Abstract 276: NOTCH1 is Essential for Ventricular Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells

Pathogenic variants in NOTCH1 have been implicated in multiple types of congenital heart defects, such as bicuspid aortic valve, Tetralogy of Fallot, and hypoplastic left heart syndrome (HLHS). However, the mechanisms by which NOTCH1 pathogenic variants cause abnormalities in human embryonic heart development are largely unknown. Here, we used CRISPR/Cas9-mediated genome editing to genetically delete NOTCH1 in human induced pluripotent stem cells (iPSCs). We found that NOTCH1 was dispensable for mesodermal and vascular endothelial differentiation of human iPSCs. Disruption of NOTCH activity promoted venous-specific gene expression but suppressed arterial-specific gene expression in iPSC-derived endothelial cells (iPSC-ECs). Intriguingly, NOTCH1 deletion significantly impaired the cardiac differentiation efficiency. In NOTCH1 homozygous knockout ( NOTCH1 -/- ) iPSC-derived cardiomyocytes (iPSC-CMs), atrial-specific genes ( NR2F2, KCNJ3 , and MYL7 ) were upregulated whereas ventricular-specific genes ( MYL2, IRX4 , and MYH7 ) were downregulated. Electrophysiological analysis by patch clamp and optical mapping indicated that atrial-like cardiomyocytes were dominant whereas the percentage of ventricular-like iPSC-CMs was dramatically reduced (<1%) in NOTCH1 -/- iPSC-CMs. In addition, mitochondrial respiration was reduced in NOTCH1 deficient iPSC-CMs compared to wild-type controls, which was likely attributed to the reduction of ventricular cardiomyocytes in NOTCH1 -/- iPSC-CMs. As NOTCH1 is primarily expressed in endothelial cells rather than cardiomyocytes, we conclude that NOTCH1 affects ventricular cardiomyocyte lineage commitment possibly through controlling cell fate determination of cardiac progenitors during human iPSC differentiation. Our study may provide novel insights into the mechanisms by which NOTCH1 mutations lead to left ventricular hypoplasia in HLHS patients.