NOTCH signaling on the underdeveloped cardiac vascularization of hypoplastic left heart syndrome in the hiPSC-derived vascularized cardiac organoids

项目总结

PROJECT SUMMARY Hypoplastic left heart syndrome (HLHS) is one of the most fatal congenital heart diseases (CHD) that occurs in 1 of every 4,344 newborns. The HLHS patients’ hearts show the severe underdevelopment of the left ventricle, heart valves, and ascending aorta, and specifically lower capillary density in the myocardium. Moreover, the genetic variant of NOTCH1 is implicated in HLHS patients showing the relevance to endocardial defects, endothelial dysfunction, and NOTCH1 signaling pathways. However, how the NOTCH1 signaling determines the underdevelopment of cardiac vasculature in the HLHS patients is still inconclusive. There is still a missing link between the pathogenesis in the cardiovascular underdevelopment and dysfunction found in the HLHS hearts and the NOTCH1 mutation. In this proposal, the genome-edited NOTCH1+/- hiPSCs will be co- differentiated in an already optimized protocol into the vascularized cardiac organoid (VCO) with cardiomyocytes, endothelial cells, smooth muscle cells, and other cardiac cells to recapitulate the underdevelopment and dysfunction of the cardiovascular system in the early development of HLHS heart with NOTCH1 mutation and to further delineate the pathogenesis of HLHS in the dynamic multicellular crosstalk of cardiovascular cells via NOTCH-DLL-JAG receptor-ligand interactions. In the preliminary results, we observed a reduced vascular formation with a smaller myocardial-like ring in VCO when NOTCH signaling was inhibited by g-secretase inhibitor, and smaller organoid size was noticed in the NOTCH1+/- VCO compared to the isogenic wildtype VCO. We will further explore the NOTCH1 signaling on vascular and cardiac formation in VCOs by measuring the cardiovascular structure, proliferation, function, and corresponding gene expressions (Aim 1). Moreover, our preliminary study also shows upregulation of NOTCH1 signaling via the NOTCH-DLL- JAG ligand-receptor interactions in the VCO compared to the non-vascularized cardiac organoid by the single- cell RNA-seq and ligand-receptor analysis. We will further investigate the ligand-receptor interactions and pseudotime trajectories of NOTCH1 signaling in the process of NOTCH1+/- VCO formation. The proteome profile over the HLHS VCO development will be uncovered by the proteomic analysis in correlation and validation with the single-cell RNA-seq data (Aim 2). The completion of the proposed project will be for better determining the NOTCH1 signaling-mediated multicellular crosstalk and downstream pathways of underdeveloped cardiac vascularization in HLHS.