Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale.

We have applied multiple site optical recording of transmembrane voltage (MSORTV) to patterned growth cultures of heart cells to analyze the effect of geometry per se on impulse propagation in excitable tissue, with cellular and subcellular resolution. Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for this application; it is internalized slowly and permits optical recording with signal-to-noise ratios as high as 40:1 (measured peak-to-peak) and average fractional fluorescence changes of 15% per 100 mV. Using a x 100 objective and a fast data acquisition system, we could resolve impulse propagation on a microscopic scale (15 microns) with high temporal resolution (uncertainty of +/- 5 microseconds). We could observe the decrease in conduction velocity of an impulse propagating along a narrow cell strand as it enters a region of abrupt expansion, and we could explain this phenomenon in terms of the micro-architecture of the tissue. In contrast with the elongated and aligned cells forming the narrow strands, the cells forming the expansions were aligned at random and presented 2.5 times as many cell-to-cell appositions per unit length. If the decrease in conduction velocity results entirely from this increased number of cell-to-cell boundaries per unit length, the mean activation delay introduced by each boundary can be estimated to be 70 microseconds. Using this novel experimental system, we could also demonstrate the electrical coupling of fibroblasts and endotheloid cells to myocytes in culture.     

Mechanisms of unidirectional block in cardiac tissues.

We used numerical solutions for cable equations representing nonuniform cardiac strands to investigate possible mechanisms of unidirectional block (UB) of action potential propagation. Because the presence of UB implies spatial asymmetry in some property along the strand, we varied membrane properties (gNa or leakage conductance), cell diameter, or intercellular resistance as functions of distance such that a propagating action potential encountered the parameter changes either gradually or abruptly. For changes in membrane properties there was very little difference in the effects on propagation for the gradual or abrupt encounter; but, for changes in cell diameter or in intercellular resistance, there were large differences leading to the production of UB over a wide range of parameter values.     

Fast calcium wave propagation mediated by electrically conducted excitation and boosted by CICR

We have investigated synchronization and propagation of calcium oscillations, mediated by gap junctional excitation transmission. For that purpose we used an experimentally based model of normal rat kidney (NRK) cells, electrically coupled in a one-dimensional configuration (linear strand). Fibroblasts such as NRK cells can form an excitable syncytium and generate spontaneous inositol 1,4,5-trisphosphate (IP(3))-mediated intracellular calcium waves, which may spread over a monolayer culture in a coordinated fashion. An intracellular calcium oscillation in a pacemaker cell causes a membrane depolarization from within that cell via calcium-activated chloride channels, leading to an L-type calcium channel-based action potential (AP) in that cell. This AP is then transmitted to the electrically connected neighbor cell, and the calcium inflow during that transmitted AP triggers a calcium wave in that neighbor cell by opening of IP(3) receptor channels, causing calcium-induced calcium release (CICR). In this way the calcium wave of the pacemaker cell is rapidly propagated by the electrically transmitted AP. Propagation of APs in a strand of cells depends on the number of terminal pacemaker cells, the L-type calcium conductance of the cells, and the electrical coupling between the cells. Our results show that the coupling between IP(3)-mediated calcium oscillations and AP firing provides a robust mechanism for fast propagation of activity across a network of cells, which is representative for many other cell types such as gastrointestinal cells, urethral cells, and pacemaker cells in the heart.     

Gap junctions in excitable cells

Gap junction channels are an integral part of the conduction or propagation of an action potential from cell to cell. Gap junctions have rather unique gating and permeability properties which permit the movement of molecules from cell to cell. These molecules may not be directly linked to action potentials but can alter nonjunctional processes within cells, which in turn can affect conduction velocity. The data described in this review reveal that, for the majority of excitable cells, there are two limiting factors, with respect to gap junctions, that affect the conduction/propagation of action potentials. These are (1) the total number of channels and (2) the selective permeability of the channels. Interestingly, voltage dependence and the time course of voltage inactivation (kinetics) are not rate limiting steps under normal physiological conditions for any of the connexins studied so far. Only specialized rectifying electrical synapses utilize strong voltage dependence and rapid kinetics to permit or deny the continued propagation of an action potential.     

Dynamic changes of cardiac conduction during rapid pacing

Slow conduction and unidirectional conduction block (UCB) are key mechanisms of reentry. Following abrupt changes in heart rate, dynamic changes of conduction velocity (CV) and structurally determined UCB may critically influence arrhythmogenesis. Using patterned cultures of neonatal rat ventricular myocytes grown on microelectrode arrays, we investigated the dynamics of CV in linear strands and the behavior of UCB in tissue expansions following an abrupt decrease in pacing cycle length (CL). Ionic mechanisms underlying rate-dependent conduction changes were investigated using the Pandit-Clark-Giles-Demir model. In linear strands, CV gradually decreased upon a reduction of CL from 500 ms to 230-300 ms. In contrast, at very short CLs (110-220 ms), CV first decreased before increasing again. The simulations suggested that the initial conduction slowing resulted from gradually increasing action potential duration (APD), decreasing diastolic intervals, and increasing postrepolarization refractoriness, which impaired Na(+) current (I(Na)) recovery. Only at very short CLs did APD subsequently shorten again due to increasing Na(+)/K(+) pump current secondary to intracellular Na(+) accumulation, which caused recovery of CV. Across tissue expansions, the degree of UCB gradually increased at CLs of 250-390 ms, whereas at CLs of 180-240 ms, it first increased and subsequently decreased. In the simulations, reduction of inward currents caused by increasing intracellular Na(+) and Ca(2+) concentrations contributed to UCB progression, which was reversed by increasing Na(+)/K(+) pump activity. In conclusion, CV and UCB follow intricate dynamics upon an abrupt decrease in CL that are determined by the interplay among I(Na) recovery, postrepolarization refractoriness, APD changes, ion accumulation, and Na(+)/K(+) pump function.     

Reflected reentry, delayed conduction, and electrotonic inhibition in segmentally depressed atrial tissues

Reflection is a subclass of reentrant cardiac arrhythmias in which reexcitation of the heart occurs as a result of to and fro electrotonically mediated transmission of impulses across a narrow zone of impaired conductivity. Although relatively well characterized in ventricular tissues, the reflection mechanism has not been studied in atrial tissues. In this study we examine the possibility of reflected reentry in segmentally depressed atrial tissues and evaluate conduction characteristics in these preparations. Narrow strips of atrial pectinate muscle or crista terminalis (canine and calf) were placed in a three-chambered bath and the central segment was superfused with an isotonic sucrose solution or an "ischemic" Tyrode's solution. Proximal to distal conduction across the 1.0- to 1.2-mm wide ischemic gap showed step delays as long as 210 ms. Reflected reentry was readily demonstrable when prominent step delays occurred during anterograde conduction of the impulse across the gap. Progressive acceleration of the stimulation rate resulted in progressively greater impairment of anterograde conduction until complete block occurred. The incidence and patterns of reflected reentry were therefore a sensitive function of the stimulation rate. Other features exhibited by these preparations include a slow recovery of excitability following the action potential, postrepolarization refractoriness, and electrotonic inhibition and summation. Our data suggest that the characteristics of conduction and reflection in segmentally depressed atrial tissues are qualitatively similar to those in ventricular tissues. The presence of electrotonic inhibition in atrial may also help to explain the functionally inexcitable zone seen in the vortex of the leading circle model of atrial flutter.     

Modèle physico-chimique de cellule excitable

A simple physico-chemical model of the excitable cell is represented by a peculiar chemically active ion exchange medium. It reveals some properties of natural cells: chemical and electrical irritability, all or none effect, summation of stimuli, electrotonus, refractory periods, periodical oscillations, action potentials, conduction of impulse. This model is based upon oversimplified assumptions, and a further investigation into more details is required.     

Influence of dynamic gap junction resistance on impulse propagation in ventricular myocardium: a computer simulation study

The gap junction connecting cardiac myocytes is voltage and time dependent. This simulation study investigated the effects of dynamic gap junctions on both the shape and conduction velocity of a propagating action potential. The dynamic gap junction model is based on that described by Vogel and Weingart (J. Physiol. (Lond.). 1998, 510:177-189) for the voltage- and time-dependent conductance changes measured in cell pairs. The model assumes that the conductive gap junction channels have four conformational states. The gap junction model was used to couple 300 cells in a linear strand with membrane dynamics of the cells defined by the Luo-Rudy I model. The results show that, when the cells are tightly coupled (6700 channels), little change occurs in the gap junction resistance during propagation. Thus, for tight coupling, there are negligible differences in the waveshape and propagation velocity when comparing the dynamic and static gap junction representations. For poor coupling (85 channels), the gap junction resistance increases 33 MOmega during propagation. This transient change in resistance resulted in increased transjunctional conduction delays, changes in action potential upstroke, and block of conduction at a lower junction resting resistance relative to a static gap junction model. The results suggest that the dynamics of the gap junction enhance cellular decoupling as a possible protective mechanism of isolating injured cells from their neighbors.     

Propagation of action potentials between parallel chains of cardiac muscle cells in PSpice simulation

Propagation of action potentials between parallel chains of cardiac muscle cells was simulated using the PSpice program. Excitation was transmitted from cell to cell along a strand of three or four cells not connected by low-resistance tunnels (gap-junction connexons) in parallel with one or two similar strands. Thus, two models were used: a 2 x 3 model (two parallel chains of three cells each) and a 3 x 4 model (three parallel chains of four cells each). The entire surface membrane of each cell fired nearly simultaneously, and nearly all the propagation time was spent at the cell junctions, thus giving a staircase-shaped propagation profile. The junctional delay time between contiguous cells in a chain was about 0.2-0.5 ms. A significant negative cleft potential develops in the narrow junctional clefts, whose magnitude depends on several factors, including the radial cleft resistance (Rjc). The cleft potential (Vjc) depolarizes the postjunctional membrane to threshold by a patch-clamp action. Therefore, one mechanism for the transfer of excitation from one cell to the next is by the electric field (EF) that is generated in the junctional cleft when the prejunctional membrane fires. Propagation velocity increased with elevation of Rjc. With electrical stimulation of the first cell of the first strand (cell A1), propagation rapidly spread down that chain and then jumped to the second strand (B chain), followed by jumping to the third strand (C chain) when present. The rapidity by which the parallel chains became activated depended on the longitudinal resistance of the narrow extracellular cleft between the parallel strands (Rol2). The higher the Rol2 resistance, the faster the propagation (lower propagation time) over the cardiac muscle sheet (2-dimensional). The transverse resistance of the cleft had no effect. When the first cell of the second strand (cell B1) was stimulated, propagation spread down the B chain and jumped to the other two strands (A and C) nearly simultaneously. When cell C1 was stimulated, propagation traveled down the C chain and jumped to the B chain, followed by excitation of the A chain. Thus, there was transverse propagation of excitation as longitudinal propagation was occurring. Therefore, transmission of excitation by the EF mechanism can occur between myocardial cells lying closely parallel to one another without the requirement of a specialized junction.     

Conduction in bullfrog atrial strands: simulations of the role of disc and extracellular resistance.

A number of fundamental properties of intercellular conduction in simulated cylindrical strands of cardiac tissue are examined. The paper is based on recent biophysical information describing the transmembrane ionic currents in bullfrog atrial cells as well as anatomical data on the structures (gap junctions) responsible for the coupling between cells in that tissue. A mathematical model of the single bullfrog atrial cell based on suction microelectrode single-cell voltage clamp data is employed, as well as a modified version of the well-known model of Heppner and Plonsey, to characterized the resistive connections between adjacent cells in a cardiac strand. In addition, the simulated cellular strand is assumed to be encased in a cylindrical, resistive endothelial sheath, thus forming an idealized atrial trabeculum; the trabeculum is immersed in an extensive volume conductor. It is possible to simulate both uniform and discontinuous conduction in this atrial strand model by appropriately changing the resistance of the intercalated discs that occur at cell boundaries. The conduction velocity achieved in the normal or control case is within the range of conduction velocities that have been measured for bullfrog atrial trabeculae using optical methods. Extracellular resistance is shown to have a significant effect on both conduction velocity and the critical value of disc resistance at which discontinuous conduction first occurs. Since the atrial cell model employed in this study is based on experimental data and can accurately simulate the atrial action potential, the transmembrane ionic currents generated by the model are capable of providing detailed information concerning the mechanisms of intercellular current spread, particularly in the region of the intercalated disc.     

Gap Junction Channels and Cardiac Impulse Propagation

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.     

Unidirectional block between isolated rabbit ventricular cells coupled by a variable resistance.

We have used pairs of electrically coupled cardiac cells to investigate the dependence of successful conduction of an action potential on three components of the conduction process: (a) the amount of depolarization required to be produced in the nonstimulated cell (the "sink" for current flow) to initiate an action potential in the nonstimulated cell, (b) the intercellular resistance as the path for intercellular current flow, and (c) the ability of the stimulated cell to maintain a high membrane potential to serve as the "source" of current during the conduction process. We present data from eight pairs of simultaneously recorded rabbit ventricular cells, with the two cells of each pair physically separated from each other. We used an electronic circuit to pass currents into and out of each cell such that these currents produced the effects of any desired level of intercellular resistance. The cells of equal size (as assessed by their current threshold and their input resistance for small depolarizations) show bidirectional failure of conduction at very high values of intercellular resistance which then converts to successful bidirectional conduction at lower values of intercellular resistance. For cell pairs with asymmetrical cell sizes, there is a large range of values of intercellular resistance over which unidirectional block occurs with conduction successful from the larger cell to the smaller cell but with conduction block from the smaller cell to the larger cell. We then further show that one important component which limits the conduction process is the large early repolarization which occurs in the stimulated cell during the process of conduction, a process that we term "source loading."     

Modulation of presynaptic store calcium induces release of glutamate and postsynaptic firing

Action potential-independent transmitter release is random and produces small depolarizations in the postsynaptic neuron. This process is, therefore, not thought to play a significant role in impulse propagation across synapses. Here we show that calcium flux through presynaptic neuronal nicotinic receptors leads to mobilization of store calcium by calcium-induced calcium release. Recruitment of store calcium induces vesicular release of glutamate in a manner consistent with synchronization across multiple active zones in the CA3 region of the rat hippocampus. This modulation of action potential-independent release of glutamate is sufficient to drive the postsynaptic pyramidal cell above its firing threshold, thus providing a mechanism for impulse propagation.     

Further study of soma,dendrite, and axon excitation in single neurons

The present investigation continues a previous study in which the soma-dendrite system of sensory neurons was excited by stretch deformation of the peripheral dendrite portions. Recording was done with intracellular leads which were inserted into the cell soma while the neuron was activated orthodromically or antidromically. The analysis was also extended to axon conduction. Crayfish, Procambarus alleni (Faxon) and Orconectes virilis (Hagen), were used. 1. The size and time course of action potentials recorded from the soma-dendrite complex vary greatly with the level of the cell's membrane potential. The latter can be changed over a wide range by stretch deformation which sets up a "generator potential" in the distal portions of the dendrites. If a cell is at its resting unstretched equilibrium potential, antidromic stimulation through the axon causes an impulse which normally overshoots the resting potential and decays into an afternegativity of 15 to 20 msec. duration. The postspike negativity is not followed by an appreciable hyperpolarization (positive) phase. If the membrane potential is reduced to a new steady level a postspike positivity appears and increases linearly over a depolarization range of 12 to 20 mv. in various cells. At those levels the firing threshold of the cell for orthodromic discharges is generally reached. 2. The safety factor for conduction between axon and cell soma is reduced under three unrelated conditions, (a) During the recovery period (2 to 3 msec.) immediately following an impulse which has conducted fully over the cell soma, a second impulse may be delayed, may invade the soma partially, or may be blocked completely. (b) If progressive depolarization is produced by stretch, it leads to a reduction of impulse height and eventually to complete block of antidromic soma invasion, resembling cathodal block, (c) In some cells, when the normal membrane potential is within several millivolts of the relaxed resting state, an antidromic impulse may be blocked and may set up within the soma a local potential only. The local potential can sum with a second one or it may sum with potential changes set up in the dendrites, leading to complete invasion of the soma. Such antidromic invasion block can always be relieved by appropriate stretch which shifts the membrane potential out of the "blocking range" nearer to the soma firing level. During the afterpositivity of an impulse in a stretched cell the membrane potential may fall below or near the blocking range. During that period another impulse may be delayed or blocked. 3. Information regarding activity and conduction in dendrites has been obtained indirectly, mainly by analyzing the generator action under various conditions of stretch. The following conclusions have been reached: The large dendrite branches have similar properties to the cell body from which they arise and carry the same kind of impulses. In the finer distal filaments of even lightly depolarized dendrites, however, no axon type all-or-none conduction occurs since the generator potential persists to a varying degree during antidromic invasion of the cell. With the membrane potential at its resting level the dendrite terminals contribute to the prolonged impulse afternegativity of the soma. 4. Action potentials in impaled axons and in cell bodies have been compared. It is thought that normally the over-all duration of axon impulses is shorter. Local activity during reduction of the safety margin for conduction was studied. 5. An analysis was made of high frequency grouped discharges which occasionally arise in cells. They differ in many essential aspects from the regular discharges set up by the generator action. It is proposed that grouped discharges occur only when invasion of dendrites is not synchronous, due to a delay in excitation spread between soma and dendrites. Each impulse in a group is assumed to be caused by an impulse in at least one of the large dendrite branches. Depolarization of dendrites abolishes the grouped activity by facilitating invasion of the large dendrite branches.     

Fast optical monitoring of microscopic excitation patterns in cardiac muscle.

Many vital processes depend on the generation, changes, and conduction of cellular transmembrane potentials. Optical monitoring systems are well suited to detect such cellular electrical activities in networks of excitable cells and also tissues simultaneously at multiple sites. Here, an exceptionally fast array system (16 x 16 photodiodes, up to 4,000,000 samples per second, 12-bit resolution) for imaging voltage-sensitive dye fluorescence, permitted real time measurements of excitation patterns at a microscopic size scale (256 pixels within an area of 1.8-8 mm2), in rat cardiac muscle in vitro. Results emphasize a recent hypothesis for cardiac impulse conduction, based on cardiac structural complexities, that is contradictory to all continuous cable theory models.     

Changes in interspike interval during propagation: Quantitative description

The intervals between nerve impulses can change substantially during propagation because of conduction velocity aftereffects of previous impulse activity. Effects of such changes on interval histograms and on statistical parameters of spike trains were evaluated for Poisson spike trains and for trains generated by a clock with added random delays. The distribution of short intervals was significantly changed during propagation for these spike trains. Substantial changes in serial correlation coefficients were found in trains with certain initial interval distributions. The relevance of these effects to neural coding is discussed.     

A device for separated and reversible co‐culture of cardiomyocytes

A novel technique is introduced for patterning and controllably merging two cultures of adherent cells on a microelectrode array (MEA) by separation with a removable physical barrier. The device was first demonstrated by separating two cardiomyocyte populations, which upon merging synchronized electrical activity. Next, two applications of this co‐culture device are presented that demonstrate its flexibility as well as outline different metrics to analyze co‐cultures. In a differential assay, the device contained two distinct cell cultures of neonatal wild‐type and β‐adrenergic receptor (β‐AR) knockout cardiomyocytes and simultaneously exposed them with the β‐AR agonist isoproterenol. The beat rate and action potential amplitude from each cell type displayed different characteristic responses in both unmerged and merged states. This technique can be used to study the role of β‐receptor signaling and how the corresponding cellular response can be modulated by neighboring cells. In the second application, action potential propagation between modeled host and graft cell cultures was shown through the analysis of conduction velocity across the MEA. A co‐culture of murine cardiomyocytes (host) and murine skeletal myoblasts (graft) demonstrated functional integration at the boundary, as shown by the progression of synchronous electrical activity propagating from the host into the graft cell populations. However, conduction velocity significantly decreased as the depolarization waves reached the graft region due to a mismatch of inherent cell properties that influence conduction. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Simulated propagation of cardiac action potentials.

We have used numerical methods for solving cable equations, combined with previously published mathematical models for the membrane properties of ventricular and Purkinje cells, to simulate the propagation of cardiac action potentials along a unidimensional strand. Two types of inhomogeneities have been simulated and the results compared with experimentally observed disturbances in cardiac action potential propagation. Changes in the membrane model for regions of the strand were introduced to simulate regions of decreased excitability. Regional changes in the intercellular coupling were also studied. The results illustrate and help to explain the disturbances in propagation which have been reported to occur at regions of decreased excitability, regions with changing action potential duration, or regions with changing intercellular coupling. The propagational disturbances seen at all of these regions are discussed in terms of the changing electrical load imposed upon the propagating impulse.     

Induction of membrane excitability in Xenopus oocytes

Immature oocytes from the African toad Xenopus laevis are not known to be excitable cells, which means that they do not generate an action potential in response to small depolarizations. However, a regenerative response is produced if successive depolarizing currents of large magnitude are applied to the oocyte membrane. This response is characterized by the occurrence of a positive transmembrane potential that can last for several minutes. The opening of voltage-dependent channels, highly selective for sodium ions, underlies the depolarization thus obtained. These channels exhibit unconventional electrophysiological and pharmacological properties, which set them apart from other types of voltage-dependent sodium channels found in excitable tissues. The opening of the oocyte sodium channels is a complex process, which includes an induction phase. During this phase, the channels change from an electrical state of inexcitability into an excitable voltage-dependent state. The induction mechanism is modulated by the temperature of the bathing medium, by the activation of enzymes (namely a phospholipase C and a protein kinase C) and by the release of calcium ions from intracellular phosphatidyl-inositol trisphosphate stores. The results summarized in this review point out the possible role that these sodium channels may play in the physiology of the oocyte.     

Effect of conditioning polarization of soma of giant neurons of mollusks on the mechanism of action-potential generation

To ascertain the properties of an excitable membrane of the soma of giant neurons of mollusks, experiments were carried out to study the effect of conditioning shift of the membrane potential on the mechanism of action-potential generation. The effect of conditioning was assessed from changes in the action-potential curve and its first derivative, as well as from the curve of transmembrane currents under voltage clamp conditions. It was found that a change in membrane potential evokes at least two reactions which have opposite effects on the mechanism of generation of action potentials. These reactions evidently have different time characteristics. One of these does not differ notably from the reaction recorded for other excitable structures, and is manifested in the activation (with hyperpolarization) or inactivation (with depolarization) of the mechanism generating action potentials. The other reaction contributes either to an increase (with depolarization) or a decrease (with hyperpolarization) in the efficiency of this mechanism. Conditioning polarization also has a marked effect on the system responsible for repolarization of the membrane during generation of action potentials. This effect is manifested in a change in the reaction of this system to tetraethylammonium ions. The specific membrane systems sustaining excitability and reacting to changes in the strength of the membrane's electrical field were found to be very inert. After a shift in the potential to a given stable level a rearrangement, lasting sometimes tens of seconds, takes place in the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 91–99, January–February, 1970.