Effects of new antiarrhythmic agent SS 68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins assessment of safety and side effects risk Accepted Manuscript Effects of[.]
Accepted Manuscript Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: assessment of safety and side effects risk Saida K Bogus, Vladislav S Kuzmin, Denis V Abramochkin, Konstantin F Suzdalev, Pavel A Galenko-Yaroshevsky PII: S1347-8613(17)30028-2 DOI: 10.1016/j.jphs.2017.01.008 Reference: JPHS 329 To appear in: Journal of Pharmacological Science Received Date: July 2016 Revised Date: 15 January 2017 Accepted Date: 30 January 2017 Please cite this article as: Bogus SK, Kuzmin VS, Abramochkin DV, Suzdalev KF, Galenko-Yaroshevsky PA, Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: assessment of safety and side effects risk, Journal of Pharmacological Science (2017), doi: 10.1016/j.jphs.2017.01.008 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: assessment of safety and side effects risk Saida K Bogusa, Vladislav S Kuzminb,c, Denis V Abramochkinb,c, Konstantin F Suzdalevd, Pavel A Galenko-Yaroshevskya, b c Kuban’ State Medical University, Sedina, 4, Krasnodar, Russia RI PT a Lomonosov Moscow State University, Leninskie gory, 1, Moscow, Russia, Pirogov Russian National Research Medical University, Ostrovityanova, 1, Moscow, Russia, Chemical Department of Southern Federal University, Zorge, 7, Rostov-on-Don, Russia SC d M AN U Running title: Effects of new antiarrhythmic drug SS-68 Corresponding author: Denis Abramochkin Address: 119991, Russia, Moscow, Leninskije gory, 1, 12, Biological faculty of Lomonosov Moscow State University, Department of human AC C EP TE D and animal physiology; e-mail: abram340@mail.ru; phone: +7-916-603-05-02 ACCEPTED MANUSCRIPT ABSTRACT The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug The first estimation of RI PT atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS68 in Purkinje fibers and myocardium of pulmonary veins have been investigated The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 SC (≥10 µmol/L) significantly decrease this parameter Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a M AN U concentration-dependent manner SS-68 (0.1-100 µmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE) Moreover, 10 µmol/L SS-68 suppressed NEinduced extra-beats and rapid firing in Purkinje fibers In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower TE D concentrations (0.1-1 µmol/L) were ineffective Also, 0.1-100 µmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins EP In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal AC C automaticity in used experimental models Key words: antiarrhythmic drug, arrhythmia, action potential, pulmonary veins, conduction ACCEPTED MANUSCRIPT Introduction Atrial and ventricular arrhythmias are still very common and dangerous cardiovascular diseases The most widely used approach in therapy of arrhythmias is based on the use of antiarrhythmic drugs Number of antiarrhythmic drugs were introduced to the clinical practice RI PT during the recent decades [1], however, most of them demonstrate numerous adverse side effects and limited effectiveness [2] Thus, the development of new antiarrhythmic drugs remains to be an actual task for experimental pharmacology SC Several natural and synthetic indole derivatives are used for biomedical purposes Indolebased pindolol and bisoprolol are used as antihypertensive agents [3] Also, both compounds M AN U demonstrate moderate antiarrhythmic effects due to their ability to block β-adrenoreceptors [4, 5] Thereby, indole derivatives are likely to be used for development of more potent antiarrhythmic drugs The compound SS-68 has been selected among numerous new derivatives of indole, which were synthesized in the Institute of Physical and Organic Chemistry of South Federal University SS-68 is an indole derivative with molecular formula C21H25ClN2 containing TE D dialkylaminopropyl group In animal models SS-68 has been demonstrated to suppress ventricular arrhythmias caused by aconitine, barium or calcium [6, 7] SS-68 reduces the rate of EP ischemia-induced arrhythmic episodes in canine ventricles during reperfusion [8] In addition, SS-68 produced antiarrhythmic effects in canine model of atrial fibrillation induced by AC C tachypacing in situ [8] Therefore, SS-68 effectively suppressed both atrial and ventricular arrhythmias in animal experiments and was proposed as potential antiarrhythmic drug However, safety of SS-68 as a potential antiarrhythmic drug has never been estimated One of the most dangerous side effects of the antiarrhythmic drugs is the induction of life-threatening arrhythmias such as ventricular fibrillation [2, 9] Proarrhythmic effects of antiarrhythmic drugs are associated with prolongation of action potential in ventricular myocardium and conduction system However, effects of SS-68 in elements of ventricular conduction system have not been investigated yet In the present study the rabbit Purkinje fibers ACCEPTED MANUSCRIPT preparations were used for that purpose since they are widely considered as very sensitive indicator of drug-induced proarrhythmic activity [10-12] Slowing of atrioventricular conduction and reduction of conduction velocity in the ventricles caused by antiarrhythmic drugs may also induce arrhythmic events [13] Alteration of RI PT conduction velocity in patients with history of myocardial infarction was a reason for withdrawal of several class I antiarrhythmic drugs from clinical usage [14] Suppression of conduction induced by class I antiarrhythmic drugs is associated with action potential upstroke velocity SC reduction [15] The present study provides the first estimation of atrioventricular conduction and action potential upstroke velocity in mammalian heart under SS-68 M AN U It is widely accepted that arrhythmogenic ectopic foci, responsible for initiation of atrial fibrillation, are frequently located in myocardium of pulmonary veins [16-18], which significantly differs from atrial myocardium in electrophysiological properties [19] Pulmonary vein myocardium is prone to triggered activity and abnormal pacemaking which may be enhanced by antiarrhythmic drugs [20, 21] Thus, effects of any potential antiarrhythmic TE D compound should certainly be tested in pulmonary vein myocardium The present study provides the first description of SS-68 effects on action potential waveform in pulmonary vein EP myocardium Rat pulmonary veins were selected as a model object due to extreme length of AC C myocardial sleeves, which simplifies obtaining the viable preparations [22] Materials and methods 2.1 Animals All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996) The experimental protocol was approved by Bioethics Committee of Moscow State University Male Wistar rats weighing 250-300g (n=33, weeks old) were purchased from “Scientific complex of biomedical technologies” animal plant (Moscow region, ACCEPTED MANUSCRIPT Russia) Totally, 33 tissue samples from a rat hearts were prepared and utilized Male rabbis of “Soviet chinchilla” breed weighing 3-3.5 kg used in the study (n=22, 16 weeks old) were obtained from the same supplier Totally 22 tissue samples from a rabbit hearts were prepared and utilized Animals were held in the animal house for weeks under a 12 h:12 h light:dark RI PT photoperiod at 20-24°C and 40-70% humidity in standard cages prior to the experiment and fed ad libitum SC 2.2 Isolated heart preparation Rats were anesthetized with intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg M AN U xylazine HCl Heparin (1000 U/kg) was added to the anesthetics solution to prevent blood coagulation in the coronary vessels of the excised heart The chest was opened and the heart was rapidly excised and placed into a bath with cold (+4ºC) Krebs-Henseleit solution that contained (in mmol/L): NaCl –118.0, KCl – 4.7, NaHCO3 – 25.0, MgSO4 – 1.2, CaCl2 – 2.5, KH2PO4 – TE D 1.2, glucose – 5.5, bubbled with carbogen (95% O2, 5% CO2), with pH 7.4 ± 0.1 2.3 Measurements of atrioventricular conduction in isolated Langendorf-perfused rat EP heart Aorta was cannulated and the heart was placed into the constant pressure (80 mmHg) AC C Langendorf perfusion system Normoxic perfusion with Krebs-Henseleit solution was carried out at 37ºC in the hearts paced (5 Hz) by bipolar platinum electrodes placed on the surface of the right atria after the dissection of sinoatrial node region Two additional pairs of bipolar teflon-coated silver electrodes were used to estimate atrioventricular conduction First pair of electrodes was located in the region of interatrial septum, another was placed on the ventricular surface close to the heart apex Electrodes were connected to the differential signal amplifier (NeuroBioLab, DL304N, Russia) Electrical signals ACCEPTED MANUSCRIPT were digitized and recorded continuously using data acquisition system (E-154, L-card, Russia) and analyzed with PowerGraph software (Powergraph 3.3, Disoft, Russia) The time intervals between negative peaks of differential signals from atrial and ventricular bipolar electrodes was accepted as approximate total time of atrioventricular conduction RI PT (Fig.1.a) 2.4 Isolated heart experimental protocol Hearts were allowed to equilibrate for 10 minutes Then SS-68 (0.1-100 µmol/L) was SC administrated during 15 with 30 of washout intervals to reduce amount of animals utilized for experiments Special time-control experiments (n=3) were performed to ensure that M AN U heart activity does not change significantly during perfusion 2.5 Isolation of Purkinje fibers Rabbits were anesthetized with ketamine/xylazine HCl mixture (25/4 mg/kg) administrated via marginal ear vein Chest was opened; heart was excised and rinsed with the TE D bath solution that contained (in mmol/L): NaCl –130.0, KCl – 4.7, NaHCO3 – 18.0, MgCl2•6H2O - 1.05, CaCl2 – 1.8, NaH2PO4, 1.2, glucose – 11, bubbled with carbogen (95% O2, 5% CO2), with EP pH 7.4 ± 0.1 A cavity of left ventricle was incised and opened, complex preparations, which included free AC C running Purkinje fibers and adjoining pieces of endocardial myocardium, were excised and carefully transferred into experimental chamber Since 90% of tested Purkinje fibers preparations demonstrated stable spontaneous firing after hour of adaptation, experiments aimed to determine the SS-68 effects in basal conditions or in norepinephrine (NE) treated Purkinje fibers were performed without electrical pacing Paced preparations of Purkinje fibers were used in additional experiments The tissue excitation was elicited by constant ms pulses (with amplitude twice above the threshold) at pacing rate of ACCEPTED MANUSCRIPT Hz A pair of the bipolar silver teflon-coated electrodes was placed on the surface of the adjoining pieces of endocardial myocardium 2.6 Experiments with isolated Purkinje fibers RI PT Three series of experiments with Purkinje fibers have been carried out in total In first series effects of SS-68 were estimated in spontaneously active Purkinje fibers SS-68 (0.1-100 µmol/L) was administrated for 15 with 30 washout intervals In second series the ability SC of SS-68 to alter NE-induced arrhythmogenic activity was tested In this series the spontaneously active Purkinje fibers were treated with 10 µmol/L NE prior to and during 15 of SS-68 M AN U administration (0.1-100 µmol/L) SS-68 and NE application periods were separated by 30 washout intervals The third series of experiments was aimed to compare the effects of SS-68, E4031 and dl-sotalol in paced Purkinje fibers SS-68 (10 µmol/L), E-4031 (10 µmol/L) and dlsotalol (30 µmol/L) were applied during 15 after hour of equilibrating continuous pacing TE D of preparations obtained from different animals 2.7 Isolation of multicellular pulmonary veins preparations EP Rats were anesthetized as described earlier The chest was opened; the heart with lung lobes was rapidly excised and rinsed with Tyrode solution that contained (in mM): NaCl 118.0, AC C KCl 2.7, NaH2PO4 2.2, MgCl2 1.2, CaCl2 1.2, NaHCO3 25.0, glucose 11.0 (bubbled with carbogen, pH 7.4 ± 0.1) To allow outflow of the solution, the outer edges of the lung lobes were trimmed The left atrium was incised at the atrioventricular border and cannulated Blood from the left atria and pulmonary veins was flushed out by injection of physiological solution Following that fascia and pulmonary arteries were removed and preparation of isolated supraventricular region including left atria, pulmonary veins and lung lobes was pinned in a preparation bowl Finally, tubular pulmonary vein preparations were isolated from one or two lung lobes Isolated pulmonary veins were cut along the axis and pinned in experimental ACCEPTED MANUSCRIPT chamber (3 ml) supplied with Tyrode solution at 10 ml min-1 (37.5°C) with inner side up pulmonary veins were paced throughout the experiment with a pair of silver Teflon-coated electrodes (pacing rate – Hz, pulse duration – ms, pulse amplitude – times threshold) since preparations lacked regular intrinsic spontaneous firing Different concentrations of SS-68 (0.1- 2.8 Microelectrode APs recording RI PT 100 µmol/L) were applied for 15 minutes SC Transmembrane potentials were recorded from pulmonary veins or Purkinje fibers with glass microelectrodes (30–45 MΩ) filled with 3M KCl connected to a high input impedance M AN U amplifier Model 1600 (A-MSystems, Sequim, WA, USA) The signals were digitized and recorded using specific hardware (E-154 analog-to-digital converter, L-card, Moscow, Russia, www.lcard.ru/) and software (PowerGraph 3.3, DISoft Company, Moscow, Russia, www.powergraph.ru/en/) The action potential duration was analyzed with MiniAnalysis 6.0.7 (Synaptosoft, Fort Lee, NJ, USA, www.synaptosoft.com) Stable impalements were maintained TE D during the entire period of drugs application Changes in the action potential upstroke velocity and action potential duration at 50% (APD50) and 90% of repolarization (APD90) were EP determined AC C 2.9 Drugs SS-68 was provided by the Department of Chemistry of Natural and High-molecular Compounds of Chemical faculty of Southern federal university, Rostov-on-Don, Russia Heparine sodium solution (5000 units/mL) was purchased from “Moscow Endocrine Plant” (Russia), xilazine HCl (Rometar) solution (20 mg/mL) was purchased from “Bioveta” (Czech Republic) Norepinephrine and dl-sotalol were purchased from Sigma (St Louis, MO, USA) E4031 was purchased from Tocris Bioscience (Bristol, United Kingdom) ACCEPTED MANUSCRIPT 2.10 Statistical analysis All data in the text and figures except the original recordings are presented as means ±SEM for number of experiments Statistica (StatSoft Inc.) was used for statistical analysis of the data Effects of SS-68 on registered parameters relative to the respective basal value of these RI PT parameters were analyzed using nonparametric one-way ANOVA test (Friedman ANOVA with further Dunn’s-Bonferroni based post hoc test for multiple comparisons in groups with repeated measurements) in all cases except experiments with rat pulmonary vein preparations In the last SC case paired t-test was applied P