Langendoff灌流兔左心房肌细胞分离模型的建立及电生理特性分析

The Modeling of Isolating Rabbit Left Atrial Myocytes and Analysis of Electrophysiological Characteristics

目的:建立离体兔左心房肌细胞的急性分离模型并分析其电生理特性。方法:健康成年家兔10只,通过Langendoff灌流系统,经冠状动脉灌流、酶解消化法分离兔左心房肌细胞,应用膜片钳全细胞模式记录细胞膜动作电位(AP)、内向L型钙通道电流(ICaL)和快钠通道电流(INa),分析APD90和APD50时程大小及随频率的适应性变化;分析ICaL和INa的电流密度-电压(I-V)曲线特征。结果:收获的单个左心房肌细胞形态良好,成功记录到典型的AP、ICaL和INa。AP静息电位为(-62.8±2.4)mV。随着刺激频率增加,APD90和APD50均相应缩短,与基础刺激频率1Hz比较,在4Hz和5Hz时明显缩短(P<0.05)。ICaL电流密度(pA/pF)为-4.79±1.28。INa电流密度(pA/pF)为-118.41±16.67。ICaL在-40mV去极化电压下开始激活,在+10mV处电流达最大峰值,反转电位在+40～+50mV范围。INa激活电位在-60mV处,-40mV处电流达最大峰值,反转电位在+20～+30mV范围。结论:此模型分离的左心房肌细胞具有正常的电生理活性和特征,可用于研究房颤的电重构及离子通道重构现象。

Objective： To study the method of acute isolating rabbit left atrial myocytes, and analysis of the electrophysiological characters thereof. Methods： Left atrial myocytes were isolated by perfusion of coronary artery and enzymatic dissociation in 10 adult rabbits. Whole-cell patch-clamp technique was used to record action potential （AP） and ionic channel currents, L-type calcium channel current （ICaL） and the fast sodium channel current （INa）. APD90 and APD50 schedule size and their adaptive changes with the frequency were analyzed. The current density- voltage （I-V） curve characteristics of ICaL and INa was also analyzed. Results： The single rabbit left atrial myocyte with normal morphology was obtained. AP and the membrane currents of ICaL and INa were successfully recorded. The resting potential was -62.8±2.4 inV. APD90 and APD50 decreased as stimulation frequency increased. APD90 and APD50 of 4 Hz or 5 Hz were significantly decreased compared with those of l Hz of stimulation frequency （P 〈 0.05）. The density of ICaL was -4.79±1.28 pA/pF, the activated potential was -40 mV, the potential peak current was ＋ 10 mV and the reversed potential was （＋40）-（＋50） mV. The density of INa was （-118.41±16.67）pA/pF, the activated potential was -60 mV, the potential peak current was -40 mV and the reversed potential was （＋20）-（＋30） mV. Conclusion： Results suggest that there were normal electrophysiological activity and features for left atrial myocytes of dissociating model, which can be used to study atrial electrical and ionic channel remodeling of atrial fibrillation.