CYTOCHALASIN B AND EMBRYONIC HEART MUSCLE: … · 2017-04-12 · SUMMARY The effec otf cytochalasin...

J. Cell Sd. 14, 163-185 (1974) 163

Printed in Great Britain

CYTOCHALASIN B AND EMBRYONIC HEART

MUSCLE: CONTRACTILITY, EXCITABILITY

AND ULTRASTRUCTURE

H. G. SACHS,* T. F. MCDONALDf AND M. SPRINGER}:Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland2I2IO, U.S.A.

SUMMARYThe effect of cytochalasin B (CB) on beating, electrical activity and cytological ultrastructure

of embryonic hearts and tissue-cultured heart preparations was investigated. The inhibition ofspontaneous pulsation and the subsequent recovery after drug washout were found to be dose-dependent and to be a function of the type of preparation. In the presence of CB, spontaneousaction potentials can be recorded from all three (intact hearts, isolated myocytes, reaggregatedcells) after cessation of all visible contractions, although the action potential duration is reduced.CB at 0-5-10 /ig/ml was found not to uncouple heart cells electrically nor to prevent cells fromaggregating and establishing new electrical coupling. In all 3 systems, however, CB does resultin disruption of myofibrillar organization. After 30 h in CB (2 /ig/ml) bundles of myofiJamentsare found to be randomly oriented, with Z-band material condensed into amorphous bodies, nolonger aligned with the myofibril, but remaining in register between the A-bands. Within thebundles the normal hexagonal cross-section of thick and thin filaments is maintained. Theunaligned Z-bodies, however, demonstrate attached thin filaments of very reduced length.Following washout of the drug, normal sarcomeric organization is restored in parallel -withresumption of spontaneous beating. The present data, therefore, are consistent with the hypo-thesis that one major effect of CB is an alteration in myofibril (myofilament) stability, and thatthis effect alone is sufficient to account for the observed inhibition of contractility.

INTRODUCTION

Cytochalasin B (CB), a fungal metabolite, has been reported to interfere with con-tractile events in a number of cell types (Wessells et al. 1971)- Considerable controversyexists concerning the mode of action of the drug, and indeed the cell processes itaffects (Burnside & Manasek, 1972; Holtzer & Sanger, 1972). A case in point is theeffect of CB on the contractility of embryonic heart cells. Wessells et al. (1971) reportedthat cultured heart cells stopped beating in the presence of CB, but Sanger & Holtzer(1972 a, b) maintained that cells continued to beat vigorously for as long as 3 days. Inthe only study on the intact embryonic heart, Manasek, Burnside & Stroman (1972)have reported that CB completely inhibits contraction. In reconciling these reports, oneis faced with several experimental variables which have not been adequately considered:

• Present address and address for correspondence: Department of Anatomy, College ofMedicine, University of Illinois at the Medical Center, Chicago, Illinois 60680, U.S.A.

t Present address: II Physiologisches Institut, Universitat des Saarlandes, 6650 Homburg/Saar, Germany.

% Present address: Anatomisches Institut der Universitat Zurich, Gloriastrasse 19, CH-8006Zurich, Switzerland.

164 H. G. Sachs, T. F. McDonald and M. Springer

(1) the doses of CB employed and the duration of exposure varied considerably; (2) thepreparations were obtained from embryos of different ages and may reflect develop-mental differences; (3) the data from various preparations may not be directly com-parable because of differences in the behaviour of heart cells in the intact tissue, andheart cells isolated in culture.

The present light-microscopic, electron-microscopic and electrophysiological studywas undertaken in an effort to clarify the effects of CB on embryonic chick heart muscle,using intact hearts, isolated tissue-cultured myocytes, and reaggregated heart cells.

MATERIALS AND METHODS

Hearts, heart cells and heart cell aggregates

Fertilized eggs of White Leghorn chickens were incubated at 37 °C for from 1-5 to 7 days.Embryos were aseptically removed from the eggs, decapitated and hearts dissected out in a poolof amniotic fluid. Heart cell cultures were prepared by standard techniques (DeHaan, 1970).Heart cell aggregates were prepared by slight variation of the techniques of Moscona (1961)from single heart cells isolated by the multiple-cycle trypsinization procedure (DeHaan, 1970).Details have been provided elsewhere (DeHaan & Sachs, 1972).

Hearts in culture were maintained under a water-saturated atmosphere of 40% OB: 5%COa:55% Nj at 37 CC. Intact 3-day hearts were removed from the embryo, while youngerhearts (11-14 somites) were observed following explanting of the whole embryo in culture (New,1955) and removal of the splanchnopleure. Cells and aggregates were incubated under a water-saturated atmosphere of 10 % O%:$ % CO8: 85 % N, at 37 °C.

Beating activity

Single cells. Single isolated cells were observed in randomly selected fields on each plate andscored as beating or not beating following a 10-s observation period at 160 x , using an invertedphase-contrast microscope.

Aggregates. Individual aggregates on the plate (100-200 per plate) were observed and scoredas beating or not beating following a 10-s observation period at either 63 or 160 x .

Intact hearts. Individual hearts were observed for 10 s at 30 x , and scored as beating if eitherthe atrial or ventricular region was contracting.

Electrophysiology

Hearts were pinned to a paraffin base in a 35-mm Falcon tissue culture dish; cells and aggre-gates readily attached to the unmodified plastic surface of Falcon culture dishes. These disheswere placed on a warm stage (37 °C) of an inverted phase-contrast microscope, and a gassingring directed a stream of 10% O s : io% COj>:8o% N, over the dish. Mineral oil was layeredover the medium to prevent evaporation (DeHaan & Gottlieb, 1968).

Transmembrane potentials were recorded using micropipette electrodes prepared by thetechnique of Tasaki et al. (1968). The intracellular pipettes were filled with 2 M KC1 and hadresistances of 50-100 MO with tip potentials less than 7 mV. The low-resistance extracellularpipettes were filled with balanced salt solution. Electrode holders (Ag/AgCl; W. P. Instrumentsand Bioelectric Instruments) were carried on Leitz micromanipulators. Signals were measuredusing a Picometric electrometer and a Tektronix storage oscilloscope. Overshoots, maximumnegative potentials and action potential durations (measured from beginning of the upstroke)were obtained from slow sweep-speed records. The maximum rate of rise was determined fromfast sweep-speed records.

Cytochalasin B and embryonic heart 165

Electron microscopy

Single cells and aggregates were fixed in the Falcon plastic dishes in cold 0-2 M cacodylate-buffered 2 % glutaraldehyde at pH 7-4 for 2-12 h, rinsed in buffer, and postfixed in 0 1 Mcacodylate-buffered 1 % OsO4 for 60 min. Following a distilled water rinse the tissues werestained for 12-20 h in 0-5 % aqueous uranyl acetate, dehydrated in a graded ethanol series andembedded in Epon 812. After polymerization at 37-5 °C, the Epon plate was separated from theFalcon plastic dish. Selected areas of the Epon plate were mounted on Lucite rods and weresectioned parallel to the surface. For identification, numbered circles were slightly engraved intothe Falcon plastic dish around selected cells or aggregates. The engraved pattern was thus raisedfrom the surface of the Epon plate after separation from the Falcon plastic dish and allowed rapidand accurate identification of the cells after fixation and embedding. The whole hearts wereprocessed similarly, but treated with propylene oxide after dehydration and embedded in plasticcapsules.

Serial sections were mounted on Formvar-coated 1 x 2 mm single-hole grids. The sectionswere stained in lead citrate (Venable & Coggeshall, 1965) and examined at 75 kV with a HitachiHU-11E-1 electron microscope.

MediaAggregates and cells were incubated in medium 8i8A! (DeHaan, 1970). This medium,

containing 1-3 rain K+, was formulated as a physiological medium to maximize the percentage ofspontaneously active cells (DeHaan, 1970), and consists of 2 0 % medium 199 (GIBCO), 7 4 %potassium-free balanced salt solution, 2 % horse serum (Colorado Serum Co.), and 4 % foetalcalf serum (GIBCO) previously dialysed against potassium-free balanced salt solution. Penicillin(100 units/ml) and streptomycin (50 fig/mi) were also present. The composition of the balancedsalt solution was (ITIM): NaCl, 1160; MgSO4.7HsO, o-8; NaH,PO4.H,O, 0 9 ; CaCl8.2HjO,i -8; NaHCO3, 26-2; and glucose, 5 5 . A number of experiments were repeated in medium with4 5 mM K+. No differences in CB action were noted from experiments with the mediumcontaining the lower concentration of K+, and the data are pooled. Hearts were cultured in amedium consisting of 50 % medium 199, 50 % potassium-free balanced salt solution with peni-cillin (100 units/ml) and streptomycin (50/ig/ml). Glucose-deficient medium, containing lessthan C2 mM glucose, was prepared with 2 % foetal calf serum, glucose-free Mi 99, and glucose-free, sucrose-substituted balanced salt solution.

Drugs

The volume of medium in the tissue culture dish was 2 ml and the volume of drug solutionadded was usually 20 fil. Cytochalasin B (Imperial Chemical Industries) was made up as a stocksolution of 1 mg/ml in dimethylsulphoxide (DMSO). Two different lots of CB were used; thefirst was obtained from Dr D. M. Fambrough whose stock was a gift from Dr H. Holtzer, andthe second was obtained directly from ICI. No difference was noted between the 2 stocks ineither potency or effect.

RESULTS

Spontaneous beating: inhibition and reversibility

Intact hearts. Intact hearts from 3-day embryonic chicks were organ cultured andtreated with CB 2 /£g/ml for 30 h or CB 10 /^g/ml for 12 h (Table 1 A). Other heartsserved as control or were incubated in medium containing DMSO 10/il/ml. Spon-taneous beating and contractile strength were well maintained in the control andDMSO-treated hearts. With 2 /ig/ml CB there was a gradual decrease in contractilestrength and by 15 h beating was barely perceptible. Nevertheless, 7 of 8 hearts werestill beating, while by 24 h only 3 were beating and by 30 h none was beating. With


Table i. The effect of cytochalasin B on the spontaneous beating activity of 2-dayembryonic chick hearts: number of hearts beating, relative contractile strength

A. Incubation with cytochalasin BTime, h . . . o 6 9 12 15 24 30

Control

CB 2 fig/ml

8/8

8/84-4-4-4-

CB io/ig/ml 8/8

B. RecoveryTime, min ...

8/8

8/8

8/8

After CB 2 fig/ml for 30 h

After CB 10 ytg/ml for 12 h

8/8

8/8

8/8

0

0/8

0/8

8/8

8/8

0/8

15

6/8

2/8

8/8

7/8

3°

7/8

7/8

8/8

3/8

8/8

0/8

90

8/8

7/8

Each group contained 8 hearts and the ratio of beating hearts to total is given in each case.The relative contractile strength of the beating hearts is denoted below the ratio where(+ + + +) indicates vigorous and ( + ) barely perceptible contractions. A, all hearts wereequilibrated for 2 h before the addition of CB 2 or io/tg/ml. B, recovery from 30 h with CB2 yttg/ml or 12 h with 10 fig/ml. Culture plates containing the hearts were washed 3 times withfresh medium.

CB the contractile strength declined more rapidly than at the lower dose andwithin 12 h all 8 hearts had stopped beating. The recovery of beating and of contractilestrength was more complete in the hearts incubated for 30 h with 2 /^g/ml than inthose incubated for 12 h in 10 /igjm\ (Table 1 B).

Manasek et al. (1972) have reported that younger embryonic hearts (11-13 somites)were more sensitive to CB inhibition of beating than older hearts (22-28 somites).Young embryonic hearts (11-14 somites) were incubated in the present study withCB 2/ig/ml or 10/ig/ml. At the lower dose all hearts (n = 6) stopped beating after7-12 h, while at 10 /tg/ml the hearts (n = 3) stopped within 3 h. Control hearts (n = 8)and hearts treated with DM SO io/^g/ml (n = 3) were beating vigorously after24 h. We conclude that the young hearts were more sensitive to CB than the 3-dayhearts (see Table 1).

It should be noted that while CB-treated hearts often exhibited various degrees ofA-V block before cessation of beating, the contractile event was usually quite synchron-ous in either the atrial or ventricular regions.

Tissue-cultured heart cells. Tissue-cultured heart cells from 4- or 7-day embryonichearts were treated with CB for 24 h. Drug treatment commenced 20—24 h aftertrypsinization and plating. The responses of 4- and 7-day cells were similar and thedata were pooled. Fig. IA presents the normalized percentages of single isolatedbeating cells during treatment with CB 0-5, 1, or 2/tg/ml. Cessation of beating wasdose-dependent; after 4 h in CB o-$fig/ml about 50% of the cells were inhibited,


100

0 s -

30 45Time, min

Fig. 1. The spontaneous beating activity of isolated single heart cells during incubationwith CB, and subsequent recovery. The data from 4- and 7-day cells were pooled andnormalized to 100 %; the mean percentage of beating cells at o h was 63 %. A, effect ofCB 0-5 /ig/ml (A), 1 /ig/ml (A), and 2/ig/ml (D) I control (O) and DMSO 10/il/ml ( • ) .B, recovery after 24 h in CB; all plates were washed 3 times with fresh medium. Dataare mean values from 2-6 plates of 2-4 separate cultures. Between 75 and 200 cells/plate were observed depending on the temporal resolution required. The time requiredfor these counts was 3-10 min and the placement of points with regard to the time axisrefers to the median of the observation point. The median (about 2 min) of the firstobservation point is denoted as o min.

while in 2 /Jg/ml more than 75 % were inhibited. After 24 h, only 3 % of the cells werebeating in CB 0-5 /Jg/ml and no beating cells were observed in CB 1 or 2 /ig/ml. In thecontrols (normal medium, and normal medium with DMSO 10 /il/ml) beating activitywas well maintained throughout the observation period.

The recovery of beating activity after 24 h of CB was quite rapid, with about 50 %recovery within 30 min of drug washout and 75 % recovery within 75 min (Fig. I B ) ;2 h later more than 90 % of the cells had resumed beating. The recovery periodrequired was dependent upon the previous dose of CB; cells which had been exposedto 0-5 /tg/ml recovered faster than those exposed to 2 /ig/ml.

Cells treated with CB 10 /^g/ml (4 plates, n = 550) were completely inhibited within8 h. After an additional 18 h in CB, they were washed thoroughly (5 times) but werestill quiescent 1 h later, in marked contrast to those recovering from CB 2 /tg/ml.Despite 3 more washes at 2-h intervals, only 3 % were beating after 8 h of recovery,compared with 103 % in control plates (values normalized, 100% = % beating cellsbefore CB). Thereafter, there was a gradual resumption of beating activity, with 12 %beating cells after 24 h of recovery, 36% after 42 h, and 90% after 60 h. This repre-sents almost complete recovery, since beating activity in the control plate at 60 hwas 93 %.

Heart cell aggregates. Aggregates formed from 4- or 7-day heart cells were similarlytreated with CB for 30 h. Drug treatment commenced 20—24 n after trypsinization

i68 H. G. Sachs, T. F. McDonald and M. Springer

100

2 60 -

Fig. 2. The spontaneous beating activity of heart cell aggregates during incubation withCB, and subsequent recovery. The data from 4- and 7-day aggregates were pooled, A,effect of CB 0-5 fig/ml (A), 2 /ig/ml ( • ) and 10 /Jg/ml ( • ) ; DMSO 10 /il/ml was notdistinguishable from control ( • ) . B, recovery after 30 h in CB; all plates were washed3 times with fresh medium. In addition, aggregates recovering from CB io/^g/mlwere again washed 3 times after 1 h of recovery. Note the break in time axis. Data aremean values from 4-6 plates of 2-3 separate cultures.

of the intact heart. The responses of 4- and 7-day aggregates were similar and the datawere pooled. Fig. 2 A shows the effect of CB 0-5, 2, and io/^g/ml on the percentage(absolute) of beating aggregates. As with the single cells, the inhibition of beating wasdose-dependent. At 0-5 /tg/ml, CB had little effect, at 2 /tg/ml only 2% of the aggre-gates were beating after 30 h, while at io//g/ml all beating had ceased within 24 b.Recovery from CB after thorough washing occurred more rapidly in aggregates thanin cells but was also dose-dependent. After 30 min nearly 90 % of the aggregates treatedwith CB 2 /tg/ml had resumed beating, but even after 1 h only 5 % of the aggregatestreated with CB io/(g/ml were active (Fig. 2 B). However, during the 6th hour ofrecovery from CB io/<g/ml the beating percentage inexplicably increased from 15 %to 89%.

Inhibition of spontaneous pulsation might result from a reduction in the uptake ofglucose. To determine whether the CB effects on beating could be accounted for byreduced glucose uptake, 7-day aggregates were incubated for 30 h in glucose-deficientmedium (see Methods). Although there was a decline in pulsation rate of approximately20 %, more than 98 % of the aggregates remained beating.

Aggregate morphology in cytochalasin B

The morphology of CB-treated aggregates was markedly different from that of thecontrols and was dose-dependent. Fig. 3 A shows the appearance of a typical controlaggregate 3 h after plating in a Falcon dish (approximately 24 h post-trypsinization).The spheroidal heart cell mass is surrounded by a ring of fibroblast-like cells which haveemerged from the aggregate. Twenty-four hours later the appearance of the control


aggregates is characterized by a flatter central core with an increased number offibre-blasts radiating outwards (Fig. 3B). Unpublished observations indicate that thevast majority of these fibroblasts have emerged from the aggregate. After 24 h inCB 2 /tg/ml (Fig. 3c) or CB 10/tg/ml (Fig. 3D) the fibroblast outgrowth is no largerthan that seen immediately before CB. At 2 /tg/ml the fibroblast cells assume a flatrectangular appearance and remain in close contact with the aggregate, but at 10 /<g/mlthey separate from the aggregate, round up and, in many cases, extend narrowprocesses outward from the central region of the cell, a process termed 'arborization'by Sanger & Holtzer (1972a).

The inhibition of fibroblast emergence from the aggregates may be related to theknown inhibitory effect of CB on cell locomotion (Carter, 1967). Recently, it has beenreported that CB prevents the sorting out of aggregated neural retina and heart cells(Maslow & Mayhew, 1972) and heart and liver cells (Steinberg & Wiseman, 1972).Control aggregates of cells from embryonic hearts display sorting out of ' myocytes'and 'fibroblasts'. The vast majority of cells without myofibrils (as determined byelectron microscopy of aggregate cross-sections at 26000 x ) are found in a 1- to 3-cell-thick layer around the outer perimeter of the aggregate (Sachs & DeHaan, 1973).Aggregates formed during 24 h in the presence of CB 2 /tg/ml had similar proportion ofnon-myofibril-containing cells as control aggregates. Counts along the 2 axes of across-section of a control aggregate showed that 21% of the cells did not containmyofibrils (n = 49 cells), while in a CB-treated aggregate 19% were non-muscle cells(n = 71). However, the control and CB-treated aggregates differed in the distributionof such cells; the majority of the outermost cells around the perimeter of controlaggregates were non-myocytes (69%, n = 44 cells), while the proportion of non-myocytes in the perimeter of aggregates formed in CB was comparable to that foundin the interior (17%, n = 54 cells). Thus, CB would appear to have prevented thesorting out of non-myocyte heart cells ('fibroblasts') and myocytes during aggregation.

Excitability in the presence of cytochalasin B

An inhibition of spontaneous action potential generation by CB could account forthe cessation of beating activity. To determine whether CB blocked excitability, intact3-day hearts, tissue-cultured cells from 7-day hearts and aggregates of cells from 7-dayhearts were treated with CB 2 /tg/ml for 30 h. All preparations had ceased beating, butelectrophysiological recordings from the 3 preparations showed typical spontaneousaction potentials (Fig. 4). Spontaneous action potentials were also recorded from 7-dayaggregates treated for 30 h with CB 10 /tg/ml and from 2-5-day aggregates after 30 hwith CB 2 /tg/ml.

It has already been noted that before hearts stopped beating in CB there was noevidence of asynchronous beating. In order to investigate whether cells in an aggregatemaintained electrical coupling (Sachs & DeHaan, 1973) in the presence of CB, 7-dayaggregates were incubated with CB 2 /^g/ml for 6 days. After 6 days, the aggregatemorphology was similar to that seen after CB 10/fg/ml for 24 h (Fig. 3D). Thefibroblasts were separated from the aggregate and many were 'arborized'. Impale-ments with 2 microelectrodes separated by about 200 /tm in aggregates of about 250 /tm


Table 2. Action potential parameters in j-day aggregates

MDP (mV) OS (mV) V^ (V s"1) APD (ms)

Control (n = 3s) -8 i ' 9± i -2 27-2±cc6 n8-3±5-3 i6s-3±2-5CB 2 /ig/ml, 30 h (n = 39) — 8o-2±i-2 26-5 ±o-6 139-5 ±8-2* n6-4±2-6**

MDP = maximum diastolic potenrial (or maximum negative potential); OS = overshoot(or maximum positive potential): F ^ = maximum rate of rise of the action potential;APD = duration of the action potential measured with respect to the membrane potential at thebeginning of the upstroke. Values are mean ± S.E. • Significantly different from control,P < 0-05. *• Significantly different from control, P < o-oi.

diameter showed that the action potentials and pacemaker potentials were essentiallysimultaneous (Fig. 5). This provides strong evidence of the maintenance of electricalcoupling even after prolonged incubation with CB.

We further wished to determine whether CB would interfere with aggregate forma-tion and establishment of electrical coupling between cells. Aggregates formed bygyration of isolated cells in the presence of CB 2 /ig/ml were often larger than controls,as has been noted by Steinberg & Wiseman (1972), but had a noticeably looser, morecellular appearance. When plated in Falcon tissue culture dishes they generally failedto attach to the plastic surface, except for some small aggregates which attached poorly.This behaviour may reflect the sparsity of fibroblasts at the exterior, since these cellsattach to the plastic surface more readily than myocytes, and provide an ' anchor' forthe aggregate. Impalements of these aggregates indicated that the ability to generatespontaneous action potentials was not impaired by aggregation in the presence of thedrug. In addition, electrical coupling between widely separated cells in the aggregatewas noted. Thus, formation of nexal-like junctions during aggregation, the probableroutes of coupling (see DeHaan & Sachs (1972) for discussion), is presumed to beunimpaired by CB treatment. Typical gap junctions were seen in micrographs ofaggregates formed in the presence of CB. When aggregates formed in CB were washedby gentle centrifugation and resuspended in fresh medium they attached rapidly tothe plastic dishes and resumed beating within a few hours.

CB treatment did not prevent electrical activity but did cause noticeable changes insome parameters of the action potential, particularly the plateau region. The effect ofCB 2 /ig/ml for 30 h on action potential parameters is presented in Table 2. There wasno significant change in either the maximum diastolic potential (MDP) or overshoot(OS) with drug treatment. However, the mean action potential duration (APD)decreased from 165 to 116 ms, and in additional experiments this decrease was fullyapparent within 15 min of incubation with CB. The increased rate of rise of the actionpotential {Vm&x) was seen in only 2 of the 4 experiments, and the data are pooled inTable 2.

Ultrastructure

Previous electron-microscopic studies (Manasek et al. 1972; Wessells et al. 1971)on heart tissue demonstrated that CB disrupted myofibrillar organization. However,


such studies failed to establish whether there was degradation of filaments or disrup-tion of filament assemblies.

Controls. A total of 10 identified beating cells in 2 different cultures were completelyserially sectioned parallel to the dish surface. After 24 h in culture, such cells werefound to have varying degrees of myofibrillar organization. Figs. 6 and 7 representtypical cells within this range. In cross-section, the myofibrils were arranged in thecharacteristic hexagonal array of thick and thin filaments.

The degree of myofibrillar organization in the heart cells reassociated into aggre-gates was generally greater than that of single isolated cells. The myofibril bundleswere thicker, the Z-material was more compact and the A- and I-bands were easilyresolved (Fig. 8), but in general there was no apparent preferential orientation of themyofibrils. A total of 10 identified beating control aggregates from 5 cultures wereexamined at 3 different levels of sectioning within each aggregate. The organizationseen in 7-day aggregates was comparable to that of the intact 3-day heart (1 control),although in the latter there was noticeable pericircumferential orientation of themyofibrils about the tubular heart, in agreement with the observations of Manasek(1968).

Multiple sections of 2 cells and 2 aggregates incubated with DM SO (io/jg/ml) for30 h were examined, and showed no change in ultrastructure.

CB-treated material. Three hours after plating, heart cell aggregates were exposed toCB (2 or io/^g/ml) for up to 30 h. Aggregates which had stopped beating wereprocessed for electron microscopy (n = 8). After 6 h in CB 2 /ig/ml, the myofibrilswere well aligned, but the Z-material was somewhat diffuse and no longer formed acompact band. Following 30 h of CB treatment the majority of the myofibrils weredisrupted, appearing as randomly oriented filament bundles of approximately sarco-mere length, often lacking Z-bands. Other myofibrils, while interrupted at the Z-band,still maintained alignment of sarcomeres (Fig. 9). The myofibrils retained thecharacteristic hexagonal array of thick and thin filaments in cross-section (inset, Fig. 9).The Z-material was seen as amorphous bodies of high electron density, frequentlyin register, but usually out of alignment with the myofilament bundles. Myofibrillardisruption in aggregates treated with CB 10 /tg/ml for 30 h was similar to that at 2 /*g/ml,but the amorphous Z-material, hereafter referred to as 'Z-bodies', was distinctlyout of alignment with the myofibrils. These Z-bodies consisted of a dense central zonewith thin filaments (6 nm diameter) extending bilaterally outward, and should not beconfused with the amorphous normal precursors of Z-bands identified in developingmuscle cells by Heuson-Stiennon (1965) and also termed 'Z-bodies'.

An important observation was that the degree of myofibrillar disruption in theoutermost muscle cells of the aggregates was indistinguishable from that of theinnermost cells, both after 6 h in CB 2 /ig/ml and after 30 h in CB either 2 or 10 /tg/ml.

Heart cells cultured for 24 h after trypsinization were then treated with CB 2 /ig/mlfor 24 h. Identified cells which stopped beating during incubation with the drug werefixed for electron microscopy. A total of 8 identified cells were serially sectioned.Myofibrillar disruption was similar to that seen in the aggregates, but in some sectionsparallel to the myofilament bundles there appeared to be no associated Z-bodies


(Fig. 11 A). However, in adjacent serial sections, the Z-bodies were often found in regis-ter but out of alignment with the bundle (Figs. 11 B, C). In addition, apparently normalthin filaments running parallel to the thick filaments but without attachment toZ-bodies are clearly demonstrated.

Three-day hearts which had ceased beating during incubation with CB 2 /ig/ml for30 h (n = 2) or 10 /tg/ml for 12 h (n = 2) were examined, and the degree of myofibril-lar disruption was similar to that observed in CB-treated cells and aggregates. In allcases, the cessation of beating was correlated with the appearance of a disruptedcontractile apparatus. While the isolated cells and those that had been reaggregated hadbeen exposed to trypsin in the process of culturing, the hearts had not been exposed tothe proteolytic treatment. The myofibrillar disruption, therefore, must be presumedto be due to CB action, and not to some aspect of the culturing procedures. DMSO(10/tl/ml, 30 h) did not produce any disruption.

With the exception of the myofibrillar disorganization the cytoplasm of CB-treatedheart preparations appeared normal. The cristae and matrix of the mitochondrion wereindistinguishable from those of controls. In addition, neither desmosomes nor gapjunctions in the aggregates and intact hearts appeared to have been affected by thedrug.

Leptomeric organelles (Thoenes & Ruska, i960) were found in normal and treatedcells, aggregates and hearts. The dense bands of these organelles had a periodicity ofapproximately 0-16 /tm, in agreement with the published values of 0-19 /im (Karlsson& Anderson-Cedergren, 1968) and 0-14 /tm (Viragh & Challice, 1969). These organel-les had the same appearance in control and CB-treated muscle. Fig. 12 shows, forexample, that an aggregate treated with CB 2 /«g/ml for 30 h shows normal leptomericorganelles, in marked contrast to the disrupted myofibrils.

The recovery of beating following prolonged incubation with CB 2 /ig/ml was rapidin cells, aggregates and hearts. Identified isolated cells which had stopped beatingduring 24 h of incubation with CB 2 /ig/ml and then resumed beating following drugwashout, were fixed either 45 min (n = 8) or 2-5 h (n = 8) after washout. After 45 minof recovery, Z-bodies were realigned with the myofibril bundles and myofibrils ofseveral sarcomeres in length were again apparent (Fig. 13). The Z-bands of thesemyofibrils, however, were still quite diffuse and often did not extend completely acrossthe myofibril bundle. After 2-5 h of recovery, there was complete reformation andrealignment of the myofibrils. Z-bands were now complete and compact, and freeZ-bodies were not seen (Fig. 14).

Similar ultrastructural reorganization accompanied the recovery of beating inaggregates (n = 8) and hearts (« = 3) following washout of CB 2 /Jg/ml. After 30 h withCB 10 /tg/ml and 45 min of recovery the myofibrillar organization of identified aggre-gates which had resumed beating (n = 2) was similar to that shown in Fig. 13. In con-trast, myofibrillar disarray remained pronounced in aggregates which were stillquiescent after 2 h of recovery (w = 8).


DISCUSSION

Cytochalasin B completely inhibited the spontaneous contractile activity of intactembryonic hearts, isolated single heart cells, and reaggregated heart cells. The exposuretime necessary for complete inhibition was dose-dependent and, in the case of intacthearts and aggregates, sensitivity was inversely related to age. This inhibition couldresult from a block of excitation, uncoupling of excitation from contraction or dis-ruption of the contractile apparatus. The ability to generate action potentials was notinhibited, and the spontaneous frequency was similar to that in controls. The drug did,however, decrease the duration of the action potential. There was significant disruptionof the myofibrillar structure with CB treatment, and this alone would be sufficientto block contraction. We have no evidence concerning any effect of CB on excitation-contraction coupling.

On a comparative basis, single isolated cells were more susceptible to CB than werereaggregated cells or the intact hearts. This may in part be due to the state of organi-zation of the myofibrils before drug treatment. Both the single cells and the reaggre-gated cells were initially derived from intact hearts by proteolytic enzyme treatment.Freshly trypsinized cells have been shown to have greatly disrupted myofibrils and, inmany cases, dissociated myofilament bundles (Fischman & Moscona, 1971; M.Springer, unpublished observations). Both the isolated ceils and aggregates used in thepresent study had between 18 and 24 h of recovery from the trauma of preparationbefore exposure to CB, a period sufficient to allow considerable myofibrillar recovery.

Comparison of the myofibrillar organization in control single cells and aggregatessuggests that cell-to-cell contact, as in aggregates, may be important in the reassemblyof trypsin-disrupted myofibrils. Indeed, this contact would also seem to be importantin the reassembly of CB-disrupted myofibrils, as evidenced by the slower recovery ofbeating in single cells exposed to CB io/ig/ml for 24 h compared with the recoveryof aggregates after exposure for 30 h. In this regard, it had been shown that reaggre-gated heart cells can repair or resynthesize tetrodotoxin-sensitive membrane alteredduring trypsin treatment or dissociation, while isolated cultured cells seem unable todo so (Sachs, McDonald & DeHaan, 1973).

The removal or alteration of Z-bands has been reported following urea treatment(Rash, Shay & Biesele, 1968), during the post-mortem period (Henderson, Goll &Stromer, 1970), following the action of a specific endogenous protein fraction (Busch,Stromer, Goll & Suzuki, 1972), or following severing of the attached tendon (Shafiq,Gorycki, Asiedu & Milhorat, 1969). None of these conditions results in the disrup-tion of myofibrillar alignment, I-bands are not removed, and only the Z-bands aregrossly affected. Embryonic heart cells in mitosis are characterized by the disarray ofmyofibrils and virtual absence of Z-material (Hay & Low, 1972). However, in mitoticcells 'actin filaments still project into the I-band for a distance that approximatesthe position of the Z-band' (Hay & Low, 1972), a configuration not observed in CB-treated muscle. Holtzer's group (Holtzer & Sanger, 1972; Holtzer, Sanger, Ishikawa& Strahs, 1972) has recently suggested that the disruption of myofibrillar organizationproduced by colcemid is identical with that produced by CB.

174 H- G- Sachs, T. F. McDonald and M. Springer

CB may affect metabolism by interfering with sugar transport. The drug has beenreported to block the uptake of deoxyglucose by chick embryo fibroblasts (Kletzien,Perdue & Springer, 1972), by rat hepatoma cells (Estensen & Plagemann, 1972), andby leukocytes and L-cell fibroblasts (Zigmond & Hirsch, 1972). In the latter case, CBreduced lactate production in the presence of exogenous glucose but not in glucose-free medium, suggesting that the glycolytic pathway was intact. In addition, Sanger &Holtzer (1972*) found a decrease in total HeLa cell glycogen content with CB. Ourresults demonstrating aggregate beating in glucose-deficient medium suggests that theinfluence of CB on beating cannot be accounted for by reduced glucose uptake. In thisconnexion it is worth noting that the inhibition of membrane ruffling and other relatedactivities induced by CB similarly cannot be accounted for by a reduced sugartransport (Yamada & Wessells, 1973).

The molecular mechanisms of action of CB are still unknown, although this is anactive area of investigation. Results presented in this study indicate that the thinfilaments and Z-material are altered by the action of the drug, and there is some sup-port for these findings. Forer, Emmerson & Behnke (1972) have reported that CB doesnot inhibit the binding of heavy meromyosin to F-actin, and this is consistent with ourelectron-microscopic demonstration of intact hexagonal arrays, suggesting unalteredbridging. On the other hand, Spudich & Lin (1972) suggest that CB acts on actin eitherby 'partial depolymerization, or by an increase in the width of the filaments, or. . .adecreased rigidity of the actin rod.' More recently Spudich (1972) has stated that CBdoes not inhibit G-actin polymerization, but does alter the morphology of actinfilaments seen in the electron microscope; in particular, the filaments often appearshorter when incubated in the presence of CB.

We propose that during recovery from CB, the Z-bodies realign with the bundles ofthick and thin filaments. Coincident with this realignment there is a disappearance offree Z-bodies. The reformation of intact myofibrils necessary for beating would requirerejoining of the thin filaments of the Z-body with those of the A-band. The foregoingassumes that Z-band region in the reconstructed myofibrils results from the realign-ment of existing Z-bodies with the adjacent myofibrils bundles rather than de novoZ-band synthesis. Although we cannot definitely rule out the latter possibility, theappearance of the myofibrils in the early stages of reorganization (Figs. 13, 14), as wellas the virtual absence of ectopic Z-bodies at 2 h after drug washout, argue stronglyagainst it.

Leptomeric organelles (Thoenes & Ruska, i960; Ovalle, 1972) were found in bothcontrol and CB-treated cells. The periodic dense bands of these organelles have beendescribed as Z-like by Karlsson & Anderson-Cedergren (1968), and in some caseshave been shown to be continuous with the Z-band of the myofibril (Viragh &Challice, 1969). They were not displaced nor did they appear to be structurally alteredby CB treatment.

Recently Lieberman, Manasek, Sawanobori & Johnson (1973) have presented theirresults on the effects of 10-40 /tg/ml CB on artificial strands of embryonic myocardialcells. Our results are not in agreement on several points. They reported an almostimmediate decrease in the rate of rise of the action potential (VmSLX) concommitant with


an increased APD, in contrast to our findings at lower doses of no change in I^ax,and a decreased APD. Furthermore, they suggest that only after excitation ceases dothey see a block of contractility. For want of any more obvious explanation, we mustconclude that the high doses of CB they employed prevented them from seeing themore subtle changes we have detailed here.

We thank Ms W. Asch and Mr W. Duncan for unfailing technical assistance, and Dr R. L.DeHaan for advice and encouragement. We also thank Dr John E. Rash for most helpful criti-cism of drafts of this manuscript. Fellowships from the Carnegie Institution of Washington(H. G. S. and T. McD.), the Canadian Heart Association (T. McD.) and the Canton ofZurich (M. S.) are gratefully acknowledged.

REFERENCES

BURNSIDE, B. & MANASEK, F. J. (1972). Cytochalasin B: problems in interpreting its effects oncells. Devi Biol. 27, 443-444.

BUSCH, W. A., STROMER, M. H., GOLL, D. E. & SUZUKI, A. (1972). Cat+-specific removal ofZ lines from rabbit skeletal muscle. J. Cell Biol. 52, 367-381.

CARTER, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature, Land. 213, 261-264.DEHAAN, R. L. (1970). The potassium sensitivity of isolated embryonic heart cells increases

with development. Devi Biol. 23, 226-240.DEHAAN, R. L. & GOTTLIEB, S. H. (1968). The electrical activity of embryonic chick heart cells

isolated in tissue culture as singlets or in inter-connected cell sheets. J. gen. Physiol. 52,643-665.

DEHAAN, R. L. & SACHS, H. G. (1972). Cell coupling in developing systems: the heart cellparadigm. In Current Topics in Developmental Biology (ed. A. A. Moscona & A. Monroy),pp. 193-228. New York: Academic Press.

ESTENSEN, R. D. & PLAGEMANN, P. G. W. (1972). Cytochalasin B: inhibition of glucose andglucosamine transports. Proc. natn. Acad. Sci. U.S.A. 69, 1430-1434.

FISCHMAN, D. & MOSCONA, A. A. (1971). Reconstruction of heart tissue from suspensions ofembryonic myocardial cells; ultrastructural studies on dispersed and reagggregated cells. InCardiac Hypertrophy (ed. N. Alpert), pp. 125-139. New York: Academic Press.

FORER, A., EMMERSEN, J. & BEHNKE, O. (1972). Cytochalasin B: does it affect actin-like fila-ments? Science, N.Y. 175, 774-776.

HAY, D. A. & Low, F. N. (1972). The fine structure of progressive stages of myocardial mitosisin chick embryos. Am. J. Anat. 134, 175-202.

HENDERSON, D. W., GOLL, D. E. & STROMER, M. H. (1970). A comparison of shortening andZ-line degradation in post mortem bovine, porcine and rabbit muscle. Am. J. Anat. 128, 117—136.

HEUSON-STIENNON, J. A. (1965). Morphog6nese de la cellule musculaire striee etudiee aumicroscope electronique. J. Microscopie 4, 657-678.

HOLTZER, H. & SANGER, J. W. (1972). Cytochalasin B: microfilaments, cell movement and whatelse? Devi Biol. 27, 444-446.

HOLTZER, H., SANGER, J. W., ISHIKAWA, H. & STRAHS, K. (1972). Selected topics in skeletalmyogenesis. Cold Spring Harbor Symp. quant. Biol. 37, 549-566.

KARLSSON, U. & ANDERSON-CEDERGREN, E. (1968). Small leptomeric organelles in intrafusalmuscle fibers of the frog as revealed by electron microscopy. J. Ultrastruct. Res. 23, 417-426.

KLETZLEN, R. F., PERDUE, J. F. & SPRINGER, A. (1972). Cytochalasin A and B. Inhibition ofsugar uptake in cultured cells. J. biol. chem. 247, 2964-2966.

LIEBERMAN, M., MANASEK, F. J., SAWANOBORI, T. & JOHNSON, E. A. (1973). Cytochalasin B:its morphological and electrophysiological actions on synthetic strands of cardiac muscle.Devi Biol. 31, 380-403.

MANASEK, F. J. (1968). Embryonic development of the heart. I. A light and electron microscopestudy of myocardial development in the early chick embryo. J. Morph. 125, 329-366.

MANASEK, F. J., BURNSIDE, B. & STROMAN, J. (1972). The sensitivity of developing cardiacmyofibrils to cytochalasin B. Proc. natn. Acad. Sci. U.S.A. 69, 308-312.


MASLOW, D. E. & MAYHEW, E. (1972). Cytochalasin B prevents specific sorting of reaggregatedembryonic cells. Science, N.Y. 177, 281-282.

MOSCONA, A. A. (1961). Rotation-mediated histogenetic aggregation of dissociated cells.A quantifiable approach to cell interactions in vitro. Expl Cell Res. 22, 455-475.

NEW, D. A. T . (1955). A new technique for the cultivation of the chick embryo in vitro.J. Embryol. exp. Morph. 3, 326—331.

OVALLE, W. K. (1972). Fine structure of rat intrafusal muscle fibers. J. Cell Biol. 52, 382-396.RASH, J. E., SHAY, J. W. & BIESELE, J. J. (1968). Urea extraction of Z-bands, intercalated disks

and desmosomes. J. Ultrastruct. Res. 24, 181-189.SACHS, H. G. & DEHAAN, R. L. (1973). Embryonic myocardial cell aggregates; volume and

pulsation rate. Devi Biol. 30, 233-240.SACHS, H. G., MCDONALD, T. F. & DEHAAN, R. L. (1973). Tetrodotoxin sensitivity of cultured

embryonic heart cells depends on cell interactions, jf. Cell Biol. 56, 255-258.SANCER, J. W. &HOLTZER, H. (1972a). Cvtochalasin B: effects on cell morphology, cell adhesion,

and mucopolysaccharide synthesis. Proc. natn. Acad. Sci. U.S.A. 69, 253-257.SANGER, J. W. & HOLTZEB, H. (19726). Cytochalasin B: Effects on cytokinesis, glycogen and

*H-D-glucose incorporation. Am. J. Anat. 135, 293-298.SHAFIQ, S. A., GORYCKI, M. A., ASIEDU, S. A., MILHORAT, A. T. (1969). Tenotomy: effect

on the fine structure of the soleus of the rat, Arch, Neurol. 20, 625-633.SPUDICH, J. A. (1972). Effects of cytochalasin B on actin filaments. Cold Spring Harbor Symp.

quant. Biol. 37, 585-593.SPUDICH, J. A. & L I N , S. (1972). Cytochalasin B, its interaction with actin and actomyosin from

muscle. Proc. natn. Acad. Sci. U.S.A. 69, 442-446.STEINBERG, M. S. & WISEMAN, L. L. (1972; Do morphogenetic rearrangements require active

cell movements? The reversible inhibition of cell sorting and tissue spreading by cytochalasinB.J. Cell Biol. 55. 606-615.

TASAKI, K., TSUKAHARA, Y., ITO, S., WAYNER, M. J. & Yu, W. Y. (1968). A simple, direct andrapid method for filling microelectrodes. Physiol. Behav. 3, 1000-1100.

THOENES, W. & RUSKA, H. (i960). Ober leptomere Myofilbrillen in der Herzmuskelzelle.Z. Zellforsch. mikrosk. Anat. 51, 560-570.

VENABLE, J. H. & COGGESHALL, R. G. (1965). A simplified lead citrate stain for use in electronmicroscopy. J. Cell Biol. 25, 407-408.

VIRAGH, S. & CHALLICE, C. E. (1969). Variations in filamentous and fibrillar organization,and associated sarcolemmal structures, in cells of the normal mammalian heart. J. Ultra-struct. Res. 28, 321-334.

WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUKNA, M. A., TAYLOR,

E. L., WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmentalprocesses. Science, N.Y. 171, 135-143.

YAMADA, K. M. & WESSELLS, N. K. (1973). Cytochalasin B: Effects on membrane rufflinggrowth cone and microspike activity, and microfilament structure not due to altered glucosetransport. Devi Biol. 31, 413-429.

ZIGMOND, S. H. & HIRSCH, J. G. (1972). Cytochalasin B: inhibition of D-2 deoxyglucosetransport into leukocytes and fibroblasts. Science, N.Y. 176, 1432-1434.

(Received 26 March 1973)

Cytochalatin B and embryonic heart 177

Fig. 3. Phase-contrast photomicrographs of heart cell aggregates, A, control 3 h afterattachment to culture dish, and B 24 h later. Note the increased fibroblast ring, c,after 24 h in CB 2 /Jg/ml. Note the flattened appearance and sparsity of fibroblastsassociated with the central core. D, after 24 h in CB 10 /ig/ml. The fibroblasts are few innumber, removed from the central core, and rounded up with many extending narrowprocesses, x 140.

C E L 14

H. G. Sachs, T. F. McDonald and M. Springer

Intact heart Cells Aggregate

\s\s\Fig. 4. Action potentials recorded from 3-day intact heart (ventricle), 7-day heart cellsin monolayer culture, and 7-day heart cell aggregates. In each case the preparation hadbeen treated with CB 2 /^g/ml for 30 h and was not visibly beating. Vertical scale =100 mV; horizontal scale = 0-58.

Fig. 5. Action potentials recorded simultaneously from 2 cells in a 7-day aggregatetreated with CB 2 /ig/ml for 6 days. The 2 microelectrodes were approximately 200 fimapart in an aggregate of about 250 fim diameter. At slow sweep speed (A) the actionpotentials are shown to occur synchronously. At higher sweep speed (B) the traces forthe rapid rising phases of the action potentials are seen to occur with less than 100 /isdelay. The 2 traces (B) were superimposed at the maximum negative potential duringthe recording. One cell has a slightly slower rising phase and a slightly smaller actionpotential amplitude. Vertical scale: A, B, 100 and 40 mV, respectively; horizontal scale:A, B, 1 s and 1 ms, respectively.

Cytochalasin B and embryonic heart

Fig. 6. Control. Single isolated beating myocyte with poorly developed myofibrils after24 h in culture, x 30000.Fig. 7. Control. Well developed myofibril in a single isolated beating myocyte after 24 hin culture, x 30000.Fig. 8. Control. Myofibrils with well developed sarcomeres in an aggregate after 48 hin culture, x 30000.


Fig. 9. Aggregate after 30 h in CB 2 /tg/ml. The myofibrils are disrupted bundles ofthick and thin filaments of approximately sarcomere-length scattered throughout thecytoplasm. Dense clumps of Z-material (Z) seem to be out of alignment with the bund-les. Junctions, polyribosomes and mitochondria appear normal, x 38600. Inset: cross-section through thick and thin filament bundle of the same aggregate, illustrating thecharacteristic hexagonal array, x 80700. Scale value, 0 1 /im.

Fig. 10. Higher magnification of 2 Z-bodies in an aggregate after CB 10 fig/ml for 30 h.Thin filaments emerge in parallel fashion on each side of the diffuse electron-densematerial, x 63 600.


fePlP


Fig. I I . Three adjacent serial sections through a single identified myocyte, treated for24 h with CB 2 /tg/ml. This cell had ceased beating after 6 h in CB. The hollowarrows point to a Z-body in register and out of alignment with the bundle of myofiJa-ments. The Z-material is hardly recognizable in A, but clearly visible in B and c.The small arrow heads point to the thin filaments, running between the thick filaments,x 63 600.


B

•v-

0-5


12

Fig. 12. Leptomeric organelles (/) in an aggregate treated for 30 h with CB 2 /Jg/ml.Typical CB-induced Z-bodies (Z) are found but the electron-dense bands of the lepto-meres are not affected by CB. x 31 800.


Fig. 13. Single isolated beating myocyte after 45 min of recovery from CB 2 /ig/ml,24-h treatment. This cell stopped beating after 6 h of CB, was still quiescent after24 h of CB but resumed beating after washing. The sarcomeres are perfectly aligned.Z-material is rather clumped, but the normal disk shape is becoming apparent (arrow-head), x 29550.Fig. 14. Single identified myocyte which was beating after 3 h of recovery from 24 hwith CB 2 /ig/ml. This cell was still beating after 6 h in CB, but was quiescent after 24 hof CB treatment. The well formed myofibril exhibits distinct sarcomeres with pro-minent compact Z-bands, some of which still show some irregularities (arrowhead),x 2955O-

Date post:	10-Jul-2020
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

CYTOCHALASIN B AND EMBRYONIC HEART MUSCLE: … · 2017-04-12 · SUMMARY The effec otf cytochalasin...

Documents