Intracellular Calcium - Gap Junctions Factor Pre-acupuncture Treatment To Improve The Role In Ischemic Arrhythmias

Coronary artery disease induced by myocardial ischemia is one of the diseases with highest mortality in the world.The ischemic arrythmia,especially the ventricular fibrillation is the main cause leading to the sudden death.The electro-acupuncture was reported to improve the ischemic arrhythmia.Our previous study showed that the cardiac arrhythmias induced by myocardial ischemia and reperfusion could be attenuated by the pretreatment of acupuncture.However,the mechanisms underlying the anti-arrhythmic effect of the pretreatment of acupuncture remain unknown.The gap junction of myocardial cell is a low resistance channel,composed of two hexamers termed connexons from neighboring cardiac muscle cell membrane.Each connexon is equivalent to a single hemi-channel.Pairs of connexons from apposing plasma membranes join to form complete channels spanning both membranes and the extracellular gap. Myocardial gap junction is very important in the cell-to-cell communication and impulse conduction.Cx43,weighing 43 kDa,is a major connexin in ventricle and play a crucial role in ensuring cardiac electricity conduction,especially the electric synchronicity.The alternation of Cx43 in quantity,the phosphorylation state,the distribution or function (remodeling) may cause the disorder of cardiac electric conductance,followed by reentrant arrhythmia.Cx43 is a functionally calcium-associated protein.The Cx43 remodeling may account for the intercellular calcium overload resulted from ischemic arrhythmia.In addition, the increase of intercellular calcium is accompanied by the calcium oscillation,which is related to the occurrance of arrhythmia.All together,both the calcium oscillation and calcium-related Cx43 are the key points in the mediation of ischemic arrythmias.The cardioprotection of ischemic preconditioning can be successfully mimicked by the repetitive stimulations of L-Ca²⁺ channel opener and beta-adrenoceptor agonist which is also the neurotransmitter released by the sympathetic terminals.Since the electro-acupuncture was shown to affect the function of sympathetic activity,the repetitive pretreatment of electro-acupuncture may also reduce the occurrence of ischemic arrhythmias as the ischemic preconditioning does,by attenuating the calcium overload and Cx43 remodeling.The aim of present study is to determine if Cx43 protein expression,Cx43 phosphorilation,intracellular Ca²⁺([Ca²⁺]_i) and its oscillation are involved in the mediation of the anti-arrhythmic effect produced by the repetitive electro-acupuncture pretreatment in the rats subjected to myocardial ischemia and reperfusion(MIR).The MIR model was produced by occlusion of the left anterior descending(LAD) coronary artery in both whole animal and isolated heart.The results of present study may elucidate the mechanisms underlying the anti-arrhythmic effect of the pretreatment of electro-acupuncture so as to provide further the scientific evidence to support the possible application of pretreatment of acupuncture in prevention and treatment of ischemic arrythmia in the future.The 130 male Sprague-Dawley(SD) rats weighing about 215±35g were randomly divided into five groups,namely,the normal control group(NC),myocardial ischemic group (IR),electro-acupuncture pretreatment+myocardial ischemic group(EA),the electro-acupuncture pretreatment+myocardial ischemia+18 beta-glycyrrhetinic acid(EAG) group and the electro-acupuncture pretreatment+myocardial ischemia+Bay K8644(EAB) group.The LAD was ligated for 30 min followed by a 10-min reperfusion in IR group.In EA group the rats were stimulated by electro-acupuncture with intensity of 1-3 mA at bilateral Neiguan points for 30 minutes once a day in 3 consecutive days before the day the LAD was ligated.In NC group the rats underwent the same operation except LAD ligation and acupuncture stimulation.The rats in EAG and EAB groups were given Cx43 antagonist and Ca²⁺ channel opener respectively 30 minutes before each time of the pretreatment of acupuncture.Other performances are the same as those in EA.The present study covers three series of experiments:1 To identify the effect of acupuncture pretreatment on ischemic arrythmia;2 To determine the role played by the[Ca²⁺]_i in mediating the anti-arrhythmic effect of acupuncture pretreatment;3 To To determine the role played by Cx43 protein expression and phosphorilation.The results are as follows:1.Effect of EA pretreatment on ischemic arrythmiasThe arrhythmic score in NC group was 0,showing that there was no cardiac arrhythmia in the group.However,the different atrial and ventricular arrhythmias occurred in IR group with an arrhythmia score of 3.75±0.25,significantly higher than that in NC group(P＜0.05); interestingly,the arrhythmia score was 0.75±0.25 in EA group,significantly lower than that in IR group,exhibiting that the incidence of arrhythmia in EA group was significantly decreased(P＜0.05) after the electro-acupuncture pretreatment.The results confirmed that the cardiac arrhythmias induced by ischemia and reperfusion can be attenuated by the repetitive acupuncture pretreatment.2 Effect of EA pretreatment on the[Ca²⁺]_i and[Ca²⁺]_i oscillation in resting and electrically-stimulated single ventrieular myocyte isolated from the perfused heart subjected to global ischemia and reperfusion 2.1 The effect of EA pretreatment on the[Ca²⁺]_i and[Ca²⁺]_i oscillation in resting single ventricular myocyte isolated from the perfused heart subjected to global ischemia and reperfusionThe average resting[Ca²⁺]_i concentration in the single ventricular myocyte was 73.67±1.08 nmol/L in NC group;in IR group the resting[Ca²⁺]_i concentration was 110.42±1.49 nmol/L,significantly higher than NC group(P＜0.01);after repetitive EA pretreatment the resting[Ca²⁺]_i in EA group was 85.42±1.56 nmol/L which is significantly lower than that in IR group(P＜0.01),showing that IR-enhanced resting[Ca²⁺]_i was attenuated by EA pretreatment.The EA-attenuated resting[Ca²⁺]_i was reversed to 110.42±1.64 and 110.833±1.41 nmol/L in EAG and EAB groups in both of which the rats were respectively administered 18 beta-glycyrrhetinic acid,a Cx43 agonist,and Bay K8644,a L-Ca²⁺ channel opener,indicating that Cx43 antagonists and Ca²⁺ channel opener can inhibit the EA pretreatment-induced attenuation of[Ca²⁺]_i.2.2 Effects of EA pretreatment on[Ca²⁺]_i oscillations in the resting single ventricular myocyte isolated from perfused hearts subjected to simulative global myocardial ischemia and reperfusionThere was no any[Ca²⁺]_i oscillations in the resting single ventricular myocyte isolated from the hearts without simulative myocardial ischemia in NC group;in IR group[Ca²⁺]_i oscillations occurred frequently in the resting myocytes isolated from the hearts subjected to global simulative myocardial ischemia.The oscillation number in the IR group was 43.83±2.68,significantly higher than NC group;after the rats were repetitively pretreated by EA the number of[Ca²⁺]_i oscillations in the resting state were significantly attenuated to 8.33±1.58 in EA group,significantly lower than IR group(P＜0.01).The EA-attenuated resting calcium oscillation was reversed to 34±2.11 and 48.75±3.34 in EAG and EAB groups in both of which the rats were respectively administered 18 beta-glycyrrhetinic acid, and Bay K8644,indicating that administration of Cx43 antagonists and Ca²⁺ channel opener prior to EA can diminish the EA pretreatment-induced the attenuation of[Ca²⁺]_i oscillation.2.3 Effects of EA pretreatment on[Ca²⁺]_i oscillations in electrically-stimulated single ventricular myocyte isolated from perfused hearts subjected to simulative global myocardial ischemia and reperfusionThe number of calcium oscillations occurred in the single ventricular myocyte stimulated electrically in NC group were 0;More frequent[Ca²⁺]_i oscillations were observed in the single cardiac cell stimulated electrically in the IR group.The oscillation number was 189.42±29.38 in the group,significantly higher than that in NC group(P＜0.01).The number of [Ca²⁺]_i oscillations induced by electric stimulation in EA group,in which the rats were pretreated by EA,significantly decreased to 10.08±3.09,which was significantly lower than IR group(P＜0.01).Similar to the resting state,Administration of 18 beta-glycyrrhetinic acid) and Bay K8644 prior to EA treatment reduced the[Ca²⁺]_i oscillation to 125.25±17.24 and 289.83±25.98 times in EAG Group and the EAB group respectively.The number of calcium oscillations of EAG Group and the EAB group were both significantly higher than that in EA group(P＜0.01).The results suggest that both Cx43 antagonist and Ca²⁺ channel opener can diminished the EA pretreatment-attenuated[Ca²⁺]_i oscillations induced by electric stimulation.Taken together,all above results indicate that[Ca²⁺]_i oscillations,L-Ca²⁺ channel and Cx43 are involved in the mediation of the anti-arrhythrnic effect of EA pretreatment.Further study is needed so as to determine which one,Cx43 protein content or Cx43 protein phosphorylation,is the key point in mediating the anti-arrhythmic effect of EA pretreatment.3 The effect of EA pretreatment on the protein content and phosphorylation of Cx43 in ventricular myocyte isolated from the heart in the rats subjected to myocardial ischemia and reperfusion3.1 The effect of EA pretreatment on the Cx43 protein content in ventricular myocyte isolated from the heart in the rats subjected to myocardial ischemia and reperfusionIn this part of study the total protein content of Cx43 in the NC group was treated as 100%.The results showed that the Cx43 protein contents were 82.25±0.02%,83.99±0.02%, 80.52±0.01%and 86.14±0.01%in IR,EA,EAG and EAB groups respectively,a little bit lower than that in NC group.However,there is no significant difference among IR,EA,EAG and EAB groups(P＞0.05),suggesting that the interventions including EA,EA+Cx43 antagonist and Ca²⁺ channel opener don't affect the Cx43 protein level.The results indicate that Cx43 protein concentration is not involved in the mediation of the anti-arrhythmic effect of EA pretreatment.3.2 The effect of EA pretreatment on Cx43 phosphorylation in ventricular myocyte isolated from the perfused hearts subjected to simulative global ischemia and reperfusion In this part of study both phosphorylated and non-phosphorylated Cx43 in the NC group were treated as 100%.The results showed that the content of nonphosphated Cx43 in IR group was 238±0.33%,which were significantly higher than NC group(P＜0.05),while the corresponding content of phosphorylated Cx43 was 42.26±0.03%,which were significantly lower than the NC group(P＜0.05).After EA pretreatment the contents of nonphosphated and phosphrylated Cx43 in the EA group were 108±0.06%and 89.21±0.25%,which were significantly lower and higher than that in IR group respectively(P＜0.05).The content of nonphosphated Cx43 in EAG Group and the EAB group were 246±0.18%and 253±0.16% respectively,both of which were significantly higher than the EA group(P＜0.05).While the content of phosphorylated Cx43 in EAG Group and the EAB group were 39.26±0.01%and 29.28±0.02%respectively,both of which were significantly lower than that in EA group (P＜0.05).Taken together,the above results indicate that EA pretreatment can attenuate the increase in non-phosphorylated Cx43 induced by ischemia and reperfusion,and enhance the contents of phosphorylated Cx43.Both Cx43 antagonists and Ca²⁺ channel opener can blunt the beneficial influence of EA treatment on the phosphorylation of Cx43.CONCLUSIONThe repetitive EA pretreatrnent can produce anti-arrhythmic effect in the rats subjected to myocardial ischemia and reperfusion.The anti-arrhythmic effect of EA pretreatment is at least partially attribute to inhibiting IR-induced enhancement of[Ca²⁺]_i concentration and oscillations,decreasing non-phosphorylated Cx43 and increasing phosphorylated Cx43 in the cardiac cells.