A.C.G. van Ginneken

A transient outward current in isolated cells from the crista terminalis of rabbit heart.

Voltage‐clamp experiments were carried out with the objective of identifying and characterizing the time‐ and voltage‐dependent properties of a transient outward current recorded in single myocytes from the crista terminalis region of the rabbit heart. A collagenase enzymic dispersion procedure similar to that described by Desilets & Horackova (1982) was used to obtain these viable individual myocytes. Transmembrane ionic currents were recorded using a single micro‐electrode voltage‐clamp technique. In experiments aimed at studying a tetrodotoxin‐resistant transient inward current, (ICa); a transient outward current was consistently recorded following blockade of ICa with Cd2+ (5 X 10(‐4) M). The time and voltage dependence of the activation and inactivation of this current were measured. Its steady‐state inactivation curve spans the voltage range ‐70 to ‐10 mV, and it is activated between ‐20 and +10 mV. The reversal potential of this transient outward current is approximately ‐75 mV in [K+]O 5 mM, suggesting that it is carried mainly by K+. This transient outward current can be inhibited completely by external application of 4‐aminopyridine (4‐AP, 3 mM). The time‐ and voltage‐dependent properties, the reversal potential, and the sensitivity to 4‐AP of this transient outward current are all very similar to those of a transient outward current first identified in molluscan neurones. Hence, we have labelled it, IA. Selective inhibition of IA and knowledge of its voltage‐ and time‐dependent properties yield specific predictions concerning its role in the action potential of isolated crista terminalis cells. Consistent with these predictions, a decrease in stimulus rate is found to decrease the duration of the action potential and vice versa; and application of effective doses of 4‐AP results in a substantial lengthening of the action potential. These results are discussed in terms of the possible physiological role of IA in subsidiary or follower pace‐maker tissue, and the anatomical and physiological heterogeneity of the sino‐atrial node region of the rabbit heart.

Pacemaker activity of the rabbit sinoatrial node. A comparison of mathematical models

In the past decade, three mathematical models describing the pacemaker activity of the rabbit sinoatrial node have been developed: the Bristow-Clark model, the Irisawa-Noma model, and the Noble-Noble model. In a comparative study it is demonstrated that these models, as well as subsequent modifications, all have several drawbacks. A more accurate model, describing the pacemaker activity of a single pacemaker cell isolated from the rabbit sinoatrial node, was constructed. Model equations, including equations for the T-type calcium current, are based on experimental data from voltage clamp experiments on single cells that were published during the last few years. In contrast to the other models, only a small amount of background current contributes to the overall electrical charge flow. The action potential parameters of the model cell, its responses to voltage clamp steps and its current-voltage relationships have been computed. The model is used to discuss the relative contribution of membrane current components to the slow diastolic depolarization phase of the action potential.

Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node

In an attempt to understand better the directional differences in conduction velocity in the rabbit sinoatrial node, a possible conductive role of the abundant connective tissue surrounding the myocytes has been investigated. In particular, starting from the finding of communicating junctions between heart muscle cells and fibroblasts in tissue culture, heterologous gap junctions were searched for in thin sections of the rabbit sinoatrial node. Within and at the edge of nodal cell clusters, fibroblasts often show thin sheet-like extensions parallel to the surface of myocytes. In contrast to the intimately contacting myocytes, fibroblast extensions are kept separated from the myocytes by the basement membrane of the latter. Besides some rare undefined membrane appositions a single tiny gap junction-like structure was found between a fibroblast and a myocyte in a tissue area in which the calculated number of gap junctions between myocytes amounts from 1.10(4) to 3.10(4). Yet, fibroblasts are linked together regularly by small gap junctions containing a wider gap than the junctions between the myocytes (1.4 +/- 0.4 nm vs. 1.0 +/- 0.4 nm, resp., P less than 0.05). As an alternative to direct electrical coupling, the possibility of interaction between fibroblasts and nodal cells by capacitive coupling has been considered. Model calculations based on the reconstruction of some fibroblast extensions parallel to nodal cells show that the current which can be transmitted from discharging nodal cells to fibroblasts is negligible. It is concluded that fibroblasts do not participate in the impulse conduction within the sinoatrial node. The origin of the directional differences in conduction velocity in the sinoatrial node must be found in the spatial arrangement of the myocytes and the distribution of the gap junctions between these cells only.

Single delayed rectifier channels in the membrane of rabbit ventricular myocytes.

In rabbit ventricular cells, the delayed rectifier current (IK) has not been extensively studied, and properties of single IK channels still need to be determined. In this study, we present data on a voltage-dependent channel in rabbit ventricular cells; the properties indicate that it is an IK channel. Patch-clamp experiments were carried out on cell-attached and inside-out patches of rabbit ventricular cells. Single-channel currents were recorded at negative potentials as inward currents with 150 mM K+ in the pipette. Voltage-dependent channel activity was only present after the return from a depolarizing test pulse, indicating activation on depolarization. Single-channel conductance calculated from the current-voltage relation was 13.1 pS (pooled data, n = 8). The shift in reversal potential of the unitary currents, determined at 150 and 300 mM K+ at the intracellular side of the membrane, showed that the channels were highly permeable to potassium ions. Increase of the duration or the amplitude of the depolarizing test pulse increased channel activity. The time constant for activation at +30 mV was 187 msec (pooled data, n = 4). Half-activation potential was -4.9 +/- 3.8 mV (mean +/- SD), and the slope factor was 7.2 +/- 3.7 mV (mean +/- SD). Current tails, reconstructed from averaged single-channel currents, revealed that the time course of deactivation decreased from 694 +/- 73 msec at -80 mV to 136 +/- 39 msec at -110 mV. Additional evidence that the channel was indeed an IK channel was provided by the observation that the channel was blocked by 10(-7) M E-4031, a class III antiarrhythmic agent that has been shown to block a component of the macroscopic IK in guinea pig heart.

Mechanisms of impulse generation in isolated cells from the rabbit sinoatrial node.

The contribution to spontaneous activity of three currents INa, If, and Isi was investigated in isolated spontaneously active SA nodal cells. It was demonstrated that isolated cells have electrophysiological properties similar to those of cells in the intact node. Evidence for the contributory role of INa to the upstroke of the action potential was obtained from membrane responsiveness curves and from the observation that Vmax was strongly reduced after the addition of 9 microM TTX to the bathing solution. The relative role of If and Isi as depolarizing diastolic membrane currents was investigated by relating the maximal density of each of these currents to the corresponding DDR during spontaneous activity. Both currents If and Isi-peak appear to be linearly related to DDR, indicating their contributory role to diastolic depolarization. Although the cells studied were heterogeneous with regard to the density of If and Isi we found no evidence for their separation into distinct groups of pacemaker cell types.

Slow inward current in aggregates of neonatal rat heart cells and its contribution to the steady state current-voltage relationship

In voltage clamped neonatal rat heart cells a transient current is observed during depolarizing potential steps, which was identified as slow inward current (Isi) by its range of activation, by its reversal potential of approximately +50 mV and by its sensitivity to D600 or low external Ca2+. ThisIsi activates too fast to be detected by the present methods, which implies that activation is completed within milliseconds. The time constant of inactivation was weakly potential dependent and less than 30 ms between −40 mV and +20 mV. Thef∞ curve ofIsi had a sigmoidal shape with 90% and 10% values near −50 mV and −10 mV respectively, half maximum was at −25 mV. From double pulse experiments an estimate was obtained of the potential dependence and amplitude of steady stateIsi. A maximum was expected around −30 mV. Steady stateIsi appears to be present indeed in steady state current voltage relations, as the relative minimum at −30 mV in such relations is abolished by 5·10−7 g/ml D600. Currents tails during hyperpolarizing steps from prepulse potentials near 0 mV are potential dependent in a way expected whenIsi contributes to these current tails by a decrease in inactivation. Moreover, the current tails are diminished by D600 or Co2+. Consequenses of steady stateIsi are discussed.

The passive electrical properties of spheroidal aggregates cultured from neonatal rat heart cells.

Membrane specific resistance and capacitance of non‐spontaneously active spheroidal aggregates, cultured from collagenase‐dissociated neonatal rat heart cells, were calculated from changes in membrane potential due to intracellularly injected rectangular hyper‐ and depolarizing current pulses during diastole. The relation between steady‐state membrane voltage displacement and injected current is linear for current pulses between +10 and ‐10 nA. No significant fall‐off of electrotonic potential is measured in an aggregate at increasing distances from the site of current injection. The aggregate membrane resistance (input resistance) was best fitted by an inverse square function of the aggregate radius. This suggests selective current flow through the outer membranes of the spheroidal aggregate. Taking this into account the membrane specific resistance was calculated to be 753 +/‐ 38 omega cm2 (S.E. of mean; n = 39). The time course of the change in membrane potential is exponential with a time constant ranging from 5 to 26 ms, depending on the aggregate radius. The aggregate membrane capacitance is calculated from the exponential transients for each aggregate and appears to be a cubic function of the radius, indicating that the membrane area of all cells in the preparation equally contributes to the input capacitance. The membrane specific capacitance is calculated to be 0.97 +/‐ 0.02 microF/cm2 (S.E. of mean; n = 100). It is concluded that myocytes in aggregates are electrically well coupled and that a resistance in series with the inner membranes, if present, is negligible compared to the membrane resistance of the internal cells. In order to explain the finding that the membrane resistance was not inversely related to the cube of the aggregate radius, it is postulated that the membrane specific resistance might be a function of aggregate radius.

Modulation of propagation from an ectopic focus by electrical load and by extracellular potassium concentration

Cardiac arrhythmias may be due to spontaneous automatic activity from an ectopic focus. We have developed a technique in which a computer model of an ectopic focus (represented by an SA Node cell model), running in real time, can be coupled by a variable conductance, G/sub c/, to a real ventricular cell. We used this technique to investigate the effects of G/sub c/, cell size, and elevated potassium on the ability of an ectopic focus to successfully drive the ventricular cell. For the hybrid cell pair there are three possible outcomes in the steady state: (1) pacing of the SAN model cell but not driving of the ventricular cell, (2) cessation of pacing, or (3) successful pacing of the SAN model cell and driving of the ventricular cell. Elevation of potassium concentration increases both the lower and upper bound of the values of G/sub c/ which define the successful pacing and driving range.