Project Summary Recent advances in stem cell science have led to accelerated progress in cardiac regeneration. Our group successfully achieved large-scale re-muscularization of the infarcted hearts of macaque monkeys by transplanting human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). These cardiomyocytes restored ejection from ~40% to ~62%, the largest restoration of cardiac function of which we are aware. This therapy is complicated by the appearance of transient ventricular arrhythmias, which last for several weeks before disappearing. Our electrophysiological studies indicate that the arrhythmias result from pacemaking activity, which in turn results from the immaturity of the hPSC-CMs at the time of transplantation. The main goal of this proposal is to enhance the maturation of hPSC-CM to make them non-arrhythmogenic, using metabolic and transcriptional reprogramming. In Aim 1 we build on our observations that modulating metabolism has wide-ranging effects on maturation, including reducing automaticity, and increasing physiological hypertrophy, force production, and more adult-like calcium cycling. The most powerful metabolic interventions, substrate switching, metabolic hormones, and energy sensing, will be systematically optimized to enhance electrical maturity in vitro. Once optimized, we will explore the underlying mechanisms, and then test whether this maturation reduces arrhythmias by transplanting them into porcine hearts. Aim 2 takes advantage of a recently generated resource, where we performed RNA-seq on a timed series of human myocardial grafts in the rat heart as they matured to adult levels in vivo. By comparing these data to immature cardiomyocytes, we identified a set of transcriptional regulators that are candidate drivers of maturation. We will perform CRISPR-based gain-of-function studies to activate these factors in vitro. Using gene expression and electrophysiology analyses, we will then identify optimal combinations to enhance maturation. If successful, these studies will solve the greatest barrier to stem cell-based heart regeneration and bring us much closer to clinical trials.