特异性增强心肌内向整流钾电流（I_（K1））-抗心律失常新途径
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摘要
研究背景:
目前临床应用的抗心律失常药物主要是阻断心肌Na~+、K~+、Ca~(2+)离子通道,但其致心律失常副作用也引起了普遍担忧,寻找抗心律失常的新靶点和新机制一直是一个研究热点。当前,众多国内外学者将目光投向了内向整流钾电流(Inward rectifier potassium current,I_(K1))。内向整流钾通道是心肌最主要的背景外向电流,参与静息电位(Resting membrane potential,RMP)的维持和心肌动作电位(Action potential,AP)3期终末的复极。其分子基础主要是内向整流钾通道基因Kir2.x亚家族。在心室肌静息膜电位附近,I_(K1)电导要远远大于除ATP敏感钾通道(I_(KATP))以外的其他离子通道,而I_(KATP)在正常情况下是不激活的。因此,调节I_(K1)必将影响心肌静息膜电位的稳定和动作电位复极,从而对心肌的兴奋性和心律失常的发生产生深刻影响。但两个因素制约了其与心律失常关系的深入研究:一是迄今没有一种高选择性的I_(K1)阻断剂或激动剂,缺乏必要的工具药;二是目前所有抗心律失常药物均为各种离子通道或受体的阻断剂,而事实上,试图通过阻断I_(K1)产生抗心律失常作用的努力一直没有取得成功。理论上讲,I_(K1)被抑制后将使膜去极化,细胞的兴奋性和自律性增高,同时有助于早后除极、延迟后除极等异常自律性的发生;作为心室肌细胞主要的背景外向电流,I_(K1)通道的抑制使膜电阻增大,放大了跨膜电流引起的膜电位波动,从而造成膜电位的不稳定;抑制I_(K1)还可延长动作电位时程(APD),虽然在一定程度上可通过延长不应期抑制折返活动,但在某些场合也易引发长QT综合征。最近,Sung和Seemann等都利用计算机软件模拟抑制I_(K1)的效应,表明降低Kir2.1通道(形成I_(K1)的主要通道)的电导,可导致动作电位终末复极延迟,静息电位减小,出现自发性的室性心律失常。大量的临床和实验研究表明,许多疾病或病理状态下发生的心律失常是由于I_(K1)被抑制造成的。形成Kir2.1通道的基因KCNJ2突变可导致Andersen-Tawil综合症(ATS)。突变基因通过I_(K1)负显性抑制效应使通道功能降低,多数患者(71%)出现QT延长,64%的患者伴有室性心律失常。Miake等利用腺病毒将突变的Kir2.1通道基因转染给在体成年豚鼠心肌细胞,由于I_(K1)密度减小,出现APD延长,静息电位减小并不稳定和明显的异常自律活动。这些研究结果都说明了抑制I_(K1)的致心律失常风险。心衰患者中有一半伴有多种形态的心律失常,并经常由此发生猝死。很多临床和动物实验的研究证明,I_(K1)的减弱是心衰时心律失常发生的重要机制。心肌缺血和心肌梗死时发生的心律失常也与I_(K1)的下降有关。Almond等报告,大鼠慢性心肌梗死模型的心肌I_(K1)电流减小20%;Pinto等证实,狗心肌缺血区心内膜下普肯野纤维静息电位减小,是由于I_(K1)电流减小造成的;Kiesecker等在人的心肌细胞证明,内皮素可显著抑制I_(K1),此作用是内皮素致心律失常的重要机制。与此同时,也有一些完全相反的研究报道。如Li等利用转基因动物造成Kir2.1基因过表达,I_(K1)通道电导增大9~10倍,导致室性早搏、房室阻滞、房颤等多种心律失常。但这种过度表达引起的后果与上述全部或部分由于I_(K1)电流减低导致的心律失常并不矛盾。
鉴于临床中存在大量因I_(K1)减弱所致的心律失常和抑制或阻断I_(K1)的致心律失常风险,Miake等曾提出,需要设计药物或基因治疗,用于防治全部或部分由于I_(K1)减低导致的心律失常。Dhamoon等也认为,不应抑制(suppression)而是调制(modulation)I_(K1),以防治心律失常。Lopatin等更是明确指出理想的抗室性心律失常药物应在静息膜电位而非动作电位峰值开放钾通道,例如作用于I_(K1)的药物(the ideal ventricular antiarrhythmic agent might be a drug that opens K~+ channels at the resting potential, but not at the peak of the action potential, i.e. one that acts on I_(K1))。很显然,这里需要适当增强I_(K1)的药物,然而至今还未见有I_(K1)选择性激动剂的报道。Xiao等曾观察到吗啡可增强I_(K1)并使膜超级化,但它同时又可显著增强L型钙电流(ICa-L)而无特异性。
Zacopride属苯甲酰胺衍生物,兼有5-HT3受体抑制剂和5-HT4受体激动剂的性质,作用于胃肠5-HT4受体促进乙酰胆碱释放,对实验动物胃肠道具有促动力作用。我们在一项比较5-HT4受体激动剂西沙比利(cisapride)、RS67506和zacopride致心律失常副作用的研究中发现,与西沙比利显著的促心律失常副作用不同,zacopride对心律和心功能均无明显影响。探究其作用的离子机制发现,西沙比利和RS67506可抑制对大鼠心室肌I_(K1),而Zacopride对大鼠心室肌I_(K1)表现出显著的激动作用,对内向电流和外向电流部分均有明显增强效应;同时表现出明确的通道选择性,对ICa-L, INa, Ito, Isus, INa/Ca, Ipump等其他影响动作电位的主要离子流均无显著作用。这提示我们Zacopride很可能是I_(K1)选择性激动剂和潜在的抗心律失常药物。
目的:
1.确证zacopride为大鼠心肌细胞内向整流钾通道(I_(K1))特异性激动剂;
2.探讨zacopride增强I_(K1)可能的受体和信号转导机制;
3.建立大鼠心脏乳头肌延迟后除极(DADs)和触发活动(TA)模型,检验zacopride对DADs和TA的影响;
4.建立大鼠离体和在体心律失常模型,检验zacopride对多种原因所致室性心律失常的影响;
5.应用低浓度Ba~(2+)(I_(K1)相对特异性阻断剂)对I_(K1)的抑制作用,证实特异性增强I_(K1)与抗心律失常效应之间的关系。
方法:
1.采用胶原酶法急性分离大鼠(或豚鼠)左心室肌细胞。选用健康成年雄性SD大鼠(体重220～250g)或豚鼠(体重280～350g),于术前15分钟腹腔静脉注射肝素(1000U/kg),戊巴比妥钠(65mg/kg,ip)麻醉后颈动脉放血,迅速开胸取出心脏,置于4oC用100%氧气饱和的无钙台氏液中修剪,然后将心脏悬挂在Langendorff灌流装置上经主动脉逆行灌流。先用无钙台氏液灌流8～10分钟,再用胶原酶液循环灌流15～20分钟。灌流过程中保持37oC恒温,灌流压80cmH2O,并持续通以100%氧气。待心室肌组织变大、变软后将左心室剪下,置于KB液中剪碎,轻轻吹打得到分散的左心室肌细胞,经150μm孔径的滤网过滤后保存于KB液中,室温放置5～6小时后进行实验。实验前先将存放于高钾KB液中的细胞逐步复钙,然后取几滴细胞悬液加入细胞池(约1ml),平放在连有微操纵仪的倒置显微镜上静置10min,待细胞充分贴壁后,用台氏液灌流,流速为2ml/min。玻璃电极内充灌电极内液,电阻约2～5 MΩ。选取杆状、横纹清晰、无自主收缩的的心肌细胞进行实验。形成高阻抗(>1GΩ)封接后,用负压破膜,进行全细胞记录。离子电流信号经Axopatch 200B膜片钳放大器、Digidate 1322A模数转换器及Pclamp 8.0采集、贮存及分析,并保存在电脑硬盘中备用。
应用全细胞膜片钳技术,在电流钳模式下记录静息膜电位(RMP)和动作电位(AP),在电压钳模式下记录心肌细胞膜L-型钙电流(ICa-L)、电压门控钠电流(INa)、内向整流钾电流(I_(K1))、瞬时外向钾电流(Ito)、持续外向延迟整流钾电流(Isus)、延迟整流钾电流(IK,豚鼠)、钠钙交换电流(INa/Ca)、钠钾泵电流(Ipump)和ATP敏感钾电流(KATP)。应用电流密度(即单位膜电容的膜电流)表示膜电流的大小。观察①Zacopride对各离子通道的电流-电压关系曲线(I-V曲线)的效应;②Zacopride对I_(K1)作用的最大增幅、最大效应浓度以及能抵消zacopride对I_(K1)最大增强效应的BaCl2浓度(用于第4部分离体心脏灌流模型的研究);③Zacopride对静息膜电位、动作电位幅度和时程的影响。④Zacopride对静息膜电位和I_(K1)作用的时间过程。
2.应用膜片钳技术,分别观察10μmol/L 5-HT4受体阻断剂RS23597-190,10μmol/L 5-HT3受体激动剂m-chlorophenylbiguanide,5μmol/L PKA抑制剂KT5720,5μmol/L PKC抑制剂GF109203x和5μmol/L PKG抑制剂KT5823对zacopride增强I_(K1)作用的影响。
3.在大鼠乳头肌建立DADs和TA的模型。取健康雄性SD大鼠14只,体重220～250克,戊巴比妥钠按65mg/kg腹腔麻醉后迅速开胸取出心脏,浸入100% O2饱和的冰台氏液中洗净残血,沿前室间沟左缘剪开左室,小心切取5～6mm长, 2～3mm宽的左室乳头肌,置于组织灌流槽中,用不锈钢针固定于槽底的硅胶板上。标本以95% O2-5% CO2混合气体平衡的台氏液循环灌流。以多导电子刺激器产生的串刺激作为诱发DADs和TA的条件刺激,每个串刺激为6个连续脉冲,脉冲周期长度(BCL)为200ms,脉宽2ms,强度为2倍阈值。玻璃电极内充以3mmol/L KCl,电阻10～20 M?。稳定1小时后,使用三维液压式微操纵器将微电极插入单个心肌细胞内,引出正常动作电位。之后于灌流液中加入异丙肾上腺素(终浓度1μmol/L),采用上述条件刺激诱发DADs和TA。Zacopride预处理组则预先用zacopride(基于膜片钳结果,浓度为激活I_(K1)最大效应浓度)灌流组织,10分钟后再给予异丙肾上腺素,用药前后采用相同的条件刺激诱发DADs和TA,观察zacopride对异丙肾上腺素诱发DADs和TA的影响,比较两组DADs和TA的发生率。
4. Zacopride对多种心律失常模型的效应
(1)大鼠离体心脏心律失常模型
1)离体心脏缺血性或再灌注性心律失常模型造模方法:SD大鼠,麻醉,开胸,取出心脏行Langendorff主动脉逆行灌流,记录心电图,结扎冠状动脉左前降支,局部缺血15分钟,松扎后再灌注15分钟。观测指标:分别累计缺血后15分钟和再灌注后15分钟内期前收缩次数,室速持续时间和发生率,室颤持续时间和发生率。
实验分组:①单纯缺血组(对照组)或单纯缺血再灌注组(对照组)
②缺血(或再灌注)+ Zacopride不同浓度组
③缺血+ Zacopride+BaCl2组:灌流液中含1.0μmol/L Zacopride与1.0μmol/L BaCl2;或再灌注+Zacopride+BaCl2组:灌流液中含0.1μmol/L Zacopride与1.0μmol/L BaCl2;
2)离体心脏乌头碱心律失常模型
造模方法:大鼠,麻醉,开胸,取出心脏行Langendorff主动脉逆行灌流,记录心电图。
实验分组:
①给药组( n=8):先将1.0μmol/L的乌头碱0.7 ml在2分钟内由侧管经主动脉逆行注入心脏,给药完成后立即改用zacopride (1.0μmol/L)–台式液灌流心脏。
②对照组(n=8):注射相同剂量乌头碱后用正常台式液灌流心脏,其它同给药组。观测指标:比较乌头碱中毒对照组与zacopride处理组室性心律失常(包括室早、室速、室颤)的持续时间。
(2)大鼠在体心律失常模型
1)在体缺血性或再灌注性心律失常
造模方法:SD大鼠,麻醉,开胸,记录心电图,结扎冠脉左前降支,局部缺血15分钟,松扎后再灌注15分钟。不同剂量的Zacopride或Lidocaine 7.5mg/kg于缺血前或再灌注前3分钟由股静脉注射,观测量效关系。观测指标:分别累计缺血后15分钟和再灌注后15分钟内心律失常潜伏期,期前收缩次数,室速持续时间和发生率,室颤持续时间和发生率。
2)在体乌头碱心律失常模型
造模方法:SD大鼠,麻醉,记录心电图,在3秒内由舌下静脉一次性注射乌头碱30μg/kg,诱导出稳定的心律失常。Zacopride处理组在注射乌头碱后立即由股静脉注射zacopride。观测指标:比较乌头碱中毒组与zacopride给药组室性心律失常(包括室早、室速、室颤)的持续时间。
结果:
1.在0.1～10μmol/L范围内,Zacopride呈浓度依赖性增强I_(K1)。1μmol/L为zacopride最大效应浓度,可以使I_(K1)外向电流密度(-60mV)增大32.4%,内向电流密度(-100mV)增大33.8%(P<0.05),该增强效应可被1.0μmol/L BaCl2消除(并非完全抑制I_(K1),)。继续增大zacopride浓度(≥10μmol/L),I_(K1)电流并不能继续增强,相反有减小的趋势,表明zacopride不会过分增强I_(K1)。
在0.1～10μmol/L范围内,Zacopride对ICa-L、INa、Ito、Isus、IK等影响心肌细胞动作电位的主要膜电流无明显影响(P>0.05),同时对心肌细胞膜INa/Ca、Ipump和KATP(仅在细胞内ATP浓度降低时开放)亦无明显作用(P>0.05),显示Zacopride对I_(K1)通道激动作用的选择性或特异性。此前尚未见到I_(K1)特异性激动剂的报道。
Zacopride对静息膜电位、动作电位幅度(Action potential Amplitude,APA )、动作电位复极50%时程(Action potential duration at 50% repolarization,APD50)以及动作电位复极90%时程(Action potential duration at 90% repolarization,APD90)的影响。在0.1～10μmol/L范围内, Zacopride可浓度依赖性增大静息电位,增加APA,缩短APD50和APD90。1μmol/L为zacopride最大效应浓度,可使心肌细胞静息膜电位由-81.3±0.9 mV增大至-87.5±1.7mV;并缩短动作电位时程,APD50由12.79±1.24ms缩短至10.17±1.51ms,APD90由32.48±2.70ms缩短至25.61±3.97 ms (n=6, P<0.01)。
2. zacopride增强I_(K1)的时间依赖性。在电流钳模式下记录静息膜电位,待膜电位稳定后开始计时。1 min后给予心肌细胞1μmol/Lzacopride,静息膜电位在2.41±0.16 min时开始增大,至5.14±0.28 min达到稳态。在电压钳模式下分别将心肌细胞钳制在-60mV和-100mV记录I_(K1),待电流大小稳定后开始计时。同样在1 min后给予心肌细胞1μmol/L zacopride,其外向电流(-60mV)在2.35±0.26 min时开始增大,至5.03±0.30 min达到稳态;内向电流(-100mV)在2.31±0.14 min时开始增大,至5.12±018 min达到稳态。静息膜电位的变化和I_(K1)激活的时间过程完全同步(n=7,各时间点P>0.05),支持静息膜电位的增大是由于zacopride激活I_(K1)所致。
3.对zacopride增强I_(K1)的受体和信号转导途径的分析。10μmol/L 5-HT4受体阻断剂RS23597-190本身可以抑制I_(K1)电流,在预先应用RS23597-190的基础上加入1μmol/L zacopride,I_(K1)电流仍可增强(P<0.05),表明5-HT4受体不是zacopride激动I_(K1)的主要途径。10μmol/L 5-HT3受体激动剂m-chloropheylbiguanide对I_(K1)电流无明显影响,也不能逆转1μmol/L zacopride对I_(K1)的增强效应(P>0.05),表明zacopride激动I_(K1)亦非依赖于5-HT3受体。此外,5μmol/L PKA阻断剂KT5720能显著抑制1μmol/L zacopride对I_(K1)的增强效应(P<0.05),而PKC阻断剂GF109203x和PKG阻断剂KT5823则对1μmol/L zacopride的效应无明显影响(P>0.05),表明zacopride激动I_(K1)很可能经由PKA介导的信号转导通路。
4. zacopride对异丙肾上腺素诱发DAD和TA的影响。1μmol/L zacopride可有效抑制1μmol/L异丙肾上腺素所诱发的DADs和TA,使其发生率由85.7%下降至28.6%(n=14, P<0.05).
5. Zacopride对大鼠离体心脏心律失常的影响。
(1)在0.1～10μmol/L浓度范围内,Zacopride可浓度依赖性抑制大鼠离体心脏缺血性心律失常的发生。在单纯缺血组,100%大鼠发生室速,持续时间8.58±3.65s;75%大鼠出现室颤,持续时间23.51±15.77s;期前收缩数173±26个。而在冠状动脉左前降支结扎前3分钟给予0.1～10μmol/L zacopride,期前收缩数目、室速室颤持续时间、室速室颤发生率均有显著降低,呈剂量依赖性(P<0.01)。zacopride抗心律失常最大效应浓度为1μmol/L,期前收缩数降低为9±4个,室速室颤持续时间分别降至2.69±2.24s和1.67±1.33s,室速室颤发生率分别降至25.0%和12.5%(P<0.01)。基于膜片钳的结果,1μmol/L BaCl2可以抵消1μmol/L zacopride对I_(K1)的增强效应,我们在冠状动脉左前降支结扎前3分钟将1μmol/L BaCl2和1μmol/L zacopride同时灌流心脏,结果期前收缩数目(45±19)、室速和室颤持续时间(7.21±4.67s,11.87±9.43s)、室速和室颤发生率(50%,56.3%)均较单纯1μmol/L zacopride灌流组有显著升高(P<0.01),支持zacopride增强I_(K1)的作用是其抑制缺血性心律失常发生的重要机制。
(2)在0.1～10μmol/L浓度范围内,Zacopride可抑制大鼠离体心脏再灌注性心律失常的发生。在单纯再灌注组,100%大鼠发生室速,持续时间21.45±4.85s;75%大鼠出现室颤,持续时间76.4±4.91s;期前收缩数为107±27个。而在冠状动脉左前降支松扎前3分钟给与zacopride,期前收缩数目、室速室颤持续时间、室速室颤发生率均有显著降低。0.1μmol/L为zacopride抗心律失常最大效应浓度,使期前收缩数降低为11±9个,室速室颤持续时间分别降至2.31±1.87s和0.88±3.57s,室速室颤发生率分别降至31.3%和12.5%(P<0.01)。在冠状动脉左前降支松扎前3分钟将1μmol/L BaCl2和0.1μmol/L zacopride同时灌流心脏,结果期前收缩数目(64±19)、室速室颤持续时间(14.21±4.67s,14.38±11.26s)、室速室颤发生率(68.8%,50%)均较单纯0.1μmol/L zacopride灌流组有显著升高(P<0.01),表明zacopride抑制再灌注性心律失常的发生也与其增强I_(K1)的作用有关。
(3)1μmol/L zacopride可显著缩短乌头碱所致大鼠离体心脏心律失常持续时间,由对照组的48.18±2.32min缩短至20.12±1.29min(n=8, P<0.01)。
6. Zacopride对麻醉大鼠在体心律失常的影响。
(1)在5～50μg/kg剂量范围内,Zacopride可剂量依赖性抑制大鼠在体缺血性心律失常的发生。在对照组,冠状动脉左前降支结扎后344.13±59.79s可出现心律失常,100%大鼠发生室速,持续时间51.18±45.61s;75%大鼠出现室颤,持续时间6.90±7.05s;期前收缩数为149±65个。而在冠状动脉左前降支结扎前3分钟给予zacopride,则期前收缩数目、室速室颤持续时间、室速室颤发生率均显著降低,呈剂量依赖性(P<0.05)。15μg/kg为zacopride抗心律失常最大效应剂量,可使心律失常潜伏期延长至475.63±99.06s,期前收缩数降低为45±42个,仅25%大鼠出现室速,持续时间降至2.09±3.91s,并完全消除了室颤的发生(P<0.05)。其抗心律失常的效应与阳性对照药利多卡因(lidocaine,7.5mg/kg)相似(P>0.05),甚至在消除室颤方面优于利多卡因(P<0.05)。
(2)在1.5～5μg/kg剂量范围内,Zacopride可抑制麻醉大鼠再灌注性心律失常的发生。在对照组,100%大鼠发生室速,持续时间13.35±8.98s;87.5%大鼠出现室颤,持续时间11.72±8.91s;期前收缩数为23±14个。1.5μg/kg为zacopride抗心律失常最大效应剂量,使室速室颤持续时间分别降至5.10±4.62s和1.25±3.54s,室速室颤发生率分别降至32.5%和12.5%(P<0.05),但对期前收缩数无明显影响。其抗心律失常的效应与阳性对照药利多卡因(7.5mg/kg)相似(P>0.05)。
(3)15μg/kg zacopride可显著缩短乌头碱所致麻醉大鼠心律失常持续时间,由对照组的56.83±3.46min降至37.57±2.62min(n=8, P<0.01)。
结论:
1. zacopride为大鼠心室肌I_(K1)特异性激动剂,在0.1～10μmol/L范围内,可显著增强I_(K1);相同浓度范围的zacopride对大鼠I_(Ca-L)、I_(Na)、I_(to)、I_(sus)、I_(Na/Ca)、I_(pump)、K_(ATP)和豚鼠I_K均无显著影响。
2.在0.1～10μmol/L范围内, Zacopride可显著增大(超极化)静息电位,增加动作电位幅度,缩短动作电位时程。
3. Zacopride对I_(K1)的激动作用经由PKA介导的信号转导通路,其效应不依赖于5-HT3和5-HT4受体。
4. zacopride可有效抑制异丙肾上腺素诱发的延迟后除极和触发活动。
5. zacopride对大鼠心脏缺血性、再灌注性及乌头碱中毒所致的心律失常均有明显抑制作用,其抗心律失常效应主要由药物增强心室肌细胞I_(K1)所介导;zacopride对缺血性和再灌注性心律失常的效应与经典药物利多卡因(lidocaine)相似,最大效应浓度为1μmol/L,远低于zacopride促胃肠动力效应和中枢效应的有效浓度。
6. Zacopride作为心室肌I_(K1)特异性激动剂,不仅是一个潜在的抗心律失常药物,也是研究I_(K1)通道功能的极有价值的工具药。
Background: The inward rectifier K~+ current (I_(K1)) plays a significant role in the final repolarization and resting phases of the ventricular action potential. The molecular basis of I_(K1) has been ascribed to members of the Kir2.x sub-family of inward rectifier K~+ channel genes. Near RMP, the ventricular I_(K1) conductance is much larger than that of any other current except the ATP-sensitive potassium current(IKATP), which is normally not active. Dysfunction of the Kir2 channel or modulation of I_(K1) would likely have profound effects on cardiac excitability and ventricular arrhythmogenesis. However, there have been no I_(K1)-specific pharmacologic agents so far.
Objective: We would recognize zacopride as a specific I_(K1) agonist in rat cardiomyocytes and detect the probable signaling pathway(s) involved in its action. Further, we attempted to justify whether enhancing, rather than blocking, I_(K1) is a feasible pathway of antiarrhythmia.
Methods and Results: By whole-cell patch-clamp technique in rat or guinea pig cardiomyocytes, zacopride at 0.1-10μmol/L enhanced I_(K1) without effects on Ca+ current (ICa-L), Na~+ current (INa), transient outward K~+ current (Ito), sustained outward delayed rectifier K~+ current (Isus), delayed rectifier K~+ current (IK), Na~+-Ca2+ exchanger (NCX) current (INa/Ca), Na~+-K~+ pump current (Ipump) and IKATP. Both inward and outward component of I_(K1) were increased by zacopride in a concentration-relative and reversible manner (P<0.05). 1.0μmol/L Zacopride showed the maximal effect on I_(K1) with the mean increment by 33.8% in inward current (at -100mV) and 32.4% in outward current (at -60mV), and the effect could be abolished by 1μmol/L BaCl2 (P<0.05). Correspondingly, zacopride at 1.0μmol/L hyperpolarized resting membrane potential (RMP) from -81.3±0.9 mV up to -87.5±1.7mV and shortened action potential duration (APD90) from 32.48±2.70 ms to 25.61±3.97 ms (n=6, P<0.01). The enhancement of I_(K1) by zacopride could not be inhibited by 5-HT4-receptor antagonist RS23597-190 and 5-HT3-receptor agonist m-chlorophenylbiguanide at 10μmol/L respectively. Furthermore, PKA inhibitor KT5720 (5μmol/L) could inhibited the effect of zacopride on I_(K1)(P<0.05), while PKC inhibitor GF109203X and PKG inhibitor KT5823 at 5μmol/L respectively were short of effects on augmented I_(K1) by zacopride (P>0.05). In rat papillary muscles, zacopride significantly decreased the incidence of delayed afterdepolarizations (DADs) and triggered activity (TA) induced by isoproterenol (Iso) from 85.7% to 28.6% (n=14, P<0.05). Langendorff-perfused hearts were subjected to either coronary artery occlusion for 15 minutes or 15 minutes followed by 15 minutes reperfusion to induce definite arrhythmia. Zacopride at different concentration (0.1-10μmol/L) was added at 3 minutes before ischemia or reperfusion manipulating to explore its possible effect on the experimental rhythm disturbances. During ischemia, the total of PVB, the duration of VT and VF, the incidence of VT and VF were concentration-relatively decreased by zacopride treatment. The largest antiarrhythmic effect appeared at the concentration of 1.0μmol/L which consisted with the most effective concentration of zacopride on I_(K1). At 1.0μmol/L, zacopride reduced the incidence of ventricular tachycardia (VT) from 100% to 25% (P<0.01), the incidence of ventricular fibrillation from 75% to 12.5% (P<0.01), the duration of VT from 8.58±3.65s to 2.69±2.24s (P<0.01), the duration of VF from 23.51±15.77s to 1.67±1.33s (P<0.01), and the number of premature ventricular beats (PVB) from 173±26 to 9±4 (P<0.01). Based on patch clamp result that 1.0μmol/L BaCl2 could abolish the I_(K1) increment induced by 1.0μmol/L zacopride, 1.0μmol/L BaCl2 co-applied with 1.0μmol/L zacopride reversed the antiarrhythmic effect of zacopride as well. In BaCl2-treatment group, the total of PVB (45±19), the duration of VT (7.21±4.67s) and VF (11.87±9.43s), the incidence of VT (50%) and VF (56.3%) were significantly increased compared with 1μmol/L zacopride-treated rats (P<0.01). Similar protection was observed during reperfusion with a most effective concentration of zacopride at 0.1μmol/L which was 10 times less than that on ischemic arrhythmia. In the presence of 0.1μmol/L zacopride, the incidence of VT declined from 100% to 31.3% (P<0.01), VF declined from 75% to 12.5%, and the duration of VT shortened from 21.45±4.85s to 2.31±1.87s, the duration of VF shortened from 76.4±4.91s to 0.88±3.57s, the number of PVB decreased from 107±27 to11±9 (n=16, P<0.01). The antiarrhythmic effects were partly abolished by 1μmol/L BaCl2 (P<0.01). The antiarrhythmic effects of zacopride were also observed in anesthetized rats underwent 15-minute occlusion or 15-minute followed by 15-minute reperfusion. Far more dramatic protection was observed during ischemia. At the dose of 15μg/kg, zacopride showed the most potent antiarrhythmic action that compared favourably with lidocaine (7.5mg/kg), a classical antiarrhythmic agent. With prophylactic usage of 15μg/kg zacopride, the onset of arrhythmia was delayed from controlled 344.13±59.79s to 475.63±99.06s after ligation of coronary artery, the number of PVB decreased from 149±65 to 45±42, the incidence of VT declined from 100% to 25%, and the duration of VT shortened from 51.18±45.61s to 2.09±3.91s (n=8, P<0.05). None rat exhibited VF while 75% rats in control group developed VF with the mean duration of 6.90±7.05s (P<0.05). Less protection was observed during reperfusion. Zacopride showed the largest protective effects at dose of 1.5μg/kg that was 10 times less than that on ischemic arrhythmias. The incidence of VT declined from controlled 100% to 32.5%, the incidence of VF declined from 87.5% to 12.5%, and the duration of VT shortened from 13.35±8.98s to 5.10±4.62s, the duration of VF shortened from 11.72±8.91s to 1.25±3.54s (n=8, P<0.05). There was no significant effect on the total of PVB (p>0.05). Zacopride also significantly shortened the duration of aconitine-elicited arrhythmias from 48.18±2.32 min to 20.12±1.29 min (n=8, P<0.01) in ex vivo hearts and from 56.83±3.46 min to 37.57±2.62 min (n=8, P<0.01) in in vivo rats.
Conclusions:
1. Zacopride is firstly recognized as a specific agonist of I_(K1) in rat ventricular myocytes. At 0.1-10μmol/L, zacopride enhanced both inward and outward component of I_(K1) in a concentration-relative manner with no significant effects on I_(Ca-L), I_(Na), I_(to), I_(sus), I_(Na/Ca), I_(pump), I_(KATP) in rat and IK in guinea pig.
2. The augmentation of I_(K1) by zacopride might be via a PKA-mediated signaling pathway which is independent on 5-HT4 and 5-HT3 receptors.
3. Zacopride at 0.1-10μmol/L could significantly hyperpolarize the resting membrane potential, increase the action potential amplitude and shorten the action potential duration.
4. Zacopride at 1μmol/L could significantly decrease the incidence of Iso-induced delayed afterdepolari zations (DADs) and triggered activity (TA) .
5. Zacopride could remarkably suppress various ventricular arrhythmias induced by ischemia, reperfusion or aconitine.
6. Zacopride, as a specific agonist of I_(K1), is not only a potential antiarrhythmic agent but also a valuable pharmacological tool in the experimental study.
目前临床应用的抗心律失常药物主要是阻断心肌Na~+、K~+、Ca~(2+)离子通道,但其致心律失常副作用也引起了普遍担忧,寻找抗心律失常的新靶点和新机制一直是一个研究热点。当前,众多国内外学者将目光投向了内向整流钾电流(Inward rectifier potassium current,I_(K1))。内向整流钾通道是心肌最主要的背景外向电流,参与静息电位(Resting membrane potential,RMP)的维持和心肌动作电位(Action potential,AP)3期终末的复极。其分子基础主要是内向整流钾通道基因Kir2.x亚家族。在心室肌静息膜电位附近,I_(K1)电导要远远大于除ATP敏感钾通道(I_(KATP))以外的其他离子通道,而I_(KATP)在正常情况下是不激活的。因此,调节I_(K1)必将影响心肌静息膜电位的稳定和动作电位复极,从而对心肌的兴奋性和心律失常的发生产生深刻影响。但两个因素制约了其与心律失常关系的深入研究:一是迄今没有一种高选择性的I_(K1)阻断剂或激动剂,缺乏必要的工具药;二是目前所有抗心律失常药物均为各种离子通道或受体的阻断剂,而事实上,试图通过阻断I_(K1)产生抗心律失常作用的努力一直没有取得成功。理论上讲,I_(K1)被抑制后将使膜去极化,细胞的兴奋性和自律性增高,同时有助于早后除极、延迟后除极等异常自律性的发生;作为心室肌细胞主要的背景外向电流,I_(K1)通道的抑制使膜电阻增大,放大了跨膜电流引起的膜电位波动,从而造成膜电位的不稳定;抑制I_(K1)还可延长动作电位时程(APD),虽然在一定程度上可通过延长不应期抑制折返活动,但在某些场合也易引发长QT综合征。最近,Sung和Seemann等都利用计算机软件模拟抑制I_(K1)的效应,表明降低Kir2.1通道(形成I_(K1)的主要通道)的电导,可导致动作电位终末复极延迟,静息电位减小,出现自发性的室性心律失常。大量的临床和实验研究表明,许多疾病或病理状态下发生的心律失常是由于I_(K1)被抑制造成的。形成Kir2.1通道的基因KCNJ2突变可导致Andersen-Tawil综合症(ATS)。突变基因通过I_(K1)负显性抑制效应使通道功能降低,多数患者(71%)出现QT延长,64%的患者伴有室性心律失常。Miake等利用腺病毒将突变的Kir2.1通道基因转染给在体成年豚鼠心肌细胞,由于I_(K1)密度减小,出现APD延长,静息电位减小并不稳定和明显的异常自律活动。这些研究结果都说明了抑制I_(K1)的致心律失常风险。心衰患者中有一半伴有多种形态的心律失常,并经常由此发生猝死。很多临床和动物实验的研究证明,I_(K1)的减弱是心衰时心律失常发生的重要机制。心肌缺血和心肌梗死时发生的心律失常也与I_(K1)的下降有关。Almond等报告,大鼠慢性心肌梗死模型的心肌I_(K1)电流减小20%;Pinto等证实,狗心肌缺血区心内膜下普肯野纤维静息电位减小,是由于I_(K1)电流减小造成的;Kiesecker等在人的心肌细胞证明,内皮素可显著抑制I_(K1),此作用是内皮素致心律失常的重要机制。与此同时,也有一些完全相反的研究报道。如Li等利用转基因动物造成Kir2.1基因过表达,I_(K1)通道电导增大9~10倍,导致室性早搏、房室阻滞、房颤等多种心律失常。但这种过度表达引起的后果与上述全部或部分由于I_(K1)电流减低导致的心律失常并不矛盾。
鉴于临床中存在大量因I_(K1)减弱所致的心律失常和抑制或阻断I_(K1)的致心律失常风险,Miake等曾提出,需要设计药物或基因治疗,用于防治全部或部分由于I_(K1)减低导致的心律失常。Dhamoon等也认为,不应抑制(suppression)而是调制(modulation)I_(K1),以防治心律失常。Lopatin等更是明确指出理想的抗室性心律失常药物应在静息膜电位而非动作电位峰值开放钾通道,例如作用于I_(K1)的药物(the ideal ventricular antiarrhythmic agent might be a drug that opens K~+ channels at the resting potential, but not at the peak of the action potential, i.e. one that acts on I_(K1))。很显然,这里需要适当增强I_(K1)的药物,然而至今还未见有I_(K1)选择性激动剂的报道。Xiao等曾观察到吗啡可增强I_(K1)并使膜超级化,但它同时又可显著增强L型钙电流(ICa-L)而无特异性。
Zacopride属苯甲酰胺衍生物,兼有5-HT3受体抑制剂和5-HT4受体激动剂的性质,作用于胃肠5-HT4受体促进乙酰胆碱释放,对实验动物胃肠道具有促动力作用。我们在一项比较5-HT4受体激动剂西沙比利(cisapride)、RS67506和zacopride致心律失常副作用的研究中发现,与西沙比利显著的促心律失常副作用不同,zacopride对心律和心功能均无明显影响。探究其作用的离子机制发现,西沙比利和RS67506可抑制对大鼠心室肌I_(K1),而Zacopride对大鼠心室肌I_(K1)表现出显著的激动作用,对内向电流和外向电流部分均有明显增强效应;同时表现出明确的通道选择性,对ICa-L, INa, Ito, Isus, INa/Ca, Ipump等其他影响动作电位的主要离子流均无显著作用。这提示我们Zacopride很可能是I_(K1)选择性激动剂和潜在的抗心律失常药物。
目的:
1.确证zacopride为大鼠心肌细胞内向整流钾通道(I_(K1))特异性激动剂;
2.探讨zacopride增强I_(K1)可能的受体和信号转导机制;
3.建立大鼠心脏乳头肌延迟后除极(DADs)和触发活动(TA)模型,检验zacopride对DADs和TA的影响;
4.建立大鼠离体和在体心律失常模型,检验zacopride对多种原因所致室性心律失常的影响;
5.应用低浓度Ba~(2+)(I_(K1)相对特异性阻断剂)对I_(K1)的抑制作用,证实特异性增强I_(K1)与抗心律失常效应之间的关系。
方法:
1.采用胶原酶法急性分离大鼠(或豚鼠)左心室肌细胞。选用健康成年雄性SD大鼠(体重220～250g)或豚鼠(体重280～350g),于术前15分钟腹腔静脉注射肝素(1000U/kg),戊巴比妥钠(65mg/kg,ip)麻醉后颈动脉放血,迅速开胸取出心脏,置于4oC用100%氧气饱和的无钙台氏液中修剪,然后将心脏悬挂在Langendorff灌流装置上经主动脉逆行灌流。先用无钙台氏液灌流8～10分钟,再用胶原酶液循环灌流15～20分钟。灌流过程中保持37oC恒温,灌流压80cmH2O,并持续通以100%氧气。待心室肌组织变大、变软后将左心室剪下,置于KB液中剪碎,轻轻吹打得到分散的左心室肌细胞,经150μm孔径的滤网过滤后保存于KB液中,室温放置5～6小时后进行实验。实验前先将存放于高钾KB液中的细胞逐步复钙,然后取几滴细胞悬液加入细胞池(约1ml),平放在连有微操纵仪的倒置显微镜上静置10min,待细胞充分贴壁后,用台氏液灌流,流速为2ml/min。玻璃电极内充灌电极内液,电阻约2～5 MΩ。选取杆状、横纹清晰、无自主收缩的的心肌细胞进行实验。形成高阻抗(>1GΩ)封接后,用负压破膜,进行全细胞记录。离子电流信号经Axopatch 200B膜片钳放大器、Digidate 1322A模数转换器及Pclamp 8.0采集、贮存及分析,并保存在电脑硬盘中备用。
应用全细胞膜片钳技术,在电流钳模式下记录静息膜电位(RMP)和动作电位(AP),在电压钳模式下记录心肌细胞膜L-型钙电流(ICa-L)、电压门控钠电流(INa)、内向整流钾电流(I_(K1))、瞬时外向钾电流(Ito)、持续外向延迟整流钾电流(Isus)、延迟整流钾电流(IK,豚鼠)、钠钙交换电流(INa/Ca)、钠钾泵电流(Ipump)和ATP敏感钾电流(KATP)。应用电流密度(即单位膜电容的膜电流)表示膜电流的大小。观察①Zacopride对各离子通道的电流-电压关系曲线(I-V曲线)的效应;②Zacopride对I_(K1)作用的最大增幅、最大效应浓度以及能抵消zacopride对I_(K1)最大增强效应的BaCl2浓度(用于第4部分离体心脏灌流模型的研究);③Zacopride对静息膜电位、动作电位幅度和时程的影响。④Zacopride对静息膜电位和I_(K1)作用的时间过程。
2.应用膜片钳技术,分别观察10μmol/L 5-HT4受体阻断剂RS23597-190,10μmol/L 5-HT3受体激动剂m-chlorophenylbiguanide,5μmol/L PKA抑制剂KT5720,5μmol/L PKC抑制剂GF109203x和5μmol/L PKG抑制剂KT5823对zacopride增强I_(K1)作用的影响。
3.在大鼠乳头肌建立DADs和TA的模型。取健康雄性SD大鼠14只,体重220～250克,戊巴比妥钠按65mg/kg腹腔麻醉后迅速开胸取出心脏,浸入100% O2饱和的冰台氏液中洗净残血,沿前室间沟左缘剪开左室,小心切取5～6mm长, 2～3mm宽的左室乳头肌,置于组织灌流槽中,用不锈钢针固定于槽底的硅胶板上。标本以95% O2-5% CO2混合气体平衡的台氏液循环灌流。以多导电子刺激器产生的串刺激作为诱发DADs和TA的条件刺激,每个串刺激为6个连续脉冲,脉冲周期长度(BCL)为200ms,脉宽2ms,强度为2倍阈值。玻璃电极内充以3mmol/L KCl,电阻10～20 M?。稳定1小时后,使用三维液压式微操纵器将微电极插入单个心肌细胞内,引出正常动作电位。之后于灌流液中加入异丙肾上腺素(终浓度1μmol/L),采用上述条件刺激诱发DADs和TA。Zacopride预处理组则预先用zacopride(基于膜片钳结果,浓度为激活I_(K1)最大效应浓度)灌流组织,10分钟后再给予异丙肾上腺素,用药前后采用相同的条件刺激诱发DADs和TA,观察zacopride对异丙肾上腺素诱发DADs和TA的影响,比较两组DADs和TA的发生率。
4. Zacopride对多种心律失常模型的效应
(1)大鼠离体心脏心律失常模型
1)离体心脏缺血性或再灌注性心律失常模型造模方法:SD大鼠,麻醉,开胸,取出心脏行Langendorff主动脉逆行灌流,记录心电图,结扎冠状动脉左前降支,局部缺血15分钟,松扎后再灌注15分钟。观测指标:分别累计缺血后15分钟和再灌注后15分钟内期前收缩次数,室速持续时间和发生率,室颤持续时间和发生率。
实验分组:①单纯缺血组(对照组)或单纯缺血再灌注组(对照组)
②缺血(或再灌注)+ Zacopride不同浓度组
③缺血+ Zacopride+BaCl2组:灌流液中含1.0μmol/L Zacopride与1.0μmol/L BaCl2;或再灌注+Zacopride+BaCl2组:灌流液中含0.1μmol/L Zacopride与1.0μmol/L BaCl2;
2)离体心脏乌头碱心律失常模型
造模方法:大鼠,麻醉,开胸,取出心脏行Langendorff主动脉逆行灌流,记录心电图。
实验分组:
①给药组( n=8):先将1.0μmol/L的乌头碱0.7 ml在2分钟内由侧管经主动脉逆行注入心脏,给药完成后立即改用zacopride (1.0μmol/L)–台式液灌流心脏。
②对照组(n=8):注射相同剂量乌头碱后用正常台式液灌流心脏,其它同给药组。观测指标:比较乌头碱中毒对照组与zacopride处理组室性心律失常(包括室早、室速、室颤)的持续时间。
(2)大鼠在体心律失常模型
1)在体缺血性或再灌注性心律失常
造模方法:SD大鼠,麻醉,开胸,记录心电图,结扎冠脉左前降支,局部缺血15分钟,松扎后再灌注15分钟。不同剂量的Zacopride或Lidocaine 7.5mg/kg于缺血前或再灌注前3分钟由股静脉注射,观测量效关系。观测指标:分别累计缺血后15分钟和再灌注后15分钟内心律失常潜伏期,期前收缩次数,室速持续时间和发生率,室颤持续时间和发生率。
2)在体乌头碱心律失常模型
造模方法:SD大鼠,麻醉,记录心电图,在3秒内由舌下静脉一次性注射乌头碱30μg/kg,诱导出稳定的心律失常。Zacopride处理组在注射乌头碱后立即由股静脉注射zacopride。观测指标:比较乌头碱中毒组与zacopride给药组室性心律失常(包括室早、室速、室颤)的持续时间。
结果:
1.在0.1～10μmol/L范围内,Zacopride呈浓度依赖性增强I_(K1)。1μmol/L为zacopride最大效应浓度,可以使I_(K1)外向电流密度(-60mV)增大32.4%,内向电流密度(-100mV)增大33.8%(P<0.05),该增强效应可被1.0μmol/L BaCl2消除(并非完全抑制I_(K1),)。继续增大zacopride浓度(≥10μmol/L),I_(K1)电流并不能继续增强,相反有减小的趋势,表明zacopride不会过分增强I_(K1)。
在0.1～10μmol/L范围内,Zacopride对ICa-L、INa、Ito、Isus、IK等影响心肌细胞动作电位的主要膜电流无明显影响(P>0.05),同时对心肌细胞膜INa/Ca、Ipump和KATP(仅在细胞内ATP浓度降低时开放)亦无明显作用(P>0.05),显示Zacopride对I_(K1)通道激动作用的选择性或特异性。此前尚未见到I_(K1)特异性激动剂的报道。
Zacopride对静息膜电位、动作电位幅度(Action potential Amplitude,APA )、动作电位复极50%时程(Action potential duration at 50% repolarization,APD50)以及动作电位复极90%时程(Action potential duration at 90% repolarization,APD90)的影响。在0.1～10μmol/L范围内, Zacopride可浓度依赖性增大静息电位,增加APA,缩短APD50和APD90。1μmol/L为zacopride最大效应浓度,可使心肌细胞静息膜电位由-81.3±0.9 mV增大至-87.5±1.7mV;并缩短动作电位时程,APD50由12.79±1.24ms缩短至10.17±1.51ms,APD90由32.48±2.70ms缩短至25.61±3.97 ms (n=6, P<0.01)。
2. zacopride增强I_(K1)的时间依赖性。在电流钳模式下记录静息膜电位,待膜电位稳定后开始计时。1 min后给予心肌细胞1μmol/Lzacopride,静息膜电位在2.41±0.16 min时开始增大,至5.14±0.28 min达到稳态。在电压钳模式下分别将心肌细胞钳制在-60mV和-100mV记录I_(K1),待电流大小稳定后开始计时。同样在1 min后给予心肌细胞1μmol/L zacopride,其外向电流(-60mV)在2.35±0.26 min时开始增大,至5.03±0.30 min达到稳态;内向电流(-100mV)在2.31±0.14 min时开始增大,至5.12±018 min达到稳态。静息膜电位的变化和I_(K1)激活的时间过程完全同步(n=7,各时间点P>0.05),支持静息膜电位的增大是由于zacopride激活I_(K1)所致。
3.对zacopride增强I_(K1)的受体和信号转导途径的分析。10μmol/L 5-HT4受体阻断剂RS23597-190本身可以抑制I_(K1)电流,在预先应用RS23597-190的基础上加入1μmol/L zacopride,I_(K1)电流仍可增强(P<0.05),表明5-HT4受体不是zacopride激动I_(K1)的主要途径。10μmol/L 5-HT3受体激动剂m-chloropheylbiguanide对I_(K1)电流无明显影响,也不能逆转1μmol/L zacopride对I_(K1)的增强效应(P>0.05),表明zacopride激动I_(K1)亦非依赖于5-HT3受体。此外,5μmol/L PKA阻断剂KT5720能显著抑制1μmol/L zacopride对I_(K1)的增强效应(P<0.05),而PKC阻断剂GF109203x和PKG阻断剂KT5823则对1μmol/L zacopride的效应无明显影响(P>0.05),表明zacopride激动I_(K1)很可能经由PKA介导的信号转导通路。
4. zacopride对异丙肾上腺素诱发DAD和TA的影响。1μmol/L zacopride可有效抑制1μmol/L异丙肾上腺素所诱发的DADs和TA,使其发生率由85.7%下降至28.6%(n=14, P<0.05).
5. Zacopride对大鼠离体心脏心律失常的影响。
(1)在0.1～10μmol/L浓度范围内,Zacopride可浓度依赖性抑制大鼠离体心脏缺血性心律失常的发生。在单纯缺血组,100%大鼠发生室速,持续时间8.58±3.65s;75%大鼠出现室颤,持续时间23.51±15.77s;期前收缩数173±26个。而在冠状动脉左前降支结扎前3分钟给予0.1～10μmol/L zacopride,期前收缩数目、室速室颤持续时间、室速室颤发生率均有显著降低,呈剂量依赖性(P<0.01)。zacopride抗心律失常最大效应浓度为1μmol/L,期前收缩数降低为9±4个,室速室颤持续时间分别降至2.69±2.24s和1.67±1.33s,室速室颤发生率分别降至25.0%和12.5%(P<0.01)。基于膜片钳的结果,1μmol/L BaCl2可以抵消1μmol/L zacopride对I_(K1)的增强效应,我们在冠状动脉左前降支结扎前3分钟将1μmol/L BaCl2和1μmol/L zacopride同时灌流心脏,结果期前收缩数目(45±19)、室速和室颤持续时间(7.21±4.67s,11.87±9.43s)、室速和室颤发生率(50%,56.3%)均较单纯1μmol/L zacopride灌流组有显著升高(P<0.01),支持zacopride增强I_(K1)的作用是其抑制缺血性心律失常发生的重要机制。
(2)在0.1～10μmol/L浓度范围内,Zacopride可抑制大鼠离体心脏再灌注性心律失常的发生。在单纯再灌注组,100%大鼠发生室速,持续时间21.45±4.85s;75%大鼠出现室颤,持续时间76.4±4.91s;期前收缩数为107±27个。而在冠状动脉左前降支松扎前3分钟给与zacopride,期前收缩数目、室速室颤持续时间、室速室颤发生率均有显著降低。0.1μmol/L为zacopride抗心律失常最大效应浓度,使期前收缩数降低为11±9个,室速室颤持续时间分别降至2.31±1.87s和0.88±3.57s,室速室颤发生率分别降至31.3%和12.5%(P<0.01)。在冠状动脉左前降支松扎前3分钟将1μmol/L BaCl2和0.1μmol/L zacopride同时灌流心脏,结果期前收缩数目(64±19)、室速室颤持续时间(14.21±4.67s,14.38±11.26s)、室速室颤发生率(68.8%,50%)均较单纯0.1μmol/L zacopride灌流组有显著升高(P<0.01),表明zacopride抑制再灌注性心律失常的发生也与其增强I_(K1)的作用有关。
(3)1μmol/L zacopride可显著缩短乌头碱所致大鼠离体心脏心律失常持续时间,由对照组的48.18±2.32min缩短至20.12±1.29min(n=8, P<0.01)。
6. Zacopride对麻醉大鼠在体心律失常的影响。
(1)在5～50μg/kg剂量范围内,Zacopride可剂量依赖性抑制大鼠在体缺血性心律失常的发生。在对照组,冠状动脉左前降支结扎后344.13±59.79s可出现心律失常,100%大鼠发生室速,持续时间51.18±45.61s;75%大鼠出现室颤,持续时间6.90±7.05s;期前收缩数为149±65个。而在冠状动脉左前降支结扎前3分钟给予zacopride,则期前收缩数目、室速室颤持续时间、室速室颤发生率均显著降低,呈剂量依赖性(P<0.05)。15μg/kg为zacopride抗心律失常最大效应剂量,可使心律失常潜伏期延长至475.63±99.06s,期前收缩数降低为45±42个,仅25%大鼠出现室速,持续时间降至2.09±3.91s,并完全消除了室颤的发生(P<0.05)。其抗心律失常的效应与阳性对照药利多卡因(lidocaine,7.5mg/kg)相似(P>0.05),甚至在消除室颤方面优于利多卡因(P<0.05)。
(2)在1.5～5μg/kg剂量范围内,Zacopride可抑制麻醉大鼠再灌注性心律失常的发生。在对照组,100%大鼠发生室速,持续时间13.35±8.98s;87.5%大鼠出现室颤,持续时间11.72±8.91s;期前收缩数为23±14个。1.5μg/kg为zacopride抗心律失常最大效应剂量,使室速室颤持续时间分别降至5.10±4.62s和1.25±3.54s,室速室颤发生率分别降至32.5%和12.5%(P<0.05),但对期前收缩数无明显影响。其抗心律失常的效应与阳性对照药利多卡因(7.5mg/kg)相似(P>0.05)。
(3)15μg/kg zacopride可显著缩短乌头碱所致麻醉大鼠心律失常持续时间,由对照组的56.83±3.46min降至37.57±2.62min(n=8, P<0.01)。
结论:
1. zacopride为大鼠心室肌I_(K1)特异性激动剂,在0.1～10μmol/L范围内,可显著增强I_(K1);相同浓度范围的zacopride对大鼠I_(Ca-L)、I_(Na)、I_(to)、I_(sus)、I_(Na/Ca)、I_(pump)、K_(ATP)和豚鼠I_K均无显著影响。
2.在0.1～10μmol/L范围内, Zacopride可显著增大(超极化)静息电位,增加动作电位幅度,缩短动作电位时程。
3. Zacopride对I_(K1)的激动作用经由PKA介导的信号转导通路,其效应不依赖于5-HT3和5-HT4受体。
4. zacopride可有效抑制异丙肾上腺素诱发的延迟后除极和触发活动。
5. zacopride对大鼠心脏缺血性、再灌注性及乌头碱中毒所致的心律失常均有明显抑制作用,其抗心律失常效应主要由药物增强心室肌细胞I_(K1)所介导;zacopride对缺血性和再灌注性心律失常的效应与经典药物利多卡因(lidocaine)相似,最大效应浓度为1μmol/L,远低于zacopride促胃肠动力效应和中枢效应的有效浓度。
6. Zacopride作为心室肌I_(K1)特异性激动剂,不仅是一个潜在的抗心律失常药物,也是研究I_(K1)通道功能的极有价值的工具药。
Background: The inward rectifier K~+ current (I_(K1)) plays a significant role in the final repolarization and resting phases of the ventricular action potential. The molecular basis of I_(K1) has been ascribed to members of the Kir2.x sub-family of inward rectifier K~+ channel genes. Near RMP, the ventricular I_(K1) conductance is much larger than that of any other current except the ATP-sensitive potassium current(IKATP), which is normally not active. Dysfunction of the Kir2 channel or modulation of I_(K1) would likely have profound effects on cardiac excitability and ventricular arrhythmogenesis. However, there have been no I_(K1)-specific pharmacologic agents so far.
Objective: We would recognize zacopride as a specific I_(K1) agonist in rat cardiomyocytes and detect the probable signaling pathway(s) involved in its action. Further, we attempted to justify whether enhancing, rather than blocking, I_(K1) is a feasible pathway of antiarrhythmia.
Methods and Results: By whole-cell patch-clamp technique in rat or guinea pig cardiomyocytes, zacopride at 0.1-10μmol/L enhanced I_(K1) without effects on Ca+ current (ICa-L), Na~+ current (INa), transient outward K~+ current (Ito), sustained outward delayed rectifier K~+ current (Isus), delayed rectifier K~+ current (IK), Na~+-Ca2+ exchanger (NCX) current (INa/Ca), Na~+-K~+ pump current (Ipump) and IKATP. Both inward and outward component of I_(K1) were increased by zacopride in a concentration-relative and reversible manner (P<0.05). 1.0μmol/L Zacopride showed the maximal effect on I_(K1) with the mean increment by 33.8% in inward current (at -100mV) and 32.4% in outward current (at -60mV), and the effect could be abolished by 1μmol/L BaCl2 (P<0.05). Correspondingly, zacopride at 1.0μmol/L hyperpolarized resting membrane potential (RMP) from -81.3±0.9 mV up to -87.5±1.7mV and shortened action potential duration (APD90) from 32.48±2.70 ms to 25.61±3.97 ms (n=6, P<0.01). The enhancement of I_(K1) by zacopride could not be inhibited by 5-HT4-receptor antagonist RS23597-190 and 5-HT3-receptor agonist m-chlorophenylbiguanide at 10μmol/L respectively. Furthermore, PKA inhibitor KT5720 (5μmol/L) could inhibited the effect of zacopride on I_(K1)(P<0.05), while PKC inhibitor GF109203X and PKG inhibitor KT5823 at 5μmol/L respectively were short of effects on augmented I_(K1) by zacopride (P>0.05). In rat papillary muscles, zacopride significantly decreased the incidence of delayed afterdepolarizations (DADs) and triggered activity (TA) induced by isoproterenol (Iso) from 85.7% to 28.6% (n=14, P<0.05). Langendorff-perfused hearts were subjected to either coronary artery occlusion for 15 minutes or 15 minutes followed by 15 minutes reperfusion to induce definite arrhythmia. Zacopride at different concentration (0.1-10μmol/L) was added at 3 minutes before ischemia or reperfusion manipulating to explore its possible effect on the experimental rhythm disturbances. During ischemia, the total of PVB, the duration of VT and VF, the incidence of VT and VF were concentration-relatively decreased by zacopride treatment. The largest antiarrhythmic effect appeared at the concentration of 1.0μmol/L which consisted with the most effective concentration of zacopride on I_(K1). At 1.0μmol/L, zacopride reduced the incidence of ventricular tachycardia (VT) from 100% to 25% (P<0.01), the incidence of ventricular fibrillation from 75% to 12.5% (P<0.01), the duration of VT from 8.58±3.65s to 2.69±2.24s (P<0.01), the duration of VF from 23.51±15.77s to 1.67±1.33s (P<0.01), and the number of premature ventricular beats (PVB) from 173±26 to 9±4 (P<0.01). Based on patch clamp result that 1.0μmol/L BaCl2 could abolish the I_(K1) increment induced by 1.0μmol/L zacopride, 1.0μmol/L BaCl2 co-applied with 1.0μmol/L zacopride reversed the antiarrhythmic effect of zacopride as well. In BaCl2-treatment group, the total of PVB (45±19), the duration of VT (7.21±4.67s) and VF (11.87±9.43s), the incidence of VT (50%) and VF (56.3%) were significantly increased compared with 1μmol/L zacopride-treated rats (P<0.01). Similar protection was observed during reperfusion with a most effective concentration of zacopride at 0.1μmol/L which was 10 times less than that on ischemic arrhythmia. In the presence of 0.1μmol/L zacopride, the incidence of VT declined from 100% to 31.3% (P<0.01), VF declined from 75% to 12.5%, and the duration of VT shortened from 21.45±4.85s to 2.31±1.87s, the duration of VF shortened from 76.4±4.91s to 0.88±3.57s, the number of PVB decreased from 107±27 to11±9 (n=16, P<0.01). The antiarrhythmic effects were partly abolished by 1μmol/L BaCl2 (P<0.01). The antiarrhythmic effects of zacopride were also observed in anesthetized rats underwent 15-minute occlusion or 15-minute followed by 15-minute reperfusion. Far more dramatic protection was observed during ischemia. At the dose of 15μg/kg, zacopride showed the most potent antiarrhythmic action that compared favourably with lidocaine (7.5mg/kg), a classical antiarrhythmic agent. With prophylactic usage of 15μg/kg zacopride, the onset of arrhythmia was delayed from controlled 344.13±59.79s to 475.63±99.06s after ligation of coronary artery, the number of PVB decreased from 149±65 to 45±42, the incidence of VT declined from 100% to 25%, and the duration of VT shortened from 51.18±45.61s to 2.09±3.91s (n=8, P<0.05). None rat exhibited VF while 75% rats in control group developed VF with the mean duration of 6.90±7.05s (P<0.05). Less protection was observed during reperfusion. Zacopride showed the largest protective effects at dose of 1.5μg/kg that was 10 times less than that on ischemic arrhythmias. The incidence of VT declined from controlled 100% to 32.5%, the incidence of VF declined from 87.5% to 12.5%, and the duration of VT shortened from 13.35±8.98s to 5.10±4.62s, the duration of VF shortened from 11.72±8.91s to 1.25±3.54s (n=8, P<0.05). There was no significant effect on the total of PVB (p>0.05). Zacopride also significantly shortened the duration of aconitine-elicited arrhythmias from 48.18±2.32 min to 20.12±1.29 min (n=8, P<0.01) in ex vivo hearts and from 56.83±3.46 min to 37.57±2.62 min (n=8, P<0.01) in in vivo rats.
Conclusions:
1. Zacopride is firstly recognized as a specific agonist of I_(K1) in rat ventricular myocytes. At 0.1-10μmol/L, zacopride enhanced both inward and outward component of I_(K1) in a concentration-relative manner with no significant effects on I_(Ca-L), I_(Na), I_(to), I_(sus), I_(Na/Ca), I_(pump), I_(KATP) in rat and IK in guinea pig.
2. The augmentation of I_(K1) by zacopride might be via a PKA-mediated signaling pathway which is independent on 5-HT4 and 5-HT3 receptors.
3. Zacopride at 0.1-10μmol/L could significantly hyperpolarize the resting membrane potential, increase the action potential amplitude and shorten the action potential duration.
4. Zacopride at 1μmol/L could significantly decrease the incidence of Iso-induced delayed afterdepolari zations (DADs) and triggered activity (TA) .
5. Zacopride could remarkably suppress various ventricular arrhythmias induced by ischemia, reperfusion or aconitine.
6. Zacopride, as a specific agonist of I_(K1), is not only a potential antiarrhythmic agent but also a valuable pharmacological tool in the experimental study.
引文
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