Abstract Or203: Charting Postnatal Heart Development Using In Vivo Single-Cell Functional Genomics

Background: The transition at birth, marked by increased circulatory demands and rapid growth, necessitates extensive remodeling of the heart’s structure, function, and metabolism. Despite substantial advancements, the regulatory mechanisms driving these changes remain poorly understood because the study of postnatal cardiac development has predominantly relied on single-cell RNA-seq that cannot fully capture the intricate cell neighborhood regulating the postnatal maturation. Method: To test this hypothesis, we performed parallel imaging-based single-cell spatial transcriptomics and single-nucleus RNA sequencing on mouse hearts at postnatal Day 0, 7, 14, and 21 (n = 2 per time point). This strategy enabled us to capture the shift from hyperplastic growth to hypertrophic maturation. Moreover, we developed an in vivo Probe-based Indel-detectable Perturb-seq (PIP-seq) platform that utilizes probe hybridization to simultaneously detect sgRNA and transcriptomic profiles from fixed single nuclei. This integrated approach allowed us to assess perturbation states alongside their transcriptional consequences directly within the native tissue environment. Results: Here, we constructed a temporal and spatial atlas of postnatal hearts by integrating temporal single-nucleus transcriptomic profiling with image-based spatial transcriptomics. The integrative spatial analyses of postnatal heart atlas revealed the coordinated effort of the cardiac cells in shaping the postnatal heart, including how capillary endothelial cells interact with cardiomyocyte through Ptprm to drive the cell cycle exit through Meis1 in the postnatal heart. Our analysis of the localized intrinsic and extrinsic regulatory mechanisms underlying postnatal cardiomyocyte maturation identified 53 candidate regulators of cardiomyocyte maturation, with 21 candidates validated by PIP-seq for their significant impact on maturation-specific transcriptional programs. Furthermore, targeted knockout of two candidates, Rreb1 and Ncl , led to reduced cardiomyocyte size and impaired heart function, directly linking transcriptional alterations to phenotypic outcomes. Conclusion: In conclusion, our study demonstrates that cell–cell communication and localized regulatory networks are pivotal in postnatal cardiac maturation. The novel PIP-seq platform extends our ability to comprehensively explore gene function and regulatory networks in vivo, offering new insights into the mechanisms governing heart maturation.