Effects of Sotalol in reperfusion-induced arrhythmias

Bátiz, L.F.; Ponce Zumino, A.; Baiardi, G.; Carrión, A.; Verdaguer, M.N.

Cátedra de Fisiología Normal, Facultad de Ciencias Médicas, U.N.Cuyo, Mendoza, Argentina

Introduction
Objectives
Material and Methods
Results
Discussion
Conclusions

Reperfusion of the ischemic myocardium is responsible for the occurrence of ventricular arrhythmias, such as ventricular tachycardia(VT) and ventricular fibrillation(VF). This arrhythmias have been suggested as a possible explanation for the sudden cardiac death, one of the major causes of mortality in the western world. This fact has driven to make several researches in order to understand the electrophysiological mechanisms involved in the initiation and the maintenance of ventricular arrhythmias during ischemia and reperfusion. Such an approach may highlight potential therapies for these life-threatening arrhythmias.
Even though in the last years there have been solid results, the electrophysiological mechanisms and principally the cellular mechanisms are still not clearly identified. Many authors coincide in that this ischemia and reperfusion-induced arrhythmias represent a complex phenomena involving both reentrant and non-reentrant arrhythmias and at cellular level, the principal effects are modifications of potassium (K⁺) and calcium (Ca⁺⁺) homeostasis: In relation to the K⁺, some authors point out that the reduction in intracellular ATP content activate ATP-sensitive potassium channels, with the corresponding exit of this ion toward the extracellular liquid; others sustain that alterations in the delayed outward rectifier current (I_K) would also be involved in this phenomenon.
In relation to the Ca⁺⁺, most of the authors coincide in that the intracellular accumulation of this ion may have an important role in initiating the arrhythmias during reperfusion favoring the delayed afterdepolarizations appearance. The increase in the concentration of the citosolic calcium is owed, on one hand, to alterations in the activity of the ionic channels (I_Ca) and the exchangers (Na⁺/H⁺ and Na⁺/Ca⁺⁺); on the other hand, it is secondary to catecholamine accumulation, free radical production and/or phospholipid catabolism. Summing up, the increase in citosolic calcium would act as a common final via of several factors involved in the genesis of these arrhythmias.
Most of these alterations at electrophysiological level lead to a reduced conduction velocity, an alteration of the rest potential (depolarization), a decrease in ventricular action potential duration (APD) and refractoriness (ERP) and may also be responsible for the initiation of afterdepolarizations.
Because of this multiplicity of events it is that it becomes difficult to get an appropriate therapy, but it suggests that the agent should possess multiple antiarrhythmic actions. Among the drugs that could satisfy that condition, the Sotalol is. This drug, classified as a class III antiarrhythmic, has very particular actions: 1- Beta-blocking activity and 2- Capacity to prolong the repolarization (increasing the APD and the ERP) of the myocardium fibers. due to an important reduction of the delayed outward rectifier current (I_K) associated with a small decrease in the inward rectifier current (I_K1).

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Objectives:

Sprague Dawley rats were used without sex distinction and with a weight that oscillated between 250 and 300 g. Its hearts were extracted quickly after the cervical dislocation and perfused, beating spontaneously, according to the technique of Langendorff (Figure 1A) with Tyrode solution (NaCl 121 mM; NaCO3H 25 mM; KCl 5 mM; NaPO4H 1.17 mM; CaCl2 2.5 mM; Glucose 5.5 mM), at 36 ºC and balanced with carbogen to pH 7.4 ± 0.05. The flow of perfusion was 8-10 ml/min.
The experimental protocol (Figure 1B) consisted in: 1 - stabilization of the preparation: 20 min, 2 - Control Period: 15 min, 3 - Period of Occlusion or Regional Ischemia (by ligation of the left anterior descending coronary artery): 10 min, 4 - Period of Reperfusion (by deocclusion): 10 min.
They were registered in continuous form: 1- the transmembrane potential of the epicardic ventricular cells (using conventional glass microelectrodes made flexible and full with a solution of KCl 3M  and the following parameters were measured (Figure 2): action potential amplitude (APA), rest potential (RP), action potential duration at the level of 90% of the repolarization (APD90) and 2- the electrogram in DII derivation (using an electrode on the tip of the left ventricle and another on the right auricle) where they were evaluated: ventricular frequency (CF), PQ interval, interval QRST and presence of during the reperfusion, which were classified in VT and VF.
The study was carried out based on the comparison of a witness group (n=11) where the Tyrode solution didn't contain Sotalol, with other two groups where the solution was added with a racemic mixture of d- and l- Sotalol 100 µM (n=11) and 200 µM (n=12), respectively.
The results were analyzed by means of Analysis of Variance, followed by test of Tukey. The incidence and types of arrhythmias were analyzed through the contingency chart using the test of square Chi.  The values of p <0,05 were considered significant.

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Figure 1

Figure 1B

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Figure 2

Analyzing the results without keeping in mind the addition of Sotalol (group witness), it was observed that: The regional ischemia caused in all the cases, a decrease of the APA and of the RP (depolarization) (Graphic 1A and 1B). The CF also diminished (Graphic 3).
In relation to DPA₉₀, it was observed a tendency to diminish during the occlusion in the group witness (mainly in the final minutes of this period) (Graphic 2A). The reperfusion made that the values of the electrophysiological parameters evaluated returned to the ones observed during the control period, excepting the CF that although it increased, it didn't reach the control values (Graphics 1, 2 and 3). The addition of Sotalol showed the following results:
Control Period: A significant increase of DPA₉₀ was observed with the addition of Sotalol 200 µM. This belonged together at electrographical level with a prolongation of the QRST interval duration (the only evaluated parameter where they were differences among the two evaluated concentrations of Sotalol) (Graphics 2A and 2B). The CF also diminished from 289,95 ± 4,31 beats/min in the witness group to 243,05 ± 13,71 beats/min with Sotalol 100 µM and 237,31 ± 3,92 beats/min with Sotalol 200 µM (Graphic 3).
Period of Regional Ischemia (Occlusion): The addition of Sotalol (both concentrations) caused a marked increase in DPA90 during this period, without modifying, like it was mentioned previously, the other electrophysiological parameters (Graphics 1, 2 and 3).
Period of Reperfusion: In this period, almost all the analyzed parameters recovered the values of the period control, except the CF (beta-blocking activity).
In relation to the incidence of life-threatening arrhythmias during this period, it was observed a tendency to diminish (90,91% in the witness group vs. 64,29% with Sotalol 200 µM), without being significant. However, the incidence of VF fell significantly with the addition of the drug (Graphic 4), the same as the duration average of the same ones and of the TV (Graphic 5A). The percentage of hearts that recovered the sinus nodal frequency was increased (Graph 5B).

Graphic 1A and 1B

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Graphic 2A and 2B

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Graphic 3

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Graphic 4

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Graphic 5A and 5B

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Discussion:

The decrease of the APA and of the RP (depolarization) caused by the regional ischemia would be due to a cellular loss of potassium (K+), although the intimate mechanisms of this fact are not clarified. The property of slowing CF would be owed mainly to beta blockade and, at least in part, might be the result of drug-induced prolongation of the action potential duration in the sinus node.
The increase in APD90 because of the addition of Sotalol coincides with the data reported by other authors, and the intimate mechanism of this fact, studied by patch clamp, indicates that Sotalol produces an important reduction of the delayed outward rectifier current (IK) associated with a small decrease in the inward rectifier current (IK1), prolonging mainly the last part of the repolarization. This fact was much more significant during the ischemia and it would be, mainly to that the Sotalol presents the phenomenon called "reverse use-dependency"(APD increases as the stimulus frequency is reduced) and, during this period they were registered the slowest frequencies.
The evident antifibrillatory effect of Sotalol would be owed fundamentally to the property of prolonging the repolarization and therefore, to increase the ERP, however other authors have also reported, an increase of the fibrillatory threshold.

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Conclusions:

The genesis and maintenance of the ventricular arrhythmias induced by ischemia and reperfusion represent a complex phenomenon where surely the reduction in APD90 due to alterations in the potassium current and the intracellular accumulation of calcium secondary to catecholamine release have a relevant role.
Sotalol demonstrates a protective effect in the reperfusion-induced arrhythmias: it showed an important tendency to diminish the incidence of these life-threatening arrhythmias and demonstrated to attenuate markedly the severity of them, diminishing the incidence of VF and the duration average of these serious arrhythmias. Also, it increased the number of hearts that recovered the sinus nodal frequency at the end of the experience.
It is important to highlight that, although we don't observe big differences among the concentrations used in this work, previous studies indicate that the effects of the Sotalol are dose-dependent

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