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

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: 1 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.

Saida K Bogusa, Vladislav S Kuzminb,c, Denis V Abramochkinb,c, Konstantin F Suzdalevd,

Pavel A Galenko-Yaroshevskya,

a Kuban’ State Medical University, Sedina, 4, Krasnodar, Russia b

Lomonosov Moscow State University, Leninskie gory, 1, Moscow, Russia,

c

Pirogov Russian National Research Medical University, Ostrovityanova, 1, Moscow, Russia, d

Chemical Department of Southern Federal University, Zorge, 7, Rostov-on-Don, Russia

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 and animal physiology; e-mail: abram340@mail.ru; phone: +7-916-603-05-02

68 in Purkinje fibers and myocardium of pulmonary veins have been investigated

The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥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 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 NE-induced extra-beats and rapid firing in Purkinje fibers In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower 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

In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models

Key words: antiarrhythmic drug, arrhythmia, action potential, pulmonary veins, conduction

an actual task for experimental pharmacology

Several natural and synthetic indole derivatives are used for biomedical purposes based pindolol and bisoprolol are used as antihypertensive agents [3] Also, both compounds 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

Indole-of indole, which were synthesized in the Institute Indole-of Physical and Organic Chemistry Indole-of South Federal University SS-68 is an indole derivative with molecular formula C21H25ClN2 containing 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 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

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

Slowing of atrioventricular conduction and reduction of conduction velocity in the ventricles caused by antiarrhythmic drugs may also induce arrhythmic events [13] Alteration of 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 reduction [15] The present study provides the first estimation of atrioventricular conduction and action potential upstroke velocity in mammalian heart under SS-68

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 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 myocardium Rat pulmonary veins were selected as a model object due to extreme length of myocardial sleeves, which simplifies obtaining the viable preparations [22]

2 Materials and methods

“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 4 weeks under a 12 h:12 h light:dark photoperiod at 20-24°C and 40-70% humidity in standard cages prior to the experiment and fed

ad libitum

2.2 Isolated heart preparation

Rats were anesthetized with intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg 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 – 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 heart

Aorta was cannulated and the heart was placed into the constant pressure (80 mmHg) 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

The time intervals between negative peaks of differential signals from atrial and ventricular bipolar electrodes was accepted as approximate total time of atrioventricular conduction (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 administrated during 15 min with 30 min of washout intervals to reduce amount of animals utilized for experiments Special time-control experiments (n=3) were performed to ensure that 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 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

pH 7.4 ± 0.1

A cavity of left ventricle was incised and opened, complex preparations, which included free 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 1 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 2 ms pulses (with amplitude twice above the threshold) at pacing rate of

2.6 Experiments with isolated Purkinje fibers

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 min with 30 min washout intervals In second series the ability

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 5 min prior to and during 15 min of SS-68 administration (0.1-100 µmol/L) SS-68 and NE application periods were separated by 30 min washout intervals The third series of experiments was aimed to compare the effects of SS-68, E-

4031 and sotalol in paced Purkinje fibers SS-68 (10 µmol/L), E-4031 (10 µmol/L) and

dl-sotalol (30 µmol/L) were applied during 15 min after 1 hour of equilibrating continuous pacing

of preparations obtained from different animals

2.7 Isolation of multicellular pulmonary veins preparations

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, 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

100 µmol/L) were applied for 15 minutes

2.8 Microelectrode APs recording

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 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 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

determined

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)

E-4031 was purchased from Tocris Bioscience (Bristol, United Kingdom)

All data in the text and figures except the original recordings are presented as means

±SEM for number of experiments Statistica 6 (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 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

case paired t-test was applied P<0.05 was adopted as the level of statistical significance

3 Results

3.1 Effect of SS-68 on atrioventricular conduction

In our experiments the duration of atrioventricular conduction in paced perfused isolated rat heart was 58±8 ms in control conditions While 0.1 and 1 µmol/L SS-68 failed to alter atrioventricular conduction, 10 µmol/L SS-68 significantly increased this parameter by 56% of control (Fig.1a,b) No atrioventricular blocks were observed during the application of 0.1-10 µmol/L SS-68 However, 100 µmol/L SS-68 rapidly increased the duration

Langendorff-of atrioventricular conduction and induced complete atrioventricular block

3.2 Effect of SS-68 on action potential waveform and automaticity in Purkinje fibers

In our experiments SS-68 induced concentration-dependent alteration of action potential duration, automaticity and action potential upstroke velocity in spontaneously beating Purkinje fibers In control conditions APD90 was 215±15 ms, the rate of spontaneous action potentials was 87±15 per minute, action potential upstroke velocity was 480±31 V/s While 0.1 µmol/L SS-68 was ineffective to alter APD90, 1 µmol/L induced significant increase of APD90 by 30.2% of control In contrast, 10 µmol/L SS-68 shortened action potentials in Purkinje fibers by 14.5% (Fig.2a, d)

in Purkinje fibers preparations (Fig.2e)

The maximal rate of action potential upstroke in Purkinje fibers was significantly decreased by 1-10 µmol/L SS-68 by 20% and by 62.5% , respectively (Fig.2c)

3.3 Effect of SS-68 on bioelectric activity of Purkinje fibers treated with norepinephrine

In our experiments 10 µmol/L NE caused single extra-beats, series of extra-beats of various lengths and episodes of rapid firing in multicellular Purkinje fibers in all cases (5 out of

5, Fig.3a) While 0.1-1 µmol/L SS-68 was unable to alter NE-induced arrhythmic activity significantly (Fig.3b), 10 µmol/L SS-68 completely suppressed both single extra-beats and episodes of rapid firing provoked by NE in all preparations of Purkinje fibers (5 out of 5, Fig.3c)

In addition, none of applied SS-68 concentrations induced intensification of NE-induced arrhythmic activity (i.e substantial action potential prolongation, multiple early afterdepolarizations)

3.4 Effects of SS-68, E-4031 and dl-sotalol in paced Purkinje fibers

In our experiments potent inhibitor of potassium rapid delayed rectifier current (IKr)

E-4031 induced significant action potential prolongation, oscillations of plateau potential and early

afterdepolarizations (Fig.4a) in paced Purkinje fibers Similarly, dl-sotalol, known as class III

antarrhytmic drug, caused dramatic action potential prolongation (> 10 times) accompanied by early and delayed afterdepolarization (Fig.4b) In contrast, SS-68 was unable to induce proarrhythmic transformations of action potentials Purkinje fibers demonstrated normal

of SS-68 (Fig.4c)

3.5 Effects of SS-68 on action potential waveform in pulmonary vein myocardium

Paced pulmonary vein myocardium demonstrates atrial-like electrical activity with fast action potential upstroke, large overshoot and resting potential close to -80 mV Control APD90

and APD50 in pulmonary vein preparations varied from 59±2 to 74±3 ms and from 24±1.4 to

28±2.6 ms (Fig 5a, b), respectively Administration of SS-68 resulted in substantial alteration of action potential waveform in rat pulmonary vein myocardium While lower concentration were

not effective, 10 and 100 µmol/L SS-68 caused significant elevation of APD90 by 12% and 60.3% and APD50 by 14.5% and 80%, respectively On the other hand, 10 and 100 µmol/L SS-68

strongly reduced maximal upstroke velocity in rat pulmonary vein preparations by 35.9% and by 97.6%, respectively, (Fig.6a,b) It should be mentioned that extremely severe, but incomplete suppression of excitability was revealed in rat pulmonary veins in response to 100 µmol/L SS-

68

4 Discussion

In the present study the effects of a new indole derivative SS-68 on atrioventricular conduction and action potential waveform in Purkinje fibers and pulmonary vein myocardium are described for the first time We have also first described the suppression of norepinephrine-induced proarrhythmic activity by SS-68 in Purkinje fibers The results concern critical aspects

of SS-68 safety and efficacy as a potential antiarrhythmic drug

We have demonstrated that micromolar concentrations of SS-68 weakly affect atrioventricular conduction in rat heart However, atrioventricular conduction was substantially altered by 10 µmol/L and higher concentration of SS-68 in our experiments This effect may be associated either with suppression of conduction in the atrioventricular node or slowing of

be sufficient for the effective termination of atrial fibrillation induced by tachypacing in animal models [8] Also, submicromolar (0.41 μmol/L) concentration of SS-68 significantly increased effective refractory period in atrial myocardium These results allow suggestion that therapeutic doses of SS-68 may be notably lower than doses which affect conduction and probably INa in cardiac tissue Thus, the risk of side effects caused by suppression of conduction by SS-68 is expected to be low This suggestion is also supported by pattern of SS-68 effects in spontaneously active rabbit Purkinje fibers It has been shown that action potential upstroke velocity in Purkinje fibers was substantially reduced only in the presence of 10 μmol/L SS-68

Most of antiarrhythmic drugs are able to alter action potential duration [1, 25, 27] Therefore, we have estimated effects of SS-68 on action potential duration in Purkinje fibers Alteration of action potentials induced by SS-68 was dose-dependent in our experiments While