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.
The proposed studies seek to develop an in vitro method to generate human Purkinje fibers from pluripotent stem cells and utilize single-cell sequencing of the trabecular myocardium to understand lineage segregation of Purkinje fibers and the working myocardium during ventricular development. These studies will determine the molecular mechanisms underlying Purkinje fiber differentiation and translate these to generate Purkinje fiber cells in vitro. The broader impact of this work will be the use of this pluripotent stem cell model to study inherited diseases of arrhythmias in a cell-type specific manner and a greater understanding of the molecular players that govern cell-fate choices during development of the ventricular conduction system.
