Plačkić Jelena, Kockskämper Jens
Faculty of Pharmacy, Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Center (BPC) Marburg, University of Marburg, Karl-von-Frisch-Str. 1, 35032, Marburg, Germany.
Methods Mol Biol. 2018;1816:39-54. doi: 10.1007/978-1-4939-8597-5_3.
High quality cardiomyocyte isolation is of critical importance for successful studies of myocardial function at the cellular and molecular level. Although previous work has established isolation procedures for various species, it still remains challenging to produce consistently a high yield of viable and healthy cardiomyocytes. The basis for the most successful and reproducible isolation of cardiomyocytes from intact hearts is the Langendorff retrograde perfusion technique. Here, we will illustrate in detail all practical aspects of the enzyme-based Langendorff isolation of rat atrial and ventricular cardiomyocytes. This includes a series of obligatory steps starting from quick aortic cannulation to rinse the heart from blood, short perfusion of the heart with Ca-free solution to dissociate cells at the level of intercalated discs, followed by longer perfusion with low Ca-containing enzyme solution in order to disrupt the extracellular matrix network, extraction of the released cardiomyocytes and gentle Ca reintroduction to allow cells to return gradually to normal cytosolic Ca levels. The average yield of intact viable ventricular myocytes that can be achieved with our protocol is ≈70% (range ≈50-90%). For atrial myocytes, in general, it is slightly (≈10%) lower than for ventricular myocytes. The yield depends on the age of the rat and the degree of cardiac remodeling such that digestion of older and more remodeled hearts (more fibrosis) usually results in lower yields. Isolated atrial and ventricular cardiomyocytes may be employed for studies of cardiomyocyte function (e.g., shortening/contraction, intracellular [Ca] transients) as well as for biochemical and molecular biological studies (e.g., immunoblotting, PCR).
高质量的心肌细胞分离对于在细胞和分子水平成功开展心肌功能研究至关重要。尽管先前的工作已经建立了针对各种物种的分离程序，但始终如一地获得高产率的存活且健康的心肌细胞仍然具有挑战性。从完整心脏中最成功且可重复地分离心肌细胞的基础是Langendorff逆行灌注技术。在此，我们将详细阐述基于酶法的大鼠心房和心室心肌细胞Langendorff分离的所有实际操作环节。这包括一系列必要步骤，从快速主动脉插管以冲洗心脏中的血液开始，用无钙溶液对心脏进行短暂灌注以在闰盘水平解离细胞，随后用含低钙的酶溶液进行更长时间的灌注以破坏细胞外基质网络，提取释放的心肌细胞并缓慢重新引入钙以使细胞逐渐恢复到正常的胞质钙水平。按照我们的方案能够实现的完整存活心室肌细胞的平均产量约为70%（范围约为50 - 90%）。对于心房肌细胞，一般而言，其产量比心室肌细胞略低（约10%）。产量取决于大鼠的年龄和心脏重塑程度，以至于对年龄较大且重塑程度更高（纤维化更严重）的心脏进行消化通常会导致产量降低。分离出的心房和心室心肌细胞可用于心肌细胞功能研究（例如，缩短/收缩、细胞内[Ca]瞬变）以及生化和分子生物学研究（例如，免疫印迹、PCR）。
