Köhncke Clemens, Lisewski Ulrike, Schleußner Leonhard, Gaertner Carolin, Reichert Saskia, Roepke Torsten K
Experimental and Clinical Research Center, Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine.
J Vis Exp. 2013 Mar 12(73):e50145. doi: 10.3791/50145.
KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca(2+) in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca(2+) buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.
KCNE基因编码一个小家族的钾通道辅助亚基，这些亚基与钾通道α亚基形成异源复合物，以改变其功能特性。在患有心律失常（如长QT综合征和/或心房颤动）的患者中发现了KCNE基因突变。然而，导致这些疾病的确切分子病理生理学仍然不清楚。在先前的研究中，这些基因中导致疾病的突变的电生理特性大多是在异源表达系统中进行研究的，我们无法确定所报道的效应是否能直接转化为天然心肌细胞中的效应。因此，在我们实验室中，我们采用了一种不同的方法。我们通过细胞电生理学直接研究基因敲除小鼠分离心肌细胞中KCNE基因缺失的效应——这是一种我们在本期《可视化实验杂志》中描述的独特技术。通过主动脉插管将基因工程改造的KCNE小鼠的心脏迅速切除并安装在Langendorff装置上。心肌中的游离Ca(2+)被乙二醇双四乙酸（EGTA）结合，然后通过用含有胶原酶的特殊低Ca(2+)缓冲液逆行灌注冠状动脉来实现心肌细胞的解离。然后可以通过显微外科技术分离心房、游离右心室壁和左心室。然后在包括洗涤程序的多步骤中，将钙缓慢添加回分离的心肌细胞中。在分离后的前6小时内，立即对外观健康且无自发收缩的心房和心室心肌细胞进行膜片钳技术的电生理分析或其他生化分析。
