摘要：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I_Ca-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I_k1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I_k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(I_to、I_k1）减小和慢钙通道失活时间常数延长所致。

关键词：自发性高血压大鼠;膜片钳技术;细胞动作电位;离 子通道;心肌肥厚

分类号：R541.3;R331.3;Q556;Q58;R318.03文献标识码：A

文章编号：1006-2866(2000)01-0074-03

The Ionic Mechanism of Prolongation of Action Potential in Hypertrophied Lef t Ventricular Myocytes from Spontaneously Hypertensive Rats

LI XunJIANG Wenping（Department of Cardiology, The First Affiliated Hospital, Suzhou Medi cal College, Suzhou, 215006）

ABSTRACT：Objective: To study the membrane ionic basis of action potential prolongation of hypertrophied left ventricular myocytes in spontaneously hypertensive rats(SHR).Methods: The single left ventricular myocytes of SHR and normotensiv e Wistar rat s were obtained enzymatically. The action potential was recorded using conven tional microelectrode technique and membrane ionic currents were recorded using patch-clamp whole cell recording technique. The action potential and membrane ionic currents of normal and hypertrophied left ventricular myocytes were compared .Results: (1)The heart weight to body weight ratio of SHR and Wistar rats was 5.66±0.46 mg/g and 3.7±0.29 mg/g, respectively(P＜0.001), and the mean cell membra ne capacitance, 280.68±67.98 pF and 189.94±56.59 pF, respectively(P＜0.05), suggesting that SHR had heart hypertrophy and hypertrophied ventricular myocytes;(2)The A PD₅₀ and APD₉₀ of action potential in SHR was significantly prolonge d compared w ith Wistar rats(21.33±1.56 ms vs 14.91±2.95 ms, P＜0.001; 164.6±7.4 m s vs 93.27±10.59 ms, P＜0.001), indicating that the left ventricular myocytes in SHR had delay ed repolarization;(3)The amplitude of I_ca-L of SHR(1944±466.8 pA) at +10 mV wa s significantly greater than that of Wistar rats(1136±383.3 pA)(P＜0.001), t he current dens ity of I_ca-L between them was not significant(6.93±1.71 pA/pF vs 6.1 9±2.85 pA/pF), b ut the slow inactivation time constant of SHR was significantly prolonged(56.01 ±13.36 ms vs 43.63±17.89 ms, P＜0.001);(4)The inward current density o f I_K1 in SHR at -120 mV was significantly lower than that in Wistar rats( 11.3± ±2.26 pA/pF vs 14.33 pA/pF, P＜0.05), but there was no difference in out ward current density at 0 mV(2.36±0.86 pA/pF vs 2.96±1.27 pA/pF);(5)There was no difference in cur rent den sity of I_k at +90 mV between SHR and Wistar rats(12.38±5.46 pA/pF vs 11.86±3.59 pA /pF);(6)The current density of Ito in SHR at +70 mV was significantly lower than that in Wistar rats(8.21±6.64 pA/pF vs 19.16±6.17 pA/pF, P＜0.001), bu t the activatio n and inac tivation time constants were similar. This indicated that the reduced Ito may re sult from the decrease of channel numbers.Conclusion: The action potential pr olongation of hypertrophied left ventricular myocytes in SHR may result from the reduction of outward repolarizing potassium currents(I_to, I_K1) and the prolongation of slow inactivation time co nstant of I_ca-L.

key words：spontaneously hypertensive rat;patch-clamp; cardiomyocytes potential action;ionic channel;myocardial hypertrophy▲

左室肥大是心脏对 压力和容量负荷过重产生适应的结果，在此过程中，心脏的许多生理 特性发生了改变，其中，动作电位时程延长则是所有心脏肥厚模型均有的电生理异常，但构 成动作电位时程延长的膜离子流机制则报道不一，研究也尚不充分。自发性高血压大鼠（SH R）是与人类高血压病最相似的动物模型，故本研究以正常血压Wistar大鼠作为对照，采用 酶解分离左心室肌细胞，应用膜片钳全细胞记录技术，比较SHR与Wistar大鼠左心室肌细胞 膜离子流差别，以探讨SHR动作电位时程延长的膜离子流基础。

MATERIALS AND METHODS

1 心室肌细胞分离: 选用16～20周龄雄性Wistar大鼠及SHR，用戊巴比妥钠行腹腔麻醉（35 mg/kg），称取 体重及心脏重量。单个左心室肌细胞的分离参照Tomita等的方法进行^［1］。

2膜片钳全细胞记录:记录电极由硬质玻璃毛胚（内径1.6 mm，壁厚0.2 mm）经二次拉制而成，充以电极内液 后阻抗2～4M ohm。用微电极操纵器将电极缓慢推向细胞，负压吸引形成高阻封接后补偿电 极电 容，负压吸破细胞膜，补偿掉电极电阻，形成全细胞记录模式。电压钳制脉冲和数据采集由Pclamp软件（美国Axon Instrument，5.5)控制，刺激信号经D/A转换器（美国Labmastar 1 600）和膜片钳放大器（美国Dagan 8900）在电极内形成钳制电压，离子流信号经8阶Basse l滤波器滤波（截止频率1kHz), 再经A/D转换后存入计算机。记录I_ca-L的保持电位 为-40 m V，阶跃10 mV，脉宽200 ms；I_K1的保持电位为-60 mV，阶跃10 mV，脉宽200 ms；I _K的保持电位为-30 mV，阶跃20 mV，脉宽4500 ms；I_to的保持电位为-60 mV，阶跃10 mV，脉宽300 ms 。每一 钳制电压下的电流强度绘制成电流电压曲线，通道激活和失活过程采用Pclamp软件以单指数 或双指数曲线拟合。

3 细胞膜电容：在形成全细胞记录后，从保持电位-60 mV，给予-10 mV的超极化阶跃脉冲 ，按以下公式计算膜电容，Cm=τ_cI₀/ΔV_m（1-I/I₀）。其中，Cm：膜电容，τ_c ：膜 电容时间常数，I0：最大膜电容电流值，ΔV_m：阶跃电压值，I：稳态膜电容电流值。

4 动作电位记录：取大鼠左心室肌，按标准微电极技术记录心室肌细胞动作电位。玻璃微 电极阻抗为10～30 Mohm（充以3 mol/L KCl），刺激频率取1 Hz。测量静息膜电位（RP，m V），动作电位振幅（APA，mv）和动作电位时程（APD₅₀、APD₉₀，ms）。

5 各种细胞营养液及测电极液体组成（单位均为mmol/L）

5.1 台氏液：NaCl 140,KCl 4,CaCl₂ 1.8, MgCl₂ 0.5, NaH₂PO₄ 0.33, HEPES 5,Glucose 5.5,pH 用NaOH校至7.4。

5.2 KB液:KOH 70,KCl 40,KH₂PO₄ 20, Glutamic acid 50,MgCl₂ 3,Taurine 20, EGTA 0.5, HEPES 10, Glucose 10, pH用KOH校至7.4

5.3 I_ca-L: 电极内液：CsCl 120,MgCl₂ 2,CaCl₂ 1,Na₂ATP 5,EGTA 10,HEP ES 10,pH用CsOH校至7.4。电极外液：Choline-Cl 120, CaCl₂ 2, MgCl₂ 2,CsCl 4,HEPES 10, Glucose 10,pH用CsOH校至7.4。

5.4 内向整流性钾流（I_K1）：电极内液：KCl 140,