Role of Mn++ in ouabain induced cardiac arrhythmias.
There is a considerable evidence that Ca++ stored in the sarcoplasmic reticulum is involved in the initiation of the contraction in the skeletal muscle fibre. In the frog ventricle, calcium moves across the membrane to initiate the contraction. The presence of a highly developed sarcotubular system in the mammalian cardiac muscle suggests that similar activating process may be occuring in the cardiac muscle. Studies conducted by Reuter,  show that Ca++ carries an appreciable current in the inward direction when Purkinje fibres are depolarised. He proposed that this inward current may be involved in the excitation-contraction coupling in the cardiac muscle.
Manganese ions have been employed to ascertain the contribution of inward calcium current to the generation of action potential in the cardiac fibres. Hagiwara and Nakajima noted that calcium spikes in the barnacle of muscle fibres is suppressed or abolished by Mn++. The same investigators also found that mane ganese had suppressive effects on the plateau of the action potential in the frog ventricle suggesting a possible competition of manganese with calcium ions. Mn++ has been shown to prevent the development of enhanced automaticity induced with digitalis.
It was therefore decided to determine the influence of manganese on calcium exchange at myocardial cell using ouabain induced cardiac arrhythmias as an experimental model.
Following experiments were conducted:
1. Induction of arrhythmias with ouabain.
2. Langendorff heart.
3. Frog heart perfusion.
Induction of arrhythmias
Since arrhythmia induced in dog is not spontaneously reversed, dog was used as an experimental model. Stray dogs of male sex weighing between 8-12 kg body weight were selected. Six dogs were used in each group.
A widely adopted method for assaying anti-arrhythmic drugs is the use of toxic doses of cardiac glycosides. Besides it being a valuable tool for screening methods, it has a great importance also from purely clinical toxicologic stand point, in view of the frequent occurrence of arrhythmias due to digitalis overdosage.
Ouabain in a dose of 110 mcg/kg has been known to produce ventricular tachycardia responding well to anti-arrhythmic agents. The early phase of this intoxication is suitable for estimating the lowest dose of anti-arrhythmic drugs still able to suspend ventricular tachycardia. Even a small dose of 7C mcg/kg of strophanthin-G is known to produce prolonged ventricular tachycardia which is suitable for measuring not only the suspension of arrhythmia by anti-arrhythmic drug but also the duration of anti-arrhythmic action.
Considering the above facts, ouabain was selected as an arrhythmogenic agent. Dogs were anaesthetised using pentobarbitone sodium in the dose of 40 mg/kg body weight administered intravenously. Electrocardiogram was recorded on Cardel's electrocardiograph. To induce arrhythmia, ouabain was administered intravenously in the dose of 70 mcg/kg body weight. If the arrhythmia did not occur within 40 minutes of administration of this dose, every further 10 minutes ouabain, 10 mcg/kg was repeated till the development of arrhythmia. Simultaneously, the carotid blood pressure was recorded on a kymograph.
Manganese was administered as manganese chloride intravenously. Two doses of manganese 0.25 mg, and 0.5 mg/ kg body weight were administered in different groups. To determine the reversal of arrhythmia after manganese administration, ECG was recorded after 1 minute, 5 minutes and 10 minutes. In case the arrhythmia was not reversed within 10 minutes of manganese administration, the dose of manganese was repeated till arrhythmia was reverted. ECG was recorded for the next two hours to see the sustained anti-arrhythmic effect of manganese.
The effect was compared with anti-arrhythmic agent Verapamil.
Isolated rabbit heart
A rabbit weighing 2 kg was killed by stunning and heart was dissected out. It was mounted on Langendorff apparatus perfused with Ringer-Locke solution aerated with oxygen. Contractions were recorded on a smoked drum by Starling's heart lever. Effect of varying concentrations of calcium were studied and its reversal with manganese was determined. The effect was compared with known calcium antagonist Verapamil.
Isolated frog heart perfusion
Carefully isolated frog's heart was perfused with frog Ringer solution by inserting a cannula in the sinus venosus. Contractions of the heart were recorded on a smoked drum. The effect of Ca ++ and its antagonism with Mn+ + was studied.
Ouabain, in a dose of 70-80 mcg/kg body weight, induced various kinds of cardiac tachyarrhythmias, like ventricular premature beats, ventricular bigemini and ventricular tachycardia.
Manganese, in the dose of 0.25 mg/kg body weight, could revert the ventricular premature beats within one minute which occurred with ouabain 80 mcg/kg [Fig. 1]. However, the Wenckebach's phenomenon persisted.
Manganese, 0.5 mg/kg body weight, could revert the ventricular bigemini which occurred with ouabain 80 mcg/kg [Fig. 2] and restore a sinus rhythm within one minute of its administration. The effect was sustained over a period of four hours. Similar effects of manganese were also confirmed in ventricular tachycardia with AN. dissociation induced with ouabain 70 mcg/kg [Fig. 3] and [Fig. 4].
Manganese produced a gradual fall in the blood pressure at a dose of 0.5 mg/kg body weight. This was returned to basal blood pressure immediately. This could be due to peripheral vascular smooth muscle relaxation occurring with Mn+ + as reported.
In Langendorff heart, it was found that manganese could decrease the force of contraction induced by calcium [Fig. 5]. This effect was also confirmed in isolated frog heart perfusion [Fig. 6]. This effect was comparable to the effect of Verapamil, a known calcium antagonist.
Thus, manganese in the dose used, could revert tachyarrhythmias induced with ouabain. Manganese also inhibited the ventricular automaticity induced with calcium in isolated rabbit and frog hearts suggesting its competition with calcium ions in cardiac fibres.
Most cells and biological membranes possess a process that actively maintains concentration gradients of sodium and potassium. Ouabain and other cardiac glycosides inhibit the membrane bound Na+ and K+ activated adenosine triphosphatase, the receptor for cardiac glycosides. Hydrolysis of adenosine triphosphate by this enzyme provides the energy for sodium pump-the system in the sarcolemma of cardiac fibres that actively extrudes sodium and transports potassium into the fibres. Ouabain binds specifically to the Na+ K+ ATPase, inhibits its enzymatic activity and impairs the active transport of sodium and potassium. As a result, there is a gradual increase in intracellular sodium and decrease in potassium. Cardiac fibres possess the mechanism for exchange of intracellular sodium for extra cellular calcium. When inhibition of the pump of ouabain causes sodium to increase, there is an augmented exchange of intracellular sodium for extra cellular calcium. This causes an increase in the net influx of calcium and presumably an increase in the concentration of calcium in the sarcoplasm. Calcium plays an important role in the genesis of oscillatory after-potentials in the His-Purkinje tissue. Ferrier has shown that oscillatory after-potentials in the His-Purkinje tissue are involved in the genesis of ouabain induced arrhythmias.
Manganese has been known to inhibit calcium permeability in the selected tissues. Manganese has been shown to inhibit influx of calcium in cardiac fibres. It is a competitor of calcium for vectorial transport. Manganese has a negative inotropic action on the isolated rabbit atrium. It blocks the uptake of radioactive calcium by the muscle cells. Manganese also acts competitively against ouabain which is known to exert some of its actions by promoting the uptake of calcium by the muscle cells. Mn++ is also known to block oscillatory after-potentials and thus inhibit transmembrane influx of calcium or release of Ca++ from internal storage sites This can be further confirmed from the fact that manganese inhibits smooth muscle contraction in response to angiotensin and noradrenaline, the effect which can be offset by increasing the concentration of calcium in the perfusion. AMP nucleosidase is activated by MnATP. Studies of the interaction of manganese and MhATP with allosteric sites of the enzyme indicate that both manganese ion and ATP interact with enzyme protein. Carvallo inferred that sarcoplasmic reticulum ATPase is a primary H+ ion pump. Calcium translocation at the myocardial fibre results secondary to H+ gradient. Manganese being a competitor of calcium inhibits the calcium pump and activates the ATPase. Chemical agents which purportedly have direct effects on active cation transport may interact with sodium-potassium ATPase system. Thus manganese has an additional action on the myocardial Na+ K+ ATPase which is stimulated by the ion which is attributable for reversion of cardiac tachyarrhythmias.
1. Mn++ reverts ouabain induced ventricular arrhythmias like random ventricular ectopics, ventricular bigemini, rapid ventricular tachycardia with AV dissociation to sinus rhythm.
2. Mn++ acts competitively with calcium.
3. Mn++ reactivates Na+ K+ ATPase which is inhibited by ouabain.