Pretreatment Of Electroacupuncture Attenuates Myocardial Injury Induced By Ischemia And Reperfusion In The Rats-involvement Of β₁-adrenergic Receptor Signaling Pathway

BACKGROUNDCardiac dysfunction induced by myocardium ischemia can cause a high mortality. Acupuncture has been shown to attenuate the myocardial injury induced by ischemia. However, in clinic, few of the patients with coronary heart disease (CHD) went to see the acupuncturist due to that the healing effect of acupuncture on the disease was not recognized popularly. Especially, acupuncture with its effective but slow and limited action on CHD was not currently considered as a first-aid therapy to the disease because CHD at acute stage is usually too urgent to be treated by acupuncture alone. Thereby, it is interesting to create a proper way to exert the beneficial effect of acupuncture, an economic and simple therapy, in the treatment and prevention of CHD. Pretreatment, i.e. treating the patients who are inclined to suffering from CHD before they are attacked by the disease, may be a better way for acupuncture to treat and prevent CHD.It was well-documented that during myocardial ischemia and reperfusion both the sympathetic activity to the heart and the cardiac responsiveness to the sympathetic influence were increased. Then the sympathetic activity could further cause over-stimulation of β1-adrenoceptor (β₁-AR), lead to an overload of intracellular calcium ([Ca²⁺]i) and cardiac injury, via a series of the signaling pathway of β₁-AR. Pretreatment with the specific β-adrenoceptor (β-AR) agonist was shown to mimic the ischemic preconditioning to protect the heart from injury induced by the subsequent ischemia. Acupuncture had been shown to increase the excitability of sympathetic nervous system in the previous study. It is highly likely that by mimicking the aforementioned pretreatment of specific P-AR agonist, repetitive pretreatment with acupuncture prior to the myocardial ischemia may also attenuate the myocardial injury of ischemia via repetitively stimulating sympathetic nervous system so as to desensitize the responsiveness of β₁-AR and its signaling pathway to the subsequently ischemic stimulation.The aim of the present study was to determine the protective effect of the pretreatment ofacupuncture in the heart of rats with myocardial ischemia and to explore the role played by the different signaling components of Pi-AR including Pi-AR, Gsa protein, adenylate cyclase (AC), cAMP, protein kinase A (PKA) and L-type Ca2+ channel in the mediation of the protective effect of EA pretreatment. METHODSThe present study covers two sections of experiments. The experiments were performed on male Wister rats weighing 278±25 g or male Sprague-Dawley (SD) rats weighing 255±17g. The rats were divided into four groups in the 1st section of experiments, namely normal control (NC) group, ischemia-reperfusion (IR) group, electro-acupuncture (EA) group and EA+propranolol (EAP) group. Before the experiments the animals in both EA and EAP groups were pretreated with EA applied at bilateral Neiguan acupoints (PC6, according to the textbook of experimental acupuncture, Neiguan acupoint is located forelimbs ) for 30 min once a day in 3 consecutive days. In the manipulation of acupuncture two needles, punctured through the skin to a depth of about 2 mm at each Neiguan acupoint with 2-3 mm apart, were connected to positive and negative poles of acupuncture apparatus. The acupoints were stimulated by EA with intensity of 5 mA and frequency of 20 Hz. Propranolol, a specific antagonist of P-AR, was administered intraperitoneally in the EAP group at dose of lOmg/kg 15 min before each EA pretreatment (once a day in 3 consecutive days). In the 2nd section of experiments, the rats were divided into three groups, namely NC group, IR group and EA group respectively. The same EA pretreatment was performed as described above.In the section one of the present study with the aim to confirm the protective effect of EA pretreatment, the elevated ST-segment of electrocardiogram (ECG), the arrhythmic score, the ratio of infarct size / risk zone, the percentage of rod-shape myocytes and [Ca2+J;oscillations induced by isoproterenol (ISO) were compared among the NC, IR, EA and EAP groups. In the section two of the study with the purpose to explore the signaling mechanism underlying the protective effect of EA pretreatment on myocardium, the contents of Pi-adrenoceptor, Gsa protein and cAMP, the changes in amplitude of electrically induced-[Ca2+];transient caused by Forskolin (a selective activator of AC) > 8-Br-cAMP (a selective activator of PKA ) and Bay K-8644 (a selective opener of L-type Ca2+ channel) were measured respectively.RESULTSSection One— Protective effects of EA pretreatment on myocardium in the rats subjected to myocardial ischemia and reperfusion1 Effects of pretreatment with electro-acupuncture (EA) on ST segment of ECG in rats with myocardium ischemia and reperfusionThe elevated ST-segment of ECG in IR group recorded 30 min after ischemia and 10 min after reperfusion was higher than NC group (PO.01 and PO.01 respectively);in EA group the elevated ST-segment was reduced (P<0.0\ and PO.01 respectively vs IR group) significantly;while in EAP group the elevated ST-segment was almost same as IR group, much higher than EA group (PO.01 and PO.01 respectively).2 Effects of pretreatment with EA on cardiac arrhythmias in the rats with myocardial ischemia and reperfusionThe arrhythmic score was 3.17±1.17 in IR group. With pretreatment of EA, the arrhythmias produced by ischemia and reperfusion were significantly blunted in EA group, which was supported by the data that the arrhythmic score in EA group was 1.10±0.99, much lower as compared with that in IR group(/><0.01). When the animals were pretreated intraperitoneally with propranolol before the treatment of EA in EAP group, the arrhythmic score was 2.97±0.99, much higher than that in EA group (PO.01). The inhibition of the EA-induced attenuation of arrhythmias by propranolol suggests an involvement of the cardiac P-adrenergic receptors.3 Effects of EA pretreatment on risk zone/infarct size in rats with myocardial ischemia and reperfusionThe ratio of infarct size / risk zone was 38.63±2.92% in IR group. The myocardial injury was significantly diminished by EA, which was presented by a comparatively lower ratio of risk zone/infarct size of 21.51±1.73 in EA group (P<0.01). However, the ratio of infarct size / risk zone was 36.62±3.70 in EAP group, being almost the same as that in IR group, much higher than EA group (P<0.01).4 Effects of EA pretreatment on the percentage of rod-shape cells isolated from perfused heartsfollowing simulative global myocardial ischemia and reperfiisionOnly 22.80±0.78%of the cells were rod-shaped in IR group, which was much lower than 73.53±0.86% in NC group (P<0.01). The myocardial injury was significantly diminished by EA, which was presented by a comparatively high percentage of rod-shape cells of 71.62±0.46 % in EA group (P<0.01). However, the percentage of rod-shape cells was 21.41±0.67% in EAP group, being almost the same as that in IR group, much lower than EA group (P<0.01).5 Effects of EA pretreatment on [Ca2+]j oscillations induced by ISO in single ventricular myocyte isolated from perfused hearts following simulative global myocardial ischemia and reperfusionThere was no [Ca2+];oscillation induced by ISO in the myocytes isolated from the hearts without simulative myocardial ischemia in NC group;in the myocytes isolated from the hearts subjected to global simulative myocardial ischemia in IR group more [Ca2+]j oscillations were induced by ISO;in EA group in which the rats were pretreated by EA, the [Ca2+]j oscillations induced by ISO were significantly blunted in the ischemic myocytes.Section Two— Involvement of pi-AR receptor signaling pathway in the mediation of protective effect of EA pretreatment on the myocardium subjected to ischemia and reperfusion in the rats1 Involvement of cardiac Pi-AR in the myocardial protection of EA pretreatment in the ratssubjected to myocardial ischemia and reperfusionThe optical density of the protein band of p\-AR in IR group was obviously enhanced as compared with that in NC group (P<0.01). In EA group, the enhancement of the protein content of myocardial pVAR induced by ischemia and reperfusion was significantly attenuated2 Involvement of Gsa proteins in the myocardial protection of EA pretreatment in the ratssubjected to myocardial ischemia and reperfusionThe optical densities of both protein bands of Gsa-large and Gsa-small in IR group were obviously enhanced as compared with that in NC group(P<0.01 and P<0.01 respectively). In EA group the densities of both protein bands were lower than that in IR group, showing that EApretreatment significantly attenuated the enhancement of the Gsa protein content induced by ischemia and reperfusion (.P<0.05 andP<0.0l respectively).3 Involvement of AC in the myocardial protection of EA pretreatment in the rats subjected to myocardial ischemia and reperfusionForskolin, a activator of AC, caused different augmentations in the amplitude of electrically-induced [Ca2+]j transient. It was 157.87% in IR group, which is significantly higher than 77.07% in NC group (PO.01). In EA group, the augmentation in the amplitude of electrically induced-[Ca2+]i transient caused by forskolin is 78.41%, which is much lower than that in IR group (PO.01).4 Involvement of cAMP in the myocardial protection of EA pretreatment in the rats subjected to myocardial ischemia and reperfusionThe concentration of cAMP was 0.36±0.07 pmol/ml in NC group. While in IR group the cAMP of ischemic myocardium was 0.62±0.09 pmol/ml, which was markedly increased as compared with the concentration in NC group (jP<0.01). The augmentation of cAMP was significantly diminished by pretreatment of EA, which was presented by a comparatively lower cAMP concentration of 0.36±0.05 pmol/ml in EA group as compared with that in IR group5 Involvement of PKA in the myocardial protection of EA pretreatment in the rats subjected to myocardial ischemia and reperfusion8-Br-cAMP, an activator of PKA, caused different augmentations in the amplitude of electrically-induced [Ca2+];transient. It was 68.92% in IR group, which is significantly higher than 54.15% in NC group (P<0.0\). In EA group, the augmentation in the amplitude of electrically-induced [Ca2+];transient caused by 8-Br-cAMP is 57.36%, which is much lower than that in IR group (PO.05).6 Involvement of L-type Ca2+ channel in the myocardial protection of EA pretreatment in the rats subjected to myocardial ischemia and reperfusionBay K-8644, a specific opener of L-type Ca2+ channel, caused different augmentations in the amplitude of electrically-induced [Ca2+]j transient. It was 78.02% in IR group, which is significantly higher than 59.06% in NC group (PO.01). In EA group, the augmentation in the amplitude of electrically-induced [Ca2+]i transient caused by forskolin is 62.17%, which is much lower than that in IR group (PO.01).CONCLUSIONThe present results showed that the elevation of ST-segment in ECG, severe arrhythmia, enhancement in infarct size were produced by regionally myocardial ischemia and reperfusion and a decrease in the percentage of rod-shaped cells and enhancement in [Ca2+]j oscillations induced by ISO was caused by simulative global ischemia and reperfusion. Further results indicated an involvement of Pi-AR and its signaling pathway. It was observed in the present study that the contents of pVAR, Gsa protein and cAMP were increased in the cardiac myocytes subjected to ischemia and reperfusion. In addition to these changes, it was also demonstrated that the response of [Ca2+]j in the myocytes subjected to I/R to the activators of AC, PKA and L-type Ca2+ channel were also enhanced. Apparently, these components of Pi-AR signaling pathway, including pVAR, Gsaprotein, AC, cAMP, PKA as well as L-type Ca2+ channel, were all involved in the mediation of cardiac dysfunction and myocardial damage induced by myocardial ischemia and reperfusion.It was exhibited in the present study that EA pretreatment can protect the heart from injury of ischemia and reperfusion, which was supported by the data showing that the I/R-induced elevation of ST segment of ECG, enhancement of myocardial infarct size, cardiac arrhythmias and [Ca2+]j oscillations following ISO stimulation in cardiac myocytes subjected to I/R were significantly attenuated after repetitive EA pretreatment. Also, the percentage of rod-shaped cells in ventricular myocytes isolated from perfused hearts following simulative global myocardium ischemia was enhanced by EA pretreatment. As we assumed, the myocardial protection produced by EA was abolished by the pretreatment of propranolol, showing that cardiac p-ARs contributed to the mediation of above effects of EA pretreatment. In the further experiments, it has been demonstrated that the ischemia-induced increment of the contents of myocardial Pi-AR, Gsa protein and cAMP production were significantly inhibited by EApretreatment. Pretreatment with EA also attenuated the increase in the response of [Ca2+];in cardiac myocytes subjected to the simulative global I/R to the activators of AC, PKA and L-type Ca2+ channel. The present results indicated that all the components of pVAR signaling pathway, including pVAR, Gsa protein, AC, cAMP, PKA as well as L-type Ca2+ channel, are involved in the mediation of protective effect of EA pretreatment on myocardium subjected to ischemia and reperfusion in the rats.