Repolarization currents in embryonic chick atrial heart cell aggregates.

Outward membrane currents in aggregates of atrial cells prepared from 7-12-d-old chick embryonic hearts were measured with the two microelectrode voltage-clamp technique. Two outward current components, Ix1 and Ix2, were found in the plateau potential range of the action potential. The Ix1 component is activated between -50 and -20 mV; the Ix2 component is activated between -15 and +20 mV. The Ix1 component inwardly rectifies, whereas Ix2 has an approximately linear current-voltage relation. These preparations lack a time-dependent pacemaker current component, even though they beat spontaneously with an interbeat interval of approximately 1 s. A mathematical model of electrical activity is described based on our measurements of time-dependent outward current, and measurements in the literature of inward current components.     

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.     

Voltage clamp analysis of embryonic heart cell aggregates

The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.     

Acquisition of synchronous beating between embryonic heart cell aggregates and layers

Synchronous beating between chick embryonic heart cell aggregates and heart cell layers was used to study the relationship between intercellular adhesion and ionic coupling. Adhesion was measured by counting the proportion of aggregates which were not to be removed from cell layers by gentle washing after a 30 min incubation. Synchrony between bound aggregates and contiguous layers was assessed by phase microscopy. The first evidence of synchrony was seen 1.5 h after addition of aggregates to layers, following which there was an increase in the percentage of aggregates beating synchronously, reaching over 50% at 7 h and slowly increasing to a maximum of 65% by 24 h. Scanning electron microscopy and autoradiography of thymidine-labeled cells suggest that synchrony does not depend on cell movement at the interface between aggregate and layer. Acquisition of synchrony can be prevented completely by inhibiting protein synthesis, although pulsation of aggregates and layers continues in proportions unchanged from controls. After reversal of protein synthesis inhibition, synchrony is acquired at a rate and to an extent closely resembling that of newly adherent controls. These data indicate that ionic coupling is neither an inevitable nor an immediate consequence of adhesion. Since ionic coupling has been shown to correlate with the presence of gap junctions, the findings suggest that gap junctions are not involved in the initial events responsible for intercellular adhesion in vitro and that their formation following adhesion in this system may depend upon protein synthesis.     

Junctional resistance and action potential delay between embryonic heart cell aggregates

Spheroidal aggregates of embryonic chick ventricle cells were brought into contact and allowed to synchronize their spontaneous beats. Action potentials were recorded with both intracellular and extracellular electrodes. The degree of electrical interaction between the newly apposed aggregates was assessed by measuring the delay or latency (L) between the entrained action potentials, and by determining directly interaggregate coupling resistance (Rc) with injected current pulses. Aggregate size, contact area between the aggregates, and extracellular potassium concentration (Ko+) were important variables regulating the time-course of coupling. When these variables were controlled, L and Rc were found to be linearly related after beat synchrony was achieved. In 4.8 mM Ko+ L/Rc = 3.7 ms/M omega; in 1.3 mM Ko+ L/Rc = 10.1 ms/M omega. We conclude that action potential delay between heart cell aggregates can be related quantitatively to Rc.     

Concanavalin A increases spontaneous beat rate of embryonic chick heart cell aggregates

The plant lectin concanavalin A (Con A), at concentrations of 5–200 μg/ml, induced a twofold to fivefold increase in spontaneous beat rate of cultured aggregates of ventricular cells from seven-day chick embryos. This response was time, dose, and temperature dependent and was accompanied by a decrease in transmembrane potential. It could be blocked or reversed by α-methyl-D-mannoside but was not reversed by dilution alone. Binding of the lectin occurred in the cold, but a temperature-dependent process was also necessary to produce the response. Divalent (succinyl) Con A did not cause a beat rate increase. Whole heart aggregates responded similarly but less intensely than ventricular aggregates. Atrial aggregates, and whole heart aggregates treated with 5 μg/ml of Con A, produced a biphasic chronotropic response, first decreasing then increasing their beat rates. These results suggest that saccharide-bearing macromolecules on the heart cell surface play a role in regulating spontaneous beat rate.     

Ultraviolet-induced alterations of beat rate and electrical properties of embryonic chick heart cell aggregates

Embryonic heart cell aggregates were irradiated with ultraviolet light at wavelengths between 260 and 310 nm. Spontaneous beat rate was monitored with the aid of a closed-circuit TV camera and, in separate experiments, electrophysiological changes were assayed by intracellular recording. The characteristic response of 7-day aggregates was an increase in spontaneous beat rate to a maximum plateau level, followed by a rather abrupt cessation of beating. Intracellular recordings during irradiation showed a marked decline in the maximum rate of rise, overshoot, and repolarization phase of the action potential, and a significant change in threshold toward zero. The action spectrum for the termination of beating peaked between 290 and 295 nm; it fell off sharply at longer wavelengths and more slowly at shorter wavelengths. The maximum increase in beat rate was increasingly greater for shorter wavelengths and exhibited no peak in the wavelength range investigated. The sensitivity of aggregates to 295-nm light, as measured by the inverse of irradiation time required to terminate beating, decreased with increasing aggregate size and external potassium concentration, was relatively independent of temperature, and increased with embryonic age. The ultraviolet-induced increase in beat rate and termination of beating are attributed to separate complementary processes, a depolarization of the membrane, and a decline in "fast" sodium conductance.     

Morphological and functional correlates of synchronous beating between embryonic heart cell aggregates and layers

We have examined correlations between morphological and functional evidence of cell coupling between aggregates of beating embryonic heart cells and underlying layers. Synchronously beating aggregate-layer pairs were compared with asynchronous pairs. Intracellular microelectrode studies demonstrated that asynchronously beating aggregate-layers could not be induced to beat synchronously by electrical stimulation of the aggregate, whereas 86% of synchronous instances showed propagation of stimulating current pulses from aggregate to layer. By freeze fracture we have found significant differences both in the number and in the total area of gap junctions between the aggregate-layer interfaces of synchronous and asynchronous preparations. The data suggest that synchronous beating is a reliable functional indication of effective ionic coupling, and requires a certain area and number of gap junction/cell.     

Current noise parameters derived from voltage noise and impedance in embryonic heart cell aggregates.

We have recorded membrane impedance and voltage noise in the pacemaker range of potentials (-70 to -59 mV) from spheroidal aggregates of 7-d embryonic chick ventricle cells made quiescent by exposure to tetrodotoxin in medium containing 4.5 mM K+. The input capacitance is proportional to aggregate volume and therefore to total membrane area. The specific membrane capacitance is 1.24 microF/cm2. The input resistance at constant potential is inversely proportional to aggregate volume and therefore to total membrane area. The specific membrane resistance in 18 k omega . cm2 at -70 mV and increases to 81 k omega . cm2 at -59 mV. The RC time constant is 22 ms at -70 mV and increases to 146 ms at -59 mV. The aggregate transmembrane small-signal impedance can be represented by a parallel RC circuit itself in parallel with an inductive branch consisting of a resistor (rL) and an inductor (L) in series. The time constant of the inductive branch (L/rL) is 340 ms, and is only weakly dependent on potential. Correlation functions of aggregate voltage noise and the impedance data were modeled by a population of channels with simple open-close kinetics. The time constant of a channel (tau s) derived from the noise analysis is 300 ms. The low frequency limit of the pacemaker current noise (SI[0]), derived from the voltage noise and impedance, increases from 10(-20) A2/Hz . cm2 at -67 mV to 10(-19) A2/Hz . cm2 at -61 mV.     

Phase resetting of the rhythmic activity of embryonic heart cell aggregates. Experiment and theory.

Injection of a current pulse of brief duration into an aggregate of spontaneously beating chick embryonic heart cells resets the phase of the activity by either advancing or delaying the time of occurrence of the spontaneous beat subsequent to current injection. This effect depends upon the polarity, amplitude, and duration of the current pulse, as well as on the time of injection of the pulse. The transition from prolongation to shortening of the interbeat interval appears experimentally to be discontinuous for some stimulus conditions. These observations are analyzed by numerical investigation of a model of the ionic currents that underlie spontaneous activity in these preparations. The model consists of: Ix, which underlies the repolarization phase of the action potential, IK2, a time-dependent potassium ion pacemaker current, Ibg, a background or time-independent current, and INa, an inward sodium ion current that underlies the upstroke of the action potential. The steady state amplitude of the sum of these currents is an N-shaped function of potential. Slight shifts in the position of this current-voltage relation along the current axis can produce either one, two, or three intersections with the voltage axis. The number of these equilibrium points and the voltage dependence of INa contribute to apparent discontinuities of phase resetting. A current-voltage relation with three equilibrium points has a saddle point in the pacemaker voltage range. Certain combinations of current-pulse parameters and timing of injection can shift the state point near this saddle point and lead to an interbeat interval that is unbounded . Activation of INa is steeply voltage dependent. This results in apparently discontinuous phase resetting behavior for sufficiently large pulse amplitudes regardless of the number of equilibrium points. However, phase resetting is fundamentally a continuous function of the time of pulse injection for these conditions. These results demonstrate the ionic basis of phase resetting and provide a framework for topological analysis of this phenomenon in chick embryonic heart cell aggregates.     

Membrane response to current pulses in spheroidal aggregates of embryonic heart cells

Hearts from chick embryos aged 4,7, or 14 days were dissociated into their component cells, and the cells allowed to reassociate in the form of smooth-surfaced spheroidal aggregates on a gyratory shaker. Records from intracellular electrodes inserted into two widely spaced cells in a spontaneously beating aggregate indicated that the action potentials occurred virtually simultaneously. In aggregates made quiescent with tetrodotoxin, the voltage response to a current pulse injected in one cell could be noted by recording with a second microelectrode at various distance from the current source. The magnitude of the response was found not to vary with distance. It is concluded that the component cells in an aggregate are normally tightly coupled electrically; the cell boundaries do not constitute an appreciable resistive barrier. Such ag-regates behave as virtually isopential systems, with properties similar to those of single spherical cells, as modeled by Eisenberg and Engel (1970. J. Gen. Physiol. 55:736-757). Passive membrane time constant ranged from 11 to 31 ms, with a mean value of 17 ms; this value did not vary with aggregate size. Input resistance (V/I) varied inversely with aggregate size, as predicted, but with much scatter in the measured values. Specific membrane resistance was calculated as either 13,000 or 800 ohm-cm2 depending on whether input resistance was attributed to the total cell surface membrane area or to the outer surface of the sphere alone. No systematic difference in passive electrical properties of aggregates composed of 4-, 7-, and 14-day cells was seen. It is concluded that these aggregates may be suitable for voltage clamp analysis of their excitable membrane properties.     

The topology of phase response curves induced by single and paired stimuli in spontaneously oscillating chick heart cell aggregates.

The topological properties of the phase resetting of biological oscillators by an isolated stimulus delivered at various phases of the cycle depend on whether the stimulus is "weak" or "strong." When multiple stimuli are delivered to the oscillator, the response to stimulation also depends on the time between the stimuli, and the rate at which the oscillator returns to an underlying limit cycle attractor. If the time between two consecutive "weak" stimuli is sufficiently short, the effects produced by the pair of stimuli may be characteristic of a single "strong" stimulus. These results are demonstrated in a model experimental system, spontaneously beating aggregates of cells derived from embryonic chick heart, and are illustrated by consideration of a simple theoretical model of nonlinear oscillators, the Poincaré oscillator.     

Development of electrical coupling and action potential synchrony between paired aggregates of embryonic heart cells

Summary Pairs of spheroidal aggregates of embryonic chick heart cells, held in suction pipettes were brought into contact and allowed to synchronize their spontaneous action potentials. Contractions were suppressed with cytochalasin B. Both intracellular and extracellular electrodes were used to analyze the development of synchrony. Electric coupling occurred in three phases. During phase I electrical interactions were absent despite close physical contact. Phase II was characterized by partial synchrony. Action potentials in the faster aggregate (F) induced small depolarizations in the other member of the pair (S). These depolarizations sometimes triggered action potentials inS depending on when during the diastolic depolarization inS they occurred. In these cases both the latency between the action potentials (L) and the fluctuations in latency (V _L) were large. At the end of phase II the aggregates often passed through a brief period when fluctuation in interbeat interval in both increased noticeably. In phase III, beginning about 8 min after initial contact, action potentials were completely entrained at a certainL. During the subsequent 20–40 minL fell along an approximately exponential time course from about 130 to <1 msec, whileV _L declined in parallel. When well-coupled aggregates were pulled apart and immediately pressed back together, they re-established synchronization according to the usual three-phase time course. Synchronized aggregates could be partially decoupled by separating them just far enough to reduce the area of mutual contact. Pairs joined only by cellular strands maintained entrained action potentials with long latencies for many minutes. These results indicate that electronic junctions form between the paired heart cell aggregates causing the gradual development of action potential synchrony.     

Muscarinic receptors and responses in intact embryonic chick atrial and ventricular heart cells

We have studied muscarinic acetylcholine (ACh) receptors in intact atrial and ventricular heart cells dissociated from 8-day chick embryos and maintained in sparse cell cultures. Two specific antagonists, [³H]quinuclidinyl benzilate (QNB) and [³H]N-methyl scopolamine (NMS), bind to surface sites with affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{s}\text{? 40}\text{and}\text{400}\text{p}\text{M}$, respectively). The concentration of [³H]QNB sites in ventricular cell cultures (460 fmole/mg protein) was comparable to the concentration of sites in atrial cultures (420 fmole/mg protein). The same result was obtained with [³H]NMS. Autoradiography following incubation in saturating concentrations of [³H]QNB shows that nearly all of the atrial and ventricular myocytes were labeled and that the distribution of grains over individual cells was uniform. The mean binding site density was 109/μm² for atrial cells 117/μm² for ventricular cells. In contrast to the antagonist binding results, microelectrode recordings from individual myocytes or from small clusters of cells showed that many more atrial myocytes (89%) were sensitive to 10^?4M carbachol than were ventricular myocytes (26%). Saline extract of embryonic brain tissue added to the culture medium did not alter the number or distribution of ligand binding sites but it produced a 2.6-fold increase in the number of carbachol-sensitive ventricular cells.