Single-Cell RNA-Seq Reveals Endocardial Defect in Hypoplastic Left Heart Syndrome

Abstract

Hypoplastic left heart syndrome (HLHS) is one of the most challenging forms of congenital heart diseases. Previous studies were mainly focused on intrinsic defects in myocardium. However, this does not sufficiently explain the abnormal development of the cardiac valve, septum, and vasculature, known to originate from the endocardium. Here, using single-cell RNA profiling, induced pluripotent stem cells, and human fetal heart tissue with an underdeveloped left ventricle, we identified a developmentally impaired endocardial population in HLHS. The intrinsic endocardial deficits contributed to abnormal endothelial to mesenchymal transition, NOTCH signaling, and extracellular matrix organization, all of which are key factors in valve formation.Consequently, endocardial abnormalities conferred reduced proliferation and maturation of cardiomyocytes through a disrupted fibronectin-integrin interaction. Several recently described HLHS de novo mutations were associated with abnormal endocardial gene and FN1 regulation and expression. Our studies provide a rationale for considering endocardial function in future regenerative strategies for HLHS.Page 5 together, our findings have uncovered a mechanism whereby a developmentally impaired endocardium can underlie the ventricular and valvular hypoplasia in HLHS. Results HLHS patient de novo mutations were highly enriched in the endocardial and endothelial populations in a developing human heart.It is well appreciated that the genetics of HLHS is complex. Previously, a set of congenital heart disease (CHD) related de novo mutations (DNMs) were reported (Jin et al., 2017), some of which were strongly associated with HLHS. To further understand the role of these HLHS-associated DNMs in the development of human heart, we first examined their expression level at single cell resolution. A normal human fetal heart at a gestational age of 83 days was micro-dissected into the left-and right-side of the heart. Each side was enzymatically dispersed as single cells, enriched with endocardial/endothelial cells using a CD144 antibody and processed for single-cell RNA-sequencing (scRNA-seq) ( Figure 1A). The majority of the cell types expected to be present in the heart were successfully retrieved by scRNA-seq analysis as shown in the UMAP projection, including a certain amount of CMs (TNNI3 + ) in addition to pan-ECs (CDH5 +