豚鼠心房肌细胞的分离与其离子流记录

为探讨豚鼠心房肌细胞的分离方法及其离子离子流记录，采用酶解技术分离豚鼠心房肌细胞，应用膜片钳全细胞记录技术，记录细胞膜Ｌ型钙流（Ｌ－Ｉｃａ）及延迟整流性钾流（Ｉｋ）。结果获得了具正常电生理活性的单个细胞及高阻封接的形成，成功记录了Ｌ－型钙流和延迟整流钾流，认为本方法分离的单个心房肌细胞具正常的电生理活性，可用于研究心房肌细胞离子通道活性。     

犬心房肌细胞分离的方法学探讨

目的建立稳定的适于膜片钳实验研究的犬心房肌细胞分离方法。方法取健康成年犬心脏12个,采用Langendorff灌流,行回旋支插管,正常台式液灌流10 s,使心脏自主收缩排除残留余血。持续高钾液灌流,同时结扎其他血管及分支,剪去其余心脏组织,待左房及左心耳充盈,无钙台氏液灌流5 min,125 U/ml胶原酶Ⅱ200ml反复灌流消化约30~45 min,后用无钙台氏液冲洗心脏5 min,剪下心房肌组织,KB液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,逐步复钙法复钙后,室温静置1 h,应用于膜片钳记录。结果分离的活性心肌细胞比率约90%,形态呈杆状、横纹清晰、膜周边光滑完整。结论采用本方法可以获得高产量与高质量的用于膜片钳离子流检测的心房肌细胞。     

卡维地洛对豚鼠心室肌细胞离子流的研究

卡维地洛是临床上广为应用的非选择性 β受体、选择性α1受体阻滞剂 ,在动物实验和临床研究中都显示具有抗心律失常作用 ,本研究系统地观察卡维地洛对豚鼠心室肌细胞离子流的直接影响 ,探讨其细胞电生理作用。1 材料与方法 :选用 2 0 0～ 3 0 0g的健康豚鼠 ,酶解法分离获得单个左心室肌细胞 ,采用膜片钳全细胞记录技术 ,膜片钳放大器为美国AxonInstruments公司产Axopatch 2 0 0B型 ,电压钳制脉冲发放和数据采集由Pclamp 6 0 4软件控制 ,根据所记录离子流的不同采用相应的电极外液和电极内液。卡维地洛 (山东齐鲁制药厂提供 )以浓度递增方…     

单个人心房肌细胞的分离、鉴定及影响因素分析

目的 探讨分离、鉴定单个人心房肌细胞的最佳方法及其影响因素.方法 人心房肌组织取自5例先天性心脏病(PCHD)、8例冠心病(CHD)、15例瓣膜性心脏病(VHD)手术患者,采用两步酶解法分离制备心房肌细胞,光学显微镜下观察细胞形态,全细胞膜片钳技术记录乙酰胆碱敏感钾电流(I K,Ach)情况.结果 两步酶解法分离所得杆状、横纹清楚、折光性好、静止、贴壁良好的心肌细胞产率为30%～50%.PCHD记录I K,Ach成功率最高(9/10个细胞),其次为CHD(6/10个细胞),VHD最低(3/10个细胞).结论 两步酶解法可用于人心房肌细胞的分离,全细胞膜片钳技术有助于细胞活性的鉴定,其分离数量和活性受酶解情况、年龄、病种等因素影响.     

采用膜片钳技术记录心室肌细胞离子流

本文报告了心室肌细胞分离方法,采用膜片钳技术,成功地记录了细胞膜钾、钙、钠离子流,初步显示背景钾离子通道活性的电压依赖性,钙、钠离子通道活性为电压和时间依赖性,钠离子通道开放速率最快,电压在-30mV。钠离子流达峰电流仅需1ms,其活性也表现电压和时间依赖性。     

大蒜素对小鼠心房肌细胞多种钾电流的阻滞效应

目的 探讨大蒜素对小鼠心房肌细胞多种钾电流的作用及其机制。方法 采取双酶消化法将单个小鼠心房肌细胞分离出来,灌流法将药物作用于细胞,采用全细胞膜片钳技术,分别记录心房肌细胞的外向钾电流(I_K,peak)、瞬时外向钾电流(I_to)和超极化激活延迟整流钾电流(I_KUr)。结果 钳制电压在+70 mV时,30μmol/L大蒜素可使小鼠心房肌细胞I_K,peak、I_to和I_KUr峰值显著降低,对I_to表现出抑制作用,该作用还呈现出一定的浓度依赖性和电压依赖性,半数抑制浓度(IC₅₀)为(21.0±3.8)μmol/L。深入研究门控机制结果显示,I_to通道稳态失活曲线在大蒜素的作用下发生左移,且失活后再次激活的时间有所延迟,对于通道的关闭态失活过程影响不大,提示大蒜素主要通过抑制通道稳态失活和失活后恢复过程,减少I_to电流密度,同时对I_KUr也有阻滞作用,进而...     

心房颤动对心房肌细胞电生理的影响

阵发性心房颤动(房颤)常会发展为慢性房颤,其发生率与基础疾病显著相关[1],同时有18%的特发性房颤会发展为持续性房颤[2]。阵发性房颤的持续时间也很重要,房颤持续时间短于2天的患者中,有31%的患者转变为慢性房颤,而房颤持续时间超过2天的患者,有46%转为慢性房颤[1]。从这些流行病学资料可以看出,不依赖于基础心脏病,房颤本身就是一种进行性疾病。　　临床上发现当房颤存在时间较短时,药物转复或直流电转复的成功率较高。在新近出现的房颤(<24h),静脉注射氟卡胺的转复率为76%～93%,而持续时间较长时转复成功率为0%～83%[3,4]。胺碘酮对于持…     

异丙肾上腺素对犬心房肌细胞动作电位及L型钙电流的影响

目的研究两种浓度的异丙肾上腺素（Isoproterenol，ISO）对犬心房肌细胞（AP）及L型钙电流（ICa，L）的作用。方法采用离体灌注和消化的方法获取心房肌细胞，用全细胞记录技术记录单个心房肌细胞AP以及ICa，L。结果低浓度ISO（10nmol／L）可延长APD，可使90％AP时程（APD90）延长34．4％，并降低AP平台期水平。高浓度ISO（1μmol／L）可减少APD，APD90减少32．1％。两种浓度的ISO均可诱发AP后除极及触发活动。10nmol／L和1μmol／L ISO分别增加ICa，L 36．7％和49．3％。结论两种浓度的ISO对心肌细胞ICa，L均有促进作用，Ca^2＋内流引起的肌浆网Ca^2＋释放可能是房性心律失常的发生机制之一。     

人心房肌细胞的培养与鉴定

目的　探索人心房肌细胞的原代及传代培养方法。方法　取心外科手术患者 (1 5～6 0岁 ,平均 2 5岁 )常规切除的右心耳 ,利用组织贴块法原代培养心房肌细胞并进行传代培养 ,对培养细胞 (取第 3代 )进行光镜、透射电镜形态学观察和免疫细胞化学鉴定 ,并绘制生长曲线。结果原代培养 1 0d左右时细胞数目可达 1 0 6～ 1 0 7/ml;此后每 4～ 5d可传代一次 ,大多可传至第 8代。透射电镜观察到典型心肌细胞的超微结构 ,免疫细胞化学分析显示 90 %以上的细胞呈α 肌动蛋白及心肌特异性肌钙蛋白Ⅰ抗体染色阳性。细胞生长曲线显示第 3代细胞在密度为 1× 1 0 6～ 8× 1 0 6/ml呈对数生长 ,倍增时间约 2 4h。结论　利用组织贴块法成功培养出人心房肌细胞 ,所得心房肌细胞纯度高并能传代培养 ,为深入研究人心房肌细胞的病理生理及分子生物学奠定了基础。     

成年家兔心房肌细胞的分离和电生理记录

目的建立一种简单稳定的成年家兔心房肌细胞的分离、培养方法并进行电生理记录。方法麻醉后取出成年家兔心脏,采用Langendorff灌流装置及急性酶裂解法分离心房肌细胞,差速贴壁法进行纯化后培养于DMEM培养基。在倒置显微镜下观察细胞形态,利用透射电镜观察细胞超微结构,用免疫荧光染色法对心房肌细胞进行鉴定,利用全细胞膜片钳技术记录动作电位和内向钙电流和外向钾电流。结果本方法分离的心房肌细胞纯度和细胞存活率较高,并使用膜电钳技术成功记录了L-型钙电流和瞬时外向钾电流。结论该方法简便有效,细胞存活率高且为进一步进行各种电生理实验打下了基础。     

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.     

颅痛定对单个豚鼠心室肌细胞缺氧后复氧早期L-Ca电流的影响

目的 :研究颅痛定 (左旋四氢巴马汀 ,L - tetrahy- dropalm atine,L - THP)对缺氧后复氧早期心室肌细胞 L - Ca电流的影响。方法 :使用全细胞膜片钳技术研究心室肌细胞 L - Ca电流的变化。结果 :经 2 0 m in的缺氧以及 5 min的复氧后 ,1,10 ,10 0 (μmol· L- 1 ) L - THP分别将 Ca- max电流从 (6 75 .75± 10 7.0 7) p A减少至 (6 45 .46±10 8.75 ) p A、(4 92 .0 8± 72 .84) p A、(376 .6 0± 71.35 ) p A ,P值分别为 >0 .0 5、<0 .0 5、<0 .0 1。结论 :L - THP能有效地减少缺氧后复氧早期心室肌细胞 L - Ca电流 ,这种作用无疑是其抗再灌注早期心律失常的电生理基础。     

Slow recovery of excitability and the Wenckebach phenomenon in the single guinea pig ventricular myocyte

The cellular mechanisms of Wenckebach periodicity were investigated in single, enzymatically dissociated guinea pig ventricular myocytes, as well as in computer reconstructions of transmembrane potential of the ventricular cell. When depolarizing current pulses of the appropriate magnitude were delivered repetitively to a well-polarized myocyte, rate-dependent activation failure was observed. Such behavior accurately mimicked the Wenckebach phenomenon in cardiac activation and was the consequence of variations in cell excitability during the diastolic phase of the cardiac cycle. The recovery of cell excitability during diastole was studied through the application of single test pulses of fixed amplitude and duration at variable delays with respect to a basic train of normal action potentials. The results show that recovery of excitability is a slow process that can greatly outlast action potential duration (i.e., postrepolarization refractoriness). Two distinct types of subthreshold responses were recorded when activation failure occurred: one was tetrodotoxin- and cobalt-insensitive (type 1) and the other was sensitive to sodium-channel blockade (type 2). Type 1 responses, which were commonly associated with the typical structure of the Wenckebach phenomenon (Mobitz type 1 block), were found to be the result of the nonlinear conductance properties of the inward rectifier current, IK1. Type 2 sodium-channel-mediated responses were associated with the so-called "millisecond Wenckebach." These responses may be implicated in the mechanism of Mobitz type 2 rate-dependent block. Single-cell voltage-clamp experiments suggest that variations in excitability during diastole are a consequence of the slow deactivation kinetics of the delayed rectifier, IK. Computer simulations of the ventricular cell response to depolarizing current pulses reproduced very closely all the response patterns obtained in the experimental preparation. It is concluded that postrepolarization refractoriness and Wenckebach periodicity are properties of normal cardiac excitable cells and can be explained in terms of the voltage dependence and slow kinetics of potassium outward currents. The conditions for the occurrence of intermittent activation failure during diastole will depend on the frequency and magnitude of the driving stimulus.     

豚鼠左心室不同区域细胞Iks电流对缺血反应的差异

目的：研究缺血时豚鼠左心室心内、外膜细胞及M细胞缓慢激活延迟整流钾通道电流（Iks)的变化特性。方法：利用全细胞膜片钳技术观察豚 鼠左心室心内、外膜细胞及M细胞Iks变化，利用不同成分浴槽液模拟细胞正常及缺血环境，观察Iks尾电流锋值的变化情况。结果：缺血时，三层细胞Iks均小于正常状态；指令电压小于0mV时，三层细胞Iks同步减小，减少率无显性差异，P＞0．05。指令电压≥0mV时，M细胞Iks减少程度明显高于心内、外膜细胞，P＜0．05；而心内、外膜细胞减少率无显性差异，P＞0．05。结论：缺血时左心室心内、外膜细胞及M细胞Iks减弱，而M细胞Iks减弱更为明显，这可能会增加心室壁电活动不均一性，诱发心律失常。     

Electrophysiological effects of flecainide acetate on stretched guinea pig left atrial muscle fibers

Summary The electrophysiological effects of flecainide acetate (3×10^–6 M) on stretched atrial tissue were investigated using guinea-pig left atrial muscle fibers. Before stretching, the resting membrane potential was not affected by flecainide at 1 Hz, although the overshoot potential (Eov) and the action potential duration at 50% repolarization (APD₅₀) were slightly but significantly decreased by 2±1 mV and 2±1 msec, respectively. The effective refractory period (ERP) was increased by 3±1 msec. The reduction of max was 20.6±1.2%. The half-maximum potential (Vh) of the relationship between max and the resting potential was shifted to become more negative by flecainide (from –60.6±2.1 mV to –63.2±1.7 mV). After 90–120 min of washout with drug-free Tyrode's solution, the tissue was mechanically stretched to 150% of its slack length. Stretching significantly decreased the max by 16.9±3.1%, along with a slight but significant increase in ERP (3±1 msec) and shifted Vh to become more negative (from –60.6±2.1 to –63.1±1.8 mV). In the presence of flecainide, max further decreased by 20.2±2.6%, and Vh shifted from –63.1±1.8 to –65.0±1.5 mV. Comparison with the control unstretched fibers showed that flecainide significantly decreased max by 34.0±2.7%, reduced the resting membrane potential by 3±1 mV, decreased Eov by 4±1 mV, and shifted Vh from –60.6±2.1 to –65.0±1.5 mV, while the APD₅₀ and ERP did not change. In conclusion, the reduction of max in the presence of flecainide was much greater in the stretched atrial muscle fibers than in the unstretched fibers, because the max-resting potential relationship was shifted towards more negative potentials by both flecainide and stretching. These results suggest that flecainide exerts a stronger antiarrhythmic action on stretched atrial muscle fibers than on normal fibers.     

Heterogeneity of sodium current in atrial vs epicardial ventricular myocytes of adult guinea pig hearts

The different sodium channel currents (I(Na)) were reported in myocardium, neuron, and skeletal muscles. To study whether I(Na) is homogeneous within the heart, we applied whole-cell voltage clamp technique to evaluate fast voltage-gated I(Na) in atrial and ventricular myocytes isolated from guinea pig heart. It was found that the density of inward I(Na) was 50% greater at -35 mV in atrial (-42.6+/-2.9 pA/pF) than in ventricular (-27.5+/-1.8 pA/pF, P<0.01) myocytes. The half activation and inactivation voltages (V(0.5)) of I(Na) in atrial myocytes were shifted 4.5+/-0.2 and 9.6+/-0.3 mV negative to those of ventricular myocytes. Time constants for I(Na) activation (tau(m)) and inactivation (tau(h)) were twice as rapid in atrial as in ventricular myocytes. The tau(m) and tau(h) were 0.34+/-0.03 and 1.36+/-0.07 ms for atrial myocytes, and 0.69+/-0.05 and 3.27+/-0.23 ms for ventricular myocytes, respectively. Recovery of I(Na) from inactivation was slower in atrial than in ventricular myocytes, whereas the development of resting state inactivation was more rapid in atrial (tau=67.5+/-4.3 ms) than in ventricular (152.8+/-7.5 ms, P<0.01) myocytes. The results reveal marked heterogeneity of I(Na) in the density and biophysical properties in atrial and ventricular myocytes, and the study suggests the potential possibility of tissue specific cardiac sodium channel isoforms.     

心钠素对豚鼠乳头肌动作电位和心室肌细胞钙通道电流的影响

应用标准玻璃微电极和全细胞膜片钳技术研究了心钠素（ＡＮＰ）对豚鼠乳头肌动作电位和心室肌细胞钙通道电流的影响及其对β受体兴奋的拮抗作用。结果表明：ＡＮＰ可明显缩短动作电位时程，尤其动作电位平台期缩短较为明显，２０ｎｍｏｌ／ＬＡＮＰ灌流心室乳头肌可明显抑制β受体兴奋所致动作电位平台期的延长。ＡＮＰ这种作用的机制与其抑制心室肌细胞钙通道电流有关。     

Left atrial fibrillation with regular right atrial activation and a single left-to-right electrical interatrial connection: multisite mapping of dissimilar atrial rhythms

We report two patients who had isolated atrial fibrillation in the left atrium and regular activation of the entire right atrium. Mapping of the arrhythmia was performed using a 64-electrode basket catheter that was inserted intravenously and deployed in the right and left atria. Both patients manifested a single, stable interatrial electrical connection conducting in a left-to-right direction, consistent with Bachmann's bundle location. The right and left sides of the interatrial septum were activated discordantly, each reflecting activation characteristics of the respective atria. A filtering effect at the level of interatrial septum was demonstrated by calculating the fibrillation intervals on both sides of the operative interatrial connection. It was concluded that differences in activation of the left and right surfaces of the interatrial septum and preferential use and the filtering effect of the interatrial connections play a significant role in explaining the differences in activation patterns of the left and right atria in patients with atrial fibrillation.