Robert L. DeHaan

Regulation of spontaneous activity and growth of embryonic chick heart cells in tissue culture.

Abstract 1. 1. The percentage of embryonic heart cells that exhibit spontaneous activity (% BC) in tissue culture is dependent upon a wide variety of environmental parameters, including mode of tissue disaggregation, composition of the culture medium, density of cell inoculum, and other culture conditions, as well as the age of the embryo from which the cells are taken. 2. 2. Dissociating 7-day hearts with three 8-minute incubation cycles in 0.05% trypsin liberates a maximal number of viable cells; best results are obtained when enough tissue is used for dissociation to bring the resultant cell suspension to 2 to 3 × 106 cells/ml or more. 3. 3. To be able to determine % BC, clls must be plated at densities low enough to prevent extensive intercellular contacts. At densities above about 100 cells/mm2 many cells make contact with neighbors, making it impossible to distinguish whether a cell is beating because of its own electrogenic activity or as a response to an impulse conducted from a pacemaking neighbor. 4. 4. The serum used in a culture medium influences, to a marked degree, the percentage of cells which beat spontaneously. A total of 56 serum samples were tested, yielding a range of effects: from those sera which were clearly toxic to those which gave “optimal” results. A sample of serum was considered optimal if cells in standard low-potassium medium, made with that serum, were healthy in appearance, and consistently yielded 40–50% BC. 5. 5. At adult serum levels of K+ (4–5 meq/l) approximately 20% of the cells in a culture from 7-day heart beat spontaneously. Increasing extracellular K+ causes some cells to cease activity. Decreasing the K+ to 1 meq/l permits a maximal number of latent pacemakers to become active. Dose-response curves relating K+ concentration to % BC have been determined with cells from embryonic hearts aged 4, 7, 12, and 18 days. 6. 6. The greatest number of spontaneously active cells is obtained from 7-day hearts. Under optimal conditions, with 1 meq/l K+ medium, activity ranged between 40 and 50% BC, with a mean of 44% BC. The greatest number of active cells ever recorded in a single plate was 64% BC. Older hearts yielded progressively fewer pacemakers, to a minimum mean of about 10% BC at 18 days. Hearts earlier than 7 days also yielded fewer spontaneously active cells. 7. 7. Four-day heart cultures are relatively insensitive to elevated K+ in the culture medium. From this it is suggested that the high intracellular Na+-content and rapid Na+-flux characteristic of early embryonic hearts represent devices whereby early pacemaker cells can maintain a ratio of sodium: potassium conductance sufficiently large to permit the onset of spontaneous activity in the high-potassium environment provided by the egg before amnion formation. 8. 8. Two types of heart cells may be distinguished morphologically in cultures: F cells, which have the appearance of typical fibroblasts; and M cells, which resemble myoblasts of skeletal muscle. F cells comprise 40–50% of the cells; M cells 50–60%. Most overt pacemakers are M cells. However, a small proportion (less than 1%) of these which meet all the morphological criteria of F cells, are also spontaneously active. 9. 9. Mitotic activity in heart cell cultures can be induced or prevented merely by the presence or absence of chick-embryo extract. Cultures can be obtained which exhibit both rapid mitosis and high % BC, by dialyzing CEE to remove K+. It is concluded that no necessary antagonism exists between growth and the maintenance of a differentiated functional state in embryonic heart cells cultured under the conditions described.

The precardiac areas and formation of the tubular heart in the chick embryo

Abstract Small fragments of mesoderm and endoderm have been transplanted from stage 5 donor embryos labeled with thymidine- 3 H to corresponding sites in nonlabeled hosts. These fragments heal in and participate in the normal development of the recipient embryo. The distribution of the implanted cells in the tubular heart at stage 12 has been determined by autoradiography, to provide a set of corresponding points in the stage 5 precardiac mesoderm and the epimyocardial and endocardial layers in the stage 12 heart. The tubular heart has been subdivided into 24 defined regions, and the boundaries of those regions have been mapped in the stage 5 mesoderm. The process of heart formation has also been traced through intervening stages by graphic reconstruction and microdissection of the mesoderm layer and by marking the epimyocardial troughs and forming heart tube with particles of iron oxide. The preepimyocardial mesoderm is organized in the stage 5 embryo in two separate regions, one on either side of the embryonic axis, with a gap of noncardiogenic mesoderm of about 0.8 mm between them. At this stage preendocardial cells are organized into areas generally similar in size and location to the preepimyocardial regions, but they are more widely scattered. Each lateral heart-forming region can be subdivided into a series of curved bands. The rostromedial band of each heart-forming region forms the truncoconal tissue of the tubular heart. The mid-portion gives rise to the ventricles and the caudolateral area of each region contributes the cells which form the atria. During cardiogenesis, the preepimyocardial mesoderm behaves as a coherent sheet. It condenses, stretches, folds, and deforms, but it does not lose its integrity as a sheet, nor its continuity with the rest of the layer of splanchnic mesoderm. It does not break up into cells or cell clusters. Endocardial cells, on the other hand, do show some evidence of dispersal as singlets and small groups during formation of the heart tube.

Synchronization of pulsation rates in isolated cardiac myocytes

Abstract Cells dissociated from seven-day embryonic chick hearts were cultured at low density in low-potassium medium for 4–24 h. Pairs of spontaneously beating myocytes, attached 2–5 μm apart to the bottom of the culture dish were observed until they first established physical contact with each other, and then until they synchronized their separate beats to a common rhythm. The cells were immediately fixed, stained and embedded on the plate, and prepared for electron microscopy. The interval between initial contact and synchrony was determined in 15 cell pairs to range from 4–38 min (mean = 16.5 min). Of 25 cell pairs that attained synchronous rhythms, only eight entrained at a rate equal to the faster member of the pair. Eleven pairs synchronized at a rhythm equal to or slower than that of the slower member; six had intermediate rates. Electron micrographs of the region of contact revealed small sites of close membrane apposition.

The potassium-sensitivity of isolated embryonic heart cells increases with development.

Abstract Spontaneously beating myocardial cells isolated from the hearts of chick embryos aged 2–18 days were cultured in medium containing 1.3, 4.2, 8.1, or 12 mM K+. At all ages tested, the percentage of beating cells (% BC) was maximal at 1.3 mM K. In cultures from 2-to 4-day hearts, isolated pacemaker cells were relatively insensitive to the level of extracellular K-concentration (K0). In contrast, the activity of most of the cells from 7- to 18-day hearts was suppressed by high Ko. The first week of heart development, therefore, represents a period of transition from a state of K-insensitivity to one of greater sensitivity of cardiac pacemakers to the level of Ko. Differences in the kinetics of pacemaker-inhibition in response to a sudden increment of Ko were used as evidence for three subpopulations of spontaneously active cells: those in which spontaneous activity was immediately and permanently suppressed; those which stopped beating initially but gradually recovered; and those which were unaffected by an increment in Ko. The relative size of each of these subpopulations was found to change in a systematic way with embryonic age. It is suggested that the change from a state of K-resistance to K-sensitivity of the embryonic pacemakers results either from a change in ionic permselectivity of the pacemaker membranes, or from an alteration in the concentration of ions inside and outside the cells during early development.

SPECIFIC MOTIFS IN THE EXTERNAL LOOPS OF CONNEXIN PROTEINS CAN DETERMINE GAP JUNCTION FORMATION BETWEEN CHICK HEART MYOCYTES

1. Gap junction formation was compared in the absence and presence of small peptides containing extracellular loop sequences of gap junction (connexin) proteins by measuring the time taken for pairs of spontaneously beating embryonic chick heart myoballs to synchronize beat rates. Test peptides were derived from connexin 32. Non‐homologous peptides were used as controls. Control pairs took 42 +/‐ 0.5 min (mean +/‐ S.E.M.; n = 1088) to synchronize. 2. Connexins 32 and 43, but not 26, were detected in gap junction plaques. The density and distribution of connexin immunolabelling varied between myoballs. 3. Peptides containing conserved motifs from extracellular loops 1 and 2 delayed gap junction formation. The steep portion of the dose‐response relation lay between 30 and 300 microM peptide. 4. In loop 1, the conserved motifs QPG and SHVR were identified as being involved in junction formation. In loop 2, the conserved SRPTEK motif was important. The ability of peptides containing the SRPTEK motif to interfere with the formation of gap junctions was enhanced by amino acids from the putative membrane‐spanning region. 5. Peptides from loop 1 and loop 2 were equivalently effective; there was no synergism between them. 6. The inclusion of conserved cysteines in test peptides did not make them more effective in the competition assay.

Differentiation of the Atrioventricular Conducting System of the Heart

The structure and function of the atrioventricular conducting system of the heart, and its relationship to the myocardium, are examined from a developmental point of view. On the basis of information derived from electron micrographic, electrophysiologic, and developmental studies of heart tissue, it is concluded that: (1) The idea of the syncytial nature of the heart lacks a sound anatomic basis. (2) Cytodifferentiation during embryonic cardiogenesis results in the development of at least 2 distinct populations of cells: those comprising the bulk of the myocardium and a second type, the specialized cells of the conductive tissue, which differs in histology, biochemistry, and physiology. (3) The common view of the myocardium as a spontaneously active tissue may require revision, since several lines of evidence appear to indicate that myocardial cells are quiescent until stimulated by an extrinsic source. Under normal circumstances, this stimulus source is the conductive tissue.

Development of cardiac beat rate in early chick embryos is regulated by regional cues

The mesoderm of each of the paired lateral heart-forming regions (HFRs) in the stage 5-7 chick embryo includes prospective conus (pre-C), ventricle (pre-V), and sinoatrial (pre-SA) cells, arranged in a rostrocaudal sequence (C-V-SA). With microsurgery we divided each HFR into three rostrocaudally arranged segments. After 24 hr of further incubation, each segment differentiated into a spontaneously beating vesicle of heart tissue to form a multiheart embryo. The cardiac vesicles in these embryos expressed left-right and rostrocaudal beat rate gradients: the left caudal pre-SA mesoderm produced tissue with the fastest beat rate of the six while the rostral vesicle formed from right pre-C was the slowest. In another operation, we prevented the HFRs from fusing in the midline by cutting through the anterior intestinal portal at stage 8, to produce cardia bifida (CB) embryos with an independently beating half-heart on each side. In these cases, the left half-heart of 87.2% of CB embryos beat faster than the right, confirming the left-right difference in intrinsic beat rate. To assess whether the future beat rate of each region is already determined in the st 5-7 HFR, we exchanged rectangular fragments of left pre-SA mesoderm and attached endoderm with right pre-C fragments to yield a left HFR with the sequence C-V-C and a right HFR with the sequence SA-V-SA. A CB operation was subsequently performed on these exchange embryos to prevent fusion of the lateral HFRs. Preconus mesoderm, transplanted to the pre-SA region, differentiated into tissue with a rapid beat rate, while pre-SA mesoderm relocated to the preconus region formed heart tissue with a slow spontaneous rate typical of the conus. In 73% of the exchange CB embryos, the left half-heart beat faster than the right, despite the origins of its mesoderm. The exchanged mesoderm developed a rate that was appropriate for its new location rather than the site of origin of the mesodermal fragment. In a third set of operations, we implanted a fragment of st 15 differentiated conus tissue into a site lateral to the left caudal HFR in st 5, 6, and 7 embryos, and subsequently performed CB operations on them. The implant caused the adjacent half-heart to develop with a slower beat rate than in unoperated or sham-operated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of Sensitivity to Tetrodotoxin in Beating Chick Embryo Hearts, Single Cells, and Aggregates

The spontaneous activity of intact embryonic heart becomes progressively more sensitive to tetrodotoxin block with increasing age of the embryo. The activity of isolated single heart cells in culture was relatively insensitive, independent of embryo age. Aggregates formed from single cells responded to tetrodotoxin in the same manner as intact hearts; aggregated cells from older hearts were sensitive.

Chapter 5 Cell Coupling In Developing Systems: The Heart-Cell Paradigm

Publisher Summary This chapter discusses the role of cell coupling in developing systems— that is, the heart-cell paradigm. The embryonic heart cells, because of their spontaneous electrical activity and contractility, provide unique advantages as a model system for the analyses of cellular communication. In some embryos, there is no electrical coupling between the first two blastomeres immediately after the cleavage is completed. As embryos develop through the early cleavage stages, electrical coupling becomes a generally observed phenomenon. The blastula represents the first stage, in which true epithelial relationships are demonstrated in the embryos. By the blastula stage, all the embryos that have been examined exhibit electrical coupling among their cells. In the early blastula, only close membrane appositions and occasional punctuate membrane fusions have been seen. As the embryo enters the mid-blastula stages, desmosomes and gap junctions become increasingly extensive. The chapter describes the culture of embryonic cardiac myocytes. Electrical coupling in tissue culture is spurred by the occurrence of such junctions in cancer models.

Cardia bifida and the development of pacemaker function in the early chick heart

Abstract Double-hearted or “cardia bifida” embryos were produced by making a mid-line cut through the tissues of the anterior intestinal portal of the 1–3 somite chick embryo, at a time when the foregut is a shallow crescent and the cardiac primordia have not yet fused in the bulboventricular region. Each lateral primordium in the operated chicks forms a separately beating tubular heart. From the time of initiation of heart beat (at 10–11 somites) to the stage of 18–19 somites, the left heart beats faster than does the right in the great majority of embryos. This observation is in consonance with the concept of “left dominance” as applied to early embryonic development. As development progresses, however, the left heart declines in rate while the right remains stable or increases in rate. By the 21–24 somite stage, the right heart usually has a faster rate than the left. It is at 19–20 somites that the sinus venosus is just forming in the heart. It is concluded that: (1) material from the left cardiac primordium is predominant in many respects over that from the right during early embryonic development and acts as pacemaker for the entire embryonic heart up to the stage of 18–19 somites. (2) At about the time the sinus venosus forms, this dominance relation reverses, material from the right primordium taking over the pacemaker function. Whether this change represents a collapse of some left-right gradient of metabolic activity, or the overriding of such a gradient by specific sinoatrial cells from the right side, is not known. It is suggested that the present results provide evidence that the definitive sinoatrial pacemaker of the adult heart is derived wholly or in the main from the right cardiac primordium.