Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction

Author’s Accepted Manuscript Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction Bianca Hemmeryckx, Yuanbo Feng, Liesbeth Frederix, Marleen Lox, Sander Trenson, Rob Vreeken, Hua Rong Lu, David Gallacher, Yicheng Ni, H. Roger Lijnen www.elsevier.com/locate/ejphar

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S0014-2999(18)30291-7 https://doi.org/10.1016/j.ejphar.2018.05.026 EJP71808

To appear in: European Journal of Pharmacology Received date: 27 February 2018 Revised date: 17 May 2018 Accepted date: 17 May 2018 Cite this article as: Bianca Hemmeryckx, Yuanbo Feng, Liesbeth Frederix, Marleen Lox, Sander Trenson, Rob Vreeken, Hua Rong Lu, David Gallacher, Yicheng Ni and H. Roger Lijnen, Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.05.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction Bianca Hemmeryckxa, Yuanbo Fengb, Liesbeth Frederixa, Marleen Loxa, Sander Trensonc, Rob Vreekend, Hua Rong Lue, David Gallachere, Yicheng Nib, H. Roger Lijnena

a

Center for Molecular and Vascular Biology, c Cardiology, Department of Cardiovascular Sciences, and b

Radiology, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium, and

d e

Metabolomics, Pharmacokinetics, Dynamics and Metabolism Discovery Sciences, and

Translational Sciences, Safety Pharmacology Research, Janssen Research & Development, Janssen Pharmaceutical NV, Beerse, Belgium

Email authors:

Declarations of interest: none. [email protected] [email protected] [email protected]

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] Address for correspondence: Bianca Hemmeryckx, PhD. Center for Molecular and Vascular Biology KU Leuven, Campus Gasthuisberg, CDG, Herestraat 49, Box 911, 3000 Leuven, Belgium Tel 32-16-372073 e-mail: [email protected]

ABSTRACT Patients with heart disease have a higher risk to develop cardiac arrhythmias, either spontaneously or drug-induced. In this study, we have used a rabbit model of myocardial infarction (MI) with severe left ventricular systolic dysfunction (LVSD) to study potential druginduced cardiac risks with N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide (flecainide). Upon ligation of the left circumflex arteries, male New Zealand White rabbits developed a large MI and moderate or severe LVSD 7 weeks after surgery, in comparison to SHAM-operated animals. Subsequently, animals were exposed to escalating doses of flecainide (0.25-4 mg/kg) or solvent. Electrocardiograms (ECG) were recorded before surgery, 1 and 7 weeks after surgery and continuously during the drug protocol. The ECG biomarker iCEB (index of Cardio-Electrophysiological Balance = QT/QRS ratio) was calculated. During the ECG recording at week 1 and week 7 post MI, rabbits had no spontaneous cardiac arrhythmias. When rabbits were exposed to escalating doses of flecainide, 2 out of 5 rabbits with MI and moderate LVSD versus 0 out of 5 solvent-treated rabbits developed arrhythmias, such as ventricular tachycardia/ventricular fibrillation. These were preceded by a marked decrease of iCEB just before the onset (from 4.09 to 2.42 and from 5.56 to 2.25, respectively). Furthermore, 1 out of 5 MI rabbits with moderate LVSD and 1 out of 7 MI rabbits with severe LVSD developed total atrioventricular block after flecainide infusion and died. This rabbit model of MI and severe LVSD may be useful for preclinical evaluation of drug (similar mechanism as flecainide)-induced arrhythmic risks, which might be predicted by iCEB. Key words: atrioventricular block; ventricular tachycardia/ventricular fibrillation; myocardial infarction; left ventricular systolic dysfunction; flecainide

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1. Introduction Patients suffering from cardiovascular diseases are more at risk to develop life-threatening arrhythmias than patients with normal hearts (Buja et al., 1993; Solomon et al., 2005). Furthermore, a variety of drugs has been associated with an increased pro-arrhythmic risk in cardiovascular disease patients (Echt et al., 1991). The anti-arrhythmic agents N-(piperidin-2ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide (flecainide) and encainide were shown to have no

clear anti-arrhythmic effects, and they increased the incidence of sudden cardiac death (Echt et al., 1991). Flecainide treatment has been discontinued in several patients due to side effects including bradyarrhythmias, heart failure and the appearance of arrhythmias such as ventricular tachycardia (VT) (Frandsen et al., 1990). Furthermore, treatment of flecainide has been shown to result in cardiogenic shock (Buss et al., 1992) and to impair LV systolic function in patients experiencing heart failure or coronary heart disease, but also in patients with normal cardiac function (Josephson et al., 1985; Legrand et al., 1985; Santinelli et al., 1993). Additional studies showed that other sodium channel blocking drugs slow down ventricular conduction, resulting in QRS widening and potentially induce cardiac arrhythmias (Lu et al., 2010). Cardiac arrhythmias induced by sodium channel blocking drugs are linked to non-QT prolongation-related nonTorsades de Pointes-like VT and ventricular fibrillation (VF) (Lu et al., 2008). Animal models with acute cardiac diseases may not be suitable to mimic cardiovascular disease in humans, as flecainide was shown to be protective against cardiac arrhythmias in these models (Ferrara et al., 1990; Lederman et al., 1989). Therefore, in the present study, we applied a recently established rabbit model of myocardial infarction (MI) with systolic dysfunction (7 weeks post MI) (Feng et al., 2018), that may be

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useful to evaluate potential drug-induced pro-arrhythmic risks in order to mimic patients with MI and heart failure. Currently, transmural dispersion of repolarization-T wave (rTp-Te) and the beat-to-beat instability of the QT interval (QT-TI) are ECG biomarkers that were introduced to detect druginduced cardiac arrhythmias (Jacobson et al., 2011; Lu et al., 2013), and they mainly focus on the repolarization of the action potential of the heart. In 2013, Lu et al. introduced a new and non-invasive electrophysiological biomarker “the index of Cardio-Electrophysiological Balance” (iCEB), which not only focusses on the depolarization phase (QT interval), but also on the repolarization phase of the action potential (QRS duration); the balance of the depolarization and repolarization or QT/QRS ratio. This biomarker predicted only drug-induced cardiac arrhythmias, which were linked to long QT-related Torsates de Pointes, but also to non-Torsades de Pointes-like VT/VF (Lu et al., 2013). Cardiac arrhythmias were not always predicted by other biomarkers such as rTp-Te and QT-TI alone. Additionally, iCEB translated to human studies from animal models and successfully distinguished patients with an increased susceptibility to cardiac arrhythmias in long QT and Brugada syndrome (Robyns et al., 2016). In the present study, we have used a rabbit model of ischemic myocardial systolic dysfunction to monitor spontaneous as well as flecainide-induced development of cardiac arrhythmias. In addition, we have evaluated whether iCEB could be used as a predictive biomarker.

2. Material and methods All animal procedures were approved by the Ethical Committee of the KU Leuven (P076/2013), and performed in accordance with the guidelines from Directive 2010/63/EU of the European

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Parliament on the protection of animals used for scientific purpose and conform the guidelines of the Declaration of Helsinki. 2.1. Animal model New Zealand White male rabbits (3 to 4 kg), 23-weeks-old, were obtained from the breeding colony of the Laboratory Animal Center of KU Leuven at Heverlee (Belgium) and from CEGAV (Saint Mars D’Egrenne, France). Animals were kept single-housed in a temperature, humidity and light-controlled (12 h night (7 pm)/day (7 am) cycle) environment and were provided with 120g of food (Ssniff K-H, V2333, ssniff Spezialdiäten GmbH, Soest, Germany) daily and had ad libitum access to drinking water. The rabbits received once a week a small bundle of hay and had a wooden block as enrichment. Animals were divided into two groups based on randomization of the animals and matching of the body weight in each group: one group (n = 12) contained rabbits with an open-chest surgery with no manipulation of the heart (SHAM group), while the other group (n = 34) contained rabbits that received a myocardial infarction induction (MI group). Body weight and drinking habits were monitored every week. Cardiac magnetic resonance imaging (cMRI) was used to determine left ventricular (LV) infarct size 24 h post-MI induction. Cine-MRI was performed to monitor cardiac function at baseline, 48 h and 7 weeks post MI. According to the LV infarct size and the severity of myocardial systolic dysfunction, as assessed by cMRI, three different MI groups were identified: MI rabbits with no (MI_NO_LVSD), moderate (MI_M_LVSD) or severe LV systolic dysfunction (MI_S_LVSD) as described previously (Feng et al., 2018). To investigate whether the biomarker iCEB predicts the severity of delayed systolic dysfunction with or without associated cardiac arrhythmias after a reperfused MI, a 12-lead ECG was obtained before surgery and at 1 week and 7 weeks post MI surgery. After 7 weeks, animals were exposed to increasing concentrations of the pro-arrhythmogenic 4

drug flecainide and drug-induced ECG changes were continuously recorded (see below). Control animals received solvent (20% cyclodextrin in 1N HCl) only. Rabbits were euthanized by an intravenous (iv) injection of 2-4 ml of sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL, USA). 2.2. Open-chest coronary left circumflex artery ligation and follow-up All surgical procedures were performed in a sterile manner in the operating suites at our animal facility. The rabbits were sedated with an intramuscular (im) injection of a mixture of NIMATEK (Ketamine Hydrochloride, Eurovet Animal Health B.V., Bladel, The Netherlands) at 15 mg/kg and XYL-M 2% (Xylazine Hydrochloride, V.M.D. n.v., Arendonk, Belgium) at 2.5 mg/kg. The rabbits were placed on a warming blanket and the chest, both sides of the groin, part of the spine, both front limbs and the left hindlimb were shaved for attachment of the ECG electrodes linked to a cardiac monitor (Accusync 71, Milford, Connecticut, USA). After sedation, rabbits were endotracheally intubated and artificial respiration was maintained through an air-driven respirator (Mark 7 respirator, Bird Corporation, CA, USA). After iv access was established, rabbits received an iv injection of a bolus of 10 mg/kg body weight Baytrill (Bayer, Diegem, Belgium) to prevent infection and of sodium pentobarbital (Abbott Laboratories) at 40 mg/kg/h to maintain anesthesia. After disinfection of the chest and placing Steri-Drape (3M, Diegem, Belgium) to sterilize the surgical field, the skin and subcutaneous tissues were cut open layer by layer along the left sternal border. Subsequently, the 4th and 5th intercostal spaces cartilages were cut and the pericardium was opened to expose the left circumflex artery branch. The left circumflex artery was ligated by a detachable knot using 2-0 silk suture (Johnson & Johnson Medical, Diegem, Belgium) at 2 mm below the left atrial appendage, of which the pullable end was left outside the thorax after closure of the thoracic cavity. Reperfusion was 5

induced by pulling the exteriorized end of the suture in a closed-chest condition after 1 h of coronary occlusion. The criteria of a successful MI were myocardial discoloration from red to dark purple in the apex, partial LV anterior wall, lateral wall, and inferior wall myocardial systolic weakness or abnormal motion, and continuous significantly convex ST elevation in ECG records. Then, thoracic incisions were closed by three separate knots (2-0 silk suture), and muscle and skin were closed as well (3-0 vicryl suture; Johnson & Johnson Medical). Similar procedures were applied for SHAM-operated animals, except for the left circumflex artery ligation. In these animals, after the open chest-surgery, the pericardium was cut and the chest closed afterwards. In the event of sustained ventricular fibrillation during coronary occlusion or reperfusion, the animals were given additionally XYLOCAINE 2% (lidocain, Eurovet Animal Health B.V.) gradually or as a bolus (1 mg/kg iv; volume between 0.5 – 2 ml dependent on severity of arrhythmia). After reperfusion, animals were allowed to recover on a warming blanket and were ventilated further until their own respiration took over. To prevent post-surgical thrombosis and inflammation 10 mg/kg Baytrill (Bayer) and 65 U/kg heparin (LEO Pharma N.V./S.A., Lier, Belgium) were administered subcutaneously 4 h post MI surgery. All animals received daily for 4 days post-surgery 65 U/kg heparin and for 2 days postsurgery 10 mg/kg Baytrill. Vetergesic (Ecuphar nv, Oostkamp, Belgium) was given for two days post-surgery twice daily (morning and evening): 0.02 mg/kg on day 1 and 0.01 mg/kg on day 2. All medication post-surgery was given subcutaneously. Every week, surgical wounds were inspected, cleaned with isobetadine (Meda Pharma nv, Brussels, Belgium) and sprayed with aluminium spray (Kela Veterinaria nv, Sint-Niklaas, Belgium) until the wound was not visible anymore. Extra hay was given daily for 2 weeks post-surgery. 2.3. Magnetic resonance imaging 6

2.3.1. Scanning Using a 16-channel phased array knee coil, cMRI was performed on the anesthetized rabbits with a 3.0T Siemens MRI scanner (Trio, Siemens, Erlangen, Germany) with a maximum gradient capability of 45 mT/m triggered by ECG and gated by respiration using a small animal monitoring and gating system (SA Instruments, Inc. Stony Brook, NY, USA). The two ECG electrodes were attached to the shaved thorax skin and to the left leg. The respiration sensor was attached to the mid-section of the abdomen of the rabbit, which was placed supinely in a holder and gas-anesthetized with 2% isoflurane in the mixture of 20% oxygen and 80% room air, through a mask connected via a tube to a ventilation instrument (Harvard Apparatus, Holliston, MA, USA). All images were acquired during free breathing of the animal. Eight short-axial slices of the LV were collected with a slice thickness of 3.0 mm without gap for cMRI sequences. Turbo spin echo sequence of black blood imaging was applied for cardiac morphology with parameters TR: 621~750 ms, TE: 15~74 ms, FOV: 240×195 mm2, FA: 180°, and in-plane resolution: 0.9×0.9 mm2. The Cine-MRI images were acquired on True fast imaging with steady state precession in the short-axis, vertical long-axis and horizontal long-axis planes for displaying cardiac contraction. Each Cine-MRI consisted of 25 frames, spaced equally across the cardiac cycle, with the acquisition time of 2.5 min, the scan parameters TR: 357 ms, TE: 1.6 ms, FOV: 240×195 mm2, FA: 60°, spatial resolution: 1.2×0.9 mm2. The contrast delayedenhancement images were acquired by a 3D segmented k-space inversion recovery turbo fast low angle shot sequence 20 min after an iv bolus injection of meglumine gadoterate [(GdDOTA) Dotarem, Guerbet, France] at 0.2 mmol/kg with parameters TR: 396 ms, TE: 1.54 ms, TI: 360 ms, FOV: 240×180 mm2, FA: 15°, and in-plane resolution: 1.1×0.8 mm2. 2.3.2. Image analysis 7

cMRI images were read using an off-line workstation with dedicated software (SyngoMR A30, Siemens). The assessment and quantification of MI size and LV function in contrast delayedenhancement images and Cine-MRI images were done using the software SEGMENT (Medviso AB, Lund, Sweden). The endocardial and epicardial borders were manually traced in the enddiastolic and end-systolic short-axis Cine images. Papillary muscles were included in the myocardium. LV end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), ejection fraction (EF), cardiac output (CO) and mass were measured according to standard methods (Feng et al., 2015). Regional LV function was assessed by measuring wall thickening from end-diastolic phase to end-systolic phase in six clockwise sectors on the mid-ventricle section of Cine images. 2.4. ECG recording Before surgery, 1 week and 7 weeks post MI surgery rabbits were anesthetized with a mixture of ketamine and xylazine (35 mg/kg and 5 mg/kg, i.m.). Body temperature was monitored after each procedure (anesthesia, shaving, attaching and detaching ECG electrodes) using a digital thermometer. It was kept stable during the experiment by placing each animal on a heating pad supported by a wooden plank in a dorsal recumbent position. The chest and the inguinal parts of the limbs were shaved and disinfected with 70% ethanol. Needle electrodes (ML1203, ADInstruments Ltd., Oxford, UK) were placed subcutaneously in the four limbs and the chest for 12-surface ECG recordings as described previously (Lu et al., 2004). The reference needle electrode was attached to the right hind limb. 12-lead ECG recordings were acquired on a Powerlab 16/35S machine equipped with a GT201 16-channel bioamplifier (ADInstruments Ltd.). The bioamplifier settings were as follows: the low-pass and high-pass filters were set at 1 kHz and 2 Hz respectively, the notch (50 or 60 Hz) filter was turned off and the sensitivity set at 8

5 mV. Furthermore, signals were sampled digitally at a frequency and a rate of 20 kHz and 1 k/s respectively and digitally smoothed using a median filter on the Labchart Pro 8.0.2. software (ADInstruments Ltd.). When the heart rate normalized, an ECG was recorded for 5 min. 2.5. Drug-induced arrhythmia protocol Rabbits were anesthetized with a mixture of ketamine and xylazine (35 mg/kg and 5 mg/kg, intramuscularly). Body temperature was kept stable by putting animals on a heating pad in a supine position. The chest, inguinal areas of the limbs and the neck area were shaved and disinfected with 70% ethanol. Rabbits were intubated using a 3.0 (body weight up to 3.5 kg) or 3.5 (body weight > 3.5 kg) endotracheal tube. Animals were ventilated using a rodent ventilator model 683 (Harvard Apparatus UK, Cambridge, UK) at a pace of 55 strokes per min and a volume of 20 ml. The right ear vein was catheterized to infuse a mixture of ketamine/xylazine (10 mg/kg and 2 mg/kg respectively, rate was calculated as the sum of volumes of ketamine/xylazine in ml/hour) during the entire study. Heparin-flushed (250 U/ml) microcatheters (polyethylene tubing (PE-90) connected to a 20ga Luer stub (Instech Laboratories Inc., Plymouth Meeting, PA, USA) were placed in the left and right carotid artery to withdraw blood and to monitor blood pressure, respectively. Before connecting the blood pressure transducer (MLT067, AD Instruments Ltd.) to the invasive microcatheter, the transducer, connected via the bridge amp to the Powerlab 16S/GT201 system (AD Instruments Ltd.), was calibrated using a manometer (Deltacal, Utah Medical Products Inc., Midvale, UT, USA) with two settings (0 and 200 mmHg). Furthermore, the temperature system (TC-1000 temperature controller with temperature probe YSI-401 and medium heating pad were connected to the bioamplifier; AD Instruments Ltd.) was also calibrated using two temperature settings (low end: 33-35°C; high end: 40-43°C) using a water bath. The left marginal ear vein was cannulated for 9

intravenous administration of the pro-arrhythmogenic compound flecainide. After connecting the blood pressure transducer to the microcatheter and after placing the temperature probe rectally, the needle electrodes were attached to the animal (see above) to record the 12-surface ECG signals. The ECG lead signals, blood pressure and temperature were then continuously recorded using the Labchart Pro 8.0.2 software and the Powerlab 16S/GT201 system as described above. The animals were allowed to stabilize (as characterized by an unchanged heart rate and mean blood pressure over time) for 15 min. Subsequently, a baseline ECG recording for 5 min was performed before the lowest drug concentration was infused. The concentrations of flecainide (J & J, Beerse, Belgium) were 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, and 4 mg/kg. The compound was infused intravenously over a period of 5 min (rate: body weight/10 in ml/min) followed by a 15 min interval between escalating doses. After the infusion of each dose, 300 μl blood was collected from the cannulated right carotid artery in a tube containing 10 μl of 1000 U/ml heparin. Blood samples were centrifuged for 2 min at 6010 g and plasma was stored at 20°C until further analysis. Animals were euthanized by an intravenous injection of 2-4 ml of sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL, USA). 2.6. ECG analysis Sixty to sixty five individual beats of the ECG in lead II were selected from 30 s before infusion of each dose (solvent or flecainide). The ECG analysis module of Labchart Pro 8.0.2 analyzed several ECG parameters including P duration, height of the R wave, the peak and end of the T wave, and the PQ, QRS and QT intervals. The QT interval was defined as the time from the onset of the QRS to the point at which the descending limb of the T wave crosses the isoelectric line. The JT interval was calculated as the QT interval minus QRS duration. iCEB was calculated according to different formulas: the ratio of QT/QRS, JT/QRS or PQ/QRS. In addition, heart 10

rate-corrected QTc and JTc intervals and iCEB calculations were obtained by using the Bazett (QT/(RR)1/2 or QTcB), Fridericia (QT/(RR)1/3 or QTcF), and Van de Water (QT-0.087[(60/heart Rate)-1] or QTcVDW) correction formulas. All ECG parameters were calculated beat to beat and then averaged. rTp-Te and instability of the QT interval (QT-TI (total), QT-STI (short-term) and (QT-LTI) long-term) were calculated as described before (Lu et al., 2013). VT was defined as two or more consecutive ventricular premature beats and VF as a ventricular tachyarrhythmia without identifiable ECG patterns. Third-degree atrioventricular (AV) block was defined as a complete absence of AV conduction. 2.7. Blood pressure monitoring and analysis The Labchart Pro 8.0.2. software contains a blood pressure module that allows monitoring and measurement of the mean, diastolic and systolic blood pressures. Individual mean blood pressure cycles (n = 60 ± 1.0) 30 s before infusion of each dose of flecainide were selected and analyzed. For each dose and for each animal the results of the different individual cycles were averaged to obtain the mean, diastolic and systolic blood pressures. 2.8. Assessment of serum flecainide concentrations Serum samples were analyzed after protein precipitation (1:4 dilution with acetonitrile) and subsequent UPLC-MS/MS analysis. Chromatographic separation of flecainide from the matrix was achieved using a BEH C18 (50 x 2.1 mm, 1.7 µm particles) column (Waters Corp., Milford, MA, USA), fitted on a Acquity UPLC system (Waters Corp.) with an aqueous generic linear gradient from 30-98% acetonitrile at pH 2 (0.1% formic acid) at a flow-rate of 0.6 ml/min. The MS-system (API 4000, AB Sciex, Concord, Ontario, Canada) was operated in electrospray positive ion mode, producing for flecainide a protonated molecule as precursor for the MS/MS 11

analysis. Quantification was achieved using selective and flecainide specific MS/MS transitions from precursor to fragment ion (dwell time 150 ms per channel). 2.9. Statistical analysis Data are shown as means ± S.E.M. for the number of animals studied. Differences between all groups in the MRI and ECG data between baseline, 1 week and 7 weeks were analyzed using the nonparametric Kruskal-Wallis statistical test. If a statistical difference was detected (P < 0.05), the difference between the individual groups was determined using the Dunn’s Multiple Comparison test. Differences between all groups in data regarding the administration of flecainide were analyzed using the Holm-Sidak Multiple Comparison test (α = 0.05). The statistical analyses were performed with GraphPad Prism 6 software (GraphPad, La Jolla, CA).

3. Results 3.1. Effect of the left circumflex artery ligation on left ventricular function assessed by MRI Left circumflex artery artery ligation, followed by a 7 week recovery period, induced different degrees of LVSD in the New Zealand White rabbits: no (MI_NO_LVSD), moderate (MI_M_LVSD), or severe (MI_S_LVSD) LVSD developed in 29.4% (10/34), 26.5% (9/34) and 44.1% (15/34) respectively of MI animals after 7 weeks. Chronic MI sizes measured by cMRI were the highest (46% of LV) for the MI_S_LVSD rabbits and statistically significantly different from chronic MI sizes of MI_M_LVSD (30% of LV) and MI_NO_LVSD (7.9% of LV) rabbits (Table 1). Cardiac MRI confirmed the different severities in systolic dysfunction: EF for the MI_S_LVSD rabbits decreased from 57% to 36% at the acute stage (48 h post MI) and to 30% at 7 weeks post MI respectively, while the reduction was less pronounced for MI_M_LVSD animals (from 57% to 44% and to 46%, respectively). In contrast to SHAM rabbits, EDV and 12

ESV in MI_M_LVSD animals increased by 10% and 47% respectively at the acute stage and by 15% and 45% respectively at the chronic stage; in MI_S_LVSD rabbits both parameters were increased by 20% and 82% respectively at the acute stage and by 36% and 125% respectively at the chronic stage (Table 1). SV in MI_M_LVSD rabbits decreased by 17% and 11% at the acute chronic stage, respectively; the decrease of SV in MI_S_LVSD animals was more pronounced (22% and 30% at the acute and chronic stage respectively, which suggests decompensation of the heart), in comparison to the SHAM group. Global cardiac function parameters did not show significant differences between SHAM and MI_NO_LVSD animals at both stages (Table 1). The ligation of the left circumflex artery led to arrhythmias (9 – 47 min after ligation) and cardiac death in 5 out of 40 animals. Three to four weeks post MI surgery one animal with a LV infarct size larger than 50% died of chronic heart failure. 3.2. ECG monitoring MI induction led to a significant elevation of the ST segment in both MI groups at 1 and 7 weeks after surgery (Fig. 1A). Furthermore, MI_M_LVSD and MI_S_LVSD rabbit ECG scans show deep Q waves and no R waves at 1 week after surgery, whereas at 7 weeks post-surgery the R wave reappeared in 6 out of 9 (67%) MI_M_LVSD rabbits, but only in 20% (3 out of 15; P = 0.0262) of the MI rabbits with severe LVSD (Figure 1A). Table 2 provides the real data at baseline and the data obtained at 1 and 7 weeks post MI as a % change of baseline. Table 2 shows the transformation of the positive R wave (amplitude: + mV) into a negative deep Q wave (amplitude: - mV) in the MI rabbit groups versus the SHAM-operated group (P < 0.05). However, no statistically significant difference was found for this parameter between both MI rabbit groups. Heart rate, PQ, QRS, QTcF, JT and JTcF intervals and the amplitude of the T wave did not change between the three groups after 1 week and 7 weeks post MI induction 13

(Table 2). The QT interval was significantly decreased in MI rabbits with severe versus moderate LVSD at the acute, 1-week post MI stage, but this difference disappeared after six additional weeks of recovery (Table 2). As no spontaneous cardiac arrhythmias were observed in the MI rabbits, iCEB calculations according to the different correction formulas were not different between the SHAM, MI_M_LVSD and MI_S_LVSD groups (data not shown). In addition, ventricular arrhythmia ECG parameters such as transmural dispersion of repolarization and QT interval stability (short-term; long-term and total) were not affected by the severity of systolic dysfunction (data not shown). 3.3. Flecainide treatment Arrhythmias such as VT/VF (2/5 in MI_M_LVSD group) and total atrioventricular (AV) block with some escape rhythms (1/5 in MI_M_LVSD and 1/7 in MI_S_LVSD groups) leading to cardiac death were induced occasionally by flecainide only in MI rabbits and not in SHAM rabbits. Non-sustained VT episodes were seen in the two MI rabbits with total AV block before the onset of the block (MI_M_LVSD animal: 16 episodes with an average duration of 910 ± 142 ms over a time span of 4 min and 20 s; MI_S_LVSD animal: thirteen episodes with an average duration of 940 ± 45 ms over a time span of 40 s). Furthermore, two episodes (average duration: 1445 ± 445 ms within 5 s) of non-sustained VT were seen in the recovery period after infusion of 4 mg/kg flecainide in another MI_M_LVSD animal. Solvent infusion did not result in the induction of arrhythmias in any group. 10-s ECG tracings illustrate the change in the ECG pattern between normal SHAM-operated animals and rabbits with a large MI after the administration of flecainide (Fig. 1B). Infusion of solvent did not significantly affect ECG parameters in any of the groups (data not shown). Furthermore, comparison of the ECG parameters of rabbits with repeated solvent 14

infusion did not reveal significant differences between SHAM-operated, MI_M_LVSD and MI_S_LVSD groups (data not shown). Before administration of flecainide, most ECG parameters were not different between the three groups (Table 3). Only, the positive R wave (amplitude: + mV) in SHAM-operated rabbits was transformed in a negative deep Q wave (amplitude: - mV) in both MI rabbit groups (P < 0.05) (Table 3). For convenience, we expressed all changes in ECG parameters induced by dosing of flecainide as a % change of baseline. Most of the investigated ECG parameters were not different between the three groups upon treatment with flecainide (Fig. 2, 3, 4). A statistically significant increase in heart rate was detected between SHAM rabbits and MI_S_LVSD rabbits during the last dosing of the drug (Fig. 2A). In addition, MI_S_LVSD animals had a reduced amplitude of the R wave as compared to SHAM rabbits during the last two doses of flecainide infusion (Fig. 2H). As reported in the literature, flecainide did dose-dependently increase the PQ, QRS and QT interval for all three groups, including the heart rate-corrected QTcF interval when compared to solvent-treated animals (Fig. 2). The JT interval and heart rate-corrected JT interval also dose-dependently increased, however this increase was less pronounced for SHAM and MI_S_LVSD rabbits (Fig. 2). The amplitude of the R wave was significantly lower for flecainide- versus solvent-treated SHAM rabbits after administration of a dose of 1 mg/kg. This was not the case for either MI groups (Fig. 2H). Low and high doses of flecainide resulted in an increase in the amplitude of the T wave for SHAM and MI_S_LVSD rabbits, respectively (Fig. 2I). Administration of flecainide was associated with a dose-dependent decrease in iCEB (all calculations) in all groups, without, however, statistically significant differences between groups (Fig. 3). The MI_M_LVSD rabbits showed an increase of iCEB (e.g. QT/QRS) after infusion of 2 mg/kg flecainide, whereas for the other two groups it kept decreasing (Fig. 3).

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For two MI_M_LVSD rabbits with VT/VF, iCEB (QT/QRS calculation) increased with 12% (3.64 to 4.09) or 71% (3.25 to 5.56) at 2 mg/kg as compared to 1 mg/kg flecainide, and then markedly decreased with 41% (4.09 to 2.42) or 60% (5.56 to 2.25) to a value below 2.5 just before the onset of arrhythmia. In two MI rabbits, flecainide at a lower dose of 1 mg/kg induced total AV block. For the MI_M_LVSD animal, iCEB (QT/QRS) fluctuated around 3.0 and then decreased markedly with 34% (3.24 to 2.14) just before the onset of the total AV block. For the MI_S_LVSD rabbit with total AV block, iCEB (QT/QRS) decreased from a value of 5.02 at baseline to 3.65 (27%) after infusion of 1 mg/kg flecainide and subsequently increased slightly to 3.83 (5%) before the onset of the conduction abnormality. Flecainide exposure did also result in a dose-dependent increase in transmural dispersion of repolarization for SHAM rabbits, but not for MI rabbits (Fig. 4A). For MI_S_LVSD rabbits, rTpTe was increased significantly only in response to flecainide versus solvent treatment at a dose of 2 mg/kg (Fig. 4A). Overall, the short-term, long-term and total QT instability measurements were not affected by flecainide treatment for either group (Fig. 4B-D). Flecainide administration reduced mean, diastolic and systolic blood pressure significantly for SHAM rabbits at doses of 2 and 4 mg/kg, and for MI_M_LVSD rabbits, a significant reduction in all three blood pressures was observed for doses of 1 and 2 mg/kg of the compound (Table 4). For MI_S_LVSD rabbits, however, only the mean and systolic blood pressures were affected significantly by flecainide at the highest dose of 4 mg/kg (Table 4). Analysis of the flecainide concentration present in the blood after administration of each dose did not reveal significant differences between the three groups (Table 5).

4. Discussion 16

Cardiovascular disease is still one of the leading mortality causes in the Western World. The prevalence of heart failure keeps increasing, due to aging of the general population and improved survival after myocardial infarction. Because of the health impact of cardiovascular diseases, it is crucial to identify a good preclinical model and biomarker to predict cardiac risk associated with drug treatment. Progress in monitoring cardiac drug safety at the preclinical level is indeed hampered by the lack of good animal models and sensitive biomarkers. We have recently developed a rabbit model of reperfused MI with delayed LVSD that may be useful for translational research on cardiovascular diseases (Feng et al., 2018). Rabbits share basic electrophysiological characteristics with humans as reviewed by Kaese et al. (2013) and are therefore frequently used to study cardiovascular and non-cardiovascular drug-related ventricular arrhythmias and the underlying electro-physiological mechanisms (Eckardt et al., 2002; Frommeyer et al., 2011; Kijtawornrat et al., 2006; Lu et al., 2006 et al; Lu et al., 2016; Milberg et al., 2002). The main benefit of a predictive biomarker to be used in preclinical drug development and in clinical practice is to reduce cardiovascular toxicity of new drugs, and ultimately to bring safe new drugs to the market. Cardiovascular safety in general indeed is still the major cause of drug attrition in early development and of withdrawal from the market (Laverty et al., 2011). Lu et al. (2013) recently proposed a new biomarker the index of Cardio-Electrophysiological Balance (iCEB) that represents a balance between depolarization (QRS) (ejection of blood from the heart) and repolarization (QT) (refilling and electrical recovery). Therefore, in the present study, we have used this new rabbit model to monitor pro-arrhythmia risks and to evaluate the potential of iCEB as a biomarker to predict spontaneous or drug-induced arrhythmia. Although at 7 weeks post MI 44% of MI rabbits showed severe LVSD, ECG recordings of these animals did not show

17

evidence of spontaneous cardiac arrhythmias, despite significant LV epicardial or transmural scars and LV remodeling. Thus, expected, iCEB as well as other biomarkers for arrhythmias such as rTp-Te and QT-TI were not different in rabbits with severe LVSD, as compared to moderate LVSD and SHAM-operated rabbits. This is in agreement with the findings of Kijtawornrat et al. (2006) who did not observe spontaneous arrhythmias four weeks after surgical ligation of both the left anterior descending and a major apical branch of the left circumflex artery in rabbits with myocardial failure. Drug-induced cardiac arrhythmias are common but incidence is very low (Heist and Ruskin, 2010). The low prevalence of drug-induced arrhythmia has been observed also in other studies: Lu and colleagues noticed that flecainide at a dose of 10 µM elicited a VT incidence of 50% in isolated Langendorff-perfused rabbit hearts (Lu et al., 2010). Furthermore, flecainide did not evoke VT in arterially perfused rabbit LV wedge preparations stimulated with a low rate of 0.5 Hz, whereas it did induce VT/VF in 4/7 LV wedge preparations when stimulated at a fast pacing rate of 2 Hz (Lu et al., 2016). In our study, escalating doses of the pro-arrhythmia drug flecainide induced VT/VF (40% or 2/5) and total AV block (1/5 or 1/7) only in MI rabbits with LVSD and not in SHAM-operated animals without MI. Flecainide dose-dependently decreased iCEB in all groups, as also shown in humans (Robyns et al., 2016). However, no statistically significant changes between rabbits with moderate or severe LVSD and SHAM-operated rabbits were observed for this biomarker. The dose-dependent effect of flecainide on rTp-Te in SHAM rabbits was more pronounced than the effect in MI rabbits (there was a tendency to an increase of rTp-Te in a dose-dependent manner but due to a large variability in both MI groups, these differences were not statistically significant). rTp-Te is a very good biomarker for drug-induced long QT and Torsades dePointes (Yan et al., 2003), but not for other types of arrhythmias such as non-Torsades dePointes-like ventricular

18

tachycardia/ventricular fibrillation (Lu et al., 2013). Therefore, it might explain why flecainide did cause no-Torsades dePointes-like ventricular tachycardia/ventricular fibrillation without significant increases in rTp-Te in MI rabbits in the present study. Furthermore, flecainide has been shown to slow conduction, increase rTp-Te and cause non-Torsades dePointes-like cardiac arrhythmias in isolated rabbit hearts (Lu et al., 2010). In addition, short-term QT instability tended to be affected dose-dependently by flecainide, but the increase in this parameter was not statistically significant due to large data variability. In the present study, the rabbits with VT/VF, iCEB (QT/QRS) was firstly increased by 12% or 71% after 2 mg/kg flecainide as compared to the previous dose and then drastically decreased by 41% or 60% just before the onset of the arrhythmia. The first increase in iCEB by flecainide is likely due to a prolongation of QT interval and the decrease in iCEB before the onset of the cardiac arrhythmias is likely due to its bad Na+ channel blocking activities (increase of QRS duration). These data support in vitro (Lu et al., 2006; Lu et al, 2013) and human (Robyns et al., 2016) data, in which bad Na+ channel blockers reduced iCEB significantly (caused an unbalance of the cardio-electrophysiological activities) and therefore caused non-Torsades de Pointes-like VT/VF. Furthermore, two MI rabbits developed total AV block in response to escalating doses of flecainide. In both animals, iCEB (QT/QRS) remained stable during the lowest doses of flecainide infusion, but before the onset of the conduction abnormality iCEB decreased substantially in one of the animals. Flecainide toxicity resulting in bradycardia, hypotension and irreversible third degree AV block has been reported only in an 82-year-old woman (Lloyd et al. 2013). Furthermore, in both animals episodes of non-sustained VT developed prior to the onset of the total AV block. The presence of non-sustained VT episodes in animals with coronary 19

artery disease and severe LV dysfunction may be a sign of impeding sudden cardiac death. The doses of flecainide used in this study were based on the dose that is routinely given to patients. In the clinic, flecainide is administered at a maximal concentration of 2 - 2.5 mg/kg over a period of 30 min. Therapeutic flecainide levels range between 0.2 and 1.0 µg/ml as reported for patients with ventricular arrhythmias (Conard et al., 1982). Occasionally, patients developed severe cardiac adverse effects such as ventricular arrhythmias when serum flecainide levels surpassed 1 µg/ml (Morganroth and Horowitz, 1984; Roden and Woosley, 1986). In our study, serum flecainide concentrations for both MI_M_LVSD rabbits with VT/VF after infusion of 2 mg/kg flecainide ranged between 3.5 and 3.6 µg/ml. Our data thus support that flecainide should be contraindicated in MI patients with LVSD (de Paolo et al., 1987; Legrand et al., 1985). However, this study has some limitations. 7 weeks post MI might not be long enough to find MIinduced changes in cardiac ion channels and therefore changes in the ECGs of rabbits with MI and severe LVSD. Anesthesia during each ECG recording might influence the outcome of the ECG measurements. Continuous ECG recording without anesthesia as in Holter ECG may be applied for future studies. The experimental number for each drug group was limited in the present study. Furthermore, only flecainide was tested and not other pro-arrhythmic drugs acting by different mechanisms (e.g. inhibition / activation of different ion channels). We shall consider these factors in future studies.

5. Conclusions We have established a rabbit model of MI with delayed LVSD, which could mimic patients with MI and LVSD and might therefore be a suitable diseased model to investigate cardiac

20

arrhythmias induced by drugs with a similar mechanism of action as flecainide. Indeed, flecainide did elicit fatal cardiac arrhythmias such as VT/VF and AV-block, which were predicted by a sharp drop in iCEB just before the onset of the cardiac arrhythmias in this diseased rabbit model. Therefore, severe decreases in iCEB might be useful for preclinical evaluation of arrhythmic risk induced by drugs that share their mode of action with flecainide.

Acknowledgements We thank Christine Vranckx, and Inge Vorsters for expert technical assistance. We are grateful to Prof. Paul Herijgers and Mieke Ginckels for access to the surgery rooms.

Funding This study was supported by the Belgian Agency for Innovation by Science and Technology (IWT) (IWT 130691).

References Buja, G., Miorelli, M., Turrini, P., Melacini, P., Nava, A., 1993. Comparison of QT dispersion in hypertrophic cardiomyopathy between patients with and without ventricular arrhythmias and sudden death. Am. J. Cardiol. 72, 973-976. Buss, J., Lasserre, J.J., Heene, D.L., 1992. Asystole and cardiogenic shock due to combined treatment with verapamil and flecainide. Lancet 340, 546. Conard, G.J., Cronheim, G.E., Klempt, H.W., 1982. Relationship between plasma concentrations and suppression of ventricular extrasystoles by flecainide acetate (R-818), a new antiarrhythmic, in patients. Arzneimittelforschung 32, 155-159.

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de Paolo, A.A., Horowitz, L.N., Morganroth, J., Senior, S., Spielman, S.R., Greenspan, A.M., Kay, H.R., 1987. Influence of left ventricular dysfunction on flecainide therapy. J. Am. Coll. Cardiol. 9, 163-168. Echt, D.S., Liebson, P.R., Mitchell, L.B., Peters, R.W., Obias-Manno, D., Barker, A.H., Arensberg, D., Baker, A., Friedman, L., Greene, H.L., Huther, M.L., Richardson, D.W., the CAST Investigators, 1991. Mortality and morbidity in patients receiving encainide, flecainide, or placebo. The Cardiac Arrhythmia Suppression Trial. N. Engl. J. Med. 324, 781-788. Eckardt, L., Breithardt, G., Haverkamp, W., 2002. Electrophysiologic characterization of the antipsychotic drug sertindole in a rabbit heart model of torsade de pointes: low torsadogenic potential despite QT prolongation. J. Pharmacol. Exp. Ther. 300, 64-71. Feng, Y., Chen, F., Yin, T., Xia, Q., Liu, Y., Huang, G., Zhang, J., Oyen, R., Ni, Y., 2015. Pharmacological effects of Cannabidiol on acute reperfused myocardial infarction in rabbits: evaluated with 3.0T cardiac magnetic resonance imaging and histopathology. J. Cardiovasc. Pharmacol. 66, 354-363. Feng, Y., Hemmeryckx, B., Frederix, L., Lox, M., Wu, J., Heggermont, W., Lu, H.R., Gallacher, D., Oyen, R., Lijnen, H.R., Ni, Y., 2018. Monitoring Reperfused Myocardial Infarction with Delayed Left Ventricular Systolic Dysfunction in Rabbits by Longitudinal Imaging. Unpublished results. Ferrara, N., Abete, P., Leosco, D., Caccese, P., Orlando, M., Landino, P., Sederino, S., Tedeschi, C., Rengo, F., 1990. Effect of flecainide acetate on reperfusion- and barium-induced ventricular tachyarrhythmias in the isolated perfused rat heart. Arch. Int. Pharmacodyn. Ther. 308, 104-114. Frandsen, F., Pless, P., Mickley, H., Moller, M., 1990. Antiarrhythmic treatment with flecainide (Tambocor). Clinical experience from 107 patients. Acta Cardiol. 45, 351-357.

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Frommeyer, G., Milberg, P., Witte, P., Stypmann, J., Koopmann, M., Lücke, M., Osada, N., Breithardt, G., Fehr, M., Eckardt, L., 2011. A new mechanism preventing proarrhythmia in chronic heart failure: rapid phase-III repolarization explains the low proarrhythmic potential of amiodarone in contrast to sotalol in a model of pacing-induced heart failure. Eur. J. Heart Fail. 13, 1060-1069. Heist, E.K., Ruskin, J.N., 2010. Drug-Induced Arrhythmia. Circulation 122, 1426-1435. Jacobson, I., Carlsson, L., Duker, G., 2011. Beat-by-beat QT interval variability, but not QT prolongation per se, predicts drug-induced torsades de pointes in the anaesthetised methoxaminesensitized rabbit. J. Pharmacol. Toxicol. Methods 63, 40-46. Josephson, M.A., Kaul, S., Hopkins, J., Kvam, D., Singh, B.N., 1985. Hemodynamic effects of intravenous flecainide relative to the level of ventricular function in patients with coronary artery disease. Am. Heart J. 109, 41-45. Kaese, S., Frommeyer, G., Verheule, S., van Loon, G., Gehrmann, J., Breithardt, G., Eckardt, L., 2013. The ECG in cardiovascular-relevant animal models of electrophysiology. Herzschrittmacherther. Elektrophysiol. 24, 84-91. Kijtawornrat, A., Nishijima, Y., Roche, B.M., Keene, B.W., Hamlin, R.L., 2006. Use of a failing rabbit heart as a model to predict torsadogenicity. Toxicol. Sci. 93, 205-212. Laverty, H., Benson, C., Cartwright, E., Cross, M., Garland, C., Hammond, T., Holloway, C., McMahon, N., Milligan, J., Park, B., Pirmohamed, M., Pollard, C., Radford, J., Roome, N., Sager, P., Singh, S., Suter, T., Suter, W., Trafford, A., Volders, P., Wallis, R., Weaver, R., York, M., Valentin, J., 2011. How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br. J. Pharmacol. 163, 675-693.

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Lederman, S.N., Wenger, T.L., Bolster, D.E., Strauss, H.C., 1989. Effects of flecainide on occlusion and reperfusion arrhythmias in dogs. J. Cardiovasc. Pharmacol. 13, 541-546. Legrand, V., Materne, P., Vandormael, M., Collignon, P., Kulbertus, H.E., 1985. Comparative haemodynamic effects of intravenous flecainide in patients with and without heart failure and with and without beta-blocker therapy. Eur. Heart J. 6, 664-671. Lloyd, T., Zimmerman, J., Griffin, G.D., 2013. Irreversible third-degree heart block and pacemaker implant in a case of flecainide toxicity. Am. J. Emerg. Med. 31, 1418.e1-1418.e2. Lu, H.R., Gallacher, D.J., Yan, G.X., 2016. Assessment of drug-induced proarrhythmia: The importance of study design in the rabbit left ventricular wedge model. J. Pharmacol. Toxicol. Methods 81, 151-160. Lu, H.R., Rohrbacher, J., Vlaminckx, E., Van Ammel, K., Yan, G.X., Gallacher, D.J., 2010. Predicting drug-induced slowing of conduction and pro-arrhythmia: identifying the 'bad' sodium current blockers. Br. J. Pharmacol. 160, 60-76. Lu, H.R., Van Ammel, K., Vlaminckx, E., De Clerck, F., 2004. QT and JT dispersion in the drug-induced long QT syndrome in anaesthetized rabbits is accurately detected by a three-lead surface ECG measurement. J. Pharmacol. Toxicol. Methods 49, 71-79. Lu, H.R., Vlaminckx, E., Hermans, A.N., Rohrbacher, J., Van Ammel, K., Towart, R., Pugsley, M., Gallacher, D.J., 2008. Predicting drug-induced changes in QT interval and arrhythmias: QTshortening drugs point to gaps in the ICHS7B Guidelines. Br. J. Pharmacol. 154, 1427-1438. Lu, H.R., Vlaminckx, E., Van de Water, A., Gallacher, D.J., 2006. Calmodulin antagonist W-7 prevents sparfloxacin-induced early afterdepolarizations (EADs) in isolated rabbit purkinje fibers: importance of beat-to-beat instability of the repolarization. J. Cardiovasc. Electrophysiol. 17, 415-422.

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Lu, H.R., Yan, G.X., Gallacher, D.J., 2013. A new biomarker--index of cardiac electrophysiological balance (iCEB)--plays an important role in drug-induced cardiac arrhythmias: beyond QT-prolongation and Torsades de Pointes (TdPs). J. Pharmacol. Toxicol. Methods 68, 250-259. Milberg, P., Eckardt, L., Bruns, H.J., Biertz, J., Ramtin, S., Reinsch, N., Fleischer, D., Kirchhof, P., Fabritz, L., Breithardt, G., Haverkamp, W., 2002. Divergent proarrhythmic potential of macrolide antibiotics despite similar QT prolongation: fast phase 3 repolarization prevents early afterdepolarizations and torsade de pointes. J. Pharmacol. Exp. Ther. 303, 218-225. Morganroth, J., Horowitz, L.N., 1984. Flecainide: its proarrhythmic effect and expected changes on the surface electrocardiogram. Am. J. Cardiol. 53, 89B-94B. Robyns, T., Lu, H.R., Gallacher, D.J., Garweg, C., Ector, J., Willems, R., Janssens, S., Nuyens, D., 2016. Evaluation of Index of Cardio-Electrophysiological Balance (iCEB) as a New Biomarker for the Identification of Patients at Increased Arrhythmic Risk. Ann. Noninvasive Electrocardiol. 21, 294-304. Roden, D.M., Woosley, R.L., 1986. Drug therapy. Flecainide. N. Engl. J. Med. 315, 36-41. Santinelli, V., Arnese, M., Oppo, I., Matarazzi, C., Maione, S., Palma, M., Giunta, A., 1993. Effects of flecainide and propafenone on systolic performance in subjects with normal cardiac function. Chest 103, 1068-1073. Solomon, S.D., Zelenkofske, S., McMurray, J.J., Finn, P.V., Velazquez, E., Ertl, G., Harsanyi, A., Rouleau, J.L., Maggioni, A., Kober, L., White, H., Van de Werf, F., Pieper, K., Califf, R.M., Pfeffer, M.A., Valsartan in Acute Myocardial Infarction Trial (VALIANT) Investigators., 2005. Sudden death in patients with myocardial infarction and left ventricular dysfunction, heart failure, or both. N. Engl. J. Med. 352, 2581-2588.

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Yan, G.X., Lankipalli, R.S., Burke, J.F., Musco, S., Kowey, P.R., 2003. Ventricular repolarization components on the electrocardiogram: cellular basis and clinical significance. J. Am. Coll. Cardiol. 42, 401-409.

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Figure 1. Effect of left ventricular systolic dysfunction (LVSD) and flecainide on electrocardiogram (ECG) recorded tracings. A) Representative bipolar, transthoracic ECGs of ketamine/xylazine-anesthetized SHAM-operated rabbits and rabbits with a myocardial infarction (MI) with moderate (MI_M_LVSD) or severe (MI_S_LVSD) left ventricular systolic dysfunction, taken prior to surgery, 1 week and 7 weeks after surgery. B) Representative, bipolar, transthoracic ECGs (10 second tracings) obtained from a SHAM-operated rabbit that received solvent, or 2 mg/kg flecainide, from a rabbit with a large myocardial infarction (MI) developing ventricular tachycardia (VT) followed by ventricular fibrillation (VF) after intravenous infusion of 2 mg/kg flecainide, and from a rabbit with a large MI developing total atrioventricular (AV) block and several episodes of non-sustained VT prior to the onset of the AV block after intravenous infusion of 1 mg/kg flecainide. Figure 2. Effect of flecainide dosing on electrocardiogram parameters obtained from SHAMoperated rabbits and from myocardial infarction (MI)-induced rabbits with moderate (MI_M_LVSD) or severe (MI_S_LVSD) left ventricular systolic dysfunction. Heart rate (A), PQ interval (B), QRS interval (C), QT interval (D), and heart rate-corrected interval QTcF according to the Fridericia formula (E), JT interval (F), and heart rate-corrected interval JTcF according to the Fridericia formula (G), the amplitude of the R wave (H) and amplitude of the T wave (I). The flecainide doses used are 0.25 mg/kg (dose 1), 0.5 mg/kg (dose 2), 1 mg/kg (dose 3), 2 mg/kg (dose 4) and 4 mg/kg (dose 5). Data are means ± S.E.M. of 5-8 animals, and are expressed as a percentage change of baseline (before drug infusion). *,%,# P < 0.05 flecainide versus solvent for SHAM-operated, MI_M_LVSD and MI_S_LVSD rabbits, respectively, and $ P < 0.05 for flecainide-treated SHAM versus MI_S_LVSD rabbits (Holm-Sidak method (α = 0.05)).

27

Figure 3. Effect of flecainide dosing on different calculations of the “index of CardioElectrophysiological Balance” (iCEB). iCEB was calculated as QT/QRS (A) or JT/QRS (B), or calculated according to the heart rate-corrected formulas: according to Bazett QTcB/QRS (C) and JTcB/QRS (D), according to Fridericia QTcF/QRS (E) and JTcF/QRS (F), and according to Van de Water QTcVDW/QRS (G) and JTcVDW/QRS (H). iCEB values were obtained from electrocardiograms of SHAM rabbits and of myocardial infarction (MI)-induced rabbits with moderate (MI_M_LVSD) or severe (MI_S_LVSD) left ventricular systolic dysfunction. The different flecainide doses used are 0.25 mg/kg (dose 1), 0.5 mg/kg (dose 2), 1 mg/kg (dose 3), 2 mg/kg (dose 4) and 4 mg/kg (dose 5). Data are means ± S.E.M. of 5-8 animals, and are expressed as a percentage change of baseline (before drug infusion). *,%,# P < 0.05 flecainide versus solvent for SHAM, MI_M_LVSD and MI_S_LVSD rabbit groups, respectively (Holm-Sidak method (a = 0.05)). Figure 4. Effect of flecainide dosing on other electrocardiogram biomarkers. Transmural dispersion of repolarization (rTp-Te) (A), short-term (QT-STI) (B), long-term (QT-LTI) (C) and total (QT-TI) (D) instability of the QT interval were obtained for SHAM rabbits and myocardial infarction (MI)-induced rabbits with moderate (MI_M_LVSD) or severe (MI_S_LVSD) left ventricular systolic dysfunction. The flecainide doses are 0.25 mg/kg (dose 1), 0.5 mg/kg (dose 2), 1 mg/kg (dose 3), 2 mg/kg (dose 4) and 4 mg/kg (dose 5). Data are means ± S.E.M. of 5-8 animals, expressed as a percentage change of baseline (before drug infusion). *,%,# P < 0.05 flecainide versus solvent for SHAM, MI_M_LVSD and MI_S_LVSD rabbit groups, respectively (Holm-Sidak method (a = 0.05)).

28

N.D.

Chronic infarct size (% LV)

Baseline

4.0 ± 0.10

56.4 ± 0.63

57.3 ± 0.45

7 weeks post MI

3.9 ± 0.14

56.4 ± 0.83

58.2 ± 0.53

48 h post MI

EDV (ml)

58.1 ± 0.55

28.9 ± 0.74abcde

44.2 ± 1.87abd

4.1 ± 0.11

35.9 ± 0.83abd

42.7 ± 1.23abd

4.0 ± 0.13

57.5 ± 1.16

57.2 ± 0.34

46 ± 0.84abc

30 ± 1.56ab

7.9 ± 1.60

58.2 ± 0.48

MI_S_LVSD

MI_M_LVSD

MI_NO_LVSD

Baseline

EF (%)

SHAM

MRI parameters

diastolic LV volume = EDV; end-systolic LV volume = ESV; stroke volume = SV; N.D. = not detectable.

= MI_NO_LVSD; MI with moderate LVSD = MI_M_LVSD; MI with severe LVSD = MI_S_LVSD; ejection fraction = EF; end-

29

P < 0.05 versus the acute stage (Kruskal-Wallis and Dunn’s multiple comparison test). MI with no left ventricular systolic dysfunction

animals. a P < 0.05 versus SHAM; b P < 0.05 versus MI_NO_LVSD; c P < 0.05 versus MI_M_LVSD; d P < 0.05 versus baseline and e

the induction of a reperfused myocardial infarction (MI) in New Zealand white rabbits. Data are presented as means ± S.E.M. of 9-15

Evaluation of cardiac function by in vivo Cine cardiac magnetic resonance imaging (cMRI) at baseline and at 48 h and 7 weeks after

Table 1.

4.8 ± 0.10

4.7 ± 0.15

7 weeks post MI

1.8 ± 0.05 2.0 ± 0.05

1.7 ± 0.06 2.0 ± 0.08

48 h post MI

7 weeks post MI

2.3 ± 0.05 2.3 ± 0.05 2.7 ± 0.07

2.3 ± 0.09 2.3 ± 0.08 2.7 ± 0.08

Baseline

48 h post MI

7 weeks post MI

SV (ml)

1.7 ± 0.06

1.6 ± 0.05

Baseline

ESV (ml)

4.1 ± 0.07

4.0 ± 0.14

48 h post MI

2.4 ± 0.15

1.7 ± 0.07abcd

1.8 ± 0.08abd

1.9 ± 0.11ab

4.6 ± 0.20abcde

3.1 ± 0.19abd

2.3 ± 0.09

3.1 ± 0.08abd

2.6 ± 0.11abd

2.3 ± 0.07

1.7 ± 0.05

6.4 ± 0.27abde

5.6 ± 0.28abd

1.7 ± 0.07

4.9 ± 0.16ab

4.5 ± 0.19ab

30

Table 2 Effect of severity of systolic dysfunction on heart rate (HR), electrocardiogram intervals and height of R and T wave in anesthetized rabbits at baseline, 1 week and 7 weeks post myocardial infarction (MI) induction. Data are means ± S.E.M. of 9-15 animals, expressed as a percentage change of baseline (before surgery). a P < 0.05 versus SHAM rabbits; b P < 0.05 versus MI_M_LVSD rabbits; c P < 0.05 versus baseline and d P < 0.05 versus 1 week post MI induction (Kruskal-Wallis test and Dunn’s multiple comparison test). MI with no left ventricular systolic dysfunction = MI_NO_LVSD; MI with moderate LVSD = MI_M_LVSD; MI with severe LVSD = MI_S_LVSD; heart rate-corrected QT interval according to the Fridericia correction formula = QTcF; heart rate-corrected JT interval according to the Fridericia correction formula = JTcF.

SHAM

MI_M_LVSD

MI_S_LVSD

Baseline (bpm)

170 ± 9.3

159 ± 9.1

168 ± 7.5

1 week post MI (% change)

9.1 ± 5.9

16 ± 6.8

33 ± 3.7c

7 weeks post MI (% change)

3.3 ± 7.7

8.3 ± 7.1

-1.1 ± 4.3d

Baseline (ms)

85 ± 3.4

84 ± 3.5

83 ± 1.3

1 week post MI (% change)

-2.5 ± 2.3

2.9 ± 2.3

-4.0 ± 2.7

7 weeks post MI (% change)

0.091 ± 3.3

-0.013 ± 3.4

1.5 ± 3.3

Heart rate

PQ interval

QRS interval

31

Baseline (ms)

32 ± 1.5

38 ± 1.5

33 ± 1.2

1 week post MI (% change)

6.0 ± 2.7

7.2 ± 4.0

-3.3 ± 4.4

7 weeks post MI (% change)

14 ± 4.4

12 ± 7.4

7.0 ± 4.3

Baseline (ms)

154 ± 5.1

158 ± 8.1

157 ± 3.5

1 week post MI (% change)

-1.6 ± 3.5

9.2 ± 5.1

-6.9 ± 2.7b

7 weeks post MI (% change)

0.98 ± 4.1

-3.0 ± 4.7

2.1 ± 4.2

Baseline (ms)

216 ± 5.8

216 ± 9.1

220 ± 4.2

1 week post MI (% change)

0.53 ± 2.0

13 ± 3.7

3.0 ± 2.9

7 weeks post MI (% change)

0.63 ± 2.3

-1.6 ± 4.1

1.9 ± 3.2

Baseline (ms)

122 ± 5.3

120 ± 7.8

124 ± 3.7

1 week post MI (% change)

-3.6 ± 3.9

9.8 ± 5.9

-7.3 ± 3.4

7 weeks post MI (% change)

-2.6 ± 4.2

-7.6 ± 5.2

1.2 ± 4.6

Baseline (ms)

171 ± 6.6

164 ± 9.3

174 ± 4.7

1 week post MI (% change)

-1.9 ± 2.5

14 ± 4.3

2.7 ± 3.8

7 weeks post MI (% change)

-3.0 ± 2.6

-6.2 ± 4.5

0.61 ± 3.6

0.35 ± 0.034

0.41 ± 0.042

0.35 ± 0.031

QT interval

QTcF interval

JT interval

JTcF

Amplitude R wave Baseline (mV)

32

1 week post MI (% change)

7.1 ± 6.8

-147 ± 33a

-183 ± 21ac

7 weeks post MI (% change)

16 ± 7.3

-113 ± 30a

-174 ± 17ac

Baseline (mV)

0.091 ± 0.013

0.082 ± 0.016

0.092 ± 0.015

1 week post MI (% change)

18 ± 18

-86 ± 106

-31 ± 40

7 weeks post MI (% change)

33 ± 23

39 ± 41

-55 ± 24

Amplitude T wave

33

Table 3 Effect of severity of systolic dysfunction on electrocardiogram (ECG) parameters obtained from anesthetized rabbits at 7 weeks post myocardial infarction (MI) induction before the administration of flecainide. Data are means ± S.E.M. of 5-8 animals. a P < 0.05 versus SHAMoperated rabbits according to the Holm-Sidak method (α = 0.05). MI with no left ventricular systolic dysfunction = MI_NO_LVSD; MI with moderate LVSD = MI_M_LVSD; MI with severe LVSD = MI_S_LVSD; heart rate-corrected QT interval according to the Fridericia correction formula = QTcF; heart rate-corrected JT interval according to the Fridericia correction formula = JTcF; transmural dispersion of repolarization = rTp-Te; short-term QT interval instability = QT-STI; long-term QT interval instability = QT-LTI; total QT interval instability = QT-TI.

ECG parameters

SHAM

MI_M_LVSD

MI_S_LVSD

Heart rate (bpm)

195 ± 7.7

175 ± 6.7

179 ± 11

PQ interval (ms)

77 ± 1.9

78 ± 2.1

80 ± 2.3

QRS interval (ms)

35 ± 2.3

37 ± 2.9

30 ± 1.9

QT interval (ms)

159 ± 3.1

171 ± 6.0

166 ± 11

QTcF interval (ms)

234 ± 3.8

244 ± 5.6

245 ± 15

JT interval (ms)

124 ± 1.8

134 ± 7.9

136 ± 9.6

JTcF interval (ms)

184 ± 2.1

192 ± 8.9

201 ± 14

Amplitude R wave (mV)

0.35 ± 0.065

-0.050 ± 0.13a

-0.28 ± 0.078a 34

Amplitude T wave (mV)

0.104 ± 0.013

0.120 ± 0.018

0.059 ± 0.027

rTp-Te (ms)

18 ± 1.2

19 ± 2.2

17 ± 1.9

QT-STI (ms)

2.8 ± 0.31

2.6 ± 0.17

3.8 ± 0.46

QT-LTI (ms)

2.6 ± 0.38

2.4 ± 0.27

2.7 ± 0.43

QT-TI (ms)

4.8 ± 0.46

4.3 ± 0.37

5.5 ± 0.61

35

Mean BP (mmHg)

92 ± 6.6

98 ± 6.5

95 ± 5.3

Systolic BP (mmHg)

88 ± 5.1

79 ± 6.2

75 ± 4.6

Diastolic BP (mmHg)

Dose 1

87 ± 6.6

84 ± 5.3

Mean BP (mmHg)

Baseline

84 ± 8.1

92 ± 8.2

72 ± 7.6

80 ± 8.3

Solvent

Solvent

different doses of flecainide

Flecainide

MI_M_LVSD

SHAM

BP measurements after infusion of

4) and 4 mg/kg (dose 5).

80 ± 14

86 ± 12

70 ± 11

76 ± 12

Flecainide

85 ± 9.8

95 ± 10

75 ± 9.5

83 ± 10

Solvent

MI_S_LVSD

36

85 ± 8.3

94 ± 6.0

77 ± 5.4

84 ± 6.2

Flecainide

method (a = 0.05). The different flecainide doses used are 0.25 mg/kg (dose 1), 0.5 mg/kg (dose 2), 1 mg/kg (dose 3), 2 mg/kg (dose

of 5-8 animals. a P < 0.05 versus solvent-treated rabbits and b P < 0.05 versus MI_M_LVSD rabbits according to the Holm-Sidak

induced rabbits with moderate (MI_M_LVSD) or severe (MI_S_LVSD) left ventricular systolic dysfunction. Data are means ± S.E.M.

Effect of flecainide dosing on mean, diastolic and systolic blood pressure (BP) in SHAM rabbits and myocardial infarction (MI)-

Table 4

102 ± 6.5

98 ± 5.2

Systolic BP (mmHg)

79 ± 5.8 98 ± 6.1

77 ± 4.5 97 ± 4.9

Diastolic BP (mmHg)

Systolic BP (mmHg)

68 ± 6.1 87 ± 5.8

76 ± 3.9 95 ± 4.3

Diastolic BP (mmHg)

Systolic BP (mmHg)

Mean BP (mmHg)

33 ± 3.8a

74 ± 8.0

96 ± 3.0

61 ± 6.6a

95 ± 4.9

Systolic BP (mmHg)

76 ± 5.7

75 ± 4.4

42 ± 6.6a

76 ± 4.5

Diastolic BP (mmHg)

Dose 5

83 ± 4.3

49 ± 6.9a

83 ± 5.0

108 ± 3.4

87 ± 3.5

96 ± 3.7

97 ± 5.1

77 ± 5.6

85 ± 5.8

96 ± 7.6

76 ± 7.4

Mean BP (mmHg)

Dose 4

76 ± 6.3

84 ± 4.4

Mean BP (mmHg)

Dose 3

87 ± 6.4

85 ± 5.0

Mean BP (mmHg)

Dose 2

84 ± 6.2

79 ± 4.4

Diastolic BP (mmHg)

34 ± 18

59 ± 8.8a

42 ± 6.9a

75 ± 8.5

86 ± 6.1

64 ± 7.3

71 ± 7.4

82 ± 10b

71 ± 8.8a

48 ± 8.2a

69 ± 2.2

62 ± 10b 54 ± 7.9a

37

43 ± 1.2a

73 ± 2.0

57 ± 2.7

63 ± 2.8

86 ± 3.0

76 ± 2.7

87 ± 8.1

70 ± 7.9

77 ± 8.4

94 ± 7.7

77 ± 7.6

70 ± 11b

88 ± 9.4

69 ± 8.6

77 ± 9.5

96 ± 9.8

76 ± 8.9

61 ± 8.7a

83 ± 12

66 ± 12

73 ± 13

90 ± 14

73 ± 12

67 ± 7.9 88 ± 6.6

27 ± 3.5a 50 ± 4.6a

69 ± 5.2 87 ± 5.4

Diastolic BP (mmHg)

Systolic BP (mmHg)

44 ± 20

27 ± 17 89 ± 6.8

68 ± 8.4

38

53 ± 1.4a

36 ± 1.4

Table 5 Flecainide concentrations in plasma samples obtained from SHAM rabbits and rabbits with a myocardial infarction (MI) developing moderate (MI_M_LVSD) or severe (MI_S_LVSD) left ventricular systolic dysfunction measured immediately after full administration of each of the gradual increasing flecainide doses. Data are means ± S.E.M. of 5-8 animals.

Different doses of flecainide

SHAM

MI_M_LVSD

MI_S_LVSD

0.25 mg/kg

0.31 ± 0.045

0.38 ± 0.094

0.24 ± 0.034

0.5 mg/kg

0.79 ± 0.21

0.76 ± 0.14

0.54 ± 0.037

1 mg/kg

1.3 ± 0.16

1.7 ± 0.31

1.3 ± 0.13

2 mg/kg

3.3 ± 0.21

3.1 ± 0.30

2.8 ± 0.32

4 mg/kg

9.2 ± 0.96

7.2 ± 0.68

8.1 ± 1.2

39

40

41

42

43