Tian, Lei

Introduction: Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdeveloped left ventricle. Early serial postmortem examinations revealed a high rate of coronary artery abnormalities in HLHS fetal hearts (e.g., thickened wall and kinking arteries). However, the intrinsic defect in HLHS coronary vessels and its genetic basis remain unclear. Methods: We profiled human fetal heart with an underdeveloped left ventricle (ULV) and induced pluripotent stem cells derived endothelial cells (iPSC-ECs) from HLHS patients at single-cell resolution. CD144 + / NPR3 - vascular ECs were selected and classified as venous, arterial, and late arterial populations. To study the arterial EC phenotypes, we generated iPSC-arterial ECs (AECs, CDH5 + CXCR4 + NT5E -/low ) derived from 3 HLHS patients and 3 age-matched controls, and evaluated their functionalities including cell cycle regulation, angiogenesis, and inflammatory response. Results: Revealed by single cell RNA-seq and subsequent gene ontology analysis, ULV late arterial EC population showed significant defects in EC development, proliferation, angiogenesis, and Notch signaling compared to the control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation judged by the reduced CXCR4 + NT5E -/low AEC progenitors. Mature HLHS iPSC-AECs showed reduced angiogenesis and enhanced G0/G1 cell cycle arrest with downregulated cell cycle-related genes (e.g., Ki67, CCND1/2 ). Healthy human aortic smooth muscle cells exhibited abnormal proliferation and synthetic phenotypes when co-cultured with HLHS iPSC-AECs. Additionally, NOTCH pathway genes (e.g., DLL4, HEY1, GJA5 ) were suppressed in both ULV AECs and HLHS iPSC-AECs. HLHS de novo variant KMT2D directly regulated the transcription of NOTCH targeted genes involved in arterial development and proliferation via H3K4me2. Intriguingly, the treatment of NOTCH ligands (Jag1, Dll1) significantly improved the proliferation of HLHS AECs. Conclusions: Our study revealed that HLHS coronary AECs were dysfunctional in angiogenesis, proliferation, and EC-SMC interaction. KMT2D-NOTCH signaling may contribute to the impaired development and proliferation of HLHS AECs.

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 transcriptomic profiling, induced pluripotent stem cells (iPSC) derived endocardial cells (iEECs), human fetal heart tissue with underdeveloped left ventricle, as well as a Xenopus model, 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, in an endocardium-myocardium co-culture system, we found that endocardial abnormalities conferred reduced proliferation and maturation of iPSC derived cardiomyocyte (iPSC-CMs) judged by Ki67 staining, contractility, sarcomere organization, and related gene expressions through a disrupted fibronectin (FN1)-integrin interaction. Several recently described HLHS de novo mutations such as ETS1 and CHD7 showed reduced binding to FN1 promoter and enhancer in HLHS vs. control iEECs based on ChIP-qPCR analysis. Additionally, we found that suppression of the ETS1 in Xenopus caused reduced endocardial FN1 expression and impaired heart development. Supplementation of FN1 or ETS1 over-expression in HLHS iEECs could rescue dysfunctions in both endocardium and myocardium in HLHS. Our studies reveal a critical role of endocardial abnormality in causing HLHS, and provide a rationale for improving endocardial function in future regenerative strategies. Schematic illustration of the endocardial and myocardial defects in HLHS.

Hypoplastic left heart syndrome (HLHS) is a single ventricle congenital heart disease that results in severe underdevelopment of the left ventricle, mitral valve, aortic valve, and ascending aorta. Early serial postmortem examinations also revealed a high rate of coronary anomalies in HLHS, which included multiple ventriculo-coronary arterial connections as well as thick-walled and kinked coronary arteries. A previous study showed that fetal hypoplastic left hearts had a reduced endothelial cell (EC) population and lower capillary density compared with normal hearts. However, the mechanism underlying coronary abnormalities associated with HLHS remains unknown. Thus, we generated induced pluripotent stem cells derived ECs (iPSC-ECs) from three HLHS patients and three age-matched controls. Single Cell RNA-Seq (scRNA-seq) profiling identified both endocardial (NPR3 + /CDH5 + ) and coronary endothelial populations (APLN + /CDH5 + ) from the heterogeneous iPSC-ECs. Intriguingly, a subcluster of the coronary endothelial cells (CECs) with cell cycle arrest was specifically enriched in HLHS patients. Further cell cycle analysis showed that 30.6% of the HLHS cells were trapped in the G1 phase, while the majority of the control CECs entered cell cycle normally. Additionally, the cell cycle differences between control and HLHS was only seen in CECs, not in the endocardial population. To verify our transcriptomic analysis, we applied negative cell sorting (NPR3 - /CDH5 + ) on iPSC-ECs to purify CECs (iCECs) and confirmed that HLHS iCECs showed profound reduction of cell cycle/proliferative genes ( KI67, PCNA, CCNA2, CCNB1 ) and abnormal induction of CCND2 , which is the hallmark of G1 phase. BrdU assays also indicated suppressed proliferation in HLHS iCECs. Furthermore, we profiled the transcriptome from a human heart with an underdevelopment left ventricle (ULV) at single cell resolution. When compared to the normal human heart, pathway enrichment analysis of differentially expressed genes in ULV hearts demonstrated reduced cell proliferation in the CEC subpopulation. Here, we identified that CECs from HLHS patients exerted proliferative defects that can potentially impede the development of vascular/capillary structure and cause related functional deficiencies. Reformation of the coronary defect provides a promising therapeutic strategy to prevent HLHS deterioration.