Human Heart Development Research Articles

Chronological and Morphological Study of Heart Development in the Rat

Adult and embryonic laboratory rats have been used as a mammalian model organism in biomedical research, descriptive and experimental cardiac embryology, and experimental teratology. There have been, however, considerable variations and discrepancies concerning the developmental staging of the rat embryo in the reported literature, which have resulted in several controversies and inconsistencies. Therefore, we carried out a careful anatomical and histological study of rat cardiac morphogenesis from the premorphogenetic period to the mature heart in a newborn pup. A correlation between the chronology and morphological features of the heart and embryo or newborn was made. We provide a simple and comprehensive guide relating the developmental timing and fate of the embryonic components of the heart and their morphological changes in the rat based on in vivo labeling studies in the chick. We also compare the timing of heart development in rats, humans, and mice.

Determination of the Human Cardiomyocyte mRNA and miRNA Differentiation Network by Fine-Scale Profiling

To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.

Differences in Copy Number Variation between Discordant Monozygotic Twins as a Model for Exploring Chromosomal Mosaicism in Congenital Heart Defects

Studies addressing the role of somatic copy number variation (CNV) in the genesis of congenital heart defects (CHDs) are scarce, as cardiac tissue is difficult to obtain, especially in non-affected individuals. We explored the occurrence of copy number differences in monozygotic (MZ) twins discordant for the presence of a CHD, as an illustrative model for chromosomal mosaicism in CHDs. Array comparative genomic hybridization was performed on peripheral blood-derived DNA obtained from 6 discordant MZ twin pairs and on sex-matched reference samples. To identify CNV differences between both twin members as well as potential CNVs in both twins contributing to the phenotype, DNA from each twin was hybridized against its co-twin, and against a normal control. Three copy number differences in 1 out of 6 MZ twin pairs were detected, confirming the occurrence of somatic CNV events in MZ twins. Further investigation by copy number and (epi)genome sequencing analyses in MZ twins, discordant for the presence of CHDs, is required to improve our knowledge on how postzygotic genetic, environmental and stochastic factors can affect human heart development.

412: Human fetal heart development during the first and second trimester

412: Human fetal heart development during the first and second trimester

Formation of the Building Plan of the Human Heart

The intricate process of formation of the heart relies on the spatiotemporal regulation of differentiation and growth of its different parts. Errors in these processes can result in hypoplasia of the chambers and incorrect alignment of the atria and great arteries with the ventricles, with such abnormalities producing the worst forms of congenital heart disease.1 Although recent advances in molecular embryology have greatly improved our understanding of normal cardiac development, our insights into the pathogenesis of human cardiac malformations remain limited.2 With the current lack of data on gene expression in humans, inferences concerning morphogenesis rely on classic studies of human cardiac development or on extrapolation of experimental data from animal studies. Most textbooks on human embryology describe the embryonic heart tube as already possessing a linear array of the definitive components, despite this so-called segmental concept being disproved some time ago by fate-mapping experiments in chickens,3,4 which are now endorsed by recent molecular lineage analyses in mice.5 Proliferation studies in chickens revealed that the primary heart tube proliferates minimally and that its growth occurs through addition of differentiating cells from visceral mesoderm at the venous and arterial poles.6 These findings underscore previous lineage studies performed in mice, which have shown that transcription factors Islet1 and Tbx1 play a crucial role in the intricate balance between the cardiac precursor state and differentiation.7,8 Subsequent to its formation, the heart tube elongates and loops, producing its inner and outer curvatures and providing the general building plan for formation of the chambers and conduction system.9 The chambers themselves form by local proliferation and differentiation at the outer curvature.10,11 At the same time, the differentiating chamber myocardium remains flanked by primary myocardium, which is prevented from further differentiation by the …

P59. Identification of novel cardiac genes through global analysis of human ESC cardiomyocyte differentiation

Heart disease is the leading cause of mortality and morbidity in industrialized nations. The genetic programs controlling early heart development are largely poorly understood. Human embryonic stem (hES) cells, with their ability to differentiate into cardiomyocytes in culture, provide an in vitro model for human heart development. Furthermore, the ability to manipulate the differentiation of pluripotent cells also requires a thorough understanding of the molecular control of cardiogenesis. We employed a transcriptome analysis on enriched cardiomyocytes derived from hES cells and identified a large number of yet uncharacterized genes. We have evaluated the biological relevance of selected candidates by in situ hybridization during early mouse development and found that all were prominently expressed in cardiac structures. An initial functional characterization of genes by loss-of-function analysis employing morpholino injections into zebrafish embryos revealed severe cardiovascular defects indicating a critical function of selected gene for developmental processes. Our data demonstrate the potential power of integrating a study of hES cell differentiation in vitro with the analysis of heart development in zebrafish in vivo that provides new insights into the molecular processes of the heart formation in vertebrates.

Neuregulin/ErbB Signaling Regulates Cardiac Subtype Specification in Differentiating Human Embryonic Stem Cells

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a "working" chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes. To demonstrate intact neuregulin (NRG)-1β/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. All experiments used hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1β, an anti-NRG-1β neutralizing antibody, or the ErbB antagonist AG1478. We used 3 independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all 3 end points, we found that inhibition of NRG-1β/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. NRG-1β/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1β/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker.

Na+/Ca2+Exchanger is a Determinant of Excitation–Contraction Coupling in Human Embryonic Stem Cell–Derived Ventricular Cardiomyocytes

In adult cardiomyocytes (CMs), the Na(+)/Ca(2+) exchanger (NCX) is a well-defined determinant of Ca(2+) homeostasis. Developmentally, global NCX knockout in mice leads to abnormal myofibrillar organization, electrical defects, and early embryonic death. Little is known about the expression and function of NCX in human heart development. Self-renewable, pluripotent human embryonic stem cells (hESCs) can serve as an excellent experimental model. However, hESC-derived CMs are highly heterogeneous. A stably lentivirus-transduced hESC line (MLC2v-dsRed) was generated to express dsRed under the transcriptional control of the ventricular-restricted myosin light chain-2v (MLC2v) promoter. Electrophysiologically, dsRed+ cells differentiated from MLC2vdsRed hESCs displayed ventricular action potentials (AP), exclusively. Neither atrial nor pacemaker APs were observed. While I(Ca-L), I(f), and I(Kr) were robustly expressed, I(Ks) and I(K1) were absent in dsRed+ ventricular hESCCMs. Upon differentiation (7+40 to +90 days), the basal [Ca(2+)](i), Ca(2+) transient amplitude, maximum upstroke, and decay velocities significantly increased (P < 0.05). The I(Ca-L) antagonizer nifedipine (1 microM) decreased the Ca(2+) transient amplitude (to approximately 30%) and slowed the kinetics (by approximately 2-fold), but Ca(2+) transients could still be elicited even after complete ICa-L blockade, suggesting the presence of additional Ca(2+) influx(es). Indeed, Ni(2+)-sensitive INCX could be recorded in 7+40- and +90-day dsRed+ hESC-CMs, and its densities increased from -1.2 +/- 0.6 pA/pF at -120 mV and 3.6 +/- 1.0 pA/pF at 60 mV by 6- and 2-folds, respectively. With higher [Ca(2+)](i), 7+90-day ventricular hESC-CMs spontaneously but irregularly fired transients upon a single stimulus under an external Na(+)-free condition; however, without extracellular Na(+), nifedipine could completely inhibit Ca(2+) transients. We conclude that I(NCX) is functionally expressed in developing ventricular hESC-CMs and contributes to their excitation-contraction coupling.

Homozygous Mutations in the 5′ Region of the JUP Gene Result in Cutaneous Disease but Normal Heart Development in Children

Desmosomes are intercellular adhesive junctions and attachment sites for the intermediate filament (IF) cytoskeleton, prominent in tissues subject to high levels of mechanical stress such as the epidermis and heart. The obligate desmosomal constituent, plakoglobin (PG), is involved in coupling transmembrane desmosomal components with IFs. PG also contributes to intercellular adhesion through adherens junctions and has additional signaling roles. To date, two mutations in the gene encoding PG, JUP, have been described, and in both instances, patients harboring pathogenic mutations suffered from arrhythmogenic right ventricular cardiomyopathy with or without skin abnormalities. We describe homozygous nonsense mutation, p.S24X, and homozygous splice site mutation, c.468G>A, in the JUP gene that results in skin fragility, diffuse palmoplantar keratoderma, and woolly hair with no symptoms of cardiomyopathy. We show barely detectable levels of PG immunostaining in skin sections from patients harboring these mutations and show that an alternative AUG codon in p.S24X mRNA translates a 42-amino-acid N-terminal truncation. We conclude that PG is required for correct maintenance of skin integrity, and the absence of heart phenotype in patients suggests that aberrant PG expression does not compromise normal human heart development in children. Our findings provide new insight into the distinct roles that PG has in the epidermis and heart.

Dissecting spatio‐temporal protein networks driving human heart development and related disorders

Aberrant organ development is associated with a wide spectrum of disorders, from schizophrenia to congenital heart disease, but systems-level insight into the underlying processes is very limited. Using heart morphogenesis as general model for dissecting the functional architecture of organ development, we combined detailed phenotype information from deleterious mutations in 255 genes with high-confidence experimental interactome data, and coupled the results to thorough experimental validation. Hereby, we made the first systematic analysis of spatio-temporal protein networks driving many stages of a developing organ identifying several novel signaling modules. Our results show that organ development relies on surprisingly few, extensively recycled, protein modules that integrate into complex higher-order networks. This design allows the formation of a complicated organ using simple building blocks, and suggests how mutations in the same genes can lead to diverse phenotypes. We observe a striking temporal correlation between organ complexity and the number of discrete functional modules coordinating morphogenesis. Our analysis elucidates the organization and composition of spatio-temporal protein networks that drive the formation of organs, which in the future may lay the foundation of novel approaches in treatments, diagnostics, and regenerative medicine.

Examining the Cardiac NK-2 Genes in Early Heart Development

The cardiac NK-2 transcription factors are the vertebrate relatives of the Drosophila tinman gene. Without the Drosophila tinman gene, fruit flies fail to form their heart ("dorsal vessel"), and mutations or altered expression of cardiac NK-2 genes may lead to abnormal heart formation in vertebrates. Although the cardiac NK-2 gene NKX2-5 is recognized as an important factor in cases of human congenital heart disease and heart development in vertebrates, the roles of the other cardiac NK-2 genes are less clear. This report reviews what is known about the cardiac NK-2 genes in cardiac development, comparing studies in several different model systems.

Global Expression Profile of Highly Enriched Cardiomyocytes Derived from Human Embryonic Stem Cells

Human embryonic stem cells (hESC), with their ability to differentiate into cardiomyocytes in culture, hold great potential for cell replacement therapies and provide an in vitro model of human heart development. A genomewide characterization of the molecular phenotype of hESC-derived cardiomyocytes is important for their envisioned applications. We have employed a lineage selection strategy to generate a pure population of cardiomyocytes (>99%) from transgenic hESC lines. Global gene expression profiling showed that these cardiomyocytes are distinct from pluripotent and differentiated hESC cultures. Pure cardiomyocytes displayed similarities with heart tissue, but in many aspects presented an individual transcriptome pattern. A subset of 1,311 cardiac-enriched transcripts was identified, which were significantly overpresented (p < .01) in the Gene Ontology (GO) categories of heart function and heart development. Focused analysis of the GO categories ion transport, sarcomere, and heart development uncovered a unique molecular signature of hESC cardiomyocytes. Pathway analysis revealed an extensive cardiac transcription factor network and novel peroxisome proliferator-activated receptor signaling components within the cardiac-enriched genes. Notably, approximately 80% of these genes were previously uncharacterized. We have evaluated the biological relevance of four candidates-Rbm24, Tcea3, Fhod3, and C15orf52-by in situ hybridization during early mouse development and report that all were prominently expressed in cardiac structures. Our results provide the fundamental basis for a comprehensive understanding of gene expression patterns of hESC cardiomyocytes and will greatly help define biological processes and signaling pathways involved in hESC cardiomyogenic differentiation and in human heart development.

Cardiac Applications for Human Pluripotent Stem Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can self-renew indefinitely, while maintaining the capacity to differentiate into useful somatic cell types, including cardiomyocytes. As such, these stem cell types represent an essentially inexhaustible source of committed human cardiomyocytes of potential use in cell-based cardiac therapies, high-throughput screening and safety testing of new drugs, and modeling human heart development. These stem cell-derived cardiomyocytes have an unambiguous cardiac phenotype and proliferate robustly both in vitro and in vivo. Recent transplantation studies in preclinical models have provided exciting proof-of-principle for their use in infarct repair and in the formation of a "biological pacemaker". While these successes give reason for cautious optimism, major challenges remain to the successful application of hESCs (or hiPSCs) to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. In this review, we describe the phenotype of hESC- and hiPSC-derived cardiomyocytes, the state of preclinical transplantation studies with these cells, and potential approaches to overcome the aforementioned hurdles.

A functional genetic study identifies HAND1 mutations in septation defects of the human heart

Heart and neural crest derivatives expressed 1 (HAND1) is a basic helix-loop-helix (bHLH) transcription factor essential for mammalian heart development. Absence of Hand1 in mice results in embryonal lethality, as well as in a wide spectrum of cardiac abnormalities including failed cardiac looping, defective chamber septation and impaired ventricular development. Therefore, Hand1 is a strong candidate for the many cardiac malformations observed in human congenital heart disease (CHD). Recently, we identified a loss-of-function frameshift mutation (p.A126fs) in the bHLH domain of HAND1 frequent in hypoplastic hearts. This finding prompted us to continue our search for HAND1 gene mutations in a different cohort of malformed hearts affected primarily by septation defects. Indeed, in tissue samples of septal defects, we detected 32 sequence alterations leading to amino acid change, of which 12 are in the bHLH domain of HAND1. Interestingly, 10 sequence alterations, such as p.L28H and p.L138P, had been identified earlier in hypoplastic hearts, but the frequent p.A126fs mutation was absent except in one aborted case with ventricular septal defect and outflow tract abnormalities. Functional studies in yeast and mammalian cells enabled translation of sequence alterations to HAND1 transcriptional activity, which was reduced or abolished by certain mutations, notably p.L138P. Our results suggest that HAND1 may also be affected in septation defects of the human hearts, and thus has a broader role in human heart development and CHD.

Characterization of a t(5;8)(q31;q21) translocation in a patient with mental retardation and congenital heart disease: implications for involvement of RUNX1T1 in human brain and heart development

The chromosome break points of the t(8;21)(q21.3;q22.12) translocation associated with acute myeloid leukemia disrupt the RUNX1 gene (also known as AML1) and the RUNX1T1 gene (also known as CBFA2T3, MTG8 and ETO) and generate a RUNX1-RUNX1T1 fusion protein. Molecular characterization of the translocation break points in a t(5;8)(q32;q21.3) patient with mild-to-moderate mental retardation and congenital heart disease revealed that one of the break points was within the RUNX1T1 gene. Analysis of RUNX1T1 expression in human embryonic and fetal tissues suggests a role of RUNX1T1 in brain and heart development and support the notion that disruption of the RUNX1T1 gene is associated with the patient's phenotype.

Endoglin (CD105) Expression in the Human Heart Throughout Gestation: An Immunohistochemical Study

Endoglin is an 180 KDa protein which plays an important role in the vascular system and cardiac embryogenesis. Indeed, monoallelic mutations in the endoglin gene are associated with the development of hereditary hemorrhagic telangiectasia type 1; moreover endoglin knockout mice die precociously because of severe arteriovenous and cardiac malformations. In this study, endoglin immunohistochemical expression was analyzed in the heart of 23 fetuses (9-38 weeks), 5 of which displayed cardiac malformations, as well as in cardiac samples from 4 preterm and 1 term infants. At the ninth week, endoglin expression was recorded in the endocardium; it extended into the epicardial and myocardial vessels by the 10th week. This pattern was maintained throughout gestation in most fetuses, but not in those with cardiac malformations. Endoglin expression up to term gestation indicates that its role in human heart development is not limited to the early gestation. Endoglin altered expression in association with cardiac defects further highlights its importance in normal cardiac embryogenesis and morphogenesis.

Myosin-activating protein kinases in human myocardium: Localization and content

Using the immunofluorescence approach, we have determined that the recently detected protein kinases, among which are RhoA-activated kinase, integrin-linked kinase, zipper interacting protein kinase, and death-associated protein kinase, which are capable of phosphorylating myosin, are localized in the Z-lines sarcomeres of human myocardium. Additionally, we studied the content of integrin-linked and zipper interacting protein kinases in human embryonic myocardium, as well as in normal and hypertrophic adult human heart. The content of these protein kinases in adult normal myocardium increases in comparison with the embryonic heart. The content of integrin-linked and zipper interacting protein kinases in hypertrophic myocardium is higher compared with the normal adult heart. The data obtained suggest the involvement of these protein kinases in the development and hypertrophy of human heart.

ZNF418, a novel human KRAB/C2H2 zinc finger protein, suppresses MAPK signaling pathway

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporal-spatial manner. Zinc finger-containing transcription factors have been implicated as critical regulators of multiple cardiac-expressed genes, and are thought to be important for human heart development and diseases. Here, we have identified and characterized a novel zinc finger gene named ZNF418 from a human embryo heart cDNA library. The gene spans 13.5 kb on chromosome 19q13.43 encompassing six exons, and transcribes a 3.7-kb mRNA that encodes a protein with 676 amino acid residues. The predicted protein contains a KRAB-A box and 17 tandem C2H2 type zinc finger motifs. Northern blot analysis indicates that ZNF418 is expressed in multiple fetal and adult tissues, but is expressed at higher levels in the heart. Reporter gene assays show that ZNF418 is a transcriptional repressor, and the KRAB motif of ZNF418 represents the basal repressive domain. Overexpression of ZNF418 in COS-7 cells inhibits the transcriptional activity of SRE and AP-1 which may be silenced by siRNA. These results suggest that ZNF418 is a member of the zincfinger transcription factor family and may act as a negative regulator in MAPK signaling pathway.

The Role of Shear Stress on ET-1, KLF2, and NOS-3 Expression in the Developing Cardiovascular System of Chicken Embryos in a Venous Ligation Model

In this review, the role of wall shear stress in the chicken embryonic heart is analyzed to determine its effect on cardiac development through regulating gene expression. Therefore, background information is provided for fluid dynamics, normal chicken and human heart development, cardiac malformations, cardiac and vitelline blood flow, and a chicken model to induce cardiovascular anomalies. A set of endothelial shear stress-responsive genes coding for endothelin-1 (ET-1), lung Krüppel-like factor (LKLF/KLF2), and endothelial nitric oxide synthase (eNOS/NOS-3) are active in development and are specifically addressed.

The area composita of adhering junctions connecting heart muscle cells of vertebrates – IV: Coalescence and amalgamation of desmosomal and adhaerens junction components – Late processes in mammalian heart development

In the adult mammalian heart, the cardiomyocytes and thus their terminally anchored myofibrillar bundles are connected by large arrays of closely spaced or even fused adhering junctions (AJs), termed “intercalated disks” (IDs). In recent years, the ID complex has attracted special attention as it has become clear that several human hereditary cardiomyopathies are caused by mutations of genes encoding ID marker proteins, in particular some that are also known as constituents of epithelial desmosomes. Previously, we have shown that in the mature myocardial ID the compositional differences between desmosome-like and adhaerens junctions are, by and large, lost and a composite hybrid structure, the area composita, is formed. We now report results from immunofluorescence and (immuno-)electron microscopic studies of heart formation during mouse embryogenesis and postnatal growth and show that the formation of the IDs with extended area composita structures is a late, primarily postnatal process. While up to birth small distinct desmosomes and AJs are resolved as predominant ID structures, areae compositae of increasing sizes and merged marker protein patterns occupy most of the IDs in the mature heart. Differences in the patterns of ID formation and amalgamation of the two ensembles of junction proteins in time and space are also demonstrated. Together with corresponding observations during rat and human heart development our results indicate that ID topogenesis and area composita formation are also late developmental processes in other mammals. We discuss the importance of the ID and the areae compositae in cardiac functions and, consequently, in cardiomyopathies and possible myocardial regeneration processes.