Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency

Abstract

BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

hiPSCs as a Unique Platform to Model Cardiogenesis in NOTCH1-Associated HLHS: hiPSCs to Model Complex Congenital Heart Defects

“…Overall, the findings of Ye et al 9 highlight the power of hiPSCs, genome editing, and single-cell RNA sequencing as a unique toolkit to gain new insights into the comprehension of the mechanisms that underlie congenital heart defects etiology. With this human model in hand, they showed that NOTCH1 genetic ablation impact early stages of human cardiogenesis by blocking ventricular-like cardiomyocyte differentiation and proliferation and inhibiting cardiac mesoderm cell differentiation into first heart field progenitors that represent the major source of left ventricle Recently, 3D self-organizing cardiac organoids have been generated.…”

“…One of the main advantages of cardiac stepwise-directed differentiation of hPSCs as a human cardiogenesis platform is the unique possibility to profile the genetic networks and mutation-associated perturbations from pluripotency through stage-specific transitions in cardiac differentiation. Here, Ye et al 9 employed single-cell RNA sequencing to interrogate for the first time the impact of NOTCH1 KO at different stages of cardiac lineage specification. While there were no major changes before day 5 of differentiation (cardiac mesoderm), the amount of second heart field progenitors and epicardial cells was increased in NOTCH1 KO cells.…”

“…To better decipher the mechanistic basis of NOTCH1 contribution to human heart development, Ye et al 9 in this issue of Circulation Research used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9)-mediated genome editing to generate NOTCH1 -deficient ( NOTCH1 knockout) hiPSCs as a disease model. Since NOTCH1 knockout mice present with severe defects in vascular morphogenesis, 10 NOTCH1 knockout hiPSCs were first differentiated into endothelial cells.…”

“…However, this does not exclude the concomitant impairment of ventricular cardiomyocyte proliferation when the NOTCH-dependent pathway is perturbed. To test that possibility, Ye et al 9 quantified early cardiomyocyte proliferation by measuring Ki67+ as well as cyclin D1 and phosphorylated RB+ cells to label proliferating cardiomyocytes. They showed that NOTCH1 ablation resulted in defective cardiomyocyte proliferation due to significant reduction of genes involved in cell cycle progression as well as downstream targets of NOTCH and WNT pathways as measured by bulk RNA-sequencing.…”

Abnormal Progenitor Cell Differentiation and Cardiomyocyte Proliferation in Hypoplastic Right Heart Syndrome

“…2 Human iPSC-derived cardiomyocytes (iPSC-CMs) were generated by sequential modulation of Wnt signaling. 3 Immunofluorescence staining indicated the intercalated…”