Epicardium and Coronary Vessels.

Congenital anomalies and acquired diseases of the coronary blood vessels are of great clinical relevance. The early diagnosis of these conditions remains, however, challenging. In order to improve our knowledge of these ailments, progress has to be achieved in the research of the molecular and cellular mechanisms that control development of the coronary vascular bed. The aim of this chapter is to provide a succint account of the key elements of coronary blood vessel development, especially in the context of the role played by the epicardium and epicardial cellular derivatives. We will discuss the importance of the epicardium in coronary blood vessel morphogenesis, from the contribution of the epicardially derived mesenchyme to these blood vessels to its role as an instructive signaling center, attempting to relate these concepts to the origin of coronary disease.

Llgl1 mediates timely epicardial emergence and establishment of an apical laminin sheath around the trabeculating cardiac ventricle.

During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.

miR-194-3p regulates epithelial-mesenchymal transition in embryonic epicardial cells via p120/ß-catenin signaling.

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor ß (TGF-ß) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin-3'UTR-MUT. In epicardial cells, TGF-ß-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences ß-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin. In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters ß-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

Protocol for the isolation and single-nuclei multiomic analyses of the human fetal epicardium.

The characterization of cell populations that reside in the outer layer of the heart has been hindered by difficulties in their isolation. Here, we present a protocol for isolation and single-nuclei multiomic analyses of the human fetal epicardium. We describe steps for microdissection, isolation, and enrichment of epicardial cells by mechanical dissociations and direct lysis. We then detail procedures for integrating transcriptome and chromatin accessibility datasets. This approach allows the analysis of diverse cell populations, marked by unique cis-regulatory elements. For complete details on the use and execution of this protocol, please refer to Travisano et al.1.

Triphenyl phosphate-induced pericardial edema is associated with elevated epidermal ionocytes within zebrafish embryos.

Triphenyl phosphate (TPHP) is an organophosphate ester-based plasticizer and flame retardant. The objective of this study was to identify the potential role of epidermal ionocytes in mediating TPHP-induced pericardial edema within zebrafish embryos. Exposure to TPHP from 24 to 72 h post fertilization (hpf) resulted in a significant increase in pericardial edema and the number of ionocytes at 72 hpf relative to time-matched embryos treated with vehicle. In addition, co-exposure of embryos to mannitol (an osmotic diuretic) blocked TPHP-induced pericardial edema and effects on ionocyte abundance. However, knockdown of ATPase1a1.4 - an abundant Na+/K+-ATPase localized to epidermal ionocytes - mitigated TPHP-induced effects on ionocyte abundance but not pericardial edema, whereas co-exposure of embryos to ouabain - a Na+/K+-ATPase inhibitor - enhanced TPHP-induced pericardial edema but not ionocyte abundance. Overall, our findings suggest that TPHP may have multiple mechanisms of toxicity leading to an increase in ionocyte abundance and pericardial edema within developing zebrafish embryos.

Every Beat You Take-The Wilms' Tumor Suppressor WT1 and the Heart.

Nearly three decades ago, the Wilms' tumor suppressor Wt1 was identified as a crucial regulator of heart development. Wt1 is a zinc finger transcription factor with multiple biological functions, implicated in the development of several organ systems, among them cardiovascular structures. This review summarizes the results from many research groups which allowed to establish a relevant function for Wt1 in cardiac development and disease. During development, Wt1 is involved in fundamental processes as the formation of the epicardium, epicardial epithelial-mesenchymal transition, coronary vessel development, valve formation, organization of the cardiac autonomous nervous system, and formation of the cardiac ventricles. Wt1 is further implicated in cardiac disease and repair in adult life. We summarize here the current knowledge about expression and function of Wt1 in heart development and disease and point out controversies to further stimulate additional research in the areas of cardiac development and pathophysiology. As re-activation of developmental programs is considered as paradigm for regeneration in response to injury, understanding of these processes and the molecules involved therein is essential for the development of therapeutic strategies, which we discuss on the example of WT1.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Characterization of a common progenitor pool of the epicardium and myocardium.

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.

Notch and Bmp signaling pathways act coordinately during the formation of the proepicardium.

BACKGROUND: The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish. RESULTS: Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling. CONCLUSIONS: Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer.

Analysis of wt1a reporter line expression levels during proepicardium formation in the zebrafish.

The epicardium is the outer mesothelial layer of the heart. It covers the myocardium and plays important roles in both heart development and regeneration. It is derived from the proepicardium (PE), groups of cells that emerges at early developmental stages from the dorsal pericardial layer (DP) close to the atrio-ventricular canal and the venous pole of the heart-tube. In zebrafish, PE cells extrude apically into the pericardial cavity as a consequence of DP tissue constriction, a process that is dependent on Bmp pathway signaling. Expression of the transcription factor Wilms tumor-1, Wt1, which is a leader of important morphogenetic events such as apoptosis regulation or epithelial-mesenchymal cell transition, is also necessary during PE formation. In this study, we used the zebrafish model to compare intensity level of the wt1a reporter line epi:GFP in PE and its original tissue, the DP. We found that GFP is present at higher intensity level in the PE tissue, and differentially wt1 expression at pericardial tissues could be involved in the PE formation process. Our results reveal that bmp2b overexpression leads to enhanced GFP level both in DP and in PE tissues.

MiR-195 enhances cardiomyogenic differentiation of the proepicardium/septum transversum by Smurf1 and Foxp1 modulation.

Cardiovascular development is a complex developmental process in which multiple cell lineages are involved, namely the deployment of first and second heart fields. Beside the contribution of these cardiogenic fields, extracardiac inputs to the developing heart are provided by the migrating cardiac neural crest cells and the proepicardial derived cells. The proepicardium (PE) is a transitory cauliflower-like structure located between the cardiac and hepatic primordia. The PE is constituted by an internal mesenchymal component surrounded by an external epithelial lining. With development, cells derived from the proepicardium migrate to the neighboring embryonic heart and progressive cover the most external surface, leading to the formation of the embryonic epicardium. Experimental evidence in chicken have nicely demonstrated that epicardial derived cells can distinctly contribute to fibroblasts, endothelial and smooth muscle cells. Surprisingly, isolation of the developing PE anlage and ex vivo culturing spontaneously lead to differentiation into beating cardiomyocytes, a process that is enhanced by Bmp but halted by Fgf administration. In this study we provide a comprehensive characterization of the developmental expression profile of multiple microRNAs during epicardial development in chicken. Subsequently, we identified that miR-125, miR-146, miR-195 and miR-223 selectively enhance cardiomyogenesis both in the PE/ST explants as well as in the embryonic epicardium, a Smurf1- and Foxp1-driven process. In addition we identified three novel long non-coding RNAs with enhanced expression in the PE/ST, that are complementary regulated by Bmp and Fgf administration and well as by microRNAs that selectively promote cardiomyogenesis, supporting a pivotal role of these long non coding RNAs in microRNA-mediated cardiomyogenesis of the PE/ST cells.

PRMT1-p53 Pathway Controls Epicardial EMT and Invasion.

Epicardial cells are cardiac progenitors that give rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during development. An integral phase of epicardial fate transition is epithelial-to-mesenchymal transition (EMT) that confers motility. We uncover an essential role for the protein arginine methyltransferase 1 (PRMT1) in epicardial invasion and differentiation. Using scRNA-seq, we show that epicardial-specific deletion of Prmt1 reduced matrix and ribosomal gene expression in epicardial-derived cell lineages. PRMT1 regulates splicing of Mdm4, which is a key controller of p53 stability. Loss of PRMT1 leads to accumulation of p53 that enhances Slug degradation and blocks EMT. During heart development, the PRMT1-p53 pathway is required for epicardial invasion and formation of epicardial-derived lineages: cardiac fibroblasts, coronary smooth muscle cells, and pericytes. Consequently, this pathway modulates ventricular morphogenesis and coronary vessel formation. Altogether, our study reveals molecular mechanisms involving the PRMT1-p53 pathway and establish its roles in heart development.

Spatiotemporal Analysis Reveals Overlap of Key Proepicardial Markers in the Developing Murine Heart.

The embryonic epicardium, originating from the proepicardial organ (PEO), provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts, and vascular smooth muscle cells. Maximizing the regenerative capacity of the adult epicardium depends on recapitulating embryonic cell fates. The potential of the epicardium to contribute coronary endothelium is unclear, due to conflicting Cre-based lineage trace data. Controversy also surrounds when epicardial cell fate becomes restricted. Here, we systematically investigate expression of five widely used epicardial markers, Wt1, Tcf21, Tbx18, Sema3d, and Scx, over the course of development. We show overlap of markers in all PEO and epicardial cells until E13.5, and find no evidence for discrete proepicardial sub-compartments that might contribute coronary endothelium via the epicardial layer. Our findings clarify a number of prevailing discrepancies and support the notion that epicardium-derived cell fate, to form fibroblasts or mural cells, is specified after epithelial-mesenchymal transition, not pre-determined within the PEO.

Epicardial cell lineages and the origin of the coronary endothelium.

The embryonic epicardium generates a population of epicardial-derived mesenchymal cells (EPDC) whose contribution to the coronary endothelium is minor or, according to some reports, negligible. We have compared four murine cell-tracing models related to the EPDC in order to elucidate this contribution. Cre recombinase was expressed under control of the promoters of the Wilms' tumor suppressor (Wt1), the cardiac troponin (cTnT), and the GATA5 genes, activating expression of the R26REYFP reporter. We have also used the G2 enhancer of the GATA4 gene as a driver due to its activation in the proepicardium. Recombination was found in most of the epicardium/EPDC in all cases. The contribution of these lineages to the cardiac endothelium was analyzed using confocal microscopy and flow cytometry. G2-GATA4 lineage cells are the most frequent in the endothelium, probably due to the recruitment of circulating endothelial progenitors. The contribution of the WT1 cell lineage increases along gestation due to further endothelial expression of WT1. GATA5 and cTnT lineages represent 4% of the cardiac endothelial cells throughout the gestation, probably standing for the actual EPDC contribution to the coronary endothelium. These results suggest caution when using a sole cell-tracing model to study the fate of the EPDC.

Functional Heterogeneity within the Developing Zebrafish Epicardium.

The epicardium is essential during cardiac development, homeostasis, and repair, and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity, and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA sequencing uncovered three epicardial subpopulations with specific genetic programs and distinctive spatial distribution. Perturbation of unique gene signatures uncovered specific functions associated with each subpopulation and established epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. Understanding which mechanisms epicardial cells employ to establish a functional epicardium and how they communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease.

Epicardium in Heart Development.

The epicardium, the outermost tissue layer that envelops all vertebrate hearts, plays a crucial role in cardiac development and regeneration and has been implicated in potential strategies for cardiac repair. The heterogenous cell population that composes the epicardium originates primarily from a transient embryonic cell cluster known as the proepicardial organ (PE). Characterized by its high cellular plasticity, the epicardium contributes to both heart development and regeneration in two critical ways: as a source of progenitor cells and as a critical signaling hub. Despite this knowledge, there are many unanswered questions in the field of epicardial biology, the resolution of which will advance the understanding of cardiac development and repair. We review current knowledge in cross-species epicardial involvement, specifically in relation to lineage specification and differentiation during cardiac development.

The Chicken as a Model Organism to Study Heart Development.

Heart development is a complex process and begins with the long-range migration of cardiac progenitor cells during gastrulation. This culminates in the formation of a simple contractile tube with multiple layers, which undergoes remodeling into a four-chambered heart. During this morphogenesis, additional cell populations become incorporated. It is important to unravel the underlying genetic and cellular mechanisms to be able to identify the embryonic origin of diseases, including congenital malformations, which impair cardiac function and may affect life expectancy or quality. Owing to the evolutionary conservation of development, observations made in nonamniote and amniote vertebrate species allow us to extrapolate to human. This review will focus on the contributions made to a better understanding of heart development through studying avian embryos-mainly the chicken but also quail embryos. We will illustrate the classic and recent approaches used in the avian system, give an overview of the important discoveries made, and summarize the early stages of cardiac development up to the establishment of the four-chambered heart.

Maternal voluntary exercise mitigates oxidative stress and incidence of congenital heart defects in pre-gestational diabetes.

Women with pre-gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia-induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre-gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes-induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial-mesenchymal transition at E12.5 was significantly lower with pre-gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non-exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre-gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.

Embryonic Chicken (Gallus gallus domesticus) as a Model of Cardiac Biology and Development.

Cardiovascular disease remains one of the top contributors to morbidity and mortality in the United States. Increasing evidence suggests that many processes, pathways, and programs observed during development and organogenesis are recapitulated in adults in the face of disease. Therefore, a heightened understanding of cardiac development and organogenesis will help increase our understanding of developmental defects and cardiovascular diseases in adults. Chicks have long served as a model system in which to study developmental problems. Detailed descriptions of morphogenesis, low cost, accessibility, ease of manipulation, and the optimization of genetic engineering techniques have made chicks a robust model for studying development and make it a powerful platform for cardiovascular research. This review summarizes the cardiac developmental milestones of embryonic chickens, practical considerations when working with chicken embryos, and techniques available for use in chicks (including tissue chimeras, genetic manipulations, and live imaging). In addition, this article highlights examples that accentuate the utility of the embryonic chicken as model system in which to study cardiac development, particularly epicardial development, and that underscore the importance of how studying development informs our understanding of disease.

Actin dynamics and the Bmp pathway drive apical extrusion of proepicardial cells.

The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.