Hypomorphic NOTCH1 Expression Alters Cardiomyocyte Cellular Architecture in Hypoplastic Left Heart Syndrome

Abstract

NOTCH1 is a protein involved in cardiac development and mutations in NOTCH1 are implicated in left sided congenital heart disease, including hypoplastic left heart syndrome (HLHS). Therapeutic advances for HLHS patients have been hampered by the absence of suitable experimental models. Here, using human induced pluripotent stem cells (hiPSCs), we have generated a series of CRISPR-edited complex heterozygous hypomorphic mutations in NOTCH1 that recapitulate mutations seen in HLHS patients. These cells demonstrate the downregulation of genes associated with mitochondria, the actin cytoskeleton, and cardiomyocyte development. In addition, these hypomorphic NOTCH1 hiPSCs cells showed an increased propensity to differentiate towards non-cardiomyocyte lineages such as fibroblasts and smooth muscle cells, a finding confirmed in HLHS patient-derived myocardial tissue. Abnormalities in sarcomeric and mitochondrial architecture contributed to decreased ATP production, abnormal calcium transients, and reduced contractility. Using a split nano-luciferase system as a NOTCH1 intracellular reporter, we screened a library of FDA-approved compounds. Auranofin, an agent currently employed for rheumatoid arthritis, was identified as a candidate drug that could rescue impaired differentiation and contractility seen in the hypomorphic NOTCH1 model. These findings point to cell-autonomous abnormalities in hypomorphic NOTCH1 pre-cardiomyocyte cells that can be leveraged to identify potential therapeutic strategies for patients with severe congenital cardiac abnormalities. HighlightsO_LICRISPR edited and patient-derived iPSCs with hypomorphic NOTCH1 expression were used to characterize a cell-based model of hypoplastic left heart syndrome. C_LIO_LIHypomorphic NOTCH1 iPSCs have abnormalities in pathways associated with mitochondrial function, actin cytoskeleton, and cardiomyocyte development. C_LIO_LIHypomorphic NOTCH1 iPSCs have less pluripotency and tend to skew differentiation away from cardiomyocytes and towards fibroblasts and smooth muscle cells. C_LIO_LINOTCH1 is required for cardiac cytoskeletal and mitochondrial architecture, and to preserve contractility and ATP production. C_LIO_LIA high-throughput drug screen identified auranofin as an agent that may benefit patients with hypoplastic left heart syndrome. C_LI