AR signal transduction pathway, while the cGMP-PDE signaling pathway of the M2AchR downstream might not be involved in this mechanism. Furthermore, EA at PC6 can make the β1 AR and M2 AchR signal transduction pathway recover to the normal excitation and keep balance between them, thus maintaining normal regulation of sympathetic and parasympathetic systems, and functionally protecting the cardiomyocytes from ischemic injury induced by ES. |
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