Diphenhydramine-mediated modulation of inward rectifier IK1 current induces conduction blocks in the rat pulmonary veins myocardium and facilitates supraventricular proarrhythmicity

Diphenhydramine-mediated modulation of inward rectifier IK1 current induces conduction blocks in the rat pulmonary veins myocardium and facilitates supraventricular proarrhythmicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diphenhydramine-mediated modulation of inward rectifier IK1 current induces conduction blocks in the rat pulmonary veins myocardium and facilitates supraventricular proarrhythmicity Yury Egorov, Alexandr A. Abramov, Tatiana S. Filatova, Oksana B. Pustovit, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5449722/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diphenhydramine (DPH) is a first-generation antihistamine drug widely used for allergy and other non-allergic conditions. It is known that DHP is not free of adverse effects including induction of tachyarrhythmias. Nevertheless, the mechanisms behind DPH proarrhythmicity is not well understood. In the present study in vivo ECG recordings in rats, microelectrode registration in ventricular, atrial and pulmonary vein (PV) isolated tissue, optical mapping of bioelectrical activity in supraventricular tissue preparations as well as patch-clamping for I K1 recordings in rat cardiac myocytes were used for analysis of mechanisms of DHP-induced proarrhythmicity. It is shown that DPH unable to alter heart rate, however, significantly increases duration of QT intervals in rats. Also, DPH induces substantial prolongation of action potentials (AP) in the rat ventricular myocardium. These effects are mediated by DPH-induced attenuation of both inward and functional outward components of inward rectifier (IK1) current. In the rat pulmonary veins the diphenhydramine causes substantial proarrhythmic changes including resting potential (RP) shift to less negative values, AP amplitude decrease and electrotonic-like responses as well as inexcitability, slowing of the conduction velocity, conduction blocks. An adrenaline partially antagonizes DPH-caused RP shift and inexcitability induction, however facilitates PV-derived ectopy and circulation of excitation in presence of DPH in the cardiac tissue of the pulmonary veins. In conclusion, DPH-induced attenuation I K1 promotes formation of the functional substrate highly prone to re-entrant conduction and adrenergically-induced ectopy in the cardiac tissue of pulmonary veins. Thus, DPH in addition to its torsadegenicity may facilitate induction of atrial fibrillation. diphenhydramine H1-dntihistamines supraventricular arrhythmia QT intervals conduction blocks pulmonary veins inward rectifier Kir2.x channels. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 I. Introduction Diphenhydramine (DPH) is a first-generation antihistamine drug developed and came into the practice more than 70 years ago. Nowadays DPH extensively used in therapy of allergy and a number of other non-allergic conditions. The medicine is frequently utilized to treat hay fever or to temporarily relive upper respiratory allergies due to its antipruritic and antitussive action. DPH exhibits inverse agonism for histamine H1 receptors (H1R) belonging to GPCR superfamily (Xia et al. 2021 ). A molecular mechanism of DPH action is based on stabilization of the inactive conformation of H1R and shifts the equilibrium towards an inactive state of the receptor (Leurs, Church, and Taglialatela 2002 ) (Shimamura et al. 2011 ). Thus, DPH suppresses constitutive agonist-independent activity of H1R and antagonizes histamine-induced H1R-mediated signals transduction in the cells (Bakker et al. 2000 ) (Nijmeijer, Leurs, and Vischer 2010 ). Multimodality of DPH action including antiallergic, antiemetic, hypotonic, sedative, anxiolytic and hypnotic properties results from its interaction with H1R on the periphery and in the central nervous system due to ability to cross blood-brain barrier (Welch, Meltzer, and Simons 2002). Diphenhydramine belongs to arylalkylamine group of compounds; represents classic derivative of ethanolamine-based antihistamines (Simons and Simons 2011 ).(Sneader 2001 ). DPH was firstly marketed in 1946 and, therefore, was introduced to usage before clinical pharmacological investigations were obligatory. Despite a long history the drug-drug interactions, toxicity and pharmacodynamics of DPH still poor investigated for populational groups with preexisting conditions (Simons and Simons 2011 ). Similarly with other first-generation H1-antihistamines DPH is not free from various adverse effects in multiple tissues. A cardiac toxicity for ethanolamine H1-antihistamines was firstly reported more than three decades ago (Church et al. 2010 ). Adverse effects in the heart of most of ethanolamine-based H1-antihistamines occur due to their potencies with respect to the prolongation of QT interval (Nia et al. 2010 ; Jo et al. 2009 ). DPH causes prolongation of QTc interval in healthy volunteers and patients suffering from coronary diseases (Khalifa et al. 1999 ). It has been reported that DPH is able to cause torsade de pointes (TdP) after the administration of standard doses increasing, therefore, a risk of life-threatening ventricular tachyarrhythmias induction (Pratt et al. 1994 ; Woosley 1996 ; Shah et al. 2015 ; Mohan et al. 2021 ). Electrocardiographic proarrhythmic signs and polymorphic ventricular tachycardia were found in cases of DPH overdose (JOSHI et al. 2004; Sype and Khan 2005 ). In 2020 FDA has issued Drug Safety Communication reporting that high doses of DPH causes serious cardiovascular complications like cardiac arrest or ventricular arrhythmia. Since the administration of DPH in the heart are accompanied by QT interval prolongation the hERG-encoded Kv11.1 potassium channels blockade and a decrease of repolarization reserve are proposed as main mechanisms underlying the proarrhythmic effects of the drug (Suessbrich et al. 1996 ; Woosley 1996 ). However, other ion channels responsible for cardiac repolarization as targets for DPH are not studied. In addition, despite revealed torsadogenic potential via ventricular repolarization disturbance (Ali et al. 2021 ; Hoffman et al. 2022 ), the influences of DPH in the supraventricular myocardium is not elucidated. A cardiac tissue in the pulmonary veins (PV) is considered recently as proarrhythmic substrate and a main source of ectopic activity initiating atrial fibrillation (Haïssaguerre et al. 1998). The myocardium in PV is highly prone to ectopic automaticity since exhibit abnormal profile of repolarizing currents (Tsuneoka et al. 2017 ). In the present study the effects of DPH on PV electrophysiology and repolarizing inwardly rectifier (I K1 ) current as potential mediator DPH-induced proarrhythmicity are investigated. II. Material and methods 2.1. Animals All experimental procedures were carried out in accordance with European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No 123, Strasbourg 1985) and approved by the Ethics Committee of the NMRCC Institute of Experimental Cardiology. Male Wistar rats weighing 240–260 g (10 weeks old; in total 64 animals were used) were provided by the Scientific complex of biomedical technologies animal plant (Moscow region, Russia). Animals were held in the NMRCC animal house for 2 weeks under a 12 h:12 h light:dark photoperiod in standard cages prior to the experiment and fed ad libitum . 2.1. In vivo ECG recordings Prior to experiments the animals were anesthetized with isoflurane-oxygen gas mixture (3.5 Vol%) delivered via precise vaporizer (VetEquip, USA). After the anesthesia induction the animals were placed onto heated surgical table and the body temperature was stabilized with aid of rectal probe and bio-tc-1 thermocontroller (Biotechnologies, Russia). To maintain proper anesthesia an isoflurane in concentration of 1.5-2 Vol% was delivered continuously via nose cone. Three disposable adhesive electrodes were located in standard positions on the skin surface [Farraj, 2011] and connected to «Animal Bio Amp» amplifier with PL3516 PowerLab 16/35 ADC (ADInstruments, New Zealand) for ECG recording in Lead II. The anesthesia was considered successful if heart rate (HR) was stabilized at 360±20 BPM in 10 min after the initiation. Control 10 min ECG recording was obtained before all further experiment. After ECG establishment and control records 300 µL (37⁰C) of physiological solution (vehicle) was injected intravenously (via the lateral tail vein) with aid of Harvard Apparatus syringe pump (PHD Ultra) with rate 300 µL/min. A diphenhydramine dissolved in physiological solution (300 µL) was injected via the lateral tail vein in dose 1 mg/kg with rate 300 µL/min 30 min after the vehicle administration. ECG was recorded continuously 60 min after DPH administration. ECG signals were analyzed with use of LabChart Pro software (ADInstruments). A heart rate was calculated on the basis of RR intervals 20 min after vehicle of DPH administration. Also, P, Q and T waves were identified and the duration of PQ, QRS and QT intervals was calculated. 2.2. Sharp microelectrode recordings of electrical activity in multicellular preparations Rats were anesthetized as described in previous paragraph. After anesthesia induction the animals were decapitated. The chest was opened and complex supraventricular tissue preparations (LA-PV) including left atrium and its appendage (LAA), pulmonary veins (PV) with lung lobes were rapidly excised. Tissue preparations were separated from surrounding fascia and fat, rinsed from a blood with Tyrode’s solution. A pulmonary vein of left lung lobe with PV branches was incised longitudinally and the preparation was pinned with endocardial side facing upward to the bottom of a 2.5 ml silicone-coated perfusion chamber. In a separate series of experiments myocardial strips (2.5x10 mm) from a right ventricle wall (RVV) was excised and fixed in the chamber to assess effects of DPH in ventricular myocardium. The experimental chamber was filled with physiological (Tyrode’s) solution of the following composition (in mM): NaCl 118.0, KCl 4.7, NaH 2 PO 4 1.2, MgCl 2 1.8, CaCl 2 1.8, NaHCO 3 25.0, glucose 11.0, pH 7.4 ±0.2, bubbled by 95% O 2 and 5% CO 2 gas mixture. The constant perfusion with flow rate of 18 mL/min at 37 °C and constant steady-state electrical pacing (CEP) with aid of silver teflon-coated electrodes connected to pulse generator A310 (WPI, USA) with 300 ms cycle length (CL) was started immediately after the preparation of LA-PV or RVV strips. Pacing electrodes were located on the endocardial surface of LAA with aim to induce physiological anterograde pattern of PV excitation. Electrically evoked action potentials (AP), spontaneous activity in a form of automatically occurring action potentials, resting membrane potential (RP or RMP) were recorded simultaneously from the endocardial side of the PV ostium (PVo) and from a distal segment of pulmonary veins (2-3 mm distally to the first order bifurcation of the PV but proximally to the intrapulmonary sites of PV) with aid of glass microelectrodes (10–20 MΩ) filled with 3 M KCl, connected to a multichannel intracellular electrometer (KS-700, WPI instruments, USA). A non-trabeculated cardiac tissue in the ostium of PV was assumed as closest region to PV exhibiting atrial-type of electrical activity due to stable RMP (-80±3 mV) and weak RMP shift in response to cholinergic or adrenergic stimulation (Egorov et al. 2015). Signals were digitized at 10 kHz sampling rate with analog-digital converter (Е-154, ADC L-card, Russia) which allowed calculate APs upstroke velocity. The APs duration at the level of 90% repolarization (APD) was calculated using LGraph2 software (L-Card, Russia). The APs upstroke velocity was calculated as maximum of the depolarization phase derivative (dV/dt max ) to determine the time moments of PVO or distal PV (PVd) excitations and quality of impalements. Only records with dV/dt max > 200 V/s were taken into account. Conduction time was calculated as a time interval between moments of PVO/PVd excitation and a moment corresponding to the pacing stimulus application. The upstroke velocity, amplitude of APs (APA) and RMP were calculated using LGraph2 software (L-Card, Russia). 2.3. Sharp microelectrode experiments protocols Following 30 min equilibration period under CEP, control electrically evoked APs were recorded in PV ostium as well as in distal PV for 10 min. In the first type of experiments with LA-PV preparations a diphenhydramine (10, 30 µM) was applied for 20 minutes under CEP after the control APs recording Stable microelectrode impalements were maintained during the whole period of DPH application. In the second type of experiments 10 min after DPH (30 µM) administration beginning an adrenaline (AD, Sigma-Aldrich, USA) in consecutively increased concentrations (1, 5 or 10 µM) was applied with 20 min intervals. In the third type of experiments CEP was terminated after period of equilibration and 10 min of DPH (30 μM) administration. Spontaneous AP and RMP was recorded in quiescent preparations for at least 10 min in presence of DPH alone (30 μM) or DPH (30 μM) in combination with adrenaline (1-10 µM, Sigma-Aldrich, USA). In the separate series of experiments electrically evoked APs were recorded in RVV strips under CEP in control conditions and at least 20 min after 10 or 30 µM of DPH administration. 2.4. Optical mapping of multicellular preparations The pattern of excitation in the pulmonary veins was analyzed using di-4-ANEPPS-based optical mapping technique (Ivanova and Kuzmin 2018; Ivanova et al. 2021). LA-PV tissue preparations were dissected as described in the section 2.2. The optical mapping rig included a photodiodes array (PDA, WuTech H-469V, Gaithersburg, MD, USA) designed for high speed data acquisition (1.63 Kfps). Macroscopic projections of the cardiac tissue preparations were transferred to the PDA with aid of the optical system including adapters and Computar V5013 (CBC Group, Japan) camera lens (focal length 50 mm, aperture ratio 1:1.3) mounted in a distance of 24 mm from a tissue surface. The optical system allowed to project the area of 5 mm in diameter to a hexagonal array of 464 PDA photodiodes. Thus, each photodiode in PDA array covered approximately a surface of 0.23 mm in diameter approximately. The view-field was projected also to the monitoring CMOS camera (MD50, M-Shot, China) used to match the mapping area and sites in the tissue preparations. An excitation light was emitted by LED (520±40 nm) arrays surrounding the experimental chamber with mapped preparation. A long-pass emission filter (λ>650 nm) was positioned in front of the camera lens. After dissection, LA-PV preparations were placed into experimental chamber and superfused at 37°C with constant flow (10 ml/min) by physiological solution of the same composition as used for microelectrode experiments. Voltage-sensitive dye di-4-ANEPPS (5 mg/ml, dissolved in DMSO) was added to physiological solution (final concentration 50 µM) for 30 min staining of the preparations. Then preparations were equilibrated for 20 min prior to a mapping procedure. After the equilibration 10 or 30 μM of diphenhydramine was applied for 20 min with 30 min washout intervals. In this series of experiments LA-PV preparations were paced by linear silver electrodes located in the myocardium adjoining to the orifice of the vein. Anterograde (from LA to PV) propagation of excitation with CL 250 ms was induced by steady-state pacing. 2.5. Optical mapping data analysis In all experiments evoked fluorescent signals (optical AP) from PV were recorded continuously for 5 s with 0.614 ms frame intervals, digitized using a data acquisition system (CardioPDA-III; RedShirtImaging, Decatur, GA, USA) and analyzed using Cardioplex (v.8.2.1, RedShirtImaging) software. The resting fluorescence was determined before each recording. The signals were processed via Savitsky-Golay filter using custom algorithm in order to remove noise and were normalized to the resting fluorescence. Also, minimal high-pass filter was applied to remove long time constant photodiode-derived basal drift. The maximum upstroke derivative (dF/dtmax) for each optical AP was calculated to determine the activation times in the mapped areas. Isochronic activation maps were constructed from activation times using an in-house developed software. The activation time (t act ) was calculated as time interval between activation of PVO and distal PV on the borders of the mapped area. An averaged conduction velocity (CV) in the PV was calculated as a ratio of the length of the mapped region (4-5.5 mm) and the activation time if the excitation wave demonstrated near linear pattern. 2.5. Whole-cell patch clamp recordings in isolated cardiomyocytes Ionic currents were recorded in enzymatically isolated rat cardiomyocytes. Prior to isolation the animals were intraperitoneally injected with heparin, then anaesthetized and decapitated as described above. The heart was excised, mounted onto a Langendorff apparatus and retrogradely perfused through aorta with nominally Ca 2+ -free physiological solution containing (in mM): NaCl 116; KCl 4; NaH 2 PO 4 1.7; NaHCO 3 25; MgCl 2 0.55; sodium pyruvate 5; taurine 20; glucose 11; 1 g/ml bovine serum albumin; pH 7.4±0.2 bubbled by 95% O 2 and 5% CO 2 gas mixture at 37 o C. After 5-7 minutes, the perfusion was switched to a solution of the same composition containing 0.5 mg/ml collagenase II (Worthington, USA), 0.025 mg/mL protease XIV (Sigma Aldrich, USA) and 6 μM CaCl 2 . After 40-50 min of enzymatic treatment the perfusion was stopped, the ventricles were minced and gently triturated to liberate the cardiomyocytes into “Kraftbrühe” medium containing (in mM): MgSO 4 3; KCl 30; KH 2 PO 4 30; EGTA 0.5; potassium glutamate 50; HEPES 20; taurine 20; glucose 10; pH 7.2 adjusted with KOH at 24 o C (Isenberg and Klockner 1982). The cardiomyocytes were stored in this medium at room temperature and used within 6 hours after the isolation. The capacitance of the ventricular myocytes used in the experiments was 151.6±4.5 (n=50) pF. Inward rectifier I K1 current in ventricular myocytes was recorded using conventional whole-cell patch clamp using a HEKA EPC-800 amplifier (HEKA Elektronik, Germany). Isolated cardiomyocytes were placed into an experimental chamber (RC-26; Warner Instrument Corp., Brunswick, CT, USA; volume 150 μl) mounted onto an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan). The cells were perfused with Tyrode’s based physiological solution. Potassium I K 1 current was recorded at room temperature (24⁰C) using K + -based solution containing (in mM): NaCl 150; KCl 3; CaCl 2 1.8; MgCl 2 1.2; glucose 10; HEPES 10; pH 7.4 adjusted with NaOH. Patch pipettes were pulled from borosilicate glass capillaries without filament (Sutter Instruments, CA, USA) and filled with a pipette solution. The pipette solution used for I K1 recording contained (in mM): KCl 140; MgCl 2 1; EGTA 5; HEPES 10; MgATP 4; Na 2 GTP 0.03; pH 7.2 adjusted with KOH at 24 o C. The resistance of filled patch-pipettes was within a range of 2-3 MΩ. Recorded current was normalized by cell capacitance and presented as current density (pA/pF). The obtained data were analyzed using Clampfit 10.3 software (Molecular Devices, USA). 2.6 Statistical analysis Statistical analysis was carried out using GraphPad Prism version 7. The normality of the groups was tested using the Shapiro-Wilk’s test where needed. Hypothesis testing was carried out using Freedman’s or one-way ANOVA with further Sidak’s post-hoc multiple comparisons where appropriate. Also, one-tailed paired Student’s t-test or Fisher’s exact test were used where appropriate. A p -value <0.05 was considered statistically significant. All results are expressed as means±SD except patch-clamp data where they are expressed as mean±SEM for n experiments and data regarding cycle length of spontaneous AP in PV where they expressed as median and IQR. III. Results 3.1. Effect of DPH on heart rate and ECG parameters A systemic delivery of DPH (1 mg/kg) did not alter a heart rate over the first 20 min of post-injection period in rats (Fig.1A, n=5). Similar HR values were observed in DPH-treated and control (vehicle-treated) rats 20 min after the injections (357±21 and 361±34 bpm, respectively, n=5, p >0.1). No P-wave alternans, tachy- or bradycardia episodes, tachyarrhythmia episodes or ventricular extrasystoles were observed in rats in vivo 0-60 min after the DPH administration (Fig.1B). Among ECG characteristics the diphenhydramine induced a significant prolongation of QT interval in rats. The duration of QT interval 20 min after the administration of 1 mg/kg of DPH was 95±16 ms (n=5) in comparison to 83±17 ms in vehicle-treated animals (n=5, Fig.1C, D). Unlike to QT, diphenhydramine was unable to alter substantially the duration of QRS and PQ interval (Fig.1D). 3.2. Effect of DPH on inward rectifier (I K1 ) current in ventricular myocytes and ventricular AP An inward rectifier I K1 current was changed by the DPH in the ventricular rat cardiomyocytes. When DPH was administered (10 µM, 20 min) both inward and outward components of I K1 were significantly reduced. At -120 mV the current densities were -9.8±3.7 (n=6) and -8.1±3.1 pA/pF (n=7) in control measurements and after DPH administration, respectively. Diphenhydramine significantly suppressed the peak value of the outward component of I K1 at -70 mV by 34% (0.53±0.20 (n=6) and 0.35±0.22 pA/pF (n=7), respectively, Fig.2A, B). In experiments with isolated tissue strips PDH caused significant prolongation of the ventricular APs. The DHP-induced increase of AP duration is time-dependent and reached 35.4% when was measured 20 min after the beginning of DPH (10 µM) administration (19.2±1.3 (n=5) and 26.0±1.0 ms (n=5) in control and under DPH action, respectively, p=0.01, Fig.2C). Unlike to AP duration, RMP remained unchanged by DPH in RVV (10 µM, 20 min): -78±4 and -80±5 mV in controls and in presence of DPH, respectively (n=5, p >0.1). 3.3. Effects of DPH on pulmonary veins excitability and action potentials A diphenhydramine applied in concentrations 10 or 30 µM (20 min) was unable to affect substantially RMP, AP amplitude or duration of electrically evoked AP in PV ostium. Nevertheless, administration of DPH in high concentration (30 µM) resulted in a significant increase of conduction time in the atrial myocardium (19±4%, р <0,001, n=6, Fig.3A-D, Table 1). In control experiments distal PV myocardium exhibits normal RMP and APs with fast upstroke, atrial-like amplitude (91±3 mV, n=6) and waveform in presence of steady-state electrical stimulation (Table 1). The administration DPH results in a pronounced alteration of bioelectrical parameters in the cardiac tissue of distal PV. High concentration of DPH (30 µM, 20 min) causes significant RMP shift (from -83±1 to -64±2 mV, n=6, р <0,001). In all experiments (n=8) DPH causes suppression of the excitability in the distal PV manifesting in a lack of active response to electrical stimulation and only electrotonic-like low-voltage potentials with amplitude 12±2 mV and negligible duration. Also, DPH significantly increased time interval between the moments of pacing stimulus application and electric response in the distal PV (Fig.3A-D). 3.4. Adrenergic modulation of DPH effects in the pulmonary vein myocardium The effects of DPH in PV was substantially altered by an adrenaline. The adrenaline partially and dose-dependently (1-10 µM, n=6) attenuated DPH-induced (30 µM) shift of RMP in the distal PV as well as restored the excitability and the amplitude of APs in the pulmonary veins (Fig.3A, B, Table 1). In presence of doth AD and DPH resting potential in the ostium of PV remained unchanged. Only maximal used concentration of AD (10 µM) reduced AP amplitude in the ostium of PV in presence of DPH (30 µM). In contrast to RMP and APA, the adrenaline changed the conduction time and AP duration in the ostium and distal PV synergically with DPH. The administration of AD dose-dependently (1-10 µM, n=6) and significantly increased DPH-induced (30 µM) elevation of the conduction time and, therefore, delayed the excitation of the distal sites of PV (Fig.3C, Table 1). This additive effect of AD and DPH was observed despite the recovery of RMP. The recovery of the DHP-suppressed excitability by AD allowed to estimate AP duration in the distal PV. In presence of AD (1-10 µ) and DPH (30 µ) the duration of electrically evoked APs in the distal PV was more than twice longer than the AP duration in control conditions. Unlike to ventricular APs the diphenhydramine (10, 30 µM) was unable to prolongate APs duration in PVO. However, adrenaline potentiated effect of DPH in the ostium of PV and caused significant prolongation of APs duration (Fig.3D, Table 1). 3.5. Modulation of pulmonary veins ectopy by DPH No re-entrant circulation of the excitation was observed in the electrically paced LA-PV preparations in presence of DPH (10, 30 µM, n=6) or adrenaline (10 µM, data not show n) used separately in these series of experiments. Nevertheless, AD (5 µM) in presence of DPH (30 µM) induced re-entrant circulation including anterograde (primary, pacing-induced) LA-to-PV and retrograde ‘echo’ PV-to-LA wave propagation in 2 out of 6 experiments (Fig.4A). DPH (10, 30 µM, n=6) is unable to induce ectopic AP (spontaneous activity) in the quiescent (non-paced) pulmonary veins. However, administration of adrenaline dose-dependently potentiated ability of DPH to elicit a spontaneous activity in PV. In our experiments, 1-10 µM of AD in presence of DPH caused PV-derived spontaneous activity capable of activating cardiac tissue in the ostium of PV and left atrium (Fig.4B). The spontaneous activity induced by a combination of AD and DPH manifested in a form of permanent, non-burst APs occurring with quasi-constant intervals (CL>3050 (n=1), 1700±430 (n=6) and 880±200 (n=6) ms for 1, 5 and 10 µM of AD, Fig.4B). When 5 or 10 µM of AD was applied spontaneous activity occurred in all cases and was accompanied by re-entrant episodes. 3.6. Effects of DPH on conduction of excitation in the pulmonary veins Rat pulmonary veins are characterized by consecutive, almost linear pattern of anterograde conduction of the excitation wave lacked of any conduction disturbances or conduction blocks when the excitation elicited by continuous electrical pacing of the left atria (CL=250 ms, Fig.5A, B). The conduction velocity in PV in control set of experiments was 0.74±0.05 mm/ms (n=5) which is very close to CV typical for ‘working’ atrial myocardium. The administration of DPH dose- and time-dependently affects pattern of excitation of PV. The conduction velocity was slightly decreased while activation time was slightly reduced 5 min after 10 µM of DPH (n=5) administration. These changes were aggravated and significant after 20 min of DPH (10 µM, n=5) administration: CV and t act reduced to 0.37±0.05 mm/ms and 13.49±0.75 ms (n=5, p <0.01), respectively. Conduction blocks were initiated by DPH (20 min, 10 µM) in 4 out of 5 experiments in PV branches at post-bifurcation level (Fig.5B-D). DPH applied in 30 µM concentration rapidly induced blockade of the electrically initiated anterograde excitation in the trunk of PV at sites adjacent to PV ostium in all experiments (n=3). IV. Discussion In the present study we have demonstrated for the first time that H1-antihistamine diphenhydramine induces substantial prolongation of AP in the rat atrial and ventricular myocardium; causes substantial proarrhythmic changes including depolarization, inexcitability, slowing of the conduction velocity, conduction blocks and facilitates PV ectopy in a presence of adrenergic stimulation in the cardiac tissue of the pulmonary veins. These effects in the rat myocardium are mediated by DPH-induced inward rectifier current IK1 attenuation. In our experiments we were unable to found episodes of atrial fibrillation or flutter as well as TdP in rats. On the other hand, in vivo experiments revealed obvious QT intervals prolongation, which is torsadogenic, in response to DPH administration. It has been demonstrated previously, that DPH in doses >500 mg induces bundle branch blocks, prolongation of QTc, increases the slope of QT-RR relationship curve, increases the duration and lowers the amplitude of T-wave. These effects are accompanied with sinus tachycardia (Zareba et al. 1997; Hestand and Teske 1977). It is known that DPH-caused QT prolongation in patients not always result in TdP. The DPH-overdose induced sinus tachycardia occurs due to its cholinolytic effect via a central attenuation of parasympathetic tone (JOSHI et al. 2004). Increased HR may reduce the likelihood of TdP since a shorter cycle length and decreased AP duration diminish the probability of proarrhythmic afterdepolarizations. Thus, intrinsic high heart rate in rats may protect ventricular myocardium against DPH-induced TdP in our experiments. It has been shown in several studies that DPH attenuates rapid component of a delayed rectifier IKr current. This effect of DPH is thought to be based on the ability of the drug to block hERG (Kcnj2) gene encoded Kv11.1 ion potassium channels. The blockade of hERG-channels by H1-antihistamines including DPH has been demonstrated in experimental studies utilizing heterologous expression systems and laboratory animals like guinea pigs (Suessbrich et al. 1996; Jo et al. 2009; Khalifa et al. 1999; Park, Kim, and Kim 2008). It is well known that numerous small molecules demonstrate an ability to act as a blocker for Kv11.1 channel due to specific amino acid residues architecture of its pore cavity interior (Sanguinetti and Tristani-Firouzi 2006; Sanguinetti and Mitcheson 2005). Many compounds among antihistamines besides DPH also exhibit Kv11.1 blocking potency (Taglialatela, Timmerman, and Annunziato 2000). The histamine receptor antagonists chlorpheniramine ‘weakly’ blocked hERG channels with IC50 values of 20.9±2.1 µM. This value for DPH was estimated as 27.1±1.5 µM (Suessbrich et al. 1996) and was also considered as rather weak. Nevertheless, prolongation of QT intervals in patients by DPH is thought to be mediated by hERG channels blockade. In our experiments we utilized tissue preparations and isolated cardiomyocytes from rat heart. It is well known that adult rat atrial and ventricular myocardium is lacked of delayed rectifier IKr. Thus, the prolongation of AP which was observed in our experiments in presence of DPH cannot be attributed with blockade of Kv11.1 channels and attenuation of IKr current. It is known that inward rectifier IK1 underlies not only a resting potential stabilization but also substantially contributes to AP repolarization in rodents. As has been mentioned above, DPH caused I K1 suppression in our experiment. The attenuation of I K1 current by several H1- an H2-antihistamines of various generations has been shown previously in experiments with geterologous expression systems (Liu et al. 2007). A reduction of functional outward component of I K1 in ventricular myocytes along with QT prolongation and changes observed in rat PV cumulatively support the suggestion that inward rectifier is a target of DPH additional to IKr contributing the drug-induced proarrhythmicity. It is demonstrated that thoracic veins exhibit differential subunit composition of heterotetrameric channels responsible for native I K1 current in the atrial and ventricular myocardium. The most abundant Kir subunit contributing to the native channels in the myocardium of rat thoracic veins is Kir2.2 while Kir2.3 is extremely minor (Ivanova et al. 2021; Melnyk et al. 2002). The inhibition by DPH assessed in Xenopus oocytes was found mainly for Kir2.3-containig channels while Kir2.1 was significantly less sensitive to antihistamines (Liu et al. 2007). It has been also shown that cardiomyocytes of thoracic veins wall are characterized by a smaller I K1 current density and reduced overall expression of Ki2.x subunits in comparison to atrial cardiomyocytes. It should be noted that the myocardium in the pulmonary veins ostium demonstrates AP and RMP level very similar to that observed in LA (Egorov et al. 2015; 2019). Thus, PVo was considered in our study as a region demonstrating atrial-like electrophysiology. Noteworthy, that DPH-induced changes in PVo electrophysiology were less prominent than in changes in distal PV and ventricular myocardium. The administration of DPH in our experiments resulted in a strong sift of the resting potential to less negative values (depolarization) in the PV myocardium while the shift of RMP was negligible in ventricular strips. Thus, weak attenuation of the small I K1 current composed mainly of Kir.2.1/Ki2.2 subunits in the PV results in distinctive functional changes in contrast to the effects observed in the cardiac tissue surrounding PV ostium or in the ventricle. In the present the alteration of I K1 by DPH was directly measured and I-V curve was reconstructed. Nevertheless, this provides little information regarding a mode by which PDH affects Kir2.x channels. We used reconstructed Cryo-EM human (KNCJ2) and rats Kir2.1 subunit structures of the tetrameric functional IKir channels (Fernandes et al. 2022) provided by ‘AlphaFold’ protein structures database (P63252, Q64273, Fig.6, A) to predict potential sites of DPH binding (https://doi.org/10.2210/pdb7ZDZ/pdb). Using free AI-based DeepSite module (https://www.playmolecule.com/deepsite/) from a software platform PlayMolecule provided by D-Acellera (UK) we analyzed Kir2.1 channel subunit and found ability of the protein to form several binding pockets outside of the pore-forming region of the tetrameric channel ensemble. Predictive analysis revealed several deep binding pockets (BP) that highly-suitable to venue small molecules like DPH. In particular, BP may be formed by a bend between transmembrane M1 α-helix and small side α-helix (Fig.6., B, site 1) as well as by an unfolded region between transmembrane helices and submembrane β-sheet (site 2) or by cytoplasmic β-sheet (site 3). Noteworthy, the BP in site 1 borders an intramembrane and submembrane cytoplasmic region of the Kir2.1. molecule. Further, we used molecular dynamics-based docking software utilizing multiobjective evolutionary optimization (http://www.swissdock.ch/; https://mcule.com/apps/1-click-docking/) to predict potential sites of interactions of the rat or human Kir2.1 subunits and DPH molecule and estimate a probability of DPH binding (Grosdidier, Zoete, and Michielin 2007). The modelling of DPH/Kir2.1 interaction revealed more than 50 clusters capable of DPH binding. Nevertheless, the clusters with a top cumulative rank and highest ΔG energy decrease are colocalized with the binding pockets predicted by the evaluation of the protein surface with aid of ‘deepsite’ software. Estimated ΔG energy of DPH interaction in the cluster corresponding to the site 1 is -7.53 kcal/mol and full fitness parameter is -2178.13 kcal/mol that demonstrates high probability of the binding. These computational data allow to suppose that DHP attenuates I K1 current not as a channel pore blocker but as channel gating modifier or via disruption of ion channel interaction with lipides of inner membrane leaflet. It should be noted that DPH-induced AP prolongation in ventricular strips was weakly voltage-dependent and reached its maximum during 10-30 minutes. This observation allows to suppose that DPH acts upon its slow accumulation in the lipide phase. It is well known that a gating of Kir2.1 channels is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Hibino et al. 2010; Xie et al. 2008). The binding of PIP2 is required to trigger conformational changes that switch the channel into the open state. A PIP2-dependent gating is possible due to structural interactions of PIP2-binding site and the constriction formed by the G-loop in the cytoplasmic domain of the channel (Fernandes et al. 2022). The binding of PIP2 leads the release of the G-loop constriction in the channel pore. The binding site for PIPI2 is located at the interface between the transmembrane (TMD) and cytoplasmic domains (CTD) and is coordinated by several positively charged amino acid residues (Whorton and MacKinnon 2011; Hansen, Tao, and MacKinnon 2011). The site occupies the space between outer M1 α-helix and interfacial small side helix (Fig.6.A). PIP2-binding site is colocalized with DP ‘site 1’ and ‘site 2’ as well as with predicted binding site for DPH (Fig.6.C, D). Thus, structurally confirmed data and in silico prediction allow us to speculate that DPH is able to enter vicinity occupied by the membrane bound PIP2 or displace PIP2 inside its binding site or at least affect PIP2-Kir2.1 interaction in some manner. Aforementioned DPH-caused disruption of PIP2-Kir2.1 may lead to the G-loop-dependent decrease of the channel availability for K + ions resulting in functional effects like RMP shift, AP prolongation. The regulation of Kir2.x subunits via modulation of its interaction with PIP2 is not surprising and was demonstrated for arachidonic acid (Wang et al. 2008). It was supposed that Kir2.3 is the most sensitive to antihistamines since the strength of channel-PIP2 interactions determines the susceptibility to the modulation and Kir2.3 subunit exhibits lowest affinity to PIP2 (Du et al. 2004). Native ventricular Kir (IK1) channels are Kir2.1-rich, and therefore, are expected to be weakly sensitive to DHP. Nevertheless, we observed strong effects of DPH-induced attenuation of I K1 in the rat ventricles. This is not contradictory since many factors and membrane components beside PIP2 may affect Kir-PIP2 interaction. Noteworthy, that Kir channels exhibit several binding sites for cholesterol (CHL) which is also considered recently as potent Kir modulator (Rosenhouse-Dantsker 2019). It is shown that PIP2 and CHL are in close interplay in Kir modulation. One of the CHL binding sites is located on the border of TMD and CTD superiorly to the side helix in Kir2.1 in close proximity to the binding site of PIP2 (Rosenhouse-Dantsker, Epshtein, and Levitan 2014) and coincides with the predicted DP and most favorable DPH docking region (Fig.6). Notable, the enrichment of Xenopus oocytes with CHL may results both in attenuation or enhancement of Kir-mediated ion current depending on subunit structures (Rosenhouse-Dantsker et al. 2010; Corradi et al. 2022). Thus, a competition of DHP and CHL for Kir2.x subunits may significantly affect I K1 current. In addition, the abundancy of CHL and composition CHL derivatives bound in membrane lipid rafts is highly variable in different cell populations. This fact introduces additional uncertainty to the estimation to the DPH inhibitory potential and to the elucidation of mechanism of DPH action. In conclusion, the allosteric modulation of Kir2.x channel responsible for IK1 current may be considered as additional mechanism of DPH-mediated proarrhythmicity. Our results demonstrate that adverse proarrhytmic effect of DPH may be expanded beyond the ventricular myocardium facilitating PV-derived profibrillatory activity. The ability of DPH to promote atrial arrhythmias can manifest mainly in a preliminary remodeled supraventricular myocardium or in cases of altered autonomic control of the heart. Declarations Conflict of interests All authors declare no conflict of interests. Personal contribution Yury V. Egorov -microelectrode experiments with pulmonary veins; Alexandr A. Abramov – in vivo experiments, manipulations with animals Yana A. Voronina – sharp microelectrode experiments with usage of isolated tissue preparations; Tatiana S. Filatova- patch-clamp experiments; Oksana B. Pustovit – isolation of tissue preparations; Andrew M. Karhov – molecular dynamics and docking modeling, Vlad S. Kuzmin – conceptualization, data analysis, manuscript writing; Funding Russian Science Foundation grant 22-15-00189 Author Contribution ury V. Egorov -microelectrode experiments with pulmonary veins;Alexandr A. Abramov – in vivo experiments, manipulations with animalsYana A. Voronina – sharp microelectrode experiments with usage of isolated tissue preparations;Tatiana S. Filatova- patch-clamp experiments;Oksana B. Pustovit – isolation of tissue preparations;Andrew M. Karhov – molecular dynamics and docking modeling,Vlad S. Kuzmin – conceptualization, data analysis, manuscript writing; Acknowledgements This study is supported by Russian Science Foundation grant 22-15-00189 to Vlad S Kuzmin. Data Availability All experimental data will be accessable in the cloud on request. 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Zareba, Wojciech, Arthur J Moss, Spencer Z Rosero, Raef Hajj-Ali, JoAnne Konecki, and Mark Andrews. 1997. “Electrocardiographic Findings in Patients With Diphenhydramine Overdose.” The American Journal of Cardiology 80 (9): 1168–73. https://doi.org/10.1016/S0002-9149(97)00634-6. Table Table 1. Effect of diphenhydramine (DPH) on resting potential (RP) and electrically evoked action potentials (AP) in the pulmonary veins ostium (PVo) and distal pulmonary veins (PVd). RP-PVo RP-PVd APA-PVo APA-PVd APD-PVo APD-PVd Т-PV o (%) Т-PVd (%) control -82±1 -82±1 99±1 91±3 66±5 54±4 0±0 0±0 DPH -82±1 -64±2*** 88±2 12±2*** 76±5 2±1*** 19±4*** 25±3*** DPH + AD(1μМ) -82±2 -75±3 92±3 77±8*** 83±6*** 103±5*** 25±9*** 71±20*** DPH + AD(5μМ) -82±1 -75±3 93±2 68±4*** 76±4 112±12*** 47±9*** 83±14*** DPH + AD(10μМ) -79±2 -65±4*** 84±2** 49±5*** 78±6*** 104±11*** 79±17*** 177±30*** APA – AP amplitude, APD – AP duration at level of 90% repolarization, AD – adrenaline. T-PVo, T-PVd - increase in the time of conduction from a left atria appendage to the pulmonary vein ostium or distal portion of PV. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5449722","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":385720200,"identity":"2b0fafdf-bf6a-457e-a482-0646bcff28b6","order_by":0,"name":"Yury Egorov","email":"","orcid":"","institution":"National Medical Research Cardiological Complex (NMRCC)","correspondingAuthor":false,"prefix":"","firstName":"Yury","middleName":"","lastName":"Egorov","suffix":""},{"id":385720201,"identity":"3ae21126-4ebc-49fc-8e64-cb668c249ee2","order_by":1,"name":"Alexandr A. Abramov","email":"","orcid":"","institution":"National Medical Research Cardiological Complex (NMRCC)","correspondingAuthor":false,"prefix":"","firstName":"Alexandr","middleName":"A.","lastName":"Abramov","suffix":""},{"id":385720202,"identity":"221b14c3-17d8-49c5-a74d-5306e8825200","order_by":2,"name":"Tatiana S. Filatova","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Tatiana","middleName":"S.","lastName":"Filatova","suffix":""},{"id":385720203,"identity":"6696e7e1-e116-415d-ad3c-f1d8fafb162f","order_by":3,"name":"Oksana B. Pustovit","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"Oksana","middleName":"B.","lastName":"Pustovit","suffix":""},{"id":385720204,"identity":"2e9f5281-7346-4233-8265-5b7b827024a7","order_by":4,"name":"Andrew M. Karhov","email":"","orcid":"","institution":"National Medical Research Cardiological Complex (NMRCC)","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"M.","lastName":"Karhov","suffix":""},{"id":385720205,"identity":"253fd4c4-8344-4c70-a653-b309eab5674d","order_by":5,"name":"Yana A. Voronina","email":"","orcid":"","institution":"National Medical Research Cardiological Complex (NMRCC)","correspondingAuthor":false,"prefix":"","firstName":"Yana","middleName":"A.","lastName":"Voronina","suffix":""},{"id":385720206,"identity":"ef680bae-85b3-472a-a949-3a7bd6fc529f","order_by":6,"name":"Vlad S. Kuzmin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACCRCRwGDDwMDM3MDMYEC8ljSgFkZStDAwHAZikBZigOSM9KcbHu44H83fDtRSUGDDoNt/xoC5sg23FmmJhLQbiWdu5844DNQywyCNwexGjgHjWTxa5CQSjt1IbLud2wDSwmNwGKiFx4CxEa+WxDaglnO58+Fazp/Br0VaIpkNqOVA7ga4lgM5+LVI9jwDaUnO3QjUchjoFx6zG2kFBxvO4dYicTz92c2fbXa5884fPvi44I+NnNn5wxsfNpTh1oICDgAxD4wxCkbBKBgFo4ACAADt5FJVi8BSbQAAAABJRU5ErkJggg==","orcid":"","institution":"National Medical Research Cardiological Complex (NMRCC)","correspondingAuthor":true,"prefix":"","firstName":"Vlad","middleName":"S.","lastName":"Kuzmin","suffix":""}],"badges":[],"createdAt":"2024-11-13 23:08:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5449722/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5449722/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71370920,"identity":"d1ea98cf-eab9-496e-bfb2-6e019f3f38bd","added_by":"auto","created_at":"2024-12-13 19:10:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1161899,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of diphenhydramine (DPH) on heart rate and ECG parameters in rats. \u003cstrong\u003eA.\u003c/strong\u003e A heart rate (HR) in DPH-treated rats. \u003cstrong\u003eB. \u003c/strong\u003eAn original representative ECG recording from the vehicle (\u003cem\u003etop\u003c/em\u003e) and DPH-treated (\u003cem\u003ebottom\u003c/em\u003e) rats showing coordinated P and R waves with no proarrhythmic events. Arrowheads mark P-waves of consecutive cardiac cycles. Green dots mark R-waves. \u003cstrong\u003eC. \u003c/strong\u003eRepresentative ECG signals obtained in control conditions and after DPH administration. T\u003csub\u003eend\u003c/sub\u003e – end of the T wave. \u003cstrong\u003eD.\u003c/strong\u003e QT, PR and QRS intervals after the administration of vehicle or DPH (1 mg/kg) in rats. One tailed paired Student’s t-test was used to determine significance in statistical comparisons. ns – nonsignificant.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/faa905e27ce0eb10d0c9152e.png"},{"id":71371020,"identity":"91a5e893-fcb7-413f-982c-6de26f1ec298","added_by":"auto","created_at":"2024-12-13 19:18:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":376754,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of diphenhydramine (DPH) on inward rectifier (I\u003csub\u003eK1\u003c/sub\u003e) current and ventricular action potentials (AP). \u003cstrong\u003eA.\u003c/strong\u003e DPH (10 µM, 20 min) caused a significant decrease of both inward and outward IK1 components in the cardiomyocytes from the rat heart ventricles. * - p\u0026lt;0.05, ** - p\u0026lt;0.01 (one-way ANOVA). N – number of animals; n – number of cells. \u003cstrong\u003eB.\u003c/strong\u003e Representative traces of IK1 from ventricular cardiomyocytes in control conditions (black) and after DPH (10 µM, 10 min) administration. Voltage test protocol is shown in inset. \u003cstrong\u003eC.\u003c/strong\u003e Representative traces of ventricular AP (\u003cem\u003eleft\u003c/em\u003e) in control conditions (\u003cem\u003eblack\u003c/em\u003e) and after DPH administration (10 µM, 10 min. \u003cem\u003ered\u003c/em\u003e) and DPH-induced AP prolongation (\u003cem\u003eright\u003c/em\u003e). \u003cem\u003eP\u003c/em\u003e value - one-way ANOVA (n=5).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/37cac0873d8f0d9cd3719d58.png"},{"id":71370922,"identity":"6c0dd9d5-671e-4c62-869f-c8133327d22c","added_by":"auto","created_at":"2024-12-13 19:10:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1006526,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of diphenhydramine (DPH, 30 µM) in pulmonary veins (PV) myocardium and modulation of DPH-induced changes by adrenaline. \u003cstrong\u003eA.\u003c/strong\u003e Diphenhydramine (30 µM) significantly reduces a resting membrane potential (\u003cstrong\u003eA, \u003c/strong\u003eRMP) and amplitude of an electrically evoked AP (\u003cstrong\u003eB, \u003c/strong\u003eAPA) in a distal portion of PV but not in PV ostium. An adrenaline (AD) partially attenuates the DPH-induced RMP and APA reduction in a dose-dependent manner. \u003cstrong\u003eC.\u003c/strong\u003e DPH significantly prolongates time intervals from the moment of electrical excitation of the left atria appendage (LAA) to the moment of the PV ostium (PVO) or distal PV (PVd) excitation. AD aggravates DPH-induced delay of the PV ostium or distal PV excitation. \u003cstrong\u003eD.\u003c/strong\u003e The duration of AP in PV ostium and distal part of PV myocardium is increased in presence of DPH when excitability and RMP are restored by the adrenaline. ** - р\u0026lt;0.01, *** - р\u0026lt;0.001 (one-way ANOVA, DPH \u003cem\u003evs\u003c/em\u003e control).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/c49eadab2d2b093a295aeaf0.png"},{"id":71371021,"identity":"f4596ea3-1ed3-40db-9bca-491994279922","added_by":"auto","created_at":"2024-12-13 19:18:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":857435,"visible":true,"origin":"","legend":"\u003cp\u003eAdrenaline attenuates diphenhydramine-induced alterations of RMP and AP in the rat pulmonary veins and facilitates PV proarrhythmicity.\u003cstrong\u003e A. \u003c/strong\u003eAdrenaline partially restores DPH-suppressed excitability of the distal pulmonary veins (\u003cem\u003etop\u003c/em\u003e) and permits an anterograde conduction of electrically evoked excitation from the ostium to the distal PV (\u003cem\u003eblue arrows\u003c/em\u003e). \u003cstrong\u003eB. \u003c/strong\u003eDPH (30 µM) facilitates automaticity in the rat pulmonary veins in presence of adrenaline. DPH+AD-induced spontaneous APs derived in distal PV (\u003cem\u003etop\u003c/em\u003e) exit the myocardial sleeve (\u003cem\u003eblue arrows\u003c/em\u003e) and elicit excitation in the ostium of PV (\u003cem\u003ebottom\u003c/em\u003e) and left atrial appendage. Retrograde (\u003cstrong\u003eA\u003c/strong\u003e) or anterograde (\u003cstrong\u003eB\u003c/strong\u003e) DPH+AD-induced ‘echo’ waves occur in PV or left atrium, respectively, and re-enters atrial myocardium or pulmonary veins in some excitation cycles (\u003cem\u003ered arrows\u003c/em\u003e). \u003cstrong\u003eC.\u003c/strong\u003e Dose-dependency of a cycle length (CL, top) and occurrence (bottom) of DPH+AD-induced spontaneous activity in PV. * - р\u0026lt;0,05 (Fisher’s exact test).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/d72663e0bb8da650b237b8e0.png"},{"id":71370921,"identity":"78610670-4eeb-474f-a92c-921a53bb88c9","added_by":"auto","created_at":"2024-12-13 19:10:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":369001,"visible":true,"origin":"","legend":"\u003cp\u003eDiphenhydramine suppresses conduction of excitation and causes conduction blocks in the rat pulmonary veins. \u003cstrong\u003eA.\u003c/strong\u003e Consecutive images of di-4-ANEPPS fluorescence obtained during an anterograde (LA to PV) excitation of the pulmonary vein of the left lung lobe in control conditions (\u003cem\u003etop\u003c/em\u003e) and after DPH administration (\u003cem\u003ebottom\u003c/em\u003e). Activated (depolarized) area is shown in yellow and red. \u003cstrong\u003eB.\u003c/strong\u003e Upstroke of the optical AP during an anterograde excitation of PV in control conditions (\u003cem\u003emiddle\u003c/em\u003e) and after DPH administration (\u003cem\u003eright\u003c/em\u003e) as registered by individual photodiodes shown in scheme on the left. \u003cstrong\u003eC.\u003c/strong\u003eIsochronic maps of PV activation in control conditions (\u003cem\u003eleft\u003c/em\u003e), 5 (\u003cem\u003emiddle\u003c/em\u003e) and 20 min (\u003cem\u003eright\u003c/em\u003e) after the beginning of DPH administration. Individual optical signals from points marked in the maps are shown in the bottom. PVO – pulmonary vein ostium. PVB – pulmonary veins branches. Black arrowhead demarks position of a conduction block. \u003cstrong\u003eD.\u003c/strong\u003e Increase of a conduction time (\u003cem\u003eleft\u003c/em\u003e) and decrease of a conduction velocity (CV, \u003cem\u003eright\u003c/em\u003e) induced by DPH in PV. \u003cem\u003eP\u003c/em\u003evalues are shown in the histograms (Freedman’s ANOVA).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/2722bc5e9b4c74a2233c803e.png"},{"id":71370924,"identity":"b8866656-d3ff-49e9-929a-0cdf62c29207","added_by":"auto","created_at":"2024-12-13 19:10:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3776553,"visible":true,"origin":"","legend":"\u003cp\u003ePotential mechanism of IK1 modulation by diphenhydramine (DPH). \u003cstrong\u003eA.\u003c/strong\u003e Cryo-EM structure of the human inward-rectifier potassium channel homotetramer constructed of four Kir2.1 domains (the product of KCNJ2 gene). CTD – cytoplasmic domain, TMD – transmembrane domain, PIP2 - phosphatidylinositol 4,5-bisphosphate binding site, CHL – cholesterol binding site, N-ths – N-terminus helix. \u003cstrong\u003eB.\u003c/strong\u003e Main potential binding pockets (yellow mesh, \u003cem\u003eleft\u003c/em\u003e) for small molecules of human Kir2.1 subunit as predicted by ‘deepsite’ software. Binding core of the binding pocket on the surface of Kir2.1 (site 1) near side helix is shown by red circle. (https://www.playmolecule.com/deepsite/). \u003cstrong\u003eC.\u003c/strong\u003e Binding of DHP in the binding site 1 as predicted by a molecular docking software. D. Surface visualization of human Kir2.1 channel with DPH docked in the predicted binding site.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/a69524dae1375505450bc8d4.png"},{"id":80629641,"identity":"0e19582f-b00e-4351-a0cc-dd088ae2a1ae","added_by":"auto","created_at":"2025-04-15 11:31:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8832330,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5449722/v1/04ea23f5-81d7-4772-9bd3-9a61e2f04d23.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diphenhydramine-mediated modulation of inward rectifier IK1 current induces conduction blocks in the rat pulmonary veins myocardium and facilitates supraventricular proarrhythmicity","fulltext":[{"header":"I. Introduction","content":"\u003cp\u003eDiphenhydramine (DPH) is a first-generation antihistamine drug developed and came into the practice more than 70 years ago. Nowadays DPH extensively used in therapy of allergy and a number of other non-allergic conditions. The medicine is frequently utilized to treat hay fever or to temporarily relive upper respiratory allergies due to its antipruritic and antitussive action.\u003c/p\u003e \u003cp\u003eDPH exhibits inverse agonism for histamine H1 receptors (H1R) belonging to GPCR superfamily (Xia et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A molecular mechanism of DPH action is based on stabilization of the inactive conformation of H1R and shifts the equilibrium towards an inactive state of the receptor (Leurs, Church, and Taglialatela \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) (Shimamura et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Thus, DPH suppresses constitutive agonist-independent activity of H1R and antagonizes histamine-induced H1R-mediated signals transduction in the cells (Bakker et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) (Nijmeijer, Leurs, and Vischer \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Multimodality of DPH action including antiallergic, antiemetic, hypotonic, sedative, anxiolytic and hypnotic properties results from its interaction with H1R on the periphery and in the central nervous system due to ability to cross blood-brain barrier (Welch, Meltzer, and Simons 2002).\u003c/p\u003e \u003cp\u003eDiphenhydramine belongs to arylalkylamine group of compounds; represents classic derivative of ethanolamine-based antihistamines (Simons and Simons \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).(Sneader \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). DPH was firstly marketed in 1946 and, therefore, was introduced to usage before clinical pharmacological investigations were obligatory. Despite a long history the drug-drug interactions, toxicity and pharmacodynamics of DPH still poor investigated for populational groups with preexisting conditions (Simons and Simons \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Similarly with other first-generation H1-antihistamines DPH is not free from various adverse effects in multiple tissues. A cardiac toxicity for ethanolamine H1-antihistamines was firstly reported more than three decades ago (Church et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Adverse effects in the heart of most of ethanolamine-based H1-antihistamines occur due to their potencies with respect to the prolongation of QT interval (Nia et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). DPH causes prolongation of QTc interval in healthy volunteers and patients suffering from coronary diseases (Khalifa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). It has been reported that DPH is able to cause torsade de pointes (TdP) after the administration of standard doses increasing, therefore, a risk of life-threatening ventricular tachyarrhythmias induction (Pratt et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Woosley \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Shah et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mohan et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Electrocardiographic proarrhythmic signs and polymorphic ventricular tachycardia were found in cases of DPH overdose (JOSHI et al. 2004; Sype and Khan \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In 2020 FDA has issued Drug Safety Communication reporting that high doses of DPH causes serious cardiovascular complications like cardiac arrest or ventricular arrhythmia.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSince the administration of DPH in the heart are accompanied by QT interval prolongation the hERG-encoded Kv11.1 potassium channels blockade and a decrease of repolarization reserve are proposed as main mechanisms underlying the proarrhythmic effects of the drug (Suessbrich et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Woosley \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). However, other ion channels responsible for cardiac repolarization as targets for DPH are not studied. In addition, despite revealed torsadogenic potential via ventricular repolarization disturbance (Ali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hoffman et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the influences of DPH in the supraventricular myocardium is not elucidated. A cardiac tissue in the pulmonary veins (PV) is considered recently as proarrhythmic substrate and a main source of ectopic activity initiating atrial fibrillation (Ha\u0026iuml;ssaguerre et al. 1998). The myocardium in PV is highly prone to ectopic automaticity since exhibit abnormal profile of repolarizing currents (Tsuneoka et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the present study the effects of DPH on PV electrophysiology and repolarizing inwardly rectifier (I\u003csub\u003eK1\u003c/sub\u003e) current as potential mediator DPH-induced proarrhythmicity are investigated.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"II. Material and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1. Animals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were carried out in accordance with European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No 123, Strasbourg 1985) and approved by the Ethics Committee of the NMRCC Institute of Experimental Cardiology.\u003c/p\u003e\n\u003cp\u003eMale Wistar rats weighing 240–260 g (10 weeks old; in total 64 animals were used) were provided by the Scientific complex of biomedical technologies animal plant (Moscow region, Russia). Animals were held in the NMRCC animal house for 2 weeks under a 12 h:12 h light:dark photoperiod in standard cages prior to the experiment and fed \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1. In vivo ECG recordings\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to experiments the animals were anesthetized with isoflurane-oxygen gas mixture (3.5 Vol%) delivered via precise vaporizer (VetEquip, USA). After the anesthesia induction the animals were placed onto heated surgical table and the body temperature was stabilized with aid of rectal probe and bio-tc-1 thermocontroller (Biotechnologies, Russia). To maintain proper anesthesia an isoflurane in concentration of 1.5-2 Vol% was delivered continuously via nose cone. Three disposable adhesive electrodes were located in standard positions on the skin surface [Farraj, 2011] and connected to «Animal Bio Amp» amplifier with PL3516 PowerLab 16/35 ADC (ADInstruments, New Zealand) for ECG recording in Lead II. The anesthesia was considered successful if heart rate (HR) was stabilized at 360±20 BPM in 10 min after the initiation. Control 10 min ECG recording was obtained before all further experiment.\u003c/p\u003e\n\u003cp\u003eAfter ECG establishment and control records 300 µL (37⁰C) of physiological solution (vehicle) was injected intravenously (via the lateral tail vein) with aid of Harvard Apparatus syringe pump (PHD Ultra) with rate 300 µL/min. A diphenhydramine dissolved in physiological solution (300 µL) was injected via the lateral tail vein in dose 1 mg/kg with rate 300 µL/min 30 min after the vehicle administration. ECG was recorded continuously 60 min after DPH administration.\u003c/p\u003e\n\u003cp\u003eECG signals were analyzed with use of LabChart Pro software (ADInstruments). A heart rate was calculated on the basis of RR intervals 20 min after vehicle of DPH administration. Also, P, Q and T waves were identified and the duration of PQ, QRS and QT intervals was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eSharp microelectrode recordings of electrical activity in multicellular preparations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were anesthetized as described in previous paragraph. After anesthesia induction the animals were decapitated. The chest was opened and complex supraventricular tissue preparations (LA-PV) including left atrium and its appendage (LAA), pulmonary veins (PV) with lung lobes were rapidly excised. Tissue preparations were separated from surrounding fascia and fat, rinsed from a blood with Tyrode’s solution.\u003c/p\u003e\n\u003cp\u003eA pulmonary vein of left lung lobe with PV branches was incised longitudinally and the preparation was pinned with endocardial side facing upward to the bottom of a 2.5 ml silicone-coated perfusion chamber. \u003c/p\u003e\n\u003cp\u003eIn a separate series of experiments myocardial strips (2.5x10 mm) from a right ventricle wall (RVV) was excised and fixed in the chamber to assess effects of DPH in ventricular myocardium.\u003c/p\u003e\n\u003cp\u003eThe experimental chamber was filled with physiological (Tyrode’s) solution of the following composition (in mM): NaCl 118.0, KCl 4.7, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.2, MgCl\u003csub\u003e2\u003c/sub\u003e 1.8, CaCl\u003csub\u003e2\u003c/sub\u003e 1.8, NaHCO\u003csub\u003e3\u003c/sub\u003e 25.0, glucose 11.0, pH 7.4 ±0.2, bubbled by 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e gas mixture. The constant perfusion with flow rate of 18 mL/min at 37 °C and constant steady-state electrical pacing (CEP) with aid of silver teflon-coated electrodes connected to pulse generator A310 (WPI, USA) with 300 ms cycle length (CL) was started immediately after the preparation of LA-PV or RVV strips. Pacing electrodes were located on the endocardial surface of LAA with aim to induce physiological anterograde pattern of PV excitation.\u003c/p\u003e\n\u003cp\u003eElectrically evoked action potentials (AP), spontaneous activity in a form of automatically occurring action potentials, resting membrane potential (RP or RMP) were recorded simultaneously from the endocardial side of the PV ostium (PVo) and from a distal segment of pulmonary veins (2-3 mm distally to the first order bifurcation of the PV but proximally to the intrapulmonary sites of PV) with aid of glass microelectrodes (10–20 MΩ) filled with 3 M KCl, connected to a multichannel intracellular electrometer (KS-700, WPI instruments, USA). \u003c/p\u003e\n\u003cp\u003eA non-trabeculated cardiac tissue in the ostium of PV was assumed as closest region to PV exhibiting atrial-type of electrical activity due to stable RMP (-80±3 mV) and weak RMP shift in response to cholinergic or adrenergic stimulation (Egorov et al. 2015).\u003c/p\u003e\n\u003cp\u003eSignals were digitized at 10 kHz sampling rate with analog-digital converter (Е-154, ADC L-card, Russia) which allowed calculate APs upstroke velocity. The APs duration at the level of 90% repolarization (APD) was calculated using LGraph2 software (L-Card, Russia). The APs upstroke velocity was calculated as maximum of the depolarization phase derivative (dV/dt\u003csub\u003emax\u003c/sub\u003e) to determine the time moments of PVO or distal PV (PVd) excitations and quality of impalements. Only records with dV/dt\u003csub\u003emax\u003c/sub\u003e \u0026gt; 200 V/s were taken into account. Conduction time was calculated as a time interval between moments of PVO/PVd excitation and a moment corresponding to the pacing stimulus application. The upstroke velocity, amplitude of APs (APA) and RMP were calculated using LGraph2 software (L-Card, Russia).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3. Sharp microelectrode experiments protocols\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing 30 min equilibration period under CEP, control electrically evoked APs were recorded in PV ostium as well as in distal PV for 10 min. In the first type of experiments with LA-PV preparations a diphenhydramine (10, 30 µM) was applied for 20 minutes under CEP after the control APs recording Stable microelectrode impalements were maintained during the whole period of DPH application. \u003c/p\u003e\n\u003cp\u003eIn the second type of experiments 10 min after DPH (30 µM) administration beginning an adrenaline (AD, Sigma-Aldrich, USA) in consecutively increased concentrations (1, 5 or 10 µM) was applied with 20 min intervals. In the third type of experiments CEP was terminated after period of equilibration and 10 min of DPH (30 μM) administration. Spontaneous AP and RMP was recorded in quiescent preparations for at least 10 min in presence of DPH alone (30 μM) or DPH (30 μM) in combination with adrenaline (1-10 µM, Sigma-Aldrich, USA). In the separate series of experiments electrically evoked APs were recorded in RVV strips under CEP in control conditions and at least 20 min after 10 or 30 µM of DPH administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4. Optical mapping of multicellular preparations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pattern of excitation in the pulmonary veins was analyzed using di-4-ANEPPS-based optical mapping technique (Ivanova and Kuzmin 2018; Ivanova et al. 2021). LA-PV tissue preparations were dissected as described in the section 2.2. The optical mapping rig included a photodiodes array (PDA, WuTech H-469V, Gaithersburg, MD, USA) designed for high speed data acquisition (1.63 Kfps). Macroscopic projections of the cardiac tissue preparations were transferred to the PDA with aid of the optical system including adapters and Computar V5013 (CBC Group, Japan) camera lens (focal length 50 mm, aperture ratio 1:1.3) mounted in a distance of 24 mm from a tissue surface. The optical system allowed to project the area of 5 mm in diameter to a hexagonal array of 464 PDA photodiodes. Thus, each photodiode in PDA array covered approximately a surface of 0.23 mm in diameter approximately. The view-field was projected also to the monitoring CMOS camera (MD50, M-Shot, China) used to match the mapping area and sites in the tissue preparations. An excitation light was emitted by LED (520±40 nm) arrays surrounding the experimental chamber with mapped preparation. A long-pass emission filter (λ\u0026gt;650 nm) was positioned in front of the camera lens.\u003c/p\u003e\n\u003cp\u003eAfter dissection, LA-PV preparations were placed into experimental chamber and superfused at 37°C with constant flow (10 ml/min) by physiological solution of the same composition as used for microelectrode experiments. Voltage-sensitive dye di-4-ANEPPS (5 mg/ml, dissolved in DMSO) was added to physiological solution (final concentration 50 µM) for 30 min staining of the preparations. Then preparations were equilibrated for 20 min prior to a mapping procedure. After the equilibration 10 or 30 μM of diphenhydramine was applied for 20 min with 30 min washout intervals. In this series of experiments LA-PV preparations were paced by linear silver electrodes located in the myocardium adjoining to the orifice of the vein. Anterograde (from LA to PV) propagation of excitation with CL 250 ms was induced by steady-state pacing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5. Optical mapping data analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all experiments evoked fluorescent signals (optical AP) from PV were recorded continuously for 5 s with 0.614 ms frame intervals, digitized using a data acquisition system (CardioPDA-III; RedShirtImaging, Decatur, GA, USA) and analyzed using Cardioplex (v.8.2.1, RedShirtImaging) software. The resting fluorescence was determined before each recording. The signals were processed via Savitsky-Golay filter using custom algorithm in order to remove noise and were normalized to the resting fluorescence. Also, minimal high-pass filter was applied to remove long time constant photodiode-derived basal drift. The maximum upstroke derivative (dF/dtmax) for each optical AP was calculated to determine the activation times in the mapped areas. Isochronic activation maps were constructed from activation times using an in-house developed software. The activation time (t\u003csub\u003eact\u003c/sub\u003e) was calculated as time interval between activation of PVO and distal PV on the borders of the mapped area. An averaged conduction velocity (CV) in the PV was calculated as a ratio of the length of the mapped region (4-5.5 mm) and the activation time if the excitation wave demonstrated near linear pattern.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5. \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eWhole-cell patch clamp recordings in isolated cardiomyocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIonic currents were recorded in enzymatically isolated rat cardiomyocytes. Prior to isolation the animals were intraperitoneally injected with heparin, then anaesthetized and decapitated as described above. The heart was excised, mounted onto a Langendorff apparatus and retrogradely perfused through aorta with nominally Ca\u003csup\u003e2+\u003c/sup\u003e-free physiological solution containing (in mM): NaCl 116; KCl 4; NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e1.7; NaHCO\u003csub\u003e3\u003c/sub\u003e25; MgCl\u003csub\u003e2\u003c/sub\u003e0.55; sodium pyruvate 5; taurine 20; glucose 11; 1 g/ml bovine serum albumin; pH 7.4±0.2 bubbled by 95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e gas mixture at 37\u003csup\u003eo\u003c/sup\u003eC. After 5-7 minutes, the perfusion was switched to a solution of the same composition containing 0.5 mg/ml collagenase II (Worthington, USA), 0.025 mg/mL protease XIV (Sigma Aldrich, USA) and 6 μM CaCl\u003csub\u003e2\u003c/sub\u003e. After 40-50 min of enzymatic treatment the perfusion was stopped, the ventricles were minced and gently triturated to liberate the cardiomyocytes into “Kraftbrühe” medium containing (in mM): MgSO\u003csub\u003e4\u003c/sub\u003e3; KCl 30; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e30; EGTA 0.5; potassium glutamate 50; HEPES 20; taurine 20; glucose 10; pH 7.2 adjusted with KOH at 24\u003csup\u003eo\u003c/sup\u003eC (Isenberg and Klockner 1982). The cardiomyocytes were stored in this medium at room temperature and used within 6 hours after the isolation. The capacitance of the ventricular myocytes used in the experiments was 151.6±4.5 (n=50) pF.\u003c/p\u003e\n\u003cp\u003eInward rectifier I\u003csub\u003eK1\u003c/sub\u003e current in ventricular myocytes was recorded using conventional whole-cell patch clamp using a HEKA EPC-800 amplifier (HEKA Elektronik, Germany). Isolated cardiomyocytes were placed into an experimental chamber (RC-26; Warner Instrument Corp., Brunswick, CT, USA; volume 150 μl) mounted onto an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003eThe cells were perfused with Tyrode’s based physiological solution. Potassium I\u003csub\u003eK\u003c/sub\u003e\u003csub\u003e1\u003c/sub\u003e current was recorded at room temperature (24⁰C) using K\u003csup\u003e+\u003c/sup\u003e-based solution containing (in mM): NaCl 150; KCl 3; CaCl\u003csub\u003e2\u003c/sub\u003e 1.8; MgCl\u003csub\u003e2\u003c/sub\u003e 1.2; glucose 10; HEPES 10; pH 7.4 adjusted with NaOH.\u003c/p\u003e\n\u003cp\u003ePatch pipettes were pulled from borosilicate glass capillaries without filament (Sutter Instruments, CA, USA) and filled with a pipette solution. The pipette solution used for I\u003csub\u003eK1\u003c/sub\u003e recording contained (in mM): KCl 140; MgCl\u003csub\u003e2\u003c/sub\u003e 1; EGTA 5; HEPES 10; MgATP 4; Na\u003csub\u003e2\u003c/sub\u003eGTP 0.03; pH 7.2 adjusted with KOH at 24\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003eThe resistance of filled patch-pipettes was within a range of 2-3 MΩ. Recorded current was normalized by cell capacitance and presented as current density (pA/pF). The obtained data were analyzed using Clampfit 10.3 software (Molecular Devices, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was carried out using GraphPad Prism version 7. The normality of the groups was tested using the Shapiro-Wilk’s test where needed. Hypothesis testing was carried out using Freedman’s or one-way ANOVA with further Sidak’s post-hoc multiple comparisons where appropriate. Also, one-tailed paired Student’s t-test or Fisher’s exact test were used where appropriate. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05 was considered statistically significant. All results are expressed as means±SD except patch-clamp data where they are expressed as mean±SEM for n experiments and data regarding cycle length of spontaneous AP in PV where they expressed as median and IQR.\u003c/p\u003e"},{"header":"III. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Effect of DPH on heart rate and ECG parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA systemic delivery of DPH (1 mg/kg) did not alter a heart rate over the first 20 min of post-injection period in rats (Fig.1A, n=5). Similar HR values were observed in DPH-treated and control (vehicle-treated) rats 20 min after the injections (357±21 and 361±34 bpm, respectively, n=5, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.1). No P-wave alternans, tachy- or bradycardia episodes, tachyarrhythmia episodes or ventricular extrasystoles were observed in rats \u003cem\u003ein vivo\u003c/em\u003e 0-60 min after the DPH administration (Fig.1B).\u003c/p\u003e\n\u003cp\u003eAmong ECG characteristics the diphenhydramine induced a significant prolongation of QT interval in rats. The duration of QT interval 20 min after the administration of 1 mg/kg of DPH was 95±16 ms (n=5) in comparison to 83±17 ms in vehicle-treated animals (n=5, Fig.1C, D). Unlike to QT, diphenhydramine was unable to alter substantially the duration of QRS and PQ interval (Fig.1D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. \u003c/strong\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003eDPH \u003c/strong\u003e\u003cstrong\u003eon inward rectifier (I\u003csub\u003eK1\u003c/sub\u003e) current in ventricular myocytes and ventricular AP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn inward rectifier I\u003csub\u003eK1\u003c/sub\u003e current was changed by the DPH in the ventricular rat cardiomyocytes. When DPH was administered (10 µM, 20 min) both inward and outward components of I\u003csub\u003eK1\u003c/sub\u003e were significantly reduced. At -120 mV the current densities were -9.8±3.7 (n=6) and -8.1±3.1 pA/pF (n=7) in control measurements and after DPH administration, respectively. Diphenhydramine significantly suppressed the peak value of the outward component of I\u003csub\u003eK1 \u003c/sub\u003eat -70 mV by 34% (0.53±0.20 (n=6) and 0.35±0.22 pA/pF (n=7), respectively, Fig.2A, B).\u003c/p\u003e\n\u003cp\u003eIn experiments with isolated tissue strips PDH caused significant prolongation of the ventricular APs. The DHP-induced increase of AP duration is time-dependent and reached 35.4% when was measured 20 min after the beginning of DPH (10 µM) administration (19.2±1.3 (n=5) and 26.0±1.0 ms (n=5) in control and under DPH action, respectively, p=0.01, Fig.2C). Unlike to AP duration, RMP remained unchanged by DPH in RVV (10 µM, 20 min): -78±4 and -80±5 mV in controls and in presence of DPH, respectively (n=5, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. \u003c/strong\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cstrong\u003eDPH on pulmonary veins excitability and action potentials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA diphenhydramine applied in concentrations 10 or 30 µM (20 min) was unable to affect substantially RMP, AP amplitude or duration of electrically evoked AP in PV ostium. Nevertheless, administration of DPH in high concentration (30 µM) resulted in a significant increase of conduction time in the atrial myocardium (19±4%, \u003cem\u003eр\u003c/em\u003e\u0026lt;0,001, n=6, Fig.3A-D, Table 1). \u003c/p\u003e\n\u003cp\u003eIn control experiments distal PV myocardium exhibits normal RMP and APs with fast upstroke, atrial-like amplitude (91±3 mV, n=6) and waveform in presence of steady-state electrical stimulation (Table 1). The administration DPH results in a pronounced alteration of bioelectrical parameters in the cardiac tissue of distal PV. High concentration of DPH (30 µM, 20 min) causes significant RMP shift (from -83±1 to -64±2 mV, n=6, \u003cem\u003eр\u003c/em\u003e\u0026lt;0,001). In all experiments (n=8) DPH causes suppression of the excitability in the distal PV manifesting in a lack of active response to electrical stimulation and only electrotonic-like low-voltage potentials with amplitude 12±2 mV and negligible duration. Also, DPH significantly increased time interval between the moments of pacing stimulus application and electric response in the distal PV (Fig.3A-D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. \u003c/strong\u003e\u003cstrong\u003e Adrenergic modulation of DPH effects in the pulmonary vein myocardium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of DPH in PV was substantially altered by an adrenaline. The adrenaline partially and dose-dependently (1-10 µM, n=6) attenuated DPH-induced (30 µM) shift of RMP in the distal PV as well as restored the excitability and the amplitude of APs in the pulmonary veins (Fig.3A, B, Table 1). In presence of doth AD and DPH resting potential in the ostium of PV remained unchanged. Only maximal used concentration of AD (10 µM) reduced AP amplitude in the ostium of PV in presence of DPH (30 µM).\u003c/p\u003e\n\u003cp\u003eIn contrast to RMP and APA, the adrenaline changed the conduction time and AP duration in the ostium and distal PV synergically with DPH. The administration of AD dose-dependently (1-10 µM, n=6) and significantly increased DPH-induced (30 µM) elevation of the conduction time and, therefore, delayed the excitation of the distal sites of PV (Fig.3C, Table 1). This additive effect of AD and DPH was observed despite the recovery of RMP.\u003c/p\u003e\n\u003cp\u003eThe recovery of the DHP-suppressed excitability by AD allowed to estimate AP duration in the distal PV. In presence of AD (1-10 µ) and DPH (30 µ) the duration of electrically evoked APs in the distal PV was more than twice longer than the AP duration in control conditions. Unlike to ventricular APs the diphenhydramine (10, 30 µM) was unable to prolongate APs duration in PVO. However, adrenaline potentiated effect of DPH in the ostium of PV and caused significant prolongation of APs duration (Fig.3D, Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. \u003c/strong\u003e\u003cstrong\u003e Modulation of pulmonary veins ectopy by DPH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo re-entrant circulation of the excitation was observed in the electrically paced LA-PV preparations in presence of DPH (10, 30 µM, n=6) or adrenaline (10 µM, \u003cem\u003edata not show\u003c/em\u003en) used separately in these series of experiments. Nevertheless, AD (5 µM) in presence of DPH (30 µM) induced re-entrant circulation including anterograde (primary, pacing-induced) LA-to-PV and retrograde ‘echo’ PV-to-LA wave propagation in 2 out of 6 experiments (Fig.4A).\u003c/p\u003e\n\u003cp\u003eDPH (10, 30 µM, n=6) is unable to induce ectopic AP (spontaneous activity) in the quiescent (non-paced) pulmonary veins. However, administration of adrenaline dose-dependently potentiated ability of DPH to elicit a spontaneous activity in PV. In our experiments, 1-10 µM of AD in presence of DPH caused PV-derived spontaneous activity capable of activating cardiac tissue in the ostium of PV and left atrium (Fig.4B). The spontaneous activity induced by a combination of AD and DPH manifested in a form of permanent, non-burst APs occurring with quasi-constant intervals (CL\u0026gt;3050 (n=1), 1700±430 (n=6) and 880±200 (n=6) ms for 1, 5 and 10 µM of AD, Fig.4B). When 5 or 10 µM of AD was applied spontaneous activity occurred in all cases and was accompanied by re-entrant episodes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Effects of \u003c/strong\u003e\u003cstrong\u003eDPH\u003c/strong\u003e\u003cstrong\u003e on conduction of excitation in the pulmonary veins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRat pulmonary veins are characterized by consecutive, almost linear pattern of anterograde conduction of the excitation wave lacked of any conduction disturbances or conduction blocks when the excitation elicited by continuous electrical pacing of the left atria (CL=250 ms, Fig.5A, B). The conduction velocity in PV in control set of experiments was 0.74±0.05 mm/ms (n=5) which is very close to CV typical for ‘working’ atrial myocardium. The administration of DPH dose- and time-dependently affects pattern of excitation of PV. The conduction velocity was slightly decreased while activation time was slightly reduced 5 min after 10 µM of DPH (n=5) administration. These changes were aggravated and significant after 20 min of DPH (10 µM, n=5) administration: CV and t\u003csub\u003eact\u003c/sub\u003e reduced to 0.37±0.05 mm/ms and 13.49±0.75 ms (n=5, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), respectively. Conduction blocks were initiated by DPH (20 min, 10 µM) in 4 out of 5 experiments in PV branches at post-bifurcation level (Fig.5B-D). DPH applied in 30 µM concentration rapidly induced blockade of the electrically initiated anterograde excitation in the trunk of PV at sites adjacent to PV ostium in all experiments (n=3).\u003c/p\u003e"},{"header":"IV. Discussion","content":"\u003cp\u003eIn the present study we have demonstrated for the first time that H1-antihistamine diphenhydramine induces substantial prolongation of AP in the rat atrial and ventricular myocardium; causes substantial proarrhythmic changes including depolarization, inexcitability, slowing of the conduction velocity, conduction blocks and facilitates PV ectopy in a presence of adrenergic stimulation in the cardiac tissue of the pulmonary veins. These effects in the rat myocardium are mediated by DPH-induced inward rectifier current IK1 attenuation.\u003c/p\u003e\n\u003cp\u003eIn our experiments we were unable to found episodes of atrial fibrillation or flutter as well as TdP in rats. On the other hand, \u003cem\u003ein vivo\u003c/em\u003e experiments revealed obvious QT intervals prolongation, which is torsadogenic, in response to DPH administration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been demonstrated previously, that DPH in doses \u0026gt;500 mg induces bundle branch blocks, prolongation of QTc, increases the slope of QT-RR relationship curve, increases the duration and lowers the amplitude of T-wave. These effects are accompanied with sinus tachycardia (Zareba et al. 1997; Hestand and Teske 1977). It is known that DPH-caused QT prolongation in patients not always result in TdP. The DPH-overdose induced sinus tachycardia occurs due to its cholinolytic effect via a central attenuation of parasympathetic tone (JOSHI et al. 2004). Increased HR may reduce the likelihood of TdP since a shorter cycle length and decreased AP duration diminish the probability of proarrhythmic afterdepolarizations. Thus, intrinsic high heart rate in rats may protect ventricular myocardium against DPH-induced TdP in our experiments.\u003c/p\u003e\n\u003cp\u003eIt has been shown in several studies that DPH attenuates rapid component of a delayed rectifier IKr current. This effect of DPH is thought to be based on the ability of the drug to block hERG (Kcnj2) gene encoded Kv11.1 ion potassium channels. The blockade of hERG-channels by H1-antihistamines including DPH has been demonstrated in experimental studies utilizing heterologous expression systems and laboratory animals like guinea pigs (Suessbrich et al. 1996; Jo et al. 2009; Khalifa et al. 1999; Park, Kim, and Kim 2008).\u0026nbsp;It is well known that numerous small molecules demonstrate an ability to act as a blocker for Kv11.1 channel due to specific amino acid residues architecture of its pore cavity interior (Sanguinetti and Tristani-Firouzi 2006; Sanguinetti and Mitcheson 2005). Many compounds among antihistamines besides DPH also exhibit Kv11.1 blocking potency (Taglialatela, Timmerman, and Annunziato 2000). The histamine receptor antagonists chlorpheniramine \u0026lsquo;weakly\u0026rsquo; blocked hERG channels with IC50 values of 20.9\u0026plusmn;2.1 \u0026micro;M. This value for DPH was estimated as 27.1\u0026plusmn;1.5 \u0026micro;M (Suessbrich et al. 1996) and was also considered as rather weak. Nevertheless, prolongation of QT intervals in patients by DPH is thought to be mediated by hERG channels blockade.\u003c/p\u003e\n\u003cp\u003eIn our experiments we utilized tissue preparations and isolated cardiomyocytes from rat heart. It is well known that adult rat atrial and ventricular myocardium is lacked of delayed rectifier IKr. Thus, the prolongation of AP which was observed in our experiments in presence of DPH cannot be attributed with blockade of Kv11.1 channels and attenuation of IKr current. It is known that inward rectifier IK1 underlies not only a resting potential stabilization but also substantially contributes to AP repolarization in rodents. As has been mentioned above, DPH caused I\u003csub\u003eK1\u003c/sub\u003e suppression in our experiment. The attenuation of I\u003csub\u003eK1\u003c/sub\u003e current by several H1- an H2-antihistamines of various generations has been shown previously in experiments with geterologous expression systems (Liu et al. 2007). A reduction of functional outward component of I\u003csub\u003eK1\u003c/sub\u003e in ventricular myocytes along with QT prolongation and changes observed in rat PV cumulatively support the suggestion that inward rectifier is a target of DPH additional to IKr contributing the drug-induced proarrhythmicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is demonstrated that thoracic veins exhibit differential subunit composition of heterotetrameric channels responsible for native I\u003csub\u003eK1\u003c/sub\u003e current in the atrial and ventricular myocardium. The most abundant Kir subunit contributing to the native channels in the myocardium of rat thoracic veins is Kir2.2 while Kir2.3 is extremely minor (Ivanova et al. 2021; Melnyk et al. 2002). The inhibition by DPH assessed in \u003cem\u003eXenopus\u003c/em\u003e oocytes was found mainly for Kir2.3-containig channels while Kir2.1 was significantly less sensitive to antihistamines (Liu et al. 2007).\u003c/p\u003e\n\u003cp\u003eIt has been also shown that cardiomyocytes of thoracic veins wall are characterized by a smaller I\u003csub\u003eK1\u003c/sub\u003e current density and reduced overall expression of Ki2.x subunits in comparison to atrial cardiomyocytes. It should be noted that the myocardium in the pulmonary veins ostium demonstrates AP and RMP level very similar to that observed in LA (Egorov et al. 2015; 2019). Thus, PVo was considered in our study as a region demonstrating atrial-like electrophysiology. Noteworthy, that DPH-induced changes in PVo electrophysiology were less prominent than in changes in distal PV and ventricular myocardium. The administration of DPH in our experiments resulted in a strong sift of the resting potential to less negative values (depolarization) in the PV myocardium while the shift of RMP was negligible in ventricular strips. Thus, weak attenuation of the small I\u003csub\u003eK1\u003c/sub\u003e current composed mainly of Kir.2.1/Ki2.2 subunits in the PV results in distinctive functional changes in contrast to the effects observed in the cardiac tissue surrounding PV ostium or in the ventricle.\u003c/p\u003e\n\u003cp\u003eIn the present the alteration of I\u003csub\u003eK1\u003c/sub\u003e by DPH was directly measured and I-V curve was reconstructed. Nevertheless, this provides little information regarding a mode by which PDH affects Kir2.x channels.\u003c/p\u003e\n\u003cp\u003eWe used reconstructed Cryo-EM human (KNCJ2) and rats Kir2.1 \u0026nbsp;subunit structures of the tetrameric functional IKir channels (Fernandes et al. 2022) provided by \u0026lsquo;AlphaFold\u0026rsquo; protein structures database (P63252, Q64273, Fig.6, A) to predict potential sites of DPH binding (https://doi.org/10.2210/pdb7ZDZ/pdb). Using free AI-based DeepSite module (https://www.playmolecule.com/deepsite/) from a software platform PlayMolecule provided by D-Acellera (UK) we analyzed Kir2.1 channel subunit and found ability of the protein to form several binding pockets outside of the pore-forming region of the tetrameric channel ensemble. Predictive analysis revealed several deep binding pockets (BP) that highly-suitable to venue small molecules like DPH. In particular, BP may be formed by a bend between transmembrane M1 \u0026alpha;-helix and small side \u0026alpha;-helix (Fig.6., B, site 1) as well as by an unfolded region between transmembrane helices and submembrane \u0026beta;-sheet (site 2) or by cytoplasmic \u0026beta;-sheet (site 3). Noteworthy, the BP in site 1 borders an intramembrane and submembrane cytoplasmic region of the Kir2.1. molecule.\u003c/p\u003e\n\u003cp\u003eFurther, we used molecular dynamics-based docking software utilizing multiobjective evolutionary optimization (http://www.swissdock.ch/; https://mcule.com/apps/1-click-docking/) to predict potential sites of interactions of the rat or human Kir2.1 subunits and DPH molecule and estimate a probability of DPH binding (Grosdidier, Zoete, and Michielin 2007). The modelling of DPH/Kir2.1 interaction revealed more than 50 clusters capable of DPH binding. Nevertheless, the clusters with a top cumulative rank and highest \u0026Delta;G energy decrease are colocalized with the binding pockets predicted by the evaluation of the protein surface with aid of \u0026lsquo;deepsite\u0026rsquo; software. Estimated \u0026Delta;G energy of DPH interaction in the cluster corresponding to the site 1 is -7.53 kcal/mol and full fitness parameter is -2178.13 kcal/mol that demonstrates high probability of the binding. These computational data allow to suppose that DHP attenuates I\u003csub\u003eK1\u003c/sub\u003e current not as a channel pore blocker but as channel gating modifier or via disruption of ion channel interaction with lipides of inner membrane leaflet. It should be noted that DPH-induced AP prolongation in ventricular strips was weakly voltage-dependent and reached its maximum during 10-30 minutes. This observation allows to suppose that DPH acts upon its slow accumulation in the lipide phase.\u003c/p\u003e\n\u003cp\u003eIt is well known that a gating of Kir2.1 channels is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Hibino et al. 2010; Xie et al. 2008). The binding of PIP2 is required to trigger conformational changes that switch the channel into the open state. A PIP2-dependent gating is possible due to structural interactions of PIP2-binding site and the constriction formed by the G-loop in the cytoplasmic domain of the channel (Fernandes et al. 2022). The binding of PIP2 leads the release of the G-loop constriction in the channel pore. The binding site for PIPI2 is located at the interface between the transmembrane (TMD) and cytoplasmic domains (CTD) and is coordinated by several positively charged amino acid residues (Whorton and MacKinnon 2011; Hansen, Tao, and MacKinnon 2011). The site occupies the space between outer M1 \u0026alpha;-helix and interfacial small side helix (Fig.6.A). PIP2-binding site is colocalized with DP \u0026lsquo;site 1\u0026rsquo; and \u0026lsquo;site 2\u0026rsquo; as well as with predicted binding site for DPH (Fig.6.C, D). Thus, structurally confirmed data and \u003cem\u003ein silico\u003c/em\u003e prediction allow us to speculate that DPH is able to enter vicinity occupied by the membrane bound PIP2 or displace PIP2 inside its binding site or at least affect PIP2-Kir2.1 interaction in some manner. Aforementioned DPH-caused disruption of PIP2-Kir2.1 may lead to the G-loop-dependent decrease of the channel availability for K\u003csup\u003e+\u003c/sup\u003e ions resulting in functional effects like RMP shift, AP prolongation. The regulation of Kir2.x subunits via modulation of its interaction with PIP2 is not surprising and was demonstrated for arachidonic acid (Wang et al. 2008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was supposed that Kir2.3 is the most sensitive to antihistamines since the strength of channel-PIP2 interactions determines the susceptibility to the modulation and Kir2.3 subunit exhibits lowest affinity to PIP2 (Du et al. 2004). Native ventricular Kir (IK1) channels are Kir2.1-rich, and therefore, are expected to be weakly sensitive to DHP. Nevertheless, we observed strong effects of DPH-induced attenuation of I\u003csub\u003eK1\u003c/sub\u003e in the rat ventricles. This is not contradictory since many factors and membrane components beside PIP2 may affect Kir-PIP2 interaction.\u003c/p\u003e\n\u003cp\u003eNoteworthy, that Kir channels exhibit several binding sites for cholesterol (CHL) which is also considered recently as potent Kir modulator (Rosenhouse-Dantsker 2019). It is shown that PIP2 and CHL are in close interplay in Kir modulation. One of the CHL binding sites is located on the border of TMD and CTD superiorly to the side helix in Kir2.1 in close proximity to the binding site of PIP2 (Rosenhouse-Dantsker, Epshtein, and Levitan 2014) and coincides with the predicted DP and most favorable DPH docking region (Fig.6). Notable, the enrichment of \u003cem\u003eXenopus\u003c/em\u003e oocytes with CHL may results both in attenuation or enhancement of Kir-mediated ion current depending on subunit structures (Rosenhouse-Dantsker et al. 2010; Corradi et al. 2022). Thus, a competition of DHP and CHL for Kir2.x subunits may significantly affect I\u003csub\u003eK1\u003c/sub\u003e current. In addition, the abundancy of CHL and composition CHL derivatives bound in membrane lipid rafts is highly variable in different cell populations. This fact introduces additional uncertainty to the estimation to the DPH inhibitory potential and to the elucidation of mechanism of DPH action.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the allosteric modulation of Kir2.x channel responsible for IK1 current may be considered as additional mechanism of DPH-mediated proarrhythmicity. Our results demonstrate that adverse proarrhytmic effect of DPH may be expanded beyond the ventricular myocardium facilitating PV-derived profibrillatory activity. The ability of DPH to promote atrial arrhythmias can manifest mainly in a preliminary remodeled supraventricular myocardium or in cases of altered autonomic control of the heart.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interests\u003c/h2\u003e\n\u003cp\u003eAll authors declare no conflict of interests.\u003c/p\u003e\n\u003ch2\u003ePersonal contribution\u003c/h2\u003e\n\u003cp\u003eYury V. Egorov -microelectrode experiments with pulmonary veins;\u003c/p\u003e\n\u003cp\u003eAlexandr A. Abramov – in vivo experiments, manipulations with animals\u003c/p\u003e\n\u003cp\u003eYana A. Voronina – sharp microelectrode experiments with usage of isolated tissue preparations;\u003c/p\u003e\n\u003cp\u003eTatiana S. Filatova- patch-clamp experiments;\u003c/p\u003e\n\u003cp\u003eOksana B. Pustovit – isolation of tissue preparations;\u003c/p\u003e\n\u003cp\u003eAndrew M. Karhov – molecular dynamics and docking modeling,\u003c/p\u003e\n\u003cp\u003eVlad S. Kuzmin – conceptualization, data analysis, manuscript writing;\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eRussian Science Foundation grant 22-15-00189\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eury V. Egorov -microelectrode experiments with pulmonary veins;Alexandr A. Abramov – in vivo experiments, manipulations with animalsYana A. Voronina – sharp microelectrode experiments with usage of isolated tissue preparations;Tatiana S. Filatova- patch-clamp experiments;Oksana B. Pustovit – isolation of tissue preparations;Andrew M. Karhov – molecular dynamics and docking modeling,Vlad S. Kuzmin – conceptualization, data analysis, manuscript writing;\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis study is supported by Russian Science Foundation grant 22-15-00189 to Vlad S Kuzmin.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll experimental data will be accessable in the cloud on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAli, Zahid, Mohammad Ismail, Fahadullah Khan, and Hira Sajid. 2021. \u0026ldquo;Association of H1-Antihistamines with Torsade de Pointes: A Pharmacovigilance Study of the Food and Drug Administration Adverse Event Reporting System.\u0026rdquo; \u003cem\u003eExpert Opinion on Drug Safety\u003c/em\u003e 20 (1): 101\u0026ndash;8. https://doi.org/10.1080/14740338.2021.1846717.\u003c/li\u003e\n\u003cli\u003eBakker, Remko A, Kerstin Wieland, Henk Timmerman, and Rob Leurs. 2000. \u0026ldquo;Constitutive Activity of the Histamine H1 Receptor Reveals Inverse Agonism of Histamine H1 Receptor Antagonists.\u0026rdquo; \u003cem\u003eEuropean Journal of Pharmacology\u003c/em\u003e 387 (1): R5\u0026ndash;7. https://doi.org/10.1016/S0014-2999(99)00803-1.\u003c/li\u003e\n\u003cli\u003eChurch, M. 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Estelle R., and Keith J. Simons. 2011. \u0026ldquo;Histamine and H1-Antihistamines: Celebrating a Century of Progress.\u0026rdquo; \u003cem\u003eJournal of Allergy and Clinical Immunology\u003c/em\u003e 128 (6): 1139-1150.e4. https://doi.org/10.1016/j.jaci.2011.09.005.\u003c/li\u003e\n\u003cli\u003eSneader, W. 2001. \u0026ldquo;Histamine and the Classic Antihistamines.\u0026rdquo; \u003cem\u003eDrug News \u0026amp; Perspectives\u003c/em\u003e 14 (10): 618\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eSuessbrich, H., S. Waldegger, F. Lang, and A.E. Busch. 1996. \u0026ldquo;Blockade of HERG Channels Expressed in \u003cem\u003eXenopus\u003c/em\u003e Oocytes by the Histamine Receptor Antagonists Terfenadine and Astemizole.\u0026rdquo; \u003cem\u003eFEBS Letters\u003c/em\u003e 385 (1\u0026ndash;2): 77\u0026ndash;80. https://doi.org/10.1016/0014-5793(96)00355-9.\u003c/li\u003e\n\u003cli\u003eSype, John W., and Ijaz A. Khan. 2005. \u0026ldquo;Prolonged QT Interval with Markedly Abnormal Ventricular Repolarization in Diphenhydramine Overdose.\u0026rdquo; \u003cem\u003eInternational Journal of Cardiology\u003c/em\u003e 99 (2): 333\u0026ndash;35. https://doi.org/10.1016/j.ijcard.2003.11.035.\u003c/li\u003e\n\u003cli\u003eTaglialatela, Maurizio, Henk Timmerman, and Lucio Annunziato. 2000. \u0026ldquo;Cardiotoxic Potential and CNS Effects of First-Generation Antihistamines.\u0026rdquo; \u003cem\u003eTrends in Pharmacological Sciences\u003c/em\u003e 21 (2): 52\u0026ndash;56. https://doi.org/10.1016/S0165-6147(99)01437-6.\u003c/li\u003e\n\u003cli\u003eTsuneoka, Yayoi, Masahiko Irie, Yusuke Tanaka, Takahiko Sugimoto, Yuka Kobayashi, Taichi Kusakabe, Keisuke Kato, Shogo Hamaguchi, Iyuki Namekata, and Hikaru Tanaka. 2017. \u0026ldquo;Permissive Role of Reduced Inwardly-Rectifying Potassium Current Density in the Automaticity of the Guinea Pig Pulmonary Vein Myocardium.\u0026rdquo; \u003cem\u003eJournal of Pharmacological Sciences\u003c/em\u003e 133 (4): 195\u0026ndash;202. https://doi.org/10.1016/j.jphs.2016.12.006.\u003c/li\u003e\n\u003cli\u003eWang, Chuan, Uyenlinh L. Mirshahi, Boyi Liu, Zhanfeng Jia, Tooraj Mirshahi, and Hailin Zhang. 2008. \u0026ldquo;Arachidonic Acid Activates Kir2.3 Channels by Enhancing Channel-Phosphatidyl-Inositol 4,5-Bisphosphate Interactions.\u0026rdquo; \u003cem\u003eMolecular Pharmacology\u003c/em\u003e 73 (4): 1185\u0026ndash;94. https://doi.org/10.1124/mol.107.043067.\u003c/li\u003e\n\u003cli\u003eWelch, Michael J, Eli O Meltzer, and F Estelle R Simons. 2002. \u0026ldquo;H1-Antihistamines and the Central Nervous System.\u0026rdquo; \u003cem\u003eClinical Allergy and Immunology\u003c/em\u003e 17: 337\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eWhorton, Matthew R., and Roderick MacKinnon. 2011. \u0026ldquo;Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G Proteins, PIP2, and Sodium.\u0026rdquo; \u003cem\u003eCell\u003c/em\u003e 147 (1): 199\u0026ndash;208. https://doi.org/10.1016/j.cell.2011.07.046.\u003c/li\u003e\n\u003cli\u003eWoosley, R L. 1996. \u0026ldquo;Cardiac Actions of Antihistamines.\u0026rdquo; \u003cem\u003eAnnual Review of Pharmacology and Toxicology\u003c/em\u003e 36 (1): 233\u0026ndash;52. https://doi.org/10.1146/annurev.pa.36.040196.001313.\u003c/li\u003e\n\u003cli\u003eXia, Ruixue, Na Wang, Zhenmei Xu, Yang Lu, Jing Song, Anqi Zhang, Changyou Guo, and Yuanzheng He. 2021. \u0026ldquo;Cryo-EM Structure of the Human Histamine H1 Receptor/Gq Complex.\u0026rdquo; \u003cem\u003eNature Communications\u003c/em\u003e 12 (1): 2086. https://doi.org/10.1038/s41467-021-22427-2.\u003c/li\u003e\n\u003cli\u003eXie, Lai‐Hua, Scott A. John, Bernard Ribalet, and James N. Weiss. 2008. \u0026ldquo;Phosphatidylinositol‐4,5‐bisphosphate (PIP \u003csub\u003e2\u003c/sub\u003e ) Regulation of Strong Inward Rectifier Kir2.1 Channels: Multilevel Positive Cooperativity.\u0026rdquo; \u003cem\u003eThe Journal of Physiology\u003c/em\u003e 586 (7): 1833\u0026ndash;48. https://doi.org/10.1113/jphysiol.2007.147868.\u003c/li\u003e\n\u003cli\u003eZareba, Wojciech, Arthur J Moss, Spencer Z Rosero, Raef Hajj-Ali, JoAnne Konecki, and Mark Andrews. 1997. \u0026ldquo;Electrocardiographic Findings in Patients With Diphenhydramine Overdose.\u0026rdquo; \u003cem\u003eThe American Journal of Cardiology\u003c/em\u003e 80 (9): 1168\u0026ndash;73. https://doi.org/10.1016/S0002-9149(97)00634-6.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Effect of diphenhydramine (DPH) on resting potential (RP) and electrically evoked action potentials (AP) in the pulmonary veins ostium (PVo) and distal pulmonary veins (PVd).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"613\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 9.28339%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRP-PVo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7492%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRP-PVd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8.95765%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAPA-PVo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0749%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAPA-PVd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4235%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAPD-PVo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAPD-PVd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eТ-PV\u003c/strong\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eТ-PVd (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003econtrol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.28339%;\"\u003e\n \u003cp\u003e-82\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7492%;\"\u003e\n \u003cp\u003e-82\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8.95765%;\"\u003e\n \u003cp\u003e99\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0749%;\"\u003e\n \u003cp\u003e91\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4235%;\"\u003e\n \u003cp\u003e66\u0026plusmn;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e54\u0026plusmn;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e0\u0026plusmn;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e0\u0026plusmn;0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDPH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.28339%;\"\u003e\n \u003cp\u003e-82\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7492%;\"\u003e\n \u003cp\u003e-64\u0026plusmn;2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8.95765%;\"\u003e\n \u003cp\u003e88\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0749%;\"\u003e\n \u003cp\u003e12\u0026plusmn;2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4235%;\"\u003e\n \u003cp\u003e76\u0026plusmn;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e2\u0026plusmn;1***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e19\u0026plusmn;4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e25\u0026plusmn;3***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDPH + AD(1\u0026mu;М)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.28339%;\"\u003e\n \u003cp\u003e-82\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7492%;\"\u003e\n \u003cp\u003e-75\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8.95765%;\"\u003e\n \u003cp\u003e92\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0749%;\"\u003e\n \u003cp\u003e77\u0026plusmn;8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4235%;\"\u003e\n \u003cp\u003e83\u0026plusmn;6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e103\u0026plusmn;5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e25\u0026plusmn;9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e71\u0026plusmn;20***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDPH + AD(5\u0026mu;М)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.28339%;\"\u003e\n \u003cp\u003e-82\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7492%;\"\u003e\n \u003cp\u003e-75\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8.95765%;\"\u003e\n \u003cp\u003e93\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0749%;\"\u003e\n \u003cp\u003e68\u0026plusmn;4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4235%;\"\u003e\n \u003cp\u003e76\u0026plusmn;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e112\u0026plusmn;12***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e47\u0026plusmn;9***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e83\u0026plusmn;14***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDPH + AD(10\u0026mu;М)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.28339%;\"\u003e\n \u003cp\u003e-79\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7492%;\"\u003e\n \u003cp\u003e-65\u0026plusmn;4***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8.95765%;\"\u003e\n \u003cp\u003e84\u0026plusmn;2**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0749%;\"\u003e\n \u003cp\u003e49\u0026plusmn;5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.4235%;\"\u003e\n \u003cp\u003e78\u0026plusmn;6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e104\u0026plusmn;11***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e79\u0026plusmn;17***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.3779%;\"\u003e\n \u003cp\u003e177\u0026plusmn;30***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAPA \u0026ndash; AP amplitude, APD \u0026ndash; AP duration at level of 90% repolarization, AD \u0026ndash; adrenaline. T-PVo, T-PVd - increase in the time of conduction from a left atria appendage to the pulmonary vein ostium or distal portion of PV.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"diphenhydramine, H1-dntihistamines, supraventricular arrhythmia, QT intervals, conduction blocks, pulmonary veins, inward rectifier, Kir2.x channels.","lastPublishedDoi":"10.21203/rs.3.rs-5449722/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5449722/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiphenhydramine (DPH) is a first-generation antihistamine drug widely used for allergy and other non-allergic conditions. It is known that DHP is not free of adverse effects including induction of tachyarrhythmias. Nevertheless, the mechanisms behind DPH proarrhythmicity is not well understood.\u003c/p\u003e \u003cp\u003eIn the present study in vivo ECG recordings in rats, microelectrode registration in ventricular, atrial and pulmonary vein (PV) isolated tissue, optical mapping of bioelectrical activity in supraventricular tissue preparations as well as patch-clamping for I\u003csub\u003eK1\u003c/sub\u003e recordings in rat cardiac myocytes were used for analysis of mechanisms of DHP-induced proarrhythmicity.\u003c/p\u003e \u003cp\u003eIt is shown that DPH unable to alter heart rate, however, significantly increases duration of QT intervals in rats. Also, DPH induces substantial prolongation of action potentials (AP) in the rat ventricular myocardium. These effects are mediated by DPH-induced attenuation of both inward and functional outward components of inward rectifier (IK1) current. In the rat pulmonary veins the diphenhydramine causes substantial proarrhythmic changes including resting potential (RP) shift to less negative values, AP amplitude decrease and electrotonic-like responses as well as inexcitability, slowing of the conduction velocity, conduction blocks. An adrenaline partially antagonizes DPH-caused RP shift and inexcitability induction, however facilitates PV-derived ectopy and circulation of excitation in presence of DPH in the cardiac tissue of the pulmonary veins.\u003c/p\u003e \u003cp\u003eIn conclusion, DPH-induced attenuation I\u003csub\u003eK1\u003c/sub\u003e promotes formation of the functional substrate highly prone to re-entrant conduction and adrenergically-induced ectopy in the cardiac tissue of pulmonary veins. Thus, DPH in addition to its torsadegenicity may facilitate induction of atrial fibrillation.\u003c/p\u003e","manuscriptTitle":"Diphenhydramine-mediated modulation of inward rectifier IK1 current induces conduction blocks in the rat pulmonary veins myocardium and facilitates supraventricular proarrhythmicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-13 19:10:48","doi":"10.21203/rs.3.rs-5449722/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7286669e-af2e-44dc-b777-8a7398809c33","owner":[],"postedDate":"December 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-15T11:23:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-13 19:10:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5449722","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5449722","identity":"rs-5449722","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}