Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Medical Center-University of Freiburg, Freiburg, Germany.
Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2021;2191:287-307. doi: 10.1007/978-1-0716-0830-2_17.
Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.
光遗传学方法已发展成为研究心脏电生理学的有力手段,研究范围从心律失常机制的研究到心脏神经支配的影响以及在健康和患病心肌中的异细胞结构和功能相互作用。最常见的是,这些研究使用表达通道视紫红质-2(ChR2)的小鼠模型,使目标细胞群体的光激活去极化成为可能。然而,每条新生成的小鼠品系都需要进行彻底的特征描述,因为不能想当然地认为细胞类型特异性的 ChR2 表达是普遍存在的,并且应该评估其在目标细胞中的激活的电生理反应。在本章中,我们描述了使用免疫组织化学评估 ChR2 特异性的详细方案,分离特定细胞群以使用膜片钳技术分析 ChR2 激活的电生理效应,以及全心脏实验以评估光学刺激的原位效应。