α1-Adrenergic Receptors Control the Activity of Sinoatrial Node by Modulating Transmembrane Transport of Chloride Anions

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Abstract

Norepinephrine (NE), which is released by sympathetic nerve endings, causes an increase in the frequency of spontaneous action potentials in the pacemaker cardiomyocytes of the sinoatrial node (SAN), also known as the “pacemaker” of the heart. This results in an increase in heart rate (HR). It is known that two types of postsynaptic adrenoreceptors (ARs), α1-AR and β-AR, can mediate the effects of NE. The role of α1-AR in the sympathetic control of heart rate and SAN automaticity, as well as the membrane mechanisms mediation the effects of α1-AR on the pacemaker, have not yet been elucidated. In this study, we utilized immunofluorescence confocal microscopy to examine the distribution of α1A-AR in the SAN of rats. Additionally, we assessed the expression of α1A-AR mRNA in the SAN tissue using RT-PCR. Furthermore, we investigated the impact of α1-AR stimulation on key functional parameters of the pacemaker, including the corrected sinus node recovery time (SNRT/cSNRT) and the SAN accommodation, using the Langendorff perfused heart technique. We also used optical mapping of the electrical activity of perfused, isolated tissue preparations to study the effect of α1-AR stimulation on the spatiotemporal characteristics of SAN excitation. We tested the effects of chloride transmembrane conductance blockade on alteration of functional parameters and pattern of SAN excitation caused by α1-AR. Fluorescent signals corresponding to α1A-AR have been identified in SAN cardiomyocytes, indicating the presence of α1A-AR at protein level. The expression of α1A-AR in SAN has been also confirmed at the mRNA level. The stimulation of α1-AR affects SAN functioning Phenylephrine (PHE) utilized as α1A-AR agonist causes a decrease in SNRT/cSNRT, as well as an acceleration of SAN accommodation. These effects were rate dependent and were observed at a high frequency of pacemaker tissue stimulation. PHE induces changes in the excitation pattern of the SAN. The effects of PHE on functional parameters and SAN excitation pattern are attenuated by Ca2+-dependent chloride channel blocker NPPB but remains unaffected by the protein kinase C inhibitor BIM. Our results suggest that cardiac α1-ARs are important for maintaining function of SAN pacemaker at high heart rates and that α1-AR signalling cascades in the SAN target Ca2+-dependent chloride channels are involved in the α1-adrenergic modulation of the electrophysiological properties of the heart pacemaker.

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About the authors

Y. A. Voronina

Lomonosov Moscow State University; National Medical Research Centre of Cardiology named after academician E.I. Chazov

Author for correspondence.
Email: voronina.yana.2014@post.bio.msu.ru
Russian Federation, Moscow, 119234; Moscow, 121552

A. V. Fedorov

Lomonosov Moscow State University

Email: voronina.yana.2014@post.bio.msu.ru
Russian Federation, Moscow, 119234

M. A. Chelombitko

Belozersky Research Institute of Physico-Chemical Biology, Moscow Lomonosov State University

Email: voronina.yana.2014@post.bio.msu.ru
Russian Federation, Moscow, 119992

U. E. Piunova

Lomonosov Moscow State University

Email: voronina.yana.2014@post.bio.msu.ru
Russian Federation, Moscow, 119234

V. S. Kuzmin

Lomonosov Moscow State University; National Medical Research Centre of Cardiology named after academician E.I. Chazov

Email: voronina.yana.2014@post.bio.msu.ru
Russian Federation, Moscow, 119234; Moscow, 121552

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Supplementary files

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2. Fig 1. EGM - electrogram, S1S1 - interstimulus interval, vVFSU - sinus node function recovery time, S1 - stimulation, aa10 - duration of the 10th cardiac cycle, aaN - duration of the last cardiac cycle.

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3. Fig 2. Expression of α1A-adrenoreceptors (α1A-AR) in the sinoatrial node of the rat heart. a - Representative confocal image showing fluorescent signals (red pseudocolor, Cy3) of antibodies specifically binding α1A-AR in the region of branches of the SAU artery. Endo, epi, endocardial and epicardial surface of the right atrial wall; EE, endocardial endothelium; SA, branches of the SAU artery; SMC, smooth muscle lining of the arterial wall. White arrows indicate cardiomyocytes in the central part at the periphery of the SAU expressing α1A-AR. Blue pseudocolor - cell nuclei (DAPI). b - Same as in a, but at higher magnification. c - Expression of α1A-AR mRNA transcripts in sinoatrial node (SAU) and left atrium (LA) tissue, unpaired t-test, p < 0.05.

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4. Fig. 3. Effect of α1-adrenoreceptor stimulation on cardiac automaticity. a - Increase in HR of isolated rat heart during 10 μM phenylephrine (PE), paired t-test, p < 0.05. b - Effect of PE on sinus node function recovery time. c - Effect of PE on corrected sinus node function recovery time (kVVFSU), two-factor ANOVA, Holm-Sidak'a multiple comparison correction, p < 0.05. d1-g3 - Representative examples of recordings demonstrating recovery of spontaneous SAU activity (after cessation of stimulation) in control and during PE action. In all panels, top is the original recording of the atrial electrogram; bottom is the frequency of spontaneous SAU excitations. e - Effect of PE on the rate of SAU accommodation. Regression equations are shown next to the curves.

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5. Fig. 4. Effect of chlorine channel blockade on α1-adrenergic effects in SAU. a - Representative examples of recordings showing the recovery of spontaneous SAU activity (after cessation of stimulation) under the action of phenylephrine (PE) (upper panel). A stepwise pattern (middle panel) of resumption of isolated heart rhythm (SAU automaticity) is observed during PE action on NPPB background. b - Effect of NPPB on PE-induced decrease, kVVFSU, two-factor ANOVA with Tukey's multiple comparison correction, p < 0.05. c - Effect of NPPB on PE-induced increase in SAU accommodation; top panel - S1S1=100 ms, bottom panel - S1S1=90 ms. One-factor ANOVA with Tukey's multiple comparison correction, p < 0.05.

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6. Fig. 5. Pattern of activation of rat SAU under α1-adrenoreceptor (α1-AR) stimulation in control conditions and under the action of the chloride conductance blocker NPPB. a - Representative examples of SAU activation in control (K) and under the action of phenylephrine (PE). The localization and area of the primary activation point (*, tPA) as well as the direction of tPA displacement (arrow) during PE action are shown. b - Primary activation points of SAU in control (K, shown in green) and during PE action (shown in blue). The displacement of tPA in the direction of the superior vena cava orifice for each experiment is shown by the arrow. c - Representative example of the pattern of activation of SAU under NPPB action, as well as PE action on the background of NPPB. NPPB causes the formation of a non-excitable zone in the SAU (shown by the shading) and the displacement of the tPA outside the non-excitable zone. NPPB does not increase the TPA area under PE action. d - Frequency of spontaneous APs (top), TPA area (center), and the value of TPA displacement (bottom) in tissue preparations of SAU under PE and NPPB action, * - statistically significant (p < 0.05) difference from control, # - statistically significant (p < 0.05) difference from PE, one-factor ANOVA with Tukey's multiple comparison correction. EPV - superior vena cava, PP - right atrial auricle and borderline scallop (delineated by black dotted line), IVC - inferior vena cava, OVF - oval fossa, EC - Eustachian valve, SA - rudimentary scallop of sinoatrial valve (shown by white dotted line); the mouths of the gender veins are delineated by black dotted line.

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