Biomedical subjects

R Wilders

Publications and source records attributed to R Wilders.

12 recordsLinked to original sources

PubMed1998

Quantitative analysis of dual whole-cell voltage-clamp determination of gap junctional conductance.

The dual whole-cell voltage-clamp technique is used widely for determination of kinetics and conductance of gap junctions. The use of this technique may, however, occasion to considerable errors. We have analysed the errors in steady state junctional conductance measurements under different experimental conditions. The errors in measured junctional conductance induced by series resistance alone, and by series resistance in combination with membrane resistance, were quantified both theoretically and experimentally, on equivalent resistive circuits with known resistance values in a dual voltage-clamp setup. We present and analyse a method that accounts for series resistance and membrane resistance in the determination of true junctional conductance. This method requires that series resistance is determined during the experiment, and involves some calculations to determine membrane resistance. We demonstrate that correction for both membrane and series resistance reduces the error in measured junctional conductance to near zero, even when membrane resistances on both sides of the gap junction are as low as 20 MOmega and the (true) junctional conductance is as high as 100 nS.

Computer Simulation↗

PubMed1998

Pacemaker synchronization of electrically coupled rabbit sinoatrial node cells.

The effects of intercellular coupling conductance on the activity of two electrically coupled isolated rabbit sinoatrial nodal cells were investigated. A computer-controlled version of the "coupling clamp" technique was used in which isolated sinoatrial nodal cells, not physically in contact with each other, were electrically coupled at various values of ohmic coupling conductance, mimicking the effects of mutual interaction by electrical coupling through gap junctional channels. We demonstrate the existence of four types of electrical behavior of coupled spontaneously active cells. As the coupling conductance is progressively increased, the cells exhibit: (a) independent pacemaking at low coupling conductances, (b) complex dynamics of activity with mutual interactions, (c) entrainment of action potential frequency at a 1:1 ratio with different action potential waveforms, and (d) entrainment of action potentials at the same frequency of activation and virtually identical action potential waveforms. The critical value of coupling conductance required for 1:1 frequency entrainment was <0.5 nS in each of the five cell pairs studied. The common interbeat interval at a relatively high coupling conductance (10 nS), which is sufficient to produce entrainment of frequency and also identical action potential waveforms, is determined most by the intrinsically faster pacemaker cell and it can be predicted from the diastolic depolarization times of both cells. Evidence is provided that, at low coupling conductances, mutual pacemaker synchronization results mainly from the phase-resetting effects of the action potential of one cell on the depolarization phase of the other. At high coupling conductances, the tonic, diastolic interactions become more important.

Action Potentials↗

PubMed1997

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

We previously developed a technique (R. Kumar, R. Wilders, R. W. Joyner, H. J. Jongsma, E. E. Verheijck, D. A. Golod, A. C. G. van Ginneken, and W. N. Goolsby. Circulation 94: 833-841, 1996) for study of a mathematical model cell with spontaneous activity, viz. a "real-time" simulation of a rabbit sinoatrial node cell (SAN model cell; R. Wilders, H. J. Jongsma, and A. C. van Ginneken. Biophys. J. 60: 1202-1216, 1991) simultaneously being electrically coupled via our "coupling clamp" [H. Sugiura and R. W. Joyner. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1591-H1604, 1992] circuit to a real, isolated ventricular myocyte. We now apply this technique to investigate effects of coupling conductance (Gc), cell size, and the modulation of membrane potential by elevated extracellular potassium concentration on the ability of an ectopic focus, represented by the SAN model cell, to successfully drive a ventricular cell. Values of Gc and the relative sizes of the two cells define three possible outcomes: 1) spontaneous pacing of the SAN model cell but not driving of the ventricular cell, 2) cessation of spontaneous pacing, or 3) pacing of the SAN model cell and driving of the ventricular cell. Below a critical size of the SAN model cell only the first two of these outcomes is possible. Above this critical size there is a range of Gc that allows successful operation of the system as an ectopic focus. Elevation of extracellular potassium concentration from 4 to 8 mM increases both the lower bound and upper bound of Gc for this range. Elevation of extracellular potassium concentration, as commonly observed in myocardial ischemia, may have effects on either inhibiting or releasing from inhibition an ectopic focus.

Animals↗

PubMed1996

Experimental model for an ectopic focus coupled to ventricular cells.

BACKGROUND: We used a mathematical model of a sinoatrial nodal cell (SAN model) electrically coupled to real ventricular cells (VCs) to investigate action potential conduction from an automatic focus. METHODS AND RESULTS: Since input resistance of a VC is less than that of an SAN cell, coupling of the SAN model, with a size factor of 1, to a VC produced either (1) spontaneous pacing at the slower rate of the SAN model but without driving (activation) of the VC for lower values of coupling conductance (Gj) or (2) inhibition of pacing of the SAN model by electrical coupling to the VC for higher values of Gj. When the SAN model was adjusted in size to be 3 to 5 times larger than a sinoatrial nodal cell, thus making effective SAN model capacitance 3 to 5 times larger and input resistance 3 to 5 times smaller, the SAN model propagated activity to the coupled VC for Gj above a critical value. When the VC was paced at 1 Hz, the coupled cell pair demonstrated a stable rhythm of alternating cycle lengths and alternating conduction directions. By increasing pacing frequency to 2 Hz, we converted this rhythm to a regular 2-Hz frequency in which each action potential originated in the VC. More complex periodic interactions were observed at intermediate cycle lengths and lower or higher values of Gj. CONCLUSIONS: The phenomena we observed demonstrate the critical role of the size of an automatic focus as well as the coupling in the propagation of activity from the focus into surrounding myocardium.

Action Potentials↗

PubMed1996

Modulating L-type calcium current affects discontinuous cardiac action potential conduction.

We have used pairs of cardiac cells (i.e., one real guinea pig ventricular cell and a real-time simulation of a numerical model of a guinea pig ventricular cell) to evaluate the effects on action potential conduction of a variable coupling conductance in combination with agents that either increase or decrease the magnitude of the L-type calcium current. For the cell pairs studied, we applied a direct repetitive stimulation to the real cell, making it the "leader" cell of the cell pair. We have demonstrated that significant delays in action potential conduction for a cell pair can occur either with a decreased value of coupling conductance or with an asymmetry in size such that the follower cell is larger than the leader cell. In both conditions we have shown that isoproterenol, applied to the real cell at very low concentrations, can reversibly decrease the critical coupling conductance (below which action potential conduction fails) for a cell pair with fixed cell sizes, or, for a fixed value of coupling conductance, increase the maximum allowable asymmetry in cell size for successful conduction. For either of these effects, we were able to show that treatment of the real cell with BayK 8644, which more specifically increases the magnitude of the L-type calcium current, was able to mimic the actions of isoproterenol. Treatment of the leader cell of the cell pair (the real cell) with nifedipine, which selectively lowers the magnitude of the L-type calcium current, had effects opposite those of isoproterenol or BayK 8644. The actions of nifedipine, isoproterenol, and BayK 8644 are all limited to conditions in which the conduction delay is on the order of 5 ms or more, whether this delay is caused by limited coupling conductance or by asymmetry in size of the cells. This limitation is consistent with the time course of the L-type calcium current and suggests that the effects of calcium channel blockers or beta-adrenergic blocking drugs, in addition to being selective for regions of the heart that depend on the L-type calcium current for the upstroke of the action potential, would also be somewhat selective for regions of the heart that have discontinuous conduction, either normally or because of some pathological condition.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1996

Action potential conduction between a ventricular cell model and an isolated ventricular cell.

We used the Luo and Rudy (LR) mathematical model of the guinea pig ventricular cell coupled to experimentally recorded guinea pig ventricular cells to investigate the effects of geometrical asymmetry on action potential propagation. The overall correspondence of the LR cell model with the recorded real cell action potentials was quite good, and the strength-duration curves for the real cells and for the LR model cell were in general correspondence. The experimental protocol allowed us to modify the effective size of either the simulation model or the real cell. 1) When we normalized real cell size to LR model cell size, required conductance for propagation between model cell and real cell was greater than that found for conduction between two LR model cells (5.4 nS), with a greater disparity when we stimulated the LR model cell (8.3 +/- 0.6 nS) than when we stimulated the real cell (7.0 +/- 0.2 nS). 2) Electrical loading of the action potential waveform was greater for real cell than for LR model cell even when real cell size was normalized to be equal to that of LR model cell. 3) When the size of the follower cell was doubled, required conductance for propagation was dramatically increased; but this increase was greatest for conduction from real cell to LR model cell, less for conduction from LR model cell to real cell, and least for conduction from LR model cell to LR model cell. The introduction of this "model clamp" technique allows testing of proposed membrane models of cardiac cells in terms of their source-sink behavior under conditions of extreme coupling by examining the symmetry of conduction of a cell pair composed of a model cell and a real cardiac cell. We have focused our experimental work with this technique on situations of extreme uncoupling that can lead to conduction block. In addition, the analysis of the geometrical factors that determine success or failure of conduction is important in the understanding of the process of discontinuous conduction, which occurs in myocardial infarction.

Action Potentials↗

PubMed1996

Model clamp and its application to synchronization of rabbit sinoatrial node cells.

A method for coupling an isolated cardiac cell to a simulated cardiac cell, i.e., the real-time solution of a mathematical model of such cell, has been developed. With this "model clamp" technique, the real cell and the model cell are coupled by any desired value of intercellular coupling conductance, producing the effect of mutual interaction by electrical coupling through gap junctional channels. We implemented the model clamp technique with our previously published model of an isolated rabbit sinoatrial node cell. We used this model clamp system to study synchronization of sinoatrial node cells with regard to the critical value of intercellular coupling conductance required for frequency entrainment and the common interbeat interval during frequency entrainment. This common interbeat interval lay between the intrinsic intervals of the real cell and the model cell, but was closer to that of the intrinsically faster beating cell. Critical coupling conductance increased with increasing difference in intrinsic interbeat interval of the real cell and the model cell and ranged between 50 and 300 pS in 11 hybrid cell pairs.

Action Potentials↗

PubMed1995

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels.

The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

Amino Acids↗

PubMed1993

Beating irregularity of single pacemaker cells isolated from the rabbit sinoatrial node.

Single pacemaker heart cells discharge irregularly. Data on fluctuations in interbeat interval of single pacemaker cells isolated from the rabbit sinoatrial node are presented. The coefficient of variation of the interbeat interval is quite small, approximately 2%, even though the coefficient of variation of diastolic depolarization rate is approximately 15%. It has been hypothesized that random fluctuations in interbeat interval arise from the stochastic behavior of the membrane ionic channels. To test this hypothesis, we constructed a single channel model of a single pacemaker cell isolated from the rabbit sinoatrial node, i.e., a model into which the stochastic open-close kinetics of the individual membrane ionic channels are incorporated. Single channel conductances as well as single channel open and closed lifetimes are based on experimental data from whole cell and single channel experiments that have been published in the past decade. Fluctuations in action potential parameters of the model cell are compared with those observed experimentally. It is concluded that fluctuations in interbeat interval of single sinoatrial node pacemaker cells indeed are due to the stochastic open-close kinetics of the membrane ionic channels.

Action Potentials↗

PubMed1992

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.

Animals↗

PubMed1992

Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.

The electrical properties of gap junctions in cell pairs are usually studied by means of the dual voltage clamp method. The voltage across the junctional channels, however, cannot be controlled adequately due to an artificial resistance and a natural resistance, both connected in series with the gap junction. The access resistances to the cell interior of the recording pipettes make up the artificial resistance. The natural resistance consists of the cytoplasmic access resistances to the tightly packed gap junction channels in both cells. A mathematical model was constructed to calculate the actual voltage across each gap junction channel. The stochastic open-close kinetics of the individual channels were incorporated into this model. It is concluded that even in the ideal case of complete compensation of pipette series resistance, the number of channels comprised in the gap junction may be largely underestimated. Furthermore, normalized steady-state junctional conductance may be largely overestimated, so that transjunctional voltage dependence is easily masked. The model is used to discuss conclusions drawn from dual voltage clamp experiments and offers alternative explanations for various experimental observations.

Animals↗

PubMed1991

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.

Action Potentials↗