Abstract
Illuminating the precise stepwise genetic programs directing cardiac development provides insights into the mechanisms of congenital heart disease and strategies for cardiac regenerative therapies. Here, we integrate in vitro and in vivo human single-cell multi-omic studies with high-throughput functional genomic screening to reveal dynamic, cardiac-specific gene regulatory networks (GRNs) and transcriptional regulators during human cardiomyocyte development. Interrogating developmental trajectories reconstructed from single-cell data unexpectedly reveal divergent cardiomyocyte lineages with distinct gene programs based on developmental signaling pathways. High-throughput functional genomic screens identify key transcription factors from inferred GRNs that are functionally relevant for cardiomyocyte lineages derived from each pathway. Notably, we discover a critical heat shock transcription factor 1 (HSF1)-mediated cardiometabolic GRN controlling cardiac mitochondrial/metabolic function and cell survival, also observed in fetal human cardiomyocytes. Overall, these multi-modal genomic studies enable the systematic discovery and validation of coordinated GRNs and transcriptional regulators controlling the development of distinct human cardiomyocyte populations.