| Background:Ischemic arrhythmia is a clinically common and dangerous arrhythmia.Current clinical antiarrhythmic drugs primarily act on Na+,K+ and Ca2+ channels,and most of them are channel blockers.When producing therapeutic advantage against arrhythmias,these channel blockers may lead to unwanted electrophysiological abnormalities and proarrhythmic risks.Cardiac inward rectifier potassium(Kir)channels constitute the inward rectifier potassium current(IK1)channels which are present in all ventricular and atrial myocytes and are important for stabilizing the resting membrane potential(RMP).IK1 channels establish the excitation threshold and modulate the final repolarization phase of the action potential(AP)in cardiomyocytes and thus exert profound effects on cardiac excitability and arrhythmogenesisA lot of research and clinical studies have shown that arrhythmogenesis in acute and chronic myocardial infarction(MI)is associated with decrease of IK1 in cardiomyocytes.We hypothesized that enhancing IK1 might hyperpolarize abnormal automatic/impaired cells and stabilize membrane potential thus suppress arrhythmias.Recently we reported that zacopride,a selective IK1 channel agonist,increased IK1 without effects on other currents in rat ventricular myocytes.We hypothesized that zacopride,the first selective and moderate agonist of the IK1/Kir2.1 channels,suppresses ischemic arrhythmias.The underlying mechanisms are ascribed to the activation of the IK1/Kir2.1 channel,maintenance of the RMP.To test this viewpoint,the present study was design to investigate the inhibitory effect of zacopride as IK1 agonist on arrhythmogenesis in acute and chronic MI;to observe the effects of zacopride on IK1,IKATP,RMP,AP,DADs in rat ventricular myocytes under hypoxic condition;to observe the effects of zacopride on IK1 current in the normal rat,the expression of Kir2.1 protein in rat ventricular myocytes of mimicked ischemia and the chronic arrhythmia.Objective:1.To prepare the acute arrhythmia model by coronary occlusion in rat and observe the effects of zacopride,a specific agonist of IK1,on it.2.Using a hypoxic perfusion appareil which can keep PO2 lower than 50 mmHg in the bash solution,to observe the effects of zacopride on IK1,IKATP,RMP,AP,DADs in rat ventricular myocytes and the effects of zacopride on the IKir2.x currents expressed in CHO cells under hypoxic condition.3.To observe the effects of zacopride on the expression of Kir2.1 protein in rat ventricular myocytes subjected to mimicked ischemia.4.To prepare the chronic arrhythmia model by coronary occlusion in rat and observe the effects of zacopride on arrhythmias after coronary occlusion during 4 weeks and Iso-induced arrhythmias after 4 weeks in the conscious rat with telemetry ECG recoding,and the expression of Kir2.1 protein in the ventricles of all tested rat.Methods:1.The preparation of acute arrhythmic models in rat by coronary occlusion,the grouping and the index measuredMethod of making model: Male SD rats were anaesthetized with sodium pentobarbital(65 mg/kg,i.p.).A left thoracotomy was performed and then a suture was placed around the proximal portion of the left coronary artery,which was then ligated for 15 minutes(min).Drug intervented in 3 min prior to coronary ligation(pretreatment),posterior to ligation(post-treatment),and 30 seconds(sec)just after the appearance of the first sustained ventricular tachycardia or ventricular fibrillation(Sus VT/VF)(therapeutic treatment).A lead II electrocardiogram(ECG)was recorded throughout the experiments.Groups of experiments: According to the time point of the drug intervention: pretreatment group;post-treatment group;therapeutic treatment group.And then according to the type and quantity of the drug intervention: control group;zac 5 μg/kg group;zac 15 μg/kg group;zac 50 μg/kg group;lidocaine(Lidoc)group.Index observed: the total of premature ventricular contraction(PVC),the duration of ventricular tachycardia(VT)and ventricular fibrillation(VF),the incidence of VT and VF,the latent period of the first Sus VT/VF,the termination time of the first Sus VT/VF.2.The recording of membrane current and membrane potential of ventricular myocardium in rat under hypoxic conditionThe ventricular myocytes were isolated via an enzymatic dissociation procedure.The hypoxic condition was created using a separate reservoir in which the bath solution bubbled with 100% N2 and PO2 was kept lower than 50 mmHg.Whole-cell patch clamp technique was used to record the inward rectifier potassium current(IK1)and ATP-sensitive potassium current(IKATP)at voltage-clamp configuration and the resting membrane potential(RMP),action potential(AP),delayed after depolarizations(DADs)at current-clamp configuration in rat ventricular myocytes under hypoxic condition.Glibenclamide(10 μmol/L)was added in the bath solution.3.The heteromeric expression of Kir2.1,Kir 2.2 and Kir 2.3 in CHO cells and the recording of membrane current under hypoxic conditionRespective rat cardiac orthologs of Kir2.1,Kir2.2 and Kir2.3 were cloned by reverse transcriptase-PCR.cDNAs were subcloned into the eukaryotic expression vector pEGFP-N1.CHO cells were transfected with genes encoding Kir2.1,Kir2.2 and Kir2.3.The hypoxic condition(PO2<50mmHg)as mentioned above was created.At voltage-clamp configuration,Whole-cell patch clamp technique was used to record the currents of Kir2.1,Kir2.2 and Kir2.3 in CHO cells under hypoxic condition.4.Preparation of simulated ischemia(SI)in rat ventricular myocytes and the groups of experimentsMethod of making model: Take three healthy adult SD rats,single left ventricular myocytes were isolated via an enzymatic dissociation procedure.Then make the final concentration of Ca2+ 1.8 mmol/L.The interventions by different concentrations of zacopride proceeded.The rat ventricular myocytes subjected to centrifuge after incubation and then simulated ischemia with mineral oil added on the upper supernatant fluid.Groups of experiment: control group;SI group;zac 0.3 μmol/L group;zac 1 μmol/L group;zac 3 μmol/L group.Index observed: The expressions of Kir2.1 protein in rat ventricular myocytes were performed by Western-blot.5.The preparation of chronic arrhythmic model in rat by coronary occlusion,the grouping and the index measured.Method of making model: The wireless implantable transmitter was secured in the abdominal cavity of male SD rat and the rat was subjected to left main coronary artery ligation or sham operation.Individual cage which the rat housed in was placed on reception board and a 24-hour continuous single-lead ECG trace was recorded and stored by the telemetry system after animals waked up.The study duration was set at 4 weeks.Groups of experiment: myocardial infarction(MI)group;MI+zac group;sham group;sham+zac group.Index observed:(1)The total number of episodes of super premature ventricular contraction(SPVC),conduction block(CB)and PVC,the duration of VT and VF,the incidence of VT and VF per day per rat during 4 weeks.(2)The arrhythmias induced by isoproterenol(Iso)in all telemetric rat before execute.Iso(1280 μg/kg)was injected intravenously into rats.The telemetric ECGs were recorded and analyzed to determine the total number of episodes of PVC,the duration of VT and VF,the incidence of VT and VF during 1 hour.(3)The expression of Kir2.1 protein in the ventricular myocardium of the telemetric rats were performed by Western-blot.6.Measuring the IK1 current week by week in normal ratThe ventricular myocytes were isolated via an enzymatic dissociation procedure in the control group and the zac group.Whole-cell patch clamp technique was used to record the current of IK1 at the end of 0,1,2,3,4 week at voltage-clamp configuration.Results:1.Zacopride suppressed acute ischemic arrhythmias in rat.5.0-50 μg/kg zacopride in a concentration-relative significantly suppressed arrhythmias,and zacopride at the dose of 15μg/kg had the greatest antiarrhythmic effect.(1)pretreatmentIn zac(15 μg/kg)group,the times for the latent period of arrhythmia were lengthened from 334.3±16.5 s to 465.5±23.2 s;the total episode number of PVC was decreased from 158±34 to 50±7;the episode duration of VT and the incidence of VT were decreased from 55.8±10.2 s,100% to 0.6±0.6 s,12.5%;respectively,the episode duration of VF and the incidence of VF were decreased from 5.1±2.0 s,62.5% to 0 s,0%,respectively,compared with control group.At the dose of 15 μg/kg,zacopride showed the most potent antiarrhythmic effect which compared favourably with that of lidocaine(7.5mg/kg),a classical antiarrhythmic drug(P>0.05).(2)post-treatmentIn zac(15 μg/kg)group,the times for the latent period of arrhythmia were lengthened from 335.6±11.8 s to 447.1±26.1 s,the total episode number of PVC was decreased from 105±18 to 14±5;the episode duration of VT and the incidence of VT were decreased from 51.9±12.3 s,100% to 1.8±0.9 s,37.5%;the episode duration of VF and the incidence of VF were decreased from4.4±1.7 s,75% to 0 s,0%,respectively,compared with control group.At the dose of 15 μg/kg,zacopride showed the most potent antiarrhythmic effect which compared favourably with that of lidocaine(P>0.05).(3)therapeutic treatment(1)There were no statistical difference(P>0.05)of the latent time of the first sus VT/VF in 5 groups.(2)In zac(15 μg/kg)group,the termination time of the first sus VT/VF was decreased from 129.8±54.5 s to 27.5±5.5 s,the total episode number of PVC was decreased from 110±21 to 26±10,the episode duration of VT was decreased from 191.4±55.4 s to 51.9±10.9 s,the episode duration of VF was decreased from 30.1±11.0 s to 0 s,respectively,compared with control group.At the dose of 15 μg/kg,zacopride showed the most potent antiarrhythmic effect which compared favourably with that of lidocaine(P>0.05).2.The effects of zacopride on IK1 current,IKATP current,RMP,AP and DADs in rat ventricular myocytes under hypoxic condition(1)Under hypoxic condition,the outward IK1 current was decreased markedly from 2.4±0.2(normoxic condition,at-60 mV)to 1.3±0.3 pA/pF(at-60 mV)and restored by 1μmol/L zacopride to 2.3±0.1 pA/pF(at-60 mV)with the mean increment by 76.9%.The IK1 current recovered by zacopride could be reversed to 1.5±0.2 pA/pF(at-60 mV)by 1μmol/L BaCl2,a relative specific blocker of IK1.(2)Under the hypoxic condition,the cardiomyocyte was clamped at potential of +20mV,after the activation of IKATP,1μmol/L zacopride did not alter the value of this current.IKATP was inhibited by the specific IKATP antagonist glibenclamide(10μmol/L).(3)The RMP was markedly decreased and restored by 1μmol/L zacopride under hypoxic condition.The recovery of RMP by zacopride was abolished by 1μmol/L BaCl2.(4)The action potential amplitude(APA)was decreased from 113.80±2.31 to 89.27±7.40 and the action potential duration(APD)was prolongated markedly(from 12.64±0.34 ms to 20.38±1.86 ms at ADP50 and from 31.8±0.7 ms to 48.6±3.6 ms at ADP90).The APA,ADP50 and ADP90 weret restored(109.20±4.14,13.60±0.17 ms,33.8±1.1 ms)by 1μmol/L zacopride,which could be reversed by 1μmol/L BaCl2 under hypoxic condition.(5)Zacopride significantly depressed the DADs induced by hypoxia or by Iso under hypoxic condition.Pretreatment with 1μmol/L zacopride decreased the incidence of DADs from 73.3% to 20.0% in the hypoxic model and from 66.6% to 13.3% in the Iso model,which were removed by co-application of 1 μmol/L BaCl2.3.The effects of zacopride on IKir2.x currents in CHO cells under hypoxic condition.(1)The outward component of transiently expressing IKir2.1 currents decreased from 7.6±0.3 pA/pF to 5.8±0.3 pA/pF(at-60 mV)in CHO cells under hypoxic condition.The efficacy of zacopride was a concentration-relative and maximized at 100μmol/L with a 27.6% increase in the outward current(from 5.8±0.3 pA/pF to 7.4±0.3 pA/pF).(2)Zacopride(100μmol/L)did not significantly affect the IKir2.2 or IKir2.3 in hypoxic CHO cells.4.Zacopride upregulated the expression of Kir2.1 protein in rat ventricular myocytes subjected to simulated ischemia.Zacopride at 0.3-3.0μmol/L restored the decreased expression of Kir2.1 protein in rat ventricular myocytes subjected to simulated ischemia.Zacopride of 1μmol/L showed the maximal effect(P<0.05).5.Zacopride suppressed chronic ischemic arrhythmias in conscious ratZacopride has significantly reduced the occurrence of ventricular arrhythmia and CB but not SPVC in the rat subjected to myocardial infarction during 4 weeks.(1)There were no significantly statistical differences in survival rate among the experimental groups(P>0.05).(2)The rate of occurrence of arrhythmia in MI group was significantly higher than that in other groups.The episode number of PVC and VT/VF has shown two peak values in the first day and the fourth week after coronary occlusion in MI group,while they maintained stablely on a lower level in MI+zac group close sham group during 4 weeks.The episode number of CB in MI group was obviously increased in the first day and the third week,but it was always kept a stable lower level in MI+zac group during 4 weeks.The episode number of SPVC showed a trend of growing in MI group during 4 weeks and a parallel growing of it was appearanced in the group of MI+ zac as well.(3)In MI+zac group,there were no significantly statistical differences in the total episode number of SPVC among the experimental groups while the total episode number of CB was decreased from 79±15 to 36±7,the total episode number of PVC was decreased from 461±137 to 90±48,the episode duration of VT was decreased from 59±26 s to 12±12 s(n=8,P<0.05),respectively,compared with MI group.6.Zacopride suppressed arrhythmias triggered by Iso in conscious rats subjected to coronary occlusion.Iso(1280 μg/kg)was injected intravenously in all telemetry rats before they were executed.Prominent arrhythmia occurred in the MI and sham groups but not in MI+zacopride and sham+zacopride groups.In MI+zacopride group,the total episode number and the incidence of PVC were decreased from 1073±172,100% to 44±29,25%,and the episode duration,the incidence of VT were decreased from 109.2±30.0,80% to 0,0%;VF were decreased from 21.8±13.4,40% to 0,0% compared with MI group.In sham+zacopride group,the total episode number and the incidence of PVC were decreased from 981±95,100% to 42±29,22.2%,and the episode duration,the incidence of VT were decreased from 8.7±6.4,22.2% to 0,0% compared with sham group.7.Zacopride restored the down-regulated Kir2.1 protein in rats subjected to MI.The expression of Kir2.1 protein in MI group was decreased significantly but increased in MI+zac group compared with sham group.The expression of Kir2.1 protein in sham+zac group was the hightest of 4 groups.8.Zacopride significantly increased the IK1 current.Zacopride gradually increased the outward IK1 current by week in the ventricular myocyte of normal rat.The outward IK1 current was obviously increased after the third week,and higher than the indexes observed of 0 week in zacopride group and the same time point in control group.Conclusion:1.As a specific agonist of IK1,zacopride significantly suppressed acute ischemic arrhythmias(in 15 min)and chronic ventricular arrhythmias(in 4 week)in rat after coronary occlusion.2.The important mechanism for suppressing ischemic arrhythmia was that zacopride restored the RMP,shortened the APD and suppressed the incidence of DADs by activating the IK1 under hypoxic condition.3.Zacopride could increase Kir2.1 but not the Kir2.2 and Kir2.3 under hypoxic condition.Zacopride activated IK1 is mediated by Kir2.1 subtype,which is same as that in normoxic condition,reported previously by us.4.Zacopride upregulated the expression of Kir2.1 protein in acute and chronic ischemic ventricular myocytes. |
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