Caffeine effects on mechanical activity in newborn rat myocardium

RAPID

Caffeine

Effects

COMMUNICATION

on Mechanical Rat Myocardium

Activity

(Receirled 1 1 iLl$p 198 1, acceped in rwised form

in Newborn

15 JU[Y 198 1)

Pharmacological properties of adult rat heart differ from those of other spccics. I;or example, a rather 101~ sensitivity of the adult rat heart to cardiac glycosides has been generally accepted for a long time; even a negative inotropic effect has been reported in presence of ouabain [L’]. C:affcinc, another lvell known positive inotropic agent, induces also a negative inotropic cffcct in adult rat heart [3, ci]. In papillary muscles from neonatal rats, Langcr ef al. [e] have shown that ouabain induces a positive inotropic effect. Its magnitude dcclincs with increasing age of the animal. To out knowledge, caffcinc efIects in newborn rat hearts have not been previously studied. The prrsrnt work was undertaken to investigate whether caffeine induced an ouabainlike positive inotropic effect in papillary muscles from neonatal rat heart, or a ncgativr one as in the adult. Caffcinc is knocvn to increase the cytoplasmic calcium concentration by releasing calcium ions from intracellular stores, mainly from the sarcoplasmic reticulum (SK), 19, 121. It has been sho\vn that the Ca efIlux from guinea-pig auricles was increased by substances which cause a release of calcium from intracellular stores [7]. l’his efllux is due to the functioning of a carrirrmediated Na-(:a exchange [I0 1. A reduction in C:a cfllux was observed when Na(:l was replaced by LiC:l, leading to a contracturc by addition of cafreinc to such ;I medium [7]. It was invrstigatcd in newborn rat myocardiurn xvhethcr caffcinc modified the Na-C:a exchange activity as dcmonstratcd in guinea-pig auricles. E’ol this purpose the functioning of this exchange was trstrd by lolvrring thr external sodium concentration and the effects of caffeine on the contrartilr activity \vere studied in presence of such a medium. If caffcintb, in normal cxtcrnal sodium concentration stimulates the (:a efTlux through the Nap<:a cxchangc, in low sodium medium containing caffeine a contracturc might br rxpcctcd. Ventricular papillary muscles were cut off from 3-day-old rat hearts. Thr muscles were mounted in a tissue bath (volume 2 ml:,, supcrfuscd at 12 mlmin with ‘l’yrodr: solution and maintained at 27’C:. One end of the muscle \vas fixed to the bottonl of the chamber and the other one attached to a hook connected to a transducer (UC: 2 Statham). The preparations were point-stimulated \vith a pair of platinum electrodes (30 beatsjmin). Stimuli were rectangular pulses, 2 ms in duration and 1.5 times thrrshold. Diastolic tension was adjusted in order to obtain the maximal dcvcloprd tension under control conditions. The composition of the control medium \vas (in mM): Na(:l, 121.4; KU, 4; CaCl,, 2.5; hIgC:l,, 1.25; NaH(:03, 19; SaH,PO,, 1.18 ; dextrose, 5.5; mannitol, 1.12; (pH 7.3). It was saturated \\ith 95”,, 0, and .5”;, CO,. LOM. sodium solution (20 rnhf) was prepared by replacing entirely Na( 11 with either equimolar LiCl or isoosmolar sucrose.

862

Rapid

Communication:

P. Jourdon

et al.

(A) -1

t

e In Caffeine

Inn!

Caffeine

5mt.4

(E)

Caffeme

5 “M

(I ml” 1

Caffeme

5 fnr.3 (IO min)

FIGURE 1. Continuous contractile activity pen-recordings of newborn rat heart. Caffeine was added at the time marked by the arrow: 1 rnM (A); 2.5 rnM (B) and 5 rnM (C). A transient positive inotropic effect is followed by a negative one. Separate contractile activity oscilloscope recordings are shown at three relevant times in (D), showing no modification in the time-course of contraction under caffeine 5 mM.

Figure 1 shows the effects of three different concentrations of caffeine: 1 mM [Figure 1 (A)], 2.5 mM [Figure 1 (B)],5 rnM [Figure 1 (C) and (D)], on the mechanical activity of a papillary muscle from a 3-day-old rat. The most striking change produced by exposure to caffeine was the increase in developed tension during the first minute of perfusion. The magnitude of this positive inotropic effect was dose-dependent. In the present experiment the maximum induced responses to 1, 2.5 and 5 mM of caffeine were 125%, 18Oo/o and 175% respectively. After this rapid increase the amplitude of the contraction progressively decreased. The positive inotropic effect was sustained during 5 min after which the decline in tension led to a negative inotropic effect. Ten minutes of caffeine superfusion were required for the preparation to be under steady-state. In the data presented in Figure 1, the amplitudes of the steady-state contraction correspond to 81% [Figure 1 (A)], 90% [Figure l(B)] and 77% [Figure 1 (C)] of the control amplitude. No modification of the resting tension was observed. [Figure l(D)] shows that the time course of the contraction was not affected by 5 mM caffeine. All the caffeine-induced modifications were almost reversible (90%) within 20 min of washing out with caffeine-free solution. Similar results were obtained in six additional experiments. To check the remote possibility that the development of adrenergic nerve endings accounted for the caffeine induced response, papillary muscles were treated with 10m6 M propranolol. The caffeine effects were not altered.

Caffeine

and Contraction

in Newborn

Rat Heart

863

By lowering the external sodium concentration from 140 to 20 mM, a contracture settled within 2 min and aftercontraction oscillations occurred. Then a progressive decline in resting tension level was observed (not shown). Fifteen to twenty minutes were required for the preparation to be under steady-state. By this time aftercontraction oscillations were still present [Figure 2(B, a)], and the twitch tension amplitude was enhanced (192% of control amplitude). Figure 2(A) shows the effects of caffeine (5 mM) on the contractile activity under steady-state in low sodium medium. An immediate increase in resting tension occurred. As the resting tension level rose, oscillations progressively disappeared [Figure 2(B)]. Under these conditions the contracture remained as long as caffeine was present in the superfusion medium. Figure 2(B, d) shows that the relaxation was incomplete. The caffeine-induced contracture was suppressed by washing out caffeine. Relaxation of the contracture also occurred in presence of caffeine, when the stimulator was turned off. If the preparation was superfused directly in low sodium containing caffeine solution, without previous stabilization in caffeine-free low sodium medium, the development of a sustained contracture occurred without apparent aftercontraction oscillations. Similar results were obtained in four additional experiments.

v-

[Nolo20mM

Plus

Coffelne

5 mt.4 coffelne

5 mt4 -

(305)

Plus

5 mt4 caffeine

(1 mm) Plus

5mM caffeme

(2 mm) 02

FIGURE 2. Continuous contractile activity pen-recordings of newborn rat heart shows a maintained increase in resting tension induced by caffeine (5 rn~) in Na-poor medium (A). Separate contractile activity oscilloscope recordings are shown at four relevant times in (B). They illustrate the delayed relaxation and the disappearance of the aftercontraction oscillations in (c) and (d). The dotted line indicates the level of the resting tension in low sodium medium without caffeine.

Our results demonstrated that caffeine, in normal Tyrode solution, enhanced the amplitude of the contraction of papillary muscles from newborn rat hearts. To our knowledge, such an increase in contraction has not been previously described in rat myocardium. Only a very brief contracture, lasting 10 s, was observed by Chapman and Leoty [I], in isolated trabeculae from either adult rat auricle or ventricle very rapidly superfused with caffeine (0.5 to 10 mM). As already shown for ouabain sensitivity by Langer et al. [S], our results demonstrate that caffeine response is different in newborn compared to the adult rat, where caffeine acts as a negative

86-f

Rapid

Communication:

P. Jourdon

et al.

inotropic agent [3, 61. Caffeine is well known to release calcium ions from the SR resulting into intracellular free calcium concentration increase [9, 121. In the newborn rat heart the SR is poorly developed [21]. However, 2 days after birth the characteristics of the calcium induced release of calcium and the parameters measuring the calcium reaccumulation by the rat ventricular SR were similar to those observed for the adult rabbit SR {for review see Fabiato and Fabiato [4]}. Thus it can be assumed that the caffeine induced positive inotropic effect observed in the present study is due to an increase in free calcium cytoplasmic concentration resulting from a calcium release from the SR. However, the enhancing of the contraction amplitude induced by caffeine is only transient; at all concentrations tested, under steady-state a negative inotropic effect was observed. One might suppose that this fact could be related to the presence of a sparse SR in newborn rat heart cells, leading to an impoverishment in calcium of the SR under caffeine. However a negative inotropic effect is always observed in adult rat myocardium, where the SR is well developed, even in the presence of a low caffeine concentration [3, 61. Thus it seems very unlikely that the poorly developed SR in newborn rat myocardium might yield alone to the negative inotropic effect observed under steady-state. In guinea-pig atria it has been shown the existence of a calcium efflux due to the functioning of a carrier-mediated Na-C:a exchange [IO]. This calcium efflux, dependent on the external sodium and calcium concentrations, is increased by substances which cause a release of C:a ions from intracellular stores [7]. In the present study lowering the external sodium concentration to 20 mM induced a contracture and oscillations. According to Glitsch and Pott [5], an increased internal calcium concentration is a prerequisite for the occurrence of these oscillations. Thus it can be assumed that the activity of the Na-C:a exchange was decreased under our conditions. Caffeine in low sodium medium induced a sustained contracture. This result is similar to those obtained in guinea-pig atria by Jundt et al. [7]. Therefore it can be suggested that in newborn rat myocardium a caIcium ef?lux via the Na-(:a exchange contributes to the regulation of the internal calcium concentration, and this cfllux is enhanced by caffeine through the increase in C:ai. This caffeine-enhanced calcium cfllux prevents the calcium overload and might explain the absence of contracture in normal Tyrode solution. Furthermore both this mechanisl? and the decrease in sarcoplasmic reticulum calcium content, due to the inhibition by caffeine of calcium re-uptake [9, 1_3] might contribute to the decrease in contraction amplitude observed in normal Tyrode solution for long lasting caffeine exposure. In conclusion, our results demonstrate that caffeine effects on the contractile activity in newborn rat myocardium differ from those previously described in adult rat hearts, and a stimulation by caffeine of the C:a extrusion, via the Na-C:a exchange, is suggested. Acknowledgements

The authors are grateful to Mrs M. Meier for her technical Lowy for his assistance in the preparation of the manuscript. Philippe

Jourdon,

Marie-Claude Znstitut

KEY

WORDS:

Caffeine;

Contraction;

Low

Na-medium.

assistance

Auclair

and

and to Dr R.

Paul

Lechat

de Pharmacologic, CNRS LA 206, 15 rue de 1’Ecole de hl~decine, 75006 Paris, France

Caffeine

and

Contraction

in

Newborn

Rat

Heart

863

REFERENCES 1.

2. 3.

-1.

5. 6. 7. 8.

Y. 10. 11.

12.

CHAPMAN, R. A. & LEOTY, C. Which of caffeine’s chemical relatives are able to evoke contractures in mammalian heart? In Recent Advances in Studies on Cardiac Structure and Illetabolism, Volume 7, Biochemislry and PharmacoloQ of &yocardial Hy,bertrofihy, Hypoxia and hlfarction, P. Harris, R. J. Bing & A. Fleckenstein, Eds, pp. -125-430. Baltimore: Univrrsit) Park Press (1976). DIETRICH, J. & DIACONO, J. Comparison between ouabain and taurine effects on isolated rat and guinea-pig hearts in low calcium medium. Lif Science 10, 499-507 (197 1). DIETRICH, J., JOURDON, P. 8-z DIACONO, J. Action de la cafeine sur les activites dlcctriquc et mecanique du coeur isole de Rat et de Cobaye: discussion des roles respectifs des mouvements de calcium et des catecholamines chez les deux especes. Thirapie 31, 525-539 (1976). FABIATO, A. & FABIATO, F. Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cells from adult human, dog, cat, rabbit, rat and frog hearts and from fetal and newborn rat ventricles. Annals of the JVeNew York Academy of Sciences 307, 491-522 (1978). GLITSCH, H. G. & POTT, L. Spontaneous tension oscillations in guinea-pig atria1 trabeculae. PJliigers Archiu 358, 1 l-25 (1975). HENDERSON, A., BRUTSAERT, D., FORMAN, R. & SONNENBLICK, E. Influence of caffeine on force development and force frequency relations in cat and in rat heart muscle. Cardiovascular Research 8, 162-172 ( 1974). JUNDT, H., PORZIG, H., REUTER, H. & STUCKI, J. W. The effect of substances releasing intracellular calcium ions on sodium dependent calcium efllux from guinea-pig auricles. Journal of Phyriology 246, 229-253 (1975). LANCER, G. .4., BRADY, .4. J., TAN, S. T. & SERENA, S. D. Correlation of the glycoside response, the force staircase and the action potential configuration in the neonatal rat heart. Circulation Research 36, 7-k-752 (1975). NAYLER, W. & HASKER, J, Effect of caffeine on calcium ion subcellular fractions of cardiac muscle. American Journal of Physiology 211, 950-954 (1966). REUTER, H. & SEITZ, N. The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. Journal of Physiology 195, 451~470 (1968). SCHIEBLER, T. H. & WOLFF, H. H. Elektronenmikroskopische Untersuchungen am Herzmuskel der Ratte wihrend der Entwicklung. ,
(1973).