A comparative study of collagenase- and brypsin-dissociated embryonic heart cells: reaggregation, electrophysioIogy, and pharmacology

I ~ I A N F . Ccsr,~a,a,~, MECHAEH. ~ R.
~ ~ I E v ~ ~ K ~ A 1 V ~ SHRIER . A ,L 4 N

Departnzerrt of f'h?sioio,q~,McC;ill I:nivc~rsir?, 365.5 Dr.leml,totzd Srr-ror, ;I.lor~tr.e~cri,Q . , C'trrrladl; H34; 11'6, P.
Kcceivcd June 2. 1982

D., M. R . ~ ; ( : E V A R A , and A . SIIRIEK. 1983. h comparative study o f collagenase- and trypsin-dissociated e!i~hryoni~ heart cells: reaggrcgatioe~,c.lcctrophysiology, and pharmacc~logy Can. J . Physiol. Pharrm~acol. 408 - 4 10. . 61: 'Il'hc elcctrophysiological and pharmacological properties of aggregates prepitred from ccEls of 7-day-old chick en-ehryohc;irt ventricles depend on the enzyme used for cell dissociation. The mean beat ratc of aggregates formed fn-on-etryp"ra-dissocik1at:c1 cclls was about 53 beats/min whereas ap.grcgatcs fornmed from collagcr~asc-dissoi:iated cclls hacl a nican heat ratc 01' mori: than twice this value. Spontaneous activity of most aggregates forn-acd from trypsin-dissociat6.d cells was inhibited by elevatie~g external potassium or by adding tetrodotoxin to the anedium. A similar response to pot;tssia~ai~ been in all aggregates formcct vll:as froin collageniise-dissocii~tedcells. However, appa-oxirnateiyhalf of the aggregaks formcd from collagenasc-dissoaait1tcc1ccl8s were tetrodotoxin insea~sitivc.Intracellular ~nicroelectroderecordings denaonstratixl that aggregates ftrrii-acd frcw cr~liugenaseciiissociated cells typically had reduced action potential tr~axilrrlalupstrol<e vclocitics and depolarizcd threshold potentials in comparison to those recorded from aggregates forancd from Irypsi~l-dissociatedcells. In thc presence of tctroaitrtoxin thc: 1naxi1na1upstroke vcllc~city aggregates formed Prom either collagenasc- or trypsin-di~sociatedce1B.s dccrcasea! markedly. In of the case of the collapca~ase-treated cclls, the spontaneoiis activity which persisted in the presence of tetrc~cioaoxin was abolished by the slow channel blocker r9-600. Computer siniuliztion of membrane depcsiiirization supports the view that aggregates f'orrnct8 from cullagenase-treated cells have a reducctf: fast inward sodium current and a significant leakagi: current. Aggregates prepareci from trylasin-dissociated ceIls display propertics which more closely resernklc those of intact 7-clay embryonic ventricular r tissue. We thcrefose concltrcfe that. contrary to previous reports, coilagca~ascis not the enzyme necessarily best stnitccf f i ~ cell elissociation in all tissrae ckilture studies.
('OLIZZA.

COLIZL,~, M . R . GCJEVAR.~, SHRIEK. D.. et A. l!183. A con-sparative study s f collagcn;asc- and trypsin-di$soci:atcd c~nbryonic heart cells: reaggreption, electrophysiology, and pharn-eacology. Can. 9. Physiol. Pharrnactsl. 61: 408-419.
Les propriitis @?ectrophysi01ogiqws pharanacologiqimc:, d'agrkgats provcnant dc ccllules dc ventricules cardiayues st ii'embryons de po~alcts 5g@s 7 jc~ursddpendent de I'cnzyrne eltilisce pour la efissociat'non ccll~slairc.Lta dr6clucncc cardiayuc de moyctlne dcs agrkgats provenant dc celllules dissoci&e\ avec de la trypsine Ctait d'cnviron 53 bitttcments/rnin alors qtre cclle dcs agrkgats provcnant de cellules dissoci6cs avec dc la collagCnase itiait au n~cainsdeux fois plus dlcvCc. O n a pu inhibcr I'nctivitk sgontance de tous Ies ayregats ti)rrnC!s par les cel8ules dissocikes avcc de la trypsine en 6lcvanl Ic potassialm cxtcrnc 011 en ajoulant de la tktrodotoxine au milieu de dissociation. On a pu observer kine rCponse au potassium sin-ailaircchez toils Ies agrkgats provenant des cellules dissoci6es avec dc la collagknase. r P ~ u t ~ f o jprcs de Ia naoiti6 dc ccs dernicrs Ctaient s, inscalsibles 2 la tktrodotsxine. Des enregistrements intracellulaircs avcc dcs microClcctr.odcs ont dknloratrk y i ~ c conmparks :u[x , agrdgaes provel-rant de csI1~81c~ dissociiks avec de la trypsine, ies agrCgats provcnant dc ccll~~lch dissoci6cs ;IVCC de ];I collag6rrase prescntaient g6nCralcment de lalias f'aikles pointes de vitcssc tlc n-acpntkc ~ L poaentiei d'action ct lies potenticis dc I seuil dkpolarisds. En prCscnce de titrodotoxinc. la poinec de vitessc des iagrCgats provenant de l'unc hsti I'aeitrc cat6goric dc cellules dirninua nettemcnt. 7'outefsis l'actisiitc.2 spontante dcs cellulcs traitbcs 5 la collag6nasc, clui s3%tait naaie~tenucen lardserzce de la t6trodotoxii-mc. rut kli~nineepar I' inhibitea~rde voic lea~te. II-hOO. tine stiti-sulation sur orcliniatcur dc la ddpolarisation rncrnbras-nairc supporte l'hypothesc clui wait que B s agn-dgatsBi~rmCs des cellules traitkes h la C C P I I ~ ~ ~ ~ I ~ aicnt e par ISC ken courant sodique entrxnt plats friible et kin coalrant dc t.ui8e important. Idesagrkgats priyarks avec des ccBlis8cs dissocides avcc dc la trypsine prCsentent des proprietks qiii ressemblent beaucomp plus ii cellcs dn tissrs vca~tric~alairc intact ~ i c s e~nhryons AgCs de 7 jotlrs. Nous concluons donc, contrairernent $ cc qui a d6.iB CtC dit. qktc la cnllag6nase n'cst pas nCccssaircrnent la tracillcurc caszyme ii ~atiliscrpour Ia dissociation cellulaire lors d'expkriences avcc la culture cellmlairc. ( 'l'radali t par lc Jo~irnal]

Inf reduction 't'issue C U I ~ B I ~ p r e p a r a t i ~havc~ been wdely ~ a w d C ~~ ~ to study r i ~ e ~ ~ ~ b r properties of develop~ngcrardiac arae niuscle. These prcp:aratiornh are nsuallv t ~ ~ a dfro111 e enry me-dissociated cellc. T r y p i n , a nonspecific. protease, IS the en/yme most cor~1111only used to di\scaciate cells fro111 the hearts of ernkrycsnic and necpa~rztal chiclc (('avanatagh 19555; Dei-laan and Gott1iet-P 1968; Fischrnar.~and Ma,\cc~na

1971; Hyde et al. 1909; 1,iebcrrnan 1967; IAot-a~pre et al. 1979; k1cI)onald et al. 1942: Robin\on and Legato 19869; Shrier and Clay 1980; SpereIaklr ancl ldcI.arnkuhl 1963; Wollenbergcr 1964). etaltaryonicmouse (Fischtnan arad Mosc.o~~a 1 ). and r-aecmatal rat 197 (iiarary kind Farleg; 1963; Jongsnna 1972: Jorag\~na al. et 1975; Masten 107 1 : I,cgrito 1972; Mark :and Stra\\er 1966: Schrinnc et al. 1977). Crude collagr.na\e has al\o beera used to dissociate e~nbryo~lhc (Ci~vanaughct a1.

1963; S m i t h ;and Berncft 1964) a n d Ladula carciaac ceBBa ( B e r r y e t a l . 1970. (.;8ick c t a / . 1074: Ktsnc~ 1909: Powell a n d Tah~i\t I 9 4 6 ; I h n i c t ;dB. 1077: C'lark et a l . 1978; R a j 4 et a1 1978; I\enherg :and K l o c k n e r IOtiO: Hu\tarn;ante ct 611. 1982, Hunnht a n d G i l e \ 1981) C a ~ l a n a u g hc t a l . (I963) ok\erved that C P L B ~ collageC nase laeltied nnore coenplete dii\oclakron of enlbryonic chick heart and waa It.\\ d a m a g i n g than t r y p \ ~ n . a\ judged by \cveral cnterrra inscluding cell appearance i t a d vrabillty. Maseon-P6vst et al. ( 1'476r a l conclucied ~ that collagenlt\c wa\ \ i ~ p e r ~ o o tryp\In for c'c~II dl\tr sociiation, b a w d o n an ultra\truut~rral \terdy o f I I ~ M born rat heart t i ~ \ \ o c ~ a t c c i the kwc~e n t y m e \ . by The p u r p o \ e of tklns \tudy wa\ to tfetcrrn~alcw h e t h e r tllerc. w e r e a n y clectrophy\na>log~ciiB r pl1;irm;i~olo~ic;11 o d l f f e r e n c c \ between the cell enenabranei ot cultured cell\ dls\ociated by t h e \ e two a g e n t \ . W c coraaparecf the p r o p m t i f i of heart cell aggregate\ prepared f~cjtn - d a j 4 old chick e m b r y o heart ventricle\ tii\\c~clatedwrth tryp\In o r c o l l a g e n a \ e . The aggregate ~raorphology. berat rate, electrophy\~cslogy,a r ~ d pharnnacology mere founcl t o d c p c n d o n t h e d i \ w c i a t s n g crlzynse u\cct In p ; l r t ~ c ~ i lar, aggregate\ preparcti f r o m collagenasc-cf~\\ocii~tt'ci cells dlap'lajcd a reduced upktrtske velocity of t h e iactlon potentla1 and a d c c r c a w d tetrotlotoxin \cn\itivlty. U n d e r t h e condltlcsna used in thi\ \ t u d y , w e c o n c l u d e that aggregate\ prepared trorn trygs\ln-cli\\a)r:lated cell\ c-lniplay clectrophy\io8og~caland plaar~aaacological propertie\ more tygslcal of nntact ?-day ucntriceilar t14sa1e than thoae prepared f r o m collagcr~ase-di4\c~ci~ited cell\. C'oanputer sxlnnuldtxlon\ bd\ecl o n ;mil ionic ~alc~ciel o m fr koltage-clamp nleaaurernents c Ehiharca and .lohn\on 1980: Clray and Shrier led81 t r ) w g g e s t that the cxperl~r-aental re\ulte can b e explained b y a r e d ~ a c e d 1,lst inward w d ~ u r ncurrent a n d an ~a~crt.;t\eti leakage c ~ a r r c n t rn collageraa\e-d1\soc1;1tec1 cell\.

1980) consistccl of 5 x 10 ' gjrizl, cr-ystallinc trypsin (Worthin~ton Dio~hciilical:245 k!/nig) arad 5 x 10 " ' m , /i dcoxyribonuclcase (LVorthingtoai; 9.8 X 1 0 ' U/nlg) in cslcium- rind magncsiurn-free phosphate bnf'fcrcd solution at pH 7 . 3 . ( h i Collagcnasc-dissociatioii rslcdium consistccl of 5 X I ! g / m l , collagcnase (Sigma; 'l'ypc 1 . 1463 B!/magb ;tiad B 5 X I 0 " g/rnL dcoxyrilaot~irclc:isc (Worthit~gtun)i t a calcium- aancf magncsiur~l-frw phosphate bnffcrcd aolution ;it pH 7.3.

b'ulrrirr rncl~licl klediurrs k l XA (Sachs and L)cilaan lCB93)cor-atainetP 20% ~ncdiurn19'1 (Granci Island Biological Conapany (GIBCO)), 20;' heat-inactivaacti taorsc scrutn (K.C. Ric,logic;~l).4% k t a l calf serum fG[BCO). and 0.5% gci~talalycin(Schering) in potassium-free Far-lc's balanced salt solution (rnil8imolar): NaCl, 1 16.0: MgSO,, 0 . 8 ; NaH2P0,, 0.0: ('aC'I1. l . t i ; NaH&'(.)13, 26.3; glucosc, 5 . 5 . 'I'he potasaiuin conccantration of the final r-micdium was aqastcti to 1.3 m M .
Drr4g.v

Tetrodotosin (T'I'X) (Signla) was dissolved in distilled watcr ( I rng/nnL) ancl diluted to final ~.onccntrationsof 1 x 10 ' y/mH. to I x 10 ' g/nlB,. Id-600. a gcncnsus gift of Kiaoli i1G Bt,~~dwigshni'cn Khein. Gern-aany), was ilisam solved in distitlcd water and dilutcd tc) a filial r.onccntration of 2 x 10 ' g/ml,. ~i/~rc~tr-o~~l~.siolo~ "I'Bic coa~tcrats of' each tlask were trnra\f'crrcd to a pla5tic tisme c~rIturc cbiska (Falcon No. 300 1 ) on the warrncd stagc of a dissecting microscope. 'il'i-ac aggrcgatcs ;dhcrcd firmly to the bottom of the dish in an hour OI. less. Tlac tclaipcratinrc was maintained at 37 . 0.5''C lay a tcrnpcraturc controller (coalt tinuously rmlonitoredj. ' h e bath ivoliairac 0.5 ~ n l , )was coiltiniiousiy p c r f ~ ~ s eat ;i r;lte of' I tnL/rnin with mcdium 8 181' d cnacdiinn~X lXA witlaout serurnj cquilibratcd with a pus lrlixturc of 5'h/r: CO-. - - 1!YJ O2 - X55i N-. Drugs were addcd to time pel-fusion rncdium. Mhythn~icallybeating ugr~:gatcs were viewed at :r magnification of x50 and thcir s i ~ c swcrc mcasureal witli an ocular reticle whosc srnallcst division represcntetl 1% prn. Measurements could bc made to about half a division. hggrcgate diameter was taken to be the nacan of' thc major and minor diiamicrcrs ira the horizontal pfanc. Aggregate volumc u a s calculated assuming Bhc aggregate to he a; prolate spheroid. d'ui~tr-actioras wcrc visi~ally tnor-mitorcdto obtain beat rates. El~ctriciilactivity was recorded intracellularly using micropipcttes filled with 3 M KC'I. Thc clectr-ode innpcdanct: ranged from 3 0 - 7 0 M i l . 'I'Bic s i ~ n a i s \vcrc recordcci using an aruplifier with negative capacity coanapraisatic~na n d the cxtracelllllar rlacclii~rn couplet8 to a currcnt-to-voitagc coalvcrter was that ralaintainsc! tkac bath ;at virtual groutat! via :ui agrar laridpc anel an A g : AgCI wire. 'T'1.r~nlcnlbrane v o l t a ~ c was arnplificd (X50) and stos-ccl eta riii:ignctic tape (1-11' 3964) for off-lint: analysis with a digital oscilloscope t Nicolet). 'rhe action potential duration was rncasurcd as the tirnc fronr inaxirmial upstrokc alclc~city( V,,,,,,) f;iO'X action potcslto tiai repolarization (APll,,,) and frotr-s V,,,,,, to maxin~um diastolic potential iiaPB),,,,!). 'I'hrcshoBd potential was cstimateti from thc brcak between diastolic aicpolari~ationand tlac

J'~-P/~[IY(IF~o~I I;ertilizccI eggs frona Whi tc Leghorn hcns wcrc iracit bated ar 17'Y' ;ma1 85<X hhurniclity. Scvcn-day-old ct-albryos mlcre rernaovcd and decapitatctf, 3rd their heart\ were ascptic:illy excised. 'I'i-ac 3pic:il portions of the heart ventricles wcrc isolated, fragnentcd, and dissociated i n t ~ ~itaglccells U S I I I ~ cnryrnatic dissociation conlhincd with rnccll;araical agitation (b1cHaaai 1070). CBnc-half thc tihhuc wah dihxociatcd in rr-yphin, atlei thc other hia8f iia collagcnase. A n inoculuni of 5 x 10i to 7 x 10' ccllh was acidcd to 3 n ~ b ,of' cultiirc rnediisrrl X18A contaia~cd in a 25-mL Erlenmc.!;er tlask. 'The 1 flask was passeti with 1 ra~ixtureof 5'2 COT- I Oc4 (I2-- 85(k Nz.scaiccl with a silicorae rubber stopper. and placcai on a g!/ratory table ( 7 0 rcvolutions/rnin~for 48.--79ti at 37C to allow xpherical aggrcgatcs to form (Sachs and DcHaan 1073; Dasl-faan anct IlcFeticc 1978).

4 10

C AN

I PIiYSTOL PHXRM4C'OL

VOB

h i . 1083

"
a

oa8

'

*
' e 8

Volume

(ym91

Frc; 2. Kate- volume plot of 'I' aggregiatcs (( 1. tl = I ( 17) and C aggregates (a, = 1 13). Arrows at '4 and B Lare drawn iz :at volumes calcu1iati.d using 800 and 200 pin diaaneters. respectively. Heat rakes were obtained by viwally monatoriny
Diameter (urn)

contractions
( EZ =

Fit;. I . 'Thc sire tiistribut~onof and C aggregates ( n = 243).

'r aggregates

403)

iraore rapid upstroke phase. Mcasurerncr-its of V,,,,,,were made with an electronic differcntiatc-lr which was linear froin 5 kl/s to 1000 V / S . In this stucdy PI always rcfers to the number of aggregates sampled and docs not represent arluitiple mciasurerncnts fro111 a singlc preparation.
('w??putor-siriliricifior!.~

The chc-tar~putntioras citrrittd out on an HP 1000 Series-F were conlputer (Hewlett---I'ackarci) locatcci in the llepartn-rent of ,4nacsthcsia Research at McGil? hlniversity.

Aggrc-'g~l~bo~t

Aggregates forrncd during 48-72 h of gyration culture i'rom trypsin-dissociatd ventric~m1:trcells ('F aggregates) ivcrc comparetl with those fornmed tron-a collagcnase-tfis~ocii~ted cells ( C aggregates). Figure I \ho\%\ the d i ~ t r l b u t i ~ r ~diameters of T aggregates of from 15 cultures and C aggregates froan 12 cultures (of whlch 8 were paired s~rlttare\).Most ( 7 2 % ) 'F aggregates rangcd in diameter between 1561 and 250 pm wherea\ nnost (70'%)C aggregate\ ranged betvlleen 50 and 150 prn: on14 7'CX7 of T aggregates were smaller 'rt d than 150 pna while only 16% of C' agh ' g dtes were greater than 208 pan. The nacarl diameter (t SD) o f T aggregates (210.3 z 54.3 k m , ra = 403) was \ignificant]? different ( P < 0.01) from that of C aggregates : (134.1 t 63.8 ptn, ra = 2 4 3 ) .
,Yponscrnsous I~t-'uf i"'hlt~ VvJhen T and C aggregates were pliasctl iar tistptae cul-

(PZ =

FK;. 3 . The distiilaurion of beat rates of I aggregates 107) and C aggregates ( n - 1 14).

hire dishes they remained spheroidal f i ~ r several imours and beat spontanecpusiy with a regular rhythm. I t was shown by Sachs and IleIHaan (1973) that aggregate beat rake ia inversely rclated TO aggregate voleln~e.'Fkmcre is evidence of a similar volunnc-kcat rate trerad for T aggrh=gated(Fig.2). Morecsver, there is good currespondence betweera the beat rates fbund here in the 10" 110' p a 3 volunre range and timcjse reported previously (Sasl-as and IIelHaan 1973). In contrast, beat rates CPC C aggregates tend to be independent of aggregate voiurme. For example, the mean beat nate (tS1)) of C aggregates with v d u m e s in the range berwcen 1 0 and IOh p m ' 4 1 15.1 k 25.2 keatsjrnin: r? = 70) is not

('01,IZZA El' hi.

TABL.~;. I . Effect of external potassium concentraticdn on spontaneous activity

Beat rate (Beatsjmin)

H .3 nlM 1 K+](,
-

2.5 rnM
23.826.8

% decrease

?' aggregates (IZ = 10'1

52.322.9 9 1 . 6 5 15.6
5

54
44

C aggregates ( n = 8)

5 1 . 2 5 12.8

Nc~rt:: Beat rates given are mearl5

SL9.

FIG.4. 'I'ypical intracellular recordings of spontaneous I activity in ' ' aggregates and C aggregates.

significantly different horn that of & aggregates in the volume range 10-0 I07Ftn' (113.1 -t 19.3 beatslmin; rr = 44). Howe\er, in the volume range 10" to 10' pnl'. the mean beat rate of C aggregates was appmxinnately double that of 'r aggregates (53.3 a 83-3 beats/mins; n = 104). Figure 3 shows the distribution of spontaneous beat rates for T and C aggregates. It has been previously demonstrated that increasing the external potassium concentration ( K t I,, will slow or abolish spontaneous activity in isolated embryonic heart cells (DeHaan 1970) and in T aggregates (IIeHaan and DcFelice 1978). Table 1 shows the decrease in I spontaneous beat rate of both '' aggregates and G aggregates 4 120- 150 prn diameter) that occurred when [K I., was increased from 1.3 to 2.5 m M . In both cases, a further elevation of [ K ' I,, to 3.5 mM resulted in irregular beat rates characteristically lowcr than 1 beatlrnin. At aKt I,, of 4.5 nmhf all spontaneous activity in both groups was arrested. It laas been previously reported that more tlaan 805% of T aggregates stopped beating in 10 g/mL TTX (McDonald et all. 1972). 'These results were replicated in five experiments b r a = 4 5 ) in which 'l'TX at doses between 10 ' and 5 x lo-' g/mL stopped the beating of 70'141 of the T aggregates. Any residual spontaneous
+

FIG.5 . (A) Superin~posed action potentials rccordeef from a T aggregate and a C aggregate. BB) I!pp@r pair of traces: superimposed upstroke phases displayed at a faster sweep speed. Vertical calibration bar is 50 my. Lower pair of traces: differentiated upstrokc phases.

beating was generally irregular and sporadic bursts s f activity were occasionally observed. In contrast, the response of C aggregates to TTX was less uniform. In six experiments ( n = 5 1 ) spontaneous activity persisted in the presence of 10-' to 5 x 10-1 g/mL TTX: in onc of these experiments spontaneous activity persisted when the TTX concentration was increased t~ 10 g/mL. In eight other experiments ( n 56) TTX at doses betweens 10-' and 5 x 10 g/rnL stopped the beating of 45% of the C aggregates. Residual activity was sporadic. Elec-trophysicr/ca~y Spontaneous electrical activity recorded frorar 'F and C aggregates is shown in Figs. 4 and 5 . 'The pooled results for the action potential parameters measured from 12 experiments ( 1 2 = 14) using 'T aggregates and frcm 1 1 experiments ( n = 25) using C aggregates are presented in Table 2. Recordings were made after impalements had stabilized, as reflected by a constant interbeat interval and stable action potential pararne-

'

('AN I It IYSIOL PHARMAC'OL '

VOL 01. IcW?

ters. Action potential of 7' and 4: aggregates whic\-ehad signil-icaritly diffcrcnt inferbeat interval\ and ntaximum upstroke velocities dicl not havc significantly ditTercnt action potential durations. The addition of 'FTX ( I 0 ' to 5 x 10 ' g,/mL) led to a prcsgrussive reduction of V,,,,, in 'r aggregateb and prolongation of the interbeat interval (Fig. 6). In addltron, there was a dcpolari~ation ira the lcvcl of the threshold potential. After several minutea tho pacemaker depolarization failed to reach thre\hold armd spontaneous acalon potentla1 generation was blc~cked. Thc mean resting potential C-f SD) of these aggregates was found to be -54.3 -+ 9.7 rnV o r = 12, A t~imilarresponse to TTX wa$ fsslnct in \ome (' aggregrates ( F I ~6). Following cessaslon of aca-ivit!/ . the mean resting potentiat of these preparation\ wat -54.9 t 7.2 ma/ (fz = 8 ) . Iiowcver, in six other C aggregate\ \pontaneou\ act~vity was serstaincd n n the presence of TTX, with reducecl up\troke veloc~tlc\ and depolarl~ed thre\hold pstentaal Bevel\ (Fig. 7 and Table 2). The add~tlonof the slow channel bIocker I)-GOO (Kohlhardt et ul. ld842B at a concentration o f 2 X 10 ' girnk tc9 a C' aggregate beating i n the pre4cnse of TTX blocked spontaneous activity 'Il'hc ability (st I>600 to block spontaneous ractavliy can also be ob\erveci in C aggregates and T aggregates in the ab\ence o f TTX: in two of three 7 aggregates and four of four C ' aggregates 11-600 (2 x 10 " g/mL\ blocked spontaneous activity. A comparison of electrlciil actlvity of the ?' aggregates hefore and after 13-600 \how\ that 11-600 reduced the ovcrjhoot, shortened the action potential duration, apd reduced the interbeat ~ n t e al, but had no r~ effect c ~ r a I/,,,,,, (Fig. 8 ) . Figure 8 al\o \ h o ~ v \that thc rc\ponne of C aggregates to 11-600 was \irnil:ar but there w;is a decrease of V ,,,,,.

Sinzulnfio/~ pact~makcr- l ~ d uf c upsrr-okc pl~cr.~r.~ Si~mulationsof the pacemaker process anif the upstroke pfiasc of the actron potential of ?' ant! 4-7 aggregates were carried c~utby nurncrical integration of a system of differentia! ec~uations ctb~aineci from voltageclamp measurements of backgroinnd (/,,,) and timedependent pacem~tker(I,,) currents in 7-day trypsindissociatcd vca~tric~~lar aggregates (Clay and Shrier 1981a ) . Measurements of the T'I'X sensitive, kist inward sodium cur-rcnt ( I N a ) made in trypsin-clissociatcd aggregates were also incorporated into the model (Ebihara ct al. 1980; Ebihara and Johnson 1980). 'The parameters of the model arc ssaiecI Bi~r a 200-p.,rra diameter apgregate which has approximately 2.15 x I OF' crmr of mcrnbranc surfacc area (Clay and Shricr I9MBa). All currents arc given in aianoamperes, voltages in millivoits, cc~nductancesin nlillisiemens, rate constarats in inverse seconds, and tinmes in seconds. The

C -+ T T X

FIG. 6. Left hand panel: spontaneous activity in T aggregates (upper trace) and C aggregates (lower trace). Right hand panel: blockade of spontaneous activity by addition of 'l'TX ( 10 ' g/mL).

~ 2 1 I,, = 220 (?')

1.I (V

tp -

I
-

+ j.2 t-

+ j3i)+

50)/( 1

exp ( - (V

+ 5(3)/25))

where

[3]

p=(1

+ cxp(-(VT

90)/25)) ' , y = 1 - p ;

Y
lhlh given by is [4]

~IP.

JNdb=0.23(V-40).

The I , , component is given by [S] with

K ' I ~ ; .7. (A) Su~~erin~poscd potentials recorded from action a continuous impalement of a single ccll within a C aggregate before (C) and after (TI'%), exposure to 10 ' p/rnL 15-n~in T'I'X. (B) Upper pair of traces: superirmposed action potential upstroke phases displayed at a fiister sweep speed. Vertical calibration bar is 50 ITIV.Lower pair of traces: differentiated upstroke phases.

JM,= ~ ~ ( ) s x ' ( - 2 ) / ( 1 M'

+ .Y

-t

x ' )

[64
and [7]

(1

+ exp(-iV +

112)/25)) ';: = 1 - ul;

X

--

SIM' -/

cHs/dt = -(cu, t P,)s

+ a,
- exp (-0.2 (V 4 55)))

with rate constants

[8]

transmeallbrane voltage is denoted by V. Ihg given by the surn of two tianc-independent curis rents:

cu,

p,

0.8 (V

+ 551161

0.059 exp (-0.105 (V t 55)).

'Thc TTX sensitive, fast inward sodium current (Ihd)is given by

I.91

PKI is given by

IKd =

m3h ( v - 40)

CAN. J. PIIYSIOL. FHARMACOL. VCIL, 61. 1983

RG. 8. Cornparison of the effects of D-600 (2 X 10 -' gjrnL) on action potentials recorded from 'r aggregates rind C aggregates. Left hand panel: (A) Superimposed action potentials recorded from continuous impalement of a cell within a $ aggregate before (T) and after (11-604)) 20-nain exposure to D-600. (B) The upper pair of traces show superimposed rapstrolte phases froan these recordings, while the Bowcr pair of traces show thc upstroke velocities. Scale: 50 tnV verlicial, 0 - 5 s horizontal (A), 5 ans horizcsntal (I%). Right hand panel: (A) Superilnposed action potentials recorc%cd from contin~~ous impaIernent of a cell within a C aggregate before (C) and after (D-600) 12-min exposure to D-6(#). (B) The upper pair of traces show wimilc the lower pair of traces show the upstroke velocities. Scale: 250 rns superimposed upstroke phases from these rec~ardings, horizontal (A:), 5 rns horizontal (B).

p,,

356 exp (0.079V)

+ 3.1

x \Oqexp(0.35V).

The net ionic membrane current is given by

L 1 21

1,0,1,' = IN', + IK?+ lhp.

FIG.9. Simulation of membrane potential during pacemaker and upstroke phases of the action potential. (A) Membrane potential when jNd 323 mS and I,lpp 0 nA. = = (B) Membrane potential when iIVa mS and I,,,,, 5 nA. = 40 =

The expression for IN,, in Eq. 9 is the same as that used by Hsdgkin and Huxley (1952) to describe I,, in nerve. 'The expressions for the rate constants a,,,and PI,, in Eq. 1 1 have the same functional form a\ in the Hodgkin-Huxley equations. The rate constant\ for ah and PI, were taken from Ebihara and Sohnwn ($980). The ganembrane voltage was obtained by numericaIBy integrating [131 d V / d t = (I,,,,, + I,,,,)/C

with

where

[ I 14

CY,, =

100(V

+ 45)/(1

- exp (-0.1 (V

al, 135 exp ( - ( V =

+ 80)/h.8)

where I,,, is the externally iqected current. The pararneter C is the input capacitance of an aggregate. wh~ch has been determined previously to be 0.023 pF for aggregates of 200 Frna diameter (Clay et al. 1979). The initial ccpnditicpn on membrane voltage was \et to be -90 mV, which closely approxinnates imaxisrlurn diastolic potential (MDP) for both TF and C aggregates. We also set the activation parameter s equ:aI to 1, since 4 5 ) ) ) s is fully activated at the action potential peak and does not change rapidiy during repoiarizatiotm. We initially set rn = 0 and h = 1 , which approxi~natesteady-~tate values of these parameters at MDP. 'The time courge of

voltage change from these initial conditic~ns was cietermined by numerically integrating Eq. 13 using the algorithm of Rush and Idarsen 1978) uith a 50-[ASintegration time step. Each sirnellation was terra~jirlated at -40 rnV In the case of T aggregates I,,,, and g,,, wtis O was set at 323 mS, whereas in C aggregates- Sy, \YiiS reduced to 60 ra1S to reflect the reduction of V,,,,,,from 120 V/s in T aggregates to 24 V/s in C aggregates. 'To simulate the more rapid pacemaker tiepolari~ritionin C aggregates. I,,, was \et to 5 nA. ?'he re\~altsshc~wnin Fig. 9 yualitativcly mimic the major differences in the eleetrophysic~logicalpra)perties c)frY(line Ab and C (line aggregates: a Inore rapid pacemaker depolari7ation and a \lower upstroke phase of C aggregates. Moreover, the simulation\ also show the depolari~ationof threshold potet-atial obseralcd in C slggregate\ (Fig. 5 ) . FanaIiy, we note that neither reduction of KN,, nor the change of I , , , alone could account for the observed differet-accs between T and C aggregates.

pletely block the generation of' spontaneo~ls ktcclon potentials In aggreyatcs of cardiac cells, whereas a scerzdy in\varct current can increaee the beat ratc (Scott 1979). Similarly, irnrnctfiarely after microelectrode ~rnpalement, rapid beat rates are often okserved. 'I'hese are presumably the result of iqjury-induced nonspecific leakage currents (Shrler and CBay 1989). 'I'hc sinmulation~spresented in this study are con\lstenb with the hypothesis that treatment wlth crude coBl:agenra\e le-ads to membrane di\ruptivn giving rise to a nonspecific membrane leakage currcnt (Fig. 9 ) . Modifications of nlembrane currents by enzyme treatment may conceivably saccount for the cfifk'erence in spontaneous activitj between C and 'I' aggregates. 'I'he fast inward s~~dlurm currcnt has a component whrch ralaintains steady leveBs of activataon in the pacc~naker vc~Itagerange (the winadow current, I , This window , ), cuprent not only al-fects actlon poterataal parameters (Atwell et al. 1979), but also pacemaker dcpolariaatic~n rate and consequently spontaneous beat rate (McAllister et al. 1975; Shrier and Clay 198'9).In con^ Discisssisn puter sirnulation\, a reduction of the fast !nw;ard sc>dau~n The results of this study indicate that collagenase- current (avh~ch accounts for- the reduced I;,,,,, in C aggretreated cells formed smaller aggregates tl~antrypsingates) results in a slowing of the pacemaker depolartreated cells. 'I'hc size of aggregates has bcen shown to ization rate. When ,ijNCIrcdelced fron-athe control value is depend upon the tissue origira of the cells uscd, their of 323 to 60 mS, spontaneous activity ceases. Changes developmental stage, thc rnodc of cell isolation, and the 1 an 7-clay in the ttane-deperadent pacemaker current (Ik, conditions of cel l rotation (Moscona 1965 1. When the ventricular T aggregate\ have been shown to be prispeed of rotation is too great. the shearing forces exceed nlarily responsnble for the ce\ration of spontaneous the mutual adhesiveness of the cells and aggregation is actiklity when ( K tI,, is clevatecl from 1.3 to 4.5 naM prevented. At slower rotation rates, aggregates form (Clay and Shrier I98 1 ( a ) . 'T'hc degree of slowing of beat from cclls which, upon contact. attach firmly enough to rates of t aggregates at elevated ( K t I,, wras sim~lar to resist the sllearing forces. Under the same conditions that found in 'I' aggregates. Furthcrrnore, ?' and C' smaller aggregates will be formed from less cohesive aggregates ceased beating at slmllar 1 K ' ,. 'I'hi5 \ugcells (Moscona 1065). 'B'he fact that C aggregates were gests that lk, is present in C' aggregate\ ancl at leaat in somc respects i\ similar to Bk, in T ~zggregatcs.A possmaller in size than T aggregates inciicates a possible decrease in cell-cell adhcsivity in C aggregates. 1-low- sible role of other cur-rent\ (primarily background curever. Cavanra~ighet al . ( 1963) found that collligenaserents) cannot be ruled out. Indeed. the ce\s;aticsn of ciissociated ceBls adlaered nwre rapidly to glass c~tlture spontaneous activity of ventricular tiswe denrang e ~ n b r y dishes and tended to flatten faster. The adhesion of cells onic development appears to be drne to a reduction of the to glass may involve a process different from that background inward sodium current I, ,,, (Clay and Shrner involved in cell -cell adhesion. It has been posttaliated BY81 R ) . Bnlthough we have shown that a nonspecific that cell recognition and selective cell adhesion arc leakage current suffices to account for the differences in naediated by interaction of spccific cell surface conr- beat rate, possible ce>ntrihttklon\of I , i,,,cLinnotbe ,and pcsnents (Moscona 1960). Furthermore, isolation techruleci out. To evaluate thc contribution of 6, and I,,, niques ( e . g . , enzyme type and concentration) and crrl- wo~rldrequire tracer-flux meastarements coupled with ture conditions can influence adhesiveness. voltage-clamp experiments. In this strstly, C aggregates on average beat more In C' aggregates, lapstroke velocities wath a mekin of' rapidly than 'I' aggregates. A comparison of the sponta- 24.3 V/s were ob3erved. suggesting that the fdst sodaneoers activity of I and 'P aggregates sl-ao\vsthat there is urn currcnt becanae rnarkcdly reduced ti~llowlangcolla( a rnarked ciiffercnce in the rate of pacen~akertiepolar- genase treatment anti cell dissociation. T T X wab effecization (Fig. 4). The rats of pacemaker depo'iarizaticpn tive In redeacing V,,,,,of these aggregate\ by a f a c t ~ ofr ~ reflects a rather fine balance of in\vartl alad outward four ('H'able 2 ) , de~nonstratingthe retention of rt 'I'TXcurrents. Indeed, it has been observed that a few nano- receptor ~nteraction.The observed depolara~ation of amperes of steady applied outward currcnt can c a n - threshold potential after 'I'TX was added (Fig. 7 ) is also

consistent with the hypothesis that TI'X blocks the fast socliuraa surrcrat in C aggregates. The ability of C aggregates to maintain spu~ataneousactivity in the presence of 'L'TX could result frona residual 'I'TX-insensitive fast sodiulra channels or alternatively from slow inward Na- Ca chaasnels. 'I'lae observation that D-600 abolished the sporstaneous action poteratials in 1"FX-treated C aggregates and slowed the upstroke phase of C.' but not T aggregates (Fig. 7) is consistent with the latter alternative. fioevevcr, it should be noted that D-600 increased beat rate and suk~sequentlyblocked sponta~GCPLIS activity in '8' rzntl C aggregates not treated witla T'FX. I'his nray be attributed to known effects of D-600 crn outward potassium c~rrrents(Kass and Tsie1.a 1975). Cultured cardiac cells which have a reduced 'TTX sensitivity or V,,,;!,have been described as dedifferentiated (Sperelakis et al. 1976). C: aggregntcs display these prc~pertiss;however, other electroplaysiologi6.bal parameters of cells at earlier stagcs of dcveIoprnerat are absent. There is rao decrease of the MDP, overshoot, or action potcrntial duratiora ira C.' aggregates to values typical of younger embryonic ventricular cells (McDonald and I>cHaan 19-72: McDonald and Sachs 1975). Conseytiently, nlc conclude that the ef'fect of cell ctissociation using crrade collagennse does not lead to a consplete dedif'ferentiation of nlernbrane fianction after 2-3 days of gyration culture. Schannc et a!. ( 1977) have deraaornstrated that ta-ypsincfissociated neonatal rat cell coloasies develop T'TXinsensitive action potentials and develop slow reThis charagc has been sponses after 4-6 days in cult~rre. attrib~atctlto the trypsir~ization procedure aa~dnot to dedifferentiaticm. Lt is conceiv;able that this type of dissociation-i~acti~ced transbr~natic~n could asso occur in 'r aggregates but with a much prolonged time ccjurse. Enzyme-dissocia~edited cells which are cult~nred as monolayers have been reported to be T7'X insensitive and to lack a East action potential upstroke (Sperefakis ant1 Lehmk~nhl 1965). However, it has beelm recently ctemonstrated that try~ssin-dissc~ciated cells in monolayer culttire can retain TTX sensitivity and a rapid upstroke velocity Ql,ornpre et al. 1979; Robinson and Legato 1980). Excessive trypsinization was s~eggested as a possible explanation for the poor recovery observed ira the earlier inonolayer cultiare studies (Kobinsoa~ and Legato 1980). Cells fr(11117-day embryonic chick heart in T'FXinsensitive monolayer cultua-es can recover TTX sensitivity when dissa~ciated reassembled into aggregates and QSachs a?. 1973). This suggests that recovery depends et on cell-celi interactions. The absence of TI'X sensitivity in some C aggregates suggests that there rmaay bc scme disruption in the recovery process normally seen in T itggregates. The beat rates of C aggregates are independent of aggregate size, and the raaean beat rate

and distrib~ation heat rates are xiralilar to those deterof mined from single ~nycpcytes obtained from 7-day embryonic hearts QSachsand DeHaan 19-73).The sinailarities bctwcen C aggregates and single cells naay bc due to loss of scpr-aae cell-cell interactions in C aggregates. H is of interest to note that 'FT'X sensitivity and t exps-essecl in rapid action potentiai upstrokes are t ~ l l y aggregates prepared froin collagenase-dissociated neonatal rat heart cells (dc: Bruijne and Jongsnlu 1980). I4owever, aggregates of 7-day embryonic chick heart cclls prepared slsing collagenase at a cc~nceratratio~t ten times higher than that used in this study demonstrated slow upstroke velocitie\ 6-32 V / s ) which were reduced (to 5 V/s) upon addition c~f 10 "/rial, 1"1'% (Mackenzie and Standen 1982). A mixture of trypsin and cc~llagenase been shown has to be effective for the isolation of viable single anyocardial cells fionm adult rats (Saclobsora 1977). On the other hand, an earlier report QLPellaan1964) indicated that tlissc~ciationof embryonic chick heart sells using con-ebinationsof collagenase arad trypsin gave less satisfactory rcsults than when trypsin was used alone. A more recent study by HIaworth et al. ( 1986))showed that exposure of adult cardiac cells to trypsin after COHlagenase treatment can be beneficial. I t was proposed that trypsin naay allow the gap-junction channels in the intact cells to close, thereby conferring upon them Ca" resistance. This approach might also be beneficial for tlac dissociation of ernbryor~ic heal? cells. However, the properties of adult and embryonic isolated cardiac cells differ: ranlike adult heart cells, embryonic heart cells in culture retain the ability to redevejop electrical ccjupiing, presumably by the formation of functional rlexal ranits (DeHaan and Hirakow 1972; Clapham et a!. 1380). The observation that T aggregates appear Inore similar clectrophysiologically to the intact vcastricle than C' aggregates is somewhat unexpecteei since it laas beera shc~wnthat trypsin can enter cardiac cells during isolatioia and can do some intracelIular damage (Kasten 197 1). However, it should be noted that highly purificd collage~maseis almo\t cc~mplctely ineffective in dissociating cardiac cells (hclasnon-PCvet et al. 1976; Kono 1969). The ability of crude ccrlllagenaw to dissociate heart rrauscle would appear to reside to a significant extent in the inapurities present in the collagenasc. In particular, the collagcnase used in our study showed a significant lcvel of protease 6264 U/rr-eg) and some low level cl~~stripai~a activity ( 1 . 1 U / ~ n g ) . contrast, tryp111 $in \Ifas present in exceedingly low c~~ncentratioi~\ 60.04 &r/~ng) and it is most uniikely that trypsin contamination would play a significant role. We have not investigated the role of the contaaninants present In crrade collagenase. 'l'here is recent evidence that pro-

tease dissociation media can have effects equivalent to 28: 169- 184. J. I98 that of crude collagenase wit11 regard to dissociation of Cnak~u, M., and A. SHKIER. 1 a . Analysis of subthreshold pacernaker currents in chick enabryonic heart cells. cardiac muscle (Bustarnante et al. 1982). It also appears J . Physiol . BI,ondon'B, 312: 47 6 -490. disthat prolonged exposure to protease-co~rtaia~ing 198 1 6. Ilcvelopmental changes in subthreshold pacesociation media may give rise to cell dannage (J. 0. rnakcr cur-rents in chick ernbryonic heart cells. J . Physiol. Bustarnante, personal communication), wlaich might (London!, 312: 49 1 -504. account for some of the effects we observed. L ~ A K I ,.A. M . , A. C ~ ~ T A DG.N I , I FH~AMINI, G . FESTUCCIA, and The ability to obtain isolated cells $'or tissue clnlt~lre '8'. T , ~ R K A N ~1977.. h-eparation and some properties of VA seems to depend o the combination of enzymes, culm isolated beating miyocytes frorn adult rabbit hcart. .I. h/lol. ture conditions, and the tissue under study. Based on Cc11. Clardiol. 9: 777 -784. our results we comciucie that crude collagenase may not DE BKIJIJNE,, and H. J . JONGSMA. J. 1980. Membrane properties of aggregates of collagenase-dissociated rat hcart always be superior to trypsin as a means of dissociating cells. Iiz Advances in myocardiology. Vol. 1. Edited by hear3 cells as previously propc~sed(Cavanaugh et al. M. Tajuddin, P. K. Das, M. Tariq, and N. S . Ilhalla. 1963: kIasso11-Pevcl ct al. 1976). In fact. aggregates Urlivcrsity Park Press, Baltimore. pp. 23 1 -- 242. prepared from trypsin-dissociate cardiac ventricular K. ct.81~ from ?'-day chick ermlbryos display functional prop- LIEHAAN, I,. 1967. Regulation of spontaneous activity and growth of cnabryonic chick heart cells in tissue culture. erties more typical of intact 7-day ventricular tissue 5 e v . Biol. 16: 216-249. than thosc prepared from collagenase-dissociated cells. 1970. The potassium sensitivity of isolated embryonic heart cells iancreases with development. Dev. Biol. 23: 226-240. % ~ E H A A NL., and L. J . DEFEI~IC'E. C)scillatory propK. , 1978. erties and excitability of the heart cell membrane. dpl Periodicitics in chemistry and biology. Edited by H . Eyring. Acadernic Prcss Inc.. New York. pp. 181 -233. LIEHAAN, . L., and S. G O ~ L I E B . 'The electricai activR 1968. ity of ernbryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets. J . Gen. PhysioI. 52: 643 - 665. IIEHA.AN, I,. , and R. H ~ R A K U W .1972. Synchronization of K. pulsation rates in isolated cardiac myocytes. Exp. Cell Res. 70: 214-220. ,&rw~La.. I . CO~IE.N,EISNER. . OHBA, C. OSEBA. D.. D. M and E ~ ~ I ~ ~ A and E. '4. JOHNSOK. L., R A , 1980. Fast sodium current 1979. Ttac steady state of TTX-sensitive ("window") in cardiac nauscle. A quantitative description. Biophys. J. sodium curretat in cardiac Pa~rkinje fibers. Pflmegers Arch. 32: 779-790. 379: 137- 142. HEIIKY, . PJ., PI. S . FRIEND. J . SctiecJE~.1970. MarM and EBKHAKA, N. SIIIGETBP. LIEBEKMAN. E. A. L., kt. and JOHNSON. phology and metabolism of intact muscle cells isolated 1980. The initial inward current in spherical clusters of chick embryonic heart cells. J . Gen. Physiol. 75: frorn adult rat heart. Circ. Kes. 26: 679-687. BLISTARIANT'E, T. CVATANARE, 'T. F. MCDONALI). J . O., aiad 437-456. 1982. A new approach to tlae isolation of adult cardiocytes. F I S C F ~ MD. A .,, and A. '4. M o s c o ~ a 197 1 . Reconstitution I~N . Can. J . Physiol. Fharnaacol. 60: 997 - 1002. of heart tissue from suspensions of enabryonic nmyocardial cclls: ultrastructural studies on dispersed and reagCAVANAI!GH, W. 1955. Pailsation, raiigration am division M. rd of dissociated chick heart cclls in vitro. J . Exp. Zool. 128: gregated cclls. In Cardiac hypertrophy . Ec1itt.d by N . W. ABpert. Academic Press h c . , New York and Loiadons. 7 3 -585. C ~ \ V A N . ~ ~D.( J .I, IW. 0. BE:KNH)T, 'T.E. S ~ I I T H . ~ ; , and 1963. pp. 125-139. Dissociation of heart cells by collagenase. Nature GI.I('K, M . R., A. H. BIJKNS, and CV. J . R~rsrw. 1974. Dispersion and isolation of beating cells from adult rat (I.,oncBon), 200: 26 I -262. C I . . ~ P H ; Z11. ,E., A . SIIKIER, K. L. IIEHAAN. ~I and ICIMO. heart. Anal. Biochem. 61: 32-34. Junctional rcsistance and action potential delay between HARAKY, and 13. FARL,EY. I.. 1963. In vitro studies on single beating heart cells. I. Growth and organization. Exp. Cell embryonic heart cell aggregates. J . Gen. Physiol. 75: 033--6.54. Wes . 29: 45 1 -465. ~:L.AKK, C;., n. J . C;ANNOS. N. BOUICIN. . S . B'A'ITT'EN, hI. G HAWOK'FII, A., 1). K. HUNTER, H . A. BEKKOFF. K. and 1980. and M. PJ. R ~ R R Y . 1978. An inmproved procedure for the 'The isolation of Ca2'-resistant myocytes from the adult rat. high-yield preparation of intact beating heart cells from the J . 1Mo1. Cell. Cardiol. 12: 735-723. adult rat. Biochemical and morphologic study. J . Mol. Hor>c;~apr, L . , and A . F. MBJXLEY. 1952. A quantitative A. Cell. Cardiol. 10: 1101 - 1121. current and its application to condescription of n ~ m b r a n c CI-AY, . K.. I.,. J . DEFEI.ICE, J and M . I.,. DEHAAN. 1979. duction and excitation in nerve. J . Physicdl. (London), 117: Current noise paranicters derived from voltage noise and 500 - 544. iinpedance in crnbryonic hcart ccll aggregates. Biophys. J . HUME,J . K., and LV. GILES.198 1 . '4ctive and passive elec-

Acknowledgements 'I'his work was supported by grants from the Medjica! Rcscaa-ch Council elf Canada arnd thz Canadian IleaI-8 Foundation. M.K.G. is a recipient of a grecioctoral traiareeship from the Canadian Heart Foundation. Wc thank $. 0. Bustamantc for helpful conversations. P. KrraJeviC for prcsgramrning assistance, K. Ro7ansky for technical assistance. and S . James and C. Parnglin for typing the manuscript.

41 8

C A N S PFlYSLOI, Pilt4RMACOL VOL 61, 1983

tric:11 properties of single bul8frog atrial cells. J. Gen. .I. Gen. Physiol. 61: 89- 109. Physiol. 78: 19-42. M ~ D O N A L T., F., and H. G. SACHS. D 1975. Electrical actiarHvr)~<, A., B. Bax)NDEr2, A. M.~TTER, P. CHENEVAL, ity in embryonic heart cell aggregates. Developl-raenta! asJ. B. Frr,~oux, and I,. GIRARDIER. !969. ~HORIC) and heteropect~ Pfluegcrs Arch. 354: 15 1 - 164. cellular junctions in cell cujtures: an electrophysioEogica1 MCDONALD, F., H. C . S ~ r ~ r ranci W. I,. DEHAAN.1972. 7'. s, and naorphological study. Prog. Brain Res. 31: 283-3 1 1 . Ilcvelc~pnicnt f sensitivity to tetrodotoxin in beating chick o ISF.NBF.RC;. and kJ. KLOCKNEK. G., 1980. Glg~cocalyxis not ernbryo hearts, single cells arltf aggregates. Science required for slow inward calcium current in isolated rat (Washington, D.C.), 176: 1248-- 1250. heart rnyocytes. Nature (London), 284: 35%- 360. MC)SCANA, A. 1960. Patterns and nsechanisnis of tissue A. J ~ c o ~ s o r S ., I,. 1977. Culture of spontaneously contracti~sg u reconstruction from dissociated cells. bn Developing cell rasyocardial cells from adult rats. Cell Struct. Funct. 2: systems and their control. Edited Oy 1). Raldnick. Korlald 1 --9. Press. New York. pp. 45-70. -1965. Recombination of dissociated ce%lsand the JQBN(;SMA. J . 1972. Synchronisatie in ialteraktic van hartH. cellen in weefsclcultuur. Ph.D. thesis. Atrasterdam. development of cell aggregates. I n Cells and tissues in Joxtisbfia, H. J.. M. h%i\s.c;c>~-Pkv~r, HOL,LANI)I'R, C.'. C. and culture. Edited by E. N. William. Academic Press, New J . rx: B K L ~ ~ J N E . Synchronization of the beating fre1975. York. pp. 489-529. quency of cultured rat heart cells. bn Ilevelopnsental and P ~ W E I ~ L .and V . W. ?'v\/IsT. 1976. A rapid technique for 'T., , physiological correlates of cardiac muscle. 6:'Llitcd iri\ the isolation and purification of rada11t cardiac rnuscjc cclls M. P,icbcr~nan and T. Sano. Raven Press, New York. having respiratory control and tolerance to calciuna. Biopp. 185- 196. chem. Biophys. Res. C:onlmun. 115: 183- 189. K ~ s s K. S . , and K. W. 'I'SBEN. , 1975. Multiple effects of RAJS, J . . M. SUNBERG, R . SCIN[IBY. I)ANEL,B.. . G. N. G calciuni antagonists on plateau currents in cardiac Purki~sje TOKNLINC;, RIBI'RFEID, S. W . J ~ l i c ~ ~ s s I97X. A P. and ox. fibres. J . Gen. Physiol. 66: 869- 192. rapid method for the isolation of viable carcliac myclcytes KASTEN. R . 197 1 . Cytology and cytochernistry of rnamF. from adult rat. Exp. (:ell Kes. 115: 183- i89. rnalian n~yaacardialcells in cultures. Acta Histochernica ROBINSON. B . , and M. J . I,EG.~To. K. I9XO. Maintained differentiation in rat cardiac rnonolayer cultures: tetrodotoxin Suppl. 9: 775-805. sensitivity and ultrastructure. J. Mol. Cell. Cardiol. 12: K O B I L H A RM .~ ~ ,. BAUER, KKACSE, A. F;LECKEND, B H. and 493-498. STEIN. 1972. Differentiation of' the transinera1br:ane Na and Ca channels in rnan~rnajian cardiac fakers by the use o T Kusrr, H . , and H. I ARSEN. 1978. A practical algorithm fhr solving dynamic rnemibrane equations. IEEE 'Trans. Biospecid inhibitors. Pfl~~egcrs Arch. 335: 309- 322. med. Eng. 25: 389--392. Kor;o, 7'. 1969. Roles of collagenases and other protocolytic SAC'IIS,. C;., and W. L. DEHAAN. H 1973. Embryoraic Inyoenzymes in the dispersal of animal tissues. Biochini. Biocardial cell aggregates: volume :and pulsation ratc. [lev. phys. Acta, 178: 397-400. Hiol. 30: 233-240. LEC~~Z~I'C),1972. LIltr-astructura! characteristics of the rat M. J . ventricular cell grown in tissue culture, with special referS A C ~ I S . C;., 'F. F. M C ~ ~ O N A L ~R. ,I > . IIEB-BA~N. H. and D 1973. 'I'ctrodotoxin sensitivity of culturcd ernbryonic heart cells ence to sarcomerogenesis. J . Mol. C'ell. Cardiol. 4: depend on cell interactions. J. Ce$l Btiol. 56: 255-258. 299-317. %I-IANNE, 0. F., E. RUI%-C~;,R~~TTL, C. RPJARL), and I). LIERERMAN, 1967. Effect of cell density :and low M on M. C ~ ~ A K T I E K . Ileternainants of electrical activity in 1977. action potentials of cultured chick heart cells. Circ. Res. clusters of c ~ ~ l t i ~cardiac cells horn nconattrl rats. J . MOB. 21: 879-802. red Cell. Cardiol. 9: 269-282. LOMI'KE, M.. 1 . PAGC~B*BOB,I.. %;nssou'r. 1979. A. and G SCOTT,S. I979. Stimulation simulations of young yet culMaintenance of kist Na channclls during primary culture of tured beating hearts. Ph. 1 . thesis. State L!niversity of New 1 embryonic chick heart cells. J. hlol. Cell. Cardiol. 11: 813-825. York. Buffalo. M.~~CKENZIE, N. 13. STANDEN. E., and 1982. The effects of SBIKIEK, and J . K. CLAY.1980. Pacernakcr currents in A., chick en~bryonic heart cells change with development. stimulation ratc on calcium-dependent action potentials Nature (1,osadon). 283: 070-67 1 . recorcied from chick embryo heart cell aggregates. J . Phys1982. A cornparison of the pacemaker p ~ ~ p u ' f i c s iol. (London ), 324: 1 - 10. of chick embryonic atrial and ventricular heart cells. MARK, . E., and F. F. STRASSER. 6 1066. Pacemaker activity J . Mcmbr. Riol. 69: 49-54,. and mitosis in cultures of newborn rat heart ventricle cells. Ski~rkr, E., arad W. D. BEIRNIIT. 'F. 1964. The establishment Exp. Cell. Res. 44: 217-213. and M f $ s s c ~ ~ - P k vM.,, J . H . JONC;SMA, J . DE BKUIJNE. of beating myocardia! cells in long term culture in fluid ~r mediura. Exp. Cell Rcs. 36: 179- 191. 1976. Collagenasc anci trypsin dissociated heart cells: a N . , and ~IL. comparative ultrastructural study. J . Mol. Cell. Cardid. 8: SPEREII.AKBS, D. L E I ~ M K I J1964. Effect of current on trtins~-r-aembrarae potentials in culturcd chick heart cells. 737 -- 757. J . Gen. Physic~l. 895-927. 47: M c A r , ~ r s r ~ K , E., D. NOBLE.and K. W. TSIEN.197.5. R. 1965. Ir~scnsitivityof cultured chick heart cells tc~ Reconstruction of the electrical activity of cardiac Purkinje autonorrsic agents and tetrodotoxin. Am. J . Plmysiol. 209: fibcrs. J . Playsiol. (London). 251: I -53. 693 - 698. ~/ICIION~ZLD, and 8.L. DEHAAN.973. Ion levels and T. F., 8 SPERELAKIS, K. SHI(?ENOB(.J, M . J . MC-LEAN. N., and 1956. membrane potential in chick heart tissue and culta~r-ed cclls.

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