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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.

Long QT syndrome (LQTS) is a detrimental arrhythmia syndrome mainly caused by dysregulated expression or aberrant function of ion channels. The major clinical symptoms of ventricular arrhythmia, palpitations and syncope vary among LQTS subtypes. Susceptibility to malignant arrhythmia is a result of delayed repolarisation of the cardiomyocyte action potential (AP). There are 17 distinct subtypes of LQTS linked to 15 autosomal dominant genes with monogenic mutations. However, due to the presence of modifier genes, the identical mutation may result in completely different clinical manifestations in different carriers. In this review, we describe the roles of various ion channels in orchestrating APs and discuss molecular aetiologies of various types of LQTS. We highlight the usage of patient-specific induced pluripotent stem cell (iPSC) models in characterising fundamental mechanisms associated with LQTS. To mitigate the outcomes of LQTS, treatment strategies are initially focused on small molecules targeting ion channel activities. Next-generation treatments will reap the benefits from development of LQTS patient-specific iPSC platform, which is bolstered by the state-of-the-art technologies including whole-genome sequencing, CRISPR genome editing and machine learning. Deep phenotyping and high-throughput drug testing using LQTS patient-specific cardiomyocytes herald the upcoming precision medicine in LQTS.

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Pulmonary atresia with intact ventricular septum (PA-IVS) is a detrimental congenital heart disease where the pulmonary valve is not appropriately developed. Several hypotheses speculated to explain the pathogenesis of this disorder contains abnormal coronary arterial development, atypical blood flow through venous valve and atretic pulmonary valve formation. Conventional treatment strategy is pulmonary valve perforation, and PA-IVS patients after treatment present with varying degrees of ventricular hypoplasia: from single ventricle palliation (1v) to 1½-ventricle palliation (1.5v) and bi-ventricle repair (2v). Mechanistic studies are required to further explain the different levels of RV hypoplasia in PA-IVS patients. Here, we generated PA-IVS-specific induced pluripotent stem cells (iPSCs) from patients with a spectrum of RV hypoplasia. PA-IVS iPSC-derived cardiomyocytes (iPSC-CMs) contracted normally and displayed sarcomeric structures with intercalated cardiac troponin T and α-actinin. Early-stage PA-IVS iPSC-CMs exhibited a variety of compromised proliferation activities, which were not able to be rescued by Wnt signaling pathway activation. Transcriptomic profiling by bulk RNA seq suggested that pathways involved in the cell cycle and mitosis were downregulated in day13 PA-IVS-1v iPSC-CMs, but not in PA-IVS-2v iPSC-CMs. However, at later stage (day20), pathways involved in the regulation of cell division and mitosis was upregulated in PA-IVS-1v cardiomyocytes, indicating a possible developmental delay in the cardiomyocyte proliferation for PA-IVS-1v. Intriguingly, differentially expressed genes between PA-IVS-2v and control CMs were mostly enriched in the pathways relevant to glucose metabolism, mitochondrial biogenesis, and muscle contraction. The differentially involved pathways between PA-IVS-1v and PA-IVS-2v cardiomyocytes suggest that an intrinsic transcriptional program may lead to divergent degrees of cardiomyocyte proliferation and ventricular growth in PA-IVS. We conclude that patient iPSC-CMs can recapitulate cardiomyocyte proliferation defects which are involved in ventricular hypoplasia in PA-IVS.