Doxorubicin-induced Automaticity in Cultured Chick...

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[CANCER RESEARCH 50, 4209-4215, July 15, 1990] Doxorubicin-induced Automaticity in Cultured Chick Heart Cell Aggregates1 Joan C. Mitrius2 and Stephen M. Vogel3 Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612 ABSTRACT The use of doxorubicin, a clinically important antitumor agent, is associated with toxic cardiac side effects, including arrhythmias and, on rare occasions, sudden death. We have used aggregates of cultured chick embryonic heart cells, a preparation free of neuronal and vascular ele ments, to investigate the direct effects of doxorubicin on automaticity. Under control conditions, normally quiescent aggregates of ventricular origin could be induced to contract spontaneously during a 24- to 48-h incubation with doxorubicin. The percentage of aggregates exhibiting automaticity was concentration dependent between 0.01 and 1 n\i doxo rubicin. At relatively high drug exposure (e.g., 1 MMdoxorubicin for 48 h), beating was shown to be dysrhythmic. Whereas control aggregates exhibited large stable resting potentials on impalement with intracellular microelectrodes, treated aggregates exhibited spontaneous action poten tials with Phase 4 depolarization. Doxorubicin, in a dose-dependent manner, increased the rate of Phase 4 depolarization, reduced the maxi mum diastolic potential, and shortened the action potential duration. In dysrhythmic aggregates, microelectrode recordings also revealed exam ples of prematuredepolarizations, extrasystoles, and dropped beats. This study provides the first cellular electrophysiological recordings of doxo- rubicin-induced automaticity and rhythm disturbances in heart muscle. The results suggest that the induction of automaticity is due to a depo larizing action of doxorubicin on the cardiac membrane. INTRODUCTION Doxorubicin, a major chemotherapeutic agent active against certain solid and hematological malignancies, is limited in its clinical use by the development of acute and chronic cardiotoxic effects (1,2). The most serious manifestation is a chronic dose- related cardiomyopathy that has been shown to increase dra matically above cumulative doses of about 550 mg/nr (3). Less well known is the tendency for doxorubicin to disturb the cardiac rhythm, sometimes resulting in life-threatening arrhyth mias and sudden death (4). The drug-induced rhythm disturb ances, which can be detected electrocardiographically in hu mans and animals, have been reported to occur acutely (within the first few hours), after long-term treatment, or within 24 to 48 h following administration of doxorubicin (5-8). The acute dysrhythmias are similar following bolus injections or contin uous infusions of doxorubicin. The occurrence of clinically significant cardiac dysrhythmias prior to the development of cardiomyopathy suggests that doxorubicin exerts early and significant effects on the membrane properties of heart cells. Previously, studies of doxorubicin have concentrated on the chronic cardiomyopathy that is characterized by ultrastructural alterations (e.g., myofibrillar loss and cytoplasmic vacuoliza- tion) and the destruction of biological membranes, apparently through free radical-dependent lipid damage (9). In general, the same histopathological changes and subcellular effects of dox orubicin that occur in humans develop in animal models (10). Also, in monolayer cultures exposed to doxorubicin, in which Received 6/26/89; revised 2/8/90. ' Supported by American Cancer Society Grant CH-437. 2Submitted in partial fulfillment to the James Scholar Program for Independ ent Study in the College of Medicine, University of Illinois. Present address: Department of Internal Medicine, Good Samaritan Medical Center, 1111 E. McDowell Road, Phoenix, AZ 85006. 3To whom requests for reprints should be addressed, at Department of Pharmacology (M/C 868). University of Illinois College of Medicine, 835 South Wolcott Avenue, Chicago, IL 60612. the myocardial cells undergo similar ultrastructural changes, spontaneous beating ceases as the cells die (11, 12). With reduced exposure to doxorubicin, arrhythmias could be pro duced in vitro (11). However, there have been no reports thus far describing the cellular electrophysiological basis of doxo- rubicin induced aberrant automaticity in heart muscle at drug concentrations used in a clinical setting. Therefore, in the present study, we analyzed the electrophysiological effects of doxorubicin, making use of an in vitro myocardial preparation, namely, chick embryonic heart cells cultured as spherical clus ters, or aggregates. Aggregates of cultured embryonic heart cells have many advantages for such a study, which have been summarized as follows, (a) Embryonic heart cells remain viable in vitro for days to weeks and exhibit contractile activity typical of the intact embryo heart, (b) The tissue culture approach makes possible the prolonged (e.g., 24-48 h) incubations with doxo rubicin that are necessary for cardiotoxic effects to occur. The effects of doxorubicin on the cultured cells are direct without the complications of drug action on neuronal and vascular tissues, (c) Recordings of both transmembrane potentials and contractions can be made in aggregates by using microelectrode and photoelectric techniques. Furthermore, in aggregates of embryonic chick hearts, many of the electrophysiological properties and pharmacological re ceptors observed are characteristic of the hearts from which the cells are isolated (13). Aggregates cultured from 7-day-old embryonic ventricles, as used in the present study, typically exhibit a quiescent resting membrane potential, tetrodotoxin- inhibitable action potentials, and an absence of automaticity, much as the intact heart (14). These cultured cells also continue to respond in vitro to catecholamines, histamine, acetylcholine, and angiotensin II (15-18). Spherical aggregates retain the tight cell-to-cell electrical coupling characteristic of cardiac muscle (19). The slow calcium inward current and the fast (tetrodo- toxin-sensitive) sodium current occur in this preparation and have been directly measured using the two-microelectrode volt age clamp (18, 20). Cell aggregates cultured from 7-day-old chick embryonic hearts are capable, under certain conditions (e.g., in low extracellular K+ solutions), of developing pace maker-like activity, i.e., spontaneously firing action potentials with diastolic (Phase 4) depolarization. The ionic currents underlying this diastolic depolarization have been analyzed and resemble those found in adult cardiac Purkinje fibers (21). In the present study, an in vitro myocardial preparation has been used as a model system to investigate the possible mech anisms by which doxorubicin produces cardiotoxicity. The ma jor findings are that doxorubicin exposure causes the appear ance of spontaneous beating, which is sometimes dysrhythmic, in normally quiescent heart cell aggregates and that the altered behavior is associated with membrane depolarization, a phe nomenon not previously described for cardiac cells of ventric ular origin. MATERIALS AND METHODS Materials Drugs. Doxorubicin was donated by Dr. Vern Verhoef on behalf of Adria Laboratories (Columbus, OH). 4209 on July 9, 2018. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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[CANCER RESEARCH 50, 4209-4215, July 15, 1990]

Doxorubicin-induced Automaticity in Cultured Chick Heart Cell Aggregates1

Joan C. Mitrius2 and Stephen M. Vogel3

Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612

ABSTRACT

The use of doxorubicin, a clinically important antitumor agent, isassociated with toxic cardiac side effects, including arrhythmias and, onrare occasions, sudden death. We have used aggregates of cultured chickembryonic heart cells, a preparation free of neuronal and vascular elements, to investigate the direct effects of doxorubicin on automaticity.Under control conditions, normally quiescent aggregates of ventricularorigin could be induced to contract spontaneously during a 24- to 48-hincubation with doxorubicin. The percentage of aggregates exhibitingautomaticity was concentration dependent between 0.01 and 1 n\i doxorubicin. At relatively high drug exposure (e.g., 1 MMdoxorubicin for 48h), beating was shown to be dysrhythmic. Whereas control aggregatesexhibited large stable resting potentials on impalement with intracellularmicroelectrodes, treated aggregates exhibited spontaneous action potentials with Phase 4 depolarization. Doxorubicin, in a dose-dependentmanner, increased the rate of Phase 4 depolarization, reduced the maximum diastolic potential, and shortened the action potential duration. Indysrhythmic aggregates, microelectrode recordings also revealed examples of prematuredepolarizations, extrasystoles, and droppedbeats. Thisstudy provides the first cellular electrophysiological recordings of doxo-rubicin-inducedautomaticity and rhythm disturbances in heart muscle.The results suggest that the induction of automaticity is due to a depolarizing action of doxorubicin on the cardiac membrane.

INTRODUCTION

Doxorubicin, a major chemotherapeutic agent active againstcertain solid and hematological malignancies, is limited in itsclinical use by the development of acute and chronic cardiotoxiceffects (1,2). The most serious manifestation is a chronic dose-related cardiomyopathy that has been shown to increase dramatically above cumulative doses of about 550 mg/nr (3). Lesswell known is the tendency for doxorubicin to disturb thecardiac rhythm, sometimes resulting in life-threatening arrhythmias and sudden death (4). The drug-induced rhythm disturbances, which can be detected electrocardiographically in humans and animals, have been reported to occur acutely (withinthe first few hours), after long-term treatment, or within 24 to48 h following administration of doxorubicin (5-8). The acutedysrhythmias are similar following bolus injections or continuous infusions of doxorubicin. The occurrence of clinicallysignificant cardiac dysrhythmias prior to the development ofcardiomyopathy suggests that doxorubicin exerts early andsignificant effects on the membrane properties of heart cells.

Previously, studies of doxorubicin have concentrated on thechronic cardiomyopathy that is characterized by ultrastructuralalterations (e.g., myofibrillar loss and cytoplasmic vacuoliza-tion) and the destruction of biological membranes, apparentlythrough free radical-dependent lipid damage (9). In general, thesame histopathological changes and subcellular effects of doxorubicin that occur in humans develop in animal models (10).Also, in monolayer cultures exposed to doxorubicin, in which

Received 6/26/89; revised 2/8/90.' Supported by American Cancer Society Grant CH-437.2Submitted in partial fulfillment to the James Scholar Program for Independ

ent Study in the College of Medicine, University of Illinois. Present address:Department of Internal Medicine, Good Samaritan Medical Center, 1111 E.McDowell Road, Phoenix, AZ 85006.

3To whom requests for reprints should be addressed, at Department of

Pharmacology (M/C 868). University of Illinois College of Medicine, 835 SouthWolcott Avenue, Chicago, IL 60612.

the myocardial cells undergo similar ultrastructural changes,spontaneous beating ceases as the cells die (11, 12). Withreduced exposure to doxorubicin, arrhythmias could be produced in vitro (11). However, there have been no reports thusfar describing the cellular electrophysiological basis of doxo-rubicin induced aberrant automaticity in heart muscle at drugconcentrations used in a clinical setting. Therefore, in thepresent study, we analyzed the electrophysiological effects ofdoxorubicin, making use of an in vitro myocardial preparation,namely, chick embryonic heart cells cultured as spherical clusters, or aggregates.

Aggregates of cultured embryonic heart cells have manyadvantages for such a study, which have been summarized asfollows, (a) Embryonic heart cells remain viable in vitro fordays to weeks and exhibit contractile activity typical of theintact embryo heart, (b) The tissue culture approach makespossible the prolonged (e.g., 24-48 h) incubations with doxorubicin that are necessary for cardiotoxic effects to occur. Theeffects of doxorubicin on the cultured cells are direct withoutthe complications of drug action on neuronal and vasculartissues, (c) Recordings of both transmembrane potentials andcontractions can be made in aggregates by using microelectrodeand photoelectric techniques.

Furthermore, in aggregates of embryonic chick hearts, manyof the electrophysiological properties and pharmacological receptors observed are characteristic of the hearts from which thecells are isolated (13). Aggregates cultured from 7-day-oldembryonic ventricles, as used in the present study, typicallyexhibit a quiescent resting membrane potential, tetrodotoxin-inhibitable action potentials, and an absence of automaticity,much as the intact heart (14). These cultured cells also continueto respond in vitro to catecholamines, histamine, acetylcholine,and angiotensin II (15-18). Spherical aggregates retain the tightcell-to-cell electrical coupling characteristic of cardiac muscle(19). The slow calcium inward current and the fast (tetrodo-toxin-sensitive) sodium current occur in this preparation andhave been directly measured using the two-microelectrode voltage clamp (18, 20). Cell aggregates cultured from 7-day-oldchick embryonic hearts are capable, under certain conditions(e.g., in low extracellular K+ solutions), of developing pacemaker-like activity, i.e., spontaneously firing action potentialswith diastolic (Phase 4) depolarization. The ionic currentsunderlying this diastolic depolarization have been analyzed andresemble those found in adult cardiac Purkinje fibers (21).

In the present study, an in vitro myocardial preparation hasbeen used as a model system to investigate the possible mechanisms by which doxorubicin produces cardiotoxicity. The major findings are that doxorubicin exposure causes the appearance of spontaneous beating, which is sometimes dysrhythmic,in normally quiescent heart cell aggregates and that the alteredbehavior is associated with membrane depolarization, a phenomenon not previously described for cardiac cells of ventricular origin.

MATERIALS AND METHODS

Materials

Drugs. Doxorubicin was donated by Dr. Vern Verhoef on behalf ofAdria Laboratories (Columbus, OH).

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ELECTROPHYSIOLOGICAL EFFECTS OF DOXORUB1CIN IN THE HEART

Culture Supplies. Fertilized eggs were obtained from Sharp Sales(West Chicago, IL). M199, fetal calf serum, and trypsin-EDTA werepurchased from GIBCO (Grand Island, NY). Trypan blue was obtainedfrom Sigma Chemical Co. (St. Louis, MO). Tissue culture dishes werepurchased from Falcon (Oxnard, CA), and bacteriological plates wereobtained from Fisher Scientific Co. (Itasca, IL). Glass coverslips (16mm2) were obtained from BélicoGlass Co. (Vineland, NJ), and NU-

serum was from Collaborative Research, Inc. (Bedford, MA).

Heart Cell Primary Culture

Preparation of Chick Cardiac Myocytes. Primary cultures of chickcardiomyocytes were prepared under sterile conditions by a modification of the method of Sachs and DeHaan (22). Hearts from chickembryos were excised on the seventh day in ovo. After discarding theatria, two slits in the apical portion of the ventricle were made tofacilitate blood removal. Eighteen ventricles were pooled at roomtemperature in culture medium composed of M199 and 10% fetal calfserum and containing antibiotics (10,000 units/ml of penicillin and 10mg/ml of streptomycin). To obtain a suspension of dissociated myo-cytes, the ventricles were transferred to a 25-ml Ehrlenmeyer flaskcontaining 0.05% trypsin-EDTA, triturated twice using a large-borepipet, and then gently agitated in a 37°Cshaker bath for 7 min. The

supernatant containing dispersed myocytes was added to an equalvolume of M199 with 10% fetal calf serum to terminate the trypsinaction. Then, the supernatant was centrifuged at 500 x g for 5 min.Since it was found that mechanical agitation of the tissue withouttrypsin could produce cell separation to some extent, the undigestedventricular tissue was next resuspended in culture medium withouttrypsin and treated in all other respects exactly as described above.Four to five additional cycles of alternating trypsin and culture mediumwere used to completely digest the ventricles. The pellets of each digestwere pooled in culture medium with the exception of the first pelletwhich was discarded. The pellets were washed twice with culturemedium, centrifuged, and resuspended in culture medium to which 5mM HEPES4 buffer (pH 7.4) was added. In several cultures examined,

viability was greater than 90% by the Trypan blue exclusion test.Separation of Myocytes from Fibroblasts. Cardiac myocytes were

separated from fibroblasts on the basis of rapid attachment of fibro-blasts to tissue culture dishes (23). From a total of 18 hearts, asuspension of 10s cells/ml was plated into 150-mm2 tissue culturedishes (Falcon) to give 400 cell/mm2 of surface area. After a 3-h

incubation in humidified 95% air:5% CO2 atmosphere, supernatantscontaining principally myocytes were collected and centrifuged at 1000x g (or 10 min.

Generation and Treatment of Aggregate Cultures. In order to establishaggregates, the dissociated cells were placed in sterile bacteriologicalplates to which the heart cells do not adhere. The dissociated cells wereplated onto 100-mm2 Petri dishes at a density of 1000 cells/mm2. After

an incubation period of 24 h, spherical aggregates were formed (50 to200 Mmin diameter). Aggregates were then separated from any remaining single myocytes by a brief centrifugation step (1000 x g for 3 min).The aggregates were then transferred to new tissue culture dishes orcoated coverslips for a second 24-h period where they were permittedto adhere. Drug treatment was initiated 24 h after aggregates wereseeded. For contraction studies (see below), aggregates were seeded in35-mm2 tissue culture dishes at a concentration of 1 to 2 aggregates/mm2 of surface area. For electrophysiological studies, aggregates were

seeded on coated coverslips according to the method of Mains and May(24). Briefly, 16-mm2 round coverslips were immersed in protamine

(2.5 mg/ml) in 0.15 M NaCl. After 30 min, excess protamine solutionwas removed by aspiration, and the coverslips were immersed in NU-serum for a period of at least 1 h. Aggregates were then seeded on theprepared coverslips at a density of 2/mm2.

Detection of Contractions and Recording of Action Potentials from Cultured Heart Cell Aggregates

Detection of Contraction in Heart Cell Aggregates. A tissue culturedish containing aggregates was transferred to the thermostatically con-

4The abbreviation used is: HEPES. 4-(2-hydroxyethyl)-l-piperazineethanesul-

fonic acid.

trolled stage (37°C)of an inverted phase-contrast microscope. The pH

was maintained at 7.4 by the inclusion of HEPES buffer in the culturemedium. Preliminary experiments demonstrated that aggregates maintain consistent beat patterns under these conditions for at least 1 h.The experimental observations (on any single tissue culture dish) tookplace in less than 40 min. In experiments in which the number ofaggregates exhibiting automaticity was counted after various experimental treatments, the aggregates were viewed at a magnification ofx200 to x400, permitting the contractions of spontaneously beatingaggregates to be detected by direct visual observation. In experimentsin which the rhythmicity of beating was analyzed, the contractions weredetected photoelectrically. To do this, the microscope image was projected, with the aid of a camera, to a television monitor at a finalmagnification of xlOOO to X2000. A photocell placed at the edge ofthe projected image was used to record changes in light intensityresulting from the pulsations of the aggregate, similar to the method ofLampidis et al. (11). The output of the photocell circuit was amplified,recorded on a pen writer (Grass Model 7 polygraph), and simultaneously digitized by means of an analogue-to-digital converter. The digitized records could be viewed on the video monitor of an IBM ATmicrocomputer. The acquisition of data (i.e., the "twitch contraction"

plus a segment of the preceding baseline) was triggered by an eventdetector with a preset threshold. The acquisition program ("pCLAMP";Axon Instruments) "kept track of" the time interval between successive

acquisitions, as this information would be needed for the subsequentcalculation of the cycle length. The data were stored on floppy diskettesfor offline analysis of the cycle length (see below).

Microelectrode Recording from Aggregates. Protamine-treated cover-slips with strongly adhering heart cell aggregates were transferred to abath (volume of 2 ml) that was attached to the stage of an invertedphase-contrast microscope. The aggregates were continuously super-fused at a rate of 6 ml/min with 100 ml of M199 medium, which wasrecirculated between a reservoir and the bath by means of a peristalticpump. The fluid was heated to 37°Cby means of an interposed

condenser. The reservoir fluid was bubbled with 95% O2-5% CO2 via afine polyethylene tube (pH 7.4). At the K* concentration in M199

(about 5.4 mmol), the vast majority of aggregates exhibited stableresting membrane potentials and no spontaneous firing, in agreementwith the previous findings of Clay and Shrier (21) for aggregatescultured from 7-day-old chick embryo hearts. When desired, the aggregates were paced at a constant frequency (1 Hz) by suprathreshold,rectangular current pulses delivered via a glass pipet (tip diameter, 50to 100 Mm) positioned within 100 Mm of the aggregate. To recordintracellular potentials, the aggregates were impaled with conventionalmicroelectrodes which were filled with 3 M KC1 and had a resistance of40 to 60 MSÃŽ.The microelectrode was electrically connected via a silver-silver chloride wire to a high input impedance preamplifier. The bathfluid was grounded via a silver-silver chloride wire, which served as areference electrode. After amplification, the action potential and asegment of the preceding baseline were digitized using a computer(IBM PC AT) equipped with an analogue-to-digital converter (Tecmar,Inc.) and software to control data acquisition (pCLAMP; Axon Instruments). Data acquisition was triggered by an event detector, much asdescribed in the preceding paragraph. The acquired data were storedon floppy diskettes. Analysis of the action potential waveform wasperformed offline, using software developed in this laboratory, whichextracts various parameters of the stored action potentials (restingpotential, maximum diastolic potential, action potential duration atselected levels of repolarization, and the peak overshoot potential). Thereader is referred to Vogel and Terzic (25) for a more detailed description of action potential analysis. The slope of the pacemaker potentialand the takeoff potential at which the action potential is initiated weredetermined for spontaneously firing aggregates. The method for estimating the takeoff potential can be found in the legend to Fig. 5. Forquiescent aggregates, the takeoff potential was considered identical tothe resting membrane potential just prior to suprathreshold electricalstimulation.

Analysis of Cycle Lengths. To examine the rhythmicity of spontaneous beating in heart cell aggregates treated with doxorubicin, thecycle length intervals between 50 and 200 successive beats were deter-

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ELECTROPHYSIOLOGICAL EFFECTS OF DOXORUBICIN IN THE HEART

mined. For purposes of data analysis, the cycle length was consideredas the time between an identical point on the rising phase of twoconsecutive beats (either photocell output or action potential). Computer programs (pCLAMP; Lotus 1-2-3) were used to automaticallymeasure the cycle lengths from the stored data and to construct a cyclelength histogram for each aggregate. Also, the mean cycle length andstandard error of the mean were calculated. The variance of the cyclelength distribution (i.e., the sum of squared deviations from the mean)was used as an index of the degree of dysrhythmia caused by doxorub-icin.

Statistics. Data were analyzed by Student's t test, and differences

between mean values at P < 0.05 were considered statistically significant.

RESULTS

Induction of Abnormal Automaticity and Dysrhythmia by Dox-orubicin in Chick Heart Cell Aggregates

Abnormal Automaticity. Evidence that doxorubicin inducesspontaneous beating in quiescent chick cardiac cell aggregatesis presented in Fig. 1. The percentage of aggregates contractingspontaneously was low (<10%) in untreated cultures, but wasincreased markedly following a 24-h incubation with doxorubicin (Fig. 1, Day 1 curve). As can be seen, the effects ofdoxorubicin on automaticity increased with drug concentrationbetween 0.01 and 1.0 /¿M;at 1.0 /¿M,over 80% of the aggregatesexamined contracted spontaneously. The effects of a 48-h incubation with doxorubicin were also tested (Fig. 1, Day 2 curve).At a doxorubicin concentration of 1.0 /¿M,a slight decrease inthe percentage of aggregates contracting was noted at 48 h (Fig.1, Day 2 curve), due perhaps to the cytotoxicity of doxorubicinat this concentration. It was therefore tested whether doxorubicin affects cell viability. To do this, a separate series of experiments was performed in which cardiac cell aggregates wereexposed to doxorubicin for 48 h and then tested for an abilityto exclude the dye, trypan blue. Failure to exclude the dye wasobserved at 1 and 10 ^M doxorubicin only in the noncontractingaggregates that presumably had ceased to function (data notshown). In contrast, the aggregates excluded trypan blue atdoxorubicin exposures of less than 1 /¿Mfor 48 h (data notshown), indicating that induction of automaticity by doxorubicin was probably not associated with destruction of the cellmembrane. Thus, only at relatively high doxorubicin concentra

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Fig. 1. Effect of doxorubicin to induce automaticity in cultured chick embryoheart cells. The percentage of aggregates contracting spontaneously is plotted asa function of the doxorubicin concentration at exposure times of 24 h (Day 1curve) and 48 h (Day 2 curve). The experimental protocol was as follows.Aggregates in tissue culture dishes were incubated without drug (control group)or with a single concentration of doxorubicin (range; 10~" to 10~6 mol). For

experimental measurements of automaticity, each tissue culture dish was transferred to the thermostatically controlled stage (37"C) of an inverted microscope.

The pH of the medium was maintained at 7.4, using a HEPES buffer. Theaggregates were viewed at a magnification of x200 to x400, permitting spontaneous twitch contractions to be detected by direct visual observation. Approximately 50 aggregates were examined from each culture dish. Two to 4 determinations were made at each doxorubicin concentration with different cultures ofaggregates. The percentages given are based on a total of 96 to 253 observationsper data point.

lions was there evidence that doxorubicin impaired cell membrane integrity.

Dysrhythmic Beating. To investigate whether doxorubicincauses dysrhythmia in cultured heart cells, the pattern of spontaneous beating was examined in a series of aggregates treatedwith doxorubicin. Both rhythmic and dysrhythmic beat patternswere observed depending on the doxorubicin concentration.Fig. 2 provides sample photocell recordings of twitch contractions from a rhythmic (Fig. 2A) and dysrhythmic aggregate(Fig. IE) that had been exposed to 0.3 or 1.0 /¿Mdoxorubicin,respectively, for 48 h. The histograms of cycle lengths in Fig. 2demonstrate a striking difference between rhythmic and dysrhythmic beat patterns. When the pattern of beating is regular,the histogram of the cycle lengths generally has a single dominant peak (Fig. 1A). In comparison, histograms associated withdoxorubicin-treated cultures that are beating irregularly havemuch broader distribution (Fig. 2B). To provide an index ofthe degree of dysrhythmia caused by doxorubicin treatment,the variance of the interval distributions was calculated. Asexpected, the variance was larger for the dysrhythmic aggregate(6.7 x IO6 ms2) than for the rhythmic aggregate (0.04 x IO6ms2). The measured variances for 92 different treated aggregates

are presented in Fig. 3. Aggregates that beat rhythmically (i.e.,the interbeat intervals are distributed unimodally) always hadvariances of less than 1.0 x IO6 ms2. Whereas the variances

were generally well below this value at doxorubicin exposuresof 0.1 or 0.3 /¿mol,an exposure to doxorubicin of 1.0 /¿M(for48 h) resulted in a mixture of variances 50% of which exceeded(often markedly) a value of 1 x IO6ms2. This confirms that this

relatively high exposure to doxorubicin can produce dysrhythmic beat patterns in cultured heart cells.

Electrophysiological Actions of Doxorubicin in Cultured HeartCell Aggregates

Spontaneous Firing and Depolarization of Doxorubicin-treatedHeart Cell Aggregates. Representative intracellular microelec-trode recordings from control and doxorubicin-treated aggregates are shown in Fig. 4. Control aggregates exhibited stableresting membrane potentials of about —70mV (Fig. 4A), but

could be excited to fire an action potential by a brief electricalstimulus (Fig. 4A, inset). The action potential was accompaniedby a twitch contraction that was easily visible under the invertedmicroscope at a magnification of x200 to x400. Doxorubucintreatment (>10~7 M for 24 to 30 h) caused the majority of

aggregates to beat spontaneously (e.g., see Fig. 1). Spontaneously firing action potentials with diastolic (Phase 4) depolarization were found upon impaling such aggregates. Therecords in Fig. 4, B to D, illustrate that, for increasing doxorubicin concentrations, there were a progressive depolarizationof the maximum diastolic potential and a steeper slope of Phase4 depolarization.

Concentration-dependent Effects of Doxorubicin on the Cardiac Action Potential. Table 1 summarizes the characteristics ofthe action potential recorded from a total of 107 aggregates.The control aggregates were paced at a standard frequency of1 Hz to elicit the action potential, as the vast majority of themdid not beat spontaneously. The maximum diastolic potential(i.e., the maximum negative potential during the diastolic interval) had a mean value under control conditions of —67±1mV (n = 37). The maximum diastolic potential of spontaneously beating aggregates in 0.1, 0.3, and 1.0 /¿Mdoxorubicinwas -63 ±3 (n = 8), -57 ±2(n = 25), and -51 ±2 mV (n =

33), respectively, and was significantly depolarized in comparison with control values at the two highest concentrations.

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ELECTROPHYSIOLOGICAL EFFECTS OF DOXORUBICIN IN THE HEART

Fig. 2. Doxorubicin-induced automaticityand dysrhythmia. Initially quiescent cell aggregates from 7-day-old chick embryo hearts wereincubated in tissue culture dishes with doxo-rubicin for 48 h. For experiments, a tissueculture dish containing treated aggregates wastransferred to the thermostatically controlledstage (37°C)of an inverted microscope. The

twitch contractions of spontaneously beatingaggregates were detected photoelectrically, asdescribed in "Methods." A and B, penwriter

records of contractions from two aggregatestreated with doxorubicin at a concentration of0.3 fimol (A) or 1.0 nmol (B). Note that dys-rhythmic beating occurred in the aggregateexposed to 1 /*Mdoxorubicin. A' and B', his

tograms of cycle lengths corresponding to therecords in A and B. Note high variability ofcycle lengths in the dysrhythmic case. Timecalibration in I applied to A-B.

4000

CYCLE LENGTH (MSEC)

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CYCLE LENGTH (MSEC)

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INDIVIDUAL AGGREGATES (number)

Fig. 3. Incidence of dysrhythmic beating in heart cell aggregates exposed todoxorubicin. Quiescent aggregates were exposed to doxorubicin in culture for 48h. Spontaneous beating that resulted from doxorubicin treatment was detectedphotoelectrically in randomly selected aggregates, and the distribution of cyclelengths over a series of beats was analyzed in each case. The variance of the cyclelength distribution is plotted for 92 different aggregates at three doxorubicinconcentrations (O.I, 0.3, and 1 /<tnol. as indicated). Dysrhythmic beating (indicated by a variance >1 x 10' ms2) occurred in about 50% of the aggregates

examined at the highest doxorubicin concentration. See text for further details.

These changes in maximum diastolic potential thus corresponded to a depolarization of 4, 10, and 16 mV at 0.1, 0.3,and 1.0 UM doxorubicin. The takeoff potential, i.e., the membrane potential at which the action potential is initiated, alsowas progressively depolarized as a function of the doxorubicinconcentration (Table 1). The slope of diastolic (Phase 4) depolarization in spontaneously beating aggregates was increased ina concentration-dependent fashion; the change in this parameter was statistically significant at 1.0 ^mol in comparison with0.1 ¿¿Mdoxorubicin. Finally, at 1.0 UM doxorubicin, there wasa small but statistically significant decrease in action potentialduration. The overshoot potential was not significantly alteredby incubations with doxorubicin.

Intracellular Microelectrode Recordings of Dysrhythmia inDoxorubicin-treated Heart Cell Aggregates. Several examplesof dysrhythmia in doxorubicin-treated aggregates are presentedin Fig. 5. Fig. SA shows an intracellular recording from anaggregate which generated action potentials at an uneven rate.The cycle length histogram, which is also given, shows severalpeaks reflecting this arrhythmic beating. The records from adifferent aggregate in Fig. 5B provide an example of a suddendeceleration in the rate of beating by the mechanism of the"dropped beat," i.e., a failure of Phase 4 depolarization to

achieve the action potential threshold for one or more beats.

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SCO(msec)OIIMC)\_^

\—»o.-•>1000

Fig. 4. Spontaneous electrical activity and depolarization in cultured chickcardiac aggregates treated with doxorubicin. Aggregates were seeded on coverslipscoated with protamine and incubated without drug (control) or with variousconcentrations of doxorubicin for 24 to 30 h. After transferring the coverslips toan experimental chamber, intracellular microelectrodes were used to recordresting and action potentials from randomly selected aggregates. In A, a stableresting membrane potential was recorded from a control aggregate. In the /nser,stimulation with suprathreshold rectangular current pulses elicited an actionpotential, demonstrating that the aggregate was excitable. In B to D, spontaneously firing action potentials with diastolic (Phase 4) depolarization wererecorded from aggregates exposed to 0.1 MM(B), 0.3 MM(O, and 1 MM(D)doxorubicin. In B, two consecutive sweeps were superimposed. The maximumdiastolic potential, which is indicated in B to D, was progressively depolarizedwith increasing doxorubicin concentration. Ordinate in all panels: mV (20 mV/div); abscissa: ms. Note different time scales in A and B and in C and D. MDP,maximum diastolic potential.

The associated histogram shows that most of the 136 cyclelengths were in the 400 to 800 ms range. The exceptions were4 unusually long interval lengths, indicating roughly 1,2 (2cases), or 4 dropped beats. Fig. 5C depicts a recording from athird aggregate in which a sudden acceleration in beating occurred, apparently by the generation of an extrasystole. In thisinstance, the extrasystole appears to have arisen from a depolarizing afterpotential (Fig. 5C1, arrow), which did not alwaysreach the action potential threshold (Fig. 5C2, arrow). In someaggregates, a temporary period of arrest occurred (for 20 to 60s) during which time the membrane potential was stable. Theresting membrane potential of such arrested cells provides anindication of the depolarizing action of doxorubicin (0.3 to 1.0

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ELECTROPHYSIOLOGICAL EFFECTS OF DOXORUBICIN IN THE HEART

Table 1 Effect of doxorubicin on the action potential in aggregates of chick ventricular cellsAggregates of chick ventricular heart cells were incubated in culture without drug (control) or exposed to doxorubicin for 24 to 30 h. Coverslips containing cultured

aggregates were transferred to an experimental chamber where they were superfused with bubbled (95% O2, 5% CO2) culture medium at 37°C.Aggregates were

impaled with conventional intracellular microelectrodes, and resting and action potentials were recorded. Control aggregates were paced, with suprathresholdrectangular current pulses, at a frequency of 1 Hz.

Concentration(>lM)ControlDoxorubicin

(0. 1)Doxorubicin(0.3)Doxorubicin

(1.0)MDP°(mV)-67

±Ie-63±3-57

±2d-51±2"Overshoot(mV)+21

±2+19±5+23

±2+17 + 2APD«,(ms)220+

12243±18191±13175+

IS*APD50(ms)174

+9202±19147±13137+

14"APD.o(ms)64

+360+948±537±3''Phase

4slope(mV/s)29

+437+453+ 8'Takeoffpotential*(mV)-66+

1-49+3-47+2"-40±2dCyclelength(s)(1.0)0.7

±0.11.0±0.10.9

+ 0.1No.

ofcellsimpaled/no.of

cultures37/108/129/533/5

a MDP, maximum diastolic potential; ADPco, action potential duration at 90% repolarization; APD50 and APD|0 defined similarly.* Takeoff potential is that potential at which action potential is initiated (see Fig. 5 legend).' Mean ±SE.* P< 0.05 versus control.*P£ 0.05 versus 0.1 ^M doxorubicin.

Fig. 5. Intracellular microelectrode recordings from cultured cardiac aggregates exhibiting dysrhythmic beating. The aggregates in Ato C were exposed to 1.0 pM doxorubicin inculture for 24 h. AI and A2, continuous recording from an aggregate showing fluctuations in action potential frequency. Time calibration in A2 applies lo Al and A2. In A3, theassociated histogram demonstrates a broaddistribution of cycle lengths in this aggregate(316 beats analyzed; variance of distributionwas 6.0 x 10' ms2). Bl, two sweeps illustratinga "dropped beat" (note long pause). In B2, the

cycle length histogram for a sequence of actionpotentials suggests that predominantlyrhythmic beating occurred in this aggregateexcept during occasional quiescent periods (arrows). Also illustrated (Bl) are several featuresof the action potential measured in this study(Table 1). First arrow in upper trace points tothe maximum diastolic potential (MDP). Thesecond arrow points to the takeoff potential atwhich the action potential is initiated. A sharptransition from Phase 4 (diastolic depolarization) to Phase 0 (action potential upstroke)occurs at the takeoff potential. This transitionpoint was estimated from the intersection ofimaginary lines (note dashed lines) projectedfrom Phase 4 and Phase 0. (The dashed lineswere slightly displaced from Phase 4 and Phase0 for illustrative purposes.) Action potentialplateau and repolarization (Phases 2 and 3)are also indicated. C, intracellular recordingfrom an aggregate that exhibited aberrant au-tomaticity in the form of an extrasystole. Depolarizing afterpotentials are seen in thesetraces that attained the action potential threshold in CI (arrow), but not in C2 (arrow).

mV

mV

10080flO4020A3111ill.!0

ó ¿two«cCYCLE

LENGTH (MSEC)

TIME (SEC)

mV

TAKEOfF POTENTIAL

CYCLE LENGTH (MSEC)

1

250

(msec)

500

jiimol). As summarized in Table 2, the quiescent resting membrane potentials of treated aggregates (-63 to -30 mV depend

ing on doxorubicin concentration) fell in a range considerablymore positive than that of the control resting potentials (—61to -75 mV).

DISCUSSION

Aggregates of cultured ventricular cardiomyocytes retainelectrophysiological characteristics similar to intact cardiacmuscle, including an absence of automaticity. Doxorubicin

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ELECTROPHYSIOLOGICAL EFFECTS OF DOXORUBICIN IN THE HEART

Table 2 Depolarizing action of doxorubicinAggregates of chick ventricular heart cells were exposed in culture to various

concentrations of doxorubicin for 24 h. Coverslips containing cultured aggregateswere transferred to an experimental chamber where they were superfused withbubbled (95% O2, 5% CO¡)culture medium at 37°C.Aggregates were impaled

with conventional intracellular microelectrodes, and resting potentials were recorded when aggregates arrested spontaneously. Control resting potentials were-67 ±l mV that ranged from -61 to -75 mV.

Cellno.1

234567Doxorubicin

concentration(/!M)0.1

0.10.31.01.01.01.0RMP*(mV)-59

-63-57-45-38-30-55

°RMP, resting membrane potential.

induced spontaneous firing accompanied by twitch contractionsin cultured aggregates at concentrations lower than thoseknown to produce cytotoxic damage. The effects of doxorubicinwere determined in vitro at concentrations (0.1 to l UM)whichcorrelate well with in vivo levels achievable during the first 24to 48 h following an i.v. bolus or continuous infusion of astandard dose of doxorubicin (26, 27). Thus, in this experimental preparation, clinically relevant concentrations of doxorubicin had a direct effect on the cardiac cells.

The data in Table 1 indicate a depolarizing action of doxorubicin in that the maximum diastolic potential was progressively shifted toward positive membrane potentials with increasing drug concentration. As confirmatory evidence, the restingpotential measured during a pause in spontaneous beating wasmarkedly depolarized in treated aggregates when comparedwith the quiescent resting membrane potential of controls (Table 2). It has been demonstrated that membrane depolarizationby electric current (19) or by hypokalemic solutions (21) caninduce automaticity in quiescent ventricular aggregates. Likewise, the induction of automaticity by doxorubicin after acontinuous exposure of 24 to 48 h could well be the result ofmembrane depolarization.

The action of doxorubicin on automaticity may represent adistinctly different effect of doxorubicin from the destructionof intracellular organelles and myofibrils associated with car-diomyopathy. Doxorubicin, at low concentrations, can producearrhythmias in vitro in the absence of ultrastructural changes(within 48 h) (11). The molar concentrations in the study ofLampidis et al. (see Ref. 11) (0.17 to 0.85 /¿M)are similar tothe range (0.1 to 1 /¿M)used in the present study to induceautomaticity and dysrhythmia in cultured chick aggregates.Much higher (>150 /UM)doxorubicin concentrations causedloss of function (e.g., cessation of beating) within 4 h alongwith the correlated ultrastructural (loss of myofibrils, swellingof mitochondria and sarcoplasmic reticulum, vacuolization, anddisruption of Z-bands) and biochemical changes (e.g., láclatedehydrogenase release from cells) (11,12). Thus, simply on thebasis of the concentration and duration of doxorubicin application, the cultured cardiac cells can be made to exhibit the twomost important aspects of the cardiotoxicity, namely, dysrhythmia and cell death.

The occurrence of dysrhythmia in humans during doxorubicin therapy is recognized. In two prospective studies usingcontinuous Holler monitoring, arrhythymias atlribulable todoxorubicin occurred in 24 lo 30% of Ihe palients within 24 hof administration (7, 28). EKG abnormalities are observed inhumans (6) and experimenlal dogs (5) during Ihe course ofdoxorubicin trealmenl (e.g., premature ventricular beats, non-

sustained ventricular tachyarrhylhmias).Il is possible lhat doxorubicin, by virtue of its depolarizing

action, could cause abnormal automat icily in Ihe inlact heartthrough conversion of latent pacemaker cells into ectopie pacemakers. In this regard, preliminary experiments in dog Purkinjefibers demonslrated lhat doxorubicin (50 /¿mol)causes a gradual membrane depolarization accompanied by markedly enhanced automalicity during a 6-h continuous exposure (29).Rather similar effecls on Iransmembrane polenlial and auto-malicily were produced in cultured aggregates of chick heartcells with considerably lower drug concentrations that wereapplied for a 24- to 48-h period (present results).

The molecular mechanism for the depolarizing action ofdoxorubicin is unknown. Doxorubicin probably does noi di-reclly affecl ionic channels, since LeMarec et al. (30) demon-slraled in cardiac Purkinje fibers the absence of any electro-physiological action of the acute application of 50 I¿Mdoxorubicin. A possible indirect mechanism lhal could give rise lomembrane depolarizalion is ihrough the formation of doxorub-icin-calalyzed free radicals (e.g., Superoxide, hydrogen peroxide, or hydroxyl radicals) (9, 31), which are known lo reacl withmembrane lipids. Doxorubicin could, as a resull, cause a generalmembrane permeabilily change and/or indireclly affecl Iheproperlies of specific ion channels lhat are embedded in the cellmembrane. These membrane aclions of doxorubicin could, inturn, give rise to depolarization.

Besides membrane depolarization, doxorubicin (at relativelyhigh concenlralions) also causes excessive lissue calcium levels,which may contribute to the cardiac toxicily (i.e., loss of func-lion and cell dealh) observed in Ihis and many olher sludies.The underlying mechanism by which doxorubicin dislurbs tissue Ca2+ levels may be through interference in various Ca2+

Iransporl processes. In Ihis regard, doxorubicin is known loinhibit certain Ca2+translocalion mechanisms in vesicular prep-

aralions of cardiac membranes. These include Ihe Na/Ca exchanger in cardiac sarcolemma (32) and Ca-ATPase activity insarcoplasmic reticulum (33). A physiological manifestation ofthe Na/Ca exchanger, which is electrogenic in heart muscle, isthe delayed afterdepolarizalion occurring in cardiac Purkinjefibers in condilions of excessive Ca2+ loading; Binah et al. (34)demonslrated an immediate inhibition of ouabain-induced delayed afterdepolarizations by 50 /¿Mdoxorubicin, suggestingthat this agenl indeed inhibits Na/Ca exchange in inlacl heartmuscle. Azuma et al. (35) reported indirecl evidence that doxorubicin, within minules, causes a Iransient increase in the slowCa2+ inward current of intacl, perfused chick embryo hearts.

Chick cardiac aggregales, because of Iheir small size andfavorable geomelry, have been used as a model system to studyIhe ionic currenls underlying pacemaker behavior (see Refs. 19and 21). Since the present invesligation establishes lhal chickcardiac aggregales exhibit doxorubicin-induced automaticily,dysrhythmia, and depolarization at clinically significant drugconcentralions, il is now possible lo examine Ihe ionic basis ofIhese effecls wilh voilage clamp methods.

ACKNOWLEDGMENTS

The authors thank Pedro Allegre and Tim O'Connell for skillful

technical assistance during the course of this study.

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ELECTROPHYSIOLOGICAL EFFECTS OF DOXORUBICIN IN THE HEART

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