Effects of buffer magnesium on positive inotropic agents in guinea pig cardiac muscle

European Journal of Pharmacology, 165 (1989) 181-189

181

Elsevier EJP 50829

Effects of buffer magnesium on positive inotropic agents in guinea pig cardiac muscle R o n n y K a f i l u d d i , R i c h a r d H . K e n n e d y * a n d E r n s t Seifen Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, U.S.A.

Received 14 March 1989, accepted 28 March 1989

Experiments examined effects of extracellular Mg 2÷ concentration (Mgo2+) on dose-dependent actions of strophanthidin, norepinephrine, Bay K-8644 and extracellular Ca 2+ (Ca 2+) in electrically stimulated atrial and ventricular muscle isolated from guinea pig heart. Mgo2+ itself elicited a concentration-dependent negative inotropic effect. Elevation of Mg 2+ between 0.6 and 12 mM increased the concentration of strophanthidin necessary to produce its toxic effects without affecting the maximum developed tension prior to toxicity. Similarly, Mg 2+ did not alter the maximum contractile force elicited by cumulative addition of norepinephrine, Bay K-8644 or Ca 2o+ , but increased their EDs0 values. These data suggest that interactions between Mg 2+ and the four positive inotropic agents were not mediated by effects on receptor binding or Na+,K+-ATPase, but rather by alterations at one or more steps involved in excitation-contraction coupling. Cardiac glycosides; Norepinephrine; BAY k 8644; Ca 2+ (extracellular); Mg 2+ (extraceUular); Cardiac muscle; (Guinea-pig)

1. Introduction

Magnesium status has a significant effect on the disturbances in cardiac rhythm that are elicited by digitalis glycosides. Hypomagnesemic patients have a higher incidence of digitalis-induced ventricular and supraventricular tachycardia (Iseri et al., 1975; Whang et al., 1985; K i m et al., 1961), and the sensitivity to cardiotonic steroid-induced arrhythmias increases in animals maintained on a magnesium-deficient diet (Vitale et al., 1963) as well as in those made acutely hypomagnesemic by hemodialysis (Seller et al., 1970; Seller, 1971; Neff et al., 1972). On the other hand, administration of

* To whom all correspondence should be addressed: Department of Pharmacology, Slot 611, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205, U.S.A.

magnesium (Mg 2+) is known to suppress these arrhythmias in both man (Szekely and Wynne, 1951) and animals (Stanbury and Farah, 1950; Seller et al., 1970; Seller, 1971; Specter et al., 1975). Mechanisms for the interaction between Mg 2÷ and cardiotonic steroids are currently unclear. Digitalis-induced arrhythmias result from both indirect effects involving the autonomic nervous system (Gillis and Quest, 1979) and direct myocardial actions which are mediated by inhibition of sarcolemmal N a + , K + - A T P a s e , the m e m b r a n e N a + - p u m p (Schwartz et al., 1975; Akera and Brody, 1977). This reduction in Na+,K+-ATPase activity causes intracellular N a ÷ accumulation, alterations in N a + / C a 2+ exchange, Ca 2+ overload, and transient depolarizations (Ferrier et al., 1973; Kass et al., 1978a; Colquhoun et al., 1981; Brown et al., 1986). A study by Tackett (1986) suggests that the .autonomic nervous system is involved in

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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the interaction between Mg 2+ and digitalis glycosides; hypomagnesemic dogs exhibit an enhanced sensitivity to glycoside toxicity that is associated with abrupt sympathetic discharge. In contrast, elevations in extracellular Mg 2÷ (Mg 2÷) terminate ouabain-induced arrhythmias in isolated, guinea pig ventricular muscle due to suppression of transient depolarizations and an increase in threshold potential, thus suggesting an interaction at the level of the myocardium (Saikawa et al., 1987). In spite of this knowledge regarding effects of Mgo2+ on digitalis-induced arrhythmogenesis, the influence of Mgo2+ on the inotropic efficacy of cardiotonic steroids has not been well established. Furthermore, the interaction between Mgo2+ and other positive inotropic agents has not been examined. Therefore, the current project was designed to monitor effects of Mg 2÷ on the responsiveness of isolated cardiac muscle to (1) dose-dependent inotropic and arrhythmogenic actions of cardiotonic steroids, and (2) direct cardiac effects of norepinephrine, extracellular Ca 2+ (CaZo+) and Bay K-8644, agents that enhance contractility by mechanisms different than that of the cardiotonic steroids. 2. Materials and methods

2.1. Isolated left atrial and right ventricular muscle preparations Hartley guinea pigs (male, 250-300 g) were killed by decapitation. After thoracotomy, hearts were immediately removed and perfused through the aorta with Krebs-Henseleit solution of the following composition (in mM): NaC1 119; NaHCO 3 25; KC1 4.7; MgC1z 1.2; glucose 11.1; and CaC12 1.8 (0.4 mM CaC12 for preparations used in Ca2o+ dose-response curves). The solution was buffered to pH 7.4 by saturation with 95% 02-5% CO2 gas, and temperature was maintained at 37 o C. Left atrial muscle and right ventricular strips (thickness less than 0.8 mm) were dissected and bathed in an oxygenated Krebs-Henseleit solution (37 o C) similar to the one described above but containing various concentrations (0.3-12 mM) of Mg o2+ . In order to prevent effects of possible

catecholamine release from the tissue (Seifen, 1974), nadolol (10 -7 M), a /3-adrenoceptor antagonist, was added to the solution in all experiments except those involving norepinephrine. An a-adrenoceptor antagonist was not included in the buffer because of the relatively minor positive inotropic action of a-adrenoceptor agonists in guinea pig cardiac muscle (Wagner and Reinhardt, 1974; Kennedy and Seifen, 1987). Each preparation was paced via platinum contact electrodes. A contraction frequency of 3.3 Hz was maintained by 0.2 ms square wave pulses set at 50% above threshold voltage. Force of isometric contraction was measured by force-displacement transducers and recorded continuously on a polygraph. After the tissue was beating for several min, a lengthtension curve was determined, and muscle length was maintained at that which elicited 90% of maximum contractile force observed at optimal length (approximately 1.0 g for atrial muscle and 1.5 g for ventricular strips). A stabilization period of 90 min was allowed before preparations were challenged by various agents. During this equilibration period, the bathing solution was changed every 15 min. Atrial and ventricular muscle were used to examine effects of Mg 2÷ on (1) developed tension and (2) dose-response curves for positive inotropic and toxic effects of strophanthidin (a relatively rapid-acting cardiotonic steroid), norepinephrine, Ca 2+ and Bay K-8644 (a dihydropyridine Ca 2+ agonist). Dose-response curves were obtained by cumulative addition to the organ bath. Consecutive addition of an agent was made only after preparations reached a steady state response to the preceding concentration. 2.2. Statistical evaluation Data were analyzed by one-way analysis of variance with Duncan's multiple range test or by Chi-square analysis. Criterion for significance was a P value less than 0.05. All data are represented as means ± S.E. 2.3. Materials Bay K-8644 was a generous gift from Miles Laboratories, Inc., New Haven, CT. Nadolol was

183 a generous gift from E.R. Squibb and Sons, Inc., Princeton, NJ. Strophanthidin, digoxin and ( - ) norepinephrine bitartrate were purchased from Sigma Chemical Co., St. Louis, MO. Other chemicals were of reagent grade. Stock solutions of Bay K-8644 and strophanthidin were prepared daily in dimethylsulfoxide (DMSO) and ethanol, respectively. Final concentrations of D M S O and ethanol in the bathing solution never exceeded 0.15%, and these concentrations had no effect on resting or developed tension. Stock solutions of norepinephrine were prepared daily in 0.01 N HC1. Resting tension, contractile force and p H were not affected by the amount of HC1 added to the bathing solution.

3. Results

3.1. Effects of Mgeo + on developed tension Initial experiments examined effects of Mgo2+ on contractile force in guinea pig atrial muscle. Following stabilization in 0.3 m M Mg o2+ , increasing concentrations of MgCI 2 were added to the bathing solution. Mg 2+ elicited a concentrationdependent negative inotropic effect (fig. 1) with an EDs0 of 6.4 + 0.7 mM. EDs0 values were determined graphically using the assumption that 20 m M Mgoz+ produced a maximal effect. The 6.0 m M Mg2o+-induced reduction in developed tension did not vary significantly whether obtained by single or cumulative addition (fig. 1), thus suggesting that cumulative exposure did not alter the sensitivity to negative inotropic effects of Mg 2+.

3.2. Effects of Mg2o + on dose-dependent actions of strophanthidin Effects of Mg 2+ on inotropic and toxic actions of cardiotonic steroids were examined in guinea pig atrial and ventricular muscle by comparing strophanthidin dose-response curves in Mg 2+ concentrations above and below that of normal (1.2 m M Mg 2÷) Krebs-Henseleit solution (fig. 2). The steroid elicited a positive inotropic effect followed by toxicity in both atrial and ventricular muscle.

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Increasing Mg 2+ appeared to shift the dose-response curve to the right. Toxic effects - including contracture, extrasystolic arrhythmias and a decline in developed tension - were first observed at 0.6/~M strophanthidin in all atrial and ventricular preparations bathed in 0.6 m M MgZo+ (as depicted by the curve ending at 0.4/zM in fig. 2), while 1.0 /~M strophanthidin was required to elicit these toxic events in tissues bathed in 1.2 or 6.4 m M Mg2o÷ . Toxic effects were first observed at 1.6/~M strophanthidin in preparations bathed in 12 m M Mg 2+, and this toxicity was expressed only by contracture and a decline in contractile force, not by extrasystoles. Since the onset of toxic events often made it impossible to obtain complete dose-response curves, it was not feasible to determine EDs0 values and thus accurately quantitare the Mg2+-induced shift. Mg 2+ had no effect on the m a x i m u m contractile force observed in the presence of strophanthidin.

3.3. Effects of Mg2o + on actions of norepinephrine The protocol for these experiments was similar to that used to monitor dose-dependent effects of strophanthidin except that the bathing solution

184 did not c o n t a i n the /3-adrenoceptor a n t a g o n i s t nadolol. C o n t r o l values for developed t e n s i o n i n these p r e p a r a t i o n s (fig. 3) w e r e n o t statistically different from those o b t a i n e d i n the presence of n a d o l o l (fig. 2), thus suggesting that Mgo2÷ h a d n o appreciable effect o n catecholamine stores i n the tissue. A d d i t i o n of n o r e p i n e p h r i n e p r o d u c e d a d o s e - d e p e n d e n t positive inotropic effect in all preparations. Mg2o+ did n o t affect m a x i m a l developed tension in the presence of n o r e p i n e p h r i n e ; however, dose-response curves were shifted to the fight. According to analysis of variance, EDs0 values for n o r e p i n e p h r i n e increased significantly i n b o t h atrial a n d ventricular muscle as Mgo2+ was raised between 0.6 a n d 12 m M . D u n c a n ' s m u l t i p l e

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range test i n d i c a t e d that EDs0 values at 12 m M Mgo2+ represented the o n l y significant difference w h e n c o m p a r e d to those o b t a i n e d i n 0.6 m M 2+, M g o , however, values at 12 m M were also sign i f i c a n t l y greater t h a n those at 1.2 a n d 6.4 m M M g 2+"

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Fig. 2. Effects of Mgo2+ on dose-dependent actions of strophanthidin in left atrial (bottom panel) and right ventricular (top panel) muscle isolated from guinea pig heart. Preparations were equilibrated for 90 re.in in Krebs-Henseleit buffer (37 o C) containing either 0.6 (triangles, n = 6), 1.2 (squares, n = 6), 6.4 (inverted triangles, n = 6) or 12 mM (diamonds, n = 6 ) Mgoz+ plus 10 -7 M nadolol before addition of strophanthidin. Dose-response curves for strophanthidin were obtained by cumulative addition. Vertical bars indicate S.E. The last point of each curve represents the highest concentration which did not elicit toxic effects.

3.4. Effects of Mgeo ÷ on actions of increasing Ca2o+ The positive i n o t r o p i c effect of CaZo+ was also e x a m i n e d i n the presence of various concentrations of M g o2+ . U n l i k e previous experiments, these p r e p a r a t i o n s were stabilized i n K r e b s - H e n s e l e i t buffer c o n t a i n i n g 0.4 i n s t e a d of 1.8 m M CaC12. C u m u l a t i v e a d d i t i o n of CaC12 elicited a positive i n o t r o p i c effect at each Mgo2+ (fig. 4). Dose-re-

185

sponse curves for Ca2o+ were shifted to the right by elevations in Mgo2+ in both atrial and ventricular preparations; analysis of variance indicated that EDs0 values (fig. 4) for Ca2o÷ increased with elevations in Mg 2+ o . EDs0s at 12 mM Mgo2+ were significantly greater than those at 0.6 mM (Duncan's multiple range test). Mgo2÷ had no significant effect on maximum developed tension observed in the presence of increasing Ca2o÷ .

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elevations in Mg2o+ shifted the Bay K-8644 dose-response curve to the right, while having no significant effect on maximum contractile force. EDs0 values (fig. 5) in atrial muscle increased with Mgo2÷ according to analysis of variance, and Duncan's multiple range test indicated that the value at 12 mM Mgo2+ was significantly greater than that at 0.6 mM. There was a similar trend in ventricular muscle; however, these changes were not statistically significant.

4. Discussion

Studies in man and animals have shown that Mg 2+ deficiency predisposes to cardiac glycoside-

186

induced arrhythmias (Iseri et al., 1975; Whang et al., 1985; Kim et al., 1961; Vitale et al., 1963; Seller et al., 1970; Seller, 1971; Neff et al., 1972) and that administration of Mg 2+ reverses these toxic dysrhythmic events (Szekely and Wynne, 1951; Stanbury and Farah, 1950; Seller et al., 1970; Seller, 1971; Neff et al., 1972; Specter et al., 1975). Results of current experiments support the hypothesis that this interaction involves direct effects on the myocardium. It has been proposed that both therapeutic and arrhythmogenic actions of cardiotonic steroids result from inhibition of Na+,K+-ATPase, the membrane Na + pump. Moderate inhibition of the Na + pump is believed to increase the depolarization-induced Na + transient (Lee and Dagostino, 1982), thus producing an increase in intracellular Ca 2+ via sarcolemmal Na+/Ca 2+ exchange (BarcenasRuiz et al., 1987) and positive inotropic actions (Akera and Brody, 1977). More extensive inhibition of the Na + pump causes intracellular Na + accumulation, intracellular Ca 2+ overload, oscillatory release of Ca 2+ from the sarcoplasmic reticulum (Fabiato and Fabiato, 1975a,b; Kass et al., 1978a) and transient inward currents through either nonspecific cation channels (Colquhoun et al., 1981) or electrogenic Na+/Ca 2+ exchange (Brown et al., 1986). These transient inward currents give rise to oscillatory afterpotentials and cause extrasystolic contractions if threshold is attained (Ferrier et al., 1973; Kass et al., 1978b). The termination of ouabain-induced arrhythmias by Mgo2+ in guinea pig ventricular muscle has been ascribed to an increase in threshold potential (Saikawa et al., 1987) as well as to suppression of transient depolarizations (Kass et al., 1978b). Mg 2+ could act at one or more of a number of sites to depress these oscillatory afterpotentials. Seller (1971) proposed that magnesium's antiarrhythmic property is mediated by reactivation of digitalis-inhibited Na+,K+-ATPase, whereas Shine and Douglas (1975) suggested that Mgo2+ suppresses acetylstrophanthidin-induced arrhythmias in rabbit interventricular septa by direct effects on K + permeability. Current results, although not definitive, suggested that the interaction between Mgo2÷ and cardiotonic steroids may also involve Mg2+-induced alterations in excitation-contrac-

tion (EC) coupling. Mgo2+ elicited a concentration-dependent negative inotropic effect similar to that demonstrated previously in tissue isolated from several species (Shine and Douglas, 1974; Vierling and Reiter, 1975; Vierling et al., 1978; Bristow et al., 1977; Stanbury and Farah, 1950). In addition, Mgoz+ antagonized the positive inotropic actions of norepinephrine, Ca2o+, Bay K8644 and strophanthidin in a similar fashion causing a rightward shift in the dose-response curves without affecting maximum contractile force. It is possible that Mgo2+ elicited these antagonistic actions by affecting receptor binding of strophanthidin, norepinephrine and Bay K-8644; however, the similarity between interactions with these three agents and that with Ca2o+ suggests a more common mechanism. In contrast to strophanthidin, Ca 2+ o , norepinephrine and Bay K-8644 enhance Ca z+ influx, at least in part, through voltage-dependent channels (Tsien, 1977). The positive inotropic action of Ca 2+ probably results from effects of an increase in the extracellular to intracellular Ca 2+ gradient (Reuter, 1979), whereas the C.a2+ agonist Bay K8644 increases the probability of Ca 2+ channel opening during membrane depolarization (Hess et al., 1984; Brown et al., 1984). Norepinephrine stimulates fl-adrenoceptors and enhances cyclic AMP-dependent phosphorylation of sarcolemmal and sarcoplasmic reticular proteins (Tada and Inui, 1983). Phosphorylation of the sarcolemmal protein increases both the opening probability of Ca 2+ channels during depolarization (Tsien et al., 1986; Trautwein and Cavalie, 1985) and slow inward Ca 2+ current (Tsien, 1973; Trautwein et al., 1982). Cyclic AMP-dependent phosphorylation of sarcoplasmic reticular phospholamban stimulates Ca 2+ uptake into the sarcoplasmic reticulum (Tada et al., 1974; Fabiato, 1981), thereby increasing relaxation rate and enhancing the amount of Ca 2+ available for release during subsequent depolarization. There is evidence to support the interaction of Mg 2+ with many steps involved in EC coupling including (1) Ca 2+ influx through voltage-dependent Ca 2+ channels, (2) sarcoplasmic reticular Ca 2+ release, (3) sarcolemmal N a + / C a 2+ exchange, (4) binding of Ca 2+ to rapidly exchangea-

187 ble stores, and (5) sarcolemmal or sarcoplasmic reticular Mg2+-dependent Ca2+-ATPase. Elevated levels of Mgo2+ decrease 45Ca 2+ uptake rate in isolated, blood perfused rat interventricular septa (Shine and Douglas, 1974). This reduced influx may be mediated by magnesium's effect on rapidly exchangeable, superficially located sarcolemmal Ca 2÷ binding sites (Langer et al., 1974; Turlapaty and Altura, 1978) or by inhibition of Ca 2÷ influx through voltage-dependent channels (Edwards, 1982; Hess et al., 1986; Lansman et al., 1986; Muller and Finkelstein, 1974). In addition, elevations in Mg 2+ increase intracellular Mg 2÷ (Mg 2+) activity within 30 min (Hess et al., 1982; Lopez et al., 1984), and experiments by White and Hartzell (1988) indicate that Mg 2+ has an inhibitory effect on voltage-gated Ca 2÷ current which is more pronounced after phosphorylation of the channel. Increased levels of Mg 2+ could also elicit effects observed in the current study by blocking Ca 2÷ release channels in the sarcoplasmic reticulum (Smith et al., 1986; Meissner et al., 1986) or by affecting sarcolemmal a n d / o r sarcoplasmic reticular Ca 2÷ pumps (Fabiato and Fabiato, 1975b; Roufogalis et al., 1982; Shigekawa et al., 1978). Mg 2÷ has been reported to inhibit N a + / C a 2+ exchange in sarcolemmal vesicles (Trosper and Phillipson, 1983; Ledvora and Hegyvary, 1983; Wakabashi and Goshima, 1981). This exchange is believed to carry a net Ca 2+ efflux under control conditions (Mullins, 1979; Barcenas-Ruiz et al., 1987). Thus, its inhibition is probably not involved in the Mg2+-induced negative inotropic effect or in the antagonistic interactions of Mg 2+ with Bay K-8644, Ca 2+ and norepinephrine. However, inhibition of N a + / C a 2÷ exchange would be expected to antagonize cardiotonic steroid-induced elevations in intracellular Ca 2÷ and the resulting increase in contractile force and arrhythmias. It is not possible in light of current data to determine if a Mg2+-induced inhibition of N a + / Ca 2÷ exchange is involved in the altered responsiveness to strophanthidin. In summary, results of this study indicate that increasing levels of Mg 2+ (1) decrease the sensitivity of isolated cardiac muscle to positive inotropic effects of strophanthidin, Ca 2+ o , norepinephrine and Bay K-8644 without affecting the

maximal developed tension elicited by each agent and (2) antagonize the direct arrhythmogenic actions of strophanthidin. Possible mechanisms for observed interactions between Mg 2+ and these positive inotropic agents include (1) blockade of voltage-dependent Ca 2÷ channels, (2) inhibition of N a + / C a 2+ exchange, (3) inhibition of sarcoplasmic reticular Ca 2÷ release channels, (4) displacement of Ca 2÷ from rapidly exchangeable, sarcolemmal binding sites and (5) actions on sarcolemmal a n d / o r sarcoplasmic reticular Ca 2+ pumps.

Acknowledgements The authors would like to thank Mr. William C. Hardwick for excellent assistance in preparation of the graphics. This work was supported by U.S. Public Health Service Grant AG05237 from the National Institute on Aging and by a Graduate Student Biomedical Research Support Grant from the University of Arkansas for Medical Sciences. R.H. Kennedy is a recipient of a Research Career Development Award from the National Institute on Aging.

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