Microphysiological Model of Human Cardiac Sympathetic Innervation

项目概要 目标：我们将开发并验证人类心脏交感神经支配的微生理学平台 人类心脏交感神经支配的体外建模并应用自主神经元规范和 它与致命的心脏病的相互作用。心脏受到自主神经系统的严重支配， 由副交感神经和交感神经组成，提供反馈控制和整体调节 心脏表现。从历史上看，针对心脏神经病的新治疗药物的开发 利用动物模型，由于稳态机制的差异，动物模型表现出各种局限性 自主神经系统的缺陷以及无法重现准确的人类疾病表型。在我们的 在拟议的工作中，我们将开发一种新型的分区 3D 微电极阵列 (MEA) 共培养平台 模拟人类交感神经支配并解决交感神经的基本问题 心脏和自主细胞的连接、相互调节和发育。此外，与 致心律失常性心肌病 (ACM) 患者来源的人诱导多能干细胞 (hiPSC)，我们 期望重现 ACM 综合征表型并检查患病的心脏交感神经支配 在我们的微生理学平台上，有助于理解神经调节以及神经元 对心脏功能和疾病的贡献。我们将利用我们团队开发的最先进的技术：(1) 高通量多模态 3D 微电极阵列，(2) 来自人类自主神经的单细胞转录组 神经元和心肌细胞在相互作用过程中发生连续的分子变化，(3) 遗传报告基因 具有等基因控制细胞的系统来定义特定的人类自主神经元群体并执行高 神经元-心脏连接的分辨率分析，（4）神经元活动的光遗传学控制 连接的心脏组织。焦点/目标：我们提出的研究重点是开发一个体外平台 研究 hiPSC 的神经心脏相互作用。我们将开发和优化分区化的 3D MEA co- 多孔培养平台，用于监测心肌细胞、交感神经细胞的电生理学特性 神经元和神经心脏连接，然后评估平台支持功能突触的能力 通过光遗传学神经元刺激形成（目标 1）。我们还将生成的发展轨迹 通过单细胞转录组分析将 hiPSC 心肌细胞连接到 hiPSC 交感神经元，如 以及神经元刺激后 hiPSC-CM 的结构和功能变化（目标 2）。此外， 我们将检查神经支配是否影响细胞命运选择（目标 2）。在目标 3 中，我们将雇用 ACM 患者- 将含有桥粒基因突变的 hiPSC/hESC 衍生到我们的微生理平台上，并 研究交感神经支配在 ACM 呈现的致病表型中的作用，这将是 体内得到验证。所提出的心脏自主神经支配体外模型可以提供广泛的应用， 包括临床前药物测试和体外疾病模型，以了解心脏自主神经的病因学 心肌病和神经病。

PROJECT SUMMARY Goal: We will develop and validate a microphysiological platform of human cardiac sympathetic innervation for in vitro modeling of the human cardiac sympathetic innervation and apply autonomic neuron specification and its interaction with a fatal cardiac disease. The heart is heavily innervated by the autonomic nervous system that consists of both parasympathetic and sympathetic nerves, providing feedback control and regulate overall cardiac performance. Historically, the development of new therapeutic agents targeting cardiac neuropathies have utilized animal models, which exhibited various limitations due to the disparity in homeostatic mechanisms of autonomic nervous systems and the inability to recapitulate accurate human disease phenotypes. In our proposed work, we will develop a novel compartmentalized 3D microelectrode array (MEA) co-culture platform to model human sympathetic innervation and address the fundamental questions on sympatho-cardiac connections, reciprocal regulation, and development of cardiac and autonomic cells. Furthermore, with arrhythmogenic cardiomyopathy (ACM) patient-derived human induced pluripotent stem cells (hiPSC), we expect to recapitulate ACM syndromic phenotypes and examine the diseased cardiac sympathetic innervation on our microphysiological platform, conducive to understanding neuromodulation as well as the neuronal contribution to heart function and disease. We will leverage state-of-art techniques developed by our team: (1) high-throughput multimodal 3D microelectrode arrays, (2) single-cell transcriptomes from human autonomic neurons and cardiac cells for a continuum of molecular changes during their interactions, (3) genetic reporter systems with isogenic control cells to define specific human autonomic neuron populations and perform high- resolution analysis of the neuron-cardiac connection, (4) the optogenetic control of neuronal activities on connected cardiac tissue. Focus/Aim: Our proposed research focuses on developing an in vitro platform to study neuro-cardiac interactions with hiPSCs. We will develop and optimize a compartmentalized 3D MEA co- culture platform in multi-well format to monitor electrophysiology properties of cardiomyocytes, sympathetic neurons and neuro-cardiac junction, followed by evaluation of the platform’s ability to support functional synapse formation with optogenetic neuronal stimulation (Aim 1). We will also generate the developmental trajectory of hiPSC-cardiomyocytes connected to hiPSC-sympathetic neurons through single cell transcriptomic analysis, as well as structural and functional changes in hiPSC-CMs following neuronal stimulations (Aim 2). Furthermore, we will examine whether the innervation affects cell fate choice (Aim 2). In Aim 3, we will employ ACM patient- derived hiPSC/hESCs harboring desmosomal gene mutations onto our microphysiological platform and investigate the role of sympathetic innervation in pathogenic phenotypes presented by ACM, which will be validated in vivo. The proposed in vitro model of cardiac autonomic innervation could provide broad applications, including preclinical drug testing and in vitro disease modeling for etiological understanding of cardiac autonomic cardiomyopathies and neuropathies.