Abstract 4121148: A functional survey of postnatal heart maturation with <i>in vivo</i> Perturb-seq in spatial and temporal resolution

Background: Cardiomyocyte maturation is a crucial process that involves intricate molecular and structural changes necessary for more vigorous and efficient heart function after birth. Understanding the regulatory mechanisms of this process is essential for leveraging these insights to develop advanced therapies for heart disease. Despite recent advancements, the limitations of current in vivo genetic and single-cell approaches - such as the low throughput of in vivo genetics, lack of spatial information in single-cell RNA-seq, and, most importantly, the lack of high-throughput combined use of both approaches - hinder the construction of a comprehensive regulatory network of cardiomyocyte maturation. Methods: Here, we established an experimental pipeline combining time-course single-nucleus RNA sequencing and spatial transcriptomics with our optimized in vivo Perturb-seq platform to establish the regulatory network underlying heart maturation. Results: Our current study has generated a postnatal murine heart atlas with 67,000 nuclei from postnatal heart. Temporal analysis of the cardiomyocyte transcriptome dynamics identifies Ankrd1+ cardiomyocyte as a transient state between proliferative and matured cardiomyocytes. We constructed a regulatory network consisting of 35 known and novel regulators for cardiomyocyte maturation. Our analysis of postnatal cardiomyocyte interactome elucidated signaling pathways’ stage-specific contribution to cardiomyocyte maturation. With the spatial transcriptomic data, we further identified signaling pathways that played unique roles in the maturation of chamber-specific cardiomyocytes. Using fixed RNA in vivo Perturb-seq established in this study, we have validated 21 novel regulators of postnatal cardiomyocyte maturation out of 53 candidate regulators from previous temporal and spatial analyses. Conclusions: We have not only created a single-cell-resolution temporal and spatial atlas during postnatal heart development but also established an in vivo Perturb-seq platform to allow the functional interrogation of key regulatory genes in a physiologically relevant context. Importantly, the proposed working model unlocks new possibilities for research, no longer limiting to a single gene or pathway, but allowing for the exploration of a network of genes. This high-resolution, high-throughput in vivo mapping and screening platform has the potential to revolutionize the way we study organogenesis and disease progression in the heart.