Studying Heart Development Research Articles

Myocardial trabeculation in embryos of Scyliorhinus canicula (Elasmobranchii, Chondrichthyans)

Currently, three types of ventricular myoarchitecture are recognized in vertebrates, namely compact, spongy (trabeculated) and mixed myocardium. Mixed myocardium, which has been recently proposed as the primitive condition in gnathostomes, is composed of two myocardial layers: an inner trabeculated and an outer compact one. The trabeculation process has been studied in teleosts, showing exclusively spongy myocardium, and mammals and birds, characterized by a compact myocardial ventricular wall. In zebrafish, mouse and chicken embryos, the trabeculae develop as luminal myocardial ridges protruding into the lumen. In mammals and birds, further compactation of trabeculae leads to the formation of a compact layer. The potential mechanisms that may contribute to the formation of the ridges are under discussion and include myocardial proliferation, endocardial invagination, and bending of the entire myocardial layer. However, no description of the development of the mixed myocardium is available.To shed some light on this issue, we have studied the heart development of an elasmobranch species with mixed myocardium, the lesser spotted dogfish (Scyliorhinus canicula; Chondrichthyes), by means of histological and immunohistochemical techniques for light microscopy, semithin sections, scanning electron microscopy and transmission electron microscopy.At the beginning of developmental stage 27 the ventricle has a single layer of cardiomyocytes lined by endocardium. At stage 28, the cardiomyocytes proliferate and stratified in several cell layers. Small spaces can be seen in between cardiomyocytes of the inner layers and the endocardium locally invaginates. At stage 29, bigger spaces lined by endocardial cells are connected with the ventricular lumen. Then, two layers, outer and inner, are recognized in the ventricular wall. The outer layer is the presumptive compact myocardium and the inner one is the presumptive trabeculated myocardium. At stage 30, the trabeculae become more complex, showing a bunch‐like branching pattern. At stage 33, the inner trabeculated layer is similar to that of the adult heart, while the outer layer starts to become thicker to form the definitive compact layer.Our results suggest that in the dogfish the intertrabecular spaces develop by connections between early intramyocardial spaces and the lumen of the ventricle through invaginations of the endocardial line. Chondrichthyans are the earliest diverged lineage of gnathostomes and, consequently, they have the most primitive cardiac design. Although chicken, mouse, and recently zebrafish have been considered powerful vertebrate models to study heart development, we propose that the trabeculation process in the dogfish is representative of the early steps of the ventricular morphogenesis in vertebrates.Support or Funding InformationThis study was supported by CGL2014‐52356‐P and FPU15/03209This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Automatic non-invasive heartbeat quantification of Drosophila pupae

The importance of studying model organisms such as Drosophila melanogaster has significantly increased in recent biological research. Amongst others, Drosophila can be used to study heart development and heartbeat related diseases. Here we propose a method for automatic in vivo heartbeat detection of Drosophila melanogaster pupae based on morphological structures which are recorded without any dissection using FIM imaging. Our approach is easy-to-use, has low computational costs, and enables high-throughput experiments. After automatically segmenting the heart region of the pupa in an image sequence, the heartbeat is indirectly determined based on intensity variation analysis. We have evaluated our method using 47,631 manually annotated frames from 29 image sequences recorded with different temporal and spatial resolutions which are made publicly available. We show that our algorithm is both precise since it detects more than 95% of the heartbeats correctly as well as robust since the same standardized set of parameters can be used for all sequences. The combination of FIM imaging and our algorithm enables a reliable heartbeat detection of multiple Drosophila pupae while simultaneously avoiding any time consuming preparation of the animals.

Cardiac Regeneration: Lessons From Development.

Palliative surgery for congenital heart disease has allowed patients with previously lethal heart malformations to survive and, in most cases, to thrive. However, these procedures often place pressure and volume loads on the heart, and over time, these chronic loads can cause heart failure. Current therapeutic options for initial surgery and chronic heart failure that results from failed palliation are limited, in part, by the mammalian heart's low inherent capacity to form new cardiomyocytes. Surmounting the heart regeneration barrier would transform the treatment of congenital, as well as acquired, heart disease and likewise would enable development of personalized, in vitro cardiac disease models. Although these remain distant goals, studies of heart development are illuminating the path forward and suggest unique opportunities for heart regeneration, particularly in fetal and neonatal periods. Here, we review major lessons from heart development that inform current and future studies directed at enhancing cardiac regeneration.

A series of robust genetic indicators for definitive identification of cardiomyocytes

Definitively identifying the cell type of newly generated cells in the heart and defining their origins are central questions in cardiac regenerative medicine. Currently, it is challenging to ascertain the myocardial identity and to track myocardial progeny during heart development and disease due to lack of proper genetic tools. This may lead to many misinterpretations of the findings in cardiac regenerative biology. In this study, we developed a set of novel mouse models by inserting double reporter genes nlacZ/H2B-GFP, mGFP/H2B-mCherry into the start codon of Tnnt2 and Myh6. nlacZ (nuclear lacZ) and mGFP (membrane GFP) are flanked by two LoxP sites in these animals. We found that the reporter genes faithfully recapitulated Tnnt2 and Myh6 cardiac expression from embryonic stage and adulthood. The reporter mice provide unprecedented robustness and fidelity for visualizing and tracing cardiomyocytes with nuclear or cell membrane localization signals. These animal models offer superior genetic tools to meet a critical need in studies of heart development, cardiac stem cell biology and cardiac regenerative medicine.

Advances in the Study of Heart Development and Disease Using Zebrafish.

Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish, Danio rerio, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications.

The Early Stages of Heart Development: Insights from Chicken Embryos.

The heart is the first functioning organ in the developing embryo and a detailed understanding of the molecular and cellular mechanisms involved in its formation provides insights into congenital malformations affecting its function and therefore the survival of the organism. Because many developmental mechanisms are highly conserved, it is possible to extrapolate from observations made in invertebrate and vertebrate model organisms to humans. This review will highlight the contributions made through studying heart development in avian embryos, particularly the chicken. The major advantage of chick embryos is their accessibility for surgical manipulation and functional interference approaches, both gain- and loss-of-function. In addition to experiments performed in ovo, the dissection of tissues for ex vivo culture, genomic, or biochemical approaches is straightforward. Furthermore, embryos can be cultured for time-lapse imaging, which enables tracking of fluorescently labeled cells and detailed analysis of tissue morphogenesis. Owing to these features, investigations in chick embryos have led to important discoveries, often complementing genetic studies in mice and zebrafish. As well as including some historical aspects, we cover here some of the crucial advances made in understanding early heart development using the chicken model.

Ventricular cell fate can be specified until the onset of myocardial differentiation

The mechanisms that govern specification of various cell types that constitute vertebrate heart are not fully understood. Whilst most studies of heart development have utilised the mouse embryo, we have used an alternative model, embryos of the frog Xenopus laevis, which permits direct experimental manipulation of a non-essential heart. We show that in this model pluripotent animal cap explants injected with cardiogenic factor GATA4 mRNA express pan-myocardial as well as ventricular and proepicardial markers. We found that cardiac cell fate diversification, as assessed by ventricular and proepicardial markers, critically depends on tissue integrity, as it is disrupted by dissociation but can be fully restored by inhibition of the BMP pathway and partially by Dkk-1. Ventricular and proepicardial cell fates can also be restored in reaggregated GATA4-expressing cells upon transplantation into a host embryo. The competence of the host embryo to induce ventricular and proepicardial markers gradually decreases with the age of the transplant and is lost by the onset of myocardial differentiation at the late tailbud stage (st. 28). The influence of the host on the transplant was not limited to diversification of cardiac cell fates, but also included induction of growth and rhythmic beating, resulting in generation of a secondary heart-like structure. Our results additionally show that efficient generation of secondary heart requires normal axial patterning of the host embryo. Furthermore, secondary hearts can be induced in a wide range of locations within the host, arguing that the host embryo provides a permissive environment for development of cardiac patterning, growth and physiological maturation. Our results have implications for a major goal of cardiac regenerative medicine, differentiation of ventricular myocardium.

Gene network analysis: from heart development to cardiac therapy.

Networks offer a flexible framework to represent and analyse the complex interactions between components of cellular systems. In particular gene networks inferred from expression data can support the identification of novel hypotheses on regulatory processes. In this review we focus on the use of gene network analysis in the study of heart development. Understanding heart development will promote the elucidation of the aetiology of congenital heart disease and thus possibly improve diagnostics. Moreover, it will help to establish cardiac therapies. For example, understanding cardiac differentiation during development will help to guide stem cell differentiation required for cardiac tissue engineering or to enhance endogenous repair mechanisms. We introduce different methodological frameworks to infer networks from expression data such as Boolean and Bayesian networks. Then we present currently available temporal expression data in heart development and discuss the use of network-based approaches in published studies. Collectively, our literature-based analysis indicates that gene network analysis constitutes a promising opportunity to infer therapy-relevant regulatory processes in heart development. However, the use of network-based approaches has so far been limited by the small amount of samples in available datasets. Thus, we propose to acquire high-resolution temporal expression data to improve the mathematical descriptions of regulatory processes obtained with gene network inference methodologies. Especially probabilistic methods that accommodate the intrinsic variability of biological systems have the potential to contribute to a deeper understanding of heart development.

Deepak Srivastava: follows his heart to study the heart.

One percent of all births worldwide are to children with a heart malformation. To better understand what has gone wrong in these children and how to prevent or correct such malformations in others, Deepak Srivastava, the Younger Family Director at the Gladstone Institute of Cardiovascular Disease and Director of the Roddenberry Stem Cell Center at Gladstone, studies the biology of heart development. He has identified several transcription factors, microRNAs, and other components that are essential for cardiogenesis in the mammalian embryo,1–5 but Srivastava’s work is relevant for heart problems affecting adults too. Heart failure after a heart attack is a major cause of death in men and women, and Srivastava is using the knowledge he gains from studying heart development to coax damaged adult hearts to regenerate. For example, his team has shown that, using a cocktail of developmental factors, heart fibroblasts—cells that form the rigid scar tissue after a myocardial infarction—can be converted into contracting cardiomyocytes both in vitro and in live mice.6,7 From caring for sick children, Srivastava is a physician at the Benioff Children’s Hospital in San Francisco, to regenerating mouse hearts, all of Srivastava’s career decisions and research interests have come down to the same basic principle, he told Circulation Research in a recent interview: do what you feel most passionate about, rather than what seems practical. This, he says, is the key to a happy career. My parents, Satish and Rani Srivastava, immigrated to the United States from India when I was less than a year old, along with my 2 older sisters. My father was a scientist and he moved to the City of Hope Medical Center in Los Angeles as a faculty member and stayed there for 8 years. So I grew up in Los Angeles for …

Methods to assess Drosophila heart development, function and aging

In recent years the Drosophila heart has become an established model for many different aspects of human cardiac disease. This model has allowed identification of disease-causing mechanisms underlying congenital heart disease and cardiomyopathies and has permitted the study of underlying genetic, metabolic and age-related contributions to heart function. In this review we discuss methods currently employed in the analysis of the Drosophila heart structure and function, such as optical methods to infer heart function and performance, electrophysiological and mechanical approaches to characterize cardiac tissue properties, and conclude with histological techniques used in the study of heart development and adult structure.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Building and repairing the heart: what can we learn from embryonic development?

Mammalian heart formation is a complex morphogenetic event that depends on the correct temporal and spatial contribution of distinct cell sources. During cardiac formation, cellular specification, differentiation, and rearrangement are tightly regulated by an intricate signaling network. Over the last years, many aspects of this network have been uncovered not only due to advances in cardiac development comprehension but also due to the use of embryonic stem cells (ESCs) in vitro model system. Additionally, several of these pathways have been shown to be functional or reactivated in the setting of cardiac disease. Knowledge withdrawn from studying heart development, ESCs differentiation, and cardiac pathophysiology may be helpful to envisage new strategies for improved cardiac repair/regeneration. In this review, we provide a comparative synopsis of the major signaling pathways required for cardiac lineage commitment in the embryo and murine ESCs. The involvement and possible reactivation of these pathways following heart injury and their role in tissue recovery will also be discussed.

Abstract 006: An Advance toward Making A Heart: Engineering Functional Human Heart Constructs with Induced Pluripotent Stem Cells and Decellularized Mouse Hearts

Background: Heart disease is the leading cause of death in the world, requiring the development of novel and personalized therapeutic strategies for disease treatment. Heart tissue engineering holds a great promise for those therapies based on the reconstruction of patient-specific cardiac muscle. A bioartificial heart was reported by reseeding rat cardiomyocytes ( CMs ) into decellularized rat heart. Whether such method could be used to make human heart constructs remained unknown. Objectives: We sought to rebuild functional heart constructs using human induced pluripotent stem ( iPS) cells and decellularized whole mouse hearts. Methods: We repopulated decellularized mouse hearts with human iPS cell derived multipotential cardiovascular progenitors ( MCPs ). MCPs represent the earliest heart progenitors during human cardiogenesis and can give rise to multiple cardiovascular lineage cells. Results: The seeded MCPs differentiated in situ into CMs, smooth muscle and endothelial cells, which reconstructed the decellularized mouse hearts. The engineered constructs exhibited myocardium and vessel-like structures, contracted spontaneously with a rate of 40~50 beats/min, exhibited intracellular Ca2+ transients and responded as expected to various drug interventions. In addition, we found heart ECM could promote proliferation and myofilament formation of CMs from the repopulated human MCPs. Therefore this study established a novel strategy of human heart tissue engineering, which could be beneficial to study heart development, and future preclinical applications.

Epicardium Formation as a Sensor in Toxicology.

Zebrafish (Danio rerio) are an excellent vertebrate model for studying heart development, regeneration and cardiotoxicity. Zebrafish embryos exposed during the temporal window of epicardium development to the aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit severe heart malformations. TCDD exposure prevents both proepicardial organ (PE) and epicardium development. Exposure later in development, after the epicardium has formed, does not produce cardiac toxicity. It is not until the adult zebrafish heart is stimulated to regenerate does TCDD again cause detrimental effects. TCDD exposure prior to ventricular resection prevents cardiac regeneration. It is likely that TCDD-induced inhibition of epicardium development and cardiac regeneration occur via a common mechanism. Here, we describe experiments that focus on the epicardium as a target and sensor of zebrafish heart toxicity.

Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Progressive Anatomical Closure of Foramen Ovale in Normal Neonatal Mouse Hearts

In the prenatal heart, right-to-left atrial shunting of blood through the foramen ovale is essential for proper circulation. After birth, as the pulmonary circulation is established, the foramen ovale functionally closes as a result of changes in the relative pressure of the two atrial chambers, ensuring the separation of oxygen depleted venous blood in the right atrium from the oxygenated blood entering the left atrium. Little is known regarding the process of anatomical closure of the foramen ovale in the postnatal heart. Genetically engineered mouse models are powerful tools to study heart development and to reveal mechanisms underlying cardiac anomalies, including defects in atrioventricular septation. Using three-dimensional reconstructions of serial sectioned hearts at early postnatal Days 2-7, we show a progressive reduction in the size of the interatrial communication throughout this period and complete closure by postnatal Day 7. Furthermore we demonstrate that fusion of the septum primum and septum secundum occurs between 4 weeks and 3 months of age. This study provides a standard timeline for morphological closure of the right-left atrial communication and fusion between the atrial septa in normal mouse hearts.

Expression of Nkx2.5 in Wild Type, Cardiac Mutant, and Thyroxine-Induced Metamorphosed Hearts of the Mexican Axolotl

Nkx2.5, a homeodomain-containing transcription factor, is known to be necessary for normal heart development in vertebrates. It is one of the earliest lineage-restricted genes expressed in cardiovascular progenitor cells and knowledge of its expression patterns has important therapeutic implications for damaged cardiomyocytes. Mexican axolotl is a unique system to study heart development for two reasons: the presence of a mutant phenotype lacking organized myofibrils due to sarcomeric tropomyosin deficiency and the ability to induce metamorphosis by administration of exogenous thyroid hormone. In this study, we cloned and sequenced the as yet uncharacterized Nkx2.5 cDNA from normal and cardiac mutant axolotl heart RNA. Comparison of cDNA sequences of Nkx2.5 from normal and mutant axolotl hearts did not show differences suggesting that loss of function mutation in Nkx2.5 is not responsible for the mutant phenotype. However, quantitative studies show higher expression of Nkx2.5 in mutant hearts raising the possibility that increased expression of Nkx2.5 may contribute to the mutant phenotype. We also evaluated quantitative changes in expression of Nkx2.5 in axolotl hearts during embryonic and postembryonic heart development induced by exogenous thyroid hormone. There is an apparent increase in Nkx2.5 transcript levels in metamorphosed hearts.

Role of myofibril-inducing RNA in cardiac TnT expression in developing Mexican axolotl

The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene ``c”, for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts. Myofibril-inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac troponin T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart.

Establishment of model of congenital heart disease with Tbx2 gene knockdown: experiment with zebrafish

To establish a model of congenital heart disease with Tbx2 gene knockdown. 1200 fertilized eggs of zebrafish (Danio rerio) were divided into 3 groups: wild type group (n = 100), Tbx2-morpholino (MO) group (n = 600, to be injected with Tbx2-MO so as to knock down the Tbx2 gene), and control MO group (n = 500, to be injected with control MO). 12, 24, 36, 48, 72, and 96 hours after fertilization the heart structure, heart rate, rhythm, contractibility, and blood flow of the embryos were observed under anatomic microscope and digital camera was used to record the findings. Whole-mount in situ hybridization was conducted to observe the expression of versican in the heart. Eight hours after fertilization 27.2% of the fertilized eggs of the Tbx2-MO group died, and 21.3%, 32.8%, and 18.7% of them showed mild, moderate, or severe developmental abnormality of heart, including hypogenetic ventricle, dilatation of atria, abnormal atrioventricular canal, slow heartbeat, arrhythmia, and blood regurgitation. Whole-mount in situ hybridization showed that versican did not decrease with the passage of time but was expressed abnormally in the atria and ventricles, and did not disappear 72 hours after fertilization; however, it was expressed normally in the statolith. MO antisense oligonucleotide knockdown is a good method for the study of heart development. Tbx2 gene plays an important role in the differentiation of atria and ventricles and formation of atrioventricular canal.

Seminars in Cell and Developmental Biology Model Systems for the Study of Heart Development and Disease

Seminars in Cell and Developmental Biology Model Systems for the Study of Heart Development and Disease