Abstract Body (Do not enter title and authors here): Background: Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect and leading cause of infant mortality. Approximately 70% of HLHS patients develop endocardial fibroelastosis (EFE), a fibrotic thickening of the ventricular endocardium that significantly limits therapeutic options and worsens clinical outcomes. Despite its clinical importance, the cellular landscape of EFE tissue and the molecular mechanisms driving its development remain poorly defined. In this study, we aim to characterize EFE-specific cellular states and their functions associated with EFE pathogenesis.
Methods: We collected human left ventricle tissue samples from three EFE patients and three age-matched healthy controls. We then performed single-nucleus multi-omics (sn-multi-omics) sequencing and conducted integrative analysis using both public and in-house computational tools.
Results: Our sn-multi-omics analysis of EFE patient samples reveal 10 major cell types and identify 2 novel EFE-specific cellular states, which accounted for ~19.8% of cells in EFE samples. The newly identified EFE-specific cells co-express marker genes from multiple lineages, such as VWF (endothelial cells) and COL1A1 (fibroblasts), indicating a mixed or transitional identity. In addition, velocity analysis of the EFE-specific cells shows an abnormal cell-fate transition from endothelial cells. Consistently, chromatin accessibility and gene expression levels of endothelial cell marker genes are significantly reduced in EFE-specific cells compared to healthy endothelial cells in controls. Functional enrichment analysis of the high expression genes from the EFE-specific cells show an enrichment of collagen production and extracellular matrix organization, highlighting a profibrotic cellular phenotype.
Conclusions: Our analyses identify previously unrecognized cellular states in EFE patient samples. The EFE-specific cells exhibit abnormal transitions from endothelial cells with dysregulations in cell identities. The transitional states display aberrant co-expression of marker genes from multiple lineages and are functionally enriched for collagen production and extracellular matrix organization, which are hallmarks of fibrotic remodeling. Therefore, the abnormal transitional cellular states and their associated marker genes may contribute to EFE pathogenesis and represent potential targets for therapeutic interventions.