diane colizza et al- a comparative study of collagenase- and brypsin-dissociated embryonic heart...

8/3/2019 Diane Colizza et al- A comparative study of collagenase- and brypsin-dissociated embryonic heart cells: reaggregati

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

I ~ I A N F . Ccsr,~a,a,~,ECHAEH.. ~ ~ ~ I E v ~ ~ K . A ,~ 1 4L V ~ NHRIERDepartnzerrt 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,

Kcceivcd June 2. 1982( 'OLIZZA. D ., M . R . ~ ; ( : E V A R A , and A . SIIRIEK.983. h comparative study o f collagenase- and trypsin-dissociated e!i~hryoheart cells: reaggrcgatioe~,c.lcctrophysiology, and pharmacc~logy Can. J . Physiol. Pharrm~acol.61: 408 - 4 10.

'Il'hc elcctrophysiological and pharmacological properties of aggregates prepitred from ccEls of 7-day-old chick en-ehryo hc;irventricles depend on the enzyme used for cell dissociation. The mean beat ratc of aggregates formed fn-on-e ryp"ra-dissocik1at:c1cclls was about 53 beats/min whereas ap.grcgatcs fornmed from collagcr~asc-dissoi:iatedcclls hacl a nican heat ratc 01' mori: thantwice this value. Spontaneous activity of most aggregates forn-acd from trypsin-dissociat6.d cells was inhibited by elevatie~external potassium or by adding tetrodotoxin to the anedium. A similar response to pot;tssia~ai~ll:as been in all aggregates formccfroin collageniise-dissocii~tedcells. However, appa-oxirnateiyhalf of the aggregaks formcd from collagenasc-dissoaait1tcc1ccl8were tetrodotoxin insea~sitivc. ntracellular ~nicroelectrode ecordings denaonstratixl that aggregates ftrrii-acd frcw cr~liugenasciiissociated cells typically had reduced action potential tr~axilrrlalupstrol


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1963; Smith ;and Berncft 1964) a n d Ladula carc iaac ceBBa( B e r r y e t a l . 1970. (.;8ick c t a / . 1074: K tsnc ~ 1909:P o w e l l a n d T ah ~i \t I 9 4 6 ; I h n i c t ;dB. 1077: C'lark e t al .1978; Raj4 e t a1 1978; I \ e n h e r g :and K l o c k n e r IOt iO:Hu\tarn;ante ct 611. 1982, Hunnht a n d G i l e \ 1981)C a ~ l a n a u g h t a l . (I963) o k \ e r v e d t h a t C P L B ~ Co l l a g e -na se l a e l t i e d nnore c oe np le t e d i i \ oc l a k r on of e n l b r y o n i cc h i c k h e a r t and w a a It .\ \ d a m a g i n g t h a n t r y p \ ~ n . a \j u d g e d by \cvera l cnte r rra insc luding ce l l app ear an ce i t a dv r a b i l l t y . M a s e o n - P 6 v s t e t al. ( 1'476r a l ~onc luc iedtha t c o l l a ge n l t \ c wa\ \ i ~ p e r ~ o ro t ry p \ I n f o r c ' c ~ I I d l \ -s o c ii a ti o n , b a w d o n an ul t ra \ t ruut~ r ra l terdy o f I I ~ M o rnr a t h e a r t t i ~ \ \ o c ~ a t c c iy the k w c ~ n t y m e \ .The p u r p o \ e of tklns \tudy w a\ t o t f e tc r rn~a lcw h e t h e rt llerc. we re an y c lec t rophy\na>log~ciiB r p l1; i rm; i~ olo~ ic ;11d l f f e r e n c c \ b e t w e e n t h e c e l l e ne n a b ra n e i ot c u l t u r e dc e l l \ d l s \ o c i a t e d by t h e \ e tw o ag en t \ . W c coraaparecf thep r o p m t i f i of h e a r t c e ll a g g r e g a t e \ p r e p a r e d f~cjtn - d a j -old c h i c k e m b r y o h e a r t v e n tr i c l e\ t i i \\ c ~ c l at e dw r th t r yp -\ In o r c o l l a g e n a \ e . The a g g r e g a t e ~ r a o r p h o l o g y . beratrate, e l e c t r o p h y \ ~ c s l o g y , r ~ d ha r nna c o logy me r e f ounc lt o d c p c n d o n t h e d i \ w c i a t s n g c r l zy n se u \cct In p ; l r t ~ c ~ i -l a r , a g g r e g a t e \ p r e p a r c t i f r o m collagenasc-cf~\\ocii~tt 'cic e l l s d l a p ' l a j c d a r e du c e d upk tr tske ve loc i t y of th e iactlonpotent la1 and a d c c r c a w d t e t ro t lo t o x in \ c n \ it i v l ty .U nde r t he c ond l t l c sna use d i n t hi \ \ t u d y , w e c o n c l u d etha t aggrega te \ prepared t rorn t rygs \ ln-c l i \ \a ) r : la ted ce l l \c - ln iplay c lec t rophy\ io8og~ca l nd p la a r ~a aa c o log i ca l r op -e r t i e \ more tygslcal of nntact ?-day ucntr iceilar t14sa1et h a n t h o a e p r e p a r e d f r o m collagcr~ase-di4\c~ci~itede l l \ .C'oan puter sxlnnuldtxlon\ bd\ecl o n ;mil i on i c ~a l c ~c i e lr o mk o l t a g e - c l a m p n l e a a u r e r n e n t s c Ehiharca an d . l ohn \on1980: Clray and S h r i e r led81 t r ) w g g e s t t h at t h e c x p e rl -~ r- ae n ta l e \ u l t e c a n b e e xp l a ine d by a r e d ~ a c e d ,lst i n w a r dw d ~ u r n ur r e nt a nd an ~a ~c r t . ; t \ e t ie a k a g e c ~ a r r c n t ncollageraa\e-d1\soc1;1tec1 e l l \ .

J ' ~ - P / ~ [ I Y ( I F ~ o ~ II;ertilizccI eggs frona Whi tc Legh orn hcns wcrc iracit batedar 17'Y ' ;ma1 85


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4 10 C A N I PIiYSTOL PHXRM4C'OL VOB h i . 1083

D i a m e t e r (urn)Fit;. I . 'Thc sire t i istr ibut~onof 'r aggregates ( E Z -= 403)an d C aggregates ( n = 243).

iraore rapid upstro ke ph ase . M casurerncr-its of V,,,,,,were madewith an electronic differcntiatc-lr which was linear froin 5 kl/sto 1000 V /S .In this stucdy PI always rcfers to the number of aggregatessampled and docs not represent arluitiple mciasurerncnts fro111a singlc preparation.('w??putor- i r i l i r ic i f ior! .~Th e chc-tar~putntiorasere citrrittd out on an HP 1000 Series-Fcon lpu ter (He wlett---I'ack arci) locatcci in the llepartn-rent of,4nacsthcsia Research at McG il? hlniversity.

Aggrc-'g~l~bo~tAggregates forrncd during 48-72 h of gyration cul-ture i'rom trypsin-dissociatd ventric~m1:trcells ('F aggre-gates) ivcrc comparetl with those fornmed tron-a col-lagcnase-tfis~ocii~tedells (C aggregates). Figure I\ho \%\ t h e d i ~ t r l b u t i ~ r ~f diameters o f T aggregatesfrom 15 cultures and C aggregates froan 12 cultures (ofwhlch 8 were paired s~rlttare\).Most ( 7 2% ) F aggre-gates rangcd in diameter between 1561 and 250 pmwherea\ nnost (70 '%)C aggregate\ ranged betvlleen 50an d 150 prn: on14 7'CX7 of T aggregates were smaller'rt dtes werehan 150 pna while only 16% of C' agh ' g dgreater than 208 pan. The nacarl diameter (t D ) o f Taggregates (210 .3 z 4.3 km , ra = 403) was \ignifi-cant]? different ( P < 0.01 f rom that of C aggregates(134.1 t 63.8 ptn, ra = 2 4 3 ) .

" I 'a % oa 8 *8 ' e 8

V o l u m e ( y m 9 1

Frc; 2 . Kate- volume plot of 'I' aggregiatcs (( 1. l = I ( 17and C aggregates (a,z = 1 13). Arrows at '4 an d B Lare draw n:at volumes calcu1iati.d using 800 and 200 pin diaaneters.respectively. Heat rakes were obtained by viw ally monator inycontractions

FK;.3 . The distiilaurion of beat rates of I aggregates( P Z = 107) an d C aggregates ( n - 1 14).hire dishes they remained spheroidal f i ~ r everal imoursand beat spontanecpusiy with a regular rhythm. I t wasshow n by Sachs and IleIHaan (1973) hat aggregate beatrake ia inversely rclated TO aggregate voleln~e.Fkmcre isevidence of a sim ilar volunnc-kcat rate trerad fo r Taggrh=gated(Fig.2 ). Morecsver, there is good currespon-dence betweera the beat rates fbund here in the10" 110' p a 3 volunre range and timcjse reported pre-viously (Sasl-as and IIelHaan 1973) . In contrast, beatrates CPC C aggregates tend to be independent of aggre-gate voiurme. For example, the mean beat nate (t 1))

,Yponscrnsous I~t- 'uf"'hlt~ of C aggregates with v d u m e s in the range berwcen 10VvJhenT and C aggregates were pliasctl ia r tistptae cul- and I O h pm ' 4 1 15.1 k 25.2 keatsjrnin: r? = 70) is not


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('01,IZZA El' hi.

TABL.~ ; .. Effect of external potassium concentraticdn on spontaneous activity-Beat rate(Beats jmin)

H .3 nlM 1K+]( , 2.5 rnM % decrease-- - - - -?' aggrega tes 52 .3 22 .9 23 .826 .8 54

(I Z = 10'1C aggregates 9 1 .6 5 15.6 5 1 . 2 5 12.8 44( n = 8)Nc~rt : : Beat rates given ar e mearl5 5 SL9.

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

significantly different horn that of & aggregates in thevolume range 10-0 I07Ftn' (113.1 -t 19.3beatslmin; rr = 44 ). Ho we \er , in the volume range 10"to 10' pnl'. the mean beat rate of C aggregates wasappmxinnately double that of 'r aggregates (53.3 a83 - 3 beats/mins; n = 104). Figure 3 shows the distri-bution of spontaneous beat rates for T and C aggregates.I t has been previously demonstrated that increasingthe external potassium concentration ( K t ,, will slow orabolish spontaneous activity in isolated embryonicheart cells (DeH aan 1970) and in T aggregates (IIeHaanand DcFelice 1978 ). Table 1 shows the decrease in

spontaneous beat rate of both 'I'aggregates and G aggre-gates 4 120- 150 prn diameter) that occurred when[ K I., was increased from 1.3 to 2.5 mM. In both case s,a further elevation of [ K , , to 3.5 mM resulted in irreg-ular beat rates characteristically lowcr than 1 beatlrnin.At aK t I,, of 4.5 nmhf all spontaneous activity in bothgroups was arrested.It laas been p reviously reported that m ore tlaan 805%ofT aggregates stopped beating in 10 g/m L TTX(McDonald et all. 1972). 'These results were replicatedin five experiments b r a = 4 5 ) in which 'l'TX at dosesbetween 10 ' nd 5 x lo-' g/mL stopped the beatingof 70'141 of the T aggregates. Any residual spontaneous

F I G .5 . ( A ) Su pe rin ~p os ed ction potentials rccordeef froma T aggregate an d a C aggregate. BB) I!pp@r pair of traces:superimposed upstroke phases displayed at a faster sweepspe ed. Vertical calibration bar is 50 my. ower pair of traces:differentiated upstrokc phases.beating was generally irregular and sporadic bursts s factivity were occasionally observed. In contrast, theresponse of C aggregates to TTX was less uniform. Insix experiments ( n = 5 1 ) spontaneous activity per-sisted in the presence of 10-' to 5 x 10-1 g/ m L TTX:in onc of these experiments spontaneous activity per-sisted when the TTX concentration was increased t~10 ' g / m L . In eight other experiments ( n - 56) TTXat doses betweens 10 -' a nd 5 x 10 g/rnL stopped thebeating of 45% of the C aggregates. Residual activitywas sporadic.Elec-trophysicr/ca~ySpontaneous electrical activity recorded frorar 'F andC aggregates is shown in Figs. 4 and 5 . 'The pooledresults for the action potential parameters measuredfrom 12 experiments ( 1 2 = 14) using 'T aggregates andfrcm 1 1 experiments ( n = 25) using C aggregates arepresented in Table 2. Recordings were made afterimpalements had stabilized, as reflected by a constantinterbeat interval and stable action potential pararne-


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('AN I I't IYSIOL PHARMAC'OL VO L 01 . I cW ?

ters. Action p otential of 7' and 4: aggregates whic\-ehadsignil-icaritly diffc rcnt inferbeat in terva l\ and ntaximumupstroke velocities dicl not havc significantly ditTercnaction potential durations.The addition of 'FTX ( I 0 ' to 5 x 10 ' g,/mL) led toa prcsgrussive reduction of V,,,,, in 'r aggregateb andprolongation of the interbeat interval (Fig. 6). In addltron, there was a dcpolari~ation r a the lcvcl of thethreshold potential. After several minutea tho pacemaker depolarization failed to reach thre\hold armdspontaneous acalon potentla1 generation was blc~ckeThc mean resting potential C -f SD) of these aggregatewas found to be -54 .3 -+ 9. 7 rnV o r = 12 ,A t~im ilar esponse to T TX wa$ fsslnct in \ome (ag gregrates ( F I ~ . ). Following cessaslon of aca-ivit!/the mean resting potentiat of these preparation\ wa-54.9 t 7 .2 ma/ (f z = 8) . I iowcver , in six other Caggregate\ \pontaneou\ act~vity was serstaincd nnthe presence o f TTX, with reducecl up\troke veloc~tland depo l a r l ~ed hre\hold pstentaal Bevel\ (F ig . 7 andTable 2) .The add~ t l onof the slow channel bIocker I)-GOO(Kohlhardt et ul . ld842B at a concentration o f 2 X10 ' i r n k tc9 a C ' aggregate beating in th e pre4cnse oTTX blocked spontaneous activity 'Il'hc ability (st I >600 to block spontaneous ractavliy can also be ob\ervecin C aggregates and T aggregates in the ab\ence oTTX: in two of three 7' aggregates and four o f four Caggregates 11-600 (2 x 10 " g/mL\ blocked spontaneous activity. A comparison of electrlciil actlvity of the?' aggregates hefore and after 13-600 \ h o w \ that 11-60reduced the ovcrjhoot, shortened the action potentiaduration, apd reduced the in te rbea t ~ n t e r ~l , but ha d noeffect c ~ r a I/,,,,,, (Fig. 8) . Figure 8 al\o \ h o ~ v \ hat thcrc\ponne of C aggregates to 11-600was \irnil:ar but the rew;is a decrease of V ,,,,,.Sinzulnf io/~ f pact~makcr- l ~ d psrr-okcp l ~ c r . ~ 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 asystem of differentia! ec~ uations tb~aineci rom voltageclamp measurements of backgroinnd (/,,,) and timedependent pacem~tker(I,,) currents in 7-day trypsindissociatcd vca~tr ic~~larggregates (Clay and Shrie1981a ) . Measurements of the T'I'X sensitive, kist inward sodium cur-rcnt ( I Na )ad e in trypsin-clissociatcdaggregates were also incorporated into the mode(Ebihara ct al . 1980; Ebihara and Johnson 1980). 'Theparameters of the model arc ssaiecI Bi~r a 200-p.,rradiameter apgregate which has approximately 2 .1 5x I OF' crmr of mcrnbranc su rfacc area (Clay and ShricI9MBa). All currents arc given in aianoamperes, voltages in m illivoits, cc~ndu ctances n nlillisiemens, rateconstarats i n inverse se co nd s, and tinmes in seco nds . Th e


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C + TTX

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 ' /mL).

K ' I ~ ; .7. ( A ) Su~~erin~poscdction potentials recorded froma continuous impalement of a single ccll within a C aggregatebefore (C) and after (TI'%),5-n~in xposure to 10 ' p/rnLT'I'X. (B ) Upper pair of traces: superirmposed action potentialupstroke phases displayed at a fiister sweep speed. Verticalcalibration bar is 50 ITIV.Lower pair of traces: differentiatedupstroke phases.transmeallbrane voltage is denoted by V.Ihgs given by the surn of two tianc-independent cur-rents:

~ 2 1 I,, = 22 0 (?') t p - I + + .2 t- + j3i)+1 . I ( V + 50) / ( 1 - exp (- V + 5(3)/25))

where[3] p = ( 1 + c x p ( - ( V T 9 0 ) / 2 5 ) ) ' , y = 1 - p ;

Y = ~ I P .lhlhs given by[4] J N d b = 0 . 2 3 ( V - 4 0 ) .The I , , component is given by[S ] JM ,= ~ ~ ( ) s x ' ( M '2 ) / ( 1 + .Y -t x')with[64 w = ( 1 + exp( - i V + 112)/25)) ';: = 1 - ul ;

X -- S I M '- /and[7] cHs/dt = -(cu, t P,)s + a,with rate constants[ 8 ] cu, = 0.8 ( V + 551161 - exp ( -0 .2 (V 4 5 5 ) ) )

p, - 0.059 exp ( -0 .105 (V t 55) ) .'Thc T T X sensitive, fast inward sodium current ( I h d ) sgiven by

PK I is given by I.91 IKd m3h ( v - 40)


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C A N . J . PIIYSIOL. FHARMACOL. VCIL, 61. 1983

RG.8. Cornparison of the effects of D-600 (2 X 10- ' jr nL ) on action potentials recorded fro m 'r aggregates rind Caggregates. 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 superim posed rapstroltephases froan these recordings, while the Bowcr pair of traces show thc upstroke velocities. Scale: 50 tnV verlicial, 0 - 5 shorizontal (A ) , 5 ans horizcsntal (I%). Right hand panel: (A) Superilnposed action potentials recorc%cd rom c o n ti n ~~ o usmpaIe-rnent of a cell within a C aggreg ate before (C ) and after (D-600) 12-min exposure to D-6(#). (B ) The upper pair of traces showsuperimposed upstroke phases from these rec~ardings,wimilc the lower pair of traces show the upstroke velocities. Scale: 250 rnshorizontal (A:), 5 rns horizontal (B ) .

FIG.9. Simulation of membrane potential during pace-maker and upstroke phases of the action potential. (A ) Mem-brane potential when jN d 32 3 mS and I,lpp 0 nA .(B ) Membrane potential when iIVa40 mS and I,,,,, 5 nA .with

where[ I 14 CY,, 100 (V + 45)/(1 - exp (-0.1 ( V + 4 5 ) ) )

al, 135exp ( - ( V + 80)/h.8)

p,, = 356 exp ( 0 . 079V )+ 3.1 x \ O q e x p ( 0 . 3 5 V

The net ionic membrane current is given byL 121 1,0,1,' = I N ' , + IK ?+ lhp.

The expression for IN , , in Eq. 9 is the same as thatused by Hsdgkin and Huxley (1952) to describe I, , innerve. 'The expressions for the rate constants a,,, nd PI ,in Eq. 1 1 have the same functional form a\ in theHodgkin-Huxley equations. The rate constant\ for ahan d PI, were taken from Ebihara and S o h n w n ($980)The ganembrane voltage was obtained by numericaIByintegrating[131 d V / d t = (I , , , , , + I,,,,)/Cwhere I,,, is the externally iq ec te d curren t. The pararn-eter C is the input capacitance of an aggregate. wh~chas been determined previously to be 0.023 pF fo raggregates of 200 Frna diameter (C lay et a l. 1979).Theinitial ccpnditicpn on membrane voltage was \et to be-90 mV, which closely approxinnates imaxisrlurn di-astolic potential (MDP) for both TF and C aggregates.We also set the activation parameter s equ:aI to 1, sinces is fully activated at the action potential peak and doesnot change rapidiy during repoiarizatiotm. We initiallyse t rn = 0 and h = 1 , which approx i~nate teady -~tatvalues of these parameters at MDP. 'The time courge of


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voltage change from these initial conditic~nswas cieter-mined by numerically integrating Eq. 13 using the algo-rithm of Rush and Idarsen 1978 ) ui th a 50-[AS ntegra-tion tim e ste p. Each sirnellation wa s terra~jirlated at-40 rnV In the cas e of T aggregates I,,,,tis O an d g,,,was set at 32 3 mS, whereas in C aggregates-Sy,YiiSreduced to 60 ra1S to reflect the reduction of V,,,,,, rom120 V /s in T aggregates to 24 V/s in C aggreg ates. 'Tosimu late the more rapid pacemaker tiepolari~ritio n n Caggregates. I,,, was \et to 5 nA . ? 'he re\~alts hc~w n nFig. 9 yualitativcly mimic the major differences in theeleetrophysic~logicalpra)perties c)frY lin e Ab and C (lineaggregates: a Inore rapid pacemaker depolari7ationan d a \lower upstroke phase of C aggregates. More-over , the s imula t ion\ a l so show the depo lar i~a t ion fthres hold potet-atial obseralcd in C slggregate\ (F ig . 5 ) .FanaIiy, we note that neither reduction of KN,, nor thechange of I , , , alone could account for the observeddifferet-accs betw een T a nd C aggregates.

DiscisssisnThe results of this study indicate that collagenase-treated cells formed sm aller aggregates tl ~ an rypsin-treated cells. 'I'hc size of aggregates has bcen shown todepend upon the tissue origira of the cells uscd, theirdevelopmental stage, thc rnodc of cell isolation, and the

conditions of cel rotation (Mos con a 1965 1. When thespeed of rotation is too great. the shearing forces excee dthe mutual adhesiveness of the cells and aggregation isprevented. At slower rotation rates, aggregates formfrom cclls which, upon contact. attach firmly enoug h toresist the sllearing forces. Under the same conditionssmaller aggregates will be formed from less cohesivecells (Moscona 1065). B'he fact that C aggregates weresmaller in size than T aggregates inciicates a possibledec reas e in cell-cell adhcsiv ity in C aggregates. 1-low-ever. Cavanra~igh t al . ( 1963) found that collligenase-ciissociated ceBls adlaered nwre rapidly to glass c~tlturedishes and tended to flatten faster. The adhesion of cellsto glass may involve a process different from thatinvolved in cell -cell adhes ion. I t has been posttaliatedthat cell recognition and selective cell adhesion arcnaediated by interaction of spccific cell surface conr-pcsnents (M osc ona 196 0). Furthe rmore , isolation tech-niques ( e. g . , enzyme type and concentrat ion) and crrl-ture conditions can influence adhesiveness.In this strstly, C aggregates on average beat morerapidly than 'I' aggregates. A comparison of the sponta-neoers act ivity of ( an d 'P aggregates sl-ao\vs hat there isa rnarked ciiffercnce in the rate of pace n~ak er iepolar -ization (Fig. 4) . The rats of pacemaker depo'iarizaticpnreflects a rather fine balance of in\vartl alad outwardcurrents . Ind eed, it has been observed that a few nano-amperes of s teady applied outward currcnt can can-

pletely block the genera tion of' sp on tan eo ~l s ktcclonpotentials In aggreyatcs of cardiac cells, whereas ascerzdy in\varct current can increaee the beat ratc (Scott19 79 ). Sim ilarly , irnrnctfiarely after microelec trode~rnpa l emen t , apid beat rates are often okserved. 'I'heseare presumab ly the result of iqjury-induced nonspecificleakage currents (S hr ler and CBay 1989). 'I'hc sinmu-lation~spresented in this study are con\lstenb with thehyp othe sis th at tre atm ent wlth cru de coBl:agenra\e le-adsto membrane di\ruptivn giving rise to a nonspecificmembrane leakage currcnt (Fig. 9 ) .Modifications of nlembrane currents by enzymetrea tme nt ma y co nce ivab ly saccount for the cfifk'erence inspontaneous act ivi t j between C and 'I' aggreg ates. 'I'hefast inward s~~dlurmurrcnt has a component whrchralaintains steady leveBs of activataon in the pacc~nakervc~Itage ange (th e winadow c urre nt, I,,, This windowcupren t not on ly al-fects actlo n poterataal para me ters(Atw ell et al. 197 9), but also pacemaker dcpolar-iaatic~n rate and consequently spontaneous beat rate(Mc Allister et al. 1975; Shrier and Clay 198'9). n con^puter sirnulation\, a reduction of the fast !nw;ard sc>dau~ncurrent (av h~c h ccoun ts for- the reduced I;,,,,, in C ag gre-gates) results in a slowing of the pacemaker depolar-ization rate. Wh en ,ijNCIs rcdelced fron-a the con trol valu eof 323 to 60 mS, pontaneous activity ceases. Changesin the ttane-deperadent pacemaker current (Ik,an 7-clayventricular T aggregate\ have been shown to be pri-nlarily responsnble for the ce\ration of spontaneousactiklity when ( K t ,, is clevatecl from 1.3 to 4.5 naM(Clay and Shrier I98 1 a ) . 'T'hc deg ree of slow ing of beatrates of t ggregates at elevated ( K t ,, wras sim~lar othat found in 'I' aggregates. Furthcrrnore, ?' and Caggregates ceased beating at slmllar 1 K ' ,. 'I'hi5 \ug-gests that lk, is present in C' aggregate\ ancl at leaat insomc respects i\ similar to Bk, in T ~zggregatcs.A pos-sible role of othe r cur-rent\ (prima rily background cur-rents) cannot be ruled out. Indeed. the ce\s;aticsn ofsponta neous activity of ventricular ti sw e denrang e~ n b ry -onic development ap pe ars to be drne to a reduction of thebackground inward sodium current I, ,,,(Clay and ShrnerBY81 R ) . Bnlthough we have shown that a nonspecificleakage curre nt suffices to account for the differences inbeat rate , possible ce>ntrihttklon\ of I,,nd i,,,cLinnotberuleci out. To evaluate thc contribution of 6, and I,, ,w o~ rld equire tracer-flux meastarements coup led withvoltage-clamp experiments.In C' aggregates, lapstroke velocities wath a mekin of'24.3 V /s were ob3erved. su ggesting that the fdst soda-urn currcnt becanae rnarkcdly reduce d ti~llowlang olla-genase treatment anti cell dissociation . T T X wab effec-tive In redeacing V,, , , ,of these aggregate\ by a f a c t ~ ~ rffour ('H'able 2 ) , de~n onstrat in g he retention of rt 'I'TX-receptor ~nterac t ion .The observed depolara~ationofthreshold potential after 'I'TX was added (F ig. 7 ) is also


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consistent with the hypothesis that TI'X blocks the fastsocliuraa surrcrat in C aggreg ates. The ability of C aggre-gates t o maintain s pu~ atan eou s ctivity in the presenceof 'L'TX cou ld resu lt frona residual 'I'TX-insen sitive fastsodiulra channels or alternatively from slow inwardNa- Ca chaasnels. 'I'lae observation that D-600 abol-ished the sporstaneou s action poteratials in 1"FX -treatedC aggregates and slowed the upstroke phase of C.' bu tno t T aggregates (Fig. 7 ) is consistent with the latteralternative. fioevevcr, i t should be noted that D-600increased beat rate and suk~sequentlyblocked sponta-~ G C P L I S ctivity in '8' rzntl C aggregates not treated witlaT'FX. I'his nray be attributed to known effects of D-600crn ou tward potassium c~ rrr en ts Kas s and Tsie1.a 1975).Cultured cardiac cells which have a reduced 'TTXsensitivity or V,,,;!,have been described as dediffer-entiated (Sperelakis et al. 1976). C: aggregntcs displaythese prc~ pertiss ; how eve r, othe r electroplaysiologi6.balparameters of cells at earlier stagcs of dcveIoprnerat areabse nt. Th ere is rao decre ase of the M D P , overshoot, oraction potcrntial duratiora ira C aggregates to values typi-cal of younger embryonic ventricular cells (McDonaldand I>cHaan 19-72: McDonald and Sachs 1975). Con-seytiently, nlc conclude that the ef'fect of cell ctis-sociation using crrade collagennse does not lead to aconsplete dedif'ferentiation of nlernbrane fianction after2-3 days of gyration culture.Schan nc et a!. ( 19 77 ) have deraaornstrated that ta-ypsin-cfissociated neonatal rat cell coloasies develop T'TX-insensitive action potentials and develop slow re-sponses after 4-6 days in cu lt~r re. his charagc has beenattrib~atctl o the trypsir~ization procedure aa~ dnot todedifferentiaticm. Lt is conceiv;able that this type ofdissociation-i~acti~cedr a n s b r ~ n a t i c ~ nould asso occur in'r aggregates but with a much prolonged time ccjurse.Enzyme-dissocia~edited cell s wh ich are cu lt~n red asmonolayers have been reported to be T7'X insensitiveand to lack a East action potential upstroke (Sperefakisant1 Lehmk~nhl1965). However, i t has beelm recentlyctemonstrated that try~ssin-dissc~ciatedells in mono-layer culttire can retain TTX sensitivity and a rapidupstroke velocity Ql,ornpre et al. 1979; Robinson andLegato 1980). Excessive trypsinization was s~eggestedas a possible explanation for the poor recovery observedira the earlier inono layer cultiare studies ( Ko bins oa~ ndLegato 1980).Ce lls fr(1111 7-day embryo nic chick hear t in T'FX -insensitive mo nolayer cultua-es can recover T T X sensi-tivity when dissa~ciated nd reassembled into aggregatesQSachs t a? . 197 3). This sugg ests that recovery depen dson cell-celi interactions. The absence of TI'X sensi-tivity in some C ag gre ga tes su gg est s that the re rmaay b cscme disruption in the recovery process normally seenin T itggregates. The beat rates of C aggregates areindependent of aggregate size, and the raaean beat rate

an d distrib~ ation f h eat rates a re xiralilar to those dete r-mined from single ~nycpcytes obtained from 7-dayembryonic hearts QSa chs nd DeHaan 19-73).The sinai-larities bctwcen C aggregates and single cells naaybc due to loss of scpr-aae ce ll- ce ll in teractio ns in Caggregates.H is of interest to note that 'FT'X sensitivity andrapid action potentiai upstrokes are t ~ l l yxps-essecl inaggregates prepared froin collagenase-dissociated neo-natal rat heart c ells (dc: Bruijne and Jongsnlu 1980).I4owever, aggregates of 7-day embryonic chick heartcclls prepared slsing collagenase at a cc~nceratratio~tentimes higher than that used in this study demon-strated slow upstroke velocitie\ 6-32 V / s ) which werereduced (to 5 V / s ) upon addition c~f10 "/rial, 1"1'%(Mackenzie and Standen 1982).A mixture of trypsin and cc~llagen ase as been shownto be effective for the isolation of viable single anyo-cardial cells fionm adult rats (Saclobsora 1977). On theother hand, an earlier report QLPellaan1964) indicatedthat tlissc~ciationof embryonic chick heart sells usingcon-ebinationso f collag ena se arad trypsin gave less satis-factory rcsults than when trypsin was used alone.A more recent study by HIaworth et a l . ( 1986))showedthat exposure of adult cardiac cells to trypsin after C O H -lagenase treatment can be beneficial. I t was proposedthat trypsin naay allow the gap-junction channels in theintact cells to clos e, thereby con ferring upon them Ca"resistance. This approach might also be beneficial fortlac dissociation of ernbryor~ic eal? cells. How ever, theproperties of adult and em bryonic isolated cardiac cellsdiffer: ranlike adult heart cells, embryonic heart cells inculture retain the ability to redevejop electrical ccju-piing, presumably by the formation of functional rlexalranits (DeHaan and Hirakow 1972; Clapham et a!.1380).The observation that T aggregates appear Inoresimilar clectrophysiologically to the intact vcastriclethan C' aggregates is somewhat unexpecteei since i t laasbeera s hc ~w n hat trypsin ca n en ter cardiac cells d uringisolatioia and can d o so me intracelIular dama ge (K asten19 71). However, it should be noted that highly purificdcollage~mase s alm o\t cc~m plctely ineffective in dis-soc iating c ardia c ce lls (hclasnon-PCvet et al . 1976;Kono1969). The ability of crude ccrlllagenaw to dissociateheart rrauscle would appear to reside to a significantextent in the inapu rities present in the collag ena sc. Inparticular, the collagcnase used in our study showed asignificant lcvel of protease 6264 U/rr-eg) and some lo wl evel c l~~ st r ip ai~activity ( 1 . 1 U / ~ n g ) .11 co ntra st, tryp-$in \Ifas present in exceedingly low c~~ncentrat i60.04 &r/~ n g ) n d it is most uniikely that trypsin con-tamination would play a significant role. We have notinvestigated the role of the contaaninants present Incrrade collagenase. 'l'here is recent evidence that pro-


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tease dissociation media c an have effects equivalent tothat of crude collagenase wit11 regard to dissociation ofcardiac muscle (Bustarnante et al. 1982). It also appearsthat prolonged exposure to pro tease-co~rta ia~ing is-sociation media may give rise to cell dannage ( J . 0 .Bustarnante, personal communication), wlaich mightaccount for some of the effects we observed.The ability to obtain isolated cells $'or tissue clnlt~lreseems to depend om the combination of enzym es, cul-ture conditions, and the tissue under study. Based onour res ults w e comciucie that cr ude collage nase may no talways be superior to trypsin as a m eans of dissociatinghear3 cells as previously propc~sed Cavanaugh et al.1963: kIasso11-Pevcl ct al. 1976). In fact. aggregatesprepared from trypsin-dissociate cardiac ventricularct.81~ ro m ?'-day chi ck ermlbryos displa y func tion al pr op-erties more typical of intact 7-day ventricular tissuethan thosc prepared from collagenase-dissociated cells.Acknowledgements

'I'his work was supported by grants from the Medjica!Rcscaa-ch Council elf Canada arnd thz Canadian IleaI-8Foundation. M.K.G. is a recipient of a grecioctoraltraiareeship from the Canadian Heart Foundation. Wcthank $. 0 . Bustamantc for helpful conversations.P. KrraJeviC for prcsgramrning assis tanc e, K . Ro 7ans kyfor technical assistance. and S . James and C . Parnglinfor typing the manuscript.,&rw~La..D .. I . CO~IE.N,. EISNER. . OHBA , nd C. O S E B A .

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418 C A N S PFlYSLOI, Pi l t4RMACOL VO L 61, 1983tric:11 properties of s ingle bul8frog atrial cells. J . G en .Physiol. 78: 19-42.H v r ) ~ < , . , B . Bax)NDEr2, A . M .~ T T E R ,. P . C H E N E V A L ,B . F r r ,~ o ux , nd I,. GI RA RD IER .969. ~ H O R I C ) and hetero-cellu lar junctions in cell cujtures : an electrophysioE ogica1and naorphological study. Prog. Brain Res. 31: 283-3 1 1 .ISF.NBF.RC;.., nd kJ. K L O C K N E K .980. Glg~cocalyx s notrequired for slow inward calcium current in isolated ratheart rnyocytes. Nature (London), 284: 35%- 60.J ~ c o ~ s o r u ,. I, . 1977. Culture of spontaneously contracti~sgrasyocardial cells from adult rats. Cell Struct. Funct. 2:1 --9.JQBN(;SMA.. J . 1972. Synchronisatie in ialteraktic van hart-cellen in weefsclcultuur. Ph. D. thesis. Atrasterdam.Joxtisbfia, H. J . . M. h% i\s .c ;c> ~-P kv~ r,.'. C. HOL,LANI)I'R,ndJ . rx: B K L ~ ~ J N E .975. Synchronization of the beating fre-quency of cultured rat heart cells. bn Ilevelopnsental andphysiological correlates of cardiac muscle. 6:'Llitcd iri\M . P,icbcr~nan and T. Sano. Raven Press, New York.pp. 185- 196.

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%I- IANNE, 0 . F., E . R U I % - C ~ ; , R ~ ~ T T L ,. RPJARL), nd I).C ~ ~ A K T I E K .977. Ileternainants of electrical activity inc lust ers of c~ ~ l t i ~ r edardiac cells ho rn nconattrl rats. J . MOBCell . Cardiol . 9: 269-282.SCOTT,S. I979. Stimulation simulations of young yet cul-tured beating hearts. Ph.11. thesis. State L!niversity of N ewYork. Buffalo.S B I K I E K ,. , and J . K. C L A Y .1980. Pacernakcr currents inchick en~bryonicheart cells change with development.Nature (1,osadon). 283: 070-67 1 .-- 1982. A cornparison of the pacemaker p ~ ~ p u ' fof chick embryonic atrial and ventricular heart cells.J . Mcmbr. Riol. 69: 49-54,.Ski~rkr,F. E., arad W . D. BEIRNIIT .964. The establishmentof beating myocardia! cells in long term culture in fluidmediura. Exp. Cell Rcs. 36: 179- 191.S P E R E I I . A K B S ,. , and D. L E I ~ M K I J ~ I L .964. Effect of currenton trtins~-r-aembraraeotentials in culturcd chick heart cells.J . Gen. Physic~l. 7: 895-927.

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COLIZXA ET A L . 4 19Membrane cation channels: changes in developing hearts, pp . 209-234.in cell cultures, an d in organ culture. In Develc~prnental rad WOI,&ENHEK(;ER,. 1964. Rhythmic and arrhythrnic con-physiological correlates of cardiac muscle. Edited b~ tractile activity of single myocardial cclls cislturcd in vitro.M . Lichcrman and T. Sano. Raven Press Inc., New York. Circ. Mes. 14- 15(Suppl. 2) : 184-201.

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