From Stem Cells to Populations—Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects

Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and environmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections have enabled genome-wide association studies (GWAS) in non-syndromic CHD. The ability to generate human to create human induced pluripotent stem cells (hiPSC) and further differentiate them to organotypic cells enables further exploration of genotype–phenotype correlations in patient-derived cells. Here we review how these technologies can be used in unraveling the genetics and molecular mechanisms of heart development.

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Genome-Wide Association Studies and Meta-Analyses for Congenital Heart Defects

Circulation Cardiovascular Genetics ◽

10.1161/circgenetics.116.001449 ◽

2017 ◽

Vol 10 (3) ◽

Cited By ~ 13

Author(s):

A. J. Agopian ◽

Elizabeth Goldmuntz ◽

Hakon Hakonarson ◽

Anshuman Sewda ◽

Deanne Taylor ◽

...

Keyword(s):

Congenital Heart Defects ◽

Congenital Heart ◽

Association Studies ◽

Heart Defects ◽

Genome Wide Association ◽

Genome Wide Association Studies ◽

Genome Wide ◽

Meta Analyses

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Semaphorin3f as an intrinsic regulator of chamber-specific heart development

10.1101/2021.05.19.444704 ◽

2021 ◽

Author(s):

Rami Halabi ◽

Paula B. Cechmanek ◽

Carrie L. Hehr ◽

Sarah McFarlane

Keyword(s):

Congenital Heart Defects ◽

Heart Development ◽

Congenital Heart ◽

Molecular Mechanisms ◽

Heart Defects ◽

Border Region ◽

Specific Gene ◽

Atrioventricular Canal ◽

Term Survival ◽

Ventricular Cardiomyocytes

During development a pool of precursors form a heart with atrial and ventricular chambers that exhibit distinct transcriptional and electrophysiological properties. Normal development of these chambers is essential for full term survival of the fetus, and deviations result in congenital heart defects. The large number of genes that may cause congenital heart defects when mutated, and the genetic variability and penetrance of the ensuing phenotypes, reveals a need to understand the molecular mechanisms that allow for the formation of chamber-specific cardiomyocyte differentiation. We find that in the developing zebrafish heart, mRNA for a secreted Semaphorin (Sema), Sema3fb, is expressed by all cardiomyocytes, whereas mRNA for its receptor Plexina3 (Plxna3) is expressed by ventricular cardiomyocytes. In sema3fb CRISPR zebrafish mutants, ventricular chamber development is impaired; the ventricles of mutants are smaller in size than their wild type siblings, apparently because of differences in cell size and not cell numbers, with ventricular cardiomyocytes failing to undergo normal developmental hypertrophy. Analysis of chamber differentiation indicates defects in chamber specific gene expression at the border between the ventricular and atrial chambers, with spillage of ventricular chamber genes into the atrium, and vice versa, and a failure to restrict bmp4a mRNA to the atrioventricular canal. The disrupted atrioventricular border region in mutants is accompanied by hypoplastic heart chambers and impaired cardiac function. These data suggest a model whereby cardiomyocytes secrete a Sema cue that, through spatially restricted expression of the receptor, signals in a ventricular chamber-specific manner to establish a distinct border between atrial and ventricular chambers that is important for functional development of the heart.

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Abnormal Cardiac Development in the Absence of Heart Glycogen

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7179-7187.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7179-7187 ◽

Cited By ~ 68

Author(s):

Bartholomew A. Pederson ◽

Hanying Chen ◽

Jill M. Schroeder ◽

Weinian Shou ◽

Anna A. DePaoli-Roach ◽

...

Keyword(s):

Cardiac Function ◽

Cardiac Muscle ◽

Heart Development ◽

Congenital Heart ◽

Cardiac Development ◽

Glycogen Synthase ◽

Heart Defects ◽

Mendelian Inheritance ◽

And Function ◽

Impaired Cardiac Function

ABSTRACT Glycogen serves as a repository of glucose in many mammalian tissues. Mice lacking this glucose reserve in muscle, heart, and several other tissues were generated by disruption of the GYS1 gene, which encodes an isoform of glycogen synthase. Crossing mice heterozygous for the GYS1 disruption resulted in a significant underrepresentation of GYS1-null mice in the offspring. Timed matings established that Mendelian inheritance was followed for up to 18.5 days postcoitum (dpc) and that ∼90% of GYS1-null animals died soon after birth due to impaired cardiac function. Defects in cardiac development began between 11.5 and 14.5 dpc. At 18.5 dpc, the hearts were significantly smaller, with reduced ventricular chamber size and enlarged atria. Consistent with impaired cardiac function, edema, pooling of blood, and hemorrhagic liver were seen. Glycogen synthase and glycogen were undetectable in cardiac muscle and skeletal muscle from the surviving null mice, and the hearts showed normal morphology and function. Congenital heart disease is one of the most common birth defects in humans, at up to 1 in 50 live births. The results provide the first direct evidence that the ability to synthesize glycogen in cardiac muscle is critical for normal heart development and hence that its impairment could be a significant contributor to congenital heart defects.

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Focused Strategies for Defining the Genetic Architecture of Congenital Heart Defects

Genes ◽

10.3390/genes12060827 ◽

2021 ◽

Vol 12 (6) ◽

pp. 827

Author(s):

Lisa J. Martin ◽

D Woodrow Benson

Keyword(s):

Genetic Variation ◽

Congenital Heart Defects ◽

Heart Development ◽

Genetic Architecture ◽

Congenital Heart ◽

Rare Variants ◽

Heart Defects ◽

Genetic Origin ◽

Genetic Studies ◽

The Ideal

Congenital heart defects (CHD) are malformations present at birth that occur during heart development. Increasing evidence supports a genetic origin of CHD, but in the process important challenges have been identified. This review begins with information about CHD and the importance of detailed phenotyping of study subjects. To facilitate appropriate genetic study design, we review DNA structure, genetic variation in the human genome and tools to identify the genetic variation of interest. Analytic approaches powered for both common and rare variants are assessed. While the ideal outcome of genetic studies is to identify variants that have a causal role, a more realistic goal for genetic analytics is to identify variants in specific genes that influence the occurrence of a phenotype and which provide keys to open biologic doors that inform how the genetic variants modulate heart development. It has never been truer that good genetic studies start with good planning. Continued progress in unraveling the genetic underpinnings of CHD will require multidisciplinary collaboration between geneticists, quantitative scientists, clinicians, and developmental biologists.

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Maternal iron deficiency does not affect the iron status of fetuses with congenital heart defects: Does it affect heart development?

International Journal of Cardiology ◽

10.1016/j.ijcard.2020.01.066 ◽

2020 ◽

Vol 306 ◽

pp. 89

Author(s):

Yuan Qin ◽

Meng Gao ◽

Wenting Jiang ◽

Anqi Li ◽

Wanqiang Xue ◽

...

Keyword(s):

Iron Deficiency ◽

Congenital Heart Defects ◽

Heart Development ◽

Congenital Heart ◽

Iron Status ◽

Heart Defects ◽

Maternal Iron

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Say NO to ROS: Their Roles in Embryonic Heart Development and Pathogenesis of Congenital Heart Defects in Maternal Diabetes

Antioxidants ◽

10.3390/antiox8100436 ◽

2019 ◽

Vol 8 (10) ◽

pp. 436 ◽

Cited By ~ 5

Author(s):

Engineer ◽

Saiyin ◽

Greco ◽

Feng

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Congenital Heart Defects ◽

Heart Development ◽

Congenital Heart ◽

Heart Defects ◽

Maternal Diabetes ◽

Embryonic Heart ◽

Pregestational Diabetes ◽

Enos Uncoupling

Congenital heart defects (CHDs) are the most prevalent and serious birth defect, occurring in 1% of all live births. Pregestational maternal diabetes is a known risk factor for the development of CHDs, elevating the risk in the child by more than four-fold. As the prevalence of diabetes rapidly rises among women of childbearing age, there is a need to investigate the mechanisms and potential preventative strategies for these defects. In experimental animal models of pregestational diabetes induced-CHDs, upwards of 50% of offspring display congenital malformations of the heart, including septal, valvular, and outflow tract defects. Specifically, the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) signaling is a major driver of the development of CHDs in offspring of mice with pregestational diabetes. NO from endothelial nitric oxide synthase (eNOS) is crucial to cardiogenesis, regulating various cellular and molecular processes. In fact, deficiency in eNOS results in CHDs and coronary artery malformation. Embryonic hearts from diabetic dams exhibit eNOS uncoupling and oxidative stress. Maternal treatment with sapropterin, a cofactor of eNOS, and antioxidants such as N-acetylcysteine, vitamin E, and glutathione as well as maternal exercise have been shown to improve eNOS function, reduce oxidative stress, and lower the incidence CHDs in the offspring of mice with pregestational diabetes. This review summarizes recent data on pregestational diabetes-induced CHDs, and offers insights into the important roles of NO and ROS in embryonic heart development and pathogenesis of CHDs in maternal diabetes.

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Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Nature Communications ◽

10.1038/s41467-021-25329-5 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Yonatan R. Lewis-Israeli ◽

Aaron H. Wasserman ◽

Mitchell A. Gabalski ◽

Brett D. Volmert ◽

Yixuan Ming ◽

...

Keyword(s):

Congenital Heart Defects ◽

Self Assembly ◽

Human Heart ◽

Congenital Heart ◽

Cardiac Development ◽

Heart Defects ◽

Cell Types ◽

Cellular Level ◽

Cardiac Tissues

AbstractCongenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.

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Hox and Tale transcription factors in heart development and disease

The International Journal of Developmental Biology ◽

10.1387/ijdb.180192sz ◽

2018 ◽

Vol 62 (11-12) ◽

pp. 837-846 ◽

Cited By ~ 8

Author(s):

Fabienne Lescroart ◽

Stephane Zaffran

Keyword(s):

Transcription Factors ◽

Congenital Heart Defects ◽

Heart Development ◽

Congenital Heart ◽

Hox Genes ◽

Heart Defects ◽

First Year ◽

First Year Of Life ◽

Cardiac Mesoderm

Hox genes are highly conserved transcription factors with critical functions during development, in particular for patterning the antero-posterior axis of the embryo. Their action is very often associated with cofactors including the TALE family transcription factors. From Drosophila to vertebrates, Hox genes have been shown to have a major role in heart development. In this review, we focus on the increasing evidence implicating the anterior Hox genes and the Tale family members during heart development both in the cardiac mesoderm and in neural crest cells. Congenital heart defects are the leading cause of death in the first year of life and a better understanding of the role of Hox and Tale factors is highly relevant to human pathologies and will provide novel mechanistic insights into the underlying defects.

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Abstract P515: Dynamics Of Cardiomyocyte Differentiation In Isogenic Induced Pluripotent Stem Cells Derived From Trisomy21 Patients Hints Molecular Pathology Of Congenital Heart Defects In Down Syndrome

Circulation Research ◽

10.1161/res.129.suppl_1.p515 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Malay Chaklader ◽

Beverly A Rothermel

Keyword(s):

Stem Cells ◽

Down Syndrome ◽

Induced Pluripotent Stem Cells ◽

Congenital Heart Defects ◽

Pluripotent Stem Cells ◽

Congenital Heart ◽

Heart Defects ◽

Congenital Defects ◽

Cardiac Progenitors ◽

Induced Pluripotent

Down syndrome (DS) is the most frequently occurring human chromosomal disorder and is responsible for a range of both congenital defects and progressive, degenerative conditions. For instance, an estimated 50% DS neonates are born with congenital heart defects (CHD) and more than 50% of DS adults develop early onset Alzheimer’s. Using induced pluripotent stem cells (iPSCs) derived from DS patients and isogenic controls we previously demonstrated the presence of a hyper-metabolic, hyper-fused mitochondrial network in trisomic iPSCs (3S-iPSCs) compared to disomic (2S-iPSCs) controls. Furthermore, mitochondrial function was normalized by siRNA depletion of RCAN1, an inhibitor of the protein phosphatase calcineurin (CN). Both CN signaling and mitochondrial metabolism have been implicated in a variety of steps during the progression from embryonic stem cells to cardiac progenitors, including self-renewal, exit from pluripotency, and commitment to cardiac verses hematopoietic lineages. Based on this, we hypothesized that the dynamics of many of these processes will be altered over the course of differentiation of 3S-iPSCs to cardiomyocytes when compared to 2S-iPSCs. Here, we investigate the temporal expression of pluripotency associated genes and lineage associated genes as well as cardiac mesoderm and mature cardiomyocyte specific genes. We also define and compare changes in CN activity, expression of specific CN isoforms, mitochondrial expansion, ROS generation, and activation of stress responses. Our study identifies early developmental and metabolic sequelae capable of contributing to CHD in DS that may result from a disruption in the normal balance in crosstalk between CN and RCAN1.

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