Project Summary Ventricular arrhythmias are a common cardiac complication that may result from inherited mutations, improper positioning of conductive cell types during development, or as a result of fibrotic scarring following a myocardial infarction. Despite the role of the His-Purkinje system in these conditions, still little is known about the developmental origins of these cells and what molecular cues drive their specification. This lack of information has hampered the generation of human pluripotent stem-cell (hPSC) based models of the ventricular conduction system (VCS) to study how these cells couple with the surrounding myocardium, and how this becomes dysregulated in disease. In Aim 1 of this proposal we seek to define a method to generate human VCS cells using hPSCs. Using a variety of physiological assays we aim to understand changes in the electrophysiological properties of these cells during differentiation towards a conductive fate, and examine the interactions of conductive cell types with hPSC-derived ventricular cardiomyocytes in various culture systems. We will also characterize the molecular changes that underlie differentiation of these cells toward a conductive fate and compare them to their fetal counterparts using RNA sequencing. In Aim 2 we will conduct single-cell RNA sequencing of progenitor cell types that give rise to the VCS and other lineages during mouse development. Using tSNE and PCA clustering algorithms we seek to profile the heterogeneity of these progenitor cell types and identify subpopulations present during differentiation of the VCS. Using temporal samples collected from various stages of ventricular development we will employ lineage trajectory algorithms to clarify lineage relationships during VCS specification. The research laid out herein uses complementary model systems to perform detailed in vitro studies of the molecular and electrophysiological properties of hPSC-derived VCS cells and combines this analysis with single-cell profiling of the developing VCS as it become specified within the native signaling environment. This work will establish a new in vitro model for future studies exploring cell-type specific contributions to arrhythmias and may identify molecular targets for gain/loss of functions studies to determine if novel regulators identified in our analysis are functionally required for VCS development.