An integrated human stem cell model for elucidating myocardial-endocardial interactions in cardiac development and disease

Hypoplastic left heart syndrome (HLHS) is a severe type of congenital heart defects, which is characterized by the underdevelopment of left side of the heart. The clinical presentation of HLHS includes hypoplasia of the left ventricle and structural defects in mitral valves, aortic valve, and ascending aorta. HLHS newborns usually

die within a week without surgical treatment. We and others have linked the pathogenic NOTCH1 mutations to HLHS and calcific aortic valve disease. It appears that abnormal NOTCH signaling interrupts the communication between myocardium and endocardium thus leads to incomplete growth of ventricular chamber. However, the

mechanisms by which NOTCH1 mutations results in hypoplasia of the left ventricle are largely unknown due to limited models for studying HLHS. Genetically engineered animals are not capable of reproducing the clinical phenotypes in HLHS patients. Previous studies have focused on the structural and electrophysiological defects

in cardiomyocytes from HLHS patient-derived induced pluripotent stem cells (iPSCs), which may not recapitulate the underlying non-cell autonomous scenarios in the hypoplastic ventricles. In this proposal, we hypothesize that NOTCH1-mediated myocardial-endocardial crosstalk is required for normal human ventricular cardiomyocyte

differentiation, and NOTCH1 mutations leads to abnormal myocardial-endocardial interactions which cause the hypoplasia of ventricular cardiomyocytes in HLHS. We will employ an integrated stem cell model using HLHS and CRISPR genome-edited iPSCs to investigate how NOTCH1 mutations lead to abnormal myocardial-

endocardial interactions in HLHS. We will design a novel co-culture platform using human iPSC-derived cardiomyocytes (iPSC-CMs) and endothelial cells (iPSC-ECs) with distinct NOTCH1 genetic composition to study the intercellular communication between endocardium and myocardium in both healthy and diseased

conditions. In Specific Aim 1, we will investigate the cellular and molecular mechanisms by which endothelial NOTCH1 deficiency suppresses human ventricular cardiomyocyte differentiation and proliferation. In Specific Aim 2, we will determine how the crosstalk between myocardium and endocardium affects ventricular

cardiomyocyte differentiation and proliferation by co-culture of human iPSC-CMs and iPSC-ECs. In Specific Aim 3, we will decipher the mechanisms by which NOTCH1 mutations results in the dysfunctional myocardial- endocardial interactions and contribute to hypoplasis of the left ventricle using genome-edited HLHS-iPSCs. The

completion of this R01 project will have a major impact on the understanding of HLHS through interactions between endocardium and myocardium using clinically relevant and patient-derived cardiomyocytes and endothelial cells.