Tomsits Philipp, Schüttler Dominik, Kääb Stefan, Clauss Sebastian, Voigt Niels
Department of Medicine I, University Hospital Munich, Campus Großhadern, Ludwig-Maximilians University Munich (LMU); Partner Site Munich, Munich Heart Alliance (MHA), DZHK (German Centre for Cardiovascular Research); Walter Brendel Center of Experimental Medicine, Ludwig-Maximilians University Munich (LMU);
Department of Medicine I, University Hospital Munich, Campus Großhadern, Ludwig-Maximilians University Munich (LMU); Partner Site Munich, Munich Heart Alliance (MHA), DZHK (German Centre for Cardiovascular Research); Walter Brendel Center of Experimental Medicine, Ludwig-Maximilians University Munich (LMU).
J Vis Exp. 2020 Nov 3(165). doi: 10.3791/61964.
Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 °C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 °C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.
小鼠模型在心律失常研究中发挥着关键作用，能够用于研究心律失常发生的关键机制，包括离子通道功能改变和钙处理异常。为此，需要高质量的心房或心室心肌细胞来进行膜片钳测量或探索钙处理异常情况。然而，目前的分离方案所获得的高质量心肌细胞产量有限，无法在同一只小鼠身上同时进行这两种测量。本文描述了一种通过逆行酶介导的Langendorff灌注法分离高质量小鼠心房和心室肌细胞的方法，以便随后对同一只动物同时进行钙瞬变和L型钙电流的测量。获取小鼠心脏，迅速将主动脉插管以排出血液。然后，心脏首先用无钙溶液（37℃）灌注，以在闰盘水平解离组织，之后用含钙量少的酶溶液灌注以破坏细胞外基质（37℃）。随后将消化后的心脏解剖成心房和心室。将组织样本切成小块，通过小心地上下吹打使其溶解。停止酶消化，将细胞逐步重新引入生理钙浓度环境。在用荧光钙指示剂加载后，制备分离的心肌细胞以同时测量钙电流和钙瞬变。此外，还讨论了分离过程中的陷阱，并提供了膜片钳方案以及按照上述方法分离的小鼠心房和心室肌细胞同时进行钙瞬变测量时L型钙电流的代表性记录。
