LncRNAs in the Dlk1-Dio3 Domain Are Essential for Mid-Embryonic Heart Development.

The Dlk1-Dio3 domain is important for normal embryonic growth and development. The heart is the earliest developing and functioning organ of the embryo. In this study, we constructed a transcriptional termination model by inserting termination sequences and clarified that the lack of long non-coding RNA (lncRNA) expression in the Dlk1-Dio3 domain caused the death of maternal insertion mutant (MKI) and homozygous mutant (HOMO) mice starting from E13.5. Parental insertion mutants (PKI) can be born and grow normally. Macroscopically, dying MKI and HOMO embryos showed phenomena such as embryonic edema and reduced heart rate. Hematoxylin and eosin (H.E.) staining showed thinning of the myocardium in MKI and HOMO embryos. In situ hybridization (IHC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed downregulation of lncGtl2, Rian, and Mirg expression in MKI and HOMO hearts. The results of single-cell RNA sequencing (scRNA-Seq) analysis indicated that the lack of lncRNA expression in the Dlk1-Dio3 domain led to reduced proliferation of epicardial cells and may be an important cause of cardiac dysplasia. In conclusion, this study demonstrates that Dlk1-Dio3 domain lncRNAs play an integral role in ventricular development.

Investigation of verapamil-induced cardiorenal dysfunction and compensatory ion regulation in zebrafish embryos.

The purpose of the present study was to investigate the development of verapamil-induced cardiorenal failure and the response of epidermal ionocytes in zebrafish embryos to this syndrome. Zebrafish embryos were exposed to verapamil for 24 h at different developmental stages (48, 72, and 96 h post-fertilization). The exposure resulted in the generation of edema in the pericardial and yolk sac regions, with more-pronounced effects observed in later-stage embryos. Cardiac parameters showed a suppressed heart rate at all stages, with a more-significant effect appearing in later stages. Verapamil also affected cardiac parameters including the end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), and cardiac output (CO), indicating negative overall effects on cardiac performance. mRNA levels of heart failure markers (nppa and nppb genes) were upregulated in verapamil-exposed embryos at all stages. Renal function was impaired as FITC-dextran excretion was suppressed. A whole-embryo ion content analysis revealed significant increases in sodium and calcium contents in verapamil-exposed embryos. The density of epidermal ionocytes increased, and the apical membrane of ionocytes was enlarged, indicating upregulation of ion uptake. In addition, mRNA levels of several ion transporter genes (rhcg1, slc9a3, atp6v1a, atp2b1a, trpv6, and slc12a10.2) were significantly upregulated in verapamil-exposed embryos. In summary, prolonged exposure to verapamil can induce cardiorenal failure which triggers compensatory upregulation of ionocytes in zebrafish embryos.

Cardiotoxicity of Cadmium and Its Effects on Heart Efficiency During Early and Late Chick Embryogenesis.

Cadmium (Cd) is a dangerous heavy metal that is non-degradable in the environment. Many organs can accumulate Cd and adversely affect organ function and health. Cd is considered as a teratogenic and embryotoxic agent. This study aims to evaluate the teratogenicity of Cd at concentrations lesser than the permissible and its effects on the heart during chick embryogenesis. Fertilized eggs of the chick Gallus domesticus were divided into; control, saline injected and four experimental groups injected with single doses of 5, 25, 50 or 75 µM of CdCl2. Histological observations of the heart before hatching and the cardiomyocytes after hatching were recorded. Morphometric measurements of heart chambers were achieved at 3, 4 and 6 days of incubation. Electrocardiograph and respiratory rate were recorded at tenth day. Different cardiac problems had been brought on by Cd. In comparison to controls, the heart looked much larger, and in certain cases, growth retardation was seen. Degeneration in heart walls and malformations of dorsal aorta were noticed. Morphometrically, the width and wall thickness of heart chambers showed significant changes. Heart beats and respiratory rate significantly decreased compared to control. The cardiotoxic effect of Cd on heart compartments structure and function was dose dependent. One of Cd toxicity is its ability to induce cellular oxidative stress. The heart in particular is sensitive to oxidative stress. Cardiac oxidative stress might intensify heart failure and promote disease progression. Calcium is one of the components that is needed for normal heart work. Cd might interfere with calcium metabolism by removing it from the body.

From promoter motif to cardiac function: a single DPE motif affects transcription regulation and organ function in vivo.

Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7 bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.

The chromatin regulator Ankrd11 controls cardiac neural crest cell-mediated outflow tract remodeling and heart function.

ANKRD11 (Ankyrin Repeat Domain 11) is a chromatin regulator and a causative gene for KBG syndrome, a rare developmental disorder characterized by multiple organ abnormalities, including cardiac defects. However, the role of ANKRD11 in heart development is unknown. The neural crest plays a leading role in embryonic heart development, and its dysfunction is implicated in congenital heart defects. We demonstrate that conditional knockout of Ankrd11 in the murine embryonic neural crest results in persistent truncus arteriosus, ventricular dilation, and impaired ventricular contractility. We further show these defects occur due to aberrant cardiac neural crest cell organization leading to outflow tract septation failure. Lastly, knockout of Ankrd11 in the neural crest leads to impaired expression of various transcription factors, chromatin remodelers and signaling pathways, including mTOR, BMP and TGF-ß in the cardiac neural crest cells. In this work, we identify Ankrd11 as a regulator of neural crest-mediated heart development and function.

Cardiac Development and Factors Influencing the Development of Congenital Heart Defects (CHDs): Part I.

The traditional description of cardiac development involves progression from a cardiac crescent to a linear heart tube, which in the phase of transformation into a mature heart forms a cardiac loop and is divided with the septa into individual cavities. Cardiac morphogenesis involves numerous types of cells originating outside the initial cardiac crescent, including neural crest cells, cells of the second heart field origin, and epicardial progenitor cells. The development of the fetal heart and circulatory system is subject to regulatation by both genetic and environmental processes. The etiology for cases with congenital heart defects (CHDs) is largely unknown, but several genetic anomalies, some maternal illnesses, and prenatal exposures to specific therapeutic and non-therapeutic drugs are generally accepted as risk factors. New techniques for studying heart development have revealed many aspects of cardiac morphogenesis that are important in the development of CHDs, in particular transposition of the great arteries.

Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.

The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.

Cardiac programming in the placentally restricted sheep fetus in early gestation.

Fetal growth restriction (FGR) occurs in 8% of human pregnancies, and the growth restricted newborn is at a greater risk of developing heart disease in later adult life. In sheep, experimental restriction of placental growth (PR) from conception results in FGR, a decrease in cardiomyocyte endowment and an upregulation of pathological hypertrophic signalling in the fetal heart in late gestation. However, there is no change in the expression of markers of cellular proliferation nor in the level of cardiomyocyte apoptosis in the heart of the PR fetus in late gestation. This suggests that FGR arises early in gestation and programs a decrease in cardiomyocyte endowment in early, rather than late, gestation. Here, control and PR fetal sheep were humanely killed at 55 days' gestation (term, 150 days). Fetal body and heart weight were lower in PR compared with control fetuses and there was evidence of sparing of fetal brain growth. While there was no change in the proportion of cardiomyocytes that were proliferating in the early gestation PR heart, there was an increase in measures of apoptosis, and markers of autophagy and pathological hypertrophy in the PR fetal heart. These changes in early gestation highlight that FGR is associated with evidence of early cell death and compensatory hypertrophic responses of cardiomyocytes in the fetal heart. The data suggest that early placental restriction results in a decrease in the pool of proliferative cardiomyocytes in early gestation, which would limit cardiomyocyte endowment in the heart of the PR fetus in late gestation. KEY POINTS: Placental restriction leading to fetal growth restriction (FGR) and chronic fetal hypoxaemia in sheep results in a decrease in cardiomyocyte endowment in late gestation. FGR did not change cardiomyocyte proliferation during early gestation but did result in increased apoptosis and markers of autophagy in the fetal heart, which may result in the decreased endowment of cardiomyocytes observed in late gestation. FGR in early gestation also results in increased hypoxia inducible factor signalling in the fetal heart, which in turn may result in the altered expression of epigenetic regulators, increased expression of insulin-like growth factor 2 and cardiomyocyte hypertrophy during late gestation and after birth.

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart.

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Inflow Tract Development.

The development of the inflow tract is undoubtedly one of the most complex remodeling events in the formation of the four-chambered heart. It involves the creation of two separate atrial chambers, the formation of an atrial/atrioventricular (AV) septal complex, the incorporation of the caval veins and coronary sinus into the right atrium, and the remodeling events that result in pulmonary venous return draining into the left atrium. In these processes, the atrioventricular mesenchymal complex, consisting of the major atrioventricular (AV) cushions, the mesenchymal cap on the primary atrial septum (pAS), and the dorsal mesenchymal protrusion (DMP), plays a crucial role.

Human Cardiac Development.

Many aspects of heart development are topographically complex and require three-dimensional (3D) reconstruction to understand the pertinent morphology. We have recently completed a comprehensive primer of human cardiac development that is based on firsthand segmentation of structures of interest in histological sections. We visualized the hearts of 12 human embryos between their first appearance at 3.5 weeks and the end of the embryonic period at 8 weeks. The models were presented as calibrated, interactive, 3D portable document format (PDF) files. We used them to describe the appearance and the subsequent remodeling of around 70 different structures incrementally for each of the reconstructed stages. In this chapter, we begin our account by describing the formation of the single heart tube, which occurs at the end of the fourth week subsequent to conception. We describe its looping in the fifth week, the formation of the cardiac compartments in the sixth week, and, finally, the septation of these compartments into the physically separated left- and right-sided circulations in the seventh and eighth weeks. The phases are successive, albeit partially overlapping. Thus, the basic cardiac layout is established between 26 and 32 days after fertilization and is described as Carnegie stages (CSs) 9 through 14, with development in the outlet component trailing that in the inlet parts. Septation at the venous pole is completed at CS17, equivalent to almost 6 weeks of development. During Carnegie stages 17 and 18, in the seventh week, the outflow tract and arterial pole undergo major remodeling, including incorporation of the proximal portion of the outflow tract into the ventricles and transfer of the spiraling course of the subaortic and subpulmonary channels to the intrapericardial arterial trunks. Remodeling of the interventricular foramen, with its eventual closure, is complete at CS20, which occurs at the end of the seventh week. We provide quantitative correlations between the age of human and mouse embryos as well as the Carnegie stages of development. We have also set our descriptions in the context of variations in the timing of developmental features.

Establishment of Cardiac Laterality.

Formation of the vertebrate heart with its complex arterial and venous connections is critically dependent on patterning of the left-right axis during early embryonic development. Abnormalities in left-right patterning can lead to a variety of complex life-threatening congenital heart defects. A highly conserved pathway responsible for left-right axis specification has been uncovered. This pathway involves initial asymmetric activation of a nodal signaling cascade at the embryonic node, followed by its propagation to the left lateral plate mesoderm and activation of left-sided expression of the Pitx2 transcription factor specifying visceral organ asymmetry. Intriguingly, recent work suggests that cardiac laterality is encoded by intrinsic cell and tissue chirality independent of Nodal signaling. Thus, Nodal signaling may be superimposed on this intrinsic chirality, providing additional instructive cues to pattern cardiac situs. The impact of intrinsic chirality and the perturbation of left-right patterning on myofiber organization and cardiac function warrants further investigation. We summarize recent insights gained from studies in animal models and also some human clinical studies in a brief overview of the complex processes regulating cardiac asymmetry and their impact on cardiac function and the pathogenesis of congenital heart defects.

Cardiac Development at a Single-Cell Resolution.

Mammalian cardiac development is a complex, multistage process. Though traditional lineage tracing studies have characterized the broad trajectories of cardiac progenitors, the advent and rapid optimization of single-cell RNA sequencing methods have yielded an ever-expanding toolkit for characterizing heterogeneous cell populations in the developing heart. Importantly, they have allowed for a robust profiling of the spatiotemporal transcriptomic landscape of the human and mouse heart, revealing the diversity of cardiac cells-myocyte and non-myocyte-over the course of development. These studies have yielded insights into novel cardiac progenitor populations, chamber-specific developmental signatures, the gene regulatory networks governing cardiac development, and, thus, the etiologies of congenital heart diseases. Furthermore, single-cell RNA sequencing has allowed for the exquisite characterization of distinct cardiac populations such as the hard-to-capture cardiac conduction system and the intracardiac immune population. Therefore, single-cell profiling has also resulted in new insights into the regulation of cardiac regeneration and injury repair. Single-cell multiomics approaches combining transcriptomics, genomics, and epigenomics may uncover an even more comprehensive atlas of human cardiac biology. Single-cell analyses of the developing and adult mammalian heart offer an unprecedented look into the fundamental mechanisms of cardiac development and the complex diseases that may arise from it.

Cardiac Development Long Non-Coding RNA (CARDEL) Is Activated during Human Heart Development and Contributes to Cardiac Specification and Homeostasis.

Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac differentiation using human pluripotent stem cells (hPSCs) has been used to uncover the intricate gene-network regulation involved in the proper formation and function of the human heart. Here, we searched for uncharacterized cardiac-development genes by combining a temporal evaluation of human cardiac specification in vitro with an analysis of gene expression in fetal and adult heart tissue. We discovered that CARDEL (CARdiac DEvelopment Long non-coding RNA; LINC00890; SERTM2) expression coincides with the commitment to the cardiac lineage. CARDEL knockout hPSCs differentiated poorly into cardiac cells, and hPSC-derived cardiomyocytes showed faster beating rates after controlled overexpression of CARDEL during differentiation. Altogether, we provide physiological and molecular evidence that CARDEL expression contributes to sculpting the cardiac program during cell-fate commitment.

miR-1 as a Key Epigenetic Regulator in Early Differentiation of Cardiac Sinoatrial Region.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

Neural Crest.

This chapter discusses the role of cardiac neural crest cells in the formation of the septum that divides the cardiac arterial pole into separate systemic and pulmonary arteries. Further, cardiac neural crest cells directly support the normal development and patterning of derivatives of the caudal pharyngeal arches, including the great arteries, thymus, thyroid, and parathyroids. Recently, cardiac neural crest cells have also been shown to indirectly influence the development of the secondary heart field, another derivative of the caudal pharynx, by modulating signaling in the pharynx. The contribution and function of the cardiac neural crest cells has been learned in avian models; most of the genes associated with cardiac neural crest function have been identified using mouse models. Together these studies show that the neural crest cells may not only critical for normal cardiovascular development but also may be involved secondarily because they represent a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Cardiac neural crest cells span from the caudal pharynx into the outflow tract, and therefore may be susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations resulting from genetic and/or environmental insults necessarily requires better understanding the role of cardiac neural crest cells in cardiac development.

Cardiac Development and Animal Models of Congenital Heart Defects.

The major events of cardiac development, including early heart formation, chamber morphogenesis and septation, and conduction system and coronary artery development, are briefly reviewed together with a short introduction to the animal species commonly used to study heart development and model congenital heart defects (CHDs).

Evolutionary Aspects of Chamber Formation and Septation.

The formed hearts of vertebrates are widely different in anatomy and performance, yet their embryonic hearts are surprisingly similar. Developmental and molecular biology are making great advances in reconciling these differences by revealing an evolutionarily conserved building plan to the vertebrate heart. This suggests that perspectives from evolution may improve our understanding of the formation of the human heart. Here, we exemplify this approach by discussing atrial and ventricular septation and the associated processes of remodeling of the atrioventricular junction and formation of the atrioventricular insulating plane.

Inter- and Intracellular Signaling Pathways.

Cardiovascular diseases, both congenital and acquired, are the leading cause of death worldwide, associated with significant health consequences and economic burden. Due to major advances in surgical procedures, most patients with congenital heart disease (CHD) survive into adulthood but suffer from previously unrecognized long-term consequences, such as early-onset heart failure. Therefore, understanding the molecular mechanisms resulting in heart defects and the lifelong complications due to hemodynamic overload are of utmost importance. Congenital heart disease arises in the first trimester of pregnancy, due to defects in the complex morphogenetic patterning of the heart. This process is coordinated through a complicated web of intercellular communication between the epicardium, the endocardium, and the myocardium. In the postnatal heart, similar crosstalk between cardiomyocytes, endothelial cells, and fibroblasts exists during pathological hemodynamic overload that emerges as a consequence of a congenital heart defect. Ultimately, communication between cells triggers the activation of intracellular signaling circuits, which allow fine coordination of cardiac development and function. Here, we review the inter- and intracellular signaling mechanisms in the heart as they were discovered mainly in genetically modified mice.