Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes.
Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes.
复制标题
亚型特异性启动子驱动的动作电位成像，用于 hiPSC 衍生心肌细胞中的精确疾病建模和药物测试。
DOI：
10.1093/eurheartj/ehw189
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发表时间：
2017-01-21
影响因子：
39.3
通讯作者：
Sinnecker D
中科院分区：
文献类型：
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作者：
Chen Z;Xian W;Bellin M;Dorn T;Tian Q;Goedel A;Dreizehnter L;Schneider CM;Ward-van Oostwaard D;Ng JK;Hinkel R;Pane LS;Mummery CL;Lipp P;Moretti A;Laugwitz KL;Sinnecker D
由人诱导多能干细胞产生的心肌细胞是了解疾病分子机制和评价心血管药物的一个不断发展的平台。该系统的一个主要局限性是它们代表了心室、心房和心室样CM的异质混合。通过表达一个电压敏感的荧光蛋白的控制下的谱系特异性启动子，我们开发了一个方便的系统，允许高通量亚型特异性光学动作电位（AP）在这些细胞中成像。这不仅可以量化患者特异性CM中的电表型，还可以对药物作用进行亚型特异性研究，这可能有助于心血管领域的药物开发和安全药理学。由人诱导多能干细胞（hiPSC）产生的心肌细胞（CM）越来越多地用于疾病建模和药物评价。然而，它们通常是基于动作电位（AP）和基因表达的心室样、心房样和髓样细胞的异质混合物。这种异质性和高通量功能表型分析方法的缺乏阻碍了其潜力的充分开发。我们的目的是开发一种方法，用于快速，连续和亚型特异性表型的hiPSC-CM的AP形态和单细胞心律失常。我们使用心脏谱系特异性启动子来驱动hiPSC-CM中电压敏感性荧光蛋白（VSFP-CR）的表达，从而实现亚型特异性光学AP记录。在1型长QT综合征的患者特异性hiPSC模型中，与同基因对照相比，突变型心室和心房样hiPSC-CM中AP延长和频繁的早期后除极明显，但在心室样hiPSC-CM中不明显，这与致病基因的选择性表达一致。此外，我们证明了连续几天探测细胞的可行性，以研究疾病表型的遗传拯救和辨别CM亚型特异性药物作用。通过将基因编码的膜电压传感器与驱动其在hiPSC-CM的主要亚型中表达的启动子相结合，我们开发了一种用于相关细胞类型中的疾病建模和药物评价的方便系统，这有可能推动hiPSC在心血管医学中的新兴用途。
Cardiomyocytes (CMs) generated from human induced pluripotent stem cells are an evolving platform to understand molecular disease mechanism and evaluate cardiovascular drugs. A major limitation of this system is that they represent a heterogeneous mix of ventricular-, atrial-, and nodal-like CMs. By expressing a voltage-sensitive fluorescent protein under the control of lineage-specific promoters, we developed a convenient system allowing high-throughput subtype-specific optical action potential (AP) imaging in these cells. This enables not only quantification of electrical phenotypes in patient-specific CMs but also subtype-specific investigation of drug effects, which may aid both drug development and safety pharmacology in the cardiovascular field. Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential. We aimed at developing a method for rapid, sequential, and subtype-specific phenotyping of hiPSC-CMs with respect to AP morphology and single-cell arrhythmias. We used cardiac lineage-specific promoters to drive the expression of a voltage-sensitive fluorescent protein (VSFP-CR) in hiPSC-CMs, enabling subtype-specific optical AP recordings. In a patient-specific hiPSC model of long-QT syndrome type 1, AP prolongation and frequent early afterdepolarizations were evident in mutant ventricular- and atrial like, but not in nodal-like hiPSC-CMs compared with their isogenic controls, consistent with the selective expression of the disease-causing gene. Furthermore, we demonstrate the feasibility of sequentially probing a cell over several days to investigate genetic rescue of the disease phenotype and to discern CM subtype-specific drug effects. By combining a genetically encoded membrane voltage sensor with promoters that drive its expression in the major subtypes of hiPSC-CMs, we developed a convenient system for disease modelling and drug evaluation in the relevant cell type, which has the potential to advance the emerging utility of hiPSCs in cardiovascular medicine.