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Effects of procaine and caffeine on the contractility of enzymatically isolated myocytes and intact cardiac tissue
Gen. Pharmac. Vol. 18, No. 6, pp. 599--604, 1987 Printed in Great Britain. All rights reserved
0306-3623/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
EFFECTS OF PROCAINE AND CAFFEINE ON THE CONTRACTILITY OF ENZYMATICALLY ISOLATED MYOCYTES AND INTACT CARDIAC TISSUE MATTI VORNANEN Department of Biology, University of Joensuu, P.O. Box 11 l, SF-80101 Joensuu, Finland (Received 22 December 1986) Abstract--l. Procaine and caffeine exerted opposite effects on the contractility of rat cardiac tissue. 2. Procaine enhanced and caffeine depressed isometric contractile force of right ventricular strips. 3. In enzymatically isolated cells, caffeine increased the frequency and decreased the amplitude of spontaneous contractions, while procaine retarded the beating rate but concomitantly raised the shortening amplitude of the myocyte. 4. Both drugs depressed the propagation velocity of the contractile waves. 5. The results are suggested to be due to the opposite effects of the two drugs on the Ca-release process of the sarcoplasmie reticulum.
INTRODUCTION The contractility of cardiac muscle is regulated by the a m o u n t of Ca that is released in the myoplasm after excitation of the sarcolemma (Allen and Kurihara, 1980). Recent experiments suggest that even during diastole the myoplasmic Ca level fluctuates (Orchard et al., 1983; Hess and Wier, 1984; Allen et al., 1984, 1985; Valdeomillos and Eisner, 1985) causing small oscillation of resting tension (Lakatta and Lappe, 1981; Stern et al., 1983; K o r t and Lakatta, 1984; N i e m a n and Eisner, 1985). These miniature oscillations o f Ca and tension can have profound effects on the excitation-dependent Ca-release and thereby on the force generated by the cardiac muscle (Allen et aL, 1985). Oscillations are presumably caused by the sarcoplasmic reticulum (SR) which spontaneously releases its Ca content. Spontaneous cycles of repeated uptake and release of Ca can be found in Ca-tolerant enzymatically isolated myocytes as propagating contractile waves (Tirri et al., 1982; Matsuda et al., 1982; Cobbold and Bourne, 1984). The contractile waves seem to be an especially conspicuous feature of adult rat heart cells (Chiesi et al., 1981). In the present study, we describe the effects of caffeine and procaine - - t w o drugs which interfere with the function of S R - - o n the wave-like contractions of enzymatically isolated rat heart myocytes and compare them to their effects on intact tissue. The results show that in both preparations, caffeine depresses and procaine enhances contractility. The contraction promoting effect o f procaine may be due to its ability to increase Ca content o f the SR by inhibiting diastolic Cafluctuations. METHODS
Isolation of ventricular myocytes Adult (3-4 months) male and female rats of Wistar strain were used in the experiments. They were maintained on a
12-hr dark, 12-hr light cycle and fed by commercial animal chow and tap water ad libitum. Single ventricular ceils were isolated by the modified method of Kao et al. (1980). This procedure involves combined perfusion and incubation of cardiac tissue with collagenase and hyaluronidase containing solution in the presence of 50#M Ca as described previously (Vornanen and Tirri, 1983; Vornanen, 1984a). For the experiments the dispersed cells were suspended in physiological solution of the following composition (mmol/1): NaCI 136, KC1 4.6, MgCI2 1.2, NaH:PO 4 1.8, CaCI2 2.5, glucose 11, buffered with 10mM Hepes-NaOH to pH 7.4. Recording of contractions Forty to sixty percent of the myocytes were Ca-tolerant as judged from their cross-striated, rod-shaped morphology in 2.5 mM Ca solution. Ceils which contracted steadily at low frequency (1-12 contractions/min) were accepted for experiments, because slowly beating cells have been found to retain their negative resting potential (Lehto et al., 1983). Recording was carried out by a microscopic video technique. For this purpose, a 100 #1 sample of myocyte suspension was transferred on a depression slide and a myocyte filling the criteria of an intact Ca-tolerant cell was selected with the aid of a phase-contrast microscope. The cell was centred in the field of vision at such magnification that the myocyte occupied almost the whole screen of the T.V. monitor, i.e. one cell at a time constituted an experimental preparation. The myocyte was inspected during the 5 rain control period to assure that its activity remained steady, after which a concentrated solution of procaine or caffeine was carefully added to give a final concentration of 1 or 5 mM. Three contractile parameters--(1) frequency of contractions, (2) amplitude of contraction (i.e. fractional shortening of the myocyte from its resting length) and (3) velocity of contraction wave--were later analysed from the video tape. Parameters (2) and (3) were determined at slowed film speed by means of stopwatch and transparent #m-scale placed on the screen. Each parameter was measured 5 times and two extreme values were neglected and the mean of the remaining three was accepted as final value. Experiments were conducted at room temperature (20°C). Membrane potentials were recorded with conventional glass microeleetrodes filled with 3 M KCI and had resistanees between 40 and 60 Mf~. Ag-AgCl-unit was used as 599
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/ Fig. 1. Two typical enzymatically isolated Ca-tolerant cells used in the experiments and an action potential elicited by hyperpolarizing current pulse in a spontaneously beating ventricular cell.
a reference electrode. Both electrodes were connected to WPI KS-700 electrometer, which was also used to inject current through the microelectrode in eliciting action potentials. For the potential recording, a few drops of myocyte suspension were placed on depression slide attached to the stage of inverted microscope (Leitz). Cells were impaled by advancing microelectrodes at a steep angle (~ 60 °) with respect to the bottom of the chamber. Signals were displayed on an oscilloscope and were photographed using a Polaroid camera.
uniform shortening of the myocyte. In physiological solution that contains 2.5 m M Ca, the frequency of these contractions was 5.1 + 0.36/min (mean _+ SEM, n = 69) and caused a 9.7 _+ 0.45% (mean + SEM, n = 61) shortening of the myocyte's resting length ( = amplitude). The velocity of the contraction wave was 110 _+ 2 ~m/sec (mean _+ SEM n = 55) at 20°C. The effects of caffeine and procaine on these spontaneous contractile waves were examined. Immediately after its addition, 5 m M caffeine Experiments on intact tissue induced few vigorous contractions at very high freRight ventricular strips of the rat heart were used for quency, but a little later (19 _+ 14 see, mean +_ SEM) isometric tension recording. Strips under 1 mm dia were the myocyte stopped the spontaneous beating and mounted in the organ bath and connected to a forceremained at the completely relaxed state (Fig. 2). At displacement transducer (Grass FT03) as described previa concentration of l mM, the effects were similar but ously (Vornanen, 1984b). The muscles were stretched to their optimum length (Lm,x) and were paced to contract at less dramatic i.e. the frequency was first markedly 0.2 Hz by square wave pulses of 5 msec duration and voltage increased but about 2 min later recovered towards the 1.5 times the threshold value. The composition of the control level remaining, however, somewhat above physiological solution used in these experiments was as this. Amplitude was also initially enhanced but subfollows (mmol/l): NaCI 127.6, KC1 4.0 NaHCO 3 12.0, sequently clearly depressed below the control value. NaH2PO 4 0.4, MgSO 4 1.5, CaC12 2.5 and glucose 11 at The velocity of the contraction wave was markedly pH 7.4. The solution was gassed all the time with a mixture diminished (Fig. 2). of 5% CO2 and 95% Oz and kept at 30°C. Isometric tension The effects of procaine were in several ways and its first derivative were recorded on paper (Grass 7D polygraph) and monitored with aid of an oscilloscope (HP opposite to those of caffeine. The frequency of contractions was in a dose-dependent manner decreased 1201B). so that many cells stopped beating totally for a few minutes after the addition of 5 r a M procaine. RESULTS Instead, the c o n t r a c t i o n s - - n o w occurring at longer Typical cylindrical, cross-striated cells used in the time intervals were prominently much greater in experiments are shown in Fig. 1. In an unselected amplitude, especially in the presence of 5 m M propopulation of myocytes (n = 12), beating spontanecaine. The effect of procaine on the velocity of the ously at the rate of 3-12 times/min, the resting contraction wave was similar to that of caffeine, i.e. potential varied between 70 and 7 8 m V (mean it was depressed (Fig. 2). + SEM, 74_+ 0.8). Action potentials with normal Caffeine and procaine also changed the resting configuration could be elicited in these cells (Fig. 1). length of the relaxed myocytes. This effect was not Two types of contraction can be distinguished in seen immediately after the drug addition but only at enzymatically isolated Ca-tolerant myocytes of the the end of the 15 min exposure. The resting length rat heart ventricles; normal action potential coupled was 99.4 and 92.3% from the control after 15min twitches triggered by electrical stimulation and sponexposure to 1 and 5 m M caffeine, respectively. Corretaneous low-frequency contractions not associated sponding values for 1 and 5 m M procaine were 99.1 with action potential firing. Both types of contraction and 94.6%. The diminished resting length during occur in the same cell. The spontaneous low- prolonged exposure to higher (5 mM) procaine and frequency beats are characterized by slowly propa- caffeine concentrations suggests that these drugs may gating contractile wave of few activated sarcomeres cause sensitization of contractile proteins to Ca. This which spreads the whole cell length, causing noneffect seems to be stronger for caffeine.
Procaine and caffeine
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Fig. 2. The effects of two concentrations of procaine (left panel) and caffeine (right panel) on the three parameters describing spontaneous contractile activity in enzymatica[ly isolated myocytes of the rat heart. The results arc means-I-SEM of 10-20 experiments. The drug addition was made at the moment 0. Temperature 20°C.
In order to see if these effects of caffeine and procaine are limited to enzymatically isolated cells or whether they occur also in intact tissue, ventricular strips were examined. Figure 3 shows original recordings about the effects of caffeine and procaine on intact tissue. Five mM caffeine causes a typical negative inotropic effect of the adult rat heart [Fig. 3(d)], whereas procaine (2 raM) clearly increased the force of contraction [Fig. 3(a) and (b)]. The mean increase in developed tension was 37 _+ 5.6% (mean + SEM, n = 8) at 2 mM procaine solution. A few minutes after the procaine addition, the contractile force began to decline, which was caused by the loss of excitability as indicated by the recovery of tension upon increased stimulus voltage. This generally prevented the usage of higher concentrations of procaine. The positive inotropic effect of procaine was associated with increased time to peak tension
and increased duration of the contraction-relaxation cycle [Fig. 3(a)]. Aftercontractions elicited by 3 × 10 -4 ouabain were clearly depressed by procaine (4 raM) [Fig. 3(c)]. DISCUSSION The enzymatically isolated cells used in the present study were Ca-tolerant, clearly cross-striated and rod-shaped. Furthermore, the myocytes retained these characteristics several hours when kept in physiological Ca concentrations (2.5 raM) at room temperature. The sarcolemma of the cells is functional as shown by their normal resting and action potentials. Although the myocytes have the characteristics of normal cardiac cells and can be electrically induced to twitch contractions (Vornanen and Tirri, 1983), they exhibit another type of contraction which
MATTI VORNANEN
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Fig. 3. Original recordings showing the effects of caffeine and procaine on isometric contractions of right ventricular strips from the rat heart. Slow speed polygraph recording Co) and fast oscilloscope recording (a) indicating the effect of 2 mM procaine on the isometric contractile force and the time course of a single contraction-relaxation cycle. (c) Reduction of aftercontraction amplitude by 4 mM procaine in oubain-treated tissue. (d) Effect of 5 mM caffeine on isometric contractile force. Temperature 30°C. appears as spontaneous contractile waves travelling from one end of the cell to the other. These wave-like contractions are not coupled to action potentials but they are associated with membrane potential fluctuations of a few mV's amplitude (Lehto et al., -1983). This kind of contractile activity is an expression of the function of the SR (Fabiato and Fabiato, 1973; Rieser et al., 1979; Dalai et al., 1979; Chiesi et al., 1981) as repeated uptake and release cycles of Ca, and is assumed to use the same pathway as the Ca-induced Ca-release process although the mechanism may not be identical with the latter (Fabiato, 1985). Wave-like contractions are generally found in mechanically and functionally skinned cardiac cells but their occurrence in Ca-tolerant
myocytes has been often regarded as a feature of Ca overloaded, damaged cells (Haworth et al., 1980; Fabiato, 1985). On the other hand, contractile waves could also be a property of intact rat heart cells because fluctuations of intracellular Ca and tension has been measured in intact, untreated cardiac cells during diastole (Allen et al., 1984, 1985). Furthermore, propagating contractile waves have been found in intact cardiac tissue without any Ca-overloading (Stern et al., 1983). Caffeine and procaine had opposite effects on these wave-like contractions. In steady state conditions, caffeine (1 mM) shortened the interval between beats and concomitantly diminished their amplitude, whereas procaine (1 and 5 mM) increased the interval between successive contractions but simultaneously enhanced their amplitude. Caffeine is generally thought to induce Ca-release from the SR and inhibit its Ca-uptake process (Weber and Herz, 1968; Blinks et al., 1972; Blayney et al., 1978; Wier and Hess, 1984). The effects of caffeine on wave-like contractions suggest that the spontaneous contractile activity is determined by the function of SR also in the Ca-tolerant myocytes. The action of caffeine was especially evident at 5 mM concentration, causing few vigorous shortenings immediately after its addition but subsequent stopping of the contractile activity at the relaxed state of the myocyte. Increased frequency and decreased amplitude of contractile waves in the presence of 1 mM caffeine suggests that this concentration of caffeine cannot totally inhibit the uptake process although it clearly enhances the release phase. Shortening of the myocytes resting length after longer caffeine exposure may be due to its ability to sensitize the myofilaments of Ca (Wendt and Stephenson, 1983). Procaine is generally thought to retard the Ca release of SR in cardiac as well as skeletal muscle (Chapman and Leoty, 1976; Hunter et al., 1982; Ford and Podolsky, 1972). Procaine's ability to increase the interval between spontaneous contractile waves in isolated myocytes could be interpreted according to this proposal. However, procaine also caused a prominent increase in the shortening amplitude, which cannot be due to the inhibition of the Carelease from the SR. If spontaneous cyclic Ca release is determined by the Ca-loading level of the SR, i.e. if the release process is initiated at a certain critical threshold level of Ca in the SR the present experiments suggest that procaine elevates this threshold value. In the presence of procaine a greater amount of Ca may be accumulated in the SR and subsequently released during the following beat. The results are in agreement with the findings of Stephenson and Wendt (1986) on functionally skinned fragments of rat heart ventricle where procaine could enhance caffeineinduced contractions. Nieman and Eisner (1985) reported that tetracaine decreased the frequency of oscillations in intact Purkinje fibres but had no effect on their amplitude, suggesting a different mode of action for this drug. Caffeine seems to have an opposite effect to that of procaine, lowering the Caloading level which is needed to trigger the release process as found in several other cardiac preparations (Wier and Hess, 1984; Nieman and Eisner, 1985). The effects of caffeine and procaine on the iso-
Procaine and caffeine metric tension of intact tissue were similar to their action on the contractile waves of enzymatically isolated cells. This indicates that the drugs change the function of the SR in the same manner in these two preparations. The negative inotropic effect of caffeine is likely due to the depletion of the SR Ca-stores. The positive inotropic effect of procaine indicates that under the operation of physiological Ca-triggering mechanism--perhaps the Ca-induced Ca-release process--this drug sooner potentiates the Ca-release than inhibits it. In contrast to the enzymatically isolated cells the effect cannot be now--because of constantly paced stimulation--due to the longer interval between suggestive contractions to allow more effective loading of the SR. The effect of procaine on the duration of isometric contraction and the velocity of contraction wave suggest that the Ca-release rate is not enhanced but rather is retarded. A possible explanation is that spontaneous inhomogenous Ca-releases, analogous to those causing contractile waves in isolated cells, occurs also in the intact tissue. Abolition of the small diastolic Cafluctuations by procaine would allow more uniform loading of the SR with Ca and greater amount of release during the next twitch. In fact, the diastolic Ca level is not constant but small fluctuations in the Ca concentration and associated myofilament movements have been found in several cardiac tissues (Lakatta and Lappe, 1981; Stern etal., 1983; Orchard et al., 1983; Allen et al., 1984, 1985; Wier and Hess, 1984; Nieman and Eisner, 1985). These asynchronous miniature contractions can lead to the suppression of twitch tension (Stern et al., 1983; Allen et al., 1984). The finding that procaine eliminates aftercontractions, which are probably synchronized miniature Ca-fluctuations, support the proposal that inhibition of diastolic Ca-fluctuations is the mechanism of procaine's action. In addition, procaine may also cause small sensitization of contractile proteins to Ca. The present results suggest that fluctuation of tension and Ca may occur in addition to enzymatically isolated cells also in intact cardiac tissue of the rat heart. It has been found earlier that the contractile waves are more easily generated in the adult rat heart myocytes than in the cardiac cells of other mammals (Chiesi etal., 1981). If spontaneous contractile waves are generated when the critical loading of the SR is achieved, the susceptibility of the rat myocytes to their occurrence cannot be due to the low threshold for Ca-induced Ca-release as found in skinned cardiac cells of the rat (Fabiato, 1982). According to the above reasoning, a more likely explanation is the more efficient uptake system ofthe SR in the rat heart compared to other mammals. This would cause loading of the SR at lower diastolic Ca level in the rat heart and subsequent release when the critical threshold level is attained. Indeed, experiments with isolated SR suggest that the uptake process of the SR is activated at lower Ca concentrations in the rat (K~ = 0.7 #M; Froechlich et al., 1978) than in other mammals (Km = 2 - 5 p M ; Solaro and Briggs 1974, Shigekawa etal., 1976). SUMMARY
The effects of caffeine and procaine on the spon-
603
taneous contractile waves of enzymatically isolated rat ventricular cells were in several respects opposite. Caffeine (1 mM) increased the frequency of contractions but decreased their amplitude, whereas procaine (1 and 5 mM) depressed beat frequency but enhanced the amplitude of shortening. Five mM caffeine, as a final effect, stopped spontaneous contractile waves in diastole. Both drugs decreased the propagation velocity of contractile waves and caused at higher concentrations (5 mM) shortening of diastolic length of the myocyte. The latter effect is probably due to the sensitization of contractile proteins to Ca. In right ventricular strips of the rat heart, caffeine (5 mM) caused negative inotropic effect whereas procaine (2 raM) increased the force of contraction. Procaine ( 4 m M ) diminished the amplitude of aftercontractions induced by ouabain (10-4M) in the intact tissue. It is suggested that improved contractility in the presence of procaine is due to its ability to abolish fluctuations of diastolic Ca and tension.
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Allen D. G. and Kurihara S. (1980) Calcium transients in mammalian ventricular muscle. Eur. Heart J. Suppl. A, 5-15. Allen D. G., Eisner D. A. and Orchard C. H. (1984) Characterization of oscillations of intracelluiar calcium concentration in ferret ventricular muscle. J. Physiol. 352, 113-128. Allen D. G., Eisner D. A., Spirolo J. S. and Smith G. L. (1985) The relationship between intracellular calcium and contraction in calcium-overloaded ferret papillary muscles. J. Physiol. 364, 169-182. Blayney L., Thomas HI, Muir J. and Henderson A. (1978) Action of caffeine on calcium transport by isolated fractions of myofibrils, mitochondria and sarcoplasmic reticulum from rabbit heart. Circulation Res. 43, 520-526. Blinks J. R., Olson C. B., Jewell B. R. and Braveny P. (1972) Influence of caffeine and other methylxanthines on mechanical properties of isolated mammalian heart muscle. Circulation Res. 30, 367-392. Chapman R. A. and Leoty C. (1976) The time-dependent and dose-dependent effects of caffeine on the contraction of the ferret heart. J. Physiol. 256, 287-314. Chiesi M., Ho M. M., Inesi G., Somlyo A. V. and Somlyo A. P. (1981) Primary role of sarcoplasmic reticulum in phasic contractile activation of cardiac myocytes with shunted myolemma. J. Cell Physiol. 91, 728-742. Cobbold P. H. and Bourne P. K. (1984) Aequorin measurements of free calcium in single heart cells. Nature 312, 444-446. Dani A. M., Cittadini A. and Inesi G. (1979) Calcium transport and contractile activity in dissociated maremarian heart cells. Am. J. Physiol. 237, C147-C155. Fabiato A. (1982) Calcium release in skinned cardiac cells: variations with species, tissues and development. Fedn. Proc. Fedn Am. Socs exp. Biol. 41, 2238-2244. Fabiato A. (1985) Spontaneous versus triggered contractions of "calcium-tolerant" cardiac cells from the adult rat ventricle. Basic Res. Cardiol. 80, Suppl. 2, 83-88. Fabiato A. and Fabiato F. (1973) Activation of skinned cardiac cells. Subcellular effects of cardioactive steroids. Eur. J. Cardiol. 112, 143-155.
Ford L. E. and Podolsky R. J. (1972) Calcium uptake and force development by skinned fibers in EGTA buffered solutions. J. Physiol. 223, 1-19. Froechlich J. P., Lakatta E. G., Beard E., Spurgeon H. A., Weisfeldt M. C. and Gerstenblith G. (1978) Studies of sarcoplasmic reticulum function and contraction duration
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in young adult and aged rat myocardium. J. mol. Cell. Cardiol. 10, 427-438. Haworth R. A., Hunter D. R. and Berkoff H. A. (1980) The isolation of Ca2+-resistant myocytes from the adult rat. J. tool. Cell. Car.diol. 12, 715-723. Hess P. and Wier W. G. (1984) Effects of caffeine on the intracellular (Ca 2+) transient, membrane currents, and contraction. J. gen. Physiol. 83, 417-433. Hunter D. R., Haworth R. A. and Berkoff H. A. (1982) Cellular calcium turnover in the perfused rat heart: modulation by caffeine and procaine. Circulation Res. 51, 363-370. Kao R. L., Christman E. W., Luh S. L., Krauhs J. M., Frank G., Tyers O. and Williams E. H. (1980) The effect of insulin and anoxia on the metabolism of isolated mature cardiac myocytes. Archs Biochem. Biophys. 203, 586-599. Kort A. and Lakatta E. G. (1984) Calcium-dependent mechanical oscillations occur spontaneously in unstimulated mammalian cardiac tissues. Circulation Res. 54, 396-404. Lakatta E. G. and Lappe D. L. (1981) Diastolic scattered light fluctuation, resting force and twitch force in mammalian cardiac muscle. J. Physiol. 314, 369-394. Lehto H., Talo A., Tirri R. and Vornanen M. (1983) Membrane potential oscillations in enzymatically isolated rat myocardial cells. Acta physiol, scand. 118, 385-391. Matsuda H., Noma A., Kurachi Y. and Irisawa H. (1982) Transient depolarization and spontaneous voltage fluctuations in isolated single cells from guinea pig ventricles. Circulation Res. 51, 142-151. Nieman C. J. and Eisner D. A. (1985) Effects of caffeine, tetracaine, and ryanodine on calcium-dependent oscillations in sheep cardiac purkinje fibers. J. gen. Physiol. 86, 877-889. Orchard C. H., Eisner D. A. and Allen D. G. (1983) Oscillations of intracellular Ca 2÷ in mammalian cardiac muscle. Nature 304, 735-738. Rieser G., Sabbadini R., Paolini P., Fry M. and Inesi G. (1979) Sarcomere motion in isolated cardiac cells. Am. J. Physiol. 236, C70-C77. Shigekawa M., Finegan J.-A. M. and Katz A. M. (1976)
Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. J. biol. Chem. 251, 6894-6900. Solaro R. L and Briggs F. N. (1974) Estimating the functional capabilities of sarcoplasmic reticulum in cardiac muscle. Circulation Res. 34, 531-540. Stephenson D. G. and Wendt I. R. (1986) Effects of procaine on calcium accumulation by the sarcoplasmic reticulum of mechanically disrupted rat cardiac muscle. J. Physiol. 373, 185-207. Stern M. D., Kort A. A., Bhatnagar C. M. and Lakatta E. G. (1983) Scattered-light intensity fluctuations in diastolic rat cardiac muscle caused by spontaneous Ca2+-dependent cellular mechanical oscillations. J. gen. PhysioL 82, 119-153. Tirri R., Vornanen M. and Lehto H. (1982) Characterization of contractions in mechanically and enzymatically isolated myocardial cells from the rat heart ventricles. Acta physiol, scand. 116, 257-263. Valdeomillos M. and Eisner D. A. (1985) The effects of ryanodine on calcium-overloaded sheep Purkinje fibers. Circulation Res. 56, 452-456. Vornanen M. (1984a) Characterization of contractions in enzymatically isolated rat ventricular myocytes: effects of ouabain and rubidium. Can. J. Physiol. Pharmac. 62, 210-216. Vornanen M. (1984b) Effects of caffeine on the mechanical properties of developing rat heart ventricles. Comp. Biochem. Physiol. 78C, 329-334. Vornanen M. and Tirri R. (1983) Effects of acetylcholine, isoprenaline, phenylephrine and dibutyryl cAMP on the contractility of isolated myocytes of the rat heart ventricles. Acta physioL scand. 118, 433-438. Weber A. and Herz R. (1968) The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J. gen. Physiol. 52, 750-759. Wendt I. R. and Stephenson D. G. (1983) Effects of caffeine on Ca-'activated force production in skinned cardiac and skeletal muscle fibers of the rat. Pflugers Arch. ges. Physiol. 398, 210-216. Wier W. G. and Hess P. (1984) Excitation--contraction coupling in cardiac Purkinje fibers. Effects of cardiotonic steroids on the intracellular (Ca 2+) transient, membrane potential, and contraction. J. gen. Physiol. 395, 395-415.
0306-3623/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
EFFECTS OF PROCAINE AND CAFFEINE ON THE CONTRACTILITY OF ENZYMATICALLY ISOLATED MYOCYTES AND INTACT CARDIAC TISSUE MATTI VORNANEN Department of Biology, University of Joensuu, P.O. Box 11 l, SF-80101 Joensuu, Finland (Received 22 December 1986) Abstract--l. Procaine and caffeine exerted opposite effects on the contractility of rat cardiac tissue. 2. Procaine enhanced and caffeine depressed isometric contractile force of right ventricular strips. 3. In enzymatically isolated cells, caffeine increased the frequency and decreased the amplitude of spontaneous contractions, while procaine retarded the beating rate but concomitantly raised the shortening amplitude of the myocyte. 4. Both drugs depressed the propagation velocity of the contractile waves. 5. The results are suggested to be due to the opposite effects of the two drugs on the Ca-release process of the sarcoplasmie reticulum.
INTRODUCTION The contractility of cardiac muscle is regulated by the a m o u n t of Ca that is released in the myoplasm after excitation of the sarcolemma (Allen and Kurihara, 1980). Recent experiments suggest that even during diastole the myoplasmic Ca level fluctuates (Orchard et al., 1983; Hess and Wier, 1984; Allen et al., 1984, 1985; Valdeomillos and Eisner, 1985) causing small oscillation of resting tension (Lakatta and Lappe, 1981; Stern et al., 1983; K o r t and Lakatta, 1984; N i e m a n and Eisner, 1985). These miniature oscillations o f Ca and tension can have profound effects on the excitation-dependent Ca-release and thereby on the force generated by the cardiac muscle (Allen et aL, 1985). Oscillations are presumably caused by the sarcoplasmic reticulum (SR) which spontaneously releases its Ca content. Spontaneous cycles of repeated uptake and release of Ca can be found in Ca-tolerant enzymatically isolated myocytes as propagating contractile waves (Tirri et al., 1982; Matsuda et al., 1982; Cobbold and Bourne, 1984). The contractile waves seem to be an especially conspicuous feature of adult rat heart cells (Chiesi et al., 1981). In the present study, we describe the effects of caffeine and procaine - - t w o drugs which interfere with the function of S R - - o n the wave-like contractions of enzymatically isolated rat heart myocytes and compare them to their effects on intact tissue. The results show that in both preparations, caffeine depresses and procaine enhances contractility. The contraction promoting effect o f procaine may be due to its ability to increase Ca content o f the SR by inhibiting diastolic Cafluctuations. METHODS
Isolation of ventricular myocytes Adult (3-4 months) male and female rats of Wistar strain were used in the experiments. They were maintained on a
12-hr dark, 12-hr light cycle and fed by commercial animal chow and tap water ad libitum. Single ventricular ceils were isolated by the modified method of Kao et al. (1980). This procedure involves combined perfusion and incubation of cardiac tissue with collagenase and hyaluronidase containing solution in the presence of 50#M Ca as described previously (Vornanen and Tirri, 1983; Vornanen, 1984a). For the experiments the dispersed cells were suspended in physiological solution of the following composition (mmol/1): NaCI 136, KC1 4.6, MgCI2 1.2, NaH:PO 4 1.8, CaCI2 2.5, glucose 11, buffered with 10mM Hepes-NaOH to pH 7.4. Recording of contractions Forty to sixty percent of the myocytes were Ca-tolerant as judged from their cross-striated, rod-shaped morphology in 2.5 mM Ca solution. Ceils which contracted steadily at low frequency (1-12 contractions/min) were accepted for experiments, because slowly beating cells have been found to retain their negative resting potential (Lehto et al., 1983). Recording was carried out by a microscopic video technique. For this purpose, a 100 #1 sample of myocyte suspension was transferred on a depression slide and a myocyte filling the criteria of an intact Ca-tolerant cell was selected with the aid of a phase-contrast microscope. The cell was centred in the field of vision at such magnification that the myocyte occupied almost the whole screen of the T.V. monitor, i.e. one cell at a time constituted an experimental preparation. The myocyte was inspected during the 5 rain control period to assure that its activity remained steady, after which a concentrated solution of procaine or caffeine was carefully added to give a final concentration of 1 or 5 mM. Three contractile parameters--(1) frequency of contractions, (2) amplitude of contraction (i.e. fractional shortening of the myocyte from its resting length) and (3) velocity of contraction wave--were later analysed from the video tape. Parameters (2) and (3) were determined at slowed film speed by means of stopwatch and transparent #m-scale placed on the screen. Each parameter was measured 5 times and two extreme values were neglected and the mean of the remaining three was accepted as final value. Experiments were conducted at room temperature (20°C). Membrane potentials were recorded with conventional glass microeleetrodes filled with 3 M KCI and had resistanees between 40 and 60 Mf~. Ag-AgCl-unit was used as 599
600
MATT1 VORNANEN
/ Fig. 1. Two typical enzymatically isolated Ca-tolerant cells used in the experiments and an action potential elicited by hyperpolarizing current pulse in a spontaneously beating ventricular cell.
a reference electrode. Both electrodes were connected to WPI KS-700 electrometer, which was also used to inject current through the microelectrode in eliciting action potentials. For the potential recording, a few drops of myocyte suspension were placed on depression slide attached to the stage of inverted microscope (Leitz). Cells were impaled by advancing microelectrodes at a steep angle (~ 60 °) with respect to the bottom of the chamber. Signals were displayed on an oscilloscope and were photographed using a Polaroid camera.
uniform shortening of the myocyte. In physiological solution that contains 2.5 m M Ca, the frequency of these contractions was 5.1 + 0.36/min (mean _+ SEM, n = 69) and caused a 9.7 _+ 0.45% (mean + SEM, n = 61) shortening of the myocyte's resting length ( = amplitude). The velocity of the contraction wave was 110 _+ 2 ~m/sec (mean _+ SEM n = 55) at 20°C. The effects of caffeine and procaine on these spontaneous contractile waves were examined. Immediately after its addition, 5 m M caffeine Experiments on intact tissue induced few vigorous contractions at very high freRight ventricular strips of the rat heart were used for quency, but a little later (19 _+ 14 see, mean +_ SEM) isometric tension recording. Strips under 1 mm dia were the myocyte stopped the spontaneous beating and mounted in the organ bath and connected to a forceremained at the completely relaxed state (Fig. 2). At displacement transducer (Grass FT03) as described previa concentration of l mM, the effects were similar but ously (Vornanen, 1984b). The muscles were stretched to their optimum length (Lm,x) and were paced to contract at less dramatic i.e. the frequency was first markedly 0.2 Hz by square wave pulses of 5 msec duration and voltage increased but about 2 min later recovered towards the 1.5 times the threshold value. The composition of the control level remaining, however, somewhat above physiological solution used in these experiments was as this. Amplitude was also initially enhanced but subfollows (mmol/l): NaCI 127.6, KC1 4.0 NaHCO 3 12.0, sequently clearly depressed below the control value. NaH2PO 4 0.4, MgSO 4 1.5, CaC12 2.5 and glucose 11 at The velocity of the contraction wave was markedly pH 7.4. The solution was gassed all the time with a mixture diminished (Fig. 2). of 5% CO2 and 95% Oz and kept at 30°C. Isometric tension The effects of procaine were in several ways and its first derivative were recorded on paper (Grass 7D polygraph) and monitored with aid of an oscilloscope (HP opposite to those of caffeine. The frequency of contractions was in a dose-dependent manner decreased 1201B). so that many cells stopped beating totally for a few minutes after the addition of 5 r a M procaine. RESULTS Instead, the c o n t r a c t i o n s - - n o w occurring at longer Typical cylindrical, cross-striated cells used in the time intervals were prominently much greater in experiments are shown in Fig. 1. In an unselected amplitude, especially in the presence of 5 m M propopulation of myocytes (n = 12), beating spontanecaine. The effect of procaine on the velocity of the ously at the rate of 3-12 times/min, the resting contraction wave was similar to that of caffeine, i.e. potential varied between 70 and 7 8 m V (mean it was depressed (Fig. 2). + SEM, 74_+ 0.8). Action potentials with normal Caffeine and procaine also changed the resting configuration could be elicited in these cells (Fig. 1). length of the relaxed myocytes. This effect was not Two types of contraction can be distinguished in seen immediately after the drug addition but only at enzymatically isolated Ca-tolerant myocytes of the the end of the 15 min exposure. The resting length rat heart ventricles; normal action potential coupled was 99.4 and 92.3% from the control after 15min twitches triggered by electrical stimulation and sponexposure to 1 and 5 m M caffeine, respectively. Corretaneous low-frequency contractions not associated sponding values for 1 and 5 m M procaine were 99.1 with action potential firing. Both types of contraction and 94.6%. The diminished resting length during occur in the same cell. The spontaneous low- prolonged exposure to higher (5 mM) procaine and frequency beats are characterized by slowly propa- caffeine concentrations suggests that these drugs may gating contractile wave of few activated sarcomeres cause sensitization of contractile proteins to Ca. This which spreads the whole cell length, causing noneffect seems to be stronger for caffeine.
Procaine and caffeine
601
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o..-o 5mH H ImM
Caffeine
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frequency
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amplitude
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120
120
110
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~" 90 80
80 I
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Fig. 2. The effects of two concentrations of procaine (left panel) and caffeine (right panel) on the three parameters describing spontaneous contractile activity in enzymatica[ly isolated myocytes of the rat heart. The results arc means-I-SEM of 10-20 experiments. The drug addition was made at the moment 0. Temperature 20°C.
In order to see if these effects of caffeine and procaine are limited to enzymatically isolated cells or whether they occur also in intact tissue, ventricular strips were examined. Figure 3 shows original recordings about the effects of caffeine and procaine on intact tissue. Five mM caffeine causes a typical negative inotropic effect of the adult rat heart [Fig. 3(d)], whereas procaine (2 raM) clearly increased the force of contraction [Fig. 3(a) and (b)]. The mean increase in developed tension was 37 _+ 5.6% (mean + SEM, n = 8) at 2 mM procaine solution. A few minutes after the procaine addition, the contractile force began to decline, which was caused by the loss of excitability as indicated by the recovery of tension upon increased stimulus voltage. This generally prevented the usage of higher concentrations of procaine. The positive inotropic effect of procaine was associated with increased time to peak tension
and increased duration of the contraction-relaxation cycle [Fig. 3(a)]. Aftercontractions elicited by 3 × 10 -4 ouabain were clearly depressed by procaine (4 raM) [Fig. 3(c)]. DISCUSSION The enzymatically isolated cells used in the present study were Ca-tolerant, clearly cross-striated and rod-shaped. Furthermore, the myocytes retained these characteristics several hours when kept in physiological Ca concentrations (2.5 raM) at room temperature. The sarcolemma of the cells is functional as shown by their normal resting and action potentials. Although the myocytes have the characteristics of normal cardiac cells and can be electrically induced to twitch contractions (Vornanen and Tirri, 1983), they exhibit another type of contraction which
MATTI VORNANEN
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b.
rain i
5 sec )
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-
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c.
10"4M OUABAIN
10"4M OUABAIN +4 mM PROCAINE
d.
Fig. 3. Original recordings showing the effects of caffeine and procaine on isometric contractions of right ventricular strips from the rat heart. Slow speed polygraph recording Co) and fast oscilloscope recording (a) indicating the effect of 2 mM procaine on the isometric contractile force and the time course of a single contraction-relaxation cycle. (c) Reduction of aftercontraction amplitude by 4 mM procaine in oubain-treated tissue. (d) Effect of 5 mM caffeine on isometric contractile force. Temperature 30°C. appears as spontaneous contractile waves travelling from one end of the cell to the other. These wave-like contractions are not coupled to action potentials but they are associated with membrane potential fluctuations of a few mV's amplitude (Lehto et al., -1983). This kind of contractile activity is an expression of the function of the SR (Fabiato and Fabiato, 1973; Rieser et al., 1979; Dalai et al., 1979; Chiesi et al., 1981) as repeated uptake and release cycles of Ca, and is assumed to use the same pathway as the Ca-induced Ca-release process although the mechanism may not be identical with the latter (Fabiato, 1985). Wave-like contractions are generally found in mechanically and functionally skinned cardiac cells but their occurrence in Ca-tolerant
myocytes has been often regarded as a feature of Ca overloaded, damaged cells (Haworth et al., 1980; Fabiato, 1985). On the other hand, contractile waves could also be a property of intact rat heart cells because fluctuations of intracellular Ca and tension has been measured in intact, untreated cardiac cells during diastole (Allen et al., 1984, 1985). Furthermore, propagating contractile waves have been found in intact cardiac tissue without any Ca-overloading (Stern et al., 1983). Caffeine and procaine had opposite effects on these wave-like contractions. In steady state conditions, caffeine (1 mM) shortened the interval between beats and concomitantly diminished their amplitude, whereas procaine (1 and 5 mM) increased the interval between successive contractions but simultaneously enhanced their amplitude. Caffeine is generally thought to induce Ca-release from the SR and inhibit its Ca-uptake process (Weber and Herz, 1968; Blinks et al., 1972; Blayney et al., 1978; Wier and Hess, 1984). The effects of caffeine on wave-like contractions suggest that the spontaneous contractile activity is determined by the function of SR also in the Ca-tolerant myocytes. The action of caffeine was especially evident at 5 mM concentration, causing few vigorous shortenings immediately after its addition but subsequent stopping of the contractile activity at the relaxed state of the myocyte. Increased frequency and decreased amplitude of contractile waves in the presence of 1 mM caffeine suggests that this concentration of caffeine cannot totally inhibit the uptake process although it clearly enhances the release phase. Shortening of the myocytes resting length after longer caffeine exposure may be due to its ability to sensitize the myofilaments of Ca (Wendt and Stephenson, 1983). Procaine is generally thought to retard the Ca release of SR in cardiac as well as skeletal muscle (Chapman and Leoty, 1976; Hunter et al., 1982; Ford and Podolsky, 1972). Procaine's ability to increase the interval between spontaneous contractile waves in isolated myocytes could be interpreted according to this proposal. However, procaine also caused a prominent increase in the shortening amplitude, which cannot be due to the inhibition of the Carelease from the SR. If spontaneous cyclic Ca release is determined by the Ca-loading level of the SR, i.e. if the release process is initiated at a certain critical threshold level of Ca in the SR the present experiments suggest that procaine elevates this threshold value. In the presence of procaine a greater amount of Ca may be accumulated in the SR and subsequently released during the following beat. The results are in agreement with the findings of Stephenson and Wendt (1986) on functionally skinned fragments of rat heart ventricle where procaine could enhance caffeineinduced contractions. Nieman and Eisner (1985) reported that tetracaine decreased the frequency of oscillations in intact Purkinje fibres but had no effect on their amplitude, suggesting a different mode of action for this drug. Caffeine seems to have an opposite effect to that of procaine, lowering the Caloading level which is needed to trigger the release process as found in several other cardiac preparations (Wier and Hess, 1984; Nieman and Eisner, 1985). The effects of caffeine and procaine on the iso-
Procaine and caffeine metric tension of intact tissue were similar to their action on the contractile waves of enzymatically isolated cells. This indicates that the drugs change the function of the SR in the same manner in these two preparations. The negative inotropic effect of caffeine is likely due to the depletion of the SR Ca-stores. The positive inotropic effect of procaine indicates that under the operation of physiological Ca-triggering mechanism--perhaps the Ca-induced Ca-release process--this drug sooner potentiates the Ca-release than inhibits it. In contrast to the enzymatically isolated cells the effect cannot be now--because of constantly paced stimulation--due to the longer interval between suggestive contractions to allow more effective loading of the SR. The effect of procaine on the duration of isometric contraction and the velocity of contraction wave suggest that the Ca-release rate is not enhanced but rather is retarded. A possible explanation is that spontaneous inhomogenous Ca-releases, analogous to those causing contractile waves in isolated cells, occurs also in the intact tissue. Abolition of the small diastolic Cafluctuations by procaine would allow more uniform loading of the SR with Ca and greater amount of release during the next twitch. In fact, the diastolic Ca level is not constant but small fluctuations in the Ca concentration and associated myofilament movements have been found in several cardiac tissues (Lakatta and Lappe, 1981; Stern etal., 1983; Orchard et al., 1983; Allen et al., 1984, 1985; Wier and Hess, 1984; Nieman and Eisner, 1985). These asynchronous miniature contractions can lead to the suppression of twitch tension (Stern et al., 1983; Allen et al., 1984). The finding that procaine eliminates aftercontractions, which are probably synchronized miniature Ca-fluctuations, support the proposal that inhibition of diastolic Ca-fluctuations is the mechanism of procaine's action. In addition, procaine may also cause small sensitization of contractile proteins to Ca. The present results suggest that fluctuation of tension and Ca may occur in addition to enzymatically isolated cells also in intact cardiac tissue of the rat heart. It has been found earlier that the contractile waves are more easily generated in the adult rat heart myocytes than in the cardiac cells of other mammals (Chiesi etal., 1981). If spontaneous contractile waves are generated when the critical loading of the SR is achieved, the susceptibility of the rat myocytes to their occurrence cannot be due to the low threshold for Ca-induced Ca-release as found in skinned cardiac cells of the rat (Fabiato, 1982). According to the above reasoning, a more likely explanation is the more efficient uptake system ofthe SR in the rat heart compared to other mammals. This would cause loading of the SR at lower diastolic Ca level in the rat heart and subsequent release when the critical threshold level is attained. Indeed, experiments with isolated SR suggest that the uptake process of the SR is activated at lower Ca concentrations in the rat (K~ = 0.7 #M; Froechlich et al., 1978) than in other mammals (Km = 2 - 5 p M ; Solaro and Briggs 1974, Shigekawa etal., 1976). SUMMARY
The effects of caffeine and procaine on the spon-
603
taneous contractile waves of enzymatically isolated rat ventricular cells were in several respects opposite. Caffeine (1 mM) increased the frequency of contractions but decreased their amplitude, whereas procaine (1 and 5 mM) depressed beat frequency but enhanced the amplitude of shortening. Five mM caffeine, as a final effect, stopped spontaneous contractile waves in diastole. Both drugs decreased the propagation velocity of contractile waves and caused at higher concentrations (5 mM) shortening of diastolic length of the myocyte. The latter effect is probably due to the sensitization of contractile proteins to Ca. In right ventricular strips of the rat heart, caffeine (5 mM) caused negative inotropic effect whereas procaine (2 raM) increased the force of contraction. Procaine ( 4 m M ) diminished the amplitude of aftercontractions induced by ouabain (10-4M) in the intact tissue. It is suggested that improved contractility in the presence of procaine is due to its ability to abolish fluctuations of diastolic Ca and tension.
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