Simulation of contraction and relaxation in a stopped rat heart using the Na-Ca metabolic system

Aleksandr Aleksandrovich Dubok; Дубок Александр Александрович; Polina Romanovna Shibkova; Шибкова Полина Романовна; Islam Bashirovich Kurbanov; Курбанов Ислам Баширович; Aleksey Vladimirovich Alabovskiy; Aлабовский Алексей Владимирович; Vladimir Vladimirovich Alabovskiy; Алабовский Владимир Владимирович; Yulia Aleksandrovna Kotova; Котова Юлия Александровна; Tatyana Aleksandrovna Bereshnova; Бережнова Татьяна Александровна; Aleksey Anatolevich Vinokurov; Винокуров Алексей Анатольевич; Irina Viktorovna Kovalenko; Коваленко Ирина Викторовна

Simulation of contraction and relaxation in a stopped rat heart using the Na-Ca metabolic system

Authors: Dubok A.A.¹, Shibkova P.R.¹, Kurbanov I.B.¹, Alabovskiy A.V.¹, Alabovskiy V.V.¹, Kotova Y.A.¹, Bereshnova T.A.¹, Vinokurov A.A.¹, Kovalenko I.V.¹
Affiliations:
1. Voronezh State Medical University named after N.N.Burdenko
Issue: Vol 14 (2025): Материалы XXI Международного Бурденковского научного конгресса 24-26 апреля 2025
Pages: 40-46
Section: Биохимия и клинико-лабораторная диагностика
URL: https://new.vestnik-surgery.com/index.php/2415-7805/article/view/10242

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Abstract

Goal. The aim of the work is to develop a method for studying the kinetics of Na-Ca metabolism by continuously recording the contractile activity of an isolated rat heart, as well as studying the effect of certain cardioactive drugs on this process.

Materials and methods of research. The experiments were performed on isolated hearts of white rats perfused through the aorta using the Langendorff method.

The results of the study. Immediately after switching on the solution containing a high concentration of potassium (15 mmol/l), a complete cardiac arrest was observed. 5 minutes after cardiac arrest, cardiac perfusion was transferred to solution No. 3, which contained a reduced concentration of NaCI (30 mmol/l) and a high level of CI (15 mmol/l). An increase in heart tone was immediately observed. Registration of an increase and then decrease in tone in the stopped rat heart caused by a decrease and then restoration of the previous concentration of NaCI in the perfusion solution. However, immediately after reaching the max

Keywords

cardiac arrest, Na-Sa exchange, isolated rat heart, maximum contraction and relaxation forces

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Introduction. Among the biochemical and biophysical complex systems, the first place is occupied by the process of muscle contraction and relaxation. Currently, this mechanism is well studied. When working on this problem, the question of choosing a research object often arises. The process of contraction and relaxation was studied on isolated myofibrils, isolated cardiomyocytes, muscle strips, isolated animal hearts, and directly on muscles in vivo in intact animals.Having one or another advantage, any of the listed objects of research is quite difficult to assess the features of the molecular processes occurring at the time of contraction and relaxation of myofilaments. For example, the study of the kinetics of ion fluxes in the sarcolemma and reticulum involved in muscle contractions.Among them, the greatest attention is paid to the mechanisms of increasing and removing calcium ions in muscles during systole and diastole. It is known that the calcium channels that open under the influence of various stimulating signals are the initiating mechanism that causes the muscle to contract. A relatively small amount of calcium, once inside the cell, activates ryanodine receptors, causing the release of sufficient calcium from the reticulum to reduce [1]. Muscle relaxation is associated with the return of calcium from myofibrils back to the reticulum or extracellular environment with the expenditure of energy.In addition to calcium transport via ion channels, another mechanism is proposed that can increase or decrease the intracellular concentration of these ions. This is a Na-Sa exchange system [2]. The transport carrier has active centers outside and inside the cells, with high affinity for calcium and sodium ions. There is competition between the two cations for the mastery of the active center. At rest, none of the cations gets preferential movement through the membrane. The transport of a competing ion occurs only when its concentration increases outside or outside the cell.The concentration of calcium inside the cardiomyocyte reacts sensitively to changes in sodium levels: a decrease in extracellular sodium or its accumulation inside the cell stimulates the influx of calcium, while an increase in the sodium gradient promotes the excretion of calcium, mainly through the Na+/Ca2+ exchanger. This makes sodium ions an important factor controlling intracellular calcium [3]. Since in many pathological heart diseases the concentration of sodium ions inside the cells increases, the level of calcium in them can also increase. With an increase in this disorder, calcium overload of the myocardium occurs, which often leads to fatal cardiac arrhythmias.At the same time, the role of Na-Sa metabolism in pathology has not been fully studied. So far, there is little information about the effect of drugs on this biochemical process. This circumstance is explained by the fact that experimental conditions have not yet been created to assess the state of this exchange process in an isolated form. In other words, such models are very attractive, in which it is possible to study the fluxes of calcium ions solely with the help of Na-Ca metabolism, without the involvement of other mechanisms of calcium transport.An important condition for studying the sodium-dependent movement of calcium ions through the muscle sarcolemma is the elimination of other calcium fluxes. Calcium ions can enter cells through slow calcium channels and are excreted by Ca-ion pumps. Fast sodium channels can be involved in the mechanism of regulation of the intracellular Ca level. The appearance of sodium ions inside cells can also change the process of Na-Ca metabolism, which is also an interfering factor in evaluating the operation of the sodium-calcium exchanger alone [4]. Therefore, the best way to eliminate these interferences, in studying only Na-Ca metabolism, is to completely turn off the sodium and calcium channels by depolarization of cardiomyocyte membranes.This technique is well known and is carried out using a high extracellular concentration of potassium ions. At the same time, there is not only channel blockage, but also a complete cardiac arrest [5].In these conditions, it is possible to cause contractions and relaxation of the heart only by increasing or decreasing the intracellular Ca level using the Na-Ca metabolic system. The purpose of the work. The purpose of this study was to study the possibility of initiating contraction and relaxation in a stopped heart using the Na-Sa exchange mechanism.Materials and methods of research. The experiments were conducted on isolated hearts of white rats perfused through the aorta using the Langendorff method.To stabilize the contractile function of the heart, perfusion was performed with the initial solution No. 1 at a rate of 9-10 ml/min per 1 g of raw weight for 10 minutes.The initial perfusion medium was an oxygenated Ringer-Locke solution (t = 37°C) containing (in mmol/L): NaCl – 140, NaHCO3 – 2.0, KCl – 5.0, Tris-ONE (pH = 7.4) – 2.0, CaCl2 – 2.0, glucose – 11. Cardiac contractions and relaxations were recorded using rubber cans connected to an electronic pressure sensor. The signals from the sensor were sent to an analog-to-digital converter. The parameters were recorded and processed using the software of the external Zet Lab module and a computer.To stop the contractions of the heart, solution No. 2 was used, identical to the initial solution, but with an increased concentration of KCl up to 15 mmol/L.The initiation of sodium-dependent calcium absorption in an isolated rat heart was carried out by perfusion with a hyponatric solution in which the sodium concentration was reduced from 140 to 30 mmol/L. The concentration of KCl in the hyponatremium solution (solution No. 3) was maintained at a high level of 15 mmol/l. To compensate for the lack of osmotic pressure, 220 mmol/l of mannitol was added to the hyponatremic solution. The concentration of mannitol was doubled relative to the missing concentration of NaCl, since mannitol, unlike NaCl, does not dissociate into individual particles.At the end of perfusion with hyponatremic medium, perfusion was performed with a solution containing 140 mmol/l NaCl and 15 mmol/l KCl.Statistical processing of the obtained results was carried out by the method of variation statistics. The paper discusses the results for which a significance level of p < 0.05 has been achieved.The results of the study. Immediately after switching on the solution containing a high concentration of potassium (15 mmol/L), complete cardiac arrest was observed (Fig. 1).However, immediately after reaching the maximum peak of contraction, a spontaneous (spontaneous) slow weakening of tone occurred (Fig. 2). After switching the solution to the previous one containing the initial concentration of NaCI (140 mmol/l), a decrease in tone in the isolated heart was observed.Since the flow of Ca ions into cardiomyocytes through electroexcitable channels was blocked, muscle contraction and relaxation were caused by an increase and then decrease in the content of calcium ions in the heart muscle due to a different mechanism. In our experiments, the regulator of changes in calcium ions, in these experiments, was exclusively Na-Ca metabolism. The resulting graphic images were processed mathematically in order to use this technique to perform the subsequent stages of the experiment. First, the rate of increase in contraction (Vccr) during Na-Sa exchange demonstrates the opposite exchange process between sodium ions, which are released to the outside and calcium ions, which enter the cells at this time. This reaction occurs due to the gain of competition by calcium in the active center of the exchanger from the outside of the myocyte. In addition, Vcr. demonstrates the selectivity of the calcium exchanger. In our experiments, it was 28.2 mmHg/sec.The rate of relaxation during the circulation of Na-Sa exchange (V relaxation). It was 21.5 mmHg/sec.The maximum contraction force during Na-Ca metabolism (h1) shows the maximum result of Na-Ca metabolism, in which sodium output leads to maximum calcium entry into cells and maximum cardiac contraction. In our experiments, it was 1339.3 mmHg. The difference between the maximum reduction indicator and the level of achievement of the spontaneous reduction process after the peak of reduction (h 2) was calculated separately. This phenomenon has not yet been described anywhere in the literature. After reaching the maximum contraction caused by excess calcium, there is a gradual weakening of myocardial tone, despite continued perfusion with hyponatremic medium.This indicates that there is a spontaneous, gradual decrease in calcium concentration in the heart. Perhaps, at this stage, compensatory mechanisms are activated that weaken contracture phenomena in the myocardium. Apparently, to eliminate the overstrain of myofibrils, the processes of removing excess calcium from the sarcoplasm are activated. Most likely, these processes are Ca pumps of the sarcoplasmic reticulum, Ca pumps of the muscle sarcolemma, as well as mitochondria, which have the ability to accumulate excess calcium in the cytoplasm of cardiomyocytes. In our calculations, the spontaneous reduction process reached 280 mmHg. Next, the time intervals were calculated, during which the muscle first contracted and then relaxed. They were: 87 seconds with contraction, 166 seconds with relaxation (Table 1). Contraction and relaxation of muscle tissue is determined by rapid fluctuations in intracellular calcium concentration. The regulation of calcium levels in cardiomyocytes is carried out through a complex of mechanisms, among which calcium channels play a key role. Activation of calcium channels induces the release of calcium ions from the sarcoplasmic reticulum, increasing its intracellular concentration. Relaxation of cardiomyocytes occurs due to the removal of calcium ions from the cytosol with the participation of calcium ATPases (Ca2+-ATPases).The involvement of another mechanism, Na-Sa metabolism, in muscle contraction has not yet been proven.

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