Embryonic Chick Heart Research Articles

Development of Cholinergic Neuroeffector Transmission in the Avian Heart

A brief outline of the ontogenesis of cholinergic neuroeffector transmission in the embryonic chick heart is given. It is important to define the development of neuroeffector transmission to evaluate the possible effects on postjunctional membrane properties. The membrane response of the aneural chick heart undergoes a qualitative change to muscarinic agonist at a time well before cholinergic innervation. This suppressed response can be revealed later in life, even in adults, when provision is made to exclude Gi/Go and or K channel activity.

Subpopulations of Cardiomyocytes in the Embryonic Chicken Heart

Subpopulations of Cardiomyocytes in the Embryonic Chicken Heart

Invasion of human brain tumors in vitro: relationship to clinical evolution

Embryonic chick heart fragments were confronted in vitro with cells from 26 freshly resected human brain tumors. The tumor-derived cells were scored according to their survival and invasiveness. Four different responses were observed: disintegration of the tumor-derived cells (Type I); survival of cells without progressive engulfing or invasion of the heart fragment (Type II); initial encircling of the heart fragment followed by invasion (Type III); and massive invasion on initial contact (Type IV). Pattern Type III or IV was seen in 11 of 14 preparations derived from malignant tumors, and pattern Type I or II was seen in seven of 10 preparations derived from benign tumors. These results suggested that "classical malignancy," based upon the histological classification and the degree of spread in vivo, correlated with invasiveness in vitro.

Optical Indications of Spontaneous Electrical Activity and Functional Organization of Pacemaking Area in the Early Embryonic Chick Heart

Optical Indications of Spontaneous Electrical Activity and Functional Organization of Pacemaking Area in the Early Embryonic Chick Heart

Differential Localization of Myosin‐Specific Antibodies in Embryonic Chick Heart Ventricle Cells during Cytokinesisa

Differential Localization of Myosin‐Specific Antibodies in Embryonic Chick Heart Ventricle Cells during Cytokinesis^a

Contraction and membrane potential in a single cell of cultured chicken embryonic heart: H.Toyota, M.Matsumura, H.Okuyama Department of Physiology, Kawasaki Medical School, Kurashiki, Japan

Contraction and membrane potential in a single cell of cultured chicken embryonic heart: H.Toyota, M.Matsumura, H.Okuyama Department of Physiology, Kawasaki Medical School, Kurashiki, Japan

Effect of glycosylation inhibitors on N-glycosylpeptides and on invasion of malignant mouse MO4 cells in vitro

Cell surface glycans are believed to play a role in tumour invasion and metastasis. Yet, we have previously shown that the inhibitors of N-linked glycan processing swainsonine (SW) and 1-deoxynojirimycin (dNM) did not prevent invasion of chick heart fragments by MO4 murine fibrosarcoma cells in organ culture. We now present biochemical evidence that these and other inhibitors of processing were indeed effective in remodeling glycans, including those expressed at the cell surface. After metabolic labeling with tritiated mannose or fucose, glycosylpeptides were obtained by Pronase treatment of material released from intact cells by trypsin. Glycosylpeptides were separated by Biogel P-10 chromatography. With all drugs tested, there was a shift towards lower molecular weight of the glycan chains. There were, however, major quantitative differences between the different drugs and also, for monensin (MON; 0.1 microgram ml-1), between fucose-labeled and mannose-labeled chains. The shift in apparent molecular weight affected mainly fucose-labeled peptides after treatment of MO4 cells with SW (0.4 microgram ml-1). The shift induced by dNM (10 mM) + SW (0.4 microgram ml-1) in both fucosylated and mannosylated chains was much larger than that induced by SW given alone. 1-Deoxymannojirimycin (dMM; 1 mM) had major effects on both mannose and fucose-labeled structures and so did N-methyl-1-deoxynojirimycin (MdNM; 2 mM) and castanospermine (CS; 100 micrograms ml-1). With the latter drugs, incorporation of fucose in complex-type glycosylpeptides was dramatically reduced. The effect of SW on fucose-labeled glycosylpeptides of embryonic chick heart was similar to that observed on MO4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Epr Spectroscopic Studies of Detection of a Carbon-Centered Free Radical During Acetylcholine-Induced and Endothelium-Dependent Relaxation of Guinea Pig Pulmonary Artery

Vascular smooth muscle relaxation by several vasodilators, including acetylcholine (Ach) and ATP, depends on the presence of intact endothelium. Ach is thought to activate muscarinic receptors on endothelium to release an endothelium-derived relaxing factor (EDRF) which brings about relaxation of smooth muscle. In order to assess the role of free radicals in the endothelium-dependent relaxation of blood vessel, we have studied the effect of a spin-trapping agent, phenyl t-butyl nitrone (PBN), on Ach-, ATP-, and sodium nitroprusside-induced relaxation of guinea pig pulmonary artery. Arterial strips were mounted in a 5-ml organ bath containing Krebs solution equilibrated with 95% O2 and 5% CO2 at 37 degrees C. After increasing vascular tone by a synthetic prostaglandin endoperoxide analog (50 ng/ml), the strips relaxed dose-dependently in response to Ach (5 x 10(-8) M), ATP (1.5 x 10(-6) M) or sodium nitroprusside (6 x 10(-9) M). Removal of the endothelium abolished the relaxation by Ach or ATP, but did not affect the relaxation by sodium nitroprusside. PBN inhibited Ach-induced relaxation of pulmonary artery dose-dependently, but had no effect on relaxations by ATP or sodium nitroprusside. PBN did not block radioligand binding to muscarinic cholinergic membrane receptors on both chick embryonic heart and guinea pig pulmonary artery endothelial cells indicating that it does not block the muscarinic receptors. Spin trapping in combination with electron paramagnetic resonance (EPR) spectral analysis revealed a carbon-centered radical with hyperfine splitting constants of aN = 16.0 G and aH beta = 3.85G in the lipid extracts of pulmonary artery (0.2-0.4 g) incubated with PBN (14 mM) and Ach (3 x 10(-6) M) for 20 min. No signal was detected when endothelium was removed. Our data suggest that the endothelium-dependent relaxation of pulmonary artery by Ach is associated with the generation of a free-radical and can be prevented by a spin-trapping agent. ATP, however, relaxes the arterial smooth muscle by a different mechanism.

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.

Quantitative Evaluation of Melanoma Cell Invasion in Three-Dimensional Confrontation Cultures In Vitro Using Automated Image Analysis

Tumor invasion is a crucial feature of tumor growth in vivo. Confrontation cultures of multicellular melanoma spheroids and embryonic chick heart fragments provide a model for invasive growth in vitro. We have developed an image analysis method, which facilitates the objective measurement of tumor cell invasion in this model. Cryostat sections of confrontation cultures were immunohistochemically stained with an antiserum directed against the stromal component for automated recognition of the stroma tissue. The slides were automatically processed by a grey level based computerized image analysis system. On Spearman's rank correlation test, 25 out of 39 parameters correlated with the reference value of invasion, which was derived from the subjective evaluation of five independent observers. Two parameters combining the stroma margin and the total amount of stroma tissue completely reproduced the judgement of the morphologists in our test set. The quantitative evaluation of tumor invasion in vitro by automated image analysis may be helpful in pharmacologic and pathogenetic studies of tumor growth.

Fetal Expression of Renin, Angiotensinogen, and Atriopeptin Genes in Chick Heart

The renin-angiotensin and atriopeptin systems play important roles in the regulation of volume and fluid homeostasis. The two systems have opposing physiologic actions in a number of tissues. Experiments were performed to determine whether there were differences in the developmental expression of the genes for renin, angiotensinogen, and atriopeptin. Using RNA dot blot analysis, we compared levels of gene activity for renin, angiotensinogen, and atriopeptin in right atria, left atria, right ventricle, and left ventricle, from 18-day in ovum and 10-day old White Leghorn chicks. In 18-day embryonic chick heart there was expression of atriopeptin mRNA predominantly in the left and right ventricles. At this age, atriopeptin message was expressed in all four cardiac chambers, left ventricle greater than right ventricle greater than right atria greater than left atria. Renin and angiotensinogen mRNA was expressed in all cardiac chambers with reduced expression in left atria. In 10-day old chicks, renin, angiotensinogen, and atriopeptin mRNA was expressed in atrial tissue with right atria greater than left atria, with no detectable expression in left ventricle, right ventricle, or skeletal muscle. Beta actin was expressed in all four cardiac chambers and skeletal muscle, and was used to normalize signals. Cardiac expression of the genes for renin and angiotensinogen during embryogenesis suggests that the renin-angiotensin system may be involved in the growth and development of the myocardium.

Effect of dipyridamole on invasion of five types of malignant cells in organ culture.

Dipyridamole (DPD) has been shown to inhibit the motility of cells in culture. We have tested the effect of DPD on the invasion in confronting organ culture of the following malignant cell lines: mouse MO4 cells; rat NBT II bladder tumor cells; human SA4 glioblastoma cells; mouse LLC H61 lung carcinoma cells; and mouse F87 C1.6T2 melanoma x lymphocyte hybrid cells. At concentrations of 20 micrograms/ml or higher, DPD inhibited the invasion of all cell types into embryonic chick heart. In serum-free culture medium the anti-invasive concentration of DPD was about ten times lower. Anti-invasive concentrations of DPD also inhibited proliferation of the malignant cells. Both inhibition of invasion and of proliferation were reversible.

Induction of an epithelial-mesenchymal transition by an in vivo adheron-like complex

The embryonic vertebrate heart consists of two epithelia: the myocardium and endothelium, separated by the myocardial basement membrane (MBM). The myocardium has been shown to induce endothelial transformation into prevalvular mesenchyme in a temporally and site restricted manner. Previously, we hypothesized that the myocardial-endothelial interaction is mediated in vivo by aggregates of 30-nm particles in the MBM which can be removed by EDTA extraction. These MBM extracts contain fibronectin and other lower Mr proteins and can initiate an epithelial-mesenchymal transition in the AV (atrioventricular canal) endothelium of embryonic chick heart in collagen gel culture. These and other data suggested that the 30-nm multicomponent particles are similar, structurally and compositionally, to multimolecular complexes, termed adherons, secreted by L6 muscle cells in culture. The purpose of this study was to (1) test whether the removal of the 30-nm particles from MBM extracts of embryonic chick hearts would remove the in vitro biological activity and (2) determine if the fractionated MBM extracts can cause AV endothelial cells to follow the same differentiation pathway observed in vivo by monitoring immunohistochemically the cell surface expression of N-CAM. Results showed that centrifugation of extract at 100,000g for 1 hr produced a supernatant fraction that was unable to initiate mesenchyme formation from AV endothelium. However, the resuspended pellet fraction did initiate differentiation of endothelium into mesenchyme. Conditioned medium from L6 skeletal muscle cultures could not substitute for the EDTA extract of embryonic heart. Endothelial cells undergoing the transition to form mesenchyme, both in vivo and in vitro, showed a concomitant decrease in N-CAM staining. This suggested that the pellet-induced formation of migrating cells in the collagen gels is not the result a novel in vitro phenomenon.

Molecular cloning and expression of chicken cardiac troponin C.

We have isolated a full length complementary DNA clone (pCTnC1) from a 19-day embryonic chicken heart library corresponding to cardiac troponin C (TnC). Sequence analysis demonstrated varying homologies with TnC complementary DNA clones isolated from developing chick skeletal muscle. Using pCTnC1 as a hybridization probe, we have determined that cardiac TnC is constitutively expressed in both atria and ventricles of the developing and adult heart. These data along with previous immunochemical studies of TnC expression demonstrate that the slow skeletal and cardiac muscle isoform is the only TnC expressed in the heart. In contrast, expression of slow skeletal and cardiac muscle TnC is developmentally regulated in skeletal muscles of the chicken. The tightly controlled expression of slow skeletal and cardiac muscle TnC in the varying myocyte types of the heart suggests a physiologically significant role of this regulatory protein.

Endocardial cell arrangement: role of hemodynamics.

This paper demonstrates the presence of areas where endocardial cells are aligned with the blood flow in three distinct regions of the embryonic chick heart: on the inferior border of the growing septum primum, on the upper wall of the primitive ventricle above the developing interventricular septum, and on the part of the atrial floor that funnels into the atrioventricular canal. While orientation in the first two areas is transitory, the number of cells aligned with the blood flow in the atrial floor increases from days 3 to 5 of incubation. It is suggested that shear stress induces endocardial cell orientation in these areas. To further explore the role of hemodynamics in endocardial cell arrangement, the left vitelline vein was severed at stages 13-14. This resulted in abnormal development of the vitelline venous system. Examination of experimental embryos at 5 days of age revealed the presence of a distinct area of endocardial cell orientation in the dorsal wall of the right atrium, near the opening of the sinus venous. Changes in the venous return to the atria may modify flow patterns, thus inducing endocardial cell orientation. This experimental procedure may be a useful model not only for exploring the effects of hemodynamics upon the endocardium, but also for investigating other aspects of heart development under altered hemodynamic conditions, without manipulating any portion of the embryonic heart.

Embryonic chicken fibroblast collagen binding proteins: distribution, role in substratum adhesion, and relationship to integrins

Collagen binding proteins (CBP) are hydrophobic, cell surface polypeptides, isolated by collagen affinity chromatography. Antibodies to CBPs inhibit the attachment of embryonic chicken heart fibroblasts to native type I collagen fibrils in a dose-dependent manner. The CBP antibodies also induce rounding and detachment of cells adherent to a planar substratum. This process of antibody-mediated substratum detachment resulted in a clustering of CBP and cell-associated extracellular matrix at the cell surface, and the rearrangement of filamentous actin. Other functional studies showed that cells grown within a three-dimensional gel of type I collagen cannot be immunostained at the cell surface with CBP antibodies. However, treatment of cultures with purified collagenase, unmasks immunoreactive sites and permits strong cell surface immunolabeling. This result suggests that collagen sterically blocks antibody access to CBP. Finally, we show that antibodies to CBP recognize purified avian integrin beta subunits; and that antibodies to avian integrins recognize a 100,000 Mr CBP. These data demonstrate that chicken embryonic fibroblasts possess surface polypeptides that mediate adhesion to type I collagen, and suggest that two of these proteins are related to the integrin family.

Decreased fucose incorporation in cell surface carbohydrates is associated with inhibition of invasion.

Invasion of malignant MO4 cells into embryonic chick heart fragments in an organ culture assay was arrested for at least 7 days when the temperature was lowered to 28 degrees C. Prolonged culturing of MO4 cells at 28 degrees C on tissue culture substrates showed no recuperation of fucose incorporation into cell surface glycopeptides. However, invasion was restored after 10 days of organ culture in confrontation with chick heart tissue at 28 degrees C. A histoautoradiographic study showed that the regained capability to invade was accompanied by an increase in fucose labeling of the MO4 cells in the invading areas. At 28 degrees C the incorporation of [3H]fucose into total cell protein was drastically reduced, whereas [3H]leucine incorporation as a measure for protein synthesis was less affected. Cell surface glycopeptides, metabolically labeled with either fucose or glucosamine at 28 degrees C, showed a time-dependent decrease in the incorporation of fucose but not of glucosamine and no changes in overall size distribution. Low temperature did not reduce fucosyltransferase activity but the relative accumulation of fucose-1-P suggested inhibited conversion towards GDP-fucose. Moreover, mouse L cells which were incapable of invading chick heart tissue appeared also deficient in fucose incorporation, owing to low levels of fucosyltransferase activity. According to the results, fucosylation of surface carbohydrates may be required for invasive capacity and restored in MO4 cells invading at 28 degrees C by metabolic cooperation with the host tissue.

Structural and elemental characterization of heart cells grown in a collagen matrix

Cultured embryonic chick heart cells have been utilized as a model system for characterization of various membrane transport mechanisms. One advantage of this system is that the cells may be grown with differing geometries to minimize diffusion limitations and to optimize the growth configuration for particular techniques, such as ion-selective microelectrode measurements, fluorescent dye indicators, patch clamp, etc. A spontaneously contracting strand of cells embedded in a collagen matrix has recently been developed for measurements of cytoplasmic free ions by nuclear magnetic resonance (NMR) spectroscopy. These same strands, which provide the large numbers of cells needed for NMR, can be subdivided into small fragments ideal for cryopreservation prior to electron probe X-ray microanalysis (EPXMA). The aims in this study were to characterize the ultrastructure of cardiac cells within the strand, to demonstrate the quality of preservation obtainable by quick freezing methods, and to quantitatively map with EPXMA the distribution of physiologically relevant elements in thin, freeze-dried cryosections of the cells.Cells were isolated by serial trypsinization of 11-day old embryonic chick hearts. Strands of cells approximately 100 cm in length and 0.2 mm in diameter were obtained by extrusion of a cell-collagen mixture through polyethylene tubing into media within a culture dish. Three to five millimeter segments of 1-day old strands which contracted spontaneously were preserved by rapid immersion in liquid nitrogen-cooled liquid propane at 〜-185°C and stored in liquid nitrogen prior to being (a) cryosectioned for subsequent EPXMA or (b) freeze-substituted for conventional transmission electron microscopy (CTEM). Segments of strands were also chemically preserved in 2.5% glutaraldehyde in 0.1 M sodium cacodylate and processed as above for comparative CTEM. Cryosections of the frozen strands were cut at <-140°C with a dry glass knife and placed directly onto pre-cooled, carbon-coated, 200 mesh, fine bar nickel grids with a precooled implement. The grids were transferred to a liquid nitrogen cooled copper well for freeze drying at 10‒3 Torr over 24 to 48 hours. Prior to EPXMA, the grids were coated with 〜1OOÅ carbon.

Tetrodotoxin differentially blocks peak and steady-state sodium channel currents in early embryonic chick ventricular myocytes

Single ventricular myocytes were dissociated from 3-day-old embryonic chick hearts and maintained in culture for 9-21 h. The whole-cell patch clamp method was used to record tetrodotoxin (TTX)-sensitive fast Na+ currents. The peak Na+ current recorded at -20 mV ranged from 10 to 70 microA/cm2. At more negative potentials, a component of the current decayed very slowly, resulting in a significant steady-state or "late" Na+ current. The origin of the late Na+ current was revealed through the examination of single Na+ channel currents recorded in outside-out membrane patches. The single Na+ channel conductance was 20 pS. A high percentage of the trials (approximately 16%) displayed multiple reopenings of a single Na+ channel, resulting in bursts of current lasting for greater than or equal to 150 ms. The frequency distributions of the Na+ channel open-times were bi-exponential. The burst-like mode of Na+ channel activity (which underlies the slowly- or non-inactivating currents recorded macroscopically), was blocked to a greater degree by TTX, compared to the peak current. The results suggest that differential blockade may occur as a result of the slow binding and increased affinity of TTX to the open Na channel.

Use of the cell-attached patch clamp technique to examine regulation of single cardiac K channels by cyclic GMP

Cyclic nucleotides play a central role in the modulation of ion channels in a variety of tissues, including the heart. In order to determine the possible role of cyclic GMP (cGMP) in the regulation of the background K channel activity of cardiac cells, the effect of 8-Br-cGMP on the inwardly-rectifying K channels of cultured ventricular myocytes from embryonic chick hearts was examined. 8-Br-cGMP (10(-4) to 10(-3) M) inhibited these single channel currents within 3 to 10 min. Spontaneous recovery of the currents occurred with prolonged (greater than or equal to 15 min) exposure to 8-Br-cGMP, but this recovery was accompanied by altered channel behavior. Thus, a new long-lasting open state of the channel appeared, in addition to the open state observed prior to 8-Br-cGMP addition. Superfusion of the cells with the muscarinic agonist carbamylcholine (10(-5) M) also resulted in inhibition of the currents, which suggests that the cGMP-mediated inhibition of these channels may occur under physiological conditions. Thus, it appears that cGMP may be an important modulator of the background K conductance (and excitability) of cardiac cells.