激动心肌内向整流钾通道的抗心律失常作用观察
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摘要
研究背景
内向整流钾通道是由7个亚家族(Kir1-Kir7)组成的超家族,广泛分布在各种组织。其中Kir2主要分布于心肌组织,是产生心肌内向整流钾电流IK1的主要通道。近年来研究发现,心肌内向整流钾通道(Kir2)对维持心肌正常的静息电位水平、正常的兴奋节律和防止心律失常的发生都具有非常重要的意义。此外,Kir2还参与了心肌动作电位3期末复极,使跨膜电位迅速恢复到静息电位水平,这对维持心肌正常的兴奋节律非常重要。因此,Kir2功能的改变,必然会影响心肌细胞的静息电位水平和动作电位的复极,继而对心肌的电活动和心律失常的发生产生影响。
很多疾病或病理情况下发生的心律失常与Kir2功能下调有关。来自动物实验和临床研究发现,心衰时并发的心律失常均与钾电流(包括内向整流钾电流IK1)的减弱有关；心肌缺血和心肌梗死时发生的心律失常也与IK1的下降有关；近年来研究证明Andersen-Tawil综合征(ATS)主要是由于编码Kir2.1通道的基因KCNJ2发生了功能缺失性突变所致,从而导致多数患者发生室性异位搏动、多形性室性心动过速并伴有QT间期延长(LQT-7)等心电改变。而且抑制IK1本身就会由于自律性增高和触发活动而诱发新的心律失常,许多实验已证实抑制或阻断IK1产生的致心律失常作用要大于它的抗心律失常作用。因此提示我们,可能通过适当激动Kir2通道来防治全部或部分由于IK1减低而导致的心律失常。然而至今还未见有IK1选择性激动剂的报道。目前应用的抗心律失常药物几乎都是各种离子通道的阻断剂,很容易发生因某些离子电流的不适当阻断而出现新的电活动紊乱,导致新的心律失常发生。因此,对激动Kir2通道、增强IK1电流产生抗心律失常效应的研究,在理论和实践上都是一个新的尝试。
我们的前期研究发现促胃肠动力药zacopride(5-HT4受体激动剂和5-HT3受体拮抗剂)对大鼠心肌IK1表现出明显的选择性增强作用,同时对大鼠心律失常模型表现出显著的抗心律失常作用。为了在广度和深度上进一步证实zacopride对IK1的选择性激动作用及其通过激动IK1发挥的抗心律失常作用,本研究以离子通道构成和动作电位形态上更接近于人的兔作为实验动物,观察了zacopride对兔心室肌各种离子通道的作用和对多种心律失常模型的抑制效应及其产生机制。
目的
观察zacopride对兔心室肌细胞内向整流钾电流(IK1)、瞬时外向钾电流(Ito)、L-型钙电流(ICa-L)、电压门控钠电流(INa)、钠钙交换电流(INa/Ca)、延迟整流钾电流(IK)的作用,以证实zacopride对兔心室肌细胞IK1的选择性激动作用,同时观察zacopride对跨膜电位的影响；如得到预期结果,将进一步利用5-HT4受体阻断剂和5-HT3受体激动剂观测zacopride激动IK1的作用与细胞膜5-HT4和5-HT3受体的关系,利用PKA、PKC、PKG的阻断剂观测zacopride激动IK1可能的信号转导通路,同时利用蛋白免疫印迹技术进一步观察zacopride对磷酸化PKA表达的影响以证实zacopride激动IK1的信号转导通路。方法
用胶原酶和蛋白酶分离成年日本大耳白兔单个心室肌细胞,采用全细胞膜片钳技术,在电压钳模式下记录zacopride对兔心肌细胞主要离子电流IK1、Ito、ICa、INa、INa/Ca、IK的作用,在电流钳模式下记录zacopride对静息膜电位(RMP)和动作电位(AP)时程的影响；应用膜片钳技术观察10μmol/L5-HT4受体拮抗剂RS23597-190和10μmol/L5-HT3受体激动剂M-chloropheylbiguanide对zacopride激动IK1的影响,此外,分别利用5μmol/LPKC抑制剂GF109203X、PKA抑制剂KT5720和PKG抑制剂KT5823分析zacopride激动IK1的信号转导途径,同时应用Western blot方法观察zacopride对磷酸化PKA表达的影响。结果
1.0.1μmol/L-10μmol/L zacopride对IK1具有浓度依赖性激动作用,1μmol/L zacopride对IK1作用最明显,继续增加浓度,其效应不再增加或略有下降。1μmol/L zacopride可使IK1内向电流密度增加43.3%(在-100 mV时,由-14.21±1.56到-20.36±2.36,P<0.05),外向电流密度增加22.1%(在-60 mV时,由8.01±0.46到9.78±0.05,P<0.05)。1μmol/LBaCl2可大部分消除上述zacopride增强IK1的作用。在上述相同的浓度范围内,zacopride对ICa-L、INa、INa/Ca、Ito、IK和IK.tail均无明显作用(P>0.05)
2.0.1-10μmol/L zacopride可浓度依赖性增大静息膜电位,缩短动作电位时程,1μmol/L为zacopride最大效应浓度,可使心肌细胞RMP由-80.4±1.1 mV增加至-85.3±0.5mV；动作电位复极90%的时程(Action potential duration at 90% repolarization, APD90)由459.6±33.2 ms缩短至385.9±23.4ms(P<0.05)。
3. Zacopride增强IK1与增大静息电位时间过程的比较。给予1μmol/L zacopride, RMP平均在给药后2.24±0.09min开始增大,到6.32±0.13min达到稳态。IK1外向电流平均在给药后2.19±0.10min开始增大,到6.36±0.16 min达到稳态,IK1内向电流平均在给药后2.30±0.27min开始增大,到6.39±0.06 min达到稳态。RMP的变化和IK1的激活时间过程基本同步(n=6,各时间点P>0.05),说明zacopride增大静息膜电位与激活IK1密切相关。
4.10μmol/L 5-HT4受体拮抗剂RS23597-190本身可以减弱IK1电流,预先应用RS23597-190后加入1μmol/L zacopride,后者仍然能够增大IK1(P<0.05),推测zacopride激动IK1可能不是由5-HT4受体介导；10μmol/L 5-HT3受体激动剂M-chloropheylbiguanide不能影响1μmol/L zacopride激动IK1的作用(P>0.05),推测zacopride也不是依赖5-HT3受体而激动IK1电流的。
5.5μmol/L特异性PKC抑制剂GF109203x或5μmol/L特异性PKG抑制剂KT5823不能消除1μmol/L zacopride激动IK1的效应(P>0.05),而5μmol/L特异性PKA抑制剂KT5720能够显著抑制1μmol/L zacopride激动IK1的效应(P<0.05),提示zacopride激动IK1的效应可能受AC/cAMP/PKA信号转导系统调节。
6. Western blot结果显示1μmol/L zacopride作用5min和10 min后,兔心肌细胞磷酸化PKA的表达量明显增加(P<0.05,与对照组相比),随着zacopride作用时间的延长,磷酸化PKA的表达量有所减少,在zacopride作用30min时,磷酸化PKA的表达量与对照组无明显差异(P>0.05,与对照组相比)。提示1μmol/L zacopride能够促进心肌细胞PKA磷酸化,进一步证实zacopride激动IK1与PKA信号转导途径有关。
目的
观察zacopride对在体心脏、离体心脏和心肌细胞水平心律失常模型的效应,包括对缺血及再灌注诱发心律失常、哇巴因诱发心律失常的效应,以及对心室肌细胞早期后除极的效应,并结合应用IK1阻断剂BaCl2的干预,分析可能的作用机制。
方法
1.在体缺血性及再灌注性心律失常模型的制备
成年日本大耳白兔,麻醉,开胸,记录心电图,结扎冠状动脉左前降支,局部缺血15min,松扎后再灌注15min。不同剂量的zacopride和5μg/kg lidocaine分别于缺血前3min和再灌注前3 min耳缘静脉注射。实验中记录各组动物室性期前收缩(VPB, ventricular premature beats)的发生次数、室速(VT, ventricular tachycardia)和室颤(VF, ventricular fibrillation)的持续时间以及发生率。
实验分组：
①缺血性心律失常组(对照组)或再灌注性心律失常组(对照组)；
②缺血前给予zacopride不同剂量(1-10μg/kg)组；
③缺血前给予5μg/kg lidocaine组;
④再灌注前给予zacopride不同剂量(1-10μg／kg)组；
⑤再灌注前给予5μg/kg lidocaine组。
2.在体心脏哇巴因(ouabain)诱发的心律失常模型的制备
成年日本大耳白兔,麻醉,记录心电图,耳缘静脉注射ouabain (10μg/kg)诱发心律失常。观察心电图,记录给予ouabain后30 min内心律失常的持续时间、VT和VF的持续时间以及发生率。
实验分组：
①对照组：耳缘静脉注射10μg/kg ouabain;
②zacopride 1μg/kg组：预先注射1μg/kg zacopride, 3 min后再注射ouabain;
③zacopride 3μg/kg组：预先注射3μg/kg zacopride,3 min后再注射ouabain;
④zacopride 10μg/kg组：预先注射10μg/kg zacopride,3 min后再注射ouabain。
3.离体哇巴因(ouabain)诱发的心律失常模型的制备
成年日本大耳白兔,麻醉,开胸取出心脏,将心脏悬挂在Langendorff灌流装置上经主动脉逆行灌流台氏液,记录心电图。平衡30min后给药,10μmol/L ouabain由侧管经主动脉逆行注入心脏诱发心律失常。观察心电图,记录给药后60min内心律失常的持续时间。包括总的心律失常持续时间、VT和VF的持续时间以及发生率。
实验分组：
①对照组：给予10μmol/L ouabain;
②zacopride 0.1μmol/L组：给予10μmol/L ouabain和0.1μmol/L zacopride;
③zacopride 1μmol/L组：给予10μmol/L ouabain和1μmol/L zacopride;
④zacopride 10μmol/L组：给予10μmol/L ouabain和10μmol/L zacopride;
⑤zacopride 1μmol/L+BaCl2组：给予10μmol/L ouabain、1μmol/L zacopride和1μmol/L BaCl2。
4.兔心室肌细胞早期后除极模型的制备
用胶原酶和蛋白酶分离成年日本大耳白兔单个心室肌细胞,应用全细胞膜片钳技术,在电流钳模式下记录动作电位,加入5μmol/L 293B,30s后再加入20 mmol/L isoproterenol,诱发早期后除极(EADs)。实验中观测EADs的发生率以及APD90的变化。
实验分组：
①对照组(早期后除极模型)：待动作电位稳定后,加入5μmol/L 293B,30 s后再加入20 mmol/L isoproterenol;
②zacopride 1μmol/L组：待动作电位稳定后,预先加入1μmol/L zacopride, 3 min后加入5μmol/L 293B,30s后再加入20 mmol/L isoproterenol。结果
1.1-10μg/kg zacopride可以浓度依赖性地抑制麻醉兔在体缺血性心律失常的发生。在对照组,室速发生率为100%,持续时间52.2±6.6s,62.5%发生室颤,持续时间9.8±3.1s,总期前收缩个数为260±13。3μg／kg为zacopride抗心律失常的最大效应剂量,该剂量下,室速发生率降低为37.5%,持续时间减少至5.1±7.9s,无室颤发生,期前收缩总个数降低为104±7(P<0.05)。其抗心律失常效应与阳性对照药利多卡因(lidocaine)相似。
2.1-10μg/kg zacopride可以浓度依赖性地抑制麻醉兔在体再灌注诱发心律失常的发生。对照组中,100%兔发生室速,持续时间34.7±3.8s,87.5%兔发生室颤,持续时间24.2±4.0s,总的期前收缩个数119±10。3μ/kg为zacopride抗心律失常的最大效应剂量,该剂量下,室速发生率降低为37.5%,持续时间减少至4.5±2.5s,室颤发生率降低为12.5%,持续时间减少为0.6±0.6s,期前收缩总个数降低为54±7(P<0.05)。其抗心律失常效应与阳性对照药利多卡因(lidocaine)相似。
3.1-10μg/kg zacopride可以浓度依赖性地抑制麻醉兔在体哇巴因诱发心律失常的发生。对照组中,100%兔发生室速,持续时间54.7±3.2s,100%兔发生室颤,持续时间40.6±3.2s,总心律失常持续时间14.4±0.9min。3μg/kg为zacopride抗心律失常的最大效应剂量,该剂量下,室速发生率降低为37.5%,持续时间减少为11.8±5.9s,室颤发生率降低为12.5%,持续时间减少为1.8±1.8s,总心律失常持续时间降低为8.5±0.7 min(P<0.05)。
4.0.1-10μmol/L zacopride可以浓度依赖性地抑制离体兔心脏哇巴因诱发心律失常的发生。对照组中,100%兔发生室速,持续时间129.6±5.6s；87.5%兔发生室颤,持续时间29.7±4.4s；总心律失常持续时间31.6±1.2 min。与zacopride激动IK1的最有效浓度一致,1μmol/L为zacopride抗心律失常的最大效应浓度,该浓度下,室速发生率降低为37.5%,持续时间减少为35.0±17.1s,室颤发生率降低为12.5%,持续时间减少为2.4±2.4s,总心律失常持续时间降低为9.3±0.5 min(P<0.05)。同时给予1μmol/L zacopride和1μmol/L BaCl2(IK1阻断剂)后,与对照组相比,室速和室颤的发生率以及持续时间无明显差异(P>0.05),总心律失常持续时间有所降低(P<0.05)；与单独给予1μmol/Lzacopride组相比,室速和室颤持续时间、室颤发生率以及总心律失常持续时间明显增加(P<0.05),表明1μmol/L BaCl2能够大部分消除zacopride的抗心律失常作用,因此推测zacopride可能通过激动IK1而发挥其抗心律失常作用。
5.在兔心室肌细胞,利用全细胞电流钳模式记录动作电位,5μmol/L 293B和20mmol/L异丙肾上腺素可诱发早期后除极(EADs)。1μmol/L zacopride可以有效抑制EADs的发生,使其发生率由81.8%下降到9.1%(n=11,P<0.05)
结论
1.在兔心室肌,zacopride (0.1-10μmol/L)对内向整流钾电流(IK1)具有选择性激动作用,而对L-型钙电流(ICa-L)、电压门控钠电流(INa)、瞬时外向钾电流(Ito)、钠钙交换电流(INa/Ca)、延迟整流钾电流(IK)及其尾流(IK,tail)均无明显影响,是首个被证实的IK1选择性激动剂。
2. Zacopride (0.1-10μmol/L)能够增大兔心室肌细胞静息膜电位(RMP),缩短动作电位时程(APD),该作用与其激动IK1相关。
3. Zacopride选择性激动IK1的作用不是经5-HT3受体和5-HT4受体介导,而可能受AC/cAMP/PKA信号转导系统调节。
4. Zacopride对兔心脏具有显著的抗心律失常作用,表现为显著抑制在体缺血以及再灌注诱发的心律失常、在体和离体哇巴因诱发的心律失常,以及心室肌细胞的早期后除极。其作用机制与zacopride对IK1的激动作用相关。
5.本研究提示激动IK1是一条新的、有效的抗心律失常途径,值得进一步深入研究。
Background
Inward 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 vivo
After 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 vivo
After 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 vitro
After 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 myocytes
Single 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.
内向整流钾通道是由7个亚家族(Kir1-Kir7)组成的超家族,广泛分布在各种组织。其中Kir2主要分布于心肌组织,是产生心肌内向整流钾电流IK1的主要通道。近年来研究发现,心肌内向整流钾通道(Kir2)对维持心肌正常的静息电位水平、正常的兴奋节律和防止心律失常的发生都具有非常重要的意义。此外,Kir2还参与了心肌动作电位3期末复极,使跨膜电位迅速恢复到静息电位水平,这对维持心肌正常的兴奋节律非常重要。因此,Kir2功能的改变,必然会影响心肌细胞的静息电位水平和动作电位的复极,继而对心肌的电活动和心律失常的发生产生影响。
很多疾病或病理情况下发生的心律失常与Kir2功能下调有关。来自动物实验和临床研究发现,心衰时并发的心律失常均与钾电流(包括内向整流钾电流IK1)的减弱有关；心肌缺血和心肌梗死时发生的心律失常也与IK1的下降有关；近年来研究证明Andersen-Tawil综合征(ATS)主要是由于编码Kir2.1通道的基因KCNJ2发生了功能缺失性突变所致,从而导致多数患者发生室性异位搏动、多形性室性心动过速并伴有QT间期延长(LQT-7)等心电改变。而且抑制IK1本身就会由于自律性增高和触发活动而诱发新的心律失常,许多实验已证实抑制或阻断IK1产生的致心律失常作用要大于它的抗心律失常作用。因此提示我们,可能通过适当激动Kir2通道来防治全部或部分由于IK1减低而导致的心律失常。然而至今还未见有IK1选择性激动剂的报道。目前应用的抗心律失常药物几乎都是各种离子通道的阻断剂,很容易发生因某些离子电流的不适当阻断而出现新的电活动紊乱,导致新的心律失常发生。因此,对激动Kir2通道、增强IK1电流产生抗心律失常效应的研究,在理论和实践上都是一个新的尝试。
我们的前期研究发现促胃肠动力药zacopride(5-HT4受体激动剂和5-HT3受体拮抗剂)对大鼠心肌IK1表现出明显的选择性增强作用,同时对大鼠心律失常模型表现出显著的抗心律失常作用。为了在广度和深度上进一步证实zacopride对IK1的选择性激动作用及其通过激动IK1发挥的抗心律失常作用,本研究以离子通道构成和动作电位形态上更接近于人的兔作为实验动物,观察了zacopride对兔心室肌各种离子通道的作用和对多种心律失常模型的抑制效应及其产生机制。
目的
观察zacopride对兔心室肌细胞内向整流钾电流(IK1)、瞬时外向钾电流(Ito)、L-型钙电流(ICa-L)、电压门控钠电流(INa)、钠钙交换电流(INa/Ca)、延迟整流钾电流(IK)的作用,以证实zacopride对兔心室肌细胞IK1的选择性激动作用,同时观察zacopride对跨膜电位的影响；如得到预期结果,将进一步利用5-HT4受体阻断剂和5-HT3受体激动剂观测zacopride激动IK1的作用与细胞膜5-HT4和5-HT3受体的关系,利用PKA、PKC、PKG的阻断剂观测zacopride激动IK1可能的信号转导通路,同时利用蛋白免疫印迹技术进一步观察zacopride对磷酸化PKA表达的影响以证实zacopride激动IK1的信号转导通路。方法
用胶原酶和蛋白酶分离成年日本大耳白兔单个心室肌细胞,采用全细胞膜片钳技术,在电压钳模式下记录zacopride对兔心肌细胞主要离子电流IK1、Ito、ICa、INa、INa/Ca、IK的作用,在电流钳模式下记录zacopride对静息膜电位(RMP)和动作电位(AP)时程的影响；应用膜片钳技术观察10μmol/L5-HT4受体拮抗剂RS23597-190和10μmol/L5-HT3受体激动剂M-chloropheylbiguanide对zacopride激动IK1的影响,此外,分别利用5μmol/LPKC抑制剂GF109203X、PKA抑制剂KT5720和PKG抑制剂KT5823分析zacopride激动IK1的信号转导途径,同时应用Western blot方法观察zacopride对磷酸化PKA表达的影响。结果
1.0.1μmol/L-10μmol/L zacopride对IK1具有浓度依赖性激动作用,1μmol/L zacopride对IK1作用最明显,继续增加浓度,其效应不再增加或略有下降。1μmol/L zacopride可使IK1内向电流密度增加43.3%(在-100 mV时,由-14.21±1.56到-20.36±2.36,P<0.05),外向电流密度增加22.1%(在-60 mV时,由8.01±0.46到9.78±0.05,P<0.05)。1μmol/LBaCl2可大部分消除上述zacopride增强IK1的作用。在上述相同的浓度范围内,zacopride对ICa-L、INa、INa/Ca、Ito、IK和IK.tail均无明显作用(P>0.05)
2.0.1-10μmol/L zacopride可浓度依赖性增大静息膜电位,缩短动作电位时程,1μmol/L为zacopride最大效应浓度,可使心肌细胞RMP由-80.4±1.1 mV增加至-85.3±0.5mV；动作电位复极90%的时程(Action potential duration at 90% repolarization, APD90)由459.6±33.2 ms缩短至385.9±23.4ms(P<0.05)。
3. Zacopride增强IK1与增大静息电位时间过程的比较。给予1μmol/L zacopride, RMP平均在给药后2.24±0.09min开始增大,到6.32±0.13min达到稳态。IK1外向电流平均在给药后2.19±0.10min开始增大,到6.36±0.16 min达到稳态,IK1内向电流平均在给药后2.30±0.27min开始增大,到6.39±0.06 min达到稳态。RMP的变化和IK1的激活时间过程基本同步(n=6,各时间点P>0.05),说明zacopride增大静息膜电位与激活IK1密切相关。
4.10μmol/L 5-HT4受体拮抗剂RS23597-190本身可以减弱IK1电流,预先应用RS23597-190后加入1μmol/L zacopride,后者仍然能够增大IK1(P<0.05),推测zacopride激动IK1可能不是由5-HT4受体介导；10μmol/L 5-HT3受体激动剂M-chloropheylbiguanide不能影响1μmol/L zacopride激动IK1的作用(P>0.05),推测zacopride也不是依赖5-HT3受体而激动IK1电流的。
5.5μmol/L特异性PKC抑制剂GF109203x或5μmol/L特异性PKG抑制剂KT5823不能消除1μmol/L zacopride激动IK1的效应(P>0.05),而5μmol/L特异性PKA抑制剂KT5720能够显著抑制1μmol/L zacopride激动IK1的效应(P<0.05),提示zacopride激动IK1的效应可能受AC/cAMP/PKA信号转导系统调节。
6. Western blot结果显示1μmol/L zacopride作用5min和10 min后,兔心肌细胞磷酸化PKA的表达量明显增加(P<0.05,与对照组相比),随着zacopride作用时间的延长,磷酸化PKA的表达量有所减少,在zacopride作用30min时,磷酸化PKA的表达量与对照组无明显差异(P>0.05,与对照组相比)。提示1μmol/L zacopride能够促进心肌细胞PKA磷酸化,进一步证实zacopride激动IK1与PKA信号转导途径有关。
目的
观察zacopride对在体心脏、离体心脏和心肌细胞水平心律失常模型的效应,包括对缺血及再灌注诱发心律失常、哇巴因诱发心律失常的效应,以及对心室肌细胞早期后除极的效应,并结合应用IK1阻断剂BaCl2的干预,分析可能的作用机制。
方法
1.在体缺血性及再灌注性心律失常模型的制备
成年日本大耳白兔,麻醉,开胸,记录心电图,结扎冠状动脉左前降支,局部缺血15min,松扎后再灌注15min。不同剂量的zacopride和5μg/kg lidocaine分别于缺血前3min和再灌注前3 min耳缘静脉注射。实验中记录各组动物室性期前收缩(VPB, ventricular premature beats)的发生次数、室速(VT, ventricular tachycardia)和室颤(VF, ventricular fibrillation)的持续时间以及发生率。
实验分组：
①缺血性心律失常组(对照组)或再灌注性心律失常组(对照组)；
②缺血前给予zacopride不同剂量(1-10μg/kg)组；
③缺血前给予5μg/kg lidocaine组;
④再灌注前给予zacopride不同剂量(1-10μg／kg)组；
⑤再灌注前给予5μg/kg lidocaine组。
2.在体心脏哇巴因(ouabain)诱发的心律失常模型的制备
成年日本大耳白兔,麻醉,记录心电图,耳缘静脉注射ouabain (10μg/kg)诱发心律失常。观察心电图,记录给予ouabain后30 min内心律失常的持续时间、VT和VF的持续时间以及发生率。
实验分组：
①对照组：耳缘静脉注射10μg/kg ouabain;
②zacopride 1μg/kg组：预先注射1μg/kg zacopride, 3 min后再注射ouabain;
③zacopride 3μg/kg组：预先注射3μg/kg zacopride,3 min后再注射ouabain;
④zacopride 10μg/kg组：预先注射10μg/kg zacopride,3 min后再注射ouabain。
3.离体哇巴因(ouabain)诱发的心律失常模型的制备
成年日本大耳白兔,麻醉,开胸取出心脏,将心脏悬挂在Langendorff灌流装置上经主动脉逆行灌流台氏液,记录心电图。平衡30min后给药,10μmol/L ouabain由侧管经主动脉逆行注入心脏诱发心律失常。观察心电图,记录给药后60min内心律失常的持续时间。包括总的心律失常持续时间、VT和VF的持续时间以及发生率。
实验分组：
①对照组：给予10μmol/L ouabain;
②zacopride 0.1μmol/L组：给予10μmol/L ouabain和0.1μmol/L zacopride;
③zacopride 1μmol/L组：给予10μmol/L ouabain和1μmol/L zacopride;
④zacopride 10μmol/L组：给予10μmol/L ouabain和10μmol/L zacopride;
⑤zacopride 1μmol/L+BaCl2组：给予10μmol/L ouabain、1μmol/L zacopride和1μmol/L BaCl2。
4.兔心室肌细胞早期后除极模型的制备
用胶原酶和蛋白酶分离成年日本大耳白兔单个心室肌细胞,应用全细胞膜片钳技术,在电流钳模式下记录动作电位,加入5μmol/L 293B,30s后再加入20 mmol/L isoproterenol,诱发早期后除极(EADs)。实验中观测EADs的发生率以及APD90的变化。
实验分组：
①对照组(早期后除极模型)：待动作电位稳定后,加入5μmol/L 293B,30 s后再加入20 mmol/L isoproterenol;
②zacopride 1μmol/L组：待动作电位稳定后,预先加入1μmol/L zacopride, 3 min后加入5μmol/L 293B,30s后再加入20 mmol/L isoproterenol。结果
1.1-10μg/kg zacopride可以浓度依赖性地抑制麻醉兔在体缺血性心律失常的发生。在对照组,室速发生率为100%,持续时间52.2±6.6s,62.5%发生室颤,持续时间9.8±3.1s,总期前收缩个数为260±13。3μg／kg为zacopride抗心律失常的最大效应剂量,该剂量下,室速发生率降低为37.5%,持续时间减少至5.1±7.9s,无室颤发生,期前收缩总个数降低为104±7(P<0.05)。其抗心律失常效应与阳性对照药利多卡因(lidocaine)相似。
2.1-10μg/kg zacopride可以浓度依赖性地抑制麻醉兔在体再灌注诱发心律失常的发生。对照组中,100%兔发生室速,持续时间34.7±3.8s,87.5%兔发生室颤,持续时间24.2±4.0s,总的期前收缩个数119±10。3μ/kg为zacopride抗心律失常的最大效应剂量,该剂量下,室速发生率降低为37.5%,持续时间减少至4.5±2.5s,室颤发生率降低为12.5%,持续时间减少为0.6±0.6s,期前收缩总个数降低为54±7(P<0.05)。其抗心律失常效应与阳性对照药利多卡因(lidocaine)相似。
3.1-10μg/kg zacopride可以浓度依赖性地抑制麻醉兔在体哇巴因诱发心律失常的发生。对照组中,100%兔发生室速,持续时间54.7±3.2s,100%兔发生室颤,持续时间40.6±3.2s,总心律失常持续时间14.4±0.9min。3μg/kg为zacopride抗心律失常的最大效应剂量,该剂量下,室速发生率降低为37.5%,持续时间减少为11.8±5.9s,室颤发生率降低为12.5%,持续时间减少为1.8±1.8s,总心律失常持续时间降低为8.5±0.7 min(P<0.05)。
4.0.1-10μmol/L zacopride可以浓度依赖性地抑制离体兔心脏哇巴因诱发心律失常的发生。对照组中,100%兔发生室速,持续时间129.6±5.6s；87.5%兔发生室颤,持续时间29.7±4.4s；总心律失常持续时间31.6±1.2 min。与zacopride激动IK1的最有效浓度一致,1μmol/L为zacopride抗心律失常的最大效应浓度,该浓度下,室速发生率降低为37.5%,持续时间减少为35.0±17.1s,室颤发生率降低为12.5%,持续时间减少为2.4±2.4s,总心律失常持续时间降低为9.3±0.5 min(P<0.05)。同时给予1μmol/L zacopride和1μmol/L BaCl2(IK1阻断剂)后,与对照组相比,室速和室颤的发生率以及持续时间无明显差异(P>0.05),总心律失常持续时间有所降低(P<0.05)；与单独给予1μmol/Lzacopride组相比,室速和室颤持续时间、室颤发生率以及总心律失常持续时间明显增加(P<0.05),表明1μmol/L BaCl2能够大部分消除zacopride的抗心律失常作用,因此推测zacopride可能通过激动IK1而发挥其抗心律失常作用。
5.在兔心室肌细胞,利用全细胞电流钳模式记录动作电位,5μmol/L 293B和20mmol/L异丙肾上腺素可诱发早期后除极(EADs)。1μmol/L zacopride可以有效抑制EADs的发生,使其发生率由81.8%下降到9.1%(n=11,P<0.05)
结论
1.在兔心室肌,zacopride (0.1-10μmol/L)对内向整流钾电流(IK1)具有选择性激动作用,而对L-型钙电流(ICa-L)、电压门控钠电流(INa)、瞬时外向钾电流(Ito)、钠钙交换电流(INa/Ca)、延迟整流钾电流(IK)及其尾流(IK,tail)均无明显影响,是首个被证实的IK1选择性激动剂。
2. Zacopride (0.1-10μmol/L)能够增大兔心室肌细胞静息膜电位(RMP),缩短动作电位时程(APD),该作用与其激动IK1相关。
3. Zacopride选择性激动IK1的作用不是经5-HT3受体和5-HT4受体介导,而可能受AC/cAMP/PKA信号转导系统调节。
4. Zacopride对兔心脏具有显著的抗心律失常作用,表现为显著抑制在体缺血以及再灌注诱发的心律失常、在体和离体哇巴因诱发的心律失常,以及心室肌细胞的早期后除极。其作用机制与zacopride对IK1的激动作用相关。
5.本研究提示激动IK1是一条新的、有效的抗心律失常途径,值得进一步深入研究。
Background
Inward 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 vivo
After 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 vivo
After 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 vitro
After 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 myocytes
Single 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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