| BackgroundInward rectifier potassium channel which is a superfamily consisting of seven subfamilies (Kirl-Kir7) is widely expressed in various tissues. Kir2x channels which are mainly distributed in cardiac tissue mediate the IK1 current in the heart. Recent studies found that cardiac inward rectifier potassium channel (Kir2) played an important role in setting the resting membrane potential and cardiac excitability. It also likely have a profound effect on prevent arrhythmogenesis. Therefore, changes of Kir2 function certainly affect resting membrane potential, action potential repolarization and cardiac excitability, and contribute to arrhythmogenesis.Many studies have shown that the pathogenesis of arrhythmias underlying much disease or pathologic status related to the decrease of Kir2 channel function. In studies both from animal models and human ventricles of heart failure, although the results of different studies often vary, the most consistent electrophysiological changes causing arrhythmias are prolongation of the action potential and reduction in various potassium current, including the inward rectifier IK1. In addition, the arrhythmias caused by myocardial ischemia and infarction were also concerned with decrease of IK1. In recent years, Andersen-Tawil syndrome (ATS) is caused by mutations in KCNJ2 which encodes the inward rectifier K+ channel (Kir2.1). Reduction of Kir2.1 caused ventricular ectopy, polymorphic ventricular tachycardia and prolongation of the QT interval (LQT-7). Furthermore, the reduction in IK1 would lead to new arrhythmia induced by enhancing autorhthmicity and triggered activity. Many studies have confirmed that the proarrhythmic effects caused by reducing or inhibiting IK1 were greater than the antiarrhythmic effects. Therefore, it suggested that a proper augmentation of IK1 is reasonable to prevent arrhythmia induced by its reduction. However, there are no selective IK1 agonists until now. Currently, as antiarrhythmic drugs, almost all of the various ion channel blockers were liable to abnormal electrical activities because of inappropriate blocking some ionic currents. In this study, we suggested a new attempt to enhancing IK1 as an antiarrhythmic pathway. Previous research in our lab revealed that zacopride, as a potent gastrointestinal prokinetic agent, specially enhanced IK1 and had no effects on other major ionic currents in rat myocytes. Meanwhile, zacopride showed significant antiarrhythmic effects in rat arrhythmia models. In order to confirm that the antiarrhythmic effects of zacopride are mediated by selectively enhancing IK1, taking rabbit which share high degree of ion channels composition and action potential similarity to human as the experimental animal, we have recognized the effects of zacopride on various ion currents in rabbit ventricular myocytes and observed the inhibitory effects on multiple arrhythmic models, then analyzed the mechanism of its effects.Objective:In order to confirm that zacopride is the selective IK1 agonist, we would investigate the effects of zacopride on inward rectifier potassium current (IK1), transient outward potassium current (Ito), L-type calcium current (ICa-L), voltage-gated sodium current (INa), sodium-calcium exchange current (INa/Ca) and delayed rectifier potassium current (IK) in rabbit ventricular myocytes. The effects of zacopride on transmembrane potentials were observed. Furthermore, we used 5-HT4-receptor antagonist RS23597-190 and 5-HT3-receptor agonist M-chloropheylbiguanide respectively to analyze whether the effect of zacopride on IK1 is mediated by 5-HT3 receptor or 5-HT4 receptor. PKC inhibitor GF109203X, PKA inhibitor KT5720 and PKG inhibitor KT5823 were used to illustrate the probable signaling pathway(s) involved in the action of zacopride on IK1-The effect of zacopride on expression of p-PKA was observed by Western blot which was used to confirm the signaling pathway.Methods:Single rabbit ventricular myocyte was obtained by enzymatic dissociation procedure with collagenase and protease. Using whole cell recording and voltage-clamp mode, the effects of zacopride on IK1, Ito, ICa, INa, INa/Ca, IK were recorded. The effects of zacopride on resting membrane potential (RMP) and action potential (AP) were recorded by current-clamp mode. In addition, the effects of RS23597-190, M-chloropheylbiguanide, GF109203X, KT5720 and KT5823 on the action of zacopride on IK1 were observed by voltage-clamp technique. Furthermore, the effect of zacopride on expression of p-PKA was observed by Western blot.Results:1. Zacopride at 0.1μmol/L-10μmol/L could concentration-dependently enhance IK1.1μmol/L zacopride showed the maximal effect on IK1 with the mean increments by 43.3% in inward current (at-100 mV, from-14.21±1.56 to-20.36±2.36, P<0.05) and 22.1% in outward current (at-60 mV, from 8.01±0.46 to 9.78±0.05, P＜0.05), which could be mostly abolished by 1μmol/L BaCl2, a recognized blocker of IK1. The effect on IK1 of 1μmol/L zacopride was most effective and at higher concentration (>1μmol/L) appeared a saturation-like, even some weakened tendency. Meanwhile, zacopride had no effects on ICa-L, INa, INa/Ca, Ito, IK and IK, tail at 0.1μmol/L-10μmol/L (P>0.05).2. Zacopride at 0.1μmol/L-10μmol/L could concentration-dependently hyperpolarize resting membrane potential (RMP) and shorten action potential duration at 90% repolarization (APD90). Zacopride at 1μmol/L showed the maximal effects on RMP (from-80.4±1.1 mV to-85.3±0.5 mV, P＜0.05) and APD90 (from 459.6±33.2 ms to 385.9±23.4 ms, P＜0.05).3. After administration of 1μmol/L zacopride, the RMP started (onset) to increase at 2.24±0.09 min and reached steady state in 6.32±0.13 min, which consisted with the time course for IKI activation (started (onset) to increase at 2.19±0.10 min and 2.30±0.27 min and reached steady state in 6.36±0.16 min and 6.39±0.06 min at-60 mV and-100 mV, respectively.). The data suggested that the RMP hyperpolarization was probably the result of the activation of IK1 induced by zacopride.4. On the basis of presence of 10μmol/L 5-HT4 receptor antagonist RS23597-190 which itself reduced IK1,1μmol/L zacopride could still increase IK1 (P<0.05). Meanwhile, the increase of IK1 by zacopride could not be affected by 10μmol/L 5-HT3 receptor agonist M-chloropheylbiguanide. The results showed that the increase of IK1 by zacopride might not be dependent on 5-HT3 receptor and 5-HT4 receptor. 5. The PKA inhibitor KT5720 at 5μmol/L could eliminate the increase of IK1 induced by zacopride (P<0.05), while 5μmol/L PKC inhibitor GF109203x and 5μmol/L PKG inhibitor KT5823 had no significant effects on the action of zacopride (P>0.05). The data suggested that increase of IK1 by zacopride might be via a PKA-mediated signaling pathway.6. The result of Western blot showed that expression of p-PKA which increased after administrating zacopride (1μmol/L) at 5 min and 10 min (P<0.05 vs control). There were no significances between groups of without and with zacopride for 30 min (P>0.05 vs control). It suggested that the phosphorylation of PKA was stimulated by zacopride. Furthermore, increase of IK1 by zacopride was related to PKA-mediated signaling pathway.In the present study, we would recognize the effects of zacopride on arrhythmic models in vivo and in vitro, including ischemia-induced arrhythmia, reperfusion-induced arrhythmia, ouabain-induced arrhythmia and EADs in rabbit ventricular myocytes. Further, the possible mechanism was analyzed.Methods:1. Preparation of ischemia and reperfusion-induced arrhythmic models in vivoAfter the adult Japanese white rabbits were anesthetized, left thoracotomy was performed to expose the heart. Myocardial ischemia was induced by occlusion of coronary artery, followed by reperfusion. All rabbits underwent 15 min coronary artery occlusion and 15 min reperfusion to induce definite arrhythmias. The standard limb II leads of the ECG were recorded. Lidocaine at dose of 5μg/kg and different doses of zacopride were injected through ear vein at 3 min before ischemia or reperfusion. The total of VPB (ventricular premature beats), the duration and incidence of VT (ventricular tachycardia) and VF (ventricular fibrillation) were observed during ischemia and reperfusion before and after administration of zacopride or lidocaine. The experimental groups as follows:①Group of ischemia-induced arrhythmia (control group) or group of reperfusion-induced arrhythmia (control group);②Groups of administrating 1-10μg/kg zacopride before ischemia;③Group of administrating 5μg/kg lidocaine before ischemia;④Groups of administrating 1-10μg/kg zacopride before reperfusion;⑤Group of administrating 5μg/kg lidocaine before reperfusion.2. Preparation of ouabain-induced arrhythmic model in vivoAfter the adult Japanese white rabbits were anesthetized, standard limbⅡleads of the ECG were recorded. Ouabain at a dose of 10μg/kg was injected through ear vein to induce definite arrhythmia. Different doses of zacopride (1-10μg/kg) were injected through ear vein at 3 min before administration of ouabain. The total arrhythmia duration, the duration and incidence of VT and VF were observed before and after administration of zacopride during 30 min.The rabbits were randomly divided into 4 groups as follows:①Control group:injection 10μg/kg ouabain;②Group of administrating 1μg/kg zacopride:inject ouabain 3 min after administrating 1μg/kg zacopride;③Group of administrating 3μg/kg zacopride:inject ouabain 3 min after administrating 3μg/kg zacopride;④Group of administrating 10μg/kg zacopride:inject ouabain 3 min after administrating 10μg/kg zacopride.3. Preparation of ouabain-induced arrhythmic model in vitroAfter heparinization and anesthetization, the rabbit hearts were quickly removed and mounted on Langendorff aortic retrograde perfusion system. Three ECG electrodes were connected to cardiac apex, right auricular and ground respectively to simulate ECG leadⅡ. After equilibration for 30 min, ouabain (10μmol/L) was administered via Y-tube into the perfusate at the entrance of aorta to induce arrhythmia. ECG changes were recorded, the total arrhythmia duration, the duration and incidence of VT and VF were observed before and after administration of zacopride during 60 min.There were 5 groups as follows:①Control group:administrate 10μmol/L ouabain; ②Group of administrating 0.1μmol/L zacopride:administrate 10μmol/L ouabain and 0.1μmol/L zacopride;③Group of administrating 1μmol/L zacopride:administrate 10μmol/L ouabain and 1μmol/L zacopride;④Group of administrating 10μmol/L zacopride:administrate 10μmol/L ouabain and 10μmol/L zacopride;⑤Group of administrating 1μmol/L zacopride and BaCl2:administrate 10μmol/L ouabain, 1μmol/L zacopride and 1μmol/L BaCl2.4. Preparation of early afterdepolarization (EAD) model in rabbit ventricular myocytesSingle rabbit ventricular myocyte was obtained by enzymatic dissociation procedure with collagenase and protease. The action potential (AP) was recorded using whole cell recording and current-clamp mode. EADs were induced by administration of 5μmol/L 293B and 20 mmol/L isoproterenol. During the experiments, incidence of EADs and APD90 were observed.There were 2 groups as follows:①Control group (EAD model):after stabilization of action potential,293B was administrated, and isoproterenol was added after 30 s.②Group of administrating 1μmol/L zacopride:after stabilization of action potential, zacopride at 1μmol/L was pre-administrated,293B and isoproterenol were added as group one after 3 min.Results:1. The antiarrhythmic effects of zacopride at 1μg/kg-10μg/kg were observed in anesthetized rabbits during ischemia. The total of VPB, duration of VT and VF, incidence of VT and VF were concentration-dependently decreased by zacopride treatment. At the dose of 3μg/kg, zacopride showed the most potent antiarrhythmic action on ischemia-induced arrhythmia which compared favourably with lidocaine, a classical antiarrhythmic agent. With usage of 3μg/kg zacopride, the number of VPB decreased from 260±13 to 104±7 (P＜0.05), the incidence of VT declined from 100% to 37.5%(P＜0.05), and the duration of VT shorterned from 52.2±6.6 s to 5.1±7.9 s (P<0.05). None rabbit exhibited VF after application of 3μg/kg zacopride while 62.5% rabbits in control group developed VF with the mean duration of 9.8±3.1 s (P＜0.05).2. The concentration-dependent inhibitory effects of zacopride at 1μg/kg-10μg/kg on reperfusion-induced arrhythmia were observed in anesthetized rabbits. Zacopride showed the largest effect at dose of 3μg/kg on reperfusion-induced arrhythmia, which compared favourably with lidocaine. With usage of 3μg/kg zacopride, the number of VPB decreased from 119±10 to 54±7 (P＜0.05), the incidence of VT declined from 100% to 37.5%(P＜0.05), the duration of VT shorterned from 34.7±3.8 s to 4.5±2.5 s (P＜0.05), the incidence of VF declined from 87.5% to 12.5%(P＜0.05), and the duration of VF shorterned from 24.2±4.0 s to 0.6±0.6 s (P＜0.05).3. Zacopride at 1μg/kg-10μg/kg had concentration-dependent inhibitory effects on ouabain-induced arrhythmia in anesthetized rabbits. At the dose of 3μg/kg, zacopride showed the most potent antiarrhythmic action on ouabain-induced arrhythmia. With usage of 3μg/kg zacopride, the total arrhythmia duration shortened from 14.4±0.9 min to 8.5±0.7 min (P＜0.05), the incidence of VT declined from 100% to 37.5%(P＜0.05), the duration of VT shorterned from 54.7±3.2 s to 11.8±5.9 s (P＜0.05), and the incidence of VF declined from 87.5% to 12.5% (P＜0.05).4. The concentration-dependent inhibitory effects of zacopride at 0.1μmol/L-10μmol/L on ouabain-induced arrhythmia were observed in Langendorff-perfused rabbit hearts. The largest antiarrhythmic effect of zacopride appeared at 1μmol/L which consisted with the most effective concentration of zacopride on IK1. At 1μmol/L, zacopride reduced the incidence of VT from 100% to 37.5%(P＜0.05), the incidence of VF declined from 87.5% to 12.5%(P＜0.05), the duration of VT was shortened from 129.6±5.6 s to 35.0±17.1 s (P＜0.05), and the total arrhythmia duration was shortened from 31.6±1.2 min to 9.3±0.5 min (P＜0.05). BaCl2(the recorgnized blocker of IK1) at low concentration co-applied with zacopride (1μmol/L) could mostly abolish the antiarrhythmic effects of zacopride, indicating the antiarrhythmic effects of zacopride, at least partially, are mediated by enhancing IK1.5. Using whole cell recording and current clamp mode, the action potentials were recorded from rabbit ventricular myocytes. Early afterdepolarizations (EADs) were induced by 5μmol/L 293B and 20 mmol/L isoproterenol, zacopride at 1μmol/L significantly decreased the incidence of EADs from 81.8% to 9.1%(n=11, P＜0.05).Conclusion:1. Zacopride is firstly recongnized as a selective agonist of IK1 in rabbit ventricular myocytes. At the concentration of 0.1-10μmol/L, zacopride could selectively enhance inward rectifier potassium current (IK1) without effects on ICa-L, INa, Ito, INa/Ca, IK and IK,tail.2. At the concentration of 0.1-10μmol/L, zacopride could hyperpolarize the resting membrane potential (RMP) and shorten the action potential duration (APD). The effect of zacopride on RMP was highly related to the activation of IK1 by zacopride in time course.3. The enhancement of IK1 by zacopride in rabbit ventricular myocytes might be via PKA-mediated signaling pathway, while is independent on 5-HT3 receptor and 5-HT4 receptor.4. Zacopride showed markedly antiarrythmic effects on multiple rabbit ventricular arrhythmias, including ischemia and reperfusion-induced arrhythmias in vivo, ouabain-induced arrhythmias in vivo and in vitro, EADs in rabbit ventricular myocytes, being relevant to the activation of IK1.5. In present study, it suggested that selective activation of IK1 is a new antiarrhythmic pathway and is worth further studying.
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