Basic Research in

Cardiology

Basic Res Cardio187:272-279 (1992)

Effect of dynorphin A(1-13) on cardiomyocytes in culture: modulation of the response to increased extracellular calcium, but no effect on intrinsic cardiac contractile frequency or the response to isoproterenol or increased extracellular potassium* S. W. Rabkin University Hospital (Shaughnessy), University of British Columbia, Vancouver, Canada

Summary: The purpose of this study was to determine whether the endogenous opioid peptide dynorphin A(1-13) has a direct effect on the heart or acts to modulate the cardiac chronotropic response to calcium, potassium, or [3-adrenergic receptor stimulation. Spontaneously contracting myocardial cell aggregates were prepared from 7-day-old chick embryos and were maintained in culture for 72 h before study. Dynorphin A(1-13), 10 Sto 10-6M, did not alter spontaneous contractile frequency. Increases in [Ca2+]o spontaneously suppressed cardiac contractile frequency, and dynorphin A(1-13) significantly (p < 0.05) enhanced this response. Nifedipine, 10-8M, antagonized the effect of increased [Ca2+]o on cardiac contractile frequency, but did not block the action of dynorphin A(1-13) to accentuate the effect of increasing [Ca2+]o. Dynorphin A(1-13) did not alter the significant (p < 0.05) increase in contractile frequency produced by beta-adrenergic receptor stimulation by isoproterenol, or the suppression in contractile frequency produced by increases in extracellular potassium ([K+]o). These data indicate that dynorphin A(1-13) does not act directly on the cardiac myocyte to alter cardiac contractile frequency or alter the response to increases in [K+]o or to isoproterenol, but that dynorphin A(1-13) does modulate the response to increases in extracellular calcium. Key words: Dynorphin A(1-13); cardiomyocytes; nifedipine; isoproterenoI; calcium; potassium

Introduction

Endogenous opioids can produce marked alterations in systemic arterial blood pressure (for review see (24)), but whether these effects are mediated through an action in the brain on central cardiac and/or vascular neural regulation, or on the peripheral autonomic nervous system, or directly on the heart, or on the systemic vasculature is uncertain. The possibility that endogenous opioids may act directly on the heart has been the subject of some controversy (3, 5, 28). This is of special importance for dynorphins, endogenous peptides that have a high binding affinity for kappa opioid receptors (2, 4, 29) and whose cardiovascular effects include sinus bradycardia after central or intravenous administration to the intact animal (6, 8, 17, 24, 34, 37). The mechanism of action of dynorphin A(1-13)-induced bradycardia has been proposed to involve a direct suppressant action on cardiac pacemaker function, an inhibition of norepinephrine release from sympathetic nerve terminals (7, 32, 33), an increase in parasympathetic tone (17) or an action within the central nervous system (6). A direct effect on the heart is possible as opioid peptides and opioid receptors have been * Funded in part by a grant from the Medical Research Council of Canada 730

Rabkin, Dynorphin A(1-13) and the heart

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identified in t h e h e a r t , including a high affinity k a p p a opioid binding site (14, 15, 36). T o help extract a p o t e n t i a l e x p l a n a t i o n for the action of d y n o r p h i n (1-13) to induce b r a d y c a r d i a , cardiac m y o c y t e s in culture are a v a l u a b l e m o d e l b e c a u s e they p e r m i t a n assessment of direct effects of a n a g e n t o n t h e cardiomyocyte. T h u s , the p u r p o s e of this study was to d e t e r m i n e w h e t h e r d y n o r p h i n A ( 1 - 1 3 ) - i n d u c e d b r a d y c a r d i a could b e m e d i a t e d by a direct action of d y n o r p h i n o n t h e c a r d i o m y o c y t e to d e c r e a s e s p o n t a n e o u s contractile frequency, or w h e t h e r d y n o r p h i n A(1-13) m o d u l a t e d t h e action of o t h e r factors such as calcium, p o t a s s i u m or b e t a a d r e n e r g i c r e c e p t o r s t i m u l a t i o n o n myocardial contractile frequency.

Materials and methods

Cell cultures Chick embryonic ventricular cells were cultured using previously described methods (25). Briefly, white Leghorn eggs were incubated in an automatic incubator (Marsh Rollex, San Diego, California, USA) for 7 days at 37.8 ~ and 87 % humidity. Hearts were then isolated under sterile conditions from the 7-day-old chick embryo. Blood and connective tissue were removed under a dissecting microscope in a solution of balanced salts (DMS8) with the following composition: NaC1 116 mM, KC1 5.4 mM, NaHaPO4 - H20 0.4 mM, Na2HPO4 97 H20 i mM and dextrose 5.6 mM. Disaggregation was carried out by 5-min digestions in 0.005 % trypsin (Gibco Laboratories, Burlington, Ontario), 0.1% BSA and 1 x 107 DNase per ml, DMS8 (Worthington Biochemicals, Frederic, New Jersey, USA) at 37.8 ~ After three digestions the digests were diluted 1:5 in culture medium and the cells centrifuged for 3 rain at 1000 x g in a clinical centrifuge (International Equipment Co., Neetham Heights, Massachusetts, USA). Cell aggregates were made following previously described methods (22, 26). Briefly, cells were collected, counted using a hemocytometer and diluted in medium in 25-ml Erlenmeyer flasks so that total volume was 3 ml with approximately 3.0 x 106 cells/flask (Falcon, Becton Dickinson, Oxnard, California, USA). Cells were maintained in medium 818A (20 % M199, 73 % DBSK buffer, 6 % fetal bovine serum, penicillin at 100 units per ml, streptomycin at 10 Ftg per ml plus fungizone 0.25 Ixg per ml). DBSK buffer had the following composition: NaC1 116 raM, NaH2PO4 - H20 1.0 mM, MgSO4.7 H20 0.8 mM, Na2HPO4 - H20 i mM, dextrose 5.6 mM, CaC12 1.8 mM and NaHCO3 26 mM. The flasks were placed on the platform of a variable speed rotator (American Rotator, Can Lab) in the incubator at 60 RPM. After 40 to 72 h, as needed, flasks were emptied into a 35 mm x 10 mm Petri dish and swirled briefly to center the aggregates, which were allowed to stick to the bottom for about 30 rain in the incubator. A pair of Petri dishes was placed in larger dishes with intake for 5 % CO2 in air, bubbled through water, on the stage of a Wilovert inverted microscope in a specially designed plexiglass compartment. Constant temperature was maintained at 37 ~ by a heat lamp with thermostat control and a temperature probe on the microscope stage. The pH of the media was 7.43. Drugs were added in the following manner: when the beating rate was constant, dynorphin A(1-13) or drug or both were added to the withdrawn medium and returned to the aggregate dish. Two ml of medium was withdrawn, placed in a Petri dish within the incubator, the drug was added, mixed well, and returned to the aggregate dish. Beating rates were recorded for each aggregate at regular intervals.

Drugs and chemicals Culture media were obtained from Gibco, Burlington, Ontario, Canada. All chemicals were analytical grade. Dynorphin A(1-13) and isoproterenol were from Sigma Chemical Co., Missouri, USA. Nifedipine was kindly provided by Miles Pharmaceuticals (Etobicoke, Canada).

Data analysis Contractile frequency was determined and the data were presented as the mean + one SD. Change and percent change from the preceding control procedure were also calculated. Hypothesis testing used analysis of variance. The level of significance was less than 5 % (p < 0.05).

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Basic Research in Cardiology, Vol. 87, No. 3 (1992)

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Fig. 1. The change in contractile frequency over a 40-min observation period for myocardial cell aggregates exposed to dynorphin A(1-13) at three different concentrations 10 -v M, 10.6 M, and 10-SM in the media. Each point represents the mean of 4-5 aggregates + 1 S.D.

Results T h e contractile f r e q u e n c y of myocardial cell aggregates is relatively stable o v e r time. A l t h o u g h each cell aggregate can b e a t at a different rate from a n o t h e r , the rate of c o n t r a c t i o n of each aggregate is relatively c o n s t a n t a n d is n o t significantly different o v e r the o b s e r v a t i o n period. T h e r e can b e a small c h a n g e after the addition of a n aliquot into t h e m e d i u m , e v e n if it is w i t h o u t drug, b u t t h a t disappears quickly. D y n o r p h i n A(1-13), in c o n c e n t r a t i o n s of 10 -a M or 10 7 M or 10 -6 M, did n o t alter cardiac contractile f r e q u e n c y o v e r

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Fig. 2. The contractile frequency after addition of isoproterenol 10 s M to the media in the absence of dynorphin A(1-13) (control; open circles or bars) or in the presence of dynorphin A(1-13) 10 6M. The change from baseline (time zero) is indicated by the bar graph in the lower part of the panel. Each point represents the mean of 4-5 aggregates _+1 S.D.

275

Rabkin, Dynorphin A(1-13) and the heart

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Fig. 3. The beating rate of aggregates exposed to increasing concentrations of calcium in the medium from 2.0, 3.0, 3.5, and 4.0 mM calcium. The results are shown for control cells not exposed to dynorphin A(1-13), and to cells exposed to 10 -6 M dynorphin A(1-13). Cessation of activity is displayed as zero. Each point represents the mean of 4-5 aggregates _+ 1 S.D. a 60-min observation period (Fig. 1). The m e a n contractile frequency over the entire observation period was very similar at all dynorphin A(1-13) concentrations, namely, 63.1 + 1.6, 64.4 _+ 1.5, and 64.5 + 2.9 beats/rain at, respectively, 10-8, 10 7, and 1 0 4 M . The percentage changes in contractile frequency were similar and approximately the same as baseline, that is, - 0 . 2 _+ 2.5 %, 1.9 + 2.4 % , and 1.9 + 4.7 % , respectively, for dynorphin A(1-13) 10-a, 10-7, and 1 0 4 M .

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Isoproterenol 10-SM, increased the contractile frequency of myocardial cell aggregates (Fig. 2). A positive chronotropic response was evident within 2 min and was sustained over the 20-min observation period (Fig. 2). The response to isoproterenol was not significantly altered by dynorphin A(1-13). The increase in contractile frequency was 47.8 _+7.5 % when exposed to isoproterenol alone, and 55.3 _+ 3.2 % when the aggregates were exposed to both dynorphin A(1-13) and isoproterenol. Cellular aggregates exposed to increasing concentrations of extracellular calcium [Ca2+]o showed a depression of contractile frequency. This was significantly (F = 8.98; p < 0.05) accentuated by dynorphin A(1-13) (Fig. 3). A n increase in [Ca2+]o to 3 mM produced a reduction in beating rate that was slightly greater in cells exposed to dynorphin A(1-13), namely, -65.3 + 3.9 %, compared to - 7 7 . 4 + 2.0% in control myocytes that were not exposed to dynorphin A(1-13). At 3.5 mM [Ca2+]o, cessation of contraction in myocytes was noted only in cells exposed to dynorphin A(1-13). A t 4 mM [Ca2+]o spontaneous contractile frequency ceased in myocytes that were or were not exposed to dynorphin A(1-13). With nifedipine (10-8 M) in the media, aggregates continued to contract, even in the presence of higher [Ca2+]o. There was, however, more variability, as reflected by the larger standard deviation (Fig. 4). Nifedipine did not prevent the early cessation of contraction observed with increased [Ca2+]o in cardiomyocytes exposed to dynorphin A(1-13), rather, there appeared to be an accentuation of the effect of dynorphin A(1-13) to suppress spontaneous cardiac contraction when [Ca2+]o was elevated. Increases in extracellular potassium concentration ([K+]o) decreased contractile frequency until aggregates stopped beating (Fig. 5). At [K+]o of 1.1 mM, normal medium, beating rate was constant. When [K+]o was increased to 2 mM, there was a significant ( p < 0 . 0 5 ) reduction in beating rate in myocytes, regardless of whether or not they were exposed to dynorphin A(1-13). The reduction in contractile frequency was - 7 5 . 6 _+ 0.8 % in aggregates exposed to dynorphin A(1-13). This was not significantly different from the reduction of -74.8_+ 2.9 % observed in cardiomyocytes that were not exposed to dynorphin A(1-13).

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Fig. 5. Cells exposed to increasing concentrations of potassium for 20-min periods starting from 1 mM and progressing up to 2, 4 mM. Beats per minute are shown as mean + SD. Aggregates with and without (control) dynorphin A(1-13) 10-6M in the media are shown. Each point represents the mean of 4-5 aggregates + 1 S.D.

Rabkin, Dynorphin A(1-13) and the heart

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When the extracellular potassium was 4 mM aggregates stopped beating, regardless of whether or not they were exposed to dynorphin A(1-13). Discussion

Dynorphin A(1-13) produces sinus bradycardia in most, but not all studies in the intact animal after its intracisternal (17), intrathecal (37) or intravenous administration (8), or after injection into the preoptic medialis nucleus (6). The present study found that dynorphin A(1-13), at concentrations from 10 8M to 10-6M, did not alter the contractile frequency of myocardial cell aggregates in culture. These results suggest that the negative chronotropic effects of dynorphin A(1-13) observed in the intact rat (6, 8, 17, 34, 37) are not due to a direct action of dynorphin A(1-13) on cardiac pacemaker cells, but, rather, are due to a central action, presumably on the autonomic nervous system. Central nervous system mechanisms are not the complete explanation for this effect of dynorphin A(1-13), as a significant bradycardia occurs in the pithed rat after intravenous dynorphin A(1-13) administration (17). Extrapolation of these results to the intact animal must take into consideration the use of chick embryonic heart cells. However, isolated cardiac cells from chick embryo contract spontaneously, like cardiac pacemaker cells in the intact animal. In contrast, cardiac myocytes isolated from the adult rat do not contract spontaneously and, although they can be induced to contract by electrical field stimulation, the comparisons are not as relevant to the issue of the potential of an agent to alter spontaneous beating in the intact heart. Isolated cardiac cells are a good model to investigate cardiac effects of drugs, and they permit the exclusion of effects on coronary vasculature and the autonomic nervous system. The chick heart model has its limitations, but it has been extensively studied and new data can be interpreted within the context of previous detailed electrophysiologic studies (30, 31). The preparation used herein, however, was cell aggregates that have properties more closely analogous to older and intact hearts (21, 31). Furthermore, data from this model has yielded responses similar to those in mammalian species (30). Importantly, this model has been found to register a response to endogenous opioids 06). An important finding of the present study is that dynorphin A(1-13) increases the suppression of spontaneous contraction produced by increases in extracellular calcium. Cardiac contractile frequency in cultured cardiac cells is inversely related to [Ca]o, as was demonstrated here and documented in previous studies (20, 23). Leu-enkephalin increases norepinephrine stimulated 45Ca2+ uptake in guinea pig atria, and the enkephalin analogue [D-Ala2]-enkephalin increases 45Ca2+ influx in contracting myocytes (16, 27). Thus, an explanation for the observed action of dynorphin A(1-13) may be that it accentuates calcium entry into the myocyte and accentuates its effects to depress automaticity at high [Ca2+]o concentrations. The mechanism by which dynorphin would increase calcium entry into the cardiomyocyte is uncertain. An action to increase calcium entry though voltage-dependent calcium channels would appear unlikely, based on the observation that the effect of dynorphin was not antagonized by the L-type voltage-dependent calcium channel antagonist nifedipine (12). Furthermore, nifedipine is active in this model, as demonstrated by the finding that it antagonized the action of increased [Ca2+]o on the cardiomyocyte. An accentuation of calcium influx into the cardiomyocyte in the presence of elevated [K+]o is also an unlikely possibility. Dynorphin A(1-13) did not alter the reduction in automaticity produced by increasing extracellular potassium. Increased [K+]o, to levels used herein, inhibits automaticity by hyperpolarizing the cardiac cell membrane, although other high concentration of K + may depolarize the cardiomyocyte (10, 35). The absence of an action of dynorphin A(1-13) on the consequences of increased [K+]o suggests that dynorphin

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may not influence K + coupled exchangers. Isoproterenol, which stimulates [31 and [32 receptors, increases the contractile frequency of myocardial cells in culture (1, 13) by increasing the steepness of the pacemaker potential and converting quiescent fibers into repetitively discharging ones (11). The present study found that dynorphin A(1-13) does not alter the chronotropic response to isoproterenol. This can be supported, in part, by the findings of Ledda et al. (18) that dynorphin A(1-13) did not alter the change in contractile tension induced by increasing concentrations of noradrenaline in isolated guinea pig atrium subjected to field stimulation. In summary, the data in this study presents compelling evidence that the negative chronotropic effect of dynorphin A(1-13) observed in the intact animal is not due to an action of dynorphin directly on the cardiac myocyte, but, rather, is due to other actions such as a central effect to increase parasympathetic tone (17), or to inhibit norepinephrine release from sympathetic nerve terminals (32, 33), or other consequences of stimulation of prejunctional opioid receptors on the adrenergic nerve terminals in the heart (18). Dynorphin A(1-13), however, does have a direct action on the heart which involves modulating the cardiomyocyte response to increases in extracellular calcium which may, in turn, have consequences on one of the many calcium-sensitive intracellular processes.

References

1. Boder GB, Johnson IS (1972) Comparative effects of some cardioactive agents on automaticity of cultured heart cells. J Molec Cell Cardiol 4:453-463 2. Charkin C, James IF, Goldstein A (1982) Dynorphin is a specific endogenous ligand of the k-opioid receptor. Science 215:413-415 3. CIo C, Muscari C, Tantini B, Pignatti C, Bernardi P, Ventura C (1985) Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration. Life Sciences 37:1327-1333 4. Corbett AD, Paterson SJ, McKnight AT, Magnon J, Kosterlitz H (1982) Dynorphin A(1-8) and dynorphin A(1-9) are ligands for the K-subtype opiate receptor. Nature 299:79-81 5. Eiden LE, Ruth JA (1982) Enkephalins modulate the responsiveness of rat atria in vitro to norepinephrine. Peptides 4:475-478 6. Feuerstein G, Faden AI (1984) Cardiovascular effects of dynorphin A(1-8), dynorphin A(1-13) and dynorphin A(1-17) mieroinjected into the preoptic medialis nucleus of the rat. Neuropeptides 5:295-298 7. Fuder H, Buder M, Riers HD, Rothacher G (1986) On the opioid receptor sub type inhibiting the evoked release of 3H-noradrenaline from guinea-pig atria in vitro. Naunyn-Schmiedeberg's Arch Pharmacol 332:148-155 8. Gautret B, Schmitt H (1985) Central and peripheral sites cardiovascular actions of dynorphin A(1-13) in rats. Europ .1 Pharm 111:263-266 9. Glatt CE, Kenner JR, Long JB, Holaday JW (1987) Cardiovascular effects of dynorphin A(1-13) in conscious rats and its modulation of morphine bradycardia over time. Peptides 8:1089-1092 10. January CT, Fozzard HA (1984) The effect of membrane potential, extracellular potassium, and tetrodotoxin on the intracellular sodium activity of sheep cardiac muscle. Circ Res 53:652 11. Kassebaum DG, Van Dyke AR (1966) Electrophysiologieal effects of isoproterenol on Purkinje fibers of the heart. Circ Res 19:940-946 12. Kohlhardt M, Fleckenstein A (1972) Inhibition of the slow inward current by nifedipine in mammalian ventricular myocardium. N-S Arch Pharmacol 298:267-276 13. Kranse EG, Halle W, Kallabis E, Wollenberger A (1970) A positive chronotropic response of cultured isolated rat heart ceils to NV 2'-O dibutyryl-3'5' adenosine monophosphate. J Molec Cell Cardiol 1:1-10 14. Krumins SA, Faden AI, Feuerstein G (1985) Opiate binding in rat hearts: modulation of binding after hemorrhagic shock. Biochem Biophys Res Commun 127:120-128

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15. Lang RE, Herman K, Dietz R, Gaida W, Ganten D, Kraft K, Unger T (1983) Evidence for the presence of enkephalins in the heart. Life Sci 32:399-406 16. Laurent S, Marsh JD, Smith TW (1986) Enkephalins increase cyclic adenosine monophosphate content, calcium uptake, and contractile state in cultured chick embryo heart cells. J Ctin Invest 77:1436-1440 17. Laurent S, Schmitt H (1983) Central cardiovascular effects of decreasing agonists dynorphin A(1-13) and ethylketocyclazocine in the anesthetized rat. Br J Pharmacol 96:165-169 18. Ledda F, Montelli L, Corti V (1985) Sensitivity of dynorphin A(1-13) of the presynaptic inhibitory opiate receptors of the guinea-pig heart. Europ J Pharm 117:377-380 19. Lee CO, Vassalle M (1983) Modulation of intracellular Na + activity and cardiac force by norepinephrine and Ca 2+. Am J Physiol 244:Cl10-Cl14 20. McCall D (1976) Effect of verapamil and of extracellular Ca and Na on contraction frequency of cultured heart cells. J Gen Physiol 68:537 21. MacDonald TD, Sachs HG (1975) Electrical activity in embryonic heart cell aggregate developmental aspects. Pfltig Arch Physiol 354:151-164 22. Myrdall SE, De Haan RL (1983) Concavolin A increases spontaneous beating rate of embryonic chick heart cell aggregates. J Cell Physiol 117:319-325 23. Rabkin SW (1989) The effect of amiloride and its analog dichlorobenzamil on the cardiac chronotropic responses of myocardial cell aggregates in culture to alterations of extracellular potassium or calcium. Gen Pharm 20:595~00 24. Rabkin SW, Redston M (1987) Cardiovascular effects of centrally administered opioid peptides considering their opioid receptor agonist properties. J Appl Cardiol 2:403-429 25. Rabkin SW, Sunga P (1987) The effect of doxorubicin (adriamycin) on cytoplasmic microtubule system in cardiac cells. J Molec Cell Cardiol 19:1073-1083 26. Rabkin SW, Sunga P, Myrdal S (1987) Effect of EGF on cardiac chronotropic responses in embryonic chick heart cells. Biochem Biophys Res Commun 146:889-897 27. Ruth JA, Cuizon JV, Eiden LE (1984) Leucine-enkephalin increases norepinephrine-stimulated chronotropy and 45Ca++ uptake in guinea-pig atria. Neuropeptides 4:185-191 28. Saunders WS, Thornhill JA (1985) No inotropic action of enkephalins or enkephalin derivatives on electrically-stimulated atria isolated from lean and obese rats. Br J Pharmac 85:513-522 29. Smith AP, Lee NM (1988) Pharmacology of dynorphin. Ann Rev Pharmacol Toxicol 28:123-140 30. Sperelakis N, Pappos AJ (1983) Physiology and pharmacology of developing heart cells. Pharm Therap 22:1-39 31. Sperelakis N, Shigenobu K (1972) Changes in membrane properties of chick embryonic hearts during development. J Gen Physiol 60:430-453 32. Starke K, Schoffel E, Iles P (1985) The sympathetic axons innervating the sinus node of the rabbit possess presynaptic K but not mu or gamma receptors. Naunyn-Schmiedeberg's Arch Pharmacol 329:206-209 33. Szabo B, Hedler L, Ensinger H, Starke K (1986) Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors. Naunyn-Schmiedeberg's Arch Pharmacol 332:50-56 34. Thornhill JA, Gregor L, Saunders WS (1989) Opiate and alpha receptor antagonists block the pressor responses of conscious rats given intravenous dynorphin. Peptides 10:171-177 35. Vassalle M (1965) Cardiac pacemaker potentials at different extra and intracellular K concentrations. Am J Physiol 208:770 36. Weihe E, McNight AT, Corbett AD, Kosterlitz HV (1985) Proenkephalin- and prodynorphinderived opioid peptides in guinea pig heart. Neuropeptides 5:453-456 37. Xie CW, Tang J, Han JS (1986) Clonidine stimulated the release of dynorphin in the spinal cord of the rat: A possible mechanism for its depressor effects. Neurosci 65:224-228 Received June 25, 1991 accepted March 24, 1992 Author's address: Dr. Simon W. Rabkin, Division of Cardiology, University of British Columbia, 4500 Oak Street, Vancouver, B.C., V6H 3N1, Canada