Arrhythmogenic cardiomyopathy (ACM) is a leading cause of sudden cardiac death among young adults. Aberrant gap junction remodeling has been linked to disease-causative mutations in plakophilin-2 ( PKP2 ). Although gap junctions are a key therapeutic target, measurement of gap junction function in preclinical disease models is technically challenging. To quantify gap junction function with high precision and high consistency, we developed a robotic cell manipulation system with visual feedback from digital holographic microscopy for three-dimensional and label-free imaging of human induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs). The robotic system can accurately determine the dynamic height changes in the cells’ contraction and resting phases, microinject drug-treated healthy and diseased iPSC-CMs in their resting phase with constant injection depth across all cells, and deposit a membrane-impermeable dye that solely diffuses between cells through gap junctions for measuring the gap junction diffusion function. The robotic system was applied toward a targeted drug screen to identify gap junction modulators and potential therapeutics for ACM. Five compounds were found to dose-dependently enhance gap junction permeability in cardiomyocytes with PKP2 knockdown. In addition, PCO 400 (pinacidil) reduced beating irregularity in a mouse model of ACM expressing mutant PKP2 (R735X). These results highlight the utility of the robotic cell manipulation system to efficiently assess gap junction function in a relevant preclinical disease model, thus providing a technique to advance drug discovery for ACM and other gap junction–mediated diseases.

中文翻译：

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机器人操作心肌细胞以识别致心律失常性心肌病的间隙连接修饰剂


致心律失常性心肌病 （ACM） 是年轻人心源性猝死的主要原因。异常的间隙连接重塑与 plakophilin-2 （PKP2） 的疾病致病突变有关。尽管间隙连接是一个关键的治疗靶点，但在临床前疾病模型中测量间隙连接功能在技术上具有挑战性。为了以高精度和高一致性量化间隙连接功能，我们开发了一种机器人细胞操作系统，该系统具有来自数字全息显微镜的视觉反馈，用于人类诱导多能干细胞衍生的心肌细胞 （iPSC-CM） 的三维和无标记成像。机器人系统可以准确测定细胞收缩和静止期的动态高度变化，在所有细胞中以恒定的注射深度显微注射处于静止期的药物处理的健康和患病 iPSC-CM，并沉积一种膜不透性染料，该染料仅通过间隙连接在细胞之间扩散，用于测量间隙连接扩散功能。机器人系统应用于靶向药物筛选，以确定间隙连接调节剂和 ACM 的潜在治疗方法。发现 5 种化合物剂量依赖性地增强 PKP2 敲除心肌细胞的间隙连接通透性。此外，PCO 400 （pinacidil） 减少了表达突变型 PKP2 （R735X） 的 ACM 小鼠模型的跳动不规则性。这些结果突出了机器人细胞操作系统在相关临床前疾病模型中有效评估间隙连接功能的实用性，从而提供了一种推进 ACM 和其他间隙连接介导的疾病的药物发现的技术。