Baumgartner Sven, Halbach Marcel, Krausgrill Benjamin, Maass Martina, Srinivasan Sureshkumar Perumal, Sahito Raja Ghazanfar Ali, Peinkofer Gabriel, Nguemo Filomain, Müller-Ehmsen Jochen, Hescheler Jürgen
Department of Internal Medicine III-Cardiology, University of Cologne, Cologne, Germany Institute of Neurophysiology, University of Cologne, Cologne, Germany.
Department of Internal Medicine III-Cardiology, University of Cologne, Cologne, Germany.
J Cardiovasc Pharmacol Ther. 2015 Jan;20(1):104-12. doi: 10.1177/1074248414536273. Epub 2014 Jun 9.
The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.
本研究的目的是调查持续电刺激是否会影响培养的胎儿心肌细胞（FCM）的电生理特性和细胞形态。在交配后第14.5天从鼠心脏中获取胎儿心肌细胞，并使用定制的刺激室在培养中进行6天的电刺激。随后，用玻璃微电极记录FCM的动作电位。进行了α-辅肌动蛋白、连接蛋白43和纽蛋白的免疫染色。通过定量逆转录聚合酶链反应分析离子通道亚基Kcnd2、Slc8a1、Cacna1、Kcnh2和Kcnb1的表达。与未刺激的对照FCM相比，电刺激的FCM的动作电位持续时间至50%和90%复极化（APD50和APD90）显著降低。与对照FCM相比，刺激的FCM中细胞排列明显更高。与对照FCM相比，刺激的FCM中连接蛋白4,3的表达显著增加。刺激的FCM的细胞长度与细胞宽度之比显著高于对照FCM。与对照FCM相比，刺激的FCM中Kcnh2和Kcnd2上调。刺激的和对照的FCM中Slc8a1、Cacna1c和Kcnb1的表达没有差异。体外电刺激FCM后观察到的APD50降低与体内FCM的电生理成熟相对应。离子通道的表达水平表明，电刺激促进了电生理成熟复杂过程的一些重要但并非所有方面。平行排列、连接蛋白43表达增加和FCM伸长是电刺激诱导的形态成熟的标志。
