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,...