Exploring mechanisms of cardiac pacemaker cell fate determination PROJECT SUMMARY Pacemaker (PM) cells reside within the sinoatrial node (SAN), which faithfully initiates over 3 billion heartbeats during the human lifespan. PM dysfunction often necessitates device implantation to prevent circulatory collapse from bradycardia. Despite the critical importance of cardiac PM function, the mechanisms by which PM cells undergo lineage commitment remain obscure. The long-term goal of our research program is to understand the mechanistic basis for cell fate determination within the cardiac conduction system. The overall objective for this proposal is to explore molecular strategies for PM cell lineage commitment. There is an urgent need to elucidate the molecular underpinnings of PM lineage commitment to understand the fundamental biology of PM fate de- termination and to inform future development of new therapeutic strategies. My lab recently reported on key mechanisms by which Hand2 regulates PM formation using conversion of fibroblasts into induced PM myocytes (iPMs) as a model system. Building upon this preliminary data, our central hypothesis is that Hand2 interacts with AP-1 to promote subtype diversity and cooperatively binds genomic targets to orchestrate PM specification. To test our central hypothesis, we propose the following Specific Aims: 1) Define the mechanisms by which Hand2 ensures cardiac subtype diversity, 2) Explore the basis for cardiac PM lineage commitment and alterna- tive fate restriction, and 3) Boost iPM reprogramming by component annotation and combinatorial perturbation. In Aim 1, we will use our iPM reprogramming system in conjunction with genomic occupancy analysis, co-im- munoprecipitation, immunocytochemistry (ICC), single-cell RNA sequencing (scRNA-seq), and confocal micros- copy to define biochemical interactions, perturb cardiac reprogramming, and characterize the resulting cell fates. In Aim 2, we will use scRNA-seq, cell fate trajectory mapping, ICC, genomic occupancy analysis, and protein- binding microarrays (PBMs) to analyze lineage regulators, alternative fate repressors, and combinatorial inter- actions during iPM reprogramming. In Aim 3, we will systematically annotate candidate factors curated from our preliminary data and the literature. In parallel, we will build PM regulatory networks from the ground-up using novel combinatorial genomic approaches that we have recently developed. Successful completion of the pro- posed project will provide critical details regarding the establishment and maintenance of PM cell identity. This contribution will be significant because it will provide detailed insight into how cell fate is accomplished and identify potential regulators and mechanisms of PM specification. Furthermore, the proposed research is inno- vative because our unique experimental approaches and multi-disciplinary research team promise to uncover new principles in cell fate determination. Taken together, w...