Voigt Niels, Zhou Xiao-Bo, Dobrev Dobromir
Institute of Pharmacology, University of Duisburg-Essen.
J Vis Exp. 2013 Jul 3(77):e50235. doi: 10.3791/50235.
The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca(2+) handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).(1) Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca(2+) concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca(2+)-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca(2+). Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca(2+) concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca(2+) and Ca(2+) buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca(2+) transient measurements, performed in these isolated myocytes.
利用膜片钳技术研究心脏离子通道的电生理特性以及探索心脏细胞钙（Ca2+）处理异常需要分离的心肌细胞。此外，使用这些技术研究患者心肌细胞的可能性对于阐明诸如心房颤动（AF）等心脏疾病的分子基础是一项非常宝贵的需求。（1）在此，我们描述一种分离人类心房肌细胞的方法，该方法适用于膜片钳研究以及同时测量细胞内钙（Ca2+）浓度。首先，将接受心脏直视手术患者的右心耳切成小组织块（“组织块法”），并在无钙溶液中冲洗。然后将组织块在含有20 μM钙（Ca2+）的胶原酶和蛋白酶溶液中消化。此后，通过对组织悬液进行过滤和离心收获分离的心肌细胞。最后，将细胞储存溶液中的钙（Ca2+）浓度逐步调整至0.2 mM。我们简要讨论了分离过程中钙（Ca2+）和钙（Ca2+）缓冲的意义，并提供了在这些分离的心肌细胞中进行的动作电位和膜电流的代表性记录，以及同时进行的钙（Ca2+）瞬变测量。
