Impact of genetic factors on antioxidant rescue of maternal diabetes-associated congenital heart disease.

Congenital heart disease (CHD) affects ~1% of live births. Although genetic and environmental etiologic contributors have been identified, the majority of CHD lacks a definitive cause, suggesting the role of gene-environment interactions (GxE) in disease pathogenesis. Maternal diabetes mellitus (matDM) is among the most prevalent environmental risk factors for CHD. However, there is a substantial knowledge gap in understanding how matDM acts upon susceptible genetic backgrounds to increase disease expressivity. Previously, we reported a GxE between Notch1 haploinsufficiency and matDM leading to increased CHD penetrance. Here, we demonstrate a cell lineage specific effect of Notch1 haploinsufficiency in matDM-exposed embryos, implicating endothelial/endocardial derived tissues in the developing heart. We report impaired atrioventricular cushion morphogenesis in matDM exposed Notch1+/- animals and show a synergistic effect of NOTCH1 haploinsufficiency and oxidative stress in dysregulation of gene regulatory networks critical for endocardial cushion morphogenesis in vitro. Mitigation of matDM-associated oxidative stress via SOD1 overexpression did not rescue CHD in Notch1 haploinsufficient mice compared to wildtype littermates. Our results show the combinatorial interaction of matDM-associated oxidative stress and a genetic predisposition, Notch1 haploinsufficiency, on cardiac development, supporting a GxE model for CHD etiology and suggesting that antioxidant strategies maybe ineffective in genetically-susceptible individuals.

In Vivo and In Vitro Approaches to Modeling Hypoplastic Left Heart Syndrome.

PURPOSE OF REVIEW: Hypoplastic left heart syndrome (HLHS) is a critical congenital heart defect characterized by the underdevelopment of left-sided heart structures, leading to significant circulatory challenges, and necessitating multiple surgeries for survival. Despite advancements in surgical interventions, long-term outcomes often involve heart failure, highlighting the need for a deeper understanding of HLHS pathogenesis. Current in vivo and in vitro models aim to recapitulate HLHS anatomy and physiology, yet they face limitations in accuracy and complexity. RECENT FINDINGS: In vivo models, including those in chick, lamb, and mouse, provide insights into hemodynamic and genetic factors influencing HLHS. In vitro models using human induced pluripotent stem cells offer valuable platforms for studying genetic mutations and cellular mechanisms. This review evaluates these models' utility and limitations, and proposes future directions for developing more sophisticated models to enhance our understanding and treatment of HLHS.

Characterization of an induced pluripotent stem cell line (NCHi013-A) from a 5-year-old male with pulmonary atresia with intact ventricular septum and a biventricular repair.

Pulmonary atresia with intact ventricular septum (PA/IVS) is a rare congenital heart defect that causes a significant decrease of blood outflow from the heart and is fatal if left untreated. iPSC line NCHi013-A was produced from peripheral blood mononuclear cells from a male child with PA/IVS using Sendai virus reprogramming. NCHi013-A displayed normal stem cell morphology, expressed markers for pluripotency, and presented ability to differentiate into cells of endoderm, ectoderm, and mesoderm lineages. The iPSC line also maintained normal karyotype, was validated for cell identity, and tested negative for transgenes and mycoplasma contamination.

Generation of an induced pluripotent stem cell line (NCHi016-A) from a 5-year-old female with pulmonary atresia with intact ventricular septum and one-and-half ventricle palliation.

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare congenital heart defect characterized by underdeveloped pulmonary valve and right ventricular hypoplasia. Neonates undergoing surgery to open pulmonary valve have a range of post-surgical ventricular recovery: single-ventricle (1v) palliation, one-and-half ventricle (1.5v) palliation, and bi-ventricular (2v) repair. PA-IVS-1.5v typically requires surgical intervention to install cavopulmonary shunt and entails partial right ventricle recovery. NCHi016-A is an iPSC line derived from a 5-year-old female with PA-IVS-1.5v using Sendai Virus reprogramming. This iPSC line shows typical iPSC morphology, has normal karyotype, expresses pluripotency markers, and has potential to differentiate into three germ layers.

Generation of iPSC line NCHi015-A from a patient with truncus arteriosus carrying heterozygous variants in KMT2D and NOTCH1.

Truncus arteriosus (TA) is a congenital heart defect where one main blood vessel emerges from the heart, instead of individual aorta and pulmonary artreries. Peripheral mononuclear cells (PBMCs) of a male infant with TA were reporogrammed using Sendai virus. The resultant iPSC line (NCHi015-A) displayed normal colony formation, expressed pluripotency markers, and differentiated into cells from three germ layers. NCHi015-A was matched to the patient's genetic profile, had normal karyotype, retained genetic variants in KMT2D and NOTCH1, and tested negative for reprogramming transgene. This iPSC line can be used for studying congenital heart defects associated with genetic variants in KMT2D and NOTCH1.

Statins improve endothelial function via suppression of epigenetic-driven EndMT.

The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.

Characterization of an induced pluripotent stem cell line NCHi011-A from a 23-year-old female with Alagille Syndrome harboring a heterozygous JAG1 pathogenic variant.

Alagille syndrome (ALGS) is a multisystem disease with high variability in clinical features. ALGS is predominantly caused by pathogenic variants in the Notch ligand JAG1. An iPSC line, NCHi011-A, was generated from a ALGS patient with complex cardiac phenotypes consisting of pulmonic valve and branch pulmonary artery stenosis. NCHi011-A is heterozygous for a single base duplication causing a frameshift in the JAG1 gene. This iPSC line demonstrates normal cellular morphology, expression of pluripotency markers, trilineage differentiation potential, and identity to the source patient. NCHi011-A provides a resource for modeling ALGS and investigating the role of Notch signaling in the disease.

Generation of iPSC line NCHi012-A from a patient with Alagille syndrome and heterozygous pathogenic variant in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant disease affecting the liver, heart and other organs with high variability. About 95% of ALGS cases are associated with pathogenic variants in JAG1, encoding the Jagged1 ligand that binds to Notch receptors. The iPSC line NCHi012-A was derived from an ALGS patient with cholestatic liver disease and mild pulmonary stenosis, who is heterozygous for a 2 bp deletion in the JAG1 coding sequence. We report here an initial characterization of NCHi012-A to evaluate its morphology, pluripotency, differentiation potential, genotype, karyotype and identity to the source patient.

Creation of iPSC line NCHi004-A from a patient with down syndrome and congenital heart defects.

Down syndrome is a congenital disorder resulting from an extra full or partial chromosome 21, which is characterized by a spectrum of systemic developmental abnormalities, including those affecting the cardiovascular system. Here, we generated an iPSC line from peripheral blood mononuclear cells of a male adolescent with Down syndrome-associated congenital heart defects through Sendai virus-mediated transfection of 4 Yamanaka factors. This line exhibited normal morphology, expressed pluripotency markers, trisomy 21 karyotype, and could be differentiated into three germ layers. This iPSC line can be used for studying cellular and developmental etiologies of congenital heart defects induced by aneuploidy of chromosome 21.

Generation of an induced pluripotent stem cell line (NCHi010-A) from a 6-year-old female with Down syndrome and without congenital heart disease.

Down syndrome is a genetic anomaly that manifests when there is a mistake during cell division, resulting in an additional chromosome 21. Down syndrome can impact cognitive capabilities and physical development, giving rise to diverse developmental disparities and an elevated likelihood of certain health issues. The iPSC line NCHi010-A was generated from peripheral blood mononuclear cells of a 6-year-old female with Down syndrome and without congenital heart disease using Sendai virus reprogramming. NCHi010-A displayed a morphology of pluripotent stem cells, expressed pluripotency markers, retained trisomy 21 karyotype, and demonstrated potential to differentiate into cells representative of the three germ layers.

Establishment of NCHi009-A, an iPSC line from a patient with hypoplastic left heart syndrome (HLHS) carrying a heterozygous NOTCH1 mutation.

Hypoplastic left heart syndrome (HLHS) is a congenital heart malformation clinically characterized by an underdeveloped left ventricle, mitral or aortic valve stenosis or atresia, and narrowed ascending aorta. Although genetic etiology of HLHS is heterogenous, recurrent NOTCH1 variants have been associated with this defect. We report generation of an iPSC line derived from a female with HLHS with a heterozygous missense NOTCH1 (c.2058G > A; p.Gly661Ser) mutation within the conserved EGF-like repeat 17. This iPSC line exhibited typical cellular morphology, normal karyotype, high expression of pluripotent markers, and trilineage differentiation potential; and can be leveraged to dissect the complex NOTCH1-mediated HLHS disease mechanism.

Precision medicine for long QT syndrome: patient-specific iPSCs take the lead.

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.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

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 (Clustered Regularly Interspaced Short Palindromic Repeats)/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.

Generation and characterization of a human induced pluripotent stem cell (iPSC) line from a patient with congenital heart disease (CHD).

Epstein-Barr virus (EBV) immortalized lymphoblastoid cell lines (LCLs) are widely used for banking. This bioresource could be leveraged for creating human iPSC lines to model diseases including CHD. We generated an LCL-derived iPSC line (NCHi001-A) from a patient with congenital aortic valve stenosis. NCHi001-A was EBV and transgenes free, exhibited stem cell-like morphology, expressed pluripotency markers, has a normal karyotype, and could be differentiated into cells of three germ layers in vitro. Relationship inference via a microarray-based analysis showed NCHi001-A is identical to the parental cell line. NCHi001-A can be used for disease modeling, drug discovery, and cell therapy development.

Generation of an induced pluripotent stem cell line NCHi003-A from a 11-year-old male with pulmonary atresia with intact ventricular septum (PA-IVS).

Pulmonary atresia with intact ventricular septum (PA-IVS) is a rare congenital heart defect defined by membranous or muscular atresia of the right ventricular outflow tract where patients display varying degrees of hypoplasia of the right ventricle. This condition results in cyanosis due to an inability of blood to flow from the right ventricle to the pulmonary arteries, thus requiring immediate surgical intervention after birth. An iPSC line was generated from peripheral blood mononuclear cells of a 11-year-old male patient diagnosed with PA-IVS through Sendai virus-mediated reprogramming. This disease-specific iPSC line was characterized by immunocytochemistry, STR analysis, karyotype analysis, and mycoplasma testing.

Characterization of an iPSC line NCHi006-A from a patient with hypoplastic left heart syndrome (HLHS).

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect characterized by underdeveloped structures on the left side of the heart, including hypoplasia of the left ventricle and stenosis or atresia of the aortic and mitral valves. Here, we generated an iPSC line from the peripheral blood mononuclear cells of a male patient with HLHS through Sendai virus-mediated transfection of 4 Yamanaka factors. This iPSC line exhibited normal morphology, expressed pluripotency markers, had a normal karyotype, and could differentiate into cells of three germ layers. This iPSC line can be used for studying cellular and developmental etiologies of HLHS.

Nanocrown electrodes for parallel and robust intracellular recording of cardiomyocytes.

Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. However, the large variability in success rate and signal strength have hindered nanoelectrodes from being broadly adopted for proarrhythmia drug assessment. In this work, we develop vertically-aligned nanocrown electrodes that are mechanically robust and achieve > 99% success rates in obtaining intracellular access through electroporation. We validate the accuracy of nanocrown electrode recordings by simultaneous patch clamp recording from the same cell. Finally, we demonstrate that nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four incremental drug doses. Our technology development provides an advancement towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.

Translational potential of hiPSCs in predictive modeling of heart development and disease.

Congenital heart disease (CHD) represents a major class of birth defects worldwide and is associated with cardiac malformations that often require surgical intervention immediately after birth. Despite the intense efforts from multicentric genome/exome sequencing studies that have identified several genetic variants, the etiology of CHD remains diverse and often unknown. Genetically modified animal models with candidate gene deficiencies continue to provide novel molecular insights that are responsible for fetal cardiac development. However, the past decade has seen remarkable advances in the field of human induced pluripotent stem cell (hiPSC)-based disease modeling approaches to better understand the development of CHD and discover novel preventative therapies. The iPSCs are derived from reprogramming of differentiated somatic cells to an embryonic-like pluripotent state via overexpression of key transcription factors. In this review, we describe how differentiation of hiPSCs to specialized cardiac cellular identities facilitates our understanding of the development and pathogenesis of CHD subtypes. We summarize the molecular and functional characterization of hiPSC-derived differentiated cells in support of normal cardiogenesis, those that go awry in CHD and other heart diseases. We illustrate how stem cell-based disease modeling enables scientists to dissect the molecular mechanisms of cell-cell interactions underlying CHD. We highlight the current state of hiPSC-based studies that are in the verge of translating into clinical trials. We also address limitations including hiPSC-model reproducibility and scalability and differentiation methods leading to cellular heterogeneity. Last, we provide future perspective on exploiting the potential of hiPSC technology as a predictive model for patient-specific CHD, screening pharmaceuticals, and provide a source for cell-based personalized medicine. In combination with existing clinical and animal model studies, data obtained from hiPSCs will yield further understanding of oligogenic, gene-environment interaction, pathophysiology, and management for CHD and other genetic cardiac disorders.