Synergism of myocardial β-adrenoceptor blockade and I1-imidazoline receptor-driven signaling: Kinase-phosphatase switching

Biochemical and Biophysical Research Communications 511 (2019) 363e368

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Synergism of myocardial b-adrenoceptor blockade and I1-imidazoline receptor-driven signaling: Kinase-phosphatase switching A.V. Maltsev a, b, *, E.V. Evdokimovskii a, Y.M. Kokoz a a b

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Moscow Region, Pushchino, Institutskaya, 3, 142290, Russia Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Butlerova 5А, 117485, Russia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2019 Accepted 10 February 2019 Available online 19 February 2019

Recently identified imidazoline receptors of the first type (I1Rs) on the cardiomyocyte's sarcolemma open a new field in calcium signaling research. In particular, it is interesting to investigate their functional interaction with other well-known systems, such as b-adrenergic receptors. Here we investigated the effects of I1Rs activation on L-type voltage-gated Ca2þ-currents under catecholaminergic stress induced by the application of b-agonist, isoproterenol. Pharmacological agonist of I1Rs (I1-agonist), rilmenidine, and the putative endogenous I1-ligand, agmatine, have been shown to effectively reduce Ca2þ-currents potentiated by isoproterenol. Inhibitory analysis shows that the ability to suppress voltage-gated Ca2þcurrents by rilmenidine and agmatine is fully preserved in the presence of the protein kinase A blocker (PKA), which indicates a PKA-independent mechanism of their action. The blockade of NO synthase isoforms with 7NI does not affect the intrinsic effects of agmatine and rilmenidine, which suggests NOindependent signaling pathways triggered by I1Rs. A nonspecific serine/threonine protein phosphatase (STPP) inhibitor, calyculin A, abrogates effects of rilmenidine or agmatine on the isoproterenol-induced Ca2þ-currents. Direct measurements of phosphatase activity in the myocardial tissues showed that activation of the I1Rs leads to stimulation of STPP, which could be responsible for the I1-agonist influences. Obtained data clarify peripheral effects that occur during activation of the I1Rs under endogenous catecholaminergic stress, and can be used in clinical practice for more precise control of heart contractility in some cardiovascular pathologies. © 2019 Elsevier Inc. All rights reserved.

Keywords: Agmatine Rilmenidine Voltage-gated calcium currents Phosphatases Cardiomyocyte

1. Introduction Arterial hypertension is socially significant disease, which affect from 15 to 14 of the Earth's population [1]. Hypertensive disease is developed as result of the disturbance between cortical and subcortical regulation of the vasomotor system and hormonal mechanisms for blood pressure control [2]. One of the most frequently prescribed drugs for treatment of hypertension are badrenoblockers [3]. Three subtypes of b-ARs are expressed in the heart, but the b1-subtype is the crucial for functioning [4]. Their

Abbreviations: I1Rs, imidazoline receptors of the first type; b-AR, b-adrenergic receptors; VGCC, voltage-gated calcium channels; PKA, protein kinase A; PKC, protein kinase C; NO, nitric oxide; STPP, serine/threonine protein phosphatases; PP1 and PP2A, protein phosphatases 1 and 2A, respectively. * Corresponding author. Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Moscow region, Pushchino, Institutskaya, 3, 142290, Russia. E-mail address: [email protected] (A.V. Maltsev). https://doi.org/10.1016/j.bbrc.2019.02.054 0006-291X/© 2019 Elsevier Inc. All rights reserved.

activation through the Ga protein subunit leads to stimulation of adenylate cyclase, an increase in the intracellular level of cyclic adenosine monophosphate (cAMP), which ultimately activates cAMP-dependent protein kinase A (PKA) [5]. Phosphorylation of the a1 subunit of the voltage-gated Ca2þ channel (VGCC) complexes of the L-type by activated PKA raises the probability and time of the channel open state, increasing the density of inward Ca2þ-currents [6]. Redundant stimulation of VGCC activity underlies adrenergic stress and the associated pathophysiological events leading to the development of arterial hypertension, maladaptive hypertrophy of the myocardium, and ultimately heart failure [7]. It is shown that badrenoblockers can be successfully applied in problems of preventing and improving the prognosis of mortality in ischemic heart disease [7]. They also increase the average life expectancy in heart failure, asymptomatic left ventricular dysfunction, and in patients who underwent myocardial infarction [8]. Because b-ARs are widely distributed in peripheral tissues, monotherapy using bblockers can cause a number of undesirable effects, for example, bronchospasm [9].

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Another receptor system that controls the development of hypertension is imidazoline transduction. Imidazoline-binding sites (receptors, IRs) were initially discovered in the rostral ventrolateral reticular nucleus of the medulla oblongata as targets for the action of clonidine, which are differed in structure from adrenoreceptors [10]. Their activation in the center of blood pressure control led to moderate bradycardia and hypotension in anesthetized animals, on the basis of which it was postulated that IRs are involved in cardiovascular function control [11]. Using immunostaining, it was established the heterogeneity of the IRs, and to be assumed that I1Rs are key targets for the regulation of blood pressure and heart rate [11]. At present, I1-agonists (moxonidine and rilmenidine) are widely used in clinical practice as second-generation antihypertensive drugs. Recently, on the plasma membrane of isolated cardiomyocytes, we have discovered the Nischarin protein, which is considered in the modern literature as the main functional component of I1Rs [12]. The detected I1-receptors are functionally active with respect to the regulation of intracellular calcium [13]. Thus, the study of I1Rs activation against the background of the hyperactivation of bAR transduction seems to be a very urgent task. 2. Materials and methods 2.1. Cell isolation The studies were carried out in accordance with the requirements of the European Convention for the Protection of Animals, 86/609/EEC, and compliance with bioethical standards. Left ventricular cardiomyocytes were obtained by enzymatic dissociation from the hearts of Wistar rats as described previously [14]. The weight of the experimental animals was 230e250 g. The basic solution contained (in mM, pH ¼ 7.25): NaCl e 80, KCl e 10, KH2PO4 e 1.2, MgSO4 e 5, glucose e 20, taurine e 50, HEPES e 10, L-arginine e 1. The hearts were subjected to retrograde perfusion according to Langendorff by DMEM þ 10 mM HEPES (pH ¼ 7.25) for washout the tissues from the blood. After stabilization of contractions hearts are perfused by a base solution with the addition of 2.5 mM EGTA to complete cardiac arrest. Then, perfusion with a solution containing 0.2 mg/ml protease, type XIV (Sigma), 1 mg/ml bovine serum albumin, BSA (V fraction, PanEco, Russia) and 140 mM CaCl2. Tissue separation was performed at 37  C, in solution with protease and collagenase IV (0.25 mg/ml, Worthington), at a stirring of 1e2 rpm. Cardiomyocytes were precipitated by centrifugation, 1 min (600e800 rpm), washed twice and resuspended in the basic solution. The cells were kept at room temperature (22e24  C) in a basic solution containing 200 mM CaCl2. 2.2. Electrophysiology Registration of voltage-gated Ca2þ-currents from cells was carried out 2e3 h after isolation, at room temperature, by the «perforated patch clamp » method in the «whole-cell » configuration. Registered electrodes (3e5 MU) were pulled out of soft molybdenum glass. The solution for microelectrodes contained (in mM, pH ¼ 7.25): CsCl e 130, MgSO4 e 5, HEPES e 10, with the addition of amphotericin B for perforating the plasma membrane (200e250 mg/ml). The extracellular solution for the chamber contained (in mM, pH ¼ 7.25): NaCl e 80; tetraethylammonium chloride, TEA-Cl e 20; CsCl e 10; KH2PO4 e 1.2; MgCl2 e 5; CaCl2 e 2; glucose e 20; HEPES e 10; L-arginine e 1. The currents were recorded using Axopatch 2B (Molecular Devices, USA). Ca2þ-currents of L-type were induced by a rectangular stimulating impulse. The supported potential on the membrane was 40 mV, stimulus þ40 mV, duration 300 ms. For the experiments the

original « BioQuest » software package was used. The data were visualized and stored using a digital-to-analog/analog-to-digital L153 (L-card, Russia). The half-inactivation times of VGCC were estimated as the average time over which the amplitude of the integral Ca2þ-current (at 0 mV) was reduced to half of its maximum peak value. 2.3. Measurements of phosphatase activity in the myocardium Serine/threonine phosphatase activity was estimated using a non-radioactive Promega kit (Promega, USA) based on the registration of a colored molybdate-phosphate complexes in accordance with the manufacturer's recommendations. After 15-min retrograde perfusion of the hearts by DMEM þ 10 mM HEPES (pH 7.2) in the absence (control) or presence of the tested drugs, the left ventricles were excised and homogenized from the calculation of 1 g of tissue per 3 ml of buffer containing in mM: sucrose e 250, bmercaptoethanol e 15, EDTA e 0.1, phenylmethylsulfonyl fluoride e 0.1, Tris-HCl e 50 (pH 7.4), after that the tissue lysate was centrifuged at 1000g for 10 min. Free phosphates were removed from the supernatant by Sephadex G-25 filtration. The phosphatase reaction values were evaluated in 50 ml samples at 37  C in a buffer containing 50 mM imidazole, 0.2 mM EGTA, 0.02% b-mercaptoethanol and 0.1 mg/ml bovine serum albumin (pH 7.2) based on the dephosphorylation of a synthetic 754 Da-phosphopeptide (RRA[pT] VA), a substrate for phosphatase-1 (PP1) and 2A (PP2A). The specificity of the phosphatase reaction was tested by the addition of calyculin A (a common inhibitor of PP1 and PP2A). The reaction was stopped by the application of molybdenum-containing buffer (50 ml), then the samples were incubated at room temperature for 15 min to color development. Absorption spectra were measured at a wavelength of 600 nm using Infinite 200 Pro plate reader (Tecan, Austria). 2.4. Statistical analysis Dispersive ANOVA analysis followed by a post-hoc t-test by the Bonferroni method was used to identify differences between the values in the groups. A value of p less than 0.05 was taken to reflect statistically significant differences and is shown in figures as * (comparison with control) or as # (comparison of two independent groups among themselves). All data (except for the original recordings of the current traces) are represented as the mean ± standard deviation of the specified number (n) of experiments performed, from at least three different animals. 3. Results 3.1. Action of I1-agonists against the background of b-adrenergic signaling stimulation The b-ARs agonist isoproterenol (1 mM) almost triply increases the voltage-gated Ca2þ-currents (up to 286.0 ± 15.0%, Fig. 1). A sharp increase in the amplitude and density of the inward Ca2þcurrents was also accompanied by significant decrease in the time of VGCC half-activation from 14.5 ± 1.9 ms in the control to 8.7 ± 1.6 ms in the presence of isoproterenol (Fig. 1e). Activation of I1Rs by millimolar concentrations of agmatine (2 mM) partially reverses the action of isoproterenol. The amplitude of Ca2þ-currents is decreased to 168.7 ± 7.6%, although it does not return to basal values (Fig. 1b and c). It is interesting that agmatine (2 mM), by itself, reducing the Ca2þ-currents by 23.8 ± 1.9%, increased its efficiency almost 1.5-fold against the background of isoproterenol. Thus, the amplitude of VGCC under the action of the same agmatine concentration in cardiomyocytes pretreated with b-agonist is

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Fig. 1. Action of I1-agonists against the background of b-adrenergic stimulation. (а), (d) e typical recordings of Ca2þ-currents under the action of isoproterenol (black circle), with further application of agmatine (gray circle) or rilmenidine (dark gray circle) are shown. (b), (e) e change in Ca2þ-current peak amplitude (at 0 mV) in time. Here and below, the duration of the drug application is indicated by a line. (c) e summary histograms showing the effect of I1-agonists on the basal level of Ca2þ currents (control, taken as 100%) alone and against the background of the b-agonist. Hereinafter, the number in parentheses indicates the number of independent experiments. * - p  0.05 of the specified group versus control, # - p  0.05 when comparing groups with each other. (f) e summarized histograms showing the average times of VGCC half-inactivation under the drug actions, n.s. e not significantly, p  0.05.

decreased by 35.4 ± 3.7% (Fig. 1c). Agmatine did not significantly affect the half-inactivation of VGCC (12.6 ± 2.2 ms, p  0.05), but was able to returns the time to reach of half-peak amplitude against the background of isoproterenol to control values (12.3 ± 1.8 vs. 14.5 ± 1.9 ms, p  0.05, Fig. 1e). The action of 10 mM rilmenidine, a much more specific I1Rs agonist than agmatine, as a whole exhibits the same tendencies (Fig. 1d and e). Thus, activation of I1Rs is useful in prolonged excessive stimulation of the b-ARs. 3.2. Involvement of protein kinase A (PKA) in the implementation of I1-agonist effects To verify the involvement of possible negative modulation of cAMP-PKA signaling, we used a specific PKA blocker, H89. The addition of H89 (5 mM) to the chamber per se resulted in significant inhibition of Ca2þ-currents by more than a third (up to 62.6 ± 5.2% from control), thus showing that the basal level of PKA activity in cardiocyte is very important for its normal functionality (Fig. 2). It should be noted that H 89 significantly increased the halfinactivation time of VGCC up to 22.5 ± 2.2 ms (p  0.05 vs. control, Fig. 2e). The delayed inactivation kinetics induced by the PKA blocker persisted even with the subsequent application of I1-agonists: agmatine (20.7 ± 2.4 ms, p  0.05 vs. H 89), and rilmenidine (22.8 ± 2.1 ms, p  0.05 vs. H 89). Against the background of H89, both agmatine and rilmenidine were able to suppress the conductivity of VGCC in an amount comparable to their effects by itself (Fig. 2c). Obtained results indicate that activation of I1Rs reduces VGCC probably not through PKA inhibition, but the some alternative signal transduction. This mechanism is cumulative with blockade of PKA-dependent phosphorylation. It is known that fluctuations in the intracellular Ca2þ are closely related to changes in the production of nitric oxide (NO) [15]. NO is able to activate cytoplasmic guanylate cyclase, and can also directly nitrosylates target proteins modulating their activity [16]. Nitrosylation of VGCC

differently modulates the channel permeability for Ca2þ. This depends on the position of the nitrosylated cysteine residue in the protein structure, the specific subtype of channels, the free Ca2þ level in cytosol, etc [17]. Thus, it is necessary to check the participation of NO signaling in the effect of I1Rs interrelations for the bAR. 3.3. Influence of I1-agonists on the Ca2þ currents does not associated with NO synthesis modulation In Suppl. Fig. 1 is presented the results of the agmatine or rilmenidine action on the background of the non-specific blocker of NO-synthase isoforms e 7NI (2 mM). At the concentration used, 7NI had practically no effect on the basal level of Ca2þ-currents (S.Fig. 1c), nor on the channel half-activation value (S.Fig. 1e). Addition of 2 mM agmatine against the background of 7NI in the experimental chamber resulted in a decrease in the amplitude of Ca2þ-currents comparable to that of agmatine alone (S.Fig. 1b). There were also no significant differences in the values of the channel half-inactivation. Similar results were obtained for rilmenidine (S.Fig. 1d and e). Obtained results indicate that NO signaling is not involved in the mechanisms of I1-agonists reduction of isoproterenol-induced VGCC. 3.4. Role of serine/threonine protein phosphatases in realization of I1-agonist effects Activation of I1Rs drives the stimulation of Ca2þ-dependent protein kinase C (PKC) [18]. PKC is one of the crucial Ca2þ-sensors, since its activity can be modulated at physiological Ca2þ-concentrations in the cytosol [19]. It is known that PKC can induce the activation of serine/threonine protein phosphatases (STPP) [20]. The dephosphorylation of VGCC by STPP is an equally important mechanism for regulating sarcolemmal Ca2þ influx than protein

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Fig. 2. Action of I1-agonists on the background of the selective PKA blockade. (a), (d) e typical recordings of Ca2þ-currents under the application of H 89 (black square) and further decrease of Ca2þ currents in the presence of agmatine (gray circle) or rilmenidine (dark gray circle) are shown. (b), (e) e original record of the changes in Ca2þ-current peak amplitude in time. (c) e summary histograms showing the effect of I1-agonists on the basal level of Ca2þ-currents alone and against the background of the PKA blocker. (f) e summarized histograms showing the average times of VGCC half-inactivation under the drug action. * - p  0.05 of the specified group versus control, # - p  0.05 when comparing groups with each other.

kinase influences [21]. Application of both PP1 and PP2A inhibitor, calyculin A (0.5 mM), to cells led to an almost twofold increase in the amplitude of Ca2þ-currents (up to 195.6 ± 7.8%), which was probably due to the shift of the kinase-phosphatase balance towards phosphorylation (Fig. 3). The value of the channel half-inactivation

in the presence of calyculin A was significantly increased to 20.8 ± 2.6 ms (vs. control, p  0.05, Fig. 3e). Agmatine (2 mM) against the background of calyculin A decreases the amplitude of the Ca2þ-currents to 188.8 ± 3.6% (Fig. 3c), which does not differ significantly from the phosphatase inhibitor action alone, and

Fig. 3. Effect of I1-agonists against inhibition of STPP activity. (a), (d) e typical recordings of Ca2þ-currents under application of calyculin A (black triangle) and further influences of agmatine (gray circle) or rilmenidine (dark gray circle). (b) e record of the changes in Ca2þ-current peak amplitude in time. (c) e summary histograms showing the effect of I1agonists on the basal level of Ca2þ-currents (control, taken as 100%) alone and against the background of a phosphatase inhibitor. (e) e histograms showing the average times of VGCC half-inactivation under the drug action. (f) e direct measurements of STPP activity in myocardium. Evaluation of samples was carried out in normalization for control samples (dotted line). * - p  0.05 of the specified group versus control, # - p  0.05 when comparing groups with each other, n.s. e not significantly, p  0.05.

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indicates the involvement of STPP in the I1-mediated signaling. In addition, agmatine prevented the calyculin A-induced slowing of the transition of VGCC to the inactivated state, returning it to control values (18.1 ± 2.6 ms, p  0.05, Fig. 3e). Similar results were obtained using rilmenidine (Fig. 3c, e). Further, direct measurements of STPP activity in myocardial tissues were performed. Rilmenidine in a dose-dependent manner increases the magnitude of the phosphatase reaction in the myocardium (Fig. 3f). At concentrations of 10 and 20 mM, it increases the total activity of STPP by 9.9 ± 1.2% and 14.3 ± 2.9%, respectively. To further validation of the phosphatase activity determination protocol, a direct NO donor e SNAP (S-nitroso-Nacetyl-penicillamine), was used. NO is known to increase the activity of PP2A, PP2B and some others [22]. SNAP (100 mM) increased the phosphatase response by 21.5 ± 7.8% (Fig. 3f), and its simultaneous application with 20 mM rilmenidine did not significantly affect this effect (19.2 ± 8.0%, p  0.05). 4. Discussion Long-term increases in plasma catecholamine concentrations that occur during psychoemotional overloads can change the kinetic characteristics of Ca2þ-currents. It was shown that the VGCC current-voltage curves in cardiomyocytes of spontaneously hypertensive rats (SHR) by about 10 mV are shifted toward hyperpolarization potentials in comparison with normotensive rats [23]. Modified VGCC activity has also been shown for atrial myocytes and smooth muscle cells of SHR arteries. In the first case it is associated with abnormal Ca2þ-entry, which can provoke arrhythmogenic phenomena, and in the second case it is related to changes in vascular tone, correlating with severity of hypertension [24,25]. In addition, in the SHR hearts an increased level of VGCC expression has been observed [23], and recent studies have shown changes in the regulation of the PKA-dependent phosphorylation status of VGCC [26]. Blockade of PKA-dependent phosphorylation or bblocker application has improved some parameters in models of arterial hypertension [27]. One of the first strategies to compensate the hypertension was the Ca2þ antagonists, dihydropyridine blockers of VGCC, which are now widely used in clinical practice. The total blockade of the VGCC conductivity is often no less critical than their excessive activation. Therefore, a more preferable (physiologically justified) strategy is the regulation of the VGCC conduction states through phosphorylation/dephosphorylation of pore-forming or regulatory subunits. In our experiments, the activation of I1Rs agmatine or rilmenidine effectively reduced the amplitude of isoproterenol-induced Ca2þcurrents, preventing the development of b-AR-mediated positive inotropic effect. It is important to note that I1-agonists returned to the control values the times of VGCC half-inactivation, which also indicates compensation of the positive chronotropic effect caused by the isoproterenol administration. Studies of possible mechanisms for realizing the effects of I1agonists have shown that their action does not involve NOdependent signaling, nor it is related to the modulation of cAMP synthesis and the cAMP-dependent level of VGCC phosphorylation. Based on the data of inhibitory analysis, as well as on the direct measurements of phosphatase activity in the myocardium, the activation of STPP seems most likely. STPP are a superfamily of ubiquitous enzymes, which is involved in maintenance of the kinase-phosphatase switching in the cell, determining the performance of its basic homeostatic functions [28]. Stimulation of I1Rs by I1-agonists leads to an increase in the basal activity of the Ca2þdependent PKC [18]. It is well known that some PKC isoforms directly or non-directly increase activity of key protein phosphatases, for example, PP1 and PP2A [29,30]. The endogenous

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modulator of PP1 is the PKC-regulated 17-kDa protein inhibitor whose phosphorylation leads to the enhancement of PP1phosphatase activity [20]. PP1 in cardiac cells is actively involved in the regulation of both systolic and diastolic functions [21]. PP2A is another STPP, which is actively involved in the cardiovascular system [28]. PP2A dephosphorylates endothelial NOsynthase at Serine-1179, reducing its enzymatic activity [12], removes the phosphorylated status from the a1 subunit of VGCC [6]. PP2A dephosphorylates phospholambane and phospholemman, which leads to inhibition of sarcoendoplasmic reticulum Ca2þATPase activity, and increase of the Ca2þ leakage to the cytosol [28]. Inhibition of PP2A-mediated signaling increases the strength of electrochemical coupling in the cardiac muscle and its contractility [29]. These data are in good agreement with the results obtained in this study, since calyculin A almost twice potentiated the amplitude of the L-type Ca2þ currents, and significantly slowed the transition of VGCC to inactivated state. Thus, I1Rs-mediated stimulation of STPP in cardiomyocytes can be considered as new additional strategy of counteracting to catecholaminergic stress produced by hyperactivation of myocardial b-adrenoceptors (Fig. 4). So, because of the non-identity of the mechanisms by which the action on the myocardium of b-adrenoblockers and I1-agonists is realized, they can be successfully combined with each other for more effective control over the heart muscle contractility. The synergism of the b-adrenergic system blockade and the activation of I1-imidazoline receptors in the present study can be used for cardiovascular function control in the complex therapy of hypertension. The well-known inhibition of cAMP-PKA-dependent signaling in cardiomyocytes, suppressing the tonic voltage-gated Ca2þ currents through a decrease in the level of L-type Ca2þ channel phosphorylation, may not be sufficient to prevent an heightened Ca2þ overload of cells. I1-agonists through the activation of I1Rs increase the serine/threonine protein phosphatase (PP1

Fig. 4. Suggested scheme of signaling events in the cardiomyocyte, showing the relationships between I1Rs and b-ARs. Designations: I1R e imidazoline receptor of the first type; PC-PLC e phosphatidylcholine-specific phospholipase C; DAG e diacylglycerol; PKC e protein kinase C; PP1 и PP2A e protein phosphatases 1 и 2А, respectively; I1-PP1 e 17 kDa PP1-inhibitory protein; eNOS e endothelial NOsynthase; sGC e soluble guanylate cyclase; GTP e guanosine triphosphate; cGMP e cyclic guanosine monophosphate; PKG e сGMP-dependent protein kinase G; b-AR e badrenoceptor; Iso e isoproterenol; Gs e stimulating subunit of G-protein; AC e adenylate cyclase; cAMP e cyclic adenosine monophoshate; PKA e cAMP-dependent protein kinase А; P e phosphate residue translocated during phosphorylationdephosphorylation. Blunt arrows designate inhibitory effects, and sharp arrows indicate stimulating influences on the specified target. “Plus” and “minus” signs denote activating and inhibiting outcomes on L-type Ca2þ channel activity, respectively, induced by particular kinases.

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and PP2A) activity. This causes the removal of excess PKAdependent phosphorylation from Ca2þ-channels of L-type, as well as a number of other (including contractile) proteins. Eventually, the signal caused by hyperstimulation of b-adrenergic receptors is compensated, and the amplitude values of voltage-gated Ca2þ currents and their kinetic characteristics return to the control values corresponding to normal cell physiology. Funding This work was supported by a grant from the RFBR: N18-01500165А (Y.M.K.). Acknowledgments Authors are grateful to Oleg Pimenov (Institute of Theoretical and Experimental Biophysics, Pushchino) for technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.054. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.054. References [1] World Health Statistics 2016, Monitoring Health for the SDGs, World Health Organization, Geneva, 2016. http://www.who.int/gho/publications/world_ health_statistics/2016/en/. [2] D. Haspula, M.A. Clark, Neuroinflammation and sympathetic overactivity: mechanisms and implications in hypertension, Auton. Neurosci. 210 (2018) 10e17. [3] C. McCune, P. McKavanagh, I.B. Menown, A review of the key clinical trials of 2015: results and implications, Cardiol. Ther. 5 (2016) 109e132. [4] K.M. Small, L.E. Wagoner, A.M. Levin, et al., Synergistic polymorphisms of b1and a2C-adrenergic receptors and the risk of congestive heart failure, N. Engl. J. Med. 347 (2002) 1135e1142. [5] S. Bryant, T.E. Kimura, C.H. Kong, et al., Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes, J. Mol. Cell. Cardiol. 68 (2014) 47e55. [6] K.D. Keef, J.R. Hume, J. Zhong, Regulation of cardiac and smooth muscle Ca2þ channels (Cav1.2a,b) by protein kinases, Am. J. Physiol. Cell Physiol. 281 (2001) 1743e1756. [7] D.M. Bers, Calcium cycling and signaling in cardiac myocytes, Annu. Rev. Physiol. 70 (2008) 23e49. [8] J. Hong, A.R. Barry, Long-term beta-blocker therapy after myocardial infarction in the reperfusion era: a systematic review, Pharmacotherapy 38 (2018) 546e554. [9] J.G. Baker, R.G. Wilcox, b-blockers, heart disease and COPD: current

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