Bode David, Guthof Tim, Pieske Burkert M, Heinzel Frank R, Hohendanner Felix
Department of Internal Medicine and Cardiology, Charité University Medicine; German Center for Cardiovascular Research (DZHK).
Department of Internal Medicine and Cardiology, Charité University Medicine.
J Vis Exp. 2018 Jul 26(137):57953. doi: 10.3791/57953.
In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease. We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling). We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.
在本文中，我们描述了一种基于Langendorff法的优化程序，用于从射血分数保留的代谢综合征（MetS）相关心力衰竭大鼠模型中分离单细胞心房心肌细胞（ACM）。MetS相关HFpEF的患病率正在上升，与心房重构和心房颤动相关的心房心肌病在临床上具有高度相关性，因为心房重构是死亡率的独立预测因素。对分离的单细胞心肌细胞进行的研究经常用于证实和补充体内研究结果。循环血管稀疏和间质组织纤维化是从该疾病动物模型中成功分离单细胞ACM的潜在限制因素。我们通过在分离过程中使用一种能够手动调节心腔内压力的装置解决了这个问题，从而大幅提高了形态和功能完整的ACM的产量。获得的细胞可用于各种不同的实验，如细胞培养和功能性钙成像（即兴奋-收缩偶联）。我们为研究人员提供了一份详细的操作步骤、优化溶液列表、准备必要设备的详细说明以及一份全面的故障排除指南。虽然该程序的初步实施可能相当困难，但成功应用将使读者能够在MetS相关HFpEF大鼠模型中进行最先进的ACM分离，以开展广泛的实验。
