Inward Rectifier Currents IK1 and IKACh in Working Myocardium of Japanese Quail (Coturnix japonica)

Birds acquired endothermy and a four-chambered heart independently from mammals in the course of evolution. Though avian embryos are widely used in experiments, little is known about the adult avian heart. Recent studies have shown that, despite a large evolutionary distance, the set of repolarizing potassium currents in avian myocardium resembles that in mammalian heart as well as that in humans. This allows for proposing birds as a potential model in experimental cardiology. The present study for the first time describes inward rectifier currents in working myocardium of quail. Using patch clamp method, we recorded main background inward rectifier current IK1 was recorded in isolated atrial and ventricular cardiomyocytes of quail. Both inward and outward components of IK1 in ventricular cells were larger than those in atrial cells, while there were no differences in voltage dependence of inward rectification. Acetylcholine and carbachol induced activation of acetylcholine-dependent inward rectifier current IKACh in atrial but not in ventricular myocytes. IKACh in atrial myocytes was sensitive to tertiapin. Constitutively active IKACh has not been detected. In multicellular preparations of the quail right atrium, carbachol induced hyperpolarization and shortening of action potentials, while no such effects were observed in preparations of the right ventricle. Activation of IKACh upon application of carbachol was dose-dependent with EC50 = 4.922 × 10–7 М. The described distribution of inward rectifier currents in avian myocardium is similar to that in mammalian species, which are widely used as model objects in experimental cardiology.


INTRODUCTION
In the course of evolution, birds, along with mammals, independently acquired homoiothermy and a four-chambered heart, which is necessary for an adequate supply of body tissues with oxygen and to maintain an optimal metabolic rate. However, in terms of their morphology and ultrastructure, avian cardiomyocytes are similar to reptile cardiomyocytes. Compared with mammalian cardiomyocytes, avian cardiomyocytes have an elongated, fusiform shape; avian myocytes lack the transverse (or T-) tubules, which are characteristic of mammals and necessary for the rapid transmission of excitation into the cardiomyocyte [1]. Despite these features, considered archaic, bird hearts are able to maintain high pressure in the systemic circulation and can surpass mammalian hearts in performance [2]. Since the contractile activity and, consequently, the pumping function of the heart directly depend on its electrical activity, the previous studies of our group were aimed to study the set of ionic currents that provide the electrical activity of the heart of birds [3,4]. It has been shown that the depolarization of the myocardium in medium-sized birds (for example, the Japanese quail, Coturnix japonica) is provided by the fast sodium current I Na [5] and the L-type calcium current I CaL [4], while repolarization is provided by the transient outward potassium current I to1 as well as fast and slow currents of delayed rectification, I Kr and I Ks [3]. The described electrophysiological phenotype differs from the set of currents in the myocardium of reptiles, which are the closest to the birds animal group in terms of evolution [6], and resembles the electrophysiological phenotype of the mammalian myocardium, in particular, the human, to a large extent [7,8]. In total, this makes the avian myocardium both a promising object for fundamental research and a potential model object for testing new cardiotropic drugs, which requires more thorough studies of various aspects of the physiology of the avian heart.
The family of potassium inward rectifier currents (I Kir ) is one of the most important groups of ionic currents in the myocardium of vertebrates and includes three members: the main background inward rectifier current I K1 , acetylcholine-dependent current I KACh , and ATP-dependent current I KATP , which activates only under ischemic conditions [9] and is not considered in this study. The main function of the inward rectifier currents is maintaining a stable negative resting potential and participation in the late repolarization of cardiomyocytes; the background current I K1 performs this function constantly, while the acetylcholine-dependent current I KACh is activated only when acetylcholine binds to M2 receptors and provides negative chrono-and inotropic effects of parasympathetic stimulation [10].
The aim of this study was to identify potassium inward rectifier currents I K1 and I KACh in the working myocardium of birds and to describe their main properties, such as current-voltage characteristics, sensitivity to blockers and agonists, and the distribution of channels in the myocardium.

MATERIALS AND METHODS
Japanese quail (Coturnix japonica, estonian variety) of both sexes at the age of 2-4 months and weighing 250-300 g were obtained from the "Orlovsky Dvorik" farm (Mytishchi, Moscow oblast, Russia). Prior to the experiments, the animals were kept for 10 days in 12 : 12 h light-dark conditions with ad libitum access to water and commercial feed for quails. All procedures were performed in accordance with Directive 86/609/EEC on the handling of laboratory animals.
Isolated ventricular and atrial myocytes of quail were obtained according to the previously described method [4]. Prior to the decapitation, the animals were injected with heparin intraperitoneally (1000 units/kg), after which they were decapitated. After the decapitation, thoracoabdominal cavity was opened, the heart was quickly excised and placed on a Langendorff apparatus for retrograde perfusion with Ca 2+ -free modified physiological saline of the following composition: 116 mM NaCl, 4 mM KCl, 1.7 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 0.55 mM MgCl 2 , 5 mM sodium pyruvate, 20 mM taurine, 11 mM glucose, 1 mg/mL bovine serum albumin; the pH was maintained at 7.4 by aeration with carbogen (95% O 2 ; 5% CO 2 ) at 42°C. After washing out the blood from the heart and 7-9 min perfusion, the heart was perfused with a solution of the same composition supplemented with collagenase II (0.4 mg/mL) and CaCl 2 (16 μM). Perfusion with enzymes-containing solution containing the enzyme was performed for 35 or 40 min to isolate atrial or ventricular cardiomyocytes, respectively. Isolated cardiomyocytes were stored for 6-8 h at a temperature of 24°C in a Kraftbrühe solution of the following composition: 3 mM MgSO 4 , 30 mM KCl, 30 mM KH 2 PO 4 , 0.5 mM EGTA, 50 mM potassium glutamate, 20 mM Hepes, 20 mM taurine, 10 mM glucose (pH 7.2) [11].
Ionic currents and action potentials in some of the experiments were recorded in isolated quail cardiomyocytes using whole-cell patch-clamp method with HEKA EPC-800 amplifier (HEKA Elektronik, Germany). Ionic currents were recorded in voltage clamp mode, while action potentials were recorded in current clamp mode. The cells were placed in an RC-26 experimental chamber (volume 150 μl, Warner Instrument Corporation, United States) and constantly perfused with a solution simulating the extracellular environment-150 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1.2 mM MgCl 2 , 10 mM glucose, 10 mM Hepes, (pH 7.4)-at a temperature of 24°C. To block calcium current and slow delayed-rectifier potassium currents, nifedipine (10 -5 M) and chromanol 293B (2 × 10 -5 M) were added to the solution [3]. Patch pipettes were made from borosilicate glass capillaries (Sutter Instruments, United States) and filled with a solution (pH 7.2) of the following composition: 140 mM KCl, 1 mM MgCl 2 , 5 mM EGTA, 4 mM Mg-ATP, 0.03 mM Na 2 -GTP, 10 mM Hepes. The resistance of the filled patch pipettes was 2.72 ± 0.47 MΩ. The voltage-clamp protocols are shown in the sidebars of the figures.
The amplitude of inward rectifier current I K1 was estimated after subtraction of the current recorded in the presence of the blocker (1 mM BaCl 2 ), which allowed us to exclude the leak current. The amplitude of the acetylcholine-dependent current I KACh was estimated as the difference in the total inward rectifier current in the presence of an agonist of acetylcholine receptors (acetylcholine or carbachol, 10 -6 M) and in control conditions; I KACh current was also identified by its sensitivity to its selective blocker-tertiapin (10 -6 M). The currents were normalized according to the cell capacity. Inward rectification of I K1 current was estimated as the ratio of the experimentally recorded current I K1 to the hypothetical non-rectifying current (see inset in Fig. 1). The dependence of this ratio on the membrane potential was expressed by the Boltzmann distribution function: where V is the holding potential, V 0.5 is themidpoint voltage, and k is the slope of the curve.
Action potentials were recorded in isolated multicellular preparations of quail ventricular and atrial myocardium using the standard microelectrode technique. The animals were decapitated, the heart was excised and retrogradely washed with Tyrode's solution: 118 mM NaCl, 2.7 mM KCl, 1.2 mM MgCl 2 , 1.8 mM CaCl 2 , 25 mM NaHCO 3 , 11 mM glucose.
The pH was maintained at 7.4 by aeration with carbogen gas. Preparations of right atrium and right ventricle of Japanese quails were used in the experiments. Myocardial preparations were placed in the experimental chamber with the endocardial side up and perfused with Tyrode's solution at 42°C. The preparations were electrically stimulated at a frequency of 6 Hz, which corresponds to the heart rate of intact animals [12]. Action potentials were recorded after the adaptation period (30 min) using glass microelectrodes filled with 3M KCl solution (electrode resistance 20-50 MΩ) and connected to a Model 1600 amplifier (A-M Systems, United States). The duration of action potentials was assessed at the level of 50% (APD50) and 90% (APD90) repolarization. Data are presented as mean ± standard error of the mean for n cells or myocardial preparations. Statistical data processing was performed using GraphPad Prism 8.0 software (GraphPad Software, United States). Depending on the experiment, to assess the statistical significance of the differences, the Student's t-test for paired or unpaired samples was used. Differences were considered significant at p < 0.05.

RESULTS AND DISCUSSION
When the holding potential was changed in linear manner from +60 to -120 mV (Fig. 1, see inset) in the presence of nifedipine and chromanol, an inwardly rectifying current without time-dependent inactivation was observed in quail isolated cardiomyocytes.
The shape of the current-voltage relation and the sensitivity of the current to 1 mM BaCl 2 allowed us to define it as the background inward rectifier current I K1 . In ventricular myocytes, the amplitude of both the inward and outward components of the current was statistically significantly higher than the amplitude of the current in the atrial cells (Figs. 1a, 1b). This ratio is typical for most vertebrates, including humans: the I K1 amplitude in atrial and ventricular quail myocytes is close to the I K1 amplitude in isolated human cardiomyocytes [9,13,14]. The difference in the amplitude of I K1 between the atria and the ventricles provides a more negative resting potential in the ventricular myocardium of quails [3]. It should be noted that, in comparison to ectothermic vertebrates, the I K1 amplitude in quail myocardium is very high [6,13], which may be a mechanism counteracting the occurence of arrhythmias at a high heart rate, which is common for birds. However, the comparison to the literature data on the distribution of I K1 in myocardium of mammals with a similar heart rate indicates that, while I K1 amplitude in ventricles is similar, the amplitude of I K1 in quail atria is noticeably lower than that in mice and guinea pigs [15,16]. It can be assumed that the role of the repolarization reserve in quail atrial myocardium of the quail is taken on by other currents: for example, a delayed rectifier, which has a high amplitude but poorly contributes in normal conditions at the same time. On the other hand, the lower I K1 amplitude in the quail atria, as compared to that in rodents, may be phylogenetically determined.
The high amplitude of I K1 current allowed us to maintain a negative membrane potential in current clamp mode in isolated quail ventricular cardiomyocytes, which, in turn, made it possible to record action potentials in individual myocytes (Fig. 1b). However, in atrial cardiomyocytes, due to the low amplitude of I K1 , the recording of action potentials using patchclamp method was impossible; therefore, in the further experiments, the action potentials were recorded using standard microelectrode technique.  The dependence of I K1 inward rectification on the holding potential did not differ between ventricular and atrial cardiomyocytes (Fig. 1d): the the midpoint voltage V 0.5 in ventricular cells was -90.51 mV and that in atrial cells was -90.49 mV. Since the total I K1 current can be mediated by several isoforms of channels characterized by different degrees of inward rectification [17], it can be assumed that the set of channels isoforms does not differ between ventricular and atrial cardiomyocytes of quail, however, this statement requires further verification using methods of molecular biology.
Carbachol (a nonhydrolyzable analog of acetylcholine) at a concentration of 10 -6 M caused an increase in both the inward and outward components of the total inward rectifier current in atrial (Fig. 2a), but not in ventricular quail cardiomyocytes (Fig. 2b). The current activated by carbachol in atrial cardiomyocytes was sensitive to selective I KACh blocker tertiapin (10 -6 M), which allowed us to identify it as I KACh [9]. In the absence of any other external influences, tertiapin did not induce changes in the total inward rectifier current in both atrial and ventricular cardiomyocytes, which indicates the absence of constitutively active I KACh current. Carbachol caused hyperpolarization and shortening of action potentials (Figs. 2c, 2e, 2f) in isolated preparations of quail right atrium, while carbachol did not cause similar changes in electrical activity in preparations of ventricular myocardium (Fig. 2d).
The relatively high frequency of heart contractions in birds in combination with a rather small amplitude of rapid delayed rectifier current I Kr [3] suggests that other currents can participate in the repolarization of quail myocardium, for example, the constitutively active I KACh current. This assumption was not confirmed; however, it is possible that the constitutively active I KACh current can be involved in the development of pathological conditions, for example, in atrial fibrillation in avian myocardium, as well as in mammalian myocardium [9]. The distribution of I KACh channels in working myocardium of quails-their presence in the atria and their absence in the ventricles-is also typical for most vertebrates [6,9,18]. Recent studies, however, suggest a possible minor expression of I KACh current channels in ventricular cardiomyocytes of some mammalian species: I KACh channels have been found in rat and human ventricular cardiomyocytes [19,20]. Thus, the possibility of detecting these channels in the ventricular myocardium of birds cannot be excluded: I KACh can be masked by a large background I K1 current in normal conditions and might be activated only if the latter is suppressed, functioning as a arrhythmias-counteracting mechanism [21]. In addition, the possibility of a heterogeneous distribution of I KACh channels cannot be Potential, mV excluded: for example, in human ventricular myocardium, I KACh channels are absent in the mid layer of the myocardium [20] and the the bulk of isolated cardiomyocytes belongs to this population.
The activation of the I KACh current in quail atrial cardiomyocytes in the presence of carbachol was dosedependent (Fig. 3a). The half-maximum effective concentration EC 50 was 4.922 × 10 -7 M (Fig. 3b). This value is close to the EC 50 values obtained in studies on other animal species, including mammals, and also humans [22,23]. The amplitude of the inward and outward I KACh components in atrial myocytes was also comparable with the data obtained in studies on other animal species [23,24].
Thus, the distribution and characteristics of the main inward rectifier currents in the working myocardium of quail are close to those in most vertebrates, especially mammals.