A novel “optical dynamic clamp” method to make iPSC-CMs a more viable platform for drug screening

诱导多能干细胞来源的心肌细胞(IPSC-CMS)具有提供 基础研究和药物开发的相关平台。这些电池相对于电流的优势 用于研究药物对电活动影响的体外模型是人类的 来源，并可直接从目标患者群体派生。然而，一个主要的障碍是 使用IPSC-CMS的原因是它们具有不成熟的表型，因此很难确定是否会导致 行为反映了成年心肌细胞的行为。已经证明，一个缺失的内向整流器 钾电流Ik1可以通过“动态钳”--一种电生理装置--人工重新引入。 一种允许根据实时反馈和分析控制电输入的技术 膜电位。这种方法使IPSC-CMS在电生理上更像成年人，使 药物对动作电位形态和动力学影响的研究。不过，这个 方法繁琐，吞吐量低，并且只能在单个小区上执行。我们的目标是改进 通过将光遗传技术与光学测绘相结合来实现这一方法 IPSC-CM击打集群，打造了一种新型的用于筛选的“光学动态夹具”平台。 要点： 光学动态夹具的设计将基于与传统夹具相同的原理 动态夹具。 ARCHT是一种超极化光生质子泵，将被用来人工补偿 在IPSC-CMS的节拍簇中缺失IK1组分，并推动其电学成熟。 使用电压敏感染料的光学标测将用于提取动作电位的信息 形态和动力学以及提供必要的实时信息来控制 动态系统。 此平台并不限制我们只能使用单个细胞，而是允许使用节拍 IPSC-CMS集群。击打簇提供了更像组织的优势，因为 单个心肌细胞将与其相邻细胞电偶联，类似于活体 条件。 重要的是，该方法的光学性质(与膜片钳相比)将使高 使用IPSC-CMS在更像组织的细胞形式中进行药物筛选，允许更多 对疾病和药物作用机制的有意义的解释。

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have the potential to provide a relevant platform for basic research and drug development. The advantages of these cells over current in vitro models used to investigate the effect of a drug on electrical activity are that they are of human origin and can be derived directly from the target patient population. However, one major obstacle to using iPSC-CMs is that they possess an immature phenotype, making it difficult to determine if resulting behavior reflects adult cardiomyocyte behavior. It has been demonstrated that a missing inward rectifier potassium current, IK1, can be artificially re-introduced via “dynamic clamp”, an electrophysiological technique that allows for control of electrical input based on real-time feedback and analysis of membrane potential. This approach makes iPSC-CMs more electrophysiologically adult-like, enabling the investigation of the effect of drugs on action potential morphology and kinetics. However, this method is tedious, low throughput, and can only be performed on single cells. We aim to improve upon this approach by applying optogenetic techniques in combination with optical mapping in iPSC-CM beating clusters to create a novel “optical dynamic clamp” platform for screening. Key points:  The design of the optical dynamic clamp will be based on the same principles of traditional dynamic clamp.  ArchT, a hyperpolarizing optogenetic proton pump, will be used to artificially compensate for the missing IK1 component in the beating clusters of iPSC-CMs and push its electrical maturity.  Optical mapping with voltage sensitive dyes will be used to extract information on action potential morphology and kinetics as well as provide the necessary real-time information to control the dynamic system.  This platform does not restrict us to using only single cells, but allows for the use of beating clusters of iPSC-CMs. Beating clusters offers the advantage of being more tissue-like since individual cardiomyocytes will be electrically coupled to its neighbors, similarly to in vivo conditions. Importantly, the optical nature of the approach (in contrast to patch clamping) will enable high- throughput drug screening using iPSC-CMs in a more tissue-like format of cells, allowing for more meaningful interpretations of disease and drug mechanisms of action.