Microphysiological Model of Human Cardiac Sympathetic Innervation

项目摘要 目标：我们将开发和验证人类心脏交感神经支配的微生理平台， 人心脏交感神经支配的体外建模和应用自主神经元特化， 与致命心脏病的相互作用。心脏主要受自主神经系统支配， 包括副交感神经和交感神经，提供反馈控制和调节整体 心脏性能从历史上看，针对心脏神经病变的新治疗剂的开发 已经利用了动物模型，其由于体内平衡机制的差异而表现出各种限制 自主神经系统和无法概括准确的人类疾病表型。在我们 建议的工作，我们将开发一种新的分区三维微电极阵列（MEA）共培养平台 建立人类交感神经支配的模型，解决交感心神经系统的基本问题， 连接、相互调节以及心脏和自主神经细胞的发育。再加上 致瘤性心肌病（ACM）患者来源的人诱导多能干细胞（hiPSC），我们 期望概括ACM综合征的表型，并检查病变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） 高通量多模式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其相互作用过程中的连续分子变化，（3）遗传报告基因 具有同基因控制细胞的系统，以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， 相连的心脏组织焦点/目的：我们提出的研究重点是开发一个体外平台， 研究hiPSC与神经心脏的相互作用。我们将开发和优化一个分区的3D MEA共同- 多孔形式的培养平台，以监测心肌细胞、交感神经细胞、 神经元和神经-心脏接头，然后评估平台支持功能性突触的能力 用光遗传学神经元刺激形成（Aim 1）。我们还将生成 通过单细胞转录组学分析，hiPSC-心肌细胞与hiPSC-交感神经元连接，如 以及神经元刺激后hiPSC-CM的结构和功能变化（目的2）。此外，委员会认为， 我们将检验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- 将携带桥粒基因突变的hiPSC/hESC衍生到我们的微生理学平台上， 研究交感神经支配在ACM呈现的致病表型中的作用，这将是 在体内验证。所提出的心脏自主神经支配的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建模，以了解心脏自主神经病变的病因学。 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.