豚鼠离体心肌缺血再灌注的电生理模型

目的 应用计算机信号采集和处理技术研究豚鼠离体心肌细胞正常、缺血及再灌注时的电生理特性. 方法离体细胞灌流,细胞内微电极记录,计算机信号采集与处理.结果 豚鼠右心室乳头肌肌细胞静息电位RP为-72±6.58 mV,动作电位峰值(APA)为112.22±8.65 mV,0相最大去极化速率(Vmax)为107.37±10.55 mV·s-1.复极达峰值电位的10%、50%、90%所需时间(APD10、APD50、APD90)分别为39.25±8.55 ms、123.45±15.25 ms和169.35±18.12 ms.缺血缺氧台氏液灌流的心肌细胞APA与Vmax降低,APD90显著缩短,与缺血缺氧前相比有显著性意义(P＜0.05或P＜0.01).再灌注2 min内可见AP缺血缺氧样变化加重, Vmax继续降低,与缺血缺氧灌流相比有显著性意义(P＜0.01).豚鼠再灌注10 min后RP与AP波形基本恢复正常.结论 计算机信号采集和处理技术在心肌细胞电生理研究方面的应用,为全面的分析心肌细胞生物电的变化及其形成机制,亦为深入研究病理及药物作用情况下对生物电的影响提供真实客观的数据.     

硫酸铜对心肌细胞电生理特性的影响

目的 观察硫酸铜（CuSO4）对离体豚鼠乳头肌细胞的电生理效应,记录离体心肌快反应细胞动作电位;研究CuSO4对家兔窦房结细胞自发电生理活动的效应及其作用机制.方法 采用标准玻璃微电极细胞内记录方法观察不同浓度CuSO4对豚鼠心肌快反应细胞动作电位的影响,对家兔窦房结自发电生理活动的效应.结果 CuSO4能明显改变豚鼠乳头肌细胞的动作电位时程（APD,APD50,APD90）,并使其明显延长,且有剂量依赖性,用药前后APA,Vmax无明显变化;1 ×10^-4,1 ×10^-3mol/L的CuSO4可使家兔窦房结呈剂量依赖性兴奋,可使心率（RPF）增快,同时可使4相自动除极速率（VDD）加快（P＜0.01）.结论 CuSO4可使豚鼠心肌细胞动作电位时程延长,可增快离体兔窦房结细胞自律性,此效应可能影响复极相Ca＋和K＋通道.     

舒必利致心律失常的电生理研究

目的：观察不同浓度舒必利对缺血兔心浦肯野纤维动作电位的影响及舒必利对豚鼠心室肌细胞钠通道电流的作用。方法：采用标准微电极技术,观察不同浓度（1～100μmol／L）舒必利对模拟缺血液灌流的离体兔心浦肯野纤维动作电位0期去极化幅值（APA）、最大除极速率（Vmax）、有效不应期（ERP）及90％动作电位时程（APD50）的影响。应用酶解法分离豚鼠单个心室肌细胞,应用全细胞膜片钳技术记录舒必利对钠通道电流（INa）的影响。结果：不同浓度的舒必利对缺血兔浦肯野纤维动作电位的APA和APD90无明显影响,对Vmax有降低趋势。舒必利（3～300μmol／L）浓度依赖性地抑制INa（IC50=10．79μmol／L,测试电压-35mv）。10μmol／L舒必利降低了INa的最大电导gmax,使半激活、失活电压负值分别减小了1．91mV（P〈0．01）和5．22mV（P〈0．01）;恢复时间常数增加,但最大激活电流可以基本恢复至给药前。结论：舒必利可轻度逆转缺血液灌流造成的心浦肯野纤维动作电位缩短,并浓度依赖性地抑制钠电流,舒必利作用于钠通道的失活态。     

麻黄果多糖对家兔心室乳头肌电活动的影响

目的 观察麻黄果多糖对离体心室乳头肌动作电位的影响。方法 玻璃微电极细胞内纪录方法．记录麻黄果多糖对乳头肌静息电位（RP）、动作电位幅值（APA）、0相最大除极速度（Vmax）、动作电位时程（APD）及复极至50％和90％（APD50、APD90）的时间的影响．并利用M受体阻断剂和β受体阻断刺，探讨其作用靶点。结果 麻黄果多糖灌流5min时．APD、APD50、APD90均明显延长．后逐渐缩短．10min时．该3项指标与对照组相比明显缩短。应用M受体阻断剂后，该效应消失。应用8受体阻断剂时．先延长后缩短的效应仍存在。结论 麻黄果多糖作用于心室乳头肌M受体，对K^＋通道有先抑制后易化的双向效应。     

人重组生长激素对豚鼠离体心脏血流动力学及心肌细胞动作电位影响

目的 :观察人重组生长激素对正常豚鼠强心作用和对心室肌细胞动作电位的影响。方法 :采用langendorff离体心脏灌流法 ,悬浮玻璃电极法。结果 :注入人重组生长激素 10min后 ,左室最大收缩压 ,左室舒张末压 ,左室内压的最大升降速率的差值分别为 (6 .7± 5 .5 )kPa ,(0 .5 0± 0 .10 )kPa ,(139± 93)ps·s- 1,明显升高 ,心率差值为 (10± 16 )次·min- 1减慢 ,动作电位振幅值 (APA) ,超射 (OS) ,复极 5 0 %及 90 %水平的动作电位时程 (APD50 ,APD90 )分别为 (13.2± 6 )mv ,(9.6± 2 .2 )mv ,(84±2 9)ms,(90± 2 2 )ms ,显著延长 (均P <0 .0 1)。结论 :人重组生长激素对正常豚鼠具有正性肌力作用 ,可使动作电位时程延长     

改良技术虫草头孢菌粉提取物对缺血性心律失常和心肌细胞动作电位的影响

目的 观察采用改良技术获得的虫草头孢菌粉（Cordyceps Cephalosporium Mycelia,CCM）提取物对豚鼠乳头肌动作电位的影响,探讨CCM提取物实验性抗室性心律失常的机制。方法 利用垂体后叶素（Pit）诱发的大鼠缺血性心律失常模型,观察用药前后心电图的改变。采用标准玻璃微电极技术,记录豚鼠乳头肌跨膜动作电位（TAP）。在频率为1.0 Hz刺激下,观察不同浓度的CCM提取物（10,30,100 mg·L^-1）对标准Tyrode液灌流心肌TAP的影响。结果 大鼠在尾静脉注射Pit后,其心电图T波波幅发生双向性改变,CCM提取物可明显减弱Pit对T波的影响,并降低Pit诱导的心律失常发生率。不同浓度的CCM提取物可浓度依赖性地降低动作电位幅度（APA）以及0相最大上升速率（Vmax）;可延长动作电位复极50%和90%的时间（APD50、APD90）;但对静息电位（RP）无明显影响。结论 改良技术获得的CCM对APA和Vmax的抑制及对心室肌APD的延长可能是其抗心律失常作用的电生理基础。     

吗啡对豚鼠心肌电生理的影响

目的观察吗啡对离体豚鼠心肌快反应动作电位(AP)和L-型钙通道的影响。方法采用玻璃微电极技术在豚鼠心室肌细胞上研究吗啡(1×10-5、1×10-4、1×10-3mol/L)对正常及异丙肾上腺素(ISO)致豚鼠心室肌细胞动作电位的作用。结果吗啡1×10-5、1×10-4与1×10-3mol/L,能缩短动作电位时程(n=10,P<0.01),呈剂量依赖性,而动作电位幅值(APA)、0相上升最大速度(Vmax)无明显变化;吗啡又能对抗由异丙肾上腺素(ISO)致豚鼠心室肌细胞动作电位;应用L型钙通道开放剂Bay K8644(0.25μmol)可增加动作电位时程(APD)、复极化50%时间(APD50)、复极化90%时间(APD90)(n=10,P<0.01),加入吗啡可完全逆转Bay K8644(0.25μmol)对乳头肌细胞的兴奋效应。结论吗啡明显缩短APD;并能对抗由ISO引起的早后除极;能对抗L型钙通道开放剂BayK644对乳头肌细胞的兴奋效应,其机理与钙拮抗有关。     

双黄连粉针剂对豚鼠心肌电生理的影响

目的探讨双黄连粉针剂对豚鼠心脏电生理的影响,以及其心脏安全性及作用机制。方法①在体实验:双黄连粉针剂325.5,1627.5和3255.0mg·kg-1分别在两组动物进行,每组15只。一组动物按照生理盐水→双黄连粉针剂325.5→1627.5mg·kg-1,另一组按照生理盐水→双黄连粉针剂3255.0mg·kg-1的顺序经颈外静脉缓慢推注,持续5min。于给生理盐水及静脉推注各剂量药物后5min记录心电图,分析P-R间期和校正QT间期(QTc)。②离体实验:按照灌流液(空白对照)→双黄连粉针剂0.3→1.5→3→9g·L-1→洗脱的顺序灌流,持续5min。于灌流5min末记录豚鼠离体心脏心电图,分析7只豚鼠的P-R间期和QTc间期;记录左心室乳头肌动作电位,分析5只豚鼠的动作电位复极50%水平时程(APD50)和90%水平时程(APD90)。结果①在体豚鼠心电图表明,双黄连粉针剂325.5和1627.5mg·kg-1显著延长P-R间期,从生理盐水处理时的(59±5)ms分别延长到(74±10)ms和(88±20)ms(P<0.05),各浓度组的QTc间期无明显变化;双黄连粉针剂3255.0mg·kg-1处理P-R间期则从生理盐水处理时的(58±5)ms延长到(133±29)ms(P<0.05),QTc从(247±16)ms延长到(301±65)ms(P<0.05),并显示明显的室内传导阻滞。②离体豚鼠心电图表明,双黄连粉针剂3和9g·L-1使空白对照组P-R间期从(84±17)ms延长到(113±39)ms和(130±23)ms(P<0.05),伴有明显的室内传导阻滞,而各浓度组的QTc间期无明显变化。双黄连粉针剂各浓度组对正常豚鼠左心室乳头肌动作电位APD50与APD90无明显作用。结论注射用双黄连粉针剂能引起豚鼠房室和室内传导阻滞,其作用机制可能是抑制心肌细胞钠通道。     

丁咯地尔对豚鼠乳头肌快反应动作电位的影响

研究丁咯地尔(BUF)对豚鼠乳头肌快反应动作电位(AP)的影响。方法采用细胞内微电极技术 ,观察不同浓度BUF对AP的影响。结果各浓度BUF使动作电位幅度(APA)明显降低 ,使APD 30,APD 50缩短 ;BUF10-4M和10-3 使APD 90 也明显缩短。5×10-3M使AP消失之后 ,还使静息电位(RP)绝对值继续降低。结论(1)BUF对豚鼠乳头肌Ca2 、Na 通道有阻滞作用 ;对K 通道的影响较复杂。(2)BUF使豚鼠乳头肌对电刺激的兴奋性降低。(3)BUF对AP的作用是可逆的。     

杜鹃花总黄酮对豚鼠心室肌细胞生物电的影响

目的观察杜鹃花总黄酮(total flavones of rhododen-dron,TFR)对离体豚鼠右心室乳头肌细胞生物电的影响,以探讨其抗心肌缺血的作用机制。方法采用标准玻璃微电极技术记录心肌细胞的静息电位以及动作电位。结果25、50mg·L-1的TFR可明显缩短动作电位复极50%、90%时程(APD50、APD90),但100、200、400mg·L-1却可明显延长APD50和APD90,200、400mg·L-1的TFR可明显降低0期的动作电位峰值(APA)及动作电位去极化最大速率(Vmax),400mg·L-1的TFR使静息电位减小,200mg·L-1TFR能延长心室肌细胞有效不应期(ERP)。结论低浓度的TFR可能有钙阻滞作用,高浓度的TFR可能对钾通道有一定的阻断作用,200mg·L-1TFR能延长心室肌细胞有效不应期。     

金属硫蛋白对缺氧和复豚鼠乳头状肌动作电位的影响

目的：研究金属硫蛋白（ＭＴ）对缺氧和复氧豚鼠乳头状肌动作电位的影响。方法：利用标准的玻璃微电极技术，将标本暴露于缺氧液（低氧，高钾，无糖，ｐＨ６．８）和ＭＴ。结果：ＭＴ（０．０２ｍｍｏｌ·Ｌ＾－１）对正常豚鼠乳头状肌的动作电位无影响；单纯将标本于缺氧液，明显使ＡＰＤ２０，ＡＰＤ５０和ＡＰＤ９０缩短，增高ＲＰ水平，降低ＡＰＡ及Ｖｍａｘ；缺氧液加ＭＴ（０．０２ｍｍｏｌ·Ｌ＾－１）后，则使氧期间的ＲＰ，     

氨氯吡咪及其衍生物DMA对豚鼠心肌细胞跨膜动作电位时程的影响

目的 :研究氨氯吡咪 (AM)及其衍生物二甲基氨氯吡咪 (DMA)对心室肌细胞跨膜动作电位时程(APD)的影响。方法 :利用全细胞膜片钳技术 ,观察 AM及其衍生物 DMA对豚鼠心肌细胞跨膜 APD的作用。结果 :Am可使 APD复极化 3 0 %、5 0 %和 90 %的时程 (APD3 0 ,APD50 ,APD90 )延长 3 0 %、5 2 %和 3 4%。 DMA可使APD复极化 APD3 0 、APD50 、APD90 的时程缩短 4 0 %、2 9%和 2 4 %。结论 :AM可使心肌细胞跨膜 APD延长 ,而DMA可使心肌细胞跨膜 APD缩短     

IHC-66对离体豚鼠心肌细胞慢反应电活动的影响

目的：为进一步观察ＩＨＣ－６６的电生理特性，探讨其抗心律失常的作用机制。方法：采用离体豚鼠窦房结、乳头状肌及部分除极化乳头状肌标本，观察了３，６－ｄｉ（ｄｉｍｅｔｈｙｌａｍｉｎｏ）－ｄｉｂｅｎｚｏｐｙｒｉｏｄｏｎｉｕｍｏｆｆｅｒｉｃＥＤＴＡ（ＩＨＣ－６６）对不同类型心肌细胞电活动的影响。结果：ＩＨＣ－６６１０μｍｏｌ·Ｌ－１使窦房结细胞动作电位４相除极速率（ＳＰ４）降低１１．４２％、增加动作电位复极９０％时程（ＡＰＤ９０）１８％、降低零相最大上升速率（Ｖｍａｘ）４７％、使部分除极化乳头状肌细胞动作电位（ＡＰ）之ＡＰＤ９０、动作电位复极５０％时程（ＡＰＤ５０）分别缩短９％、１３％，Ｖｍａｘ降低５８％，同时，ＩＨＣ－６６（１０、３０、５０μｍｏｌ·Ｌ－１）呈剂量依赖性降低Ｂａ２＋（２ｍｍｏｌ·Ｌ－１）诱发的自发性动作电位振幅（ＡＰＡ）与Ｖｍａｘ，缩短ＡＰＤ５０、ＡＰＤ９０，明显减慢自发性电活动的频率。结论：ＩＨＣ－６６的电生理学作用与其对慢内相电流的阻滞有关。     

Comparison of guinea-pig ventricular myocytes and dog Purkinje fibres for in vitro assessment of drug-induced delayed repolarization

INTRODUCTION: QT interval prolongation and Torsade de Pointes (TdP) arrhythmias are recognised as a potential risk with many drugs, most of which delay cardiac repolarization by inhibiting the rapidly activating K(+) current (I(Kr)). The objective of this study was to compare the effects of compounds on cardiac action potentials recorded from guinea-pig ventricular myocytes and dog Purkinje fibres. METHODS AND RESULTS: Effects of dofetilide, sotalol, cisapride, terfenadine, haloperidol and sparfloxacin, compounds known to cause QT prolongation (positive controls), and nifedipine and verapamil, not associated with QT prolongation (negative controls) were studied on intracellular action potentials recorded from guinea-pig isolated ventricular myocytes (VM) and dog isolated Purkinje fibres (PF). Prolongation of action potential duration (APD) by sotalol, dofetilide and sparfloxacin was concentration-dependent and of greater magnitude in dog PF compared to guinea-pig VM. The maximum prolongation of APD in guinea-pig VM at 0.5 and 1 Hz was approximately 25% and this was associated with complete inhibition of I(Kr) by dofetilide. Effects on APD of cisapride and haloperidol in both preparations, and terfenadine in guinea-pig VM, were biphasic, consistent with inhibition of multiple ion channels. There was no effect of terfenadine on APD in dog PF. Haloperidol increased APD by more than 25% in guinea-pig VM, consistent with effects on additional repolarizing currents. The negative controls shortened APD to a greater extent in guinea-pig VM compared to dog PF. In general, the positive control drugs increased action potential triangulation (APD(40-90)) to a greater extent than APD(90). CONCLUSION: Guinea-pig isolated VM may be more sensitive for detecting APD prolongation with compounds inhibiting multiple ion channels and action potential triangulation (APD(40-90)). Effects on repolarizing currents other than I(Kr) were also distinguished in guinea-pig VM.     

A comprehensive cardiotoxicity assay by combining high-throughput multiple ion channel screening with human cardiomyocyte-based action potential validation

OBJECTIVE Cardiotoxicity refers to drug-induced arrhythmia such as Torsades de pointes.Current single ion channel(hERG)-based assay generates-30% false results.The aim is to establish an advanced in vitro cardiotoxicity assay by incorporating high throughput multiple cardiac ion channel screening and human cardiomyocytes-based validation.METHODS Effects of drugs were tested on multiple cell lines expressing hERG,Nav1.5 and Cav1.2 by automated patch clamping.Subsequently,the results were validated with human pluripotent stem cell(hPSC)-derived cardiomyocytes(hPSC-CMs)in which ion currents and action potentials were measured by manual patch clamping.RESULTS We have tested the cardiotoxicity of monomers extracted from various medical herbs.Mitragynine is the major bioactive compound isolated from kratom,a therapeutic herb from the rain forest of South East Asia.As a popular stimulant,it has been associated with a number of acute fatal incidences.We observed a typical torsadogenic hazard of mitragynine.It exerted a strong hERG inhibition in hERG-HEK293 cell line(IC50:5.2 μmol·L-1)and hPSC-CMs(IC50:0.91 μmol·L-1)without affecting other cardiac ion channels.Moreover,it caused a significant prolongation of action potential duration(APD)in hPSC-CMs(a-32.5%increase in APD at 50 and 90%repolarization).On the other hand,deoxylelephantopin,apotential anti-cancer reagent,demonstrated low cardiotoxicity.It exerted a week inhibition on hERG in HEK293 cells with an IC50 of 87.2 μmol·L-1,while the effective concentrations for suppressing the growth of cancer cells ranges from 2 to 20μmol·L-1.At 100μmol·L-1,deoxylelephantopin showed no effects on Cav1.2 and Nav1.5 and it failed to alter APD in hPSC-CMs.CONCLUSION We have successfully tested a newin vitro cardiotoxicity assay strategy which incorporates multiple cardiac ion channels screening and hPSC-CMs validation.This new strategy could facilitate the effective and efficient evaluation of existing and new drugs/reagents for potential pro-arrhythmic risk.     

Effect of altered repolarization course induced by antiarrhythmic drugs and constant current pulses on duration of premature action potentials in canine cardiac Purkinje fibers

The purpose of this study was to elucidate the mechanism of the upward shift of the electrical restitution curve, i.e., the lengthening of premature action potential duration (APDt) expressed as percentage of basic APD, induced by class I antiarrhythmic drugs in dog Purkinje fibers. In this study, six class I antiarrhythmic drugs lengthened APDt at a diastolic interval of 20 ms by 2.5-14.1%. The drugs also decreased the ratio of APD at 50% to APD at 90% of repolarization from 70.8 +/- 1.8% (n = 60) to 47.4-60.8%. The relation between the decrease in the ratio of APD50 to APD90 of the basic AP and lengthening of the normalized APDt was linear (r = 0.92; p less than 0.01). We attributed the lengthening of normalized APDt to the decreased ratio of APD50 to APD90, and applied repolarizing current pulses in short (less than or equal to 2 mm) fibers to simulate the drug-induced decrease in the ratio of APD50 to APD90. The altered repolarization course of basic AP by the current pulse during late plateau and early phase 3 caused APDt lengthening. The relation between the current-induced decrease in the ratio of APD50 to APD90 of the basic AP and the lengthening of normalized APDt was linear (r = 0.91; p less than 0.01). The slope of regression line describing this relation was similar to that in the presence of drugs. These results suggest that lengthening of the normalized APDt by class I antiarrhythmic drugs results from a more rapid repolarization during phase 2 of the preceding basic AP, possibly due to lesser influence of the delayed outward rectifying current. The lengthening of APDt by class I drugs may contribute to their antiarrhythmic action.     

Electrophysiologic effect of dipfluzine on guinea pig papillary muscles in vitro

用细胞内微电极技术记录正常和部分除极豚鼠乳头肌动作电位,研究双苯氟嗪（３－３０μｍｏｌ·Ｌ－１）对豚鼠乳头肌细胞电生理的影响．结果表明,双苯氟嗪轻度缩短正常乳头肌动作电位的平台期（ＰＰＤ）,５０％和９０％复极化时程（ＡＰＤ５０和ＡＰＤ９０）,但对静息电位（ＲＰ）,动作电位幅度（ＡＰＡ）,超射（ＯＳ）和０相最大上升速率（Ｖｍａｘ）均无显著影响．在高Ｋ＋部分去极化的豚鼠乳头肌标本,双苯氟嗪可轻度降低慢反应动作电位的ＯＳ,ＡＰＡ和Ｖｍａｘ,并缩短ＰＰＤ,ＡＰＤ５０和ＡＰＤ９０,对ＲＰ同样无显著影响．在相同浓度下,双苯氟嗪对离体豚鼠乳头肌动作电位的影响与氟桂利嗪无显著差别．提示双苯氟嗪在较高浓度时,可轻度缩短豚鼠乳头肌的复极过程及降低部分除极乳头肌的ＯＳ,ＡＰＡ和Ｖｍａｘ．     

赛庚啶对豚鼠乳头状肌的收缩性与动作电位的影响

Cyp对豚鼠孔头状肌有浓度依赖性负性肌力作用,提高浴液钙浓度此作用减弱,对高K~+除极后ISO恢复的收缩抑制作用更强。较高浓度的Cyp使频率依赖性正阶梯现象取消和翻转。对乳头状肌AP,Cyp浓度依赖性地使APD_(20),APD_(50),APD_(90)和ERP缩短。灌流液中Ca~(2+)降低或升高分别使该作用增强或减弱,K~+的改变对其作用影响较小。提示Cyp对豚鼠乳头状肌收缩性和AP的作用可能主要是抑制心肌跨膜Ca~(2+)内流所致。