Developing Chick Heart Research Articles

Inotropic effects of histamine on developing chick heart: release of transmitters from autonomic nerve terminals.

Inotropic effects of histamine were examined in isolated ventricular preparations from late embryonic and hatched chick hearts. In 19 day-old embryonic preparations, histamine had little effect on the contractile force. In preparations from 1 to 2 day-old hatched chick, histamine produced a transient decrease in contractile force followed by a sustained increase. The negative and positive responses were antagonized by atropine and propranolol, respectively, but not by histamine antagonists terfenadine, cimetidine or thioperamide. Acetylcholine produced positive inotropic responses in the embryo while negative responses were observed after hatching. In myocardium of hatched chicks, compound 48/80, which releases histamine from mast cells, produced a transient decrease in contractile force followed by a sustained increase with a similar magnitude and time course to the case of exogenously applied histamine. The negative and positive responses were inhibited by atropine and propranolol, respectively, but not by terfenadine, cimetidine or thioperamide, which was similar to the case with the responses to histamine. The present results suggest that histamine, either applied exogenously or released from myocardial store sites, produces negative and positive inotropic responses in hatched chick myocardium which are due to release of acetylcholine and norepinephrine, respectively, from autonomic nerve terminals.

Effects of Serum to Cushion Mesenchymal Cell Migration of the Developing Chick Heart in Vitro

Summary Migration of cardiac mesenchymal cells of chick embryos was examined in vitro. The conotruncal cushion and atrioventricular cushion cells of 6‐day‐old chick embryos were cultured on collagen gel in Dulbecco's modified Eagle's medium with chicken serum of calf serum, or no serum. After 48 hr of incubation, the culture was fixed with 4% formaldehyde, and photographed. The migration area of mesenchymal cells spread from the transplanted tissue was measured by digitizing the photographs. Then, the migration distance of mesenchymal cells was computed from the area with a personal computer.Mesenchymal cells cultured with chicken serum migrated a significantly longer distance than those with calf serum or no serum. Under the designed conditions, the migration distance of the mesenchymal cells of the conotruncal cushion was longer than that of the atrioventricular cushion.These results show that certain factor(s) in serum stimulates migration activity of cardiac mesenchymal cells, but the mammalian factor(s) is not cross‐reactive to cardiac mesenchymal cells of birds. Different effects of the serum from different species on the migration activity of chick cardiac mesenchymal cells were shown in the present results. At the same time, it was revealed that migration activity of mesenchymal cells of the conotruncal cushion is different from that of the atrioventricular cushion.

Chronotropic and inotropic effects of histamine in developing chick heart: differential mechanisms before and after hatching.

Chronotropic and inotropic effects of histamine were examined in isolated atrial and ventricular preparations from embryonic and hatched chicken hearts. Histamine produced positive chronotropic and inotropic responses both in embryonic and hatched hearts. The responses to histamine in middle embryonic myocardia, which were observed in the micromolar range, were antagonized by H2 antagonists but not by H1, H3 antagonists and propranolol. Isobutyl-methylxantine, an inhibitor of phosphodiesterase, produced a leftward shift of the concentration-response curve for the chronotropic effect of histamine in the embryo. The responses to histamine in myocardia from hatched chicks, which were observed in the milimolar range, appeared concurrently with the responses to tyramine during development and were antagonized by beta adrenoceptor antagonists but not by any of the histamine antagonists. The positive inotropic response to histamine in hatched ventricular preparations were greatly attenuated by reserpine pretreatment or in the presence of desipramine. Thus, we demonstrated that exogenously applied histamine produces positive chronotropic and inotropic responses in developing chicken hearts and that the mechanisms are different between embryonic and hatched chicks: direct action on H2 receptors in the embryonic heart and release of norepinephrine from sympathetic nerve terminals in hatched hearts.

Differential expression of muscarinic receptor mRNAS and their subtypes during development of embryonic chick heart and retina

Differential expression of muscarinic receptor mRNAS and their subtypes during development of embryonic chick heart and retina

Myocardial Regulation of Transforming Growth Factor-β Expression by Outflow Tract Endothelium in the Early Embryonic Chick Heart

We have demonstrated previously that the epithelial-mesenchymal transformation of cardiac endothelium in early chick heart development is induced by EDTA-soluble (ES) extracellular molecules synthesized by the myocardium of specific regions, i.e., the outflow tract (OT) and atrioventricular (AV) canal. Polyclonal antibodies (ES3) prepared against these molecules recognized two major bands, 28 and 46 kDa, in immunoblots and blocked the transformation of OT endothelial cells into mesenchyme in a three-dimensional collagen gel culture system. The studies of Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1516-1520 (1991)) and Potts and Runyan (Dev. Biol. 134, 392-401 (1989)) indicate that transforming growth factor (TGF)-β expression is necessary for the formation of mesenchyme from cardiac endothelium. In this study, we used ES3 antibodies to test the hypothesis that TGF-β expression by transforming endothelial cells is regulated by ES antigens. OT and AV endothelial cells treated with embryonic cardiocyte conditioned medium (CCM), which elicits epithelial-mesenchymal transformation, were shown by immunohistochemistry to increase expression of TGF-β1-like protein immediately prior to and during their transformation in culture. Endothelium from a nontransforming region of the heart (i.e., ventricle) did not express detectable levels of TGF-β under similar conditions. The staining pattern for TGF-β1-like protein was characterized by a distinct particulate or granular distribution within the Golgi and cytoplasm and at cell surfaces. However, when endothelial transformation was blocked by immunoadsorption of ES proteins from CCM, increased staining for TGF-β was not observed. These findings suggest an inductive relationship between myocardially derived ES proteins and TGF-β expression by chick heart endothelial cells which is requisite for their transformation into cushion mesenchyme.

Fate of the atrioventricular endocardial cushions in the developing chick heart.

To determine the fate of the atrioventricular endocardial cushions in cardiac development, we used staining methods for extracellular fibronectin, which is abundant in the endocardial cushions, and actin, which is abundant in the myocytes. White Leghorn chick embryo hearts were harvested at Hamburger and Hamilton stages 26 to 36, and serial sections of the atrioventricular valve region were stained. Before atrioventricular valve formation, fibronectin and actin staining reveal separation between the fibronectin-rich endocardial cushions and the actin-rich myocardial layer. The developing mitral valve leaflets at all of the observed stages contain a fibronectin-rich matrix but no actin-rich myocytes. In contrast, the tricuspid band includes both fibronectin matrix and actin-rich cells. We conclude that the mitral valve leaflets in the chick form predominantly from the endocardial cushion tissue, and the tricuspid band receives contributions from both the endocardial cushions and surrounding myocardium.

N-cadherin localization in early heart development and polar expression of Na +, K +-ATPase, and integrin during pericardial coelom formation and epithelialization of the differentiating myocardium

N-cadherin, a Ca 2+-dependent cell adhesion molecule, has been localized previously to the mesoderm during chick gastrulation and to adherens junctions in beating avian hearts. However, a systematic study of the dynamic nature of N-cadherin localization in the critical early stages of heart development is lacking. The presented work defines the changes in the spatial and temporal expression of N-cadherin during early stages of chick heart development, principally between Hamburger and Hamilton stages 5–8, 18–29 hr of development. During gastrulation N-cadherin appears evenly distributed in the heart forming region. As development proceeds to form the pericardial coelom (stages 6, 7, and 8, i.e., between 22 and 26 hr of development) N-cadherin localization becomes restricted to the more central areas of the mesoderm. The localization also shows a periodicity that correlates closely with the distance between foci of cavities that eventually coalesce to form the coelom. This distribution suggests that N-cadherin may have a function in the sorting out of somatic and splanchnic mesoderm cells to form the coelom. This separation of the mesoderm in the embryo for the first time physically delineates the precardiac mesoderm population. Concomitant with cell sorting during coelom formation, the precardiac cells change shape and show a distinct polarity as conveyed by (1) the apical expression of N-cadherin on precardiac cell surfaces lining the pericardial coelom, (2) the primarily lateral expression of Na +, -K + -ATPase, and (3) an enrichment of integrin (β1 subunit) on basal cell surfaces. The somatic mesoderm cells apparently down-regulate N-cadherin expression. N-cadherin is also absent from the precardiac cells close to the endoderm. The latter cells eventually form the endocardium, i.e., the endothelial lining of the heart. By contrast, in the tubular, beating heart N-cadherin is found throughout the myocardium. In summary, immunolocalization patterns of N-cadherin during early cardiogenesis suggest that this cell adhesion molecule has a major role in the dynamics of pericardial coelom formation. Subsequently, its continued expression during cell differentiation of the cardiomyocyte to form the myocardium, but not endocardium, suggests N-cadherin is an essential morphoregulatory molecule in heart organogenesis.

Potentiating effects of forskolin on the cardiovascular teratogenicity of ephedrine in chick embryos

The effects of forskolin on the teratogenicity of ephedrine in the developing chick heart were studied. Forskolin was administered to 4-day chick embryos (Hamburger-Hamilton stage 24) together with ephedrine at doses at which each agent alone caused minimal embryotoxicity. The embryos were examined for malformations on day 14 of incubation. The frequency of malformed embryos exposed to ephedrine (0.5 or 5 μmol) alone was 8 and 26%, respectively, and significantly increased to 47–72% in the presence of 1 nmol forskolin. Forskolin (1 nmol) alone did not induce a significant number of cardiac malformations. These results suggest that the increase in cAMP through stimulation of adenylate cyclase by forskolin is associated with the potentiation of ephedrine-induced cardiovascular malformations in the chick.

Immunolocalization of basic fibroblast growth factor during chicken cardiac development

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.

Histogenesis of the semilunar valves: an immunohistochemical analysis of tenascin and type-I collagen distribution in developing chick heart valves

The development of the semilunar valves takes place in association with septation of the outflow tract in the embryonic heart. Although numerous studies have focused on this process, the causal mechanisms of valvular development remain obscure. This paper reports an immunohistochemical analysis of tenascin and type-I collagen distribution in developing chick heart valves. Tenascin is a glycoprotein that is present on some embryonic extracellular matrices. It plays several significant roles in tissue differentiation, cell growth, and tissue interactions; it is also important for the formation of specific zones of connective tissue that fulfill mechanical functions. Our results show that tenascin is present during valvular morphogenesis and histogenesis, and that its distribution is associated with zones specialized in bearing mechanical loads.

Distribution of vitronectin in the embryonic chick heart during endocardial cell migration.

In the early phase of heart development, the endocardial cells migrate into the truncal swellings and atrioventricular (AV) cushions, and become mesenchymal cells. Vitronectin is a glycoprotein which is thought to mediate cell migration. The present study demonstrates by immunohistochemistry the distribution of vitronectin in order to elucidate its contribution to endocardial cell migration in the developing chick heart. At Hamburger and Mamilton's stage 23, the network of fibrillar material filled the extracellular space of both truncal swellings and AV cushions. The fibrillar network has been thought to be a matrix for endocardial cell migration. The network was stained with the anti-vitronectin antibody. At stage 29, the swellings and cushions were packed with mesenchymal cells, though immunoreactivity to the antibody was still observed in the extracellular matrix. The myocardium facing the AV cushions reacted to the antibody, but the myocardium surrounding the truncus arteriosus did not. The intensity of the immunohistochemical staining of the myocardium facing the AV cushions increased and reached a peak at stages 24 to 26, and then became weak by stage 29. The endocardial sheet, aortico-pulmonary septum and developing tunica media of the great arteries were not stained by the antibody at any stage. These results strongly suggest that vitronectin is involved in the migration of endocardial cells, and that the myocardium facing the AV cushions produces vitronectin.

Protein kinase C (calcium/phospholipid-dependent protein kinase) in developing chick heart: selective localization to atrium versus ventricle and changes in activity levels during cardiogenesis

Activity levels of calcium/phospholipid-dependent protein kinase were examined in preparations of atria and ventricles from embryonic chick hearts at various stages of development. Activity of protein kinase C was much higher in atria than ventricles. Protein kinase C activity underwent a progressive increase in atria during cardiogenesis, being highest just prior to hatching, followed by a profound decrease in activity after hatching. In contrast, activity of cyclic AMP-dependent protein kinase (protein kinase A), while also higher in atria than ventricles, remained relatively constant at the developmental stages examined, likewise decreasing following hatching. These progressive changes in atrial protein kinase C activity suggest a potential regulatory role for this enzyme in cardiogenesis.

Protein kinase C (calcium/phospholipid-dependent protein kinase) in developing chick heart: selective localization to atrium versus ventricle and changes in a

Activity levels of calcium/phospholipid-dependent protein kinase were examined in preparations of atria and ventricles from embryonic chick hearts at various stages of development. Activity of protein kinase C was much higher in atria than ventricles. Protein kinase C activity underwent a progressive increase in atria during cardiogenesis, being highest just prior to hatching, followed by a profound decrease in activity after hatching. In constrast, activity of cyclic AMP-dependent protein kinase (protein kinase A), while also higher in atria than ventricles, remained relatively constant at the developmental stages examined, likewise decreasing following hatching. These progressive changes in atrial protein kinase C activity suggest a potential regulatory role for this enzyme in cardiogenesis.

Nodose placode provides ectomesenchyme to the developing chick heart in the absence of cardiac neural crest.

The cardiac neural crest provides both ectomesenchyme and parasympathetic postganglionic neurons to the developing heart. Ablation of cardiac neural crest results in persistent truncus arteriosus, a condition in which the conotruncal and aorticopulmonary septa do not form in the developing heart. Parasympathetic postganglionic neurons are abundantly present in hearts with persistent truncus arteriosus, which indicates a "regeneration" of the neural component of the cardiac neural crest without comparable restitution of the ectomesenchymal component. The neural component has been shown to be provided by cells from the nodose placode following ablation of the cardiac neural crest. This investigation has shown that ectomesenchymal cells are also supplied to a limited extent by the nodose placode which normally has no ectomesenchymal derivatives. Although placode-derived ectomesenchyme helps to strengthen the wall of the cardiac outflow vessel, it is not competent to induce conotruncal and aorticopulmonary septal closure.

Myosin ATPase activity during avian cardiac and skeletal muscle development

The myosin ATPase activity and myosin light chain composition in developing chick heart and skeletal muscles were studied and compared. Embryonic myosin was purified and characterized from day 7 to day 19 of embryogenesis. Embryonic cardiac myosin generally showed the same Ca 2+-activated myosin ATPase activity level as the adult value. In comparison, pooled pectoralis and hindlimb skeletal muscles from day 10 through day 19 showed myosin ATPase activities that were all significantly less than the adult counterpart. The myosin light chain pattern of embryonic cardiac myosin remained relatively constant like the myosin ATPase activity, whereas developmental changes were observed in skeletal myosin light chains.

Morphogenesis of the truncus arteriosus of the chick embryo heart: movements of autoradiographic tattoos during septation.

In order to trace tissue movements during septation of the embryonic truncus arteriosus into aortic and pulmonary cardiac outlets, the cephalic margin of the developing tubular heart of chick embryos was tattooed at Hamilton-Hamburger Stages 20-22 using diffusion micropipettes filled with 0.5% agarose and radioactive macromolecular precursors (tritiated thymidine, uridine, and leucine). Following further incubation for 2, 48, or 96 hours, the locations of such tattoos were determined by autoradiography of sectioned tissue and computer reconstruction of the developing outflow tract. Two hours after tattooing, radiolabeled cells were clustered at the right distal margin of the myocardial tube, as intended. Two days later, during septation of the outflow tract into the two arterial streams, label was concentrated along the posterior margin of the myocardium, between the developing aortic and pulmonary valve anlagen to the embryo's right and left, respectively. Four days following tattooing, as truncal septation neared completion, remaining label was found primarily to the left of the aortic valve ring posterior to the pulmonary outlet. The movements of thymidine tattoos during septation were demonstrated in a series of 31 embryos, 14 fixed at 2 hours, 12 at 2 days, and 5 at 4 days following tattooing; similar results were seen in uridine and leucine labeled hearts. The motion of such tattoos in the developing chick heart suggests that the left side of the definitive semilunar valve ring derives from the right distal margin of the primitive tubular heart and that normal morphogenesis of the great arterial streams involves both retraction and rotation of the embryonic truncus arteriosus.

Development of physiological responsiveness to glucagon during embryogenesis of avian heart

Glucagon increases contractility of the heart muscle by stimulation of adenylate cyclase activity and elevation of cAMP. We have investigated the specific time of onset of glucagon sensitivity of heart muscle during development of the chick embryo. Using both isolated heart preparation and cultured cardiac cells, we have found that the contractile response to glucagon cannot be detected prior to Day 4 of development. Binding studies, carried out with heart cells prepared from 3-, 5-, 7-, and 11-day chick embryos, showed a significant increase in the number of glucagon binding sites between Days 3 and 5. Scatchard analysis showed that for Day 5 cells maximum binding capacity was 0.56 pmole/mg of protein with K d of 16.0 n M, while for Day 3, maximal binding was only 0.16 pmole/mg with K d of 15.1 n M. Therefore in this 2-day interval there was a marked increase in the receptor number, without changes in the receptor affinity. Since hormonal stimulation of adenylate cyclase depends on the presence of the regulatory component ( N s), we have used cholera toxin-induced chronotropic effect as an assay for functional N s. No response to cholera toxin could be detected prior to Day 4 of chick heart development. Therefore, emergence of the cholera toxin sensitivity correlates well with the onset of responsiveness to glucagon. We conclude that as the heart develops it acquires a physiological responsiveness to glucagon. The acquisition of the hormonal sensitivity correlates with the increase in the receptor number and the functional levels of regulatory component.

Extracellular matrix from embryonic myocardium elicits an early morphogenetic event in cardiac endothelial differentiation

A critical step in early cardiac morphogenesis can be faithfully duplicated in culture using a hydrated collagen substratum, and thereby serves as a useful model system for studying the molecular mechanisms of cell differentiation. Results from previous work suggested that the myocardium in the atrioventricular canal (AV) region of the developing chick heart secretes extracellular proteins into its associated basement membrane, which may function to promote an epithelial-mesenchymal transition of endothelium to form prevalvular fibroblasts ( E. L. Krug, R. B. Runyan, and R. R. Markwald, 1985, Dev. Biol. 112, 414–426 ; C. H. Mjaatvedt, R. C. Lepera, and R. R. Markwald, 1987, Dev. Biol., in press ). In the present study we show that an EDTA-soluble extract of embryonic chick hearts can substitute for the presence of myocardium, the presumptive stimulator tissue, in initiating mesenchyme formation from AV endothelium in culture. Ventricular endothelium was unresponsive to this material in keeping with observed in situ behavior. AV endothelial cells did not survive beyond 4–5 days when cultured in the absence of either the EDTA-soluble heart extract, myocardial conditioned medium, or the myocardium itself. Antibody prepared against a particulate fraction of the EDTA-solubilized heart extract immunohistochemically localized this material to the myocardial basement membrane. In addition, conditioned medium from embryonic myocardial cultures effectively induced mesenchyme formation. Neither a variety of growth factors nor a sarcoma basement membrane preparation were effective in promoting mesenchyme formation indicating a selectivity of the responding embryonic AV endothelial cells to myocardial basement membrane. These observations reflect a truly inductive phenomenon as there was (1) an absolute dependence on the presence of the stimulating substance/tissue and (2) retention, in culture, of both the temporal and regional characteristics observed in situ. This is in contrast to the results of others investigating the cytodifferentiation of committed cells whose phenotypic expression can be either accelerated or diminished but not obligatorily regulated by a specific agent, thus making the interpretation of data difficult, if not irrelevant, to the study of differentiation. The results of this study provide direct experimental support for the hypothesis that extracellular matrix can indeed serve as a direct stimulator or “secondary inducer” of cytodifferentiation.

Sarcolemmal Na−Ca exchange and sarcoplasmic reticulum calcium uptake in developing chick heart

Ontogenetic changes in calcium transport mediated by the sarcolemmal Na-Ca exchanger and by the sarcoplasmic reticulum calcium pump were studied in crude membranes from chick heart. Transport activities were evaluated per mass of membrane protein and heart tissue. Relative to unit heart mass Na-Ca exchange activity increases linearly from embryonic day 4 to day 10 of newborn stage. The overall increase is about 20-fold. An excellent correlation exists between activity of sodium gradient-induced calcium uptake and ouabain-sensitive (Na,K)-ATPase in crude membranes of embryonic, newborn and adult hearts. In the same membrane preparations active calcium uptake into vesicles of sarcoplasmic reticulum increases about 3-fold from embryonic day 4 to embryonic day 7, and then increases continuously until day 20. This is followed by a 3-fold elevation in reticular calcium accumulation at hatching on day 21. Maximal sarcoplasmic reticulum calcium transport activity reached at day 10 after hatching is 40- to 50-fold greater than activity values at embryonic day 4. In adult hearts the activities of both Na-Ca exchange and reticular calcium uptake drop to levels characteristic for the late embryonic period. This comparative study of sarcolemmal sodium gradient-dependent calcium flux and reticular calcium sequestration demonstrates that during chick heart differentiation the two calcium transport systems do not develop in parallel. Na-Ca exchange appears to play a greater role in calcium control of embryonic as compared to newborn and adult hearts. By contrast, the contribution of sarcoplasmic reticulum calcium transport to cardiac calcium movements becomes more predominant during and after hatching.

Characterization of conotruncal malformations following ablation of "cardiac" neural crest.

Neural crest cells from the cranial region of the neural fold populate the outflow tract of the developing chick heart. Removal of this region of premigratory neural crest has been shown previously to result in a high percentage of conotruncal malformations. The present study was undertaken to define more precisely the regions of premigratory neural crest which are needed for normal conotruncal development. Various regions and lengths of premigratory cranial neural crest were ablated using microcautery. Three defects in conotruncal development were significantly correlated with the neural crest ablation. These were high ventricular septal defect, single outflow vessel originating from the right ventricle, and single outflow vessel overriding the ventricular septum.