Apelin receptor knockdown in hESC-cardiomyocytes. Knockdown Of Apelin Receptor Reduces Cardiomyocyte Differentiation Efficiency And Contractility Of Human Embryonic Stem Cell-Derived Cardiomyocytes

Background: The apelin receptor, a G protein-coupled receptor (GPCR), has emerged as a key regulator of cardiovascular development, physiology and disease. However, there is a lack of suitable human in vitro models to investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem-cell derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease. Methods: Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterised the consequences of either apelin receptor knockdown prior to hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). Results: hESC-CMs expressed the apelin signalling system at a similar level to adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, which was associated with asynchronous contraction. Apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness. Conclusions: We have successfully knocked down, for the first time, the apelin receptor to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of a failing cardiomyocyte, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.

背景：apelin受体（apelin receptor）作为一类G蛋白偶联受体（G protein-coupled receptor, GPCR），业已被证实为心血管发育、生理稳态及疾病进程的关键调控因子。然而，当前缺乏适用于研究心血管细胞类型中apelin能系统（apelinergic system）的合适人类体外模型。本研究首次采用人类胚胎干细胞衍生心肌细胞（human embryonic stem-cell derived cardiomyocytes, hESC-CMs）与新型诱导型敲低系统，探究apelin受体在心肌细胞发育中的核心作用，并通过敲低apelin受体功能构建疾病模型，分析其功能缺失带来的一系列后果。
方法：本研究检测了hESC及hESC-CMs中apelin受体及其配体的表达水平。通过sOPTiKD系统构建了携带靶向apelin受体的四环素诱导型短发夹RNA（short hairpin RNA, shRNA）的hESC细胞系。借助表型实验，分别表征了两类敲低策略的表型效应：一是在hESC-CM分化前实施apelin受体敲低（早期敲低），二是在3D工程化心脏组织中实施敲低（晚期敲低）。
结果：hESC-CMs中apelin信号系统的表达水平与成体心脏基本一致。早期apelin受体敲低会降低心肌细胞分化效率，延长电压传感时长，该改变与心肌收缩不同步现象密切相关。Apelin受体敲低会对3D工程化心脏组织的收缩特性产生不利影响，降低收缩能力并增加组织僵硬度。
结论：本研究首次成功敲低apelin受体，证实其在hESC-CM分化过程中发挥关键调控作用。在3D工程化心脏组织中开展的apelin受体敲低实验，可重现衰竭心肌细胞的表型特征，为构建心力衰竭模型及测试新型治疗策略提供了极具潜力的实验平台。