Effects of Magnesium on the Isolated, Blood-Perfused Atrial and Ventricular Preparations of the Dog Heart

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1 Effects of Magnesium on the Isolated, Blood-Perfused Atrial and Ventricular Preparations of the Dog Heart S Yasuyuki FURUKAWA, M.D. and Shigetoshi CHIBA, M.D. SUMMARY The effects of magnesium chloride were investigated on pacemaker activity and atrial contractility, using isolated, blood-perfused canine atrial and ventricular preparations with heparinized arterial blood led from the support dogs. Magnesium chloride injected directly into the sinus node artery produced dose-related negative chronotropic and inotropic effects in isolated right atria. In small doses (0.1-1mg), magnesium chloride caused only a negative chronotropic effect without significant negative inotropic changes. The threshold dose for inducing the negative chronotropic response to magnesium chloride was approximately 0.3-1mg, but that for the negative inotropic response 1-3mg. The duration of the negative inotropic response was usually shorter than that of the negative chronotropic response. These negative effects were not inhibited by atropine which completely blocked the acetylcholineinduced effects. Magnesium chloride also produced a dose-dependent negative inotropic effect in the isolated, blood-perfused left ventricular preparation in relatively high dose ranges. Moreover, magnesium chloride produced an uniform depression of contraction amplitude at all frequencies (2-3.5Hz) examined on the frequency-force relationship. Additional Indexing Words: Isolated blood-perfused atrium Isolated blood-perfused left ventricle Chronotropism Inotropism Magnesium chloride INCE Miller and Van Dellen (1938)1) observed an early acceleration in heart rate followed by a slowing in the anesthetized dog with increasing concentration of magnesium, it has been reported that an administration of magnesium caused directly to stabilize and slow SA nodal tissue.2)-5) On the other hand, the effects of magnesium on cardiac contractility were not great provided in a normal calcium concentration.6) Recently, Shine in 19797) described that the species variation in response to magnesium was particularly important for interpretation of experiments in myocardial preparations. In the present study, the effects of magnesium chloride on SA nodal From the Department of Pharmacology, Faculty of Medicine, Shinshu University, Matsumoto. Address for reprint: Prof. Dr. Shigetoshi Chiba, Department of Pharmacology, Faculty of Medicine, Shinshu University, Matsumoto 390, Japan. Received for publication May 29,

2 240 FURUKAWA, AND CHIBA Jpn. Heart J. M arch 1981 pacemaker activity and atrial contractility were directly examined, using the isolated and blood-perfused canine atrial preparation which developed by Chiba et al.8),9) The effects of magnesium chloride on ventricular contractility were also studied by use of the isolated left ventricular preparation.10),11) Moreover, we examined for observing whether the frequencyforce relationship was modified or not by the infusion of magnesium chloride. MATERIALS AND METHODS Twelve adult mongrel dogs, weighing 9 to 20Kg, were anesthetized with sodium pentobarbital, 30mg/Kg i.v. After treatment with 200 units/kg i.v. of sodium heparin, the right atrium was excised and plunged into Tyrode solution at about 4 to 10 Ž. The isolated right atrium was perfused with heparinized blood led from the carotid artery of support dog by the aid of a pump (Harvard Apparatus 1210). A pneumatic resistance was placed in parallel with the perfusion system so that a constant perfusion pressure of 100mmHg was obtained. The atrium was suspended in a bath filled with blood at a constant temperature of 37 Ž. Atrial rate was measured with a tachometer (Nihon-Kohden RT-5) which was triggered by the potential wave of atrial electrograms, and isometric tension development and the maximum rate of tension development were measured with a force displacement transducer (Grass FTO3B). The frequency-force relationships were investigated by the use of electrical pacing through bipolar electrodes sewn to the free atrial wall. Frequency of stimulation was changed from 2 to 3.5Hz at a suprathreshold voltage (2v) and I msec duration. The atrial muscle was subjected to a resting tension of 2Gm. The wet weight of the isolated right atrial muscle was 7 to 15Gm. The details of this preparation were described in previous papers.8)'9) In the 4 experiments, a part of the left ventricular muscle, weighing about 16 to 25Gm, was excised and perfused with heparinized blood through the anterior descending branch of the left coronary artery, using the same perfusion system as for the isolated, blood-perfused atrium. Each ventricular muscle was electrically paced at 1.5 to 3.5Hz and the isometric tension development and the maximum rate of tension development were recorded on an ink-writing rectigraph. The method of isolated, blood-perfused left ventricular muscle preparation was previously described by Chiba in ) and ) Sixteen support dogs, weighing 10 to 18Kg, were anesthetized with 30mg/Kg i.v. of sodium pentobarbital. Sodium heparin, 500units/Kg i.v., was administered at the beginning of the perfusion and 200units/Kg was added at 1-hour intervals. Drugs used in these experiments were MgCl2.6H2O and CaCl2.2H2O (Wako), acetylcholine chloride (Daiichi), and atropine sulfate (Tanabe). The drugs were dissolved in 0.9% saline before use. The amount of drug solution injected was 0.01 to 0.03ml in a period of 4sec or magnesium chloride was continuously administered into the cannulated sinus node artery or the anterior descending branch at a rate of 0.35 to 7mg/min with an infusion-withdrawal pump (Harvard Apparatus 901).

3 Vol.22 No.2 MAGNESIUM CHLORIDE ON DOG HEART 241 RESULTS Effects of magnesium chloride and calcium chloride on chronotropism and inotropism in isolated atria: When magnesium chloride was injected into the cannulated sinus node artery, negative chronotropic and inotropic effects were dose-relatedly induced as shown in Fig. 1. The threshold dose was approximately 0.3-1mg of MgCl2 E6H2O for inducing the negative inotropic effects. In every case, the duration of the negative chronotropic response to magnesium chloride Fig. 1. Effects of increasing doses of MgCl2 E6H2O and CaCl2 E2H2O on an isolated, blood-perfused canine atrial preparation. Fig. 2. Chronotropic and inotropic effects induced by magnesium chloride (lower) and calcium chloride (upper) in spontaneously beating dog atria. The control sinus rate was 99 }3.5beats/min (mean }SEM) in 10 atrial preparations. Vertical bars represent SEM. CF=contractile force; HR=heart rate.

4 242 FURUKAWA, AND CHIBA Jpn. Heart J March 1981 was longer lasting than that of the negative inotropic one. The duration of negative inotropic effects, which was calculated from one-half its peak at reducing to one-half its peak at returning, was 0.6 }0.15min at a dose of 10mg of MgCl6 E6H2O in 10 experiments. The duration of negative chronotropic effects was 2.4 }0.45min. When calcium chloride was administered into the sinus node artery, positive inotropic effect was markedly produced from a dose level of 0.1mg. A larger dose of calcium chloride (3mg) caused a slight positive chronotropic effect with a much greater positive inotropic effect. Effects of calcium and magnesium chloride on atrial myocardium are summarized in Fig. 2. Since Critelli et al (1977)12) reported the difference in the effect on myocardial contractility between a single injection and continuous infusion of magnesium solution, we also examined a continuous infusion of magnesium chloride at 5 different rates in 5 preparations. Summarized data are shown in Fig. 3. These results indicate no difference between a single injection and a continuous infusion of magnesium. Effects of atropine on MgCl2- and ACh-induced negative chronotropic and inotropic responses: When ACh was injected into the sinus node artery, negative chronotropic and inotropic responses were induced at a dose of 1ƒÊg. The treatment with 3ƒÊg of atropine significantly suppressed ACh-induced responses but did not Fig. 3. Effects of an infusion of magnesium chloride on the 5 isolated dog atria. The control sinus rate was 102 }5.3beats/min in 5 isolated atria. Vertical lines represent SEM. CF=contractile force; HR=heart rate.

5 Vol.22 No.2 MAGNESIUM CHLORIDE ON DOG HEART 243 Table I. Effect of Atropine on Responses to ACh and MgCl2ý6H2O in 5 Isolated, Blood-Perfused Canine Atria Control sinus rate was 100 }5.2beats/min (mean }SEM) in 5 atrial preparations. Values represent mean }SEM. Comparisons with control values (paired t-test). *p<0.05, **p< influence negative responses to 10mg of MgCl2-6H2O in 5 atria. Summarized data are shown in Table I. Effects of MgCl2 E6H2O on the isolated left ventricle: When magnesium chloride was injected into the anterior descending branch of the left coronary artery of the isolated left ventricle paced electrically, a negative inotropic response was dose-relatedly induced at doses of 1-30mg. Effects of MgCl2 E6H2O on the frequency force relationship of the isolated canine atrium and ventricle: Effects of magnesium chloride on the frequency-force relationship were examined in atrial and ventricular preparations. Magnesium chloride suppressed the tension development to almost the same degree at each frequency examined (2-3.5Hz) in the atrial muscle as shown in Fig. 4. Summarized data are shown in Fig. 5. At a frequency of 3.5Hz, pulsus alternans frequently appeared after the treatment with magnesium chloride. Fig. 4. Effects of an infusion of 7mg/min of MgCl2 E6H2O on the frequency-force response pattern of an isometrically contracting dog atrial muscle, stimulated at 2 to 3.5Hz, 2v, and 1msec duration.

6 244 FURUKAWA, AND CHIBA Jpn. Heart J. March 1981 Fig. 5. Effects of the infusion of MgCl2 E6H2O (3.5-7mg/min) on the frequency-force relationship in 6 isolated canine atria, stimulated at 2 to 3.5Hz. Open circles show the control developed tension before magnesium infusion and closed circles show the developed tension after magnesium infusion. +=2 of 6 experiments show the pulsus alternans. DISCUSSION Concerning the chronotropism, negative chronotropic responses due to the injection of magnesium were observed by a number of investigators.2),13), Hashimoto et al (1974)5) also demonstrated that magnesium have a direct effect to stabilize and slow SA nodal tissue on the blood-perfused dog SA node preparation in situ. As reviewed by Mordes and Wacker (1977),6) it was well known that excess magnesium caused direct sinus deceleration with indirect sinus acceleration reflexed by hypotension. In the present study, magnesium chloride caused dose-dependent negative chronotropic effects in the isolated and blood-perfused atrial preparation. In the electrophysiological studies, Toda and West (1967)4) observed that magnesium increased spontaneous cycle duration in the rabbit atrium, i.e., the decrease in threshold potential was followed by a decrease in the rate of slow diastolic depolarization. More recently, Woods et al (1979)15) reported that doubling (Mg++) consistently slowed the sinus rate without changes in sinus node action potential in the dog atrium. On inotropism, it was indicated that mechanical function was well preserved after pacemaker cells had been markedly depressed by excess magnesium as reviewed by Engbaek.16) In general, the effects of magnesium on contractility were not great in the isolated heart when calcium concentration was normal.17)-20) Shine and Douglas (1974)21) reported that increasing concentration of magnesium depressed tension development in isolated and blood-perfused rat interventricular septa. Although it was already reported that magnesium chloride depressed the tension development in the dog ventricle,3)'22) in the dog atrial muscle there was no available information for any inotropic effects of magnesium. In the present experiments, we demonstrated

7 Vol.22 No.2 MAGNESIUM CHLORIDE ON DOG HEART 245 that excess magnesium produced dose-related negative inotropic response to relatively higher dose levels which induced profound sinus depression in either atrial or ventricular muscle. Critelli et al12) reported the difference in effect on myocardial contractility between a single injection and a continuous infusion of magnesium chloride in patients, i.e., the former produced negative inotropic effects but the latter did not. When magnesium was administered in the whole animal, a single injection might cause sudden hemodynamic changes which in turn might cause excitation of reflex mechanisms. In the present study, since extracardiac factors were completely excluded, direct cardiac depressive effects could be observed in both a single and continuous administration of magnesium chloride. Thus, it is concluded that magnesium chloride has direct cardiac depressant properties, i.e., clearly negative chronotropic and slightly negative inotropic properties, because these negative effects were not influenced by treatment with adequate doses of atropine. In 1975, Bayer et al23) reported that in cat papillary muscles verapamil depressed the contraction amplitudes at a higher pacing rate, while leaving them nearly unchanged at low frequencies. It was also reported that verapamil and nifedipine suppressed tension development at higher frequencies than at low frequencies, whereas manganese, pentobarbital and ethanol produced a rather uniform depression of contraction at all tested frequencies in isolated dog atrial and ventricular preparations.10),11),24),25) In this study, we demonstrated that the effect of magnesium chloride on the frequency-force relationship, which produced an uniform depression of contraction at all examined frequencies, was similar to effects of manganese, pentobarbital and ethanol. Differences in the patterns among these calcium antagonistic substances may be due to different sites of actions on the movement of cardiac intracellular calcium. REFERENCES 1. Miller JR, Van Dellen TR: Electrocardiographic changes following the intravenous administration of magnesium sulfate. J Lab Clin Med 23: 914, Stanbury JB: The blocking action of magnesium on sympathetic ganglia. J Pharmacol Exp Ther 93: 52, Stanbury JB, Farah A: Effects of the magnesium ion on the heart and on its response to digoxin. J Pharmacol Exp Ther 100: 445, Toda N, West TC: Interaction between Na, Ca, Mg, and vagal stimulation in the S-A node of the rabbit. Am J Physiol 212: 424, Hashimoto K, Suzuki Y, Chiba S: Influence of calcium and magnesium ions on the sinoatrial node pacemaker activity of the canine heart. Tohoku J Exp Med 113: 187, 1974

8 246 FURUKAWA, AND CHIBA Jpn. Heart J. M arch Mordes JP, Wacker WC: Excess magnesium. Pharmacol Rev 29: 273, Shine KI: Myocardial effects of magnesium. Am J Physiol 237: 413, Chiba S, Kimura T, Hashimoto K: Muscarinic suppression of the nicotinic action of acetylcholine on the isolated, blood-perfused atrium of the dog. Naunyn-Schmiedeberg's Arch Pharmacol 289: 315, 1975a 9. Chiba S, Yabuuchi Y, Hashimoto K: Comparison of the effects of norepinephrine and acetylcholine between intraarterial and extravascular administration to the isolated, bloodperfused canine atrium. Jap J Pharmacol 25: 433, 1975b 10. Chiba S: Effect of pentobarbital, verapamil and manganese on the frequency-force relationship of the isolated atrium and ventricle of the dog heart. Fur J Pharmacol 40: 225, Chiba S: Effect of verapamil on frequency-force relationship in isolated dog left ventricular muscle. Jap J Pharmacol 27: 175, Critelli G, Ferro G, Peschle C, Perticone Fr, Rengo Fr, Condorelli M: Myocardial contractility after injection or prolonged infusion of magnesium sulphate. Acta Cardiol 32: 65, Grantham JJ, Tu WH, Schloerb PR: Acute magnesium depletion and excess induced by hemodialysis. Am J Physiol 198: 1211, Specter MJ, Schweizer E, Goldman RH: Studies on magnesium's mechanism of action in digitalis-induced arrhythmias. Circulation 52: 1001, Woods WT, Katholi RE, Urthaler F, James TN: Electrophysiological effects of magnesium on cell in the canine sinus node and false tendon. Circulat Res 44: 182, Engbaek U: The pharmacological actions of magnesium ions with particular reference to the neuromuscular and the cardiovascular system. Pharmacol Rev 4: 396, Garb S: The effects of potassium, ammonium, calcium, strontium and magnesium on the electrogram and myogram of mammalian heart muscle. J Pharmacol Exp Ther 101: 317, Schmidt HD, Schmier J, Schmitz S: Chronotrope Wirkung von Calcium and Magnesium am isolierten Hundeherzen. Pfluegers Arch 284: 316, Kovacs T, O'Donnell JM: An analysis of calcium-magnesium antagonism in contractility and ionic balance in isolated trabecular muscle of rat ventricle. Pfluegers Arch 360: 267, Levin RM, Haugaard N, Hess ME: Opposing actions of calcium and magnesium ions on the metabolic effects of epinephrine in rat heart. Biochem Pharmacol 25: 1963, Shine KI, Douglas AM: Magnesium effects on ionic exchange and mechanical function in rat ventricle. Am J Physiol 227: 317, Bristow MR, Daniels JR, Kernoff RS, Harrison D: Effect of D600, practolol, and alterations in magnesium on ionized calcium concentration-response relationships in the intact dog heart. Circulat Res 41: 574, Bayer R, Hennekes R, Kaufmann R, Mannhold R: Inotropic and electrophysiological actions of verapamil and D600 in mammalian myocardium. Naunyn-Schmiedeberg's Arch Pharmacol 290: 49, Chiba S, Furukawa Y, Kobayashi M: Effect of nifedipine on frequency-force relationship in isolated dog left ventricular muscle. Jap J Pharmacol 28: 783, Kobayashi M, Furukawa Y, Chiba S: Effect of ethanol on frequency-force relationship in isolated right atrial muscle of the dog. J Stud Alcohol 40: 892, 1979