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Anisotropic Conduction in Monolayers ofNeonatal Rat Heart Cells Cultured

on Collagen SubstrateVladimir G. Fast, Andre G. Kleber

Abstract Anisotropic impulse conduction was studied inneonatal rat heart cell monolayers produced by culturing cellson a growth-directing substrate of collagen. Monolayers con-sisting of parallel-oriented cells without visible intercellularclefts were selected for experiments; cell lengths and widthswere 65.8±+12.5 and 12.2+3.2 gm (n=49), respectively. Actionpotential upstrokes were measured by using 12 photodiodesselected within a lOx10 diode array and a voltage-sensitivedye (RH-237). The size of the area sensed by a single diodewas 14x 14 gim. High-density multiple recordings (resolution,up to 15 gim) demonstrated the variability of local activationdelays and of the maximal rate of rise of the action potentialupstroke (Vm.), which are presumably related to the micro-scopic cellular architecture. Mean macroscopic conduction

A nisotropy of cardiac muscle plays an importantrole in the propagation of excitation waves andin the initiation and maintenance of cardiac

arrhythmias. Sano et all presented the first evidencethat tissue anisotropy caused directional differences inimpulse spread with fast velocity in a direction parallelto fiber orientation and slow velocity in a directionperpendicular to the fiber axis. Later, Clerc2 demon-strated that intracellular resistance in ventricular trabe-cula was 10 times larger in a direction transverse to fiberorientation than in a longitudinal direction. In accor-dance with continuous cable theory, conduction velocitywas three times larger in the longitudinal than in thetransverse direction in the same experiments. Morerecently Spach et a13 presented results on anisotropicpropagation that could not be explained by continuouscable theory. They found that the maximal rate of riseof the action potential upstroke (Vm:) was larger in thetransverse than in the longitudinal direction. Moreover,premature impulses propagating in the longitudinaldirection were blocked, while they continued to propa-gate in the transverse direction. Both the higher trans-verse Vma and the dependence of block on fiber orien-tation were interpreted as reflecting a higher safetyfactor for transverse than for longitudinal propagation.The subject of directional differences in Vm,, and theoccurrence of unidirectional conduction block was ad-dressed in a large number of studies.4-12

Received March 30, 1994; accepted June 10, 1994.From the Department of Physiology, University of Berne

(Switzerland).Correspondence to V.G. Fast, PhD, Department of Physiology,

University of Berne, Buihlplatz 5, CH-3012, Berne, Switzerland.©3 1994 American Heart Association, Inc.

velocities measured over distances of 135 gm were 34.6+4.5and 19.0+4.3 cm/s (mean+SD, n=13, P<.0001) in longitudi-nal and transverse directions, respectively. The anisotropicvelocity ratio was 1.89+0.38 (n=13). Mean Vmna was notsignificantly different in two directions (122.0+17.4 V/s longi-tudinally versus 125.2±15.6 V/s transversely, n= 13, P=NS). Inconclusion, we developed an anisotropic cell culture modelsuitable for studying impulse conduction with cellular resolu-tion. The anisotropic velocity ratio was close to values mea-sured in vivo. By contrast, Vmax was not dependent on thedirection of propagation. (Circ Res. 1994;75:591-595.)Key Words * ventricular myocytes * cell culture *

action potential propagation * voltage-sensitive dyes *collagen

It has been suggested that the effects of anisotropy onVmaxand the safety factor are attributed to the presenceof recurrent spatial discontinuities of axial resistancerelated to cellular borders.3'13 Experimental verificationof this hypothesis was hampered until now by the com-plex three-dimensional geometry of cardiac tissue and bythe lack of techniques available for high-resolution map-ping of activation spread. In the present study, wedeveloped a technique that allowed growth of anisotropictwo-dimensional monolayers of heart cells on a mechan-ically treated substrate of collagen. An optical methodthat allows for the recording of membrane potential withcellular resolution14 was used to assess the directionaldifferences in conduction velocity and VmEIx.

Materials and MethodsCell CultureCollagen Coating

Extracellular matrix protein collagen type IV from humanplacenta (Sigma) was dissolved in phosphate buffer at aconcentration of 20 to 50 gg/mL. Two milliliters of thesolution was applied to one side of the glass coverslips (22-mmdiameter, 0.14-mm thickness, Haska) for 1 hour at roomtemperature. Afterward, the coverslips were rinsed with dis-tilled water and air-dried. A growth-directing substrate wasobtained by gently rubbing the collagen coat with a fine brush.This mechanical treatment produced an adhesion matrixleading to a parallel alignment of the cultured cells. Myocyteswere isolated from neonatal Wystar rats (2 days old) by usinga procedure reported in detail previously.15 In brief, theventricles of the excised hearts were dissociated with trypsin(0.1%). The dispersed cells were suspended 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, KH2P04 0.5, andNaH2PO4 0.3 and containing 20 U/mL penicillin, 20 gg/mLvitamin B12, and 10% neonatal calf serum. The cell suspension

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592 Circulation Research Vol 75, No 3 September 1994

FIG 1. Phase-contrast pictures of isotropic (A) and anisotropic (B) cell monolayers (bar=50 gm).

was preplated to reduce the fibroblast content15 and thendiluted to 3x 10' cells per milliliter. Two milliliters of cellsuspension was placed into a well containing the collagen-coated coverslip. The cultures were incubated in cell mediumM199 containing 5% serum at 37°C in a humidified atmo-

sphere containing 1.5% CO,. Medium changes were per-formed every second day. Measurements were made betweenthe third and eighth day.

Voltage-Sensitive Dye StainingCoverslips were transferred into the experimental chamber

mounted on a vibration-free table. Cells were superfused at a

rate of 2 mL/min with a solution composed of (mmol/L) NaCI150, KCI 5, CaCI2 1.2, MgCl2 1, NaHCO 5.8, HEPES 5, andglucose 5. pH was 7.4, and temperature was 34°C to 35°C. Thevoltage-sensitive styryl dye RH-237 (Molecular Probes) was

used to measure transmembrane potential changes. The dyewas stored in a 2-mmol/L stock solution of dimethyl sulfoxide(DMSO) and diluted to yield a final dye concentration of 2gmol/L in Tyrode's solution (final DMSO concentration,0.1%). Cells were superfused with the dye solution for 4 to 6minutes.

Optical Recording of Impulse ConductionOptical SetupAn optical system to record the fluorescence change of

voltage-sensitive dye was used as described previously14 withseveral modifications. The system included an inverted micro-scope (Axiovert 35M) with a Plan-Neofluar objective (x40;numerical aperture, 1.3 Oil, Zeiss) and a filter set with a

band-pass exciting filter (530 to 585 nm), dichroic mirror (600nm), and a low-pass emitting filter (>615 nm). Cells were

exposed to excitation light for milliseconds. The fluores-cence emitted by the dye was measured by using a 10x 10

photodiode array (Centronic) located in the image plane of themicroscope. An area of 14 x 14 gm2 of cell culture corre-

sponded to each photodiode. The photocurrents from 96diodes were converted to voltage, and 12 channels selected outof 96 were directed into second-stage amplifiers with a sample-and-hold circuit for subtraction of DC signals. Signals were

sampled at a rate of 100 kHz per channel and 12 bits resolutionand stored on a personal computer (Macintosh IIfx, AppleComputer). To eliminate high-frequency noise, signals were

digitally filtered using a gaussian low-pass filter'6 with a cut-offfrequency of 2 kHz. The activation times were determined atthe level of 50% change of optical upstroke.

Electrical StimulationElectrical stimulation was performed via a bipolar electrode

composed of the conducting core of a glass pipette (tipdiameter, 50 to 70 gm) filled with the superfusion solution anda silver wire coiled around the pipette tip. Cells were stimu-

lated with rectangular pulses (duration, 1 millisecond; doublethreshold strength). To ensure that electrical stimulation didnot interfere with propagation measurements, the stimulationelectrode was placed > 1 mm from the measurement site.

Measurements ofActivation Map and VmaxActivation maps were constructed by using linear interpo-

lation between diodes. At a given measuring site and with agiven position of the diode array, impulse conduction wasmeasured in the direction parallel to the long cell axes and inthe transverse direction by changing the location of thestimulation electrode. To calculate Vm,x, signals were scaledto 100-mV amplitude, filtered with a cutoff frequency of 2kHz, and digitally differentiated. Repetitive exposures ofcells to excitation light resulted in the reduction of Vma. by6% (phototoxic effect). To compensate for this effect in the

comparison of longitudinal with transverse velocity, theorder of measurements was alternated from experiment toexperiment.

MorphologyBefore each recording, cell morphology was observed in red

light (filter, >630 nm), and the photodiodes were positionedover a chosen area. Pictures showing the bright-field illumina-tion image of the cells and of the photodiodes and thephase-contrast picture of cells (magnifications of x20 andx40) were made by a video camera (WV-CD50, Panasonic)and a videographic printer (UP-811, Sony). This procedureavoided phototoxic damage of cells. Morphological parametersof cells were measured from photographs. Cell length wastaken as the longest straight line within a cell contour. Thelongest line within the contour perpendicular to the long axiswas taken as the cell width. Statistical data are expressed asmean+SD. Two-tailed nonpaired Student's t test was used forcomparison, and a value of P<.05 was considered statisticallysignificant.

ResultsMorphology of Cell Monolayers

Fig 1 demonstrates partial overviews of isotropic (panelA) and anisotropic (panel B) cell monolayers. The culturedepicted in panel A was grown on a coverslip coated withcollagen; the culture shown in panel B was grown on acoverslip that was mechanically treated after collagencoating to obtain anisotropic cell growth. In isotropicmonolayers, the cell shapes were polymorphic, rangingfrom almost polygonal or round to elongated. Altogether,cells formed a dense network showing no preferentialdirection of cell orientation. By contrast, the cells grownon directed collagen substrate (panel B) were typically ofelongated shape and oriented in one direction. The aver-

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Fast and Kieber Anisotropic Conduction in Cell Culture 593

i5 2\ H FE

'~~~~~~~~~~F-0)11 10

FIG 2. Impulse conduction in an anisotropiccell culture. A, Phase-contrast image of thecell culture. The white rectangles correspondto the localization of the 12 photodiodes. Anindividual diode covers a cell membrane areaof 14x 14 pM2. B, lsochrone map duringlongitudinal impulse spread. Numbers withinrectangles indicate local activation time (inmicroseconds). lsochrones are drawn at aninterval of 100 microseconds. C, lsochronemap recorded during transverse conduction.

age cell length and width were 65.8±12.5 and 12.2±3.2gm (n-49), respectively.

Conduction in Isotropic MonolayersIn isotropic cultures, macroscopic conduction was not

dependent on the direction of the propagation wave.

The mean value for conduction velocity was 24.8±3.1cm/s (n =14). Vmtx in these measurements was 121.4±8.8V/s, when assuming a value of 100 mV for actionpotential amplitude. 14,l5

Conduction in Anisotropic MonolayersActivation Maps During Longitudinaland Transverse Conduction

Fig 2 demonstrates an example of impulse propagationin an anisotropic cell culture. In this experiment, 12recording points were distributed over the whole area

covered by the 10x10 diode array (150x150 pm2) toassess the general pattern of excitation spread. Panel Ashows the anisotropic cell monolayer with the localizationof photodiodes; panels B and C show the activation mapsduring longitudinal and transverse conduction, respec-tively. No clefts were visible on the culture image. Theinterdiode distance varied between 40 and 60 gim. Prop-agation in panel B was obtained by stimulation of themonolayer at a point located -.-1 mm to the right of themeasurement area. The map of longitudinal propagationdemonstrates that the general direction of propagationcoincided with the cell orientation. The shape of theisochrones revealed small nonuniformities in longitudinal

activation. Overall longitudinal conduction velocity mea-

sured between peripheral diodes (activation times at threeproximal and three distal diodes were averaged)amounted to 38.6 cm/s. Transverse propagation in thesame culture is shown on panel C. In this case, the culturewas stimulated by the electrode located 1 mm below themeasurement area. The general direction of propagationslightly deviated from the transverse axis of the monolayer.The overall conduction velocity was 23.3 cm/s, which was

1.7 times smaller than longitudinal velocity. Measure-ments of Vmax revealed significant variability during bothlongitudinal and transverse conduction. Vma. varied in therange of 123 to 177 and 94 to 169 V/s, respectively.However, there was no significant difference betweenmean V max values measured during longitudinal(149.1+15.4 V/s) and transverse (135.9+25.4 V/s, n= 12recording points, P=NS) propagation.

Fig 3 shows an example of another type of measure-

ment, in which recording points were aligned in a row torecord activation sequences at a higher spatial resolu-tion (interdiode distance, 15 gim) and to comparetransverse with longitudinal V,,, along a single axis.Action potential upstrokes were smooth in both trans-verse (panel B) and longitudinal (panel C) directionsshowing no discontinuities or "humps." At the same

time, the activation sequence measured along the trans-verse axis was rather nonuniform. The local conductiontimes between neighboring diodes varied from 60 to 160microseconds. In most cases, a single diode collectedlight from two cells; therefore, it was not possible to

A1

kt~~~~~~~~~1

B

1 10

11 12 13 14

c ~~~~1

1 0.5 1 1.5 1 2.5 1 3.5

time (msec)

FIG 3. Activation sequences oftransverse and longitudinal conduc-tion. A, Phase-contrast image of thecell culture and photodiodes. Num-bers within rectangles indicate photo-diode numbers. B, Normalized opti-cal upstrokes recorded by thephotodiodes shown in panel A duringtransverse conduction. C, Normal-ized optical upstrokes recorded fromthe same localization during longitu-dinal conduction.

B

C

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594 Circulation Research Vol 75, No 3 September 1994

attribute the delay between two diodes to a given cellborder with certainty. The measurements, however, areconsistent with the variability of conduction delaysintroduced by intercellular junctions.14 The averagetransverse conduction velocity measured between di-odes 1 and 10 was 18.0 cm/s, and the average longitu-dinal conduction velocity between diodes 11 and 12 was37.5 cm/s, ie, 2.1 times larger than transverse velocity.The 10 diodes located along the transverse axis wereactivated within 80 microseconds during longitudinalspread. This was consistent with the wave front nonlin-earity shown in Fig 2. As in the experiment presented inFig 2, there was substantial variability of Vm,: it variedfrom 116 to 158 V/s (longitudinal conduction) and from114 to 143 V/s (transverse conduction). However, themean values of longitudinal and transverse Vmax wereonly slightly different: 142.6±+10.9 and 130.7±9.5 V/s(n= 12), respectively.From all the measurements in anisotropic cultures,

the average conduction velocity amounted to 34.6±4.5and 19.0±+4.3 cm/s in longitudinal and transverse direc-tions, respectively (n=13 experiments, P<.0001). Themean anisotropic velocity ratio was 1.89±0.38. In con-trast to anisotropic conduction velocity, Vmax was notdifferent in two directions. From all the experiments,the average of mean Vmax values was 122.0±17.4 V/sduring longitudinal conduction and 125.2±15.6 V/s(n= 13, P=.64 [P=NS]) during transverse conduction.

DiscussionAnisotropy of cardiac tissue plays an important role

in impulse propagation and generation of cardiac ar-rhythmias. So far, the effects of anisotropy have beenstudied in isolated tissue preparations including atrialtissue,3 papillary muscle,23,13 and sheets of ventricularmyocardium.6,7,10,'7-2' In the whole heart, anisotropy wasassessed on the epicardial surface of the intactheart,22-24in a thin rim of subepicardial muscle survivingafter the intramural infarction,25 or after cryodestruc-tion of intramural layers.12'2526 Typically, a sheet ofviable tissue in such experiments consists of many celllayers, which complicates the assessment of impulseconduction at the cellular level. A possible solution toavoid the complexity of three-dimensional heart tissueis to use cultured cell monolayers.14 Thus, we developeda technique allowing the production of two-dimensionalanisotropic monolayers of heart cells. This was achievedby coating glass coverslips with collagen, which is knownfor its ability to promote cell adhesion, and by scratch-ing the collagen substrate with a brush. This mechanicaltreatment produced cell adhesion patterns in whichcells formed an anisotropic structure. Mean cell length(66 ,um) and cell width (12 ,um) in anisotropic mono-layers were slightly smaller than values found in adultrat myocytes (cell length, 73 to 98 ,um; cell width, 15 to18 Atm27) and similar to the values found in culturedstrands of neonatal rat myocytes grown in channelsformed by a photosensitive polymer.1415 The degree ofthe structural anisotropy given by the ratio of cell lengthto width as well as by cell alignment was similar to themyocardial structure in vivo. The method of multisiteoptical recording of action potential upstrokes, whichwas validated in our previous work,14 was used for thefirst time to characterize the directional differences in

Conduction Velocity AnisotropyLongitudinal conduction velocity reported for adult

ventricular myocardium is 43 to 63 cm/s, transversevelocity is 16 to 29 cm/s, and the anisotropic velocityratio is 1.7 to 3.5.2,3,12,18-20,24-26 It is generally believedthat the directional differences in conduction velocityare due to the increased axial resistivity in transversedirection. Our mean values for longitudinal and trans-verse conduction velocities in anisotropic dense cellmonolayers are somewhat lower (34 and 19 cm/s, re-spectively), but the mean anisotropic ratio (1.9) fallswithin the range known for the adult tissue. The poten-tial reasons for the lower velocity in neonatal rat heartcell cultures with respect to adult myocardial tissueinclude a larger surface-to-volume cell ratio and a lowerdegree of electrical coupling in cultured monolayers.They have been discussed elsewhere in detail.14

Local Nonuniformity of ConductionTwo types of anisotropic conduction were reported

previously,17 characterized either by smooth shapes ofextracellular potentials in both longitudinal and trans-verse directions (uniform anisotropy) or by smoothlongitudinal conduction and fractionated electrogramsin transverse direction (nonuniform anisotropy). Theintracellular recordings of transmembrane potential inpapillary muscle demonstrated the presence of notcheson the upstroke when propagation occurred in thetransverse direction but not in the longitudinal direc-tion.3 A similar observation was made in ventricularmuscle treated with the uncoupling agent heptanol.7Correlating structure to function revealed that thepresence of connective tissue separating bundles ofmyocytes was associated with fractionated extracellularelectrograms.28 More recently, another type of nonuni-formity was described, which was characterized by smallfluctuations in extracellular potentials that became ap-parent only in the second time derivative of the signalsand were observed on a small time scale.21 This indi-cates that nonuniformities of propagation may occur atseveral scales. Our results support this idea: even indense cell monolayers, conduction was nonuniform on amicroscopic scale. This was most obvious in transversedirection, where the activation time varied from 60 to160 microseconds, ie, threefold between diodes located15 Atm apart. Such a variability is consistent with thevariability of conduction delays at intercellular junc-tions.14 However, discontinuities or "notches" in theinitial or terminal portions of the action potentialupstrokes were not apparent with the inhomogeneityintroduced by cell borders.

Tissue Anisotropy and Vmax

It has been shown in most studies that Vmax is larger inthe transverse than in the longitudinal direction. Thevalues for Vmax reported in the literature were 94 to 140V/s for longitudinal and 123 to 180 V/s for transverseconduction, resulting in a transverse-to-longitudinal ra-tio of 1.3 to 1.4.3 6,1o,19,21 These directional differences inVmax have been attributed to the presence of the recur-rent spatial discontinuities of axial resistivity.3 It waspostulated that in the transverse direction, cardiactissue behaves as loosely coupled, relatively isolated

conduction velocity and Vmaxm membrane patches, each patch being nearly isopotential

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Fast and Klber Anisotropic Conduction in Cell Culture 595

and therefore having larger Vm than during continuouslongitudinal conduction.3 The anatomic substrate ex-plaining such recurrent discontinuities is not preciselyknown yet. The present study demonstrates that it ispossible to grow networks of cardiac cells with structuralanisotropy and anisotropic conduction velocity but nodirectional-dependent differences in Vm17. Therefore,the discontinuities introduced by cell borders alone donot explain the directional differences of Vmax observedin vivo. This is consistent with the simulation study29showing that the effects of discrete cell length on Vmaxare negligible in homogeneous one-dimensional cablewhen cell length is much less than the electrotonic spaceconstant. Another type of discontinuity caused by ar-rangement of cardiac cells into unit bundles30 andarrangement of these bundles into fascicles (which bothare separated by connective and vascular tissue) may beresponsible for the directional differences of Vm,, invivo. This would be consistent with the observation thatthe higher margin of safety for transverse longitudinalconduction is only observed in "nonuniform" anisotro-pic tissue, ie, in the presence of connective tissue sheetsseparating the fiber bundles.31

AcknowledgmentsThis study was supported by the Swiss National Science

Foundation and the Swiss Heart Foundation. We wish to thankRegula Fluickiger, Denis de Limoges, Hans-Ulrich Schweizer,and Juirg Burkhalter for their technical assistance.

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V G Fast and A G Klébersubstrate.

Anisotropic conduction in monolayers of neonatal rat heart cells cultured on collagen

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1994 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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