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

项目总结 左心发育不全综合征(HLHS)是最致命的先天性心脏病之一，发生在 每4344个新生儿中就有一个。HLHS患者的心脏显示左心室严重发育不全， 心脏瓣膜和升主动脉，特别是心肌中的毛细血管密度降低。此外， NOTCH1的基因变异与HLHS患者有关，显示与心内膜缺陷有关， 内皮功能障碍和NOTCH1信号通路。然而，NOTCH1信令如何确定 HLHS患者的心脏血管系统发育不良仍不确定。仍然有一名失踪人员 心血管发育不良的发病机制与HLHS的功能障碍之间的联系 心脏和NOTCH1突变。在这项提案中，基因组编辑的NOTCH1+/-HiPSCs将被联合 在已经优化的方案中分化为带血管的心脏器官(VCO)， 心肌细胞、内皮细胞、平滑肌细胞和其他心肌细胞 高血压性心脏病早期心血管系统发育不全和功能障碍 NOTCH1突变，并进一步阐明HLHS在动态多细胞串扰中的发病机制 心血管细胞通过Noch-Dll-JAG受体-配体相互作用。在初步结果中，我们观察到 当Notch信号被抑制时，VCO中血管形成减少，心肌样环变小 通过g-分泌酶抑制剂，NOTCH1+/-VCO中观察到较小的类有机物尺寸 同基因野生型压控振荡器。我们将进一步探讨NOTCH1信号在血管和心脏形成中的作用 通过测量心血管结构、增殖、功能和相应的基因表达 (目标1)。此外，我们的初步研究还显示，NOTCH1信号通过NOTCH-DLL- JAG配体-受体在VCO中的相互作用与非血管心脏器官的比较 细胞RNA-seq和配体-受体分析。我们将进一步研究配体-受体的相互作用和 NOTCH1信号在NOTCH1+/-VCO形成过程中的伪时间轨迹蛋白质组 相关的蛋白质组学分析将揭示HLHS VCO发育的概况 用单细胞RNA-seq数据进行验证(目标2)。拟建项目的完成情况将会更好 NOTCH1信号介导的多细胞串扰及其下游通路的确定 高位低位高位高位患者心脏血管发育不全。

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.