Butova X A, Myachina T A, Khokhlova A D
Institute of Immunology and Physiology Ural Branch of Russian Academy of science, Ural Federal University, Yekaterinburg Russia.
MethodsX. 2020 Dec 19;8:101189. doi: 10.1016/j.mex.2020.101189. eCollection 2021.
Single cardiomyocytes are widely used for investigations of the cellular and molecular mechanisms of regulation and modulation of cardiac performance. Intact cardiomyocytes allow one to study in detail cell function avoiding the effects of extracellular matrix and neighboring cells. The most established protocols of cardiomyocyte isolation are based on the isolated heart perfusion using a Langendorff-apparatus or on intraventricular perfusion using a syringe. However, the yield of single cardiomyocytes obtained by these methods may be low due to the cell injury following non-uniform enzyme digestion of connective tissue in different heart chambers. Moreover, isolation of atrial cardiomyocytes is challenging because of their small size and complex geometric shape. Here we present a new protocol for simultaneous isolation of high quality cardiomyocytes from the atria, ventricular free walls and interventricular septum. The protocol is based on the combination of the Langendorff perfusion method with the intraventricular and intra-atrial injection technique taking into account the collagen content variation between the different heart chambers. Obtained cells demonstrate rod-shaped morphology, a clear and regular sarcomere striation pattern and rat-specific frequency-dependence of contraction and calcium transient parameters. Our protocol provides gentle cell isolation that increases the yield of single cardiomyocytes suitable for biophysical researches .
单个心肌细胞被广泛用于研究心脏功能调节和调控的细胞及分子机制。完整的心肌细胞使人们能够详细研究细胞功能，避免细胞外基质和相邻细胞的影响。最成熟的心肌细胞分离方案基于使用Langendorff装置进行离体心脏灌注或使用注射器进行心室内灌注。然而，由于不同心腔结缔组织酶消化不均匀导致细胞损伤，这些方法获得的单个心肌细胞产量可能较低。此外，心房心肌细胞的分离具有挑战性，因为它们体积小且几何形状复杂。在此，我们提出一种从心房、心室游离壁和室间隔同时分离高质量心肌细胞的新方案。该方案基于Langendorff灌注法与心室内和心室内注射技术的结合，同时考虑了不同心腔之间胶原蛋白含量的差异。获得的细胞呈现杆状形态、清晰规则的肌节条纹模式以及大鼠特异性的收缩频率依赖性和钙瞬变参数。我们的方案提供了温和的细胞分离方法，提高了适合生物物理研究的单个心肌细胞的产量。
