Introduction
including a hyperpolarization-activated current of mixed ionic nature, known as ‘funny current’, If, or ‘pacemaker current’.
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are molecular correlates known as ‘funny current’ (If) activated by hyperpolarization. These ion channels can conduct sodium and potassium, and their permeability can change when interacting with cyclic nucleotides, mainly cyclic adenosine monophosphate (cAMP). It is known that cesium ions in millimolar concentrations can act as HCN blockers1. The proteins that form these channels are sufficiently widespread in the body; their significant contribution to many physiological functions has been shown, the main of which are the myocardial spontaneous electrical activity or intrinsic pacemaker activity, as well as the modulation of interneuronal connections in the brain. The participation of HCN in the regulation of heart rate, stimulation of neurons, integration of dendrites, mechanisms of learning, and memorization, as well as sensory systems, have been studied in detail2. Currently, HCN in peripheral tissues remains an attractive research subject.
Dario Di Francesco1-2 was the first to describe the peculiarity of HCN with some skepticism. Evolutionary and ontogenetic approaches to the study of HCN have provided evidence for a highly conserved and generic origin of this channel, as well as its premature functional and molecular expression in embryonic stem cells3. Despite the high interest in these channels, to date, the therapeutic value of specific modulators has been demonstrated only for cardiac pathology. Indeed, the widespread distribution of HCNs in various tissues and their participation in many physiological processes require the discovery of selective substances to prove that these channels can be used as drug targets and to develop new drugs.
The HCN channels belong to the superfamily of pore-loop cation channels. HCN channels are complexes consisting of the assembly of four alpha subunits that are arranged around the central pore. In mammals, four genes encoding HCN subunits were cloned1-4. All HCN monomers have the same basic structural scheme, consisting of six alpha-helices, a transmembrane domain, and two cytosolic domains at the ends of the NH and COOH4. The structure of HCN channel expression has been studied in several animal and human species at the tissue and single-cell levels. HCN subunits are highly expressed in the central and peripheral nervous system5. All four isoforms of HCN channels were found in the heart differently expressed in the departments6. The HCN channels are permeable to Na+ and K+ and conduct a mixed cation current. Membrane hyperpolarization is necessary and sufficient to activate the HCN channels; cAMP act as co-stimulatory modulators that facilitate voltage-dependent HCN channel activation6. The physiological role of HCN channels in the working healthy myocardium is currently subject of research. HCN-mediated current in human atrial and ventricular cardiomyocytes is similar to that obtained in sinus node cells, but its role should diverge, since healthy atrial and ventricular cardiomyocytes have a stable membrane resting potential and do not lead to spontaneous electrical activity 7.
Various experimental and epidemiological studies have identified numerous risk factors, such as hypertension, diabetes, and smoking, in the development of coronary heart disease8. An increase in heart rate may favor the progression of myocardial ischemia8. Heart rates during ischaemia and reperfusion are possible determinants of reperfusion arrhythmias. Ng9 used ivabradine, a selective If current inhibitor, to assesse the effects of heart rate reduction during ischaemia-reperfusion on reperfusion ventricular arrhythmias and showed that the antiarrhythmic effect of ivabradine may reflect slower development of ischaemia-induced electrophysiological changes.
The objective of this research is to study the effect of blockade of If-currents on heart inotropy in rats with modeled myocardial infarction.
Material and Methods
The myocardial infarction model reproduction technique
To reproduce myocardial infarction in experimental animals, the classical model developed in 1960 by Selye was used10.
The experiments were conducted on 18 white outbred rats, weighing 200-250 grams. The animal was anesthetized with ether by placing the rat under a glass cover. After that, the animal limbs were fixed to the operating table with rubber bands. Additional anesthesia may be required during preparation for thoracotomy; if necessary, an ether-soaked gauze pad was placed over the airway of the rat. After opening the chest, anesthesia was no longer applied. To improve the vision of the operating field, lamp lighting was used. The chest of the animal was shaved and treated with an antiseptic. A skin incision was made on the left side of the rat's chest. They spread the pectoral muscles. Blunt curved forceps were then dipped between the fifth and sixth ribs and expanded to about 15 mm. At this time, a rapid heartbeat was observed. Now air enters the chest cavity, and breathing becomes impossible, but this does not pose a serious threat to the life of the rat, since the ligation takes 60-90 seconds. After exteriorization, the heart ventricles were fixed with the thumb and forefinger of the left hand. The anterior branch of the left coronary artery was visually found, to which a ligature (Premiline 6/0, 2xDR12) was placed 0.5-1 mm below its exit from under the auricle of the heart and ligated, the heart was returned to the chest cavity. The muscles were shifted; the skin was sutured and treated with an antiseptic solution. Immediately after shifting the muscles, the animal began to breathe, and in a few minutes after the operation regained consciousness and began to actively move around the cage10.
The effect of the hyperpolarization-activated current blockade on an isolated heart with modeled myocardial infarction was studied on the 54th day of the formation of heart failure, indicating the adaptation of the cardiovascular system to heart failure caused by ligation of the left coronary artery.
Isolated heart specimen preparation
The ex vivo experiments were carried out according to the standard Langendorff isolated heart technique. For anesthesia, a 25% urethane solution (800 mg/kg) was used intraperitoneally. Then the heart was isolated, washed in Krebs-Henselite solution pre-cooled to +4oC. The heart behind the aorta was ligated with a cannula, and retrograde perfusion was performed at a constant pressure of 55-60 mm Hg and a temperature of 370C using the Langendorff System (Australia). To register the pressure in the left ventricular cavity, a latex balloon connected to a catheter and a pressure transducer was used. The studied parameters of the isolated heart were recorded and processed using the LabChart Pro V8 software. During the experiment, the change in the pressure wave amplitude (PWA), the maximum rate of increase of the pressure wave (dP/dtmax), and the maximum rate of drop of the pressure wave (dP/dtmin) in response to the addition of ZD7288 (Sigma), a hyperpolarization-activated current blocker, into the perfusion solution were calculated.
Statistical analysis of the results was carried out using paired and unpaired Student's t-tests. Values were considered significant for a value of approximately p<0.05.
Results and Discussion
To identify the effects of If blockade in an isolated heart with a model of myocardial infarction, experiments were carried out in the presence of ZD7288 blocker at concentrations of 10-9, 10-7, 10-5 M.
As shown in figures 1A 1B and 1C, the application of ZD7288 to a perfused solution at a concentration of 10-9 M led to a change in the studied parameters of the heart. The initial values of PWA were 40.2 ± 14.8 mm Hg. During the 4th minute, PWA increased to 45.2 ± 16.7 mm Hg. (p≤0.05). By the 8th minute of the experiment, PWA was 47.8 ± 16.1 mm Hg. During the 15th minute of observation, PWA increased to 49.8 ± 16.5 mm Hg. During the final minute of the experiment, PWA was 57.6 ± 17.2 mm Hg.
dP/dtmax before application to the perfused solution ZD7288 was 1082 ± 336 mmHg/sec and increased to 1199 ± 404 mm Hg/sec during the first minute of observation. By the 7th minute of the experiment, dP/dtmax increased to 1201 ± 428 mm Hg/sec. During the 12th minute of observation, dP/dtmax was 1268 ± 421 mm Hg/sec. During the final minute of observation, dP/dtmax decreased to 1419 ± 418 mm Hg/sec.
dP/dtmin after injection of the If blocker increased from 713 ± 222 mm Hg/sec up to 764 ± 254 mm Hg/sec. By the 5th minute of observation, dP/dtmin was 689 ± 249 mm Hg/sec. Then dP/dtmin increased to 720 ± 261 mm Hg/sec and 810 ± 260 mm Hg/sec during the 10th and 15th minutes of observation. By the final minute of observation, dP/dtmin increased to 909 ± 267 mm Hg/sec.
The addition of the If blocker to the perfused solution at a concentration of 10-7 M caused an increase in PWA from 38.3 ± 10.2 mm Hg up to 42.6 ± 13.2 mmHg by the 5th minute of observation. During the 10th minute of the experiment, PWA increased to 47.8 ± 15 mm Hg. Then PWA continued to increase and by the 15th and 20th minutes reached 50 ± 15.2 mm Hg and 53.5 ± 14.4 mm Hg, respectively (p≤0.05).
Application of ZD7288 caused an increase in dP/dtmax from 1053 ± 242 mmHg/sec up to 1207 ± 353 mm Hg/sec by the 5th minute of observation. dP/dtmax by the 10th minute of the experiment increased to 1295 ± 413 mm Hg/sec. By the 15th and 20th minutes of the experiment, dP/dtmax increased to 1326 ± 413 mm Hg/sec and 1387 ± 393 mm Hg/sec.
Figure 1A. The effect of If blockade with ZD7288, at concentrations of 10-9 M, 10-7 M and 10-5 M on the pressure wave amplitude of rat isolated heart with modeled myocardial infarction. The ordinate is the pressure wave amplitude the abscissa is the experiment time (minutes). The significance is indicated in comparison with the initial values. *p≤0.05.
Figure 1B. The effect of I. blockade at concentrations of 10-9 M, 10-7 M and 10-5 M on the maximum rate of pressure wave rise (dP/dtmax) of rat isolated heart with a myocardial infarction model. The ordinate axis is the maximum rate of rising of the pressure wave (dP/dtmax, in %), the abscissa axis is the experiment time (minutes). The significance is indicated in comparison with the initial values. *p≤0.05.
Figure 1C. The effect of I. blockade at concentrations of 10-9 M, 10-7 M and 10-5 M on the maximum rate of pressure wave drop (dP/dtmin) of of rat isolated heart with with a myocardial infarction model. The ordinate axis is the maximum rate of the pressure wave drop (dP/dtmin, %), the abscissa axis is the experiment time (minutes). The significance is indicated in comparison with the initial values. *p≤0.05.
Application of the If blocker in an isolated heart with modeled myocardial infarction caused an increase in dP/dtmin from 560 ± 112 mm Hg/sec up to 653 ± 184 mm Hg/sec to 5 minutes of the experiment. Then, during the 10th minute of observation, dP/dtmin was 692 ± 214 mm Hg/sec. By the 15th minute of observation, dP/dtmin increased to 711 ± 216 mm Hg/sec. At the final minute of observation, dP/dtmin increased to 743 ± 205 mm Hg/sec.
Meanwile, as shown in figures 1A 1B and 1C, the addition of the If blocker to the perfused solution at a concentration of 10-5 M caused a decrease in the studied parameters of an isolated heart with a model of myocardial infarction. In effect, before the addition of ZD7288 to the perfusion solution, PWA was 13.9 ± 4.5 mmHg, and in the first minute after the injection of the substance, it decreased to 12.1 ± 4.5 mm Hg. (p≤0.05). The minimum decrease in PWA to 7.5 ± 2.8 ml/min was observed during the 4th minute of the experiment. Then, during the 10th minute, PWA slightly increased to 8.81 ± 3.7 ml/min, and during the 15th minutes, up to 9 ± 2.9 ml/min. During the final minute of observation, PWA was 8.5 ± 2.8 ml/min.
The addition of the If blocker to the solution caused a decrease in dP/dtmax from 339 ± 112 mm Hg/sec, up to 304 ± 106 mm Hg/sec. (p≤0.05). During the 5th minute, the lowest dP/dtmax value was recorded, 187 ± 26 mm Hg/sec. By the 10th minute of the experiment, dP/dtmax increased to 197 ± 31 mm Hg/sec. During the 15th minute, dP/dtmax increased to 202 ± 29 mm Hg/sec. During the final minute of the experiment, dP/dtmax was 191 ± 24 mm Hg/sec.
After adding the IF blocker to the solution, dP/dtmin decreased from 251 ± 61 mm Hg/sec up to 226 ± 60 mm Hg/sec. (p≤0.05). By the 5th minute, dP/dtmin decreased to 159 ± 15 mm Hg/sec. Then, during the 10th and 15th minutes of observation, dP/dtmin decreased to 156 ± 23 mm Hg/sec. and 155 ± 19 mm Hg/sec. During the final minute of the experiment, dP/dtmin was 149 ± 13 mm Hg/sec.
Figure 2. Comparative analysis of the pressure wave amplitude of the Langendorff isolated heart of adult rats of the control group and with modeled myocardial infarction, with If current blocker application, at concentrations of 10-9 М, 10-7 М and 10-5 М. The ordinate is the change in the pressure wave amplitude (PWA) (%), the abscissa is the concentration of the ZD7288 blocker (M). The significance is indicated in comparison with the initial values. *p≤0.05.
During the experiment, the peculiarities of the inotropic function of an isolated heart with a model of myocardial infarction during hyperpolarization-activated If blockade were observed. Application of the If blocker at concentrations of 10-9 M caused an increase in the pressure wave amplitude by 43%, and at concentrations of 10-7 M – in PWA by 40% (p≤0.05). Perfusion of the If blocker (10-5 M) reduced PWA by 49%. dP/dtmax increased upon perfusion of ZD7288 at concentrations of 10-9 M and 10-7 M by 31%, and dP/dtmin increased by 27% and 32%, respectively. The addition of the drug to the solution (10-5 M) caused a decrease in dP/dtmax by 44%, and dP/dtmin by 41%.
A comparative analysis of the results of the study revealed that an isolated heart with a model of myocardial infarction develops a positive inotropic effect in response to adding the ZD7288 blocker at concentrations of 10-9 M and 10-7 M; while in healthy animals, a positive inotropic effect is observed only when the minimum concentration of the blocker is added. ZD7288 at a concentration of 10–5 M decreased the pressure wave amplitude in an isolated heart with a model of myocardial infarction and in a healthy rat heart; however, in a heart with a myocardial infarction model, the decrease in the pressure wave amplitude was more pronounced10(Figure 2).
Earlier, in our studies on healthy animals, the effect of If blockade on heart rate and the force of contraction of myocardial strips was shown. The age-related characteristics of the heart rate response11,12 and inotropy to the hyperpolarization-activated If blockade13,14 were studied.
Studies aimed at identifying factors causing myocardial infarction suggest that impaired coronary blood supply is not the only factor in the pathogenesis of the disease typical of human myocardial infarction. Myocardial infarction rarely occurs in perfectly healthy young people; it is possible that the development of cardiac muscle pathology is caused by biochemical changes leading to wear and tear of the myocardium and coronary arteries, in particular, chronic atherosclerosis and myocardial fibrosis, which usually precede the development of myocardial infarction in humans. Recent literature shows that the If density in cardiomyocytes changes not only with age but also in pathophysiological hypertrophy 15,16.
Conflict of Interests
The author declares that the provided information has no conflicts of interest.
Anna Mihailovna Kuptsova anuta0285@mail.ru
