© 1996 American Heart Association, Inc.
Articles |
Spatial Changes in Transmembrane Potential During Extracellular Electrical Shocks in Cultured Monolayers of Neonatal Rat Ventricular Myocytes
the Department of Physiology, University of Bern (Switzerland).
Correspondence to Anne M. Gillis, MD, FRCPC, Division of Cardiology, Department of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1. E-mail agillis@cvr.ucalgary.ca.
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Abstract |
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This study investigated the role of different types of discontinuities in tissue architecture on the spatial distribution of the transmembrane potential. Specifically, we tested the occurrence of so-called "secondary sources," ie, localized hyperpolarizations and depolarizations during the application of extracellular electrical shocks (EESs). Changes in transmembrane potential relative to action potential amplitude (
Vm/APA) were measured in patterned cultures of neonatal rat myocytes, stained with voltage-sensitive dye (RH-237), by optical mapping (96-channel photodiode array, 6- to 30-µm resolution) during the application of EES (field strength, 8 to 22 V/cm; duration, 6 ms). Across narrow cell strands (width, 218±59 [mean±SD] µm), EES applied during the relative refractory period produced a linear and symmetrical profile of Vm/APA (-65±23% maximal hyperpolarization versus +64±15% maximal depolarization). In contrast, the profile of Vm/APA was asymmetrical when EESs were applied during the action potential plateau (-95±32% versus +37±14%). At high magnification, no secondary sources were observed at the borders between cells. In dense isotropic cell monolayers or in monolayers and strands showing intercellular clefts, secondary sources were frequently observed. Intercellular clefts of the size of one to several myocytes were sufficient to produce secondary sources of the same magnitude as those that elicited action potentials in dense cell strands. There was a close correlation between the location of secondary sources during EES and localized conduction slowing during propagation. Thus, densely packed cultured cell strands behave as an electrical continuum with no secondary sources occurring at cell borders. Small intercellular clefts can create secondary sources of sufficient magnitude to exert a stimulatory effect.
Key Words: optical mapping • electrical shock • cell culture • secondary source • defibrillation
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Introduction |
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Extracellular electrical shocks delivered across the heart are commonly used to terminate ventricular tachyarrhythmias. Although the effects of defibrillation shocks on ventricular activation and repolarization have been studied extensively in single cells, perfused hearts, and whole animal preparations, the mechanism by which an extracellular electrical pulse terminates the arrhythmia is not completely understood.1 2 3 4 5 6 7 8 9 10 11 12 It has been shown that EESs depolarize or hyperpolarize excitable portions of the myocardium.3 6 11 12 If a critical mass is excited, the newly created wave fronts of activation are likely to block the multiple wavelets of the fibrillating heart, thereby interrupting reentrant pathways.13 14 In addition, EES-induced prolongation of the action potential may lead to defibrillation by prolonging the refractory period.3 4 5 6 7 9 10
Assuming that cardiac cells are well coupled to form an electrically continuous structure, the effect of application of an extracellular electrical field can be predicted from linear cable theory.15 16 17 In a continuous cable, changes in Vm are confined to the regions closely adjacent to the stimulation electrodes, the so-called "primary current sources." Between these regions of transmembrane current flow, no changes in Vm occur. Although application of this theory is adequate for the determination of macroscopic passive electrical properties in geometrically well-defined tissue, such as cylindrical trabecula or arterially perfused papillary muscles,16 it does not suffice to explain the circumscribed hyperpolarizations and depolarizations observed during EES in whole hearts.3 6 12 Therefore, the equivalent electrical circuit models used to explain the effects of EES were modified by the introduction of resistive nonuniformities into the electrically continuous medium.18 19 20 21 Such resistive barriers force electrical current to redistribute between the extracellular and intracellular spaces before and beyond the obstacle. As a consequence, depolarizing and hyperpolarizing regions, so-called "secondary sources or sinks," develop at resistive barriers. In the simple case of a linear model, the secondary sources appear as sawtoothlike changes in Vm superimposed on the primary source voltage profile.18 19 20 21 In single isolated ventricular myocytes, hyperpolarizations and depolarizations are observed at opposite cell ends exposed to the poles of an electrical field.22 23 However, the role of cell boundaries as secondary sources in an electrically coupled cellular network has not been determined. Theoretically, the strength of such sources will be related to the density, distribution pattern, and electrical conductances of gap junctions.19 Alternatively, secondary sources of transmembrane current flow may arise at resistive boundaries formed by connective tissue sheets and the vascular tree that divide the cellular network into strands.24 25
Assessment of the effect of EES at a microscopic level involves the solution of two major technical problems. First, the classic measurement of electrical activity by direct recording of extracellular electrograms or transmembrane action potentials with electrodes was hampered by interference with the strong electrical fields producing electrical artifacts. This problem has been overcome by the introduction of optical techniques measuring the Vm change from the fluorescence of a voltage-sensitive dye.26 The second problem relates to the spatial resolution of Vm measurements in three-dimensional tissue. Although measurements from myocardial areas as small as 30 µm have recently been obtained in perfused rabbits hearts,12 such measurements will average fluorescent light emitted from a large number of cells located at the surface and in the depth of the ventricular wall.3 6 27
The purpose of the present study was to investigate the role of different types of microscopic resistive discontinuities within a cellular network in the production of localized hyperpolarizations and depolarizations (secondary sources). Accordingly, we assessed the distribution of changes in Vm in cultured cell monolayers using optical measurements of voltage-sensitive dye fluorescence and a high-resolution multisite optical mapping system.28 29 30 31 32 33 The cellular networks were cultured to produce resistive barriers similar to the structural discontinuities responsible for the generation of localized hyperpolarization and depolarization during application of defibrillatory shock in vivo. In this way, effects of simulated boundaries of whole-cell bundles could be separated from the effects of boundaries between individual cells.
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Cell Cultures
Cell cultures were prepared according to previously published procedures.34 In brief, hearts from 8 to 10 neonatal rats (Wistar, 1 to 2 days old) were excised, the ventricles were minced with scissors, and the resulting small tissue pieces were dissociated in Hanks' balanced salt solution (without Ca2+ and Mg2+, GIBCO) containing trypsin (0.1%, Boehringer) and pancreatin (60 µg/mL, Sigma). The dispersed cells were, after centrifugation, resuspended in medium M199 (GIBCO) with an ionic composition of (mmol/L) NaCl 137, KCl 5.4, CaCl2 1.3, MgSO4 0.8, NaHCO3 4.2, KH2PO4 0.5, and NaH2PO4 0.3. The medium was supplemented with penicillin (20 U/mL), streptomycin (20 µg/mL), vitamin B12 (2 µg/mL), and 10% neonatal calf serum. The cell suspension was preplated in order to reduce the fibroblast content, and the myocytes remaining in the suspension were seeded at a density of 1.9x103 cells/mm2 on conditioned coverslips (see below). The cultures were kept in an incubator at 35°C in a humidified atmosphere containing 1.2% CO2. Medium exchanges were performed on the first day after seeding and every other day thereafter with supplemented medium M199 containing a reduced concentration of serum (5%).
Preparation of Patterned Cell Cultures
Experiments were performed with either isotropic monolayer cultures or linear cell strands. Isotropic cell monolayers were obtained using previously described techniques.29 32 Glass coverslips (diameter, 22 mm; thickness, 0.14 mm; Haska) were coated with collagen type IV (human placenta, Sigma), which was dissolved in phosphate buffer at a concentration of 20 µg/mL. Two milliliters of this solution was applied to one side of the coverslip for 1 hour at room temperature. Linear cell strands were obtained using previously described photolithographic techniques.34 In short, coverslips (same brand as above) were coated with a photoresist (KTFR, Kodak), which prevented the adhesion of myocytes. Linear patterns inserting into large areas located either in the center or at the periphery of the coverslip were etched into the photoresist, and the resulting photoresist-free regions were coated with collagen (same solution as used for growing monolayers). After they were seeded, cells preferentially attached to the photoresist-free regions, thereby forming 2- to 3-mm-long and 30- to 300-µm-wide cell strands, which inserted into large monolayer cell areas. The large cell areas served to condition the medium and acted as pacemakers for the cells in the strands, thereby increasing their degree of phenotypic differentiation.34 Measurements were performed between day 3 and day 12 in culture. Coverslips were transferred into the experimental chamber, which was mounted on the microscope. Cells were superfused with a Tyrode's solution with a composition of (mmol/L) NaCl 150, KCl 5.0, CaCl2 1.2, MgCl2 1.0, NaH2PO4, HEPES 5.0, and glucose 5.0. The pH of the solution was 7.4, and the temperature was kept constant at 
34°C.
Multisite Optical Recordings of the Vm Change
Vm changes were measured from the change in fluorescence of the voltage-sensitive dye RH-237 (Molecular Probes). The dye was stored in a 2 mmol/L stock solution of dimethyl sulfoxide and diluted to yield a final dye concentration of 1.5 µmol/L in Tyrode's solution. Before the optical measurement, the cell cultures were superfused with the dye solution for 5 minutes. The setup used for multisite optical recording is illustrated in Fig 1A
and has been described elsewhere in detail.29 31 32 The recording system included an inverted microscope (Axiovert 35M, Zeiss) equipped for epifluorescence and a 100-W arc mercury lamp as a light source. After passage through a heat filter, the excitation light was sent through a band-pass excitation filter (Zeiss, 530 to 585 nm) and was reflected by a dichroic mirror (600 nm) to the objective and the preparation. The emitted fluorescence passed a low pass–emitting filter (>615 nm). Cells were exposed to excitation light for 80 to 200 ms. An electromechanical shutter and shutter driver (UniBlitz VS25 and UniBlitz T132, Vincent Associates) were used to control illumination.
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The fluorescence emitted by the preparation was measured using a 10x10 photodiode array (Centronic) located in the image plane of the microscope. Spatial resolutions obtained were 30 µm (x20 magnification), 15 µm (x40), 9.5 µm (x63), 7.0 µm (x86), or 6.0 µm (x100). The photocurrents from the 96 diodes were converted to voltages by custom-built current-to-voltage converters, and the signals were then fed to second-stage amplifiers with a sample-and-hold circuit for subtraction of background fluorescence. The signals from 96 channels were multiplexed into 12 channels using a custom-made multiplexer and then digitized using three analog-to-digital converter cards (four channels, 1-MHz digitization rate, 12-bit resolution, NB-A2000, National Instruments) installed in a Quadra 840av computer (Apple Computer). The sampling rate was 25 kHz for each of the 96 channels. High-frequency noise was eliminated by digital filtering using a gaussian low-pass filter with a frequency cutoff of 1.0 to 2 kHz.29 32
Experimental Procedure and Application of EESs
Before each recording, the cell and tissue morphology was observed under red illumination (low-pass filter, >630 nm), and the photodiodes were positioned over a selected area. Both phase-contrast and bright-field images of the cells (with an overlaid grid corresponding to the photodiode locations) were recorded by a video camera.
Electrical stimulation was performed via a bipolar electrode composed of a glass pipette (tip diameter, 50 to 80 µm) filled with Tyrode's solution and a silver wire coiled around the pipette tip. Cells were stimulated with rectangular pulses (duration, 1 ms; double threshold current) delivered from a digital impulse generator (Master-8, AMPI) at a basic cycle length of 700 ms. The impulse generator was also used to synchronize the shutter opening, the data acquisition, and the application of EESs. The stimulation electrode was positioned >1 mm from the recording site.
EESs were delivered using a custom-built device that produced truncated exponential pulses. Shocks were 6 ms in duration, and the tilt was 48%. The power source and the defibrillator output were isolated from ground. EESs were applied via two pairs of platinum plate electrodes positioned at opposite ends of the bath, as illustrated in Fig 1B
. The transverse and longitudinal electrodes were 2.1 cm and 1.6 cm apart, respectively. The dimensions of the electrodes were 1.2 cmx0.2 cm (transverse) and 1.5 cmx0.2 cm (longitudinal). The defibrillating device was triggered by the stimulus and could produce EESs at preselected times during the cardiac cycle. The shock strengths were chosen to yield an electric field strength of 6 to 55 V/cm. To minimize the effects of phototoxicity on the action potential characteristics, a maximum of three recordings were made at the same site.29 32 35 At the completion of 10 experiments, the extracellular field strength was measured as the potential difference between two extracellular microelectrodes, which were positioned 1.1 mm apart. The extracellular electrode pair was oriented so that the line between them was parallel to the axis of the electrical field. The field strength was calculated from the potential difference recorded in the middle of the shock waveform, ie, 3 ms after the leading edge of the waveform. The field strength was measured in the center of the bath, in the four corners of the bath, and at locations near the stimulation electrodes along the central transverse and longitudinal axes. The electrical field strengths measured over a range of voltages delivered between the transverse and longitudinal electrodes in the center of the bath are shown in Table 1. The relationship between voltage and the measured field strength was linear (r=.99, P<.001). The electrical field strengths measured at different regions in the bath during a shock with a peak voltage of 50 V are shown in Table 2. The field strength was homogeneous throughout the bath. The coefficient of variation was 22%, 12%, and 6% for shocks with peak voltages of 25, 50, and 150 V, respectively.
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Data Analysis
The optical recordings (200 ms) were corrected for changes in fluorescence intensity caused by photobleaching of the voltage-sensitive dye. For that purpose, a linear fit to baseline fluorescence, obtained before and after the signal, was subtracted from the original recording. Examples of action potentials recorded during the application of EESs are shown in Fig 2. These were recorded in a cell strand at x40 magnification (15-µm interdiode distance). The APA was taken as the difference in fluorescence intensity measured before the onset of the action potential and immediately after the action potential reached the plateau. The change in fluorescence induced by the EES, Vm, was determined as the difference between light intensities measured before and 3 ms after the onset of the EES. Most of the capacitive charge flow is terminated by this time. Selecting a later time during the 6-ms pulse did not change the results qualitatively. Shock-induced changes in membrane potential, Vm, were expressed as a change in fluorescence intensity relative to APA, Vm/APA. In such a way, the spatial variability in fluorescence intensity due to inhomogeneous dye staining and nonuniformity of illumination was eliminated. The local activation times were determined at 50% of the APA using linear interpolation between the nearest sampling points.30 31 Activation maps and isopotential maps illustrating the EES-induced change in Vm/APA were constructed using linear interpolation between the diodes.
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Distribution of Cx43
In two experiments, the distribution of gap junctions was assessed by immunohistochemistry after the completion of the electrophysiological recordings. The cultures were fixed at room temperature in a PBS solution containing 4% paraformaldehyde and were then, after washing, immersed for 30 minutes in PBS containing 0.15% Triton X-100 (Sigma) and 3% goat serum (Pierce). Thereafter, the preparations were incubated overnight at 4°C with antibodies against Cx43 at a concentration of 2 µg/mL in PBS, as previously described36 (antibodies were courtesy of Dr D. Gros, Marseilles, France). Subsequently, the preparations were washed in PBS and incubated with the secondary antibody (Cy3-conjugated goat anti-rabbit IgG, Jackson ImmunoResearch Laboratories) for 150 minutes at room temperature. After a final wash in PBS, the preparations were mounted in PBS and observed on the same microscope used for the electrophysiological recordings. In order to assess the pattern of Cx43 distribution in the areas previously characterized electrophysiologically, the culture areas concerned were exactly aligned under bright-field illumination, and pictures were taken in the epifluorescence mode. To evaluate the effects of the fixation on the microarchitecture of the preparations, phase-contrast images were obtained from identical areas before and after fixation. Comparison of the cell borders and of the locations of the nuclei demonstrated that the fixation procedure caused no significant distortions in the architecture of the preparations.
Cell-Permeability Studies
High-intensity electrical field stimulation may induce abnormalities of cell function by electroporation of cell membranes.37 38 39 Accordingly, the effects of shocks on electroporation were evaluated from the uptake of the fluorescent dye Lucifer yellow.40 41 Lucifer yellow (potassium salt, Sigma) was dissolved in HEPES buffer and added to the bath at a final concentration of 2 mg/mL. For these experiments, a monolayer growth pattern that consisted of a rectangular area of cell adherence measuring 15.2 mmx3.2 mm was produced. No visible discontinuities were observed on examination of the phase-contrast images of these cell preparations. In control measurements, Lucifer yellow was added to the bath for 1.5 minutes, and then the cells were washed with superfusate and immediately observed under fluorescence microscopy (excitation filter, 450 nm; emission filter, 510 nm) with the x40 objective. The dye was added to the bath again, and a shock (field strength, 8 V/cm) was delivered in the transverse plane. The residual dye was removed by superfusion, and fluorescence microscopy was repeated. Approximately 10 minutes later, this sequence was again repeated for a second shock (field strength, 22 V/cm [n=3] or 43 V/cm [n=1]). Images of the fluorescent fields were recorded at the cell borders immediately adjacent to the anode and cathode and in the center of the bath along the mid transverse plane.
Statistical Analysis
Data were expressed as mean±SD. Differences were compared using the two-tailed paired or unpaired t test where appropriate. They were considered statistically significant at P<.05.
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Effects of Defined Tissue Boundaries on the Spatial Distribution of
Vm During EESIn order to mimic defined resistive boundaries, cells were grown in strands 150 to 300 µm in width. Strands of this width preferentially show a parallel alignment of the cells close to the boundary and a more random orientation in the middle of the strand (Fig 3A
and Reference 33). In a 300-µm strand, the width of the diode array at x20 magnification (30-µm interdiode distance) matched the strand width, as shown by superimposition of the diode array with the phase-contrast picture in Fig 3A. The effects of an EES (field strength, 8 V/cm; duration, 6 ms) delivered 10 ms after a stimulus are shown in Fig 3B through 3D: Fig 3B depicts the isopotential map of the relative change in Vm, Vm/APA, 3 ms after the onset of an EES. Individual recordings of the changes in Vm along a transverse axis of the strand are shown in Fig 3C. The profile of Vm/APA along a transverse axis (corresponding to the row of diodes from 1 to 10) is illustrated in Fig 3D. The EES induced a hyperpolarization of Vm in cells facing the anode and a depolarization in the cells facing the cathode. The shape of Vm corresponded to the truncated exponential field pulse in depolarized areas (modified by the initial capacitive charge flow11 ) and exhibited a slow decreasing phase in the hyperpolarized zone. As indicated in Fig 3B, the potential distribution was mostly uniform with isopotential lines running perpendicular to the electrical field. Small localized deviations from the parallel alignment of the isopotentials were observed only occasionally. In this cell preparation, when the EES was applied during the plateau phase of the action potential, the magnitude of maximal hyperpolarization (-140% of APA) measured at the border of the strand facing the anode was 4.7 times the magnitude of the maximal depolarization (+30% of APA) measured at the border of the strand facing the cathode. The transition from hyperpolarization to depolarization was gradual and resulted in a linear potential profile (Fig 3D).
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To assess whether the asymmetry of the Vm/APA profile depended on the phase of the cardiac cycle, EESs were also applied late during repolarization. In the cell preparation depicted in Fig 4A through 4C, an EES was applied 80 ms after the stimulus. Panels D through F present the effects of an early shock (10 ms after the stimulus) applied in the same culture at identical diode locations. In accordance with the cell preparation shown in Fig 3, the isopotential lines showed an almost parallel alignment perpendicular to the electrical field (Fig 4A and 4D), with minor local deviations. Also, the voltage profiles parallel to the electrical field were linear (Fig 4C and 4F). In the case of EES application during late repolarization, the voltage profile was symmetrical (Fig 4C), and the shape of both EES-induced hyperpolarizations and depolarizations corresponded to the truncated exponential. Such a symmetrical and linear voltage profile is in accordance with linear cable theory. Whereas the drop of membrane potential is exponential if an extracellular field is applied at a resistive boundary of a cablelike structure, which has a width at least three times longer than the length constant 
,15 16 the membrane potential change approaches linearity at a width of <. In our case, the width (218±59 µm) was 0.6.42 The linear profiles are also typical for the simulated secondary sources, where the distance between the resistive discontinuities is much less than .18 19 20 21 Similar to the cell preparation shown in Fig 3, application of the EES during the plateau phase of the action potential produced an asymmetrical profile of Vm/APA, with a large hyperpolarized area and a smaller depolarized area (Fig 4D). EES applied during the relative refractory period or in diastole initiated excitatory responses. The time of activation of the whole-cell strand was shorter than the duration of the EES. This and the analysis of the action potential upstrokes indicated that the cells being hyperpolarized were excited by electrotonic interaction from the cathodal make response (before being excited by their own anodal break).
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In a total of 16 cell preparations, application of EES (electrical field strength, 8 V/cm) during the plateau phase of the action potential produced consistent asymmetry of the voltage profiles with a hyperpolarization (Vm/APA) of -95±32% at the strand border facing the anode and a depolarization of +37±14% at the strand border facing the cathode. Application of EESs in 16 cell preparations during the repolarizing phase of the action potential produced a symmetrical voltage change with a hyperpolarization of -65±23% at the strand border facing the anode and a depolarization of +64±15% at the strand border facing the cathode. Taking an average APA of 100 mV,28 34 the means corresponded to -65 and +64 mV, respectively. The total change in Vm/APA from one strand border to the other was independent of the time of application of the EES (132% during the plateau phase, 129% during repolarization). In a single-cell preparation, an EES of 11 V/cm was applied across a strand of only 60 µm in width during the plateau phase. This produced a maximal hyperpolarization of -27% and a maximal depolarization of 9%, thus confirming the theoretically predicted dependence of the source magnitude on the strand width, ie, the distance between the resistive boundaries.
Effects of Cell Borders on the Spatial Distribution of Vm During EES
The role of the borders of individual cells within the cellular network for the spatial distribution of Vm was evaluated in 12 cell preparations. The change in Vm during EES within individual cells and across cell borders was studied at high resolution (x86 and x100 magnification) in narrow myocyte strands (30 to 60 µm in width). As expected, shocks induced hyperpolarization of Vm in cells close to the anode and depolarization in cells close to the cathode. Fig 5 illustrates the relation between Vm/APA and the borders of individual cells (located near the cathode) during application of an EES along the main axis of the cell strand. The grid of measuring diodes is superimposed on the phase-contrast image in Fig 5A, and a map of the individual signals is displayed in Fig 5B. Fig 5C illustrates selected action potentials measured in areas confined to the cytoplasm or overlapping with cell borders. At all measuring sites, the membranes were depolarized during the EES. There was no abrupt transition from hyperpolarization to depolarization, and no major gradients in Vm/APA were detectable between measuring sites localized in different cells. A second cell preparation in which the EES produced hyperpolarization is shown in Fig 6. Similar to Fig 5, the gradients in Vm/APA between diode pairs did not depend on whether diode pairs were located within the cytoplasm or across cell borders. Also, a change in polarity was never observed within the same cell or across cell borders.
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Gradients in Vm/APA between pairs of measuring sites that were either located within the same cell or separated by a cell border were quantitatively analyzed in a total of 12 cell preparations. In all these experiments, the cells were densely packed within cardiac strands, and no intercellular clefts separating individual cells were apparent on the microscopic images. Only those diode pairs that showed no overlap with cell borders were selected. The average gradient in Vm/APA along the electrical field between two diodes within the same cell was 6±5% (n=25) and between diodes separated by a cell border was 5±5% (n=25). This confirmed the qualitative observations in Figs 5 and 6 showing that the cell borders in densely packed cultured strands did not introduce secondary sources.
Effect of EES in Cultured Cell Monolayers
Although the spatial distribution of Vm/APA induced by EES was consistent with continuous cable properties in narrow strands of cultured myocytes with dense cell growth, this was often not the case in large cell monolayers or in long strands if shocks were delivered in parallel to the long axis. In the region that was at least two to three space constants from the anode and cathode (ie, the "midinterpolar region"43 ), three patterns of Vm/APA distribution during EES were observed (field strengths, 8 to 22 V/cm): (1) Areas showing either hyperpolarization or depolarization throughout the mapped region (150 µmx150 µm or 300 µmx300 µm) were observed in 12 of 28 measurements. (2) Areas showing an abrupt transition from hyperpolarization to depolarization were observed in 10 of 28 measurements. (3) Areas of minimal or no change in Vm during EES were found in 6 of 28 measurements. Cellular growth was dense, and no separation of cell borders (intercellular clefts) was detectable in these cases.
Fig 7 illustrates an example of Vm recorded over an area of 150 µmx150 µm in a cell monolayer near the center of the perfusion bath, ie, remote from the location of the primary sources. No gross structural discontinuities were evident on examination of the phase-contrast image of the cells (Fig 7A). Stimulation of the monolayer from a location 0.4 mm at the right of the mapping area produced propagation from right to left, as depicted on the isochronal map in Fig 7B. On this map, four areas of localized slowing of conduction were present, the total conduction time through the monolayer was 0.7 ms. The change in Vm/APA during an EES, with the electrical field being directed vertically, is illustrated in the color map of Fig 7C. Although the cell layer was dense, there were three circumscribed zones of hyperpolarization as great as -60% within the depolarized zone in the upper part of the isopotential map. Superimposition of the isochronal map with the isopotential map demonstrates that the three focal transitions from hyperpolarization to depolarization coincided with sites of localized conduction slowing. In addition to the three focal secondary sources, there was a gradual transition in Vm/APA from -160% to +80%, or of 200 mV within a distance of 150 µm. This more gradual change was likely to correspond to the transition from hyperpolarization to depolarization occurring in between two secondary sources in the midinterpolar region (corresponding to the zone in the middle of the cell strand in Fig 4C and 4F).19
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Fig 8 shows a cell preparation in which circumscribed secondary sources were absent from the region with an area of 150 µmx150 µm mapped by the diode array. The phase-contrast image of the culture, illustrating the dense cellular arrangement, is presented in Fig 8A. The isochronal map of conduction was relatively homogeneous (Fig 8B), and the total conduction time through this area was 0.5 ms. The maximum hyperpolarization of -40% induced by the EES was observed at the left border. It declined to -10% at the left border. The changes in Vm of selected action potentials are shown in Fig 8D and demonstrate the relatively homogeneous decrease in hyperpolarization throughout the mapped area. This continuous decrease of hyperpolarization indicated that the mapped region was located on the cathodal side of a resistive discontinuity, ie, a secondary source.
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The superimposition of areas showing slow conduction with circumscribed secondary sources, as illustrated in Fig 7, suggests localized discontinuities in resistance as the underlying mechanism. Such discontinuities have been shown to be related either to the presence of intercellular clefts or individual myocytes partially lacking gap junctions.32 A comparison of the location of small intercellular clefts and Cx43 distribution with isochronal and isopotential maps from an area within a cell strand of 180 µm in width is shown in Fig 9. In contrast to the strands shown in Figs 3 through 6, this strand exhibited a circumscribed area with reduced cell density and intercellular clefts. The phase-contrast picture of the fixed preparation (Fig 9A) revealed the presence of several intercellular clefts forming a U-shaped obstacle across the strand. As shown in Fig 9B, this was accompanied by a lack of Cx43 fluorescence at the sites of the clefts. Whereas the intercellular clefts in the lower half were relatively large, the clefts in the upper half had the size of approximately one myocyte. The resulting resistive discontinuities produced a corresponding "U-shaped" zone of conduction slowing during propagation from left to right, as depicted in the isochronal map in Fig 9C. In accordance with Fig 7, the localized conduction slowing superimposed with an abrupt transition from hyperpolarization to depolarization during the application of an EES 20 ms after the stimulated action potential (Fig 9D). The steepest gradient of Vm in the upper half of the mapped area was 120% of APA per 30 µm. Assuming a value of 100 mV for APA, this corresponds to a voltage gradient of 40 V/cm, which is much larger than electrical field strength (8 V/cm). This gradient is due to the resistive obstacle at that site and is equivalent to the abrupt voltage change across a resistive discontinuity in the simulated "sawtooth" pattern.18 19 20 21 Thus, relatively small-sized resistive obstacles may induce significant stepwise transitions from hyperpolarization to depolarization in Vm. This was confirmed in another cell preparation (not shown) in which an intercellular cleft of the size of an average myocyte (60 µmx15 µm) was present in an isotropic monolayer culture in the midinterpolar region, ie, at a distance >0.5 cm from the shock delivering electrodes. An EES of 8 V/cm applied 16 ms after the stimulus produced a maximal depolarization of 20% at the cleft border facing the anode and a maximal hyperpolarization of -50% at the cleft border facing the cathode.
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Effects of Shocks on Cell Permeability
It has been shown that extensive electrical fields can induce electroporation of cell membranes.37 38 39 41 If such damage correlates with the amount of hyperpolarization and depolarization, this effect should occur close to the shock electrodes (primary source) and, depending on source strength, also at secondary sources. In order to assess a potential contribution of electroporation to the changes in Vm induced by EES, we tested the cellular uptake of the fluorescent dye Lucifer yellow as a function of the applied field strength and the location within the electrical field in four monolayer preparations. During the control measurements of fluorescence and after shocks with a field strength of 8 V/cm, intracellular accumulation of Lucifer yellow was not observed. As illustrated in the bottom panels of Fig 10, accumulation of the dye occurred in the areas close to the anode and cathode after shocks with a field strength 
22 V/cm. In all four cell preparations, including one experiment when shocks with a field strength of 8 and 43 V/cm were applied, electroporation and subsequent intracellular accumulation of Lucifer yellow were absent in the center of the cultures, ie, in the midinterpolar region.
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Mechanisms of Defibrillation
Although implantable cardioverter defibrillators are commonly used in the management of survivors of sudden cardiac death, the mechanisms of defibrillation are not completely understood and may be multifactorial. A number of mechanisms have been proposed: (1) Defibrillation shocks may simultaneously excite a critical mass of myocardium, thereby blocking propagation of waves or wavelets, and abolish the arrhythmia.1 2 13 14 (2) More recent studies have suggested that defibrillation shocks produce prolongation of the action potential and ventricular refractoriness.3 4 5 6 7 8 9 10 11 12 Prolongation of refractoriness would then prevent new impulse propagation and resumption of fibrillation after the original fibrillating wave fronts have been extinguished. (3) It has also been suggested that shocks evoke a simultaneous excitation followed by synchronous repolarization. The consequent absence of spatial gradients in repolarization would make the tissue resistant to reinitiation of reentry by a subsequent stimulus. The latter hypothesis assumes that local areas of hyperpolarization and depolarization occur within cells or unit bundles and initiate a new action potential.3 6 Whatever the mechanism of defibrillation, the EESs must produce changes in the Vm beyond the tissue immediately adjacent to the shock electrodes (primary sources). It has been suggested that secondary sources due to the presence of resistive discontinuities may play a crucial role.18 19 Therefore, it is important to assess the relationship between the myocardial microscopic structures and the sites where the relevant changes in membrane potential occur.
Cardiac Cell Cultures as a Model for Studying Defibrillation
Previously, neonatal rat heart cells grown in cell cultures have been used to study impulse conduction at a microscopic level.28 29 30 31 32 33 44 Such cultures manifest some electrophysiological characteristics similar to intact cardiac tissue.28 34 42 45 In narrow strands and in anisotropically grown cultures, the degree of connectivity (number of cells connected to an individual cell), the functional anisotropy ratio (ratio of longitudinal-to-transverse conduction velocity), and the average longitudinal-to-transverse cell diameter ratio are very similar to those found in adult canine ventricle.32 However, neonatal cell cultures also have some distinct morphological features. Gap junctions are regularly distributed within the cell perimeter in neonatal hearts, whereas they are more concentrated at cell ends in adult myocardial tissue.32 46 47 In this respect, neonatal rat heart tissue more closely resembles remodeled adult tissue found in the border zone of chronic myocardial infarction.48 49 50
An advantage of the cell cultures is that the effects of cell borders on the formation of secondary sources during EES can be separated from the effects of larger resistive discontinuities. In vivo, resistive discontinuities likely include structures such as the microvasculature and connective tissue, which separate fiber bundles.24 25 Such barriers also have been reported to increase with age and in cardiac hypertrophy.24 51 Therefore, designing cultures with and without such discontinuities may contribute to our understanding of the mechanism of defibrillation. Moreover, the measurements in two-dimensional cultured strands and monolayers avoid the limitations to action potential measurements on the surface of intact hearts, which are due to interference with fluorescence changes from deeper intramural layers.3 6 12 However, two properties of monolayer cultures may limit the extrapolation of the present results to intact adult tissue: (1) The large bulk solution above the cell monolayers represents a medium of very high conductance, whereas the extracellular space in intact myocardium is restricted. Macroscopic cable analysis has shown that the extracellular space resistance in the longitudinal direction to the fiber axis is approximately equal in magnitude to the resistance of the intracellular compartment.16 Importantly, the anisotropy of the extracellular space may affect the change in membrane potential in a complex way, because the ratio of extracellular-to-intracellular resistance is different in the longitudinal compared with the transverse direction.27 52 53 This direction-dependent change in extracellular-to-intracellular resistance ratio induces field sources close to extracellular electrodes during point application of electrical fields.27 53 Therefore, depending on the type of stimulation, the role of the extracellular resistance must be considered in addition to the resistive discontinuities investigated in the present study. (2) The role of resistive discontinuities and cell-to-cell coupling in the generation of secondary sources in two-dimensional cultures may differ in three-dimensional tissue in vivo (see below).
Effects of EES in Cardiac Strands
In narrow cell strands, EES produced large hyperpolarizations and depolarizations at the strand boundaries. The Vm profiles in the axis of the electrical field closely resembled the profiles predicted from continuous cable theory. The profile linearity is expected because the width of the strand was <1.21 54 The magnitude of Vm was sufficient to initiate a new action potential when the shock was applied during the relative refractory period. The changes in Vm are consistent with the results obtained in single isolated myocytes, and the results support the concept that a densely packed cardiac strand functions as an electrically continuous medium. When single cells with a length of 130 µm were placed in an electrical field of 20 to 40 V/cm, the maximal Vm observed at opposite cell poles ranged from ±200 to ±400 mV.22 23 This corresponds to the ±80 mV changes in Vm observed at a field strength of 8 V/cm used in our experiments. In the present experiments, the average distance between the strand borders was 218 µm (on average, 15 cells in width). This distance was similar to the distance between resistive discontinuities created by connective tissue sheets in vivo.24 25
EES applied during the early plateau induced an asymmetrical voltage change with a marked hyperpolarization and a small depolarization. Other investigators have observed this phenomenon and have discussed the activation of ionic repolarizing currents as the possible underlying mechanism.11 12 The asymmetrical voltage profile is voltage dependent, since it was not observed when EES was applied in the repolarization phase. In our experiments, the maximal hyperpolarization observed during EES could exceed the resting membrane potential to levels negative to the assumed K+ equilibrium potential (eg, action potential 1 in Figs 3C and 4E). This suggests that activation of a repolarizing K+ current is not the only underlying mechanism contributing to the asymmetry of the voltage profile. The total spatial gradient in Vm was independent of the time during the cardiac cycle when the EES was applied. In Fig 4, the total voltage change was 190 to 200 mV across a strand 250 µm in width. This corresponds to a gradient of 7.6 to 8 V/cm, which is very close to the field strength applied during the EES (8 V/cm), indicating that the extracellular field gradient was the main determinant of the maximal changes in Vm between two resistive barriers.22
Effects of Cell Borders and Microscopic Resistive Barriers
The hypothesis that individual cell borders may create secondary sources responsible for defibrillation has been discussed in several experimental and theoretical studies.3 6 12 18 19 20 21 22 Optical mapping in two-dimensional cell cultures allowed us to directly assess this issue. The present experiments show that no major gradients in Vm occur across cell borders in dense cell strands during the application of EES. The absence of such gradients and the homogeneity of the potential distribution observed during EES shown in Figs 3 and 4 most likely involve the process of "lateral averaging," which has been proposed for microscopic propagation.28 Local currents flowing between cells that are perpendicular to the direction of propagation partially cancel the effects of localized resistive discontinuities that are presented by the individual gap junctions. Whether these findings can be extrapolated to adult heart tissue remains unknown. Since the overall anisotropic conduction velocity ratio and the degree of connectivity are very similar (see above) to those in adult canine ventricle, a similar behavior would be predicted in adult tissue. However, normal adult tissue has a different distribution of gap junctions around the cell perimeter, with a pronounced concentration at the cell poles.46 This difference is particularly expressed in tissue with a high anisotropy ratio, such as the crista terminalis.55 Whether secondary sources produced by such resistive discontinuities can exert an excitatory effect remains to be shown. Moreover, such gradients are likely to be diminished in the case of current flow in three-dimensional structures. In canine ventricular myocardium, the number of myocytes connected on average to an individual myocyte increases from 5.5 in two-dimensional slices to 11.3 in three-dimensional tissue.32 55 This increase in connectivity will increase the degree of electrotonic interaction and average the effect of the discontinuities imposed by the individual gap junctions in a way similar to that proposed for the mechanism of microscopic propagation.28
As has been shown in computer simulations,21 electrical cell-to-cell uncoupling will increase the magnitude of secondary sources at cell borders. Secondary sources at cell borders will be strongest in the extreme case of total uncoupling, which is equivalent to a single isolated myocyte being placed into an extracellular electrical field.22 23 In some pathophysiological settings, such as acute regional ischemia56 and chronic infarction,57 cell-to-cell uncoupling occurs in circumscribed regions. In the latter situation, surviving relatively well-coupled and rapidly conducting strands are embedded within scar tissue. Such areas are expected to be more strongly affected by an EES than is normal myocardium.
We have previously shown that the expression of gap junctions in myocytes grown in dense anisotropic monolayers is inhomogeneous at certain locations.32 At least one myocyte showing a decreased density of gap junctions at cell borders and leading to localized slowing of conduction was found in 20% of microscopic observation fields (x40 magnification).32 A similar heterogeneity of gap junction expression is likely to underlie the presence of secondary sources of hyperpolarizations and depolarizations (Fig 7) in the midinterpolar region of dense monolayers. The close relationship between the conduction pattern and the isopotential pattern at a microscopic level was a consistent finding in our experiments. Theoretically, the explanation for this correlation is straightforward, because Vm during both EES and impulse propagation depends on the same resistive network. This indicates that the location of secondary sources may be predicted from isochronal maps, which are technically easier to obtain than isopotential maps.
The rare observation of no change in Vm in such macroscopically dense networks is explained by the fact that resistive discontinuities must be separated by more than four to six space constants or 700 to 1400 µm in order to fully lose electrotonic influence on neighboring tissue. The size of the resistive barriers observed in our experiments that led to transition from hyperpolarization to depolarization ranged in size from one to several cells. However, the resistive obstacles were not complete, since impulse propagation was slowed but not fully blocked. The resulting hyperpolarizations and depolarizations were similar in magnitude to the changes that exerted an excitatory effect in the cell strands shown in Fig 4. Although a systematic analysis relating obstacle size to the magnitude of secondary sources was not carried out in this series of experiments, these results indicate that resistive obstacles present in normal myocardium, including connective tissue sheets that separate transmural layers, may represent sites from which excitation may emerge during EES.
EES and Electroporation
Electroporation has been described as a method to introduce large molecules into cells.39 41 However, it may also be considered as a state of cell damage, because large transmembrane conductances that are not present in the physiological state are created. Therefore, for the application of EES, it is important to know the range of field strength that produces a defibrillatory effect without causing cell damage. In our experiments, we tested the uptake of the fluorescent dye Lucifer yellow, which is a molecule with an Mr of 457. Lucifer yellow is impermeable to normal cell membranes but diffuses across gap junctions and has therefore been used for the assessment of cell-to-cell coupling.40 In our experiments, uptake of Lucifer yellow induced by EES was confined to the regions close to the anode and close to the cathode. This finding indicates that (1) membrane damage with electroporation is related to changes in Vm and (2) both large hyperpolarizations and large depolarizations present at the anode and cathode (primary sources) produce electroporation. The other potential hypothesis, ie, that the large field gradient outside the cell membrane per se might cause a hyperpermeable state, cannot explain the results, because the field strength was relatively uniform inside the bath (Table 1). The field strength at which uptake occurred (22 V/cm) is in accordance with the aftereffects, such as transient delayed afterdepolarizations, observed in guinea pig papillary muscle.38
Summary
In summary, optical mapping of Vm in cultured neonatal rat myocytes during EES has shown the following: (1) In narrow and dense cardiac strands (218 µm average width), transverse application of EES during the relative refractory period produced hyperpolarizations and depolarizations, which are consistent with the behavior of a well-coupled cellular network and can be simulated by linear cable theory. The magnitude of the Vm changes was sufficient to elicit action potentials. EES applied during the absolute refractory period produced larger hyperpolarizations than depolarizations. (2) In dense cell strands, no secondary sources were induced by borders between individual cells. (3) In monolayer cultures, areas showing hyperpolarization and depolarization remote from the shock electrodes alternated with areas where no changes in membrane potential occurred. Areas of abrupt transition from hyperpolarization to depolarization colocalized with areas of slow conduction. Resistive discontinuities of the size of an average myocyte in cell strands and monolayers produced secondary sources of significant strength to initiate action potentials. (4) EES of 22 V/cm induced electroporation of cell membranes. In confluent monolayers, this membrane damage was confined to the tissue adjacent to both the anode and cathode, ie, to the sites of the primary sources.
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Acknowledgments |
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This study was supported by the Swiss National Science Foundation, the Swiss Heart Foundation, and the Medical Research Council of Canada (PG 11188). Dr Gillis is a Scholar of the Alberta Heritage Foundation for Medical Research and was a recipient of a Visiting Scientist Award from the Heart and Stroke Foundation of Canada during the tenure of this work. We would like to express our gratitude to Regula Fluckiger and Lilly Lehmann for their help with the laboratory work and to Denis De Limoges for the construction of the defibrillating device.
Received April 3, 1996; accepted July 11, 1996.
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N. Chattipakorn, B. H. KenKnight, J. M. Rogers, R. G. Walker, G. P. Walcott, D. L. Rollins, W. M. Smith, and R. E. Ideker Locally Propagated Activation Immediately After Internal Defibrillation Circulation, April 14, 1998; 97(14): 1401 - 1410. [Abstract] [Full Text] [PDF]
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V. G. Fast, S. Rohr, A. M. Gillis, and A. G. Kleber Activation of Cardiac Tissue by Extracellular Electrical Shocks : Formation of `Secondary Sources' at Intercellular Clefts in Monolayers of Cultured Myocytes Circ. Res., February 23, 1998; 82(3): 375 - 385. [Abstract] [Full Text] [PDF]
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Y. Cheng, K. A. Mowrey, V. Nikolski, P. J. Tchou, and I. R. Efimov Mechanisms of shock-induced arrhythmogenesis during acute global ischemia Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2141 - H2151. [Abstract] [Full Text] [PDF]
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V. G. Fast and E. R. Cheek Optical Mapping of Arrhythmias Induced by Strong Electrical Shocks in Myocyte Cultures Circ. Res., April 5, 2002; 90(6): 664 - 670. [Abstract] [Full Text] [PDF]
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