乙酰胆碱对犬右室三层心肌细胞L型钙电流不均一性的影响

目的测定犬右室三层心肌细胞上的L型钙电流(ICa,L),并研究其对自主神经递质乙酰胆碱的反应。方法经酶解法分离获得犬右室三层心肌细胞,应用全细胞膜片钳技术,记录并比较三层心肌细胞的ICa,L,以及应用2μmol/L乙酰胆碱前后电流-电压曲线的差异。结果ICa,L的峰值电流密度外膜下大于M细胞,而M细胞又大于内膜下心肌细胞,分别为-4.896±1.907pA/pF(n=31),-3.406±0.904pA/pF(n=37),-2.788±0.756pA/pF(n=33)(P<0.05)。使用乙酰胆碱后,右室外膜下及M细胞的峰值电流密度减小[-4.921±1.023pA/pF vs -3.462±0.997pA/pF(n=12);-3.803±1.115pA/pF vs -2.959±0.883pA/pF(n=13),P均<0.05]。心内膜下心肌细胞用药前后无差异(P>0.05)。结论ICa,L在犬右室三层心肌细胞存在不均一性,乙酰胆碱可以减小心外膜下、M细胞的ICa,L,对内膜下心肌细胞的ICa,L无影响。     

人右心室瞬间外向钾电流电不均一性的初步研究

目的探讨人右心室复极1相瞬间外向钾电流(Ito)的跨壁电不均一性。方法应用全细胞钳制技术,对人右心室心外膜下(Epi)细胞、中间层(M)细胞和心内膜下(Endo)细胞复极1相Ito离子流的电流强度、密度和动力学过程进行研究。结果人右心室三层细胞之间存在明显的Ito离子流电不均一性,在37℃、0·2Hz和除极化试验电压为+70mV时,Epi细胞和M细胞峰值Ito离子流的密度分别为(13·2±2·6)pA/pF,和(8·9±3·1)pA/pF,远大于Endo细胞(2·7±0·35)pA/pF,(P<0·01);在37℃条件下,人右心室Epi细胞和M细胞Ito离子流的激活和失活过程符合Boltzmann分布,两组细胞的激活和失活V1/2、斜率因子K等均差异无统计学意义(P>0·05)。结论在人右心室跨越室壁的3层细胞间,特别是Epi细胞与Endo细胞之间、M细胞与Endo细胞之间,复极1相存在明显的Ito离子流强度差异和强大的Ito离子流梯度。     

兔上腔静脉肌袖细胞动作电位及L型钙电流特征

研究兔上腔静脉肌袖细胞(SVCM)动作电位和L型钙电流(ICa,L)特征及异丙肾上腺素(Iso)对其影响。通过全心灌流酶解方法来分离兔SVCM,利用全细胞膜片钳技术,记录10nmol/LIso干预前后SVCM及右房心肌细胞(RAM)的动作电位、钙电流。结果:与RAM比较,SVCM动作电位复极50%时间(APD50),复极90%时间(APD90)明显要长(175.11±8.21msvs77.78±7.74ms,354.39±16.40msvs173.69±11.44ms,P均<0.05),Iso明显延长两者动作电位时程。SVCMICa,L较RAM大(4.04±0.74pA/pFvs2.75±0.33pA/pF,P<0.05),Iso明显增加ICa,L峰值,SVCMICa,L变化较RAM明显(4.04±0.74pA/pFvs7.14±0.77pA/pF;2.75±0.33pA/pFvs4.72±0.86pA/pF,P<0.05)。结论:SVCM动作电位及钙离子通道与RAM存在差异,可能是触发或驱动局灶性心房颤动的基础,Iso在其中发挥重要的作用。     

人右心室心外膜下细胞瞬间外向钾电流的初步研究

两例终末期心脏来源于心脏移植的受体病人 ,用酶解分离法获得心外膜下 (Epi)细胞 ,在全细胞钳制条件下观察人右心室Epi细胞瞬间外向钾电流 (Ito1 )的电生理特性。结果发现 :①Epi细胞具有强大的Ito1 ,在 0 .2Hz、+70mV和 37℃条件下 ,Epi细胞的峰值Ito1 离子流强度和密度分别达 1 940± 440pA、1 2 .9± 2 .6pA/pF ;②温度对Ito1 强度的影响明显 ,在 +70mV、0 .2Hz、2 2℃和 37℃条件下 ,Epi细胞峰值Ito1 强度和密度分别为 1 1 90± 31 0pA和 7.7±1 .8pA/pF、1 960± 465pA和 1 3 .1± 2 .8pA/pF ,差异有显著性 (P均 <0 .0 1 ) ;③Epi细胞Ito1 离子流强度表现出明显的频率依赖性 ,在 37℃和 +70mV、刺激频率分别为 0 .2 ,0 .5 ,1和 2Hz时 ,Epi细胞Ito1 离子流强度分别为 1 91 0±42 0 ,1 660± 360 ,1 4 1 0± 2 50 ,830± 1 4 0pA ,差异均有显著性 (P均 <0 .0 1 )。结论 :人右心室Epi细胞存在强大的Ito1 ,此为人右心室Epi细胞复极 1期一个突出的电生理特点 ,可能是Brugada综合征等疾病所致恶性心律失常的重要离子基础之一。     

咪达普利对陈旧性心肌梗死代偿区心肌T型钙电流的作用

应用咪达普利(Imi)对家兔陈旧性心肌梗死(OMI)模型进行干预,探讨其对OMI心室肌细胞T型钙电流(ICaT)的影响。选择健康家兔按体重随机分为OMI、假手术组与Imi组。OMI组:采用冠状动脉前降支结扎法制备OMI模型;假手术组:手术与OMI组同,只是不结扎冠状动脉,同样喂养8周;Imi处理组:对OMI家兔口服Imi0.625mg·kg-1·d-1连续8周进行干预。取心脏分离左室游离壁代偿区三层心肌细胞,全细胞膜片钳技术记录ICaT。结果显示,家兔OMI代偿区心肌细胞发生心肌肥厚,细胞膜ICaT密度较假手术组明显增加,当刺激电位为-30mV时,其电流密度从0.35±0.02pA/pF增至2.36±0.12pA/pF(P<0.01)。Imi组代偿区心肌细胞膜ICaT密度降至0.83±0.11pA/pF。ICaT的激活曲线左移,激活曲线斜率增加。Imi组二者改变均减小。但3组心肌细胞ICaT的失活曲线和失活后恢复曲线基本不变。结论:OMI家兔代偿区发生心肌肥厚,ICaT明显增加,Imi可以逆转家兔OMI代偿区心肌肥厚,降低ICaT的电流密度。     

索他洛尔对兔在体心脏左心室壁心肌复极不均一性影响的实验研究

目的　观察索他洛尔对兔在体心脏左心室壁各层心肌复极的影响 ,以证实在体心肌 M细胞的存在 ,探讨其与心律失常的关系。　方法　采用单相动作电位 (m onophasic action potential,MAP)记录技术 ,同步记录 12只开胸兔左心室外膜心肌 (epicardium ,Epi)、中层心肌 (m id- myocardium ,Mid)和心内膜心肌 (endocardium ,Endo)的 MAP,静脉注射索他洛尔后 ,测量 3层心肌 MAP的复极时限和跨心室壁心肌复极离散度 (transm ural dispersion of repolarization,TDR)。　结果　 1用药前 Epi、Mid、Endo的 MAP10 0 %复极时限 (APD1 0 0 )分别为 (136± 16 )、(15 2± 19)、(15 0± 2 0 ) m s,TDR为 (17± 8) m s。每间隔 30 m in静脉注射索他洛尔 0 .5、1.0、1.5和 2 .0 m g· kg- 1后发现 ,索他洛尔剂量依赖性延长 3层心肌的 APD1 0 0 ,其中以延长 Mid的 APD1 0 0 更为明显 ,对 Epi和 Endo的 APD1 0 0 延长程度相近 ,使 TDR增加 ;至静脉注射 2 .0 mg· kg- 1 后 ,Epi、Mid、Endo的 APD1 0 0 分别为 (177± 30 )、(2 34± 32 )、(194± 30 ) ms,TDR为 (5 7± 15 ) ms(P <0 .0 5 ) ;2索他洛尔剂量依赖性地增加尖端扭转性室性心动过速 (torsade depointes,TDP)的发生率。　结论　在体兔心肌存在 M细胞。索他洛尔增加兔     

索他洛尔对豚鼠心室肌动作电位及延迟整流钾电流的作用特性

目的　研究索他洛尔对豚鼠乳头状肌动作电位 (AP)和单个心室肌细胞延迟整流钾电流的作用及索他洛尔致心律失常的可能机制。方法　用标准微电极技术和全细胞膜片钳技术 ,分别测定豚鼠乳头状肌AP和单个心室肌细胞离子电流。结果　在索他洛尔浓度为 10 0 μmol/L时可明显延长APD ,使APD2 0 和APD90 分别延长13.33%和 19.71%。且该作用随BCL增加而增加 ,呈现出逆频率依赖性特点。在单个心室肌细胞的实验中10 0 μmol/L索他洛尔仅对IKr有阻滞作用 ,使IKr及IKr,tail的幅值从 (0 .6 8± 0 .2 7) pA/pF和 (0 .94± 0 .30 ) pA/ pF降至(0 .4 7± 0 .18) pA/ pF和 (0 .6 0± 0 .32 )pA/ pF ;且此作用也呈逆频率依赖性。结论　索他洛尔对心肌电生理的逆频率依赖性的作用特性可能是其诱发尖端扭转性室速 (TdP)等心律失常的机制之一。     

心力衰竭患者M2乙酰胆碱受体的自身抗体对豚鼠心肌细胞L型钙通道的影响

目的观察心力衰竭患者M2乙酰胆碱能受体的自身抗体(Abs)阳性血清以及M2乙酰胆碱能受体激动剂碳酰胆碱对心肌细胞的影响.方法应用全细胞钳制技术,定量观察并比较碳酰胆碱和心力衰竭患者Abs阳性血清对豚鼠单个心室肌细胞的L型钙电流强度的影响.结果M2受体激动剂碳酰胆碱使预先由异丙肾上腺素增大的L型钙电流峰值电流强度和标准电流密度从(2111.65±203.13)pA和(18.83±1.14)pA/pF下降为(1230.87±208.14)pA(P＜0.01)和(10.72±1.06)pA/pF(P＜0.01),M2受体阻滞剂阿托品可以阻断碳酰胆碱的这一作用.同样,心力衰竭患者的Abs阳性血清能使L型钙电流的峰值电流强度由(1995.21±195.13)pA下降至(636.42±110.07)pA,P＜0.01,标准电流密度由(18.13±1.03)pA/pF下降至(5.54±0.81)pA/pF,P＜0.01;阿托品也可阻断此作用.结论心力衰竭患者Abs阳性血清对豚鼠心室肌细胞的作用与碳酰胆碱相似,对心肌细胞M2受体有"激动剂样"效应,能使心肌细胞的L型钙电流减小,产生负性肌力作用;阿托品可以阻断它们对豚鼠心室肌细胞的作用.     

自发性高血压大鼠左心室肌细胞动作电位延长的离子机制

目的:研究自发性高血压大鼠(SHR)左心室肌细胞动作电位时程延长的膜离子流基础.方法:应用酶解方法分离获得正常血压Wistar大鼠和SHR的左心室肌细胞,采用玻璃微电极技术记录动作电位,膜片钳全细胞记录膜离子流,对比正常心室肌细胞和肥大心室肌细胞间动作电位及膜离子流差别.结果:(1)SHR和Wistar大鼠的心脏/体重比分别为5.66±0.46 mg/g和3.7±0.29 mg/g (P<0.001) ,细胞平均膜电容分别为280.68±67.98 pF 和189.94±56.59 pF(P<0.05).提示SHR 心脏肥厚、心肌细胞增大;(2)SHR动作电位APD50和APD90较Wistar大鼠明显延长(21.33±1.56 ms vs 14.91±2.95 ms,P<0.001; 164.6±74 ms vs 93.27±10.59 ms,P<0.00 1),说明SHR心室肌细胞存在复极延迟;(3)SHR的平均ICa-L幅值显著大于Wistar大鼠,分别为1944±466.8 pA和1136±33.3 pA(P<0.001),电流密度二者间无差异(6.932±1.7 1 pA/pF vs 6.19±2.85 pA/pF) ,但SHR的慢失活时间常数明显延长(56.01±13.36 ms vs 43.63±17.89 ms,P<0.001);(4)S HR的Ik1内向电流密度显著小于Wistar大鼠(11.3±2.26 pA/pF vs 14.33 pA/pF,P<0.05),外向电流密度二者间差异无显著性(2.36±0.86 pA/pF vs 2.96±1.27 pA/pF);(5)SHR的Ik密度与Wista r大鼠间无差别(12.38±5.46 pA/pF vs 11.86±3.59 pA/pF);(6)SHR的Ito密度显著地低于Wistar 大鼠(+70 mV时, 8.21±6.64 pA/pF vs 19.16±6.17 pA/pF, P<0.001).但通道的激活和失活时间常数二者无差异,提示Ito的降低可能仅是通道数减少所致.结论:SHR左心室肌细胞动作电位时程延长系外向复极钾流(Ito、Ik1)减小和慢钙通道失活时间常数延长所致.     

犬右心室心外膜下细胞瞬间外向钾电流特性的研究

目的　探讨犬右心室心外膜下 (Epi)细胞复极 1期瞬间外向钾电流 (Ito1)的电生理特性。方法　应用全细胞钳制技术 ,对右心室Epi细胞复极 1期Ito1的电流强度、动力学过程和动作电位切迹进行定量观察。结果　 (1)犬右心室Epi细胞具有强大的Ito1,其激活过程呈明显的电压依赖性 ,在37℃、0 2Hz和去极化试验电压为 +70mV时 ,Epi细胞的峰值Ito1强度和密度分别达 (4 15 0± 1780 )pA和 (31± 11 4)pA/pF ,其激活和失活动力学过程符合Boltzmann分布 ;(2 )犬右心室Epi细胞Ito1具有明显的频率依赖性 ,即在基础刺激周长增加时 ,Ito1明显增大 ,并和动作电位的“尖峰 穹隆”幅度的增加相对应 ;(3)温度对Ito1强度的影响明显 ,在去极化试验电压为 +70mV、0 2Hz和温度为 2 2℃、37℃条件下 ,右心室Epi细胞的Ito1强度和密度分别为 (2 380± 10 5 0 )pA与 (18± 7 6 )pA/pF、(4 15 0± 1780 )pA与 (31± 11 4)pA/pF ,差异明显。结论　犬右心室Epi细胞存在强大的Ito1,这种具有明显电压依赖性、频率依赖性和温度依赖性的Ito1及其参与介导的“尖峰 穹隆”状动作电位图形是右心室Epi细胞复极1期一个突出的电生理特点。     

L型钙通道在LQTl发病机制中作用的实验研究

目的 分析L型钙电流(IcaL)在犬三层心室肌细胞中的特点,探讨其在LQTl发病机制中的作用.方法 成年杂种犬14只,体重13～15 kg,雌雄不拘.分离犬三层心室肌细胞,采用全细胞膜片钳技术记录动作电位(AP)和ICaL,依次用Chromanol 293B(50ìμmoL/L)阻断慢激活延迟整流性钾电流(IKs)模拟LQTl,用异丙肾上腺素(100 nmo/L)激活13肾上腺素受体(β-AR),观察AP和,ICaL的变化.分三层取少量心室肌组织,采用实时定量逆转录聚合酶链反应(RT-PCR)技术,检测各层L型钙通道a1C亚单位的mRNA含量.结果 正常情况下,犬三层心室肌细胞ICaL电流密度差异无统计学意义[外层(4.253±0.782)pA/pF,中层(4.392±0.714)pA/pF,内层(4.182±0.665)pA/pF,P＞0.05],而中层心室肌细胞动作电位时限(APD)较内层和外层的长[外层(721.48±26.59)ms,中层(911.80±31.24)ms,内层(783.52±25.27)ms,P＜0.05];阻断IKs后ICaL电流密度没有变化,而APD均明显延长[(外层(835.21±27.34)ms,中层(1089.21±30.55)ms,内层(830.64±27.12)ms,与阻断IKs前相比,P＜0.05)];β-AR兴奋使三层心室肌细胞ICaL显著增加,且三者变化差异无统计学意义[(外层(5.654±0.756)pA/pF,中层(5.458±0.702)pA/pF,内层(5.600±0.819)pAZpF,P＞0.05].但β-AR兴奋使外层和内层心室肌细胞APD缩短,中层心室肌细胞APD延长,三者变化差异有统计学意义[外层(792.63±26.71)ms,中层(1127.85±32.10)ms,内层(811.32±27.52)ms,P＜0.05].实时定量RT-PCR结果显示,三层心室肌细胞中alC亚单位的mRNA含量差异无统计学意义(外层0.112±0.019,中层0.077±0.018,内层0.109±0.012,P＞0.05).结论 L型钙通道在犬三层心室肌中的分布没有差异,在LQTl模型中,Iso使三层心室肌细胞,ICaL均匀增加,推测ICsL本身没有引起LQTl复极不稳定.     

Characteristics of IK and its Response to Quinidine in Experimental Healed Myocardial Infarction

INTRODUCTION: Mechanisms and drug treatment of serious ventricular arrhythmias in patients with healed myocardial infarction (HMI) are incompletely understood, in part because the electrophysiology and pharmacology of myocytes from noninfarcted regions of HMI hearts are not well characterized. METHODS AND RESULTS: We studied the delayed rectifier potassium current (I(K)) and quinidine responsiveness of single left ventricular subendocardial myocytes isolated from the region remote to the border zone of healed infarct myocardium (4 to 6 mm from scar edge) in cat hearts 2 months after coronary artery occlusion. Subendocardial cells isolated from corresponding regions of normal cat hearts provided controls. I(K) activation and tail currents were recorded using whole cell, voltage clamp techniques. Membrane capacitance of cells remote to HMI (187 +/- 7 pF) was significantly greater than normal (155 +/- 6 pF; P < 0.001). Action potential durations (APDs) recorded from myocytes in remote regions were prolonged (APD90 = 247 +/- 10 msec) compared to normal (214 +/- 11 msec; P < 0.05). Quinidine (1 microM) significantly prolonged APD90 in normal cells but not in remote cells. Density of I(K) (tail current) was significantly decreased in remote cells (3.1 +/- 0.3 pA/pF) compared to normal (3.9 +/- 0.3 pA/pF; P < 0.05), and voltage-dependent activation of I(K) was shifted in the positive direction. Quinidine had significantly less incremental blocking effect on I(K) already blunted by regional hypertrophy compared to its effect on normal cells in remote cells. IC50 shifted to 0.95 microM in remote cells compared with 0.50 microM in normal cells. CONCLUSION: Cells in noninfarct region remote from the scar are hypertrophied and display altered electrophysiology. Their reduced I(K) responsiveness to quinidine may explain, in part, failure of quinidine to prolong APD in such cells. Moreover, dispersion of repolarization may be decreased by the effect of quinidine on normal cells.     

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.     

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）.     

Difference between electrical remodelling after pulmonary veins and right atrium appendage pacing.

OBJECTIVE: Pulmonary veins (PVs) are important sources of paroxysmal atrial fibrillation (AF). Rapid atrial pacing changes atrial electrophysiology, and facilitates the induction and maintenance of AF. The purpose of our study was to evaluate the changes in atrial effective refractory period (AERP) proprieties and in ionic currents in PVs myocytes from dogs subjected to rapid atrial pacing in PVs and right atrial appendage (RAA) and to relate these changes to the ability to induce AF. METHODS: Twelve mongrel dogs in normal sinus rhythm were paced from the superior left PVs or RAA at 500 bpm for 4 h. Electrophysiological studies were conducted to determine the changes in AERP, dispersion, and rhythm. Ionic currents were evaluated using patch clamp technique in single PVs myocytes in sham-operated dogs, and the results were compared with those from PVs and RAA pacing groups. RESULTS: The presence of rapid atrial pacing was associated with a marked shortening in AERP in both PVs and RAA pacing group with a marked increase in AERP dispersion in PVs pacing. Both L-type calcium current (I(Ca,L)) and the transient outward current (I(to)) were reduced in both groups with an increased significance in PVs pacing group. The density of I(Ca,L) was decreased significantly from (-6.03 +/- 0.63) pA/pF in the control group to (-3.21 +/- 0.34) pA/pF in the PVs pacing group and (-4.75 +/- 0.41) pA/pF in the RAA pacing group (n = 6, P < 0.05), whereas the density of I(to) was decreased significantly from (8.45 +/- 0.71) pA/pF in the control group to (5.21 +/- 0.763) pA/pF in the PVs pacing group and (6.84 +/- 0.69) pA/pF in the RAA pacing group (n = 6, P < 0.05). CONCLUSION: Our findings provide likely ionic mechanisms of shortened repolarization in induced atrial tachycardia with a decrease in I(Ca,L) and I(to) densities, which is the likely mechanism for a decrease in action potential duration rate adaptation in the canine rapid pacing model more pronounced in the PVs pacing group underlying the crucial role of PVs in initiating AF.     

缬草单萜氧化物对兔单个心室肌细胞L-型钙电流的影响

利用全细胞膜片钳记录技术研究30μg/L和100μg/L缬草单萜氧化物(VMO)对兔单个心室肌细胞L型钙电流(ICaL)和动作电位的影响。结果:30μg/L和100μg/L的VMO使兔心室肌细胞ICaL峰值由6.04±0.59pA/pF分别减至3.99±0.31pA/pF和2.31±0.24pA/pF(n=8,P<0.01);VMO使ICaL的电流电压曲线上移,但不改变其激活电位、电位峰值和反转电位;VMO还使钙电流失活曲线左移。30μg/LVMO可使动作电位时程(APD)明显缩短,APD50和APD90分别缩短了50.3%和29.6%(n=16,P<0.05),而静息电位和动作电位幅值无明显改变。结论:VMO对LCaL具有浓度依赖性阻滞作用。这可能是其对心血管作用的重要机制之一。