小鼠不同电特性的心肌细胞Kir2.1、SCN5a和SCN1b通道基因的表达:膜片钳结合单细胞RT-PCR技术研究

Expression of Kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with various electric properties: patch clamp combined with single cell RT-PCR study

本文旨在应用膜片钳技术结合单细胞逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)技术研究小鼠胚胎心肌细胞的不同电生理学特点的分子生物学基础。应用胶原酶B消化分离得到小鼠胚胎早期(E10.5)单个心肌细胞,先用膜片钳技术记录单个胚胎心肌细胞的动作电位,随即应用玻璃微电极吸取该细胞内容物,进一步应用RT-PCR技术检测该单个心肌细胞离子通道基因Kir2.1、SCN5a和SCN1b的表达,以GAPDH为内参基因。全细胞电流钳技术记录到心室肌样细胞、心房肌样细胞和起搏样细胞的动作电位。心室肌样细胞和心房肌样细胞动作电位的最大舒张期电位(maximum diastolic potential,MDP)较起搏样细胞更负,0期最大除极速度(maximum velocity of depolarization,Vmax)更快;心室肌样细胞动作电位的MDP相对心房肌样细胞更负。相对应的,心室肌样细胞的Kir2.1、SCN5a、SCN1b三种基因表达均较强;心房肌样细胞的SCN5a表达较强,而SCN1b和Kir2.1相对心室肌样细胞表达稍弱;而起搏样细胞的上述三种基因表达均很弱。不同类型心肌细胞动作电位的基本特点与其基因表达密切相关,应用膜片钳技术结合单细胞RT-PCR技术可以敏锐检测到特定电生理特点的分子生物学基础。

This study is to explore a new method of investigating molecular basis for electrophysiological properties of early fetal cardiomyocytes. Single embryonic cardiomyocytes of mouse early developmental heart （E10.5） were obtained by a collagenase B digestion approach. After recording spontaneous action potential using whole cell patch clamp technique, the single cell was picked by a glass micropipette, followed by a standard RT-PCR to explore the expression levels of several ion channel genes. Three phenotypes of cardiomyocytes were demonstrated with distinct properties： ventricular-like, atrial-like, and pacemaker-like action potentials. Ventricular-like and atrial-like cells were characterized with much negative maximum diastolic potential （MDP） and a higher Vmax （maximum velocity of depolarization） compared to pacemaker-like cells. MDP of ventricular-like cells was the most negative. In parallel, stronger expression of SCN5a, SCN1b and Kir2.1 were observed in ventricular-like and atrial-like cells compared to that of pacemaker-like cells, where Kir2.1 in ventricular-like cells was the most abundant. Cardiomyocytes with distinct electrophysiological properties had distinct gene expression pattern. Single cell RT-PCR combined with patch clamp technique could serve as a precise detector to analyze the molecular basis of the special electrophysiological characteristics of cardiomyocytes.