绝对不应期电刺激对正常豚鼠和慢性心力衰竭豚鼠心室肌细胞动作电位及钠离子-钙离子交换的影响

目的:探讨绝对不应期电刺激(ARPES)对正常豚鼠和慢性心力衰竭(衰竭)豚鼠心室肌细胞动作电位(AP)及钠离子-钙离子(Na -Ca2 )交换的影响.方法:应用膜片钳技术中电流钳记录ARPES对AP时程的影响,再以不同的AP电压钳记录细胞膜Na -Ca2 交换电流.结果:①ABPES延长AP时程,以APD30最为显著(P＜0.01),差异有统计学意义.②与正常豚鼠心室肌细胞比较,衰竭豚鼠心室肌细胞AP的平台期明显不同,表现在APD90变化(P＜0.05)及APD50变化(P＜0.01),差异有统计学意义.③分别以基础刺激(S1)下的AP(APS1)和ARPES下的AP(APARPES)为测试电压,记录AP电压钳下的细胞膜Na -Ca2 交换电流,在正常豚鼠心室肌细胞,APARPES电压钳记录的单位膜电容下的外向电流强度的整合值高于APS1电压钳记录的相应值,而单位膜电容下的内向电流强度的整合值无显著变化.在衰竭豚鼠心室肌细胞,APARPES电压钳记录的单位膜电容下的外向电流强度的整合值明显高于APS1电压钳记录的相应值,而单位膜电容下的内向电流强度的整合值无显著变化.外向电流峰值的增加更为明显.结论:ARPES延长正常豚鼠和衰竭豚鼠心室肌细胞AP时程,对心室肌细胞膜Na -Ca2 交换电流的影响可能是其增强整体心脏收缩功能的机制之一.     

绝对不应期电刺激对心力衰竭豚鼠心室肌细胞钙离子流的影响

目的探讨绝对不应期电刺激(ARPES)对心力衰竭(简称心衰)心室肌细胞收缩和钙离子流的作用。方法应用单细胞收缩动缘检测仪,在单纯基础刺激和一定延时后同时给予ARPES,观察细胞收缩并测定以F360/F380反映细胞内钙瞬变。应用膜片钳技术中记录单纯基础刺激和ARPES(延迟后10ms刺激)所诱发动作电位(APSA1,APARPES),并比较动作电位时程(APD)。分别以APSA1和APARPES为测试电压,记录AP电压钳下的细胞膜L型钙通道电流(ICa-L)。结果①ARPES使单个心衰心室肌细胞收缩幅值增高15.53%±5.31%(P<0.05),收缩和舒张速度峰值均增加(分别增加10.60%±3.02%,23.2%±8.26%,P<0.05或0.01,n=6);F360/F380幅值增加16.82%±7.03%(P<0.01,n=6)。②ARPES延长动作APD。③与APSA1电压钳记录的ICa-L相比,APARPES电压钳记录的ICa-L减弱程度明显减少,其单位膜电容下的电流强度的整合值增加(P<0.01)。结论ARPES提高衰竭心肌收缩力与增强单个心室肌细胞钙瞬变相关。     

艾司洛尔对心室肌细胞动作电位及L-型钙离子通道的影响

目的 观察艾司洛尔对豚鼠心室肌细胞动作电位(AP)和L-型钙离子通道的影响.方法 Langendorff离体心脏逆向灌流法分离豚鼠心室肌细胞,随机选取心室肌细胞分为正常对照组和艾司洛尔(50 μmol/L和100 μmol/L)组.应用全细胞电流钳模式记录心室肌细胞AP,应用电压钳模式记录L-型钙离子通道电流(ICa-L).结果 艾司洛尔(100 μmol/L )可使心肌细胞AP时程APD20、APD50明显缩短(P<0.05),心室肌细胞ICa-L峰值电流明显降低(P<0.05).结论 艾司洛尔缩短心室肌细胞AP时程和抑制钙通道可能是其抑制交感风暴的机制之一.     

兔界嵴细胞离子通道特性研究

目的研究生理状态下及异丙肾上腺素灌流对兔界嵴(CT)与梳状肌(PM)细胞动作电位(AP)及钠电流(INa)、短暂外向钾电流(Ito)、L型钙电流(ICa-L)、延迟整流钾电流(IK)及内向整流性钾电流(IK1)的影响,探讨CT与房性心律失常的关系。方法酶解法分离兔CT及PM细胞,利用全细胞膜片钳技术,记录生理状态下及异丙肾上腺素灌流后CT与PM细胞AP及INa、Ito、ICa-L、IK及IK1的变化。结果①生理状态下,CT细胞动作电位时程(APD)较长,可见明显的平台期;PM细胞AP形态与普通心房肌细胞相似,1期复极迅速,平台期短,类似三角形。②生理状态下,CT细胞Ito电流密度比PM细胞明显降低(7.13±0.38 pA/pF vs 10.70±0.62 pA/pF,n=9,P<0.01),而INa、Ito、ICa-L、IK及IK1则无明显差别。③异丙肾上腺素灌流时CT与PM细胞APD20、APD50、APD90均延长(n=8,P<0.01);指令电位+50 mV时,CT与PM细胞Ito电流密度均减少(n=9,P<0.01)而IK均增加(n=8,P<0.05);指令电位+10 mV时,CT与PM细胞ICa-L电流密度均增加(n=9,P<0.01);IK1在两种心肌细胞均无明显差异。结论 CT与PM细胞AP差异与Ito有关。异丙肾上腺素灌流时ICa-L与IK增强,Ito抑制使CT与PM细胞APD延长,触发机制可能是CT参与房性心律失常的机制之一。     

异丙酚对家兔心室外膜心肌细胞动作电位和L-型钙电流的影响

目的研究异丙酚对家兔左、右心室心外膜心肌细胞动作电位和L-型钙电流的影响。方法酶解法分离家兔左、右心室心外膜心肌细胞。全细胞膜片钳技术记录左、右心室心外膜心肌细胞动作电位和L-型钙电流(ICa-L)在使用异丙酚前后的变化。结果在电流钳制下,左、右心室心外膜心肌细胞动作电位都具有从0期到4期的动作电位形态,2相平台期有心外膜心肌细胞特有的穹窿样突起。异丙酚使右室心外膜心肌细胞动作电位失去2相平台期穹窿样突起,呈三角形尖锥锋形。左、右心室心外膜心肌细胞动作电位时程复极化50%和90%(APD50和APD90)在异丙酚作用后都明显缩短,其中右室心外膜心肌细胞APD50和APD90缩短最为明显(P<0.05或0.01)。在电压钳制下,异丙酚使左、右心室心外膜心肌细胞ICa-L在同一指令电位下,电流幅度均明显减小,但右室心外膜心肌细胞ICa-L的减小幅度明显强于左室同层ICa-L的减小幅度(P<0.01)。异丙酚还使左、右心室心外膜心肌细胞ICa-L的I-V曲线上移,并且使右室心外膜心肌细胞ICa-L的I-V曲线处在所有I-V曲线最上部。结论异丙酚对右室心外膜心肌细胞动作电位和ICa-L的影响程度明显强于左室,从而引起左、右心外膜心肌细胞电不均一性。     

溶血磷脂酸对豚鼠心室肌动作电位及延迟整流钾电流的影响

目的观察溶血磷脂酸（LPA）对离体豚鼠心室乳头肌动作电位及心室肌细胞延迟整流钾电流的影响。方法采用标准玻璃微电极技术记录豚鼠乳头肌动作电位。应用全细胞电压钳方法记录心室肌细胞延迟整流钾电流（Ik）。结果LPA0．1、1．0、10umol/L可浓度依赖性增加心室肌动作电位幅度（APA）（P〈0．05，P〈0．01，P〈0．01），延长动作电位50％、90％时程（APD50、APD90）（P〈0．05），钾通道阻断剂TEA可部分阻断LPA对APD50的延长作用。LPA0．1、1．0、10umol／L可明显抑制Ik（P〈0．05）。结论LPA可增加豚鼠心脏乳头肌动作电位幅值、延长动作电位时程，并抑制豚鼠心室肌细胞Ik。     

鼠巨细胞病毒心肌炎自身免疫致室性心律失常机制的研究

目的探讨自身免疫机制在鼠巨细胞病毒(MCMV)诱导BALB/c鼠心肌炎室性心律失常中的作用。方法将无病原体4周龄BALB/c鼠60只,随机分为实验组(36只)和对照组(24只);实验组腹腔注射MC-MVTCID50悬液,对照组腹腔注射3T3细胞裂解液;记录所有小鼠心电图,观察心肌病理及炎性因子表达情况,检测血清抗心脏β1受体自身抗体滴度,膜片钳观察抗β1受体抗体对心肌细胞动作电位(AP)及L型钙通道(ICa-L)电流的影响。结果实验组心肌炎发病率69.4%,表现为心肌细胞变性或灶性坏死,炎性因子白细胞介素-1β和肿瘤坏死因子-α蛋白阳性表达,血管周围大量炎症细胞浸润。实验组心律失常累计发生率达50%,第6周起该组血清抗β1受体抗体滴度显著增高(与对照组比,P<0.01),且心律失常发生亦增加。1∶100抗β1受体抗体使小鼠心室肌细胞APD50和APD90延长15.3%和5.8%,使ICa-L密度显著增加。结论MCMV可能通过诱导抗β1受体自身抗体产生,增加ICa-L电流导致室性心律失常发生。     

牵拉刺激对豚鼠心室肌动作电位时程的影响及其机制

目的本实验观察了牵拉刺激对离体豚鼠左心室乳头肌动作电位的影响,为探讨心肌牵张激活离子通道电流的特点提供实验依据。方法实验采用标准微电极细胞内记录技术引导豚鼠心室乳头肌细胞动作电位,用微机化生理信号采集分析系统(NSA-Ⅲ)记录并处理信号。结果①牵拉心肌可加快整个复极过程,APD20、APD50和APD90均有缩短(P<0.05)。牵拉作用呈心肌长度依赖性。牵拉刺激对RP和APA无显著性影响(P>0.05)。②内向整流钾通道(IK1)阻断剂BaCl2(100μmol/L)可明显减弱因牵拉刺激导致的动作电位时程缩短。应用格列本脲、奎尼丁和氯化钆与未用药组比较无显著差异(P>0.05)。结论①在豚鼠心室肌存在牵拉刺激激活的外向电流,这种电流加快复极过程,使动作电位时程明显缩短。②该电流可能与内向整流钾通道(IK1)有关,但是与ATP敏感性钾通道和IKr无关。     

不同浓度的乙醇对离体豚鼠心肌动作电位的影响

目的 观察不同浓度的乙醇对豚鼠心肌电生理特性的影响。方法 采用标准玻璃微电极细胞内记录技术,记录离体豚鼠心肌细胞的动作电位。观察12.5,25.0,50.0,100.0及200.0 mmol/L乙醇对心房肌和心室乳头肌动作电位各参数的改变。结果 12.5~100.0 mmol/L 乙醇对心室乳头肌细胞的细胞膜静息电位（RMP）、动作电位幅值（APA）、动作电位时程（APD）、AP复极至50%的时程（APD50）及AP复极至90%的时程（APD90）等参数无明显影响;而200.0 mmol/L乙醇可显著降低APA（P<0.05）,50.0~200.0 mmol/L乙醇可明显延长心房肌细胞APD,APD50和APD90（P<0.05或P<0.01）。结论 乙醇对离体豚鼠心房肌、心室乳头肌电生理特性的影响有着明显差异。心房肌APD的延长可能与心房肌中分布的特殊离子通道被阻滞有关。     

新生大鼠心肌细胞短暂外向钾电流下降与心肌肥大的相关性研究

在某些心力衰竭患者中常观察到心肌细胞短暂外向钾电流 (Ito)下降和动作电位时程 (APD)延长 ,笔者主要探讨Ito下降与心肌肥大的关系及内在机制。用Ito阻断剂 ,4 氨基吡啶 (4 AP) ,处理新生大鼠心室肌细胞 ,观察作为心肌肥大指标的细胞膜电容和3 H 亮氨酸 (3 H Leu)掺入量 ,同时测Ito振幅和APD。结果 :Ito振幅下降近 5 0 %(1 5 0 .3± 1 8.6pA ,n =7vs 74 .0± 1 1 .5pA ,n =1 1 ,P <0 .0 5 )。APD50 (5 0 %复极 )显著的延长 (75 .8± 1 4 .1ms,n =7vs2 0 1 .7± 2 3.5ms,n =1 1 ,P <0 .0 5 )。膜电容和3 H Leu掺入量分别增加 4 7%和 31 % (P均 <0 .0 5 )。L 型钙通道阻断剂维拉帕米 ,能抑制 4 AP诱导的APD延长以及膜电容和3 H 亮氨酸掺入量的增加。环孢素A(CsA)也可抑制 4 AP诱导的膜电容和3 H Leu掺入量的增加 ,但对APD影响不明显。结论 :Ito下降通过延长APD ,致细胞内钙增加 ,激活钙调神经磷酸酶反应途径 ,可能引起心肌肥大     

醛固酮对心室肌细胞动作电位及L型钙通道的影响

目的研究醛固酮对心室肌细胞动作电位及L型Ca2+通道的影响,探讨其致心律失常的机制。方法分离Wister大鼠心室肌细胞,随机分为对照组和Ald组,采用全细胞膜片钳记录方法记录动作电位时程(APD)、L型钙电流(ICa-L)。结果 Ald组APD较对照组显著延长(P<0.05)。Ald组ICa-L电流密度峰值较对照组显著增大[-(9.73±0.90)pA/pF vs-(7.07±0.83)pA/pF,P<0.01]。Ald组与对照组比较,I-V曲线显著下移。结论醛固酮可能通过增加L型Ca2+通道的电流密度,延长心肌细胞APD,参与醛固酮的致心律失常作用。     

The ionic mechanisms of long QT interval in diabetic rabbits

Objective Abnormal QT prolongation associated with arrhythmias is considered the major cardiac electrical disorder and a significant predictor of mortality in diabetic patients. The precise ionic mechanisms for diabetic QT prolongation remained unclear. The present study was designed to analyze the changes of ventricular repolarization and the underlying ionic mechanisms in diabetic rabbit hearts. Methods Diabetes was induced by a single injection ofalloxan （145mg/kg, Lv. ）. After the development of diabetes （10 weeks）, ECG was measured. Whole-cell patch-clamp technique was applied to record the action potential duration （APD50, APD90）, slowly activating outward rectifying potassium current （IKs）, L-type calcium current （ICa-L） and inward rectifying potassium current （IK1）. Results The action potential duration （APD50 and APD90） of ventricular myocytes was obviously prolonged from 271.5＋32.3 ms and 347.8＋36.3 ms to 556.6~72.5 ms and 647.9~72.2 ms respectively （P〈 0.05）. Meanwhile the normalized peak current densities of IKs in ventricular myocytes investigated by whole-cell patch clamp was smaller in diabetic rabbits than that in control group at test potential of＋50mV （1.27~0.20 pA/pF vs 3.08~0.67 pA/pF, P〈0.05）. And the density of the ICa-L was increased apparently at the test potential of 10 mV （-2.67~0.41 pA/pF vs -5.404-1.08 pA/pF, P〈0.05）. Conclusion Ventricular repolarization was prolonged in diabetic rabbits, it may be partly due to the increased L-type calcium current and reduced slow delayed rectifier K＋ current （IKs） （J Geriatr Cardio12010; 7：25-29）.     

哇巴因对家兔左心房后壁动作电位和L型钙电流的影响

目的探讨哇巴因对家兔左心房后壁(LAPW)和左心耳底部(LAA)心肌细胞动作电位和L型钙电流(I_(Ca-L))的影响。方法酶解法分离家兔的LAPW和LAA组织的心肌细胞。全细胞膜片钳技术记录各个部位心肌细胞动作电位和I_(Ca-L)的变化。结果在电流钳制下,LAPW的动作电位时程复极比50%(APD_(50))和动作电位时程复极比90%(APD_(90))明显长于LAA的APD_(50)和APD_(90)(均为P<0.05)。哇巴因(1μmol/L)使LAPw和LAA心肌细胞动作电位形态呈三角形尖锥峰形,APD_(50)和APD_(90),以及静息电位和动作电位幅值幅度均明显短于各自的对照组(均为P<0.01);LAPW心肌细胞APD_(50)和APD_(90)还明显短于LAA心肌细胞加药前后的APD_(50)和APD_(90)(均为P<0.05)。在电压钳制方式下,正常组LAPW的I_(Ca-L)的电流密度明显小于LAA的I_(Ca-L)的电流密度(P<0.05)。但在1μmoL/L哇巴因作用下,它们各自I_(Ca-L)的幅度有明显增加,LAPW的I_(Ca-L)最大电流密度为(-11.13±0.99)pA/pF,LAA的I_(Ca-L)为(-8.86±0.51)pA/pF(P<0.01)。在对照组中,LAPW的I_(Ca-L)的I-V曲线位于最底部,经哇巴因作用后,LAPW的I_(Ca-L)的I-V曲线上移到其他I-V曲线的最上部。结论家兔LAPW和LAA心肌细胞存在离子流特异性差异,哇巴因加重LAPW和LAA的离子流大小异质性和离子电流的重新分配,这可能成为心房颤动易发性的启动因素和维持条件。     

二十二碳六烯酸对Sprague-Dawley大鼠心室肌细胞动作电位和瞬时外向钾离子流的作用

目的 探讨二十二碳六烯酸(DHA)对Sprague-Dawley大鼠心窜肌细胞动作电位(AP)和瞬时外向钾离子流(Ito)的作用.方法 采用酶消化法获得大鼠耐钙心室肌细胞,以全细胞膜片钳技术分别记录加入 DHA 10、20、40、60、80、100、120 和200 μmol/L 后大鼠心室肌细胞AP和Ito的变化.结果 (1)当 DHA 浓度大于30 μmol/L时,动作电位时程(APD)逐渐延长,且呈浓度依赖性(P<0.05);当 DHA 浓度在0～30 μmoL/ L 时,随着 DHA 浓度增加,APD 延长不明显(P>0.05);加入不同浓度DHA后,在5 min内 APD 随时间延长而逐渐延长,5 min后 APD 基本固定.(2)加入不同浓度DHA 后,随着 DHA 浓度增加,Ito逐渐降低,DHA对Ito呈浓度依赖性阻滞(P<0.05).DHA 对Ito半效抑制浓度为58.3 μmoL/ L.结论 当加入不同浓度 DHA 后,APD 随着 DHA 浓度增加而逐渐延长,Ito逐渐降低,DHA 对 AP 和Ito的影响可能足其抗心律失常作用的机制之一.     

Comparative pharmacology of guinea pig cardiac myocyte and cloned hERG (I(Kr)) channel

INTRODUCTION: This study used whole-cell, patch clamp techniques on isolated guinea pig ventricular myocytes and HEK293 cells expressing cloned human ether-a-go-go-related gene (hERG) to examine the action of drugs causing QT interval prolongation and torsades de pointes (TdP) in man. Similarities and important differences in drug actions on cardiac myocytes and cloned hERG I(Kr) channels were established. Qualitative actions of the drugs on cardiac myocytes corresponded with results obtained from Purkinje fibers and measurement of QT interval prolongation in animal and human telemetry studies. METHODS AND RESULTS: Adult guinea pig ventricular myocytes were isolated by enzymatic digestion. Cells were continuously perfused with Tyrode's solution at 33-35 degrees C. Recordings were made using the whole-cell, patch clamp technique. Action potentials (APs) were elicited under current clamp. Voltage clamp was used to study the effect of drugs on I(Kr) (rapidly activating delayed rectifier potassium current), I(Na) (sodium current), and I(Ca) (L-type calcium current). Dofetilide increased the myocyte action potential duration (APD) in a concentration-dependent manner, with a pIC50 of 7.3. Dofetilide 1 microM elicited early afterdepolarizations (EADs) but had little affect on I(Ca) or I(Na). E-4031 increased APD in a concentration-dependent manner, with a pIC50 of 7.2. In contrast, 10 microM loratadine, desloratadine, and cetirizine had little effect on APD or I(Kr). Interestingly, cisapride displayed a biphasic effect on myocyte APD and inhibited I(Ca) at 1 microM. Even at this high concentration, cisapride did not elicit EADs. A number of AstraZeneca compounds were tested on cardiac myocytes, revealing a mixture of drug actions that were not observed in hERG currents in HEK293 cells. One compound, particularly AR-C0X, was a potent blocker of myocyte AP (pIC50 of 8.4). AR-C0X also elicited EADs in cardiac myocytes. The potencies of the same set of drugs on the cloned hERG channel also were assessed. The pIC50 values for dofetilide, E-4031, terfenadine, loratadine, desloratadine, and cetirizine were 6.8, 7.1, 7.3, 5.1, 5.2, and <4, respectively. Elevation of temperature from 22 to 35 degrees C significantly enhanced the current kinetics and amplitudes of hERG currents and resulted in approximately fivefold increase in E-4031 potency. CONCLUSION: Our study demonstrates the advantages of cardiac myocytes over heterologously expressed hERG channels in predicting QT interval prolongation and TdP in man. The potencies of some drugs in cardiac myocytes were similar to hERG, but only myocytes were able to detect important changes in APD characteristics and display EADs predictive of arrhythmia development. We observed similar qualitative drug profiles in cardiac myocytes, dog Purkinje fibers, and animal and human telemetry studies. Therefore, isolated native cardiac myocytes are a better predictor of drug-induced QT prolongation and TdP than heterologously expressed hERG channels. Isolated cardiac myocytes, when used with high-throughput patch clamp instruments, may have an important role in screening potential cardiotoxic compounds in the early phase of drug discovery. This would significantly reduce the attrition rate of drugs entering preclinical and/or clinical development. The current kinetics and amplitudes of the cloned hERG channel were profoundly affected by temperature, significantly altering the potency of one drug (E-4031). This finding cautions against routine drug testing at room temperature compared to physiologic temperature when using the cloned hERG channel.     

银杏叶提取物对模拟缺血条件下兔心室肌细胞动作电位及ATP敏感性钾通道的影响

研究银杏叶提取物 (EGb)对模拟缺血条件下兔心室肌细胞三磷酸腺苷敏感性钾通道电流 (IKATP)及跨膜动作电位时程 (APD)的影响 ,以探讨其抗缺血性心律失常作用的电生理机制。采用酶解法分离获取兔心室肌细胞 ,将其分为正常对照、持续缺血、缺血预处理以及含EGb液 (15 ,30 ,6 0 ,12 0 μg/L)灌流 4组。用全细胞膜片钳技术 ,记录不同条件下的IKATP和跨膜动作电位。结果 :①与持续缺血组比较 ,缺血预处理以及EGb(12 0 μg/L)可使单个心室肌细胞APD50 、APD90 明显缩短 (n =5 ,P <0 .0 5 )。EGb处理组与缺血预处理组相比较 ,对APD的影响无显著差异 ;②与持续缺血组比较 ,缺血预处理和EGb(12 0 μg/L)均可以使IKATP由 112 4± 15 3pA增至 344 0± 2 0 5和 2 95 9± 12 9pA(n =5 ,P <0 .0 5 ) ,使得IKATPI V曲线抬高 ;③增大的电流均可被Glibenclimide阻断。结论 :EGb可开放细胞膜ATP敏感性钾通道、缩短APD ,产生类似心肌缺血预适应的病理生理过程。     

Effects of intracellular calcium elevation on action potential and L-type calcium current of normal and chronically infarcted rat ventricles

The present work investigated the effects of raising [Ca+2]i levels on action potential (AP) and L-type calcium current (I(Ca.L)) of normal and chronically infarcted rat ventricles. Experiments were performed by conventional electrophysiology and whole-cell patch-clamp techniques. In the former, APs were recorded in ventricular strips subjected to different pacing rates or elevation of [Ca+2]o levels. In the latter, I(Ca.L) was studied in isolated myocytes in the absence of an intracellular Ca+2 chelator. The acceleration of heart rate (6 to 240 beats/min) reduced AP duration measured at 20%, 50%, and 90% repolarization (APD20, APD50, and APD90) in the infarcted group, and increased APD20 and APD50 in the control group. Rising [Ca+]o (1.25 to 5.0 mmol/L) induced a decrease of APD20 and APD50 in both groups. Voltage clamp revealed a smaller I(Ca.L) density at approximately -17 mV in myocytes from infarcted ventricles (-1.86 +/- 0.37 vs -3.98 +/- 0.65 pA/pF, P < .05), and the appearance of a non-K+ outward current coupled to I(Ca.L). The results suggest the participation of a Ca+2-activated outward current in the repolarization of normal and infarcted rat ventricles.