Abstract Body: Introduction and Hypothesis:
Hypoplastic Left Heart Syndrome is a rare congenital heart defect characterized by underdeveloped left-sided heart structures, specifically the left ventricle (LV). NOTCH1 regulates cardiac cell specification and first heart field development, with mutations contributing to Hypolastic Left Heart Syndrome and potentially promoting LV hypoplasia. Previous animal studies exploring the role of NOTCH1 in the LV fail to capture the structural complexity of human disease. We leveraged both two and three-dimensional human induced pluripotent stem cell (iPSC)-based cellular models to study the role of NOTCH1 in cardiac cell lineage differentiation during first heart field development.
Methods:
We used CRISPR-edited NOTCH1 homozygous knockout (KO) iPSC lines and isogenic controls (WT) to generate LV-like iPSC-derived cardiomyocytes and cardioids. We measured cardioid diameter and recorded the onset of formation of chamber-like cavities in cardioids derived from both KO and WT. Cardioids were collected at D3.5 and D5.5 during first heart field progenitor patterning for bulk RNA sequencing. In parallel, iPSC-derived LV-like cardiomyocytes were generated and subjected to immunofluorescent analysis to assess the role of NOTCH1 on left ventricle development.
Results and Conclusions:
Preliminary results of cardioid generation reveal NOTCH1-deficient organoids had reduced overall cardioid growth and delayed cavity formation. LV-specific cardioids with NOTCH1 KO displayed later onset of contraction, along with slower rhythm. RNA sequencing analysis of D3.5 and D5.5 LV cardioids undergoing cardiac progenitor specification revealed a downregulation of vasculature and endocardial-related genes, such as PECAM1, ELN, and VCAM1. NOTCH1 KO cardioids showed significant dysregulated pathways associated with WNT signaling and endothelial-to-mesenchymal transition. Immunofluorescent analysis of LV-cardioids and cardiomyocytes support findings from RNA sequencing, including delayed WNT signaling and cardiomyocyte proliferation defects. These results suggest that NOTCH1 dysfunction disrupts first heart field development possibly through dysregulated endocardial differentiation and endothelial-to-mesenchymal transition during early cell lineage specification of cardiac progenitors.