Initial development of conduction pattern of spontaneous action potential in early embryonic precontractile chick heart

The conduction of spontaneous action potentials in the 7-10 somite embryonic developing chick hearts was monitored optically using a potential-sensitive merocyanine-rhodanine dye. Spontaneous optical action signals from 5 to 12 different regions of the primitive heart were recorded simultaneously. Short delays were observed among firing times of the absorption signals which were nearly synchronized among the different regions. From these delays, we estimated the conduction velocity of the spontaneous excitatory waves. Usually, in the 7-somite to the beginning of the 9-somite stage, (i) excitatory waves conducted radially over one side of the prebeating heart, at a uniform rate; (ii) the "radially" spreading electrical wave slowed considerably within the primordial fusion line at the midline of the heart; and (iii) this delay disappeared in the later period of the 9-somite stage to the 10-somite stage. These observations suggest that electrical coupling among the cells within the primordial fusion line is poor during the 7 to 9-somite stage, and that the coupling is strengthened by the late 9th or 10th somite stage.     

Effects of calcium on electrical propagation in early embryonic precontractile heart as revealed by multiple-site optical recording of action potentials

The effects of Ca2+ on electrical propagation in early embryonic precontractile chick hearts were studied optically using a voltage-sensitive merocyanine-rhodanine dye. Spontaneous optical signals, corresponding to action potentials, were recorded simultaneously from 25 separate regions of the eight-to-nine-somite embryonic primitive heart, using a square photodiode array. Electrical propagation was assessed by analyzing the timing of the signals obtained from different regions. Electrical propagation in the heart was suppressed by either lowering or raising extracellular Ca2+. Similar effects were produced by a Ca2+ ionophore (A23187). We have also found that electrical propagation across the primordial fusion line at the midline of the heart was enhanced by increasing, and depressed by lowering, external Ca2+. One possible interpretation is that intercellular communication in the embryonic precontractile heart is regulated by the level of the intracellular Ca2+ concentration, and it is suggested that intercellular communication across the primordial fusion line strongly depends on external Ca2+.     

Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of ³H-glucosamine-derived labeled UDP-N-acetyl-d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. ³H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.     

Functional pacemaking area in the early embryonic chick heart assessed by simultaneous multiple-site optical recording of spontaneous action potentials

Pacemaking areas in the early embryonic chick hearts were quantitatively assessed using simultaneous multiple-site optical recordings of spontaneous action potentials. The measuring system with a 10- X 10- or a 12 X 12-element photodiode array had a spatial resolution of 15-30 microns. Spontaneous action potential-related optical signals were recorded simultaneously from multiple contiguous regions in the area in which the pacemaker site was located in seven- to nine-somite embryonic hearts stained with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). In the seven- to early eight-somite embryonic hearts, the location of the pacemaking area is not uniquely determined, and as development proceeds to the nine-somite stage, the pacemaking area becomes confined to the left pre-atrial tissue. Analysis of the simultaneous multiple-site optical recordings showed that the pacemaking area was basically circular in shape in the later eight- to nine-somite embryonic hearts. An elliptical shape also was observed at the seven- to early eight-somite stages of development. The size of the pacemaking area was estimated to be approximately 1,200-3,000 micron2. We suggest that the pacemaking area is composed of approximately 60-150 cells, and that the pacemaking area remains at a relatively constant size throughout the seven- to nine-somite stages. It is thus proposed that a population of pacemaking cells, rather than a single cell, serves as a rhythm generator in the embryonic chick heart.     

Development of electrical rhythmic activity in early embryonic cultured chick double-heart monitored optically with a voltage-sensitive dye

Double-hearted embryos were produced by whole-embryo culture of chick embryos which were microsurgically cut through the tissue of the anterior intestinal portal at the 1- to 6-somite developmental stage, at the time when the cardiac primordia have not yet fused in the bulboventricular region. The cultured embryos were removed from an incubator usually at the 7- to 10-somite stages of development, and then spontaneous electrical action potentials and/or contractions were optically recorded simultaneously from both the right and left half-hearts, using a 10 X 10- element photodiode matrix array together with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). At the 7- to 8-somite stages, spontaneous action potentials were detected from bilateral prebeating half-hearts or sometimes from one half-heart. In each half-heart, the first spontaneous beating was often observed in the half-heart of the 9 somite embryos. In the beating half-hearts regular activity was always observed, while in the prebeating half-hearts at the 7- to 8-somite stages, both the regular and irregular rhythms of action potentials were detected, and the incidence of occurrence of regular activity significantly outnumbered that of the irregular rhythm. The heart rate in the left half-heart was faster than that in the right half-heart in the great majority of the prebeating and beating double-hearted embryos.     

Development of the fast sodium current in early embryonic chick heart cells

Summary Single ventricle cells were dissociated from the hearts of two-, theree-, four-, or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 m cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (I _Na). Voltage dependence, and kinetics ofI _Na were applied with patch electrodes in the wholecell clamp configuration.I _Na was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture condition, Between the 2d and 7d stages, the rate of current inactivation doubled an channel density increased about eighfold. At all stages tested,I _Na was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance.     

Development of the fast sodium current in early embryonic chick heart cells

Single ventricle cells were dissociated from the hearts of two-, three-, four- or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 micron cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (INa). Voltage command protocols designed to investigate the magnitude, voltage dependence, and kinetics of INa were applied with patch electrodes in the whole-cell clamp configuration. INa was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture conditions. Between the 2d and 7d stages, the rate of current inactivation doubled and channel density increased about eightfold. At all stages tested, INa was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory Na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance.     

Regional epicardial strain in the embryonic chick heart during the early looping stages

Epicardial strains were measured in Hamburger-Hamilton stage 11 and 12 embryonic chick hearts (1.6-2.0 days of incubation). These stages include part of the early phase of cardiac looping, as the initially straight heart tube bends and twists to form a curved c-shaped tube. By analyzing the motion of microbeads placed on the myocardial surface, we measured strains near the outer curvature, in the central region, and near the inner curvature of the primitive ventricle. No significant differences in strain were found between stages. Relative to end diastole, all three regions shortened by about 10% during systole in the circumferential direction, and the outer curvature shortened longitudinally by about 5%. In contrast, and unlike strains in older hearts, the inner curvature and central regions elongated by approximately 5-10% in the longitudinal direction during systole. These results are consistent with microstructural data and suggest that the material properties of the outer curvature are relatively isotropic, whereas the properties of the central and inner curvature regions are orthotropic, with contractile stress exerted primarily in the circumferential direction.     

Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart

Complete understanding of the ontogenesis and early development of electrical activity and its related contraction has been hampered by our inability to apply conventional electrophysiological techniques to the early embryonic heart. Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes. Optical signals from voltage-sensitive dyes have provided a new and powerful tool for monitoring changes in membrane potential in a wide variety of living preparations. With this technique it is possible to make optical recordings from cells which are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor for the first time spontaneous electrical activity in pre-fused cardiac primordia in early chick embryos at the 6- and early 7-somite stages of development; we have been able to determine that the time of initiation of the heartbeat is the middle period of the 9-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the 3-somite stage. This article describes ionic properties of the spontaneous action potential and genesis of excitation-contraction coupling in the early embryonic chick and rat hearts. In addition, an improved view of the ontogenetic sequence of spontaneous electrical activity and its implications for excitation-contraction coupling in the early embryonic heart are proposed and discussed.     

Inhibition of a voltage-dependent cation channel in sarcoplasmic reticulum vesicles by caesium studied by using a potential-sensitive cyanine dye

The effect of caesium on the cation transport system in sarcoplasmic reticulum vesicles has been analysed kinetically through Tris+ influx. The Tris+ influx was measured by following the change in K+ diffusion potential due to the mutual diffusion between K+ and Tris+ in the presence of valiomycin using a potential probe; 3,3'-dipropylthiadicarbocyanine iodide. The main results were as follows. (1) Tris+ influx increased when membrane potential became inside-negative. This suggests that Tris+ permeates through the channel which has a voltage-dependent gate. (2) Cs+ reacted with the cation transport system only from the outside of the vesicle and inhibited Tris+ influx. The inhibition follows a single-site titration curve with a voltage-dependent dissociation constant of 18 mM at -60 mV. The inhibition can be explained by assuming that Cs+ binds to a site located about 45% of the way through the membrane from the outside of the vesicle in the open state of the channel. These results are in good agreement with those reported by Coronado and Miller (Coronado, R. and Miller, C. (1979) Nature 288, 495-497), which were gained electrically by using sarcoplasmic reticulum vesicles incorporated into an artificial planar phospholipid bilayer.     

A membrane potential-sensitive dye for vascular smooth muscle cells assays

Changes in membrane potential of rat aorta smooth muscle cells were investigated using the bis-oxonol sensitive probe DIBAC2(3). We compared the changes in membrane potential induced by a high external KCl concentration in aorta smooth muscle cells from normotensive 2 kidney (2K) and from renal hypertensive 2 kidney-1 clip (2K-1C) rats. The spectral properties of the membrane potential were first characterized in aqueous buffers and in cultured smooth muscle cells from 2K and 2K-1C rat aortas. Fluorescence emission and the images were recorded using a laser scanning confocal microscope. The relationship between fluorescence intensity (FI) and membrane potential (psi(m)) as a function of the increasing extracellular KCl concentration was linear in the 5-40 mmol/L KCl range in both 2K and 2K-1C rat aorta cells. Cell membranes from 2K-1C rat aorta cells were more depolarized (-55 mV) than 2K rat aorta cells (-65 mV). The results show that in 2K-1C aorta cells only 10 mmol/L KCl was needed to induce complete membrane depolarization while in 2K cells 40 mmol/L KCl was needed to induce a similar effect. This study clearly shows that the method is suitable to measure the membrane potential in cultured smooth muscle cells.     

Quantitation of corneal endothelial potentials using a carbocyanine dye

The carbocyanine dye, diS-C₃-(5) was used to quantitate the plasma membrane potential of the bullfrog corneal endothelium. It was shown that valinomycin hyperpolarized the endothelial cell and that in the presence of the ionophore the membrane potential largely reflected the K⁺ equilibrium potential. Using calibration curves constructed by changing medium K⁺ concentration in the presence of valinomycin, and nigericin and ouabain to abolish ion gradients and electrogenic pump activity, the cell membrane potential was calculated to be 28.6 ± 4.2 mV. The major source of this potential was a K⁺ diffusion potential, and the membrane Na⁺ conductance reduced the cell potential to less than the apparent K⁺ equilibrium potential of 51.5 ± 5.1 mV. About 20% of the cell potential could be ascribed to the rheogenic (Na⁺+K⁺)-ATPase.     

CEPU-1 expression in the early embryonic chick brain

We describe the expression pattern of CEPU-1, a cell adhesion molecule of the immunoglobulin superfamily, in the early chick embryo brain. An initially broad domain of expression, encompassing forebrain, midbrain and anterior hindbrain, is subsequently narrowed down to a ring-shaped domain at the midbrain-hindbrain boundary, co-localizing precisely with the expression of Wnt1 at the isthmus. In addition, CEPU-1 is expressed in the dorsal aspect of rhombomere 4 and its emigrating neural crest cells. Later in development, we also find CEPU-1 expression in other parts of the developing nervous system such as sensory ganglia and in the ventral aspect of forebrain, midbrain and hindbrain.     

Channel currents during spontaneous action potentials in embryonic chick heart cells. The action potential patch clamp.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.     

Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

Summary Changes in the fluorescence intensity of the dye 3-3 dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10^–9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg²⁺ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.Presented in part at the 17th Annual Cell Biology Meeting.Cell Biol. 75:103a, 1977.     

Chloride action potentials and currents in embryonic skeletal muscle of the chick

Chloride-dependent action potentials were elicited from embryonic skeletal muscle fibers of the chick during the last week of in ovo development. The duration of the action potentials was extremely long (greater than 8 sec). The action potentials were reversibly blocked by the stilbene derivative, SITS, a specific blocker of chloride permeability. Using patch clamp pipettes, in which the intracellular chloride concentration was controlled and with other types of ion channels blocked, the membrane potential at the peak of the action potential closely coincided with the chloride equilibrium potential calculated from the Nernst equation. These data indicate that activation of a chloride-selective conductance underlies the long duration action potential. The occurrence of the chloride-dependent action potential was found to increase during embryonic development. The percentage of fibers that displayed the action potential increased from approximately 20% at embryonic day 13 to approximately 70% at hatching. Chloride-dependent action potentials were not found in adult fibers. The voltage and time-dependent currents underlying the action potential were recorded under voltage clamp using the whole-cell version of the patch pipette technique. The reversal potential of the currents was found to shift with the chloride concentration gradient in a manner predicted by the Nernst equation, and the currents were blocked by SITS. These data indicate that chloride ions were the charge carriers. The conductance was activated by depolarization and exhibited very slow activation and deactivation kinetics.