Microphysiological Model of Human Cardiac Sympathetic Innervation

项目总结 目的：建立并验证人体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模拟和应用自主神经规范和 它与一种致命的心脏病相互作用。心脏受到自主神经系统的高度神经支配， 由副交感神经和交感神经组成，提供反馈控制和整体调节 心脏功能。从历史上看，针对心脏神经疾病的新治疗药物的开发 都使用了动物模型，由于体内平衡机制的差异，这些模型表现出了各种局限性 自主神经系统和无法概括准确的人类疾病表型。在我们的 提出的工作，我们将开发一种新型的分区三维微电极阵列(MEA)共培养平台 人体交感神经支配模型及交感心脏研究的基本问题 心脏和自主神经细胞的连接、相互调节和发育。此外，有了 致心律失常心肌病(ACM)患者来源的人诱导多能干细胞(HiPSC)，WE 期望总结ACM综合征的表型，并检查病变的心脏交感神经支配 在我们的微生理平台上，有助于了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技术：(1) 高通量多模式三维微电极阵列，(2)人类自主神经单细胞转录本 神经元和心肌细胞在相互作用过程中分子变化的连续体，(3)遗传报告 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-心脏联系的分辨率分析，(4)神经活动的光遗传控制 相连的心脏组织。焦点/目标：我们建议的研究重点是开发一个体外平台 研究神经-心脏与HiPSCs的相互作用。我们将开发和优化划分的3D MEA co- 用于监测交感心肌细胞电生理特性的多孔培养平台 神经元和神经-心脏连接，然后评估平台支持功能性突触的能力 用光发生神经元刺激形成(目标1)。我们还将生成以下发展轨迹 HiPSC-通过单细胞转录分析连接到HiPSC交感神经元的心肌细胞，AS 以及神经元刺激后HiPSC-CMS的结构和功能变化(目标2)。此外， 我们将研究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衍生的hiPSC/hESCs转移到我们的微生理平台上 研究交感神经支配在ACM致病表型中的作用 在体内进行了验证。所建立的心脏自主神经支配体外模型具有广阔的应用前景。 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.