Abstract Body: Congenital heart defects affect nearly 1 in 100 live births and represent the most common birth defect worldwide. Among the most severe forms is Single Ventricle Disease, a condition where one ventricular chamber fails to develop, leaving a single ventricle to support the entire circulation. The left and right ventricles arise from distinct developmental lineages, the first and second heart fields, and acquire unique molecular identities early in embryogenesis through chamber-specific transcriptional programs driven by transcription factors such as TBX5, HAND1, and HAND2. Disruption of these programs is thought to contribute to severe ventricular malformations, yet the mechanisms that establish and maintain ventricular chamber identity in the human heart remain poorly understood. Progress has been limited by reliance on animal models, particularly mouse, which differ from humans in cardiac cellular composition, electrophysiology, metabolism, and developmental timing, leaving critical aspects of human ventricular development unresolved.
To address this gap, we established a multimodal workflow combining human fetal heart tissue collection, ex vivo MRI, 3D morphological reconstruction, and VisiumHD spatial transcriptomics to map ventricular development across gestation (8–20 weeks). We have profiled multiple developmental stages in the short-axis orientation, enabling temporally resolved analysis of how ventricular structure and molecular identity are refined during growth and maturation. Already at 8 weeks, spatial transcriptomics reveals spatially restricted left–right ventricular signatures. These include both known cardiac regulators and novel candidate genes, highlighting previously unrecognized components of human ventricular identity. Importantly, chamber-specific programs are not uniformly distributed across cell types: clear left–right distinctions are observed in cardiomyocytes, fibroblasts, and endocardial cells, but not in endothelial or epicardial cells. This indicates that ventricular identity is established in a cell-type-specific manner early in development. Several components of this program are not conserved in mouse datasets, suggesting human-specific mechanisms governing ventricular specification. In Single Ventricle Disease, using human tissue, we are investigating whether disruption of these early programs underlies failure to establish distinct ventricular identities, linking structure and function in severe congenital heart defects.