Embryonic Chick Heart Research Articles

Computational Model for Early Cardiac Looping

Looping is a vital event during early cardiac morphogenesis, as the initially straight heart tube bends and twists into a curved tube, laying out the basic pattern of the future four-chambered heart. Despite intensive study for almost a century, the biophysical mechanisms that drive this process are not well understood. To explore a recently proposed hypothesis for looping, we constructed a finite element model for the embryonic chick heart during the first phase of looping, called c-looping. The model includes the main structures of the early heart (heart tube, omphalomesenteric veins, and dorsal mesocardium), and the analysis features realistic three-dimensional geometry, nonlinear passive and active material properties, and anisotropic growth. As per our earlier hypothesis for c-looping, actin-based morpho-genetic processes (active cell shape change, cytoskeletal contraction, and cell migration) are simulated in specific regions of the model. The model correctly predicts the initial gross morphological shape changes of the heart, as well as distributions of morphogenetic stresses and strains measured in embryonic chick hearts. The model was tested further in studies that perturbed normal cardiac morphogenesis. The model, taken together with the new experimental data, supports our hypothesis for the mechanisms that drive early looping.

Bone Marrow Cells Transdifferentiate to Cardiomyocytes When Introduced into the Embryonic Heart

Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long-term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co-incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription-polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker alpha-cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial-specific beta-galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of beta-galactosidase-positive cells, indicating the presence of bone marrow-derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.

Myosin-based contraction is not necessary for cardiac c-looping in the chick embryo

During the initial phase of cardiac looping, known as c-looping, the heart bends and twists into a c-shaped tube with the convex outer curvature normally directed toward the right side of the embryo. Despite intensive study for more than 80 years, the biophysical mechanisms that drive and regulate looping remain poorly understood, although some investigators have speculated that differential cytoskeletal contraction supplies the driving force for c-looping. The purpose of this investigation was to test this hypothesis. To inhibit contraction, embryonic chick hearts at stages 10-12 (10-16 somites, 33-48 h) were exposed to the myosin inhibitors 2,3-butanedione monoxime (BDM), ML-7, Y-27632, and blebbistatin. Experiments were conducted in both whole embryo culture and, to focus on bending alone, isolated heart culture. Measurements of heart stiffness and phosphorylation of the myosin regulatory light chains showed that BDM, Y-27632, and blebbistatin significantly reduced myocardial contractility, while ML-7 had a lesser effect. None of these drugs significantly affected looping during the studied stages. These results suggest that active contraction is not required for normal c-looping of the embryonic chick heart between stages 10 and 12.

Morphogenetic adaptation of the looping embryonic heart to altered mechanical loads

The biophysical mechanisms that drive and regulate cardiac looping are not well understood, but mechanical forces likely play a central role. Previous studies have shown that cardiac torsion, which defines left-right directionality, is caused largely by forces exerted on the heart tube by a membrane called the splanchnopleure (SPL). Here we show that, when the SPL is removed from the embryonic chick heart, torsion is initially suppressed. Several hours later, however, normal torsion is restored. This delayed torsion coincides with increased myocardial stiffness, especially on the right side of the heart. Exposure to the myosin inhibitor Y-27632 suppressed both responses, suggesting that the delayed torsion is caused by an abnormal cytoskeletal contraction. This hypothesis is supported further by computational modeling. These results suggest that the looping embryonic heart has the ability to adapt to changes in the mechanical environment, which may play a regulatory role during morphogenesis.

Embryonic Myocardium Shows Increased Longevity as a Functional Tissue When Cultured in the Presence of a Noncardiac Tissue Layer

A major aim of regenerative medicine is the construction of bioengineered organs and tissue for transplantation into human patients; yet living tissue is dynamic, and thus arranging cellular and extracellular constituents into an architecture resembling normal adult organs may not be sufficient to maintain tissue stability. In this study, we used cultures of embryonic chick heart tissue as a model to explore how newly formed cardiac tissue constructs can sustain their morphological structure and functional capabilities over extended periods. During the initial days of incubation, embryonic cardiac explants will thrive as beating three-dimensional tissue aggregates. However, within the first week of culture, cardiac aggregates lose their contractile function and flatten. After 2 weeks of incubation, the cardiac cells will have spread out into a homogeneous monolayer and dedifferentiated to a noncardiac phenotype. In contrast, when the embryonic heart tissue was co-cultured with a noncardiac cell layer obtained from adult bone marrow, the cardiac aggregates maintained their contractile function, three-dimensional tissue morphology, and myocyte phenotype for a full month of incubation. The capacity of this noncardiac cell layer to sustain the phenotype and morphology of the cardiac explants was partially replicated by treatment of the heart tissue with conditioned media from bone marrow cells. These findings are discussed in regard to the importance of adjacent cell layers for facilitating organogenesis in the developing embryo and having potential utility in producing stable bioengineered tissue constructs.

Expression patterns of tbx genes during chick atrial development

T-box gene family encodes a set of transcription factors that are involved in various developmental processes. We cloned T-box genes, tbx2, −5 and −20, from chick embryonic heart, and examined their expression patterns in the developing atrium from stage 14 to 27 by in situ hybridization. In the looped heart at stages 14 – 16, tbx2, −5 and −20 were expressed in the atrioventricular (AV) canal, in which valvuloseptal endocardial cushion tissue would later develop. These three genes were also expressed in the dorsal mesentery, through which pulmonary veins connect to the left atrium. During the atrial septation at later stages, tbx5 was expressed in the primitive auricles, sinus venosus and caval veins. In the developing atrial septum, tbx5 and −20 were expressed in the spina vestibuli, and tbx2 and −5 in the primary septum. tbx20 was expressed in the AV endocardial cushion tissue. Results suggest that differentially expressed tbx2, −5, −20 may play an important role in the atrial development, such as septation and pulmonary venous development.

SPATIAL AND TEMPORAL EXPRESSION OF SYNDECAN – 2 IN CHICK HEART DEVELOPMENT

Syndecan – 2 (syn2) is a member of the syndecan family of cell surface heparan sulfate (HS) proteoglycans. While various heparan sulfate proteoglycans mediate events such as cell adhesion, growth factor binding, syn2 has been shown to participate in organizing fibrillar matrices and in axis determination during development. To examine the distribution of syn2 during cardiogenesis, embryonic chick hearts were subjected to immunohistochemical analysis using a syn2-specific polyclonal antibody (R1891). Upon examination of paraffin-embedded thick sections, syn2 exhibited a developmental and a tissue-type specific pattern of expression. Although syn2 was previously reported in the condensing mesenchyme of murine limb tissue, syn2 was not detected in the chick mesenchymal tissue, in either the atrioventricular or outflow tract regions. Interestingly, cardiac muscle throughout the heart expressed syn2 in a developmentally regulated pattern. Syndecan-2 was noticeably absent in early hearts (Hamburger-Hamilton stages 12–14); however, by stages 15–17, cardiac muscle began to exhibit detectable syn2 staining. During later stages of cardiogenesis (stages 19 – 24), syn2 became widely expressed throughout all cardiac muscle. These results demonstrate the presence and the developmental pattern of syn2 during cardiogenesis. From previous studies on the role of syn2 during other developmental processes, it is possible that this proteoglycan is also functioning as a matrix organizer or as an axis-determining molecule during critical stages of cardiac morphogenesis.

Non‐invasive investigations of early embryonic cardiac blood flow with optical coherence tomography

Objective The purpose of this investigation was to develop techniques to non-invasively measure blood flow velocities in the tubular embryonic chick heart with optical coherence tomography (OCT) technologies. The early chick and mammalian embryonic heart is extremely small (< 2mm) and, once the heart begins to beat, continually moves. A system with high spatial and temporal resolution is necessary to accurately image such small organs. We have devised methods for using OCT, an imaging device capable of high spatial (2–15 microns) and temporal (10–100 frames per second) resolution, for investigations of early embryonic structure and function. We present investigations of structure and blood flow in embryonic chick hearts from stage 8 (prior to the embryonic heart beat) through stage 13, after onset of the heartbeat and after cardiac looping has begun. Color Doppler investigations revealed that flow through the atrioventricular canal, the precursor of the atrioventricular valves, was laminar, while flow through the outflow tract, precursor of the aortic and pulmonary valves, appeared more complex. These findings suggest that blood flow and blood flow velocity may play a role in the divergent developmental pathways taken by the atrioventricular vs. the outflow tract endocardial cushions. Support: AHA 0365348B (FR).

TGFβ-mediated RhoA expression is necessary for epithelial-mesenchymal transition in the embryonic chick heart

Endothelia in the atrioventricular canal (AVC) of the embryonic heart undergo an epithelial-mesenchymal transition (EMT) and migrate into the underlying extracellular matrix. We explore here whether RhoA mediates this EMT. RhoA was detected in all cells of the chick heart during the stages studied. Expression was elevated when EMT was actively occurring. Explants treated with C3 exoenzyme in collagen gel cultures showed a significant decrease in mesenchymal cell numbers. siRNA was used to inhibit RhoA mRNA, and both activated endothelial and mesenchymal cells decreased significantly with treatment. Loss of RhoA produced a reduction of RhoB, cyclin-b2, and beta-catenin messages showing that these genes are regulated downstream of RhoA. In contrast, runx-2 was not reduced. Inhibition of TGFbeta3 or TGFbeta2 activity caused a large reduction of RhoA message. These data place RhoA in TGFbeta regulated pathways for both endothelial activation and mesenchymal invasion and demonstrate a functional requirement during EMT.

Flow and blood shear stress in a chicken embryonic heart, experiments and numerical modeling

Flow and blood shear stress in a chicken embryonic heart, experiments and numerical modeling

Sema3D, Sema3F, and Sema5A are expressed in overlapping and distinct patterns in chick embryonic heart

An increasing number of axon guidance cues have been shown recently to play important roles in the development of non-neural tissues. Semaphorins comprise one of the largest conserved families of axon guidance factors. We analyzed the expression patterns of Sema3D, Sema3F, and Sema5A genes in the chick embryonic heart by in situ hybridization. All three genes are expressed in the cardiac cushion regions, both in the mesenchymal cells, and epithelial cells in the endocardial layer, during the period of cardiac remodeling. In addition to the overlapping expression patterns in the cardiac cushion regions, these genes also exhibit distinct expression patterns in the developing heart: Sema3D is additionally expressed in the tips of the ventricular trabeculae; Sema3F is expressed in a subset of cells scattered throughout the ventricles; and Sema5A is expressed in the newly formed atrioventricular valves. The overlapping and distinct expression patterns of these genes suggest that they may play important roles in heart development.

Differential levels of tissue hypoxia in the developing chicken heart

Tissue hypoxia plays a critical role in normal development, including cardiogenesis. Previously, we showed that oxygen concentration, as assessed by the hypoxia indicator EF5, is lowest in the outflow tract (OFT) myocardium of the developing chicken heart and may be regulating events in OFT morphogenesis. In this study, we identified additional areas of the embryonic chicken heart that were intensely positive for EF5 within the myocardium in discrete regions of the atrial wall and the interventricular septum (IVS). The region of the IVS that is EF5-positive includes a portion of the developing central conduction system identified by HNK-1 co-immunostaining. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor 1alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor 1 (HIF-1) heterodimer. The pattern of the most intensely EF5-stained myocardial regions of the atria and IVS resemble the pattern of the major coronary vessels that form in later stages within or immediately adjacent to these particular regions. These vessels include the sinoatrial nodal artery that is a branch of the right coronary artery within the atrial wall and the anterior/posterior interventricular vessels of the IVS. These findings indicate that a portion of the developing central conduction system and the patterning of coronary vessels may be subject to a level of regulation that is dependent on differential oxygen concentration within cardiac tissues and subsequent HIF-1 regulation of gene expression.

Confocal Imaging of the Embryonic Heart: How Deep?

Confocal microscopy allows for optical sectioning of tissues, thus obviating the need for physical sectioning and subsequent registration to obtain a three-dimensional representation of tissue architecture. However, practicalities such as tissue opacity, light penetration, and detector sensitivity have usually limited the available depth of imaging to 200 microm. With the emergence of newer, more powerful systems, we attempted to push these limits to those dictated by the working distance of the objective. We used whole-mount immunohistochemical staining followed by clearing with benzyl alcohol-benzyl benzoate (BABB) to visualize three-dimensional myocardial architecture. Confocal imaging of entire chick embryonic hearts up to a depth of 1.5 mm with voxel dimensions of 3 microm was achieved with a 10x dry objective. For the purpose of screening for congenital heart defects, we used endocardial painting with fluorescently labeled poly-L-lysine and imaged BABB-cleared hearts with a 5x objective up to a depth of 2 mm. Two-photon imaging of whole-mount specimens stained with Hoechst nuclear dye produced clear images all the way through stage 29 hearts without significant signal attenuation. Thus, currently available systems allow confocal imaging of fixed samples to previously unattainable depths, the current limiting factors being objective working distance, antibody penetration, specimen autofluorescence, and incomplete clearing.

Global Organization of Dynamics in Oscillatory Heterogeneous Excitable Media

An oscillatory heterogeneous excitable medium undergoes a transition from periodic target patterns to a bursting rhythm driven by the spontaneous initiation and termination of spiral waves as coupling or density is reduced. We illustrate these phenomena in monolayers of chick embryonic heart cells using calcium-sensitive fluorescent dyes. These results are modeled in a heterogeneous cellular automaton in which the neighborhood of interaction and cell density is modified. Parameters that give rise to bursting rhythms are organized in distinct zones in parameter space, leading to a global organization that should be applicable to the dynamics in a large class of excitable media.

Absence of Msx2 Does Not Affect Cardiac Conduction or Rescue Conduction Defects Associated with Nkx2‐5 Mutation

The transcription factors governing embryonic development of the AV conduction system are largely unknown. Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause AV conduction defects, which are associated with anatomic hypoplasia of the conduction system. In situ expression patterns of Msx2 in the mouse and chick embryonic heart have suggested a developmental function for this transcription factor. Homozygous Nkx2-5 knockout mouse embryos express Msx2 ectopically throughout the myocardium, suggesting Msx2 affects conduction system development through a transcriptional cascade starting with Nkx2-5. Several observations support a model in which Msx2 negatively regulates formation of the conduction system and inappropriate Msx2 up-regulation causes the conduction defects associated with Nkx2-5 mutation. We obtained surface ECGs and performed intracardiac electrophysiologic studies in Msx2 knockout mice and in Nkx2-5 wild-type and heterozygous null mutant mice in an Msx2 null mutant background. Msx2 null mutant mice had normal cardiac conduction and no increased vulnerability to inducible arrhythmia. Absence of Msx2 did not alter the conduction defects observed in heterozygous Nkx2-5 knockout mice. Msx2 likely does not contribute to development of the conduction system. Abnormal Msx2 expression likely does not cause the AV conduction defects present in Nkx2-5 knockout mice.

New anti-invasive compounds: Results from the Indo-Belgian screening program

Abstract The development of anti-invasive and anti-metastatic drugs is a major challenge in current cancer research. A model to study invasion in vitro consisted of organotypic confronting cultures of human MCF-7/6 mammary cancer cells with embryonic chicken heart fragments. Untreated cultures show occupation and destruction of the normal heart fragments within eight days. Inhibition of invasion was observed after addition of the following alkaloids and (poly)phenolics to the culture medium: (+)-catechin, tangeretin, xanthohumol, 3,7-dimethoxyflavone, prenylated chalcones, a pyrazole derivative, an isoxazolylcoumarin, and a prenylated desoxybenzoin. The action targets have been found both in the cancer cells and the surrounding host tissue. We hope that the further characterization of these targets will eventually lead to the development of clinically applicable anti-invasive drugs.

Mofarotene-induced inhibition of melanoma cell motility by increasing vinculin-containing focal contacts

Tumour cell motility, which is dependent on the organization of the cytoskeleton, is considered to play an important role in the spread of malignant melanoma. Therefore, retinoids, which are modulators of cytoskeletal organization, may affect the motile activity of melanoma cells. In this study, the effects of the arotinoid mofarotene on single cell motility and vinculin organization of the highly metastatic melanoma cell line K-1735-M2 were determined. Melanoma cells were cultivated in a temperature- and CO2-controlled microincubator, which was located on the microscope stage. Cell movements were evaluated quantitatively from time-lapse video recordings using an IBAS image analysis system. Vinculin distribution was evaluated using confocal laser scanning microscopy and a specially developed computerized image analysing program. In addition, melanoma cell invasion was tested on the embryonic chick heart model. Although 10 microM mofarotene did not reduce the translocative movements of melanoma cells, it significantly inhibited stationary motility, including fast plasma membrane movements and changes in shape. Mofarotene also showed a pronounced effect on the organization of vinculin-containing cell-substratum adhesion plaques. In retinoid-treated cells, the numbers of vinculin plaques per cell, and particularly those in the marginal areas of the cells, were significantly increased compared with untreated controls. Furthermore, the compound reduced the invasiveness of melanoma cells in a three-dimensional tissue culture model. In conclusion, our data demonstrate that mofarotene, an already almost forgotten synthetic retinoid, shows interesting effects on melanoma cells, which may be relevant for a slowdown of tumour spread.

Systolic and Diastolic Ventricular Function Assessed by Pressure-Volume Loops in the Stage 21 Venous Clipped Chick Embryo

Cardiac pressure-volume relations enable quantification of intrinsic ventricular diastolic and systolic properties independent of loading conditions. The use of pressure-volume loop analysis in early stages of development could contribute to a better understanding of the relationship between hemodynamics and cardiac morphogenesis. The venous clip model is an intervention model for the chick embryo in which permanent obstruction of the right lateral vitelline vein temporarily reduces the mechanical load on the embryonic myocardium and induces a spectrum of outflow tract anomalies. We used pressure-volume loop analysis of the embryonic chick heart at stage 21 (3.5 d of incubation) to investigate whether the development of ventricular function is affected by venous clipping at stage 17, compared with normal control embryos. Steady state hemodynamic parameters demonstrated no significant differences between the venous clipped and control embryos. However, analysis of pressure-volume relations showed a significantly lower end-systolic elastance in the clipped embryos (slope of the end-systolic pressure-volume relation: 5.68 +/- 0.85 versus 11.76 +/- 2.70 mm Hg/microL, p < 0.05), indicating reduced contractility. Diastolic stiffness tended to be increased in the clipped embryos (slope of end-diastolic pressure-volume relation: 2.74 +/- 0.56 versus 1.67 +/- 0.21, p = 0.103), but the difference did not reach statistical significance. The results of the pressure-volume loop analysis show that 1 d after venous obstruction, development of ventricular function is affected, with reduced contractility. Pressure-volume analysis may be applied in the chick embryo and is a sensitive technique to detect subtle alterations in ventricular function.

Cytoskeletal actin degradation induced by lovastatin in cardiomyocytes is mediated through caspase‐2

The objective of this study was to test the hypothesis that cytoskeletal actin fragmentation is mediated through caspase-2, specifically examining the ability of a caspase-2 inhibitor to interfere with actin fragmentation, in comparison with a caspase-3 inhibitor. Cardiomyocytes were cultured from embryonic chick heart. The fine structural element of cellular F-actin was visualized by staining cardiomyocytes with NBD-phallacidin. Lovastatin induced a dramatic and concentration-dependent loss of intact F-actin. The selectivity of this effect of lovastatin was demonstrated by the absence of similar changes in F-actin when cardiomyocytes were treated with the apoptotic stimulus palmitate, the metabolism of which produces acetyl CoA, the early substrate of cholesterol synthesis, through the mevalonate pathway. FACS analysis of NBD-phallacidin-stained cells was used to quantify the amount of F-actin loss. Actin fragmentation produced by lovastatin was operative through a caspase-2 pathway, as the caspase-2 inhibitor, z-VDVAD-fmk, significantly blocked lovastatin-induced changes in F-actin, but the caspase-3 inhibitor, Ac-DEVD-CHO, did not. Interruption of the mevalonate pathway was in part responsible for lovastatin's action, as the downstream metabolite mevalonate partially reversed the effect of lovastatin on actin fragmentation. These data indicate a previously unrecognized link between cytoskeletal actin and caspase-2.

Transitions in ventricular activation revealed by two-dimensional optical mapping.

While cardiac function in the mature heart is dependent on a properly functioning His-Purkinje system, the early embryonic tubular heart efficiently pumps blood without a distinct specialized conduction system. Although His-Purkinje system precursors have been identified using immunohistological techniques in the looped heart, little is known whether these precursors function electrically. To address this question, we used high-resolution optical mapping and fluorescent dyes with two CCD cameras to describe the motion-corrected activation patterns of 76 embryonic chick hearts from tubular stages (stage 10) to mature septated hearts (stage 35). Ventricular activation in the tubular looped heart (stages 10-17) using both calcium-sensitive fluo-4 and voltage-sensitive di-4-ANEPPS shows sequentially uniform propagation. In late looped hearts (stages 18-22), domains of the dorsal and lateral ventricle are preferentially activated before spreading to the remaining myocardium and show alternating regions of fast and slow propagation. During stages 22-26, action potentials arise from the dorsal ventricle. By stages 27-29, action potential breakthrough is also observed at the right ventricle apex. By stage 31, activation of the heart proceeds from foci at the apex and dorsal surface of the heart. The breakthrough foci correspond to regions where putative conduction system precursors have been identified immunohistologically. To date, our study represents the most detailed electrophysiological characterization of the embryonic heart between the looped and preseptated stages and suggests that ventricular activation undergoes a gradual transformation from sequential to a mature pattern with right and left epicardial breakthroughs. Our investigation suggests that cardiac conduction system precursors may be electrophysiologically distinct and mature gradually throughout cardiac morphogenesis in the chick.