Characterization of contractions in enzymatically

Characterization of contractions in enzymatically isolated rat ventricular myocytes: effects of ouabain and rubidium E ~ ~ , ~ T T~ 'IO R N A N E N Deparfm~)ntof Biology, Utzm'l-srsm'rycd .Poc)nsuu, P . 0 . Box 11 I , SF-80401 Soensuu 4 0 , Finlurzd Received February 15, 1983

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Guangzhou Jinan University on 06/05/13 For personal use only.

V o ~ ~ n l u s ;M, ~. ,1984. Characterization of contractions in enzymatically isolateci rat ventricular myocytes: effects of ouabain

and rubidium. Can. J . Physiol. Pharinacol. 62: 253 -258. The role of sarcolesnn~aand cspecialiy sodium pump activity in the control of phasic contractile activity of Ca2' tolorant rnyocytes was studied using ouabain and rubidium as sodiuni purnp inhibitors. Initially. ouabaiin increased both the amplitude of she~i-teningand the frequency of phasic contractions. Latcr, the amplitude began to decline whereas the frequency of beating continued to rise, often tcrirminating in a steady contracturc of thc myocyte. Rubidium caused a rapid rise of beating frequency, frequency and the which reached its full effect within 1-5 rnjil and remained steady after that. ' h e stimulation of ce~ntractia~n inhibition of Na'-K ' ATPase were correlated in the case of oraabain but not in the case of rubidium. The results suggest that the stimulation of phasic cantractions may be caused by increased uptake of ccllular calciuna through Na' -Ca' exchange as a cclnsequence elf sodium purnp inhibition and (or) depolarization of the sarcolemrna by ouabain and rubiditana. VC)MKANEN, rCI. 1984. Characterization of ce~ntractionsin enzyrnaticalIy isolated rat ventricular myocytes: effects of ouabain and rubidium. Can. 5 . Physiol. PharmacoB. 62: 253-258. On a examine 1c r6Bc du sarcole~nmeet sp@craIc~nent de I'activitk de la pompe B sodiuni dans le contrcile dc I'activitk contractile phasique des n~yocytestolkrants au Ca2' et ce, en utilisant I'ouabainc et le rubidiuna comme inhibitcurs de pompe 2 sodium. Initialernent, l'e~uabaineaugmenta tant I'amplitude que la di-kquence des contractions phasiques. Plus tard, I'amplitude commenGa a diminucr alors quc Ia frkquence dc kattcmcnts continua d'augrnenter. se terminant souvent en une contraction continue du myocyte. l,e rubidiurn provoqua une elevation rapide de la frkquence de battements qui attengnat son plein effet en 1-5 rniir pour ensuite demeurer constante. La stin~ulationde la frecluence de contraction et %'inhibitionde la Na' -K ' A'TPase furent correlees lors de I'utilisation de l'ouabaine; toutefois, on n'observa pas le mi?nme phinomene avec I'ernploi du rubidium. Les rCsultats suggerent yue la stiniulation des contractions phasiques peut resulter d'unc absorption accrue de calcirlrn ccllulaire induite par l'kchange NaA-Ca', et que cec1 est unc consequence de l'inhibitisn dc la psrnpe h sodium et (OU)de la dkpolarisation du sarcolcn-amc par l'ouabaine et Ie rubidium. ['H'raduit par le journal]

Intrsduetisn Many slightly different methods have bccn developed for enzymatic isolation of nlam~naliaraventricular rnyocytes (for review see Dow et al. 198 1). 'H'hcse preparations differ from each other with regard to their Ca2' tolerance and spontaneous beating. Tlae iwlated rat ventricular cells of our preparation, which tolcrate rnillimerlar concentration of Cag', show steady spontaneous contractile activity of very low frequency (k'ornanen and Tirri 1983). The contraction of these myocytes is ;a slowly propagating band of a few shortened sarcomeres, whicl~proceeds from one end of the myclcyte to the other, as described by Kieser ct al. ( 1979). This type of contraction. called phasic contraction is not coupled to the action potential (AP) (Riescr ct al. 1979; Lehto et al. 1983), but it is caused by a calcium-induced calcium release process (Fabiato and Fabiato 1972, 1975; Chiesi et al. 1981). The frequency of phasic ccmtractions is regulated by the activity of sarcoplasmic reticulum (SR) and is directly dependent o m the cellular C a t concentration (Chiesi et al. 1981; Dani et al. 1979). In spite of spontaneous beating the rrayocytes of our preparation have intact sarcolernma (SE) which retain a proper resting potential and can generate action potentials (AP) connected to norinal twitch contractions (Lehto et al. 1983). Until now it is unexplained why phasic contractions occur also in Cagt-tolerant rnyocytes. In the present study we try to clarify how SI, and especially sc~diumpump activity cc~ntrolthe phasic contractile activity. The stinnulation of phasic contractile activity by ouabain and rubidium (Mb') in isolated rat ventricular myocytes is described.

2.5 rng sodium heparin by injection into the tail vein. After being slightly anaesthetired with diethy1 ether the animal was killed by cervical dislocation. The heart was quickly excised and transferred to a perfusion solution of the following composition (rnrno%/L):NaCl, 136: KCI, 4.6; MgClz. 1.2; NaPK,PC),, 1.8; glucose, 1 I , buffered with 10 mM PKEPbS-NaBW to pH 7.4. After that the asolation of ventricular myocytes was performed by a combined pea-fus~onand incubation technique using collagenase ('Type IA. Sigma) and hyaluronidase (Type I-S, Sig~raa)as descrabed earlier (Vornanen and Tixi 1983). Throughe~utthe ~\olaaionprocedure the solutions were gassed with pure oxygen and the ten~peraturewas kept at 37°C. 'The dissociated rnyocyte5 were suspended in a perfusion solution containing 2.5 nnM Ca". In some expcrinnents different CaC:12 (0.005-5.0 m M ) and KC1 (0.6-3.2 mM) concentrations were uwd, as rrmenticsned ~n the Result\. No compensation was made for the minor changes uf K ' and Ca2' to n ~ a i n t a the i ~ ~osmolapity of the solaltions.

Methods

Isolatiopl cf heart wrcolemrna and deter~rainntionc$iVa' -K' ATPcrp;e activiQ The sarcolemma from rat heart ventricles were isolated using the

bsnlks rinn ($vennic-salar myoc'yte~ Aeiult rnale Wistar rats, 3-4 months in age, were heparinized with

Recording of beating Only those cells which beat steadily with a frequency of 12 beats/ rnln or slower were acccpted for experiments as the slowly contracting nlyocytes have the most negatlve menmbrane potential of -80 to -90 I - ~ V (Lehto et al. 1983). Recording of the bcating was carried out either by using a photoamplifier connected to a chart recorder or by storing the beating on video tape with the aid of a phaw-contrast naicroscope, a TV set, and a videotape recorder as described earlier (Vornanen and Tirri 1983). Changes in contraction amplitude were measured from the photoamplifier recordings. When the videotape reccsrdcr was used, a 100-pL sample of myocytes was placed on a depression slide and the beating of I -3 myocytes in the field of vision was recorded f ~ 5r~ n i nbefore ouaba~nor Rb ' was added in amounts (sf 50 FL. Thc beating was recorded for 1 min after addition and thereafter in 5-rnin intervals for 30 nain. 'l'he experimental temperature wa\ 20°C.

254

CAN. J . I'HYSIOL PtIARMACOI,. VOL. 62. 1984


lmin

3 . 1 0 ~Ouabain ~

FIG. 1. A photoamplifier recording showing the effect of the application of 3 X 1 0 4 M ouabain on thc phasic contractior~sof an isolated ventricular myocyte. The time elapsed from the moment of ouabain addition (arrow) is shown above the recording. hypotolaic shock - LiBr treatment method (McNamara et a!. 1974) as described by Panagia et ale (1982). The final sarcolemmal sediment was suspended in B mM Tris-WC1 (pH 7.0) and was used as the enzyme preparation. l'he ATPase activity was measured by suspending 110- 170 p g sarcolennmal protein in the iancubation medium which contained 50 mM Tris -HC1 (pH 7 . 3 , 4 anM MgC12, 100 1nA4 NaCl, and 20 mM KCI in a final volurne of 1.5 ml,. The Na' -Kf ATPase activity was obtained as the difference betwecn the total ATPase activity and the activity in the presence of 1 n1M ouabain. The ATPase reaction was started after 15 min preincubation at 37 or 20°C by adding Tris-ATP (4 mM final concentration) and the inorganic phosphate liberated during 15 min of incubation was measured according to the method s f Atkinson et al. (1973). The protein content of the sarcolemmal preparations was determined by the procedure of Lowry et al. (1951) using bovine semnl albumin as the standard. Determination of (:a2' The Ca2' concentration of the nominally Ca2'-free solution (contaminating Ca2') was determined after removing the cells by centrifugation (18 min, 4000 rpnn) by using an atomic emission spectrometer (Spectraspan III B) . Drugs and .stcabistics The drugs used wcre: ouabain (G-strophanthin, Merck) and RbCl (Sigma), which were dissolved in a perfusion solution with appropriate concentrations of Ca2' and K ' . Statistical differences were calculated using Student's t-test, P < 0.05 being regarded as a significant difference.

Results Eflect of ouabain and Wb' on phasic cspztractike 6lctivity About 30-50% of ventricular rnyocytes of the rat heart were elongated and showed clear cross-striations in 2.5 rnM Ca" solution. The myocytes of the present preparation contract spontaneously at a mean rate of 5.4 2 0.3 beats/min (+ SE, n = 91) at 20°C. The contraction is a slowly ( 108 iz 3 ~ r n / s , mean k SE, n = 3 % ) propagating , wave of a few activated sarcomeres proceeding from one end sf the myocyte to the other. During this phasic contraction the whole myscyte shortens approximately 10% (10.1 5 0.7, mean iz SE, pz = 37) of its resting length. Buabain (3 X M ) increased both the amplitude of the

O u a b a i n , 3 ~ 1 0M ~~ FIG. 2. A histogram showing the effect of 3 X lOpJ M ouaba~non the arnplikude of phasic contl.actions. Tlne arnplitlsdcs are means of all the contractions occurring during a 2-min period and are expressed as percentages of the control value. 'The results are the meaa~sof four photoamplifier recordings similar to that in Fig. 1 . 'l'ke bars show + SE and asterisks indicate significant difference ( * P .( 0.05, *"$ .r 0.0 1) from the control value.

shortening and the frequency s f the phasic contractions described above (Fig. 1). The rnaxi~nalincrease in amplitude was 45% above the control value, wlnich was achieved about 12 rnin after adding the drug, after which it decHiaaed steadily (Fig. 2).


I

I

0

I

10

I

20

I

1

30MiN

0

40

20

30 MIN

IM, n = 41), (3 X I0 ' FIG. 3. (A) 'The effect of difkrent ouabain concentrations on the rate of phasic contractions: 2 (3 X (3 x 10 " M , 11 = If)), P 4 (3 X 10 -'M,rl = 1 1 ) . Inhibition of Na'-K+ A'1'Pase (X-X) and a net increase in beating M.n = a@), rate 30 man after ouabain addition are shown in the insert as a function of ouabain dose. (B) The effect of 3 X 10 rki ouabain on the rate of (5.0 mM, n = 28), n = 41), H(0.2 mM, n = 23), phasic contractions at dirfcrent Ca2- concentrations: (0.005 mM, = 10). In the insert the net response aftcr 30 rnin is expressed as a function of the Cai' concentration. Arrows indicate the time sf ouabaia-1addition and the bars show -+ SE.

There was also a moderate increase in the beating rate concomitantly with the rise in amplitude (Figs. I and 3A). A f e r 1520 min, when the amplitude was already declining the beating frequency still continued to rise, even more steeply than before. The increase in beating rate was clearly dose-dependent (Fig. 3A insert), the half-maximal effect being achieved at about 3 x 10-' M. The same ouabain doses caused steady contractures when incubation was continued longer than 30 min (Fig. 4). The freqiieney response of ouabain was clearly dependent on the extracellular Ca2+ concentration (Fig. 3B). The response appeared to saturate at 2.5 rnM Ca2+ and did not increase further at the 5 mM Ca2+ solution, the half-maximal effect being achieved at absiit 0.6 mM. In low Ca2+solutions (5 and 200 p M ) , the beating rate reached a peak value after 1520 rnin and then began to decline slowly, whereas at higher Ga2' concentrations there was a continuous and steeper rise s f frequency often terminating in steady contractures. The effect of external K ' concentration on the response of myocytes to 3 x 10 -4 M ouabain was studied in a solution containing 2.5 mM @a2+.Figure 5 shows that the response to suabaim increased with increasing K' concentration.

Wb+, which is also known to inhibit cardiac Na+-Kt ATPase in the presence of Na and K ' (Ku et al. 1975), caused a dose-dependent stimulation of the beating rate (Fig. 6A). Typically, Rb' caused a steep rise in beating frequency so that maximal stimulation was achieved most often within % -5 min after its application. Thereafter, the response remained steady for the rest of the experiment. The efficiency of Wb+ was determined by external Ca2+ (Fig. 6B) in a similar way to ouabain response. Saturation was achieved at 2.5 mM and half-maximal effect was reached at about % mM. Inhibition of Na ' -K ' A T P ~ S by P ouabuin anti Bib The inhibition of Na -K+ ATPase activity by ouabain and Rb+ was measured to find out whether it correlated with the stimulation s f beating frequency. The ouabain inhibitable Na+-K+ ATPase activity was 6.45 -+ 0.64 pmol P, .mg protein h (mean -+ SE, n = 4) at 37"C, whereas the activity at 20°C was 37.7% s f that at 37'C (2.43 -+ 0.13 pmo% P,/mg protein-' h-', n = 4). Ouabain inhibited the Na '-Kt ATPase activity dose-dependently and it fairly well correlated with the stimulation of phasic beating (Fig. 3A insert). Rbt (2- 15 mM) inhibited the Na+-K+ ATPase activity by +

'

'


256

CAN. 5. PHYSIOI.. PHRK14ACO1,. VOL.. 62, 1984

B

7

I

I

6 5 --log dose (M)

B

4

t

3

FIG.4. The steady ccmtractiares c;aused by different concentrations of o~aabain.The results arc expressed a\ perccntages of cells which have developed aneversible contractaare 45 min alker ouabain addition. The nurnber of ce1ls is given above thc sysnbols.

20.6-36.6%, but the inl~ibition was mot clearly dosedependent and it did not correlate with the stimulatic>n of beating (Fig. 6A insert).

Discussion The contraction sf intact rnyscardial cells is initiated by an action potential that triggers release of Ca2' into the myoplasrn. The source of Ca2' and the triggering mechanisrs~are still partly unclear. One of the proposed hypotheses is the calciurninduced calcium release from SR (Fabiato and Fabaato 1979). The phasic cr~ntractionis not coupled to AP (Rieser et al. 1979; Lehto et al. 19831, and it occurs in electrjcally shunted and skinned rnyocytes (Rieser et al. 1979: Fabaato and Fabiato 1972, 1975), which are freely permeable to Ca". The Ca'+-tolerant myocytes of our preparation retain membrane potentials of -60 to -90 rnV (at 23°C) and they can also generate normal twitch contractions coupled to APs (Lehto et al. 1983; Voriaanen and Tirri 1983). However, these Ga" -tolerant myocytes contract phasically at a slow frequency of about 5 beats/min (at 20°C) in 2.5 mrW Ca" solution. Hntracellular ionic concentrations and the phasic contractile activity are obviously regulated by SL as also shown by the stimulating effects of ouabain and Rb' . Stimulatic~nof beating and inhibition of Na ' - K ' ATPase by ouakain were fairly well correlated. Instead the inhibition of N a ' - K ' ATPase by Rb' was almost independent of Rb' ccancentration, although it stimulated phasic contractions in a dose-dependent rnanner. 'T'hese observations suggest that the

FIG.5 . 'The effect of 3 X 10 M oteabain o n the rate of phasic co~ltractioalsat different K' concentrations: 0-0 (4.6 mM, n = 411, [ H I ( 2 . 3 (1.15 111~44, n = 151, M (0.t'~mM, n = 7). In the incert the resutts are expressed as a function of K ' zonzentratron. where thc response is a net increase in the beating rate 38) rnin after ouabain addition (indicated with an arrow). The bars show .i SE.

stimulation of beating induced by rxlabain may be causcci by sodiunr pump inhibition; whereas the effect o f Wb' seems n ~ r ~ r e likely to be duc to the depolarization of the sarcolernnaa, although it also causes a moderate inhibition of Na' - K t ATPase. Stirnulatilag effects of both ouabain and Rb ' are dependent tsn extracellular ~ a 'in a similar concentration range, i. e.. the half-maxirn~al effect was achieved at about 1 m!V. Possible mutes for extracellular c a 2' entry arc Na ' -Ca exchange and the slow calcium channel. Depolarization of sarcc~lcmrnaby Rb ' wo~aldstinlulate Na ' -Ca exchange, which is sensitive to naembrane potential (blaallins 1979) or calcium channels would be opened. The opening of slow ca1ciur1-a cirannels is not probable sincc these channels are activated at a rnernbranc potential more positive than -50 rnV (lsenberg and Kliickner H980), whereas in myocytes beating at low frequency the potentid is -80 to -90 naV QLehtoct al. 1983), which is not likely to be dcpolarized to -50 mV by 2- 15 m.44 Rb'. In addition phasic contractions are not eliminated by the (;low channel blocker, verapamjil (Vornanen and Tirri 1983). Inhibition of the sodium purnlp by ouabain would stinaulate Wa ' - Ca exchange through the rise of intracellular Na ' and possibly also by depolarization. The fact that the csuakain response was strengthened at higher K ' concentrations is incowsistent with the sodiura~pump inhibition, because binding tsf ouabain to Nat - K L ATPase is inhibited by K t . However, the enhanced effect of ouabain at higher K ' concentrations may kc +

A

+


VORNANEN

(16) m M , n = 13), 6. (A) The cffcct of different Mb ' concentrations on the rate of phasic contractions:' &C (I5 mM, n = 23), te 5 min after Rb+ 45 mM, n = I I ) , M(2 mM, n = 29). Inhibition of N a t - K t A'H'Pase ( X - X ) and a net increase in bea the insert as a function of Rb+ dose. (B) 'I'he effect of 5 rnM RbLon the rate of phasic contractions at different CaZ+ (5.0 mM. n = 1 1 ) . (XI(2.5 mh4, rz = 13), H(0.2 mM. rz BO), U--n (0.005 m M . n -- I 1). In the insert the concentrations: net response after 5 rnin of Rb ' addition is expressed as a function of Ca2+concentration. Arrows indicate the time of Kb ' addition and the bars show 1: SE. 1-

due to the additive effects of K depolarization and sodium pump inhibition by csuabain. It has been recently shown that CaL+uptake by isolated rat rnyocytes is increased in high K t solution owing to the stimulation of potential dependent N a ' -Cat exchange (Desilets and IIorackova 1982). So it seems possible that the stimulation of phasic contractions are caused by enhanced Na' -Cat exchange as a consequence of sodium pump inhibition and (or) depolarization of the sarcolemnma by ouabain and Rb' . Thc occurrence of phasic contractions in calcium-tolerant rnyocytes may be caused by an elevated Nat/K ' ratio, for it has been shown that rat heart cells contain elevated levels of Nat and decreased intracellular K ' when prepared in the cold or subjected to low Ca2- perfusion (Altschuld et al. 1980; Hohl et al. 1983; glheung et al. 1982). Such N a ' -loaded myocytes are hypersensitive to Ca2+ but can be made tolerant by incubation at higher temperatures where a low Na' /K ' ratio is restored QHohlet al. 1983). In our dissociation procedure the cells are not subjected to low temperature. but during the low Ca2+(50 p % a ) perfusion (8- I3 rnin), Na ' accunmulation may occur. When subjected to physiological concentration of Ca2' the intracellular Na' would be exchanged for extracellular Ca2+ with the result that intracellullar Ca" rises sufficiently to trigger the calcium-induced calcium release.

ALI'SCHI:LB,R . , L. GIDB,A. ANSM, F. A. KKUGER.and G. P. BRIERLEY. 1980. Calcium tolerance of isolated sat heart cells. J. Mol. Cell. Cardid. 12: 1383- 1395. AI'KINSON, A . , A. D. ~ X T ~ Nand DY A.. G. Low.. 1973. 'The detcrmination of inorganic orthophoyhate in biological systems. Biochim. Biophys. Acta, 320: I95 -2(14. CHEUNG, J. Y . , I. @. THOMPSON, and J . V. BONVERI'E.1982. Effects of extracellular calcium removal and anoxia on isolatcd rat myocytes. Am. 9. Physiol. 243: 6184-C'1968. CHIESI,M., M. M. WO, G . [NESI. A. V . SOMLYCI, and A. P. SOMLYCP. 195 1 . Primary rolc of sarcoplasmic reticuhm in phasic contractile activation of cardiac myocytcs with shunted myolernma. J. Cell. Bio1. 91: 728-752. DANI,A. M., A. CBTTADINI, and G. INESI.1979. Calcium transport and c6ntractile activity in dissociated mammalian heart cells. Am. J. Physiol. 6: CB47-6155. DESILETS, M . , and M. MOKACKOVA. 1982. Nas -dependence of 4'Ca2+ uptake in adult rat isohtcd cells. Bicschim. Biophys. Acta, 921: 144- 157. DOW,J. W . , N. G. L. HARD IN^;, and T. POWELL.1981. Isolated cardiac rnyocytes. I. Preparation of adult rnyocytes and their homology with the intact tissue. Cardiovasc. Wcs. 15: 483-5 13. FABIAI'O.A., and F. FARIATO. 1972. Excitation -contraction coupling of isolated cardiac fibers with disrupted or closed sarcolemmas. Calcium dcperdent cyclic and tonic contractions. Circ. Kes. 31: 293-307.


25 8

CAN. J. PWYSIOL. PHlaRMACOk,. VOL. 62, 1984

1975. Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of skinned cardiac cells. J. Physiol. (London), 249: 457 -5 B 7. 1979. Calcium and cardiac excitation-contraction coupling. Annu. Rev. Physiol. 41: 473-484. HOWL,6 . M., R. A. ALTSCHULI), and G. P. BRIERLEY. 1983. Effects of calcium on the permeability of isolated adult rat hear? cclls to sodium. Arch. Biochern. Biophys. 221: 197-205. ISENBERG, @. , and U . K I ~ ~ C K N 1980. E R . Glycocalyx is not required for slow inward current in isolated rat heart myocytes. Nature (London), 284: 358-360. Ku, D., T. AKERA,and T. M. BRODY.l975. Effects of monovalent cations cln cardiac Na',K'-ATPase activity and on contractile force. Naunyn-Schrniedeberg's Arch. Pharmacol. 290: 1 I 3 - 1 3 1. LEHTO,H . , A. TALO,W. TIRRB, and M. VOWNANEN. 1983. Membrane potential oscillations in enzymatically isolated rat nayocardial cells. Acta Physiol. Scaaad. 118: 385 -39 B . LOWRY,0. H., N. J . ROSENBROU(;H, A. L. FARR,and R . J.

RANDALL. 1951. Protein measurement with the Folin phenol reagents. J. Biol. Chem. 193: 265-275. MCNAMARA, e>. B., p. v. SULAKHE, J. N . SIN(;H,and N . S . D H A I . ~ , . ~ . 1974. Properties of heart sarcolernrnal Na ' -K '-ATPase. J . Biochem. (Tokyo), 75: 795 - 803. MULLINS, L. J . 1979. The generation of electric currents in cardiac fibers by Na/Ga exchange. Am. J. Physiol. 236: G 103-C71 10. PANAGIA, V . , J. M. J . LAMERS,P. K . SINC>.~L, and N. S. DHALLA. 1982. Ca"- and Mg2'-dependent ATPase activities in the deoxycholate-treated rat heart sarcolernrna. Int. J. Biochern. 14: 387 -397. RIESEW, G . , R. SAWB~ZDINI, P. PAOLINI. M. FRY,and G . BNESI.19779. Sarcomere motion in isolated cardiac cells. Am. J . Physiol. 2366 8 ) : C70-C77. VORNANEN, M., and R. 'PIRRI.1983. Effects of acetylcholine, isoprenaline, phenyiephrine and dibutyryl-CAMP on the contractility of isolated myocytes of the rat heart ventricles. Acta Physnol. Scand. 118: 433-438.