scles the greater concentration of Ca2+ takes part in realization of the first contraction that provides increase in registered contraction amplitude . Our experiment on intact rats myocardium showed, that in all used rest periods the amplitude of first contraction exceeded basic level (Figure 4) Maximum amplitude increment in intact muscles after 60 s rest period was 87.8% ± 12.12%. However, the amplitude increment speed (T) after 30 and 60 s rest periods significantly decreased. This fact testifies the saturation of the SR with calcium ions and greater amount of calcium ejected from the SR in the time of contraction.
The same impacts used on muscles treated with amiodarone resulted in reliable potentiation strengthening (р < 0.01). As it is shown on Figure 4 intensity of this effect was increased with increasing of rest period. However, t(T50) index calculated for intact and amiodarone treated muscles hadn’t significant differences and was 11.87 ± 0.761 and 10.56 ± 0.637 s, respectively. This fact shows that after treatment of muscles with amiodarone the speed of the Са2+ uptake from cardiomyocyte myoplasm into the SR didn’t change. Taking into account existing conceptions about influence of “рost-rest” test on SR condition we can suggest, that amiodarone promotes more effective “holding” of Са2+ in the SR and/or prevents free Са2+ leakage current from the SR and does not change SR Са2+-ATPase activity.
Treatment of intact papillary muscles with caffeine results in canceling of inotropic reaction potentiation on
Figure 3. Influence of caffeine and amiodarone on postextrasystolic twitches amplitude of rat’s papillary muscles. Postextrasystolic contraction amplitude is expressed as percentage of steady state contractions. *Significant difference (p < 0.01) comparing to Control. #Significant difference (p < 0.01) between Amiodarone and Caffeine + Amiodarone.
Figure 4. Effect of amiodarone and caffeine on mechanical restitution of rat papillary muscles. *Significant difference (p < 0.01) comparing to control. #Significant difference (p < 0.01) between Amiodarone and Caffeine + Amiodarone.
the rest periods (Figure 4). This figure shows that in muscles treated with caffeine already after 20 s rest period restitution curve begins to decrease. These results point out that after caffeine action the SR capacity, as an intracellular Са2+ depot, decreases and depletion happens already at the 20 s rest period. On the background of developed caffeine effects, treatment of muscles with amiodarone didn’t provide recovery of “normal” muscle reaction on post-rest test action (Figure 4). However, amiodarone eliminates depletion of the Са2+ pool, located in the SR. On background of caffeine amiodarone prevents (p < 0.05) suppression of contraction muscle response after rest period of 20 and more seconds. Listed data agrees to our concept, that amiodarone potentiating effect on inotropic reactions of papillary muscles during “postrest” test is connected with the SR functional activity.
Another index of intracellular Са2+ recirculation is assessment of recovery of basic muscle contractility after “post-rest” test [21,22,28]. Recirculation of intracellular Са2+ fraction on isolated muscles is estimated by potentiation decay coefficient (DPC). This index is calculated from the curve of amplitude linear regression of “nth” contraction against next “n + 1st” contraction [21,22]. In our study recovery of amplitude up to basic values happened to the 9-th contraction both in a control conditions and on amiodarone background (Figure 5).
It turned out, that DPC in control and after treatment with amiodarone didn’t show significant difference (62.9% + 2.85% and 64.1% + 3.31%). This result shows that both in intact heart muscle and in one treated with amiodarone up to 62% of calcium ions from myoplasm is resequestered back to the SR, and 38%—is ejected into extracellular spaces by Na+/Ca2+-exchanger [21,22]. These results agree with previous analysis of the results and show, that amiodarone do not influence on the SR Са2+-ATPase activity.
It is shown in experiment that myocardium cells have a homeostatic mechanism which provides an optimal contraction response in different frequency modes [18,30]. As rule rise of contraction frequency leads to increase of entry of sodium and calcium ions from surroundings into a cell . Positive dependence in increase of contraction force with increase in the frequency of stimulation has been shown for majority of mammalians [31-33]. Exception is the myocardium of rat. A force of rat heart contraction decreases with increasing of stimulation frequency [33,34]. Negative force-frequency dependence of isolated rat myocardium by all appearance can be connected with the fact that process of myocardium contraction in this animals is provided, mainly with Са2+ entering at excitation of cardiomyocites from the SR . In this conditions shortening of intervals between electric stimulus leads to reduction of the time of Са2+ transporttation from uptake places to release places into the cardiomyocites SR. As a result not all Са2+ succeeds to reach terminal cisterns to the time of next contraction and to be used in next contraction response . Besides, inactivated Са2+-channels of cardiomyocytes SR are not recover completely for the short period of time between contractions [30,35]. Our study showed, that amiodarone is able to prevent contractility decay at increasing of stimulation frequency in intact rat myocardium, although it has weak negative inotropic effect, that amiodarone induces entry of greater quantity of calcium ions during contraction. Because this antyarrhythmic drug is able to block partially Са2+ and Nа+-channels resulting weak negative inotropic effect, we can exclude possibility of additional income of calcium ions into the cell from extracellular spaces. As it is known, the main calcium pool, taking part in the process of rat cardiomyocytes contraction, is intracellular calcium stored in the SR . Therefore, it is logically to suggest, that amiodarone is able to influence on work of SR Са2+-transporting systems. Probably, that increase of Са2+ transportation rate from uptake places to release places inside the structure crease of calcium ions during systole, i.e. mobilization of
Figure 5. Decay in post-rest potentiation in rat papillary muscle after 60 second rest period. *Value significantly different (0.01) from Control.
available stock of intracellular Са2+ takes place. So, our investigations allow to make an assumption that amiodarone is able to influence not only on activity of transmembrane channels and receptors, but to modulate intracellular homeostasis of calcium ions, too.
In addition because of amiodarone promotes increase of the time constant t1(T50) we can speak that this drug promotes acceleration of Са2+ transport inside the SR resulting increase of Са2+ in the places of its release from the SR. Transport of calcium ions inside cardiomyocites SR from uptake places to ejection places and time of Са2+-channels reactivation is estimated with help of the time constant t1(T50) [21,22].
According to the literature, the effect of PEC potentiation of inotropic response of cardiac muscle is associated with income of additional Ca2+ into myoplasm from the extracellular space during an extraordinary contraction AP . Calcium ions entered into cardiomyocytes are deposited in the sarcoplasmic reticulum, that increases the SR contribution in the first postextrasystolic cycle of contraction-relaxation. It is believed that the effect of PEC potentiation reflects ability of the sarcoplasmic reticulum of cardiomyocytes to accumulate additional quantity of Ca2+. So, increasing the PEC potentiation after treatment of rat papillary muscles with amiodarone demonstrates the presence of additional calcium ions stored in the SR in comparison with the control, and may be stimulated by increase in functional ability of the SR of cardiomyocytes.
A number of studies carried out using this test showed that the inotropic response of muscle strips on renewal of electrical stimulation after a rest period characterizes ability of the cardiomyocytes SR to accumulate and to hold calcium ions [21,22,27,28]. The observed phenomenon of increment of the amplitude in response to renewal of stimulation is connected with the fact that in the rest period dissociation of the Ca2+ complexes with Ca2+- binding proteins promotes inflow of additional calcium ion quantity from the sarcoplasma into the cardiomyocytes SR and increase of Ca2+ level deposited in the terminal cisterns . In addition, increase of the rest interval duration provides the time required to move the calcium ions from the uptake places to places of their release from the SR and, as a consequence, leads an increase of Ca2+ pool in terminal cisterns; later it is released during the first rest period after contraction [30,35]. Since the reactivation of Ca2+-channel SR also takes time, increase of rest interval duration between contractions may contribute to increase in activated Ca2+ channels of cell membranes and SR membrane, thus providing a greater number of calcium ions entering into cytosol from the terminal cisterns of reticulum and extracellular space. Perfusion of the muscle strips with amiodarone increased potentiation effect in the whole range of used rest intervals and the saturation threshold of the cardiomyocytes SR with calcium ions that testifies that amiodarone is able to be accumulated and to prevent calcium ion leakage current from the SR.
It is known, that caffeine is used in experimental studies to inhibit functional activity of the cardiomyocytes SR [36,37]. Caffeine effects were connected with partial inhibition of Са2+-ATPase and blockade of ryanodine receptors in open state that promotes leakage of calcium ions from the SR . Such caffeine action causes short time rise of calcium ions in cardiomyocytes myoplasm and increase of cardiomyocyte contractility, then depletion of intracellular calcium depot and, accordingly, decrease of contraction activity happens [36,38]. In addition, inhibition of amiodarone-induced increment of PEC amplitude and post-rest contraction with caffeine also testify of this hypothesis. Calculation of calcium ions recirculation coefficient from rhythm-inotropic dependence curve allow us to suggest that amiodarone effects on SR function are not related to modulation of SR’s Са2+- ATP-ase, but most likely are stipulated either by activetion of Са2+ transport from uptake places to ejection places inside this intracellular structure or by limitation of Са2+ leakage current from the SR.
Thus, our results allow reveal one more property of III class antiarrhythmic drug amiodarone associated with modulation of intracellular calcium ions homeostasis. That property is undoubtedly positive and probably allows to this drug to be high effectiveness in therapy of life-threatening arrhythmias.