Remodeling of ventricular repolarization in experimental right ventricular hypertrophy

    Remodeling of ventricular repolarization in experimental right ventricular hypertrophy S.N. Kharin, V.V. Krandycheva, A.S. Tsvetkova, K.V. Shumikhin PII: DOI: Reference:

S0022-0736(17)30134-6 doi: 10.1016/j.jelectrocard.2017.05.008 YJELC 52418

To appear in:

Journal of Electrocardiology

Please cite this article as: Kharin SN, Krandycheva VV, Tsvetkova AS, Shumikhin KV, Remodeling of ventricular repolarization in experimental right ventricular hypertrophy, Journal of Electrocardiology (2017), doi: 10.1016/j.jelectrocard.2017.05.008

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Remodeling of ventricular repolarization in experimental right ventricular hypertrophy

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S.N. Kharin, PhDa,b,*, V.V. Krandycheva, PhDa,b, A.S. Tsvetkova, PhDa, K.V. Shumikhin,

Laboratory of Cardiac Physiology, Institute of Physiology of the Komi Scientific Centre of

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a

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PhDb

the Ural Branch of the Russian Academy of Sciences, 50 Pervomayskaya Street, GSP-2,

b

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Syktyvkar, Komi Republic, 167982, Russian Federation Department of Physiology, Medical Institute, Federal State Budget Educational Institution of Higher Education «Syktyvkar State University named after Pitirim Sorokin», 11

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Babushkin Street, Syktyvkar, Komi Republic, 167000, Russian Federation

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* Corresponding author at: Institute of Physiology, 50 Pervomayskaya Street, GSP-2,

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Syktyvkar, Komi Republic, 167982, Russian Federation E-mail address: [email protected]

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Abstract BACKGROUND

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To understand electrophysiological mechanisms that underlie the progression of

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compensated right ventricular hypertrophy (RVH) to heart failure, the purpose of the study was to evaluate remodeling of ventricular repolarization in connection with hemodynamic

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abnormalities and vulnerability of the heart ventricles to arrhythmias in RVH rats with pulmonary arterial hypertension (PAH) and heart failure.

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METHODS

PAH followed by heart failure was induced by monocrotaline in adult female Wistar rats. Unipolar epicardial electrograms and cardiac hemodynamic parameters were recorded in

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situ. Vulnerability to ventricular arrhythmias was measured as the threshold dose of aconitine required to produce sustained ventricular tachycardia. Histological examination of

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the heart ventricles was performed. Activation–recovery intervals (ARIs) and ARI dispersions were used as indices of durations and heterogeneity of repolarization

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respectively to assess ventricular repolarization.

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RESULTS AND CONCLUSIONS The development of compensated RVH was characterized by the dramatic prolongation of repolarization against the less expressed increase in repolarization heterogeneity, whereas the dramatic increase in repolarization heterogeneity against the less expressed but inhomogeneous prolongation of repolarization occurred in the progression of compensated RVH to heart failure. These changes increased vulnerability of the failing heart but not the compensated heart to aconitine-induced ventricular arrhythmias.

Keywords:

Ventricular

repolarization;

hypertension; Heart failure; Animal model

Right

ventricular

hypertrophy;

Pulmonary

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Introduction Normal pulmonary circulation is characterized by a low blood pressure. In pulmonary

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arterial hypertension (PAH), blood pressure in the pulmonary circuit is increased due to

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structural and functional changes of pulmonary vessels [1,2]. Right ventricular hypertrophy (RVH) develops as an adaptation of the heart to increased wall stress in pressure overload of

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the right ventricle due to PAH, and becomes inadequate in progressive pulmonary vascular

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disease resulting in deterioration of cardiac function. A progressive increase in pulmonary arterial pressure leads to heart failure and unfavorable prognosis for patients [3–6]. PAH and RVH increase risk of ventricular arrhythmias. Ventricular arrhythmogenesis may be attributable to an increase in repolarization heterogeneity [7,8].

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The most commonly used model of pressure overload RVH is the monocrotaline (MCT) rat model of PAH and heart failure. In MCT-treated rats, reactivity of pulmonary arteries to

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vasopressors and vasodilators is reduced [9], and hemodynamic disturbances and myocardial

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injury are similar to those in humans with PAH due to pulmonary vascular disease [2]. The electrophysiological mechanisms underlying the progression of compensated RVH

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to heart failure, are not well known. In the present study, we have tried to evaluate remodeling of ventricular repolarization in connection with hemodynamic changes and vulnerability of the heart ventricles to arrhythmias in RVH rats with MCT-induced PAH and heart failure.

Methods Experimental animals Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edn (see http://grants.nih.gov/grants/olaw/guide-for-the-care-and-

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use-of-laboratory-animals.pdf, accessed 10 May 2016), and the study protocol was approved by the Bioethics Committee of the Institute of Physiology of the Komi Science Centre of the

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Ural Branch of the Russian Academy of Sciences. Female Wistar rats (body weight, 190–

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230 g) were used in the study. Rats were kept in groups of two to three under a 12 h light– dark cycle at 20–24°C, and had free access to food and water.

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Drugs

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MCT (Sigma, LLC “Sigma-Aldrich Rus”, Moscow, Russia) was used to induce PAH followed by RVH. Aconitine (Sigma, “Chimmed”, Moscow, Russia) was used to evaluate vulnerability to life-threatening ventricular arrhythmias. Tiletamine–zolazepam (Zoletil® 100; Virbac S.A., Carros, France) and xylazine (Xyla; Interchemie, Castenray, The

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Netherlands) were used for anesthesia.

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Study design

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Sample sizes were determined in accordance to Dell et al., 2002 [10], based on the previous experimental data and taking into account that there will be three animal group

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(control, MCT-induced PAH, MCT-induced heart failure). Eighty-two rats were randomly divided into two groups. Twenty-seven animals were the control group. Fifty-five rats were injected intraperitoneally with a single dose of 60 mg/kg of MCT. Further procedures were performed from fifth to eight weeks after injection. For electrophysiological

measurements

followed

by

haemodynamic

ones,

rats

were

anaesthetized with tiletamine–zolazepam (1 ml/kg, i.m.) and xylazine (0.5 mg/kg, i.m.), intubated, and artificially ventilated. The heart was exposed through a midsternal approach and the pericardium was excised. The body temperature of the animals was maintained at 38–38.5°C. Warm (38–39°C) saline was applied intermittently to the heart to moisten the epicardium and prevent surface cooling. On completion of haemodynamic measurements,

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the rats were sacrificed using cervical dislocation. For the administration of aconitine, rats were anaesthetized as mentioned above, intubated, and artificially ventilated. Afterwards, the

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left jugular vein was exposed and cannulated with a peripheral intravenous catheter

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(Flexican 26G; Apexmed International B.V., Amsterdam, the Netherlands), and aconitine was infused until ventricular fibrillation occurred. After rats had been sacrificed, the heart

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and lungs were immediately excised, rinsed with saline, blotted dry, and weighed. Before weighing, the atria were discarded; the right ventricle was separated from the left one. The

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interventricular septum was considered part of the left ventricle for left ventricular mass calculation purposes. Excised organs were stored in 20% buffered formalin for histological analysis.

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Electrophysiological measurements

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Epicardial mapping of the heart ventricles was performed under sinus rhythm. A square array of 64 electrodes spaced 0.5 mm apart was used and sequentially superimposed on the

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left ventricular base, left ventricular apex, right ventricular apex and right ventricular base. For 1–2 min, unipolar epicardial electrograms were recorded from 256 sites on the

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ventricular surface in reference to Wilson’s central terminal. Standard bipolar limb lead ECG was recorded with the application of subcutaneous needle electrodes. The signals were isolated, amplified, multiplexed and recorded by a custom-designed 144-channel computerized mapping system with a bandwidth of 0.05–1000 Hz at a sampling rate of 4000 Hz. Haemodynamic measurements A flowprobe (2PSB2252; Transonic Systems, Ithaca, NY, USA) was placed around the ascending aorta, which was gently isolated from the pulmonary artery. Lubricating gel was used to replace the air space between the probe acoustic window and the aorta. The

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flowprobe cable was connected to a transit-time flow module (Type 700; Hugo Sachs Elektronik, March, Germany; Transonic Systems) of a recording system (Hugo Sachs

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Elektronik–Harvard Apparatus, March–Hugstetten, Germany).

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For the measurement of blood pressure, polyethylene catheters (inner diameter, 0.6 mm), filled with heparinized saline, were introduced into the heart ventricles through their

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free walls. The catheters were connected to pressure transducers (SP844; MEMSCAP, France). Intraventricular pressure and standard bipolar limb lead electrocardiograms were

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recorded by a Prucka Mac-Lab 2000 system (GE Medical System GmbH, Germany). Measurements of vulnerability to arrhythmias

Vulnerability to ventricular arrhythmias was measured as the threshold dose of aconitine

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required to produce sustained (≥3 s) ventricular tachycardia. Aconitine (10 μg/ml) was

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continuously infused (0.2 ml/min) from a remote syringe placed in a programmable pump (SN-50C6; Sino Medical-Device Technology Co., Ltd, Shenzhen, China) while ECG was

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continuously recorded and timed. A dose of aconitine was calculated using the following formula: Threshold dose (μg/kg) = 10 μg/ml × 0.2 ml/min × time required for inducing

Histology

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arrhythmia (min) / body weight (kg).

Hearts and lungs were fixed with 10% buffered formalin and then embedded in paraffin. The middle third of the heart ventricles and the basal part of the lungs were used for further analysis. Serial sections of the organs were cut at thickness of 5 μm and stained with hematoxylin and eosin (heart, lungs) and Van Gieson’s stain (heart). Van Gieson’s staining was used to detect cardiac fibrosis. Light microscopy of two to three sections of each organ of each rat was performed by the blinded expert with a MIKMED-6 microscope (LOMO, Saint Petersburg, Russia).

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Data analysis The ratio of right ventricular weight to left ventricular weight was used as an index of

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RVH. Cardiac output was measured as the mean blood flow in the ascending aorta. The

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maximal systolic pressure, dP/dtmax (contractility) and dP/dtmin (lusitropy) were measured. The activation-recovery interval (ARI), an index of local repolarization duration, was

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calculated as the difference between local repolarization time and local activation time from

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each ventricular electrogram. Local activation time and local repolarization time were determined as dV/dtmin during QRS complex and dV/dtmax during ST-T complex, respectively (Fig. 1) [11]. The determinations were done automatically, inspected by the blinded experimenter and corrected if necessary. The dispersion of ARIs was quantified as

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the difference between the longest and shortest ARI and was used as the index of repolarization heterogeneity. ARI dispersions were calculated over the entire ventricular

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Statistical analysis

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epicardium (global dispersions) and each ventricle (local dispersions).

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All analyses were performed using statistica version 7.0 (Statsoft, Tulsa, OK, USA). All data are presented as means ± SD. Comparisons were performed using the Kruskal–Wallis test followed by Dunn’s test. P < 0.05 was considered statistically significant.

Results The rat mortality rate due to MCT injection was 16%. Rats (n=9) were dead within the period from 3 to 6 weeks after the injection. The survivors (n=46) showed the following clinical signs: paleness, cyanosis, weakness, lassitude, dyspnea, and body weight loss or less bodyweight gain compared with the control group. Intensity of these signs was various. In 44 of the survivor rats, the ratio of right ventricular weight to left ventricular weight

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increased approximately two-fold. Two rats did not developed RVH and were excluded from the further analysis. In addition, three rats with RVH were excluded from the further

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analysis, because hemodinamic measurements were unsuccessful due to technical reasons.

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In controls (n=19), right ventricular pressure and cardiac output were ≤33 mmHg and ≥25 ml/min, respectively. Based on the analysis of the hemodynamic parameters, the rats

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with RVH (n=28) were divided into three groups (Table 1). In the test group 1 (TG1; criteria were right ventricular pressure >33 mmHg and cardiac output ≥25 ml/min), cardiac output

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did not differ from that in the control group. In TG1, pulmonary pressure was elevated, but right ventricular performance improved substantially (contractility and lusitropy of the right ventricle were increased two-fold in comparison with the control group) and left ventricular

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performance was preserved. In the other two groups, RVH was greater than in TG1, and left ventricular performance worsened. In the test group 2 (TG2; >33 mmHg, <25 ml/min),

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pulmonary pressure was still elevated, but right ventricular pump function was deteriorated in comparison with TG1. In contrast to TG2, in the test group 3 (TG3; ≤33 mmHg), right

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ventricular systolic pressure was reduced to the level of that in the control group, and

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significant worsening of right ventricular performance occurred. Intensity of clinical signs increased from TG1 to TG3. In MCT-treated rats, structural damages in lung and cardiac tissues were observed (Table 2). These damages in TG3 were more extensive than in TG1. Figure 2 shows examples of hematoxylin-eosin-stained lung and myocardial tissues from control and MCTtreated rats. ARIs in RVH rats were prolonged in both ventricles (Table 3). ARI prolongation was inhomogeneous. ARIs in the right ventricle were prolonged significantly greater than ARIs in the left ventricle (142% vs. 66%) that resulted in decreasing interventricular differences in ARIs. ARIs in TG2 and TG3 were more prolonged than in TG1. However, the ARI

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prolongation in TG1 as compared with controls was significantly greater than the ARI prolongation in TG2 and TG3 as compared with TG1 (91% vs 28–51%).

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In TG1, the global ARI dispersion was increased due to the increase of the ARI

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dispersion on the right ventricle (Table 3) and the difference in the ARI dispersion between the ventricles was decreased. The left ventricular ARI dispersion did not change in TG1

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despite the significant prolongation of left ventricular repolarization. The increase of the global and local ARI dispersions in TG2 and TG3 were greater than that in TG1. The

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augmentation of the global and local ARI dispersions in TG1 as compared with controls was significantly less than the augmentation of those in TG2 and TG3 as compared with TG1 (for the global ARI dispersion: 21% vs 85–115%).

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There was no statistically significant difference in electrophysiological changes between TG2 and TG3. In all MCT-treated groups, the electrophysiological changes were more

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expressed in the right ventricle.

Vulnerability to aconitine-induced sustained (>3 s) ventricular tachycardia was

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significantly enhanced in rats with worsened left ventricular pump function (the combined

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group from TG2 and TG3). Vulnerability to aconitine-induced sustained ventricular tachycardia in TG1 did not differed from that in controls (Table 4). Figure 3 shows an example of ventricular tachycardia typically found in the rats in this study.

Discussion This study investigated relationships between changes of cardiac hemodynamics and ventricular repolarization in RVH caused by PAH followed by heart failure. For this purpose, the MCT rat model was used. This model adequately reflects changes occurring in the lungs and heart during the development of PAH and right ventricular heart failure in humans [2,12,13]. In addition, the present study is the first to describe epicardial

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repolarization of the in situ heart ventricles in MCT-induced RVH. The major findings of the study reported here are as follows: 1) the degree of the ARI prolongation and the increase in

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repolarization heterogeneity are related to severity of cardiac hemodynamic disturbances in

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RVH rats; 2) the prolongation of ventricular repolarization is the adaptation to the increase of afterload; 3) the increase in repolarization heterogeneity is maladaptive and

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arrhythmogenic.

Prolongation of ventricular repolarization occurs in left ventricular hypertrophy of

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different genesis [14–19] and is related to remodeling of potassium [14,20] and calcium [21] currents. There are regional differences in the influence of hypertrophy on ion currents in ventricular myocytes [21–23]. The ARI prolongation in RVH rats in our study was in

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consistent agreement with action potential prolongation [24–29] and related to changes of calcium transport [24,30], remodeling of potassium channels [24–26,29], and enhancement

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of the late inward sodium current [29] that are observed in pressure overload RVH. The increased ARI dispersion was in consistent agreement with the increased action potential

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dispersion in MCT-treated rats reported for other studies [26,27]. The more prominent

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electrical remodeling of the right ventricle observed in our study, has been demonstrated in MCT-induced

pulmonary

hypertension

in

rats

by

other

researches

[26,29].

Electrophysiological remodeling of the heart ventricles in MCT-induced RVH was similar to that in left ventricular hypertrophy induced by renovascular hypertension [31] and in heart failure [17–19]. The hemodynamic disturbances in the RVH rats correlated with myocardial injuries, which was similar to those reported by other researches [32–34], and the general status of the rats. In the TG1 rats, the improved right ventricular performance was in agreement with other findings [24,35] and was determined by RVH resulted from MCT-induced structural and functional changes of pulmonary vessels [3,36]. The results evidenced compensatory

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response of the heart in the TG1 rats that was accompanied by increasing duration and heterogeneity of repolarization. Prolongation of repolarization was likely related, at least in

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the right ventricle, to an increase in L-type calcium current density [24] that was in

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agreement with increasing contractility of the right ventricle. Interestingly, the ARI prolongation in the pressure overloaded right ventricle (142%) (the present study) was

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significantly greater than the ARI prolongation in the pressure overloaded left ventricle (72%) [31]. Such expressed prolongation of ARIs in the pressure overloaded right ventricle

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may be attributable to its features. In contrast to the left ventricle, the right one works in other mechanical conditions and its thin wall is more sensitive to mechanical stress [37]. Taking into account the difference in the increase of the ARI dispersion between the right

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ventricle and both ventricles (52% vs 21%), one might assume that the prolongation of left ventricular repolarization in TG1 restrained the increase of ventricular repolarization

therefore, was adaptive.

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heterogeneity, which is a substrate for the development of ventricular arrhythmias [7,8], and,

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The hearts of the TG2 rats were evidently in a decompensated stage, end-stage heart

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failure was observed in the TG3 rats. This was in agreement with findings reported for pressure overload RVH by other researches [5,38,39] and was likely attributable to atrophic changes of ventricular myocardium due to pressure overload right ventricular failure [39,40]. In decompensation and heart failure, the prolongation of repolarization in both ventricles was likely attributable to remodeling of the transient outward potassium current [24,40], prolongation of left ventricular repolarization resulted in the great increase of the left ventricular ARI dispersion, and the increase in repolarization heterogeneity of each ventricle contributed to the increase in repolarization heterogeneity of the heart ventricles. There are regional differences in repolarization durations of the heart ventricles in normal rats [17–20,31]. It has been shown, there are regional differences in overload-

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induced changes of repolarization durations of the heart ventricles that results in increasing of repolarization heterogeneity [21,26,31,41]. The prolongation of ventricular repolarization

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in MCT-induced RVH reported for our study was accompanied by the increase of

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repolarization heterogeneity. However, the ratio of the global ARI dispersion augmentation to the ARI prolongation in the failing heart was significantly greater than that in

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compensated RVH. This dramatic increase of ventricular heterogeneity in the failing heart was likely contributed by the increased regional differences in prolongation of repolarization

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in the failing heart as compared with compensated RVH and resulted in significantly increased vulnerability of the heart ventricles to fatal arrhythmias. One might supposed, that decompensation of the heart function and increasing of electrical instability of the heart

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ventricles were attributable to increasing of repolarization heterogeneity due to intraventricular inhomogeneity of repolarization prolongation. In the compensated heart,

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there was the significantly less increase of repolarization heterogeneity due to the interventricular difference in repolarization prolongation but not intraventricular

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inhomogeneity of repolarization prolongation that did not dramatically affect ventricular

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arrhythmogenicity.

The limitations of the present study concern two major points. First, injuries in tissues were not quantified. Visually, however, injuries in tissues were more extensive in TG3 than in TG1. Second, to keep the intact thorax and heart, the hemodynamic and electrophysiological measurements were not performed in the MCT-treated rats used in the ventricular arrhythmias experiments. That is why, these rats were divided only into two tested groups based on intensity of clinical signs and tissue injuries, which differed weakly between TG2 and TG3.

Conclusion

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The present study demonstrated ventricular repolarization in situ, cardiac hemodynamics and vulnerability of the heart ventricles to arrhythmias in pressure overload RVH resulting

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from MCT-induced PAH. The development of compensated RVH was characterized by the

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dramatic prolongation of repolarization against the less expressed increase in repolarization heterogeneity, whereas the dramatic increase in repolarization heterogeneity against the less

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expressed but inhomogeneous prolongation of repolarization occurred in the progression of compensated RVH to heart failure. These changes increased vulnerability of the failing heart

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but not the compensated heart to aconitine-induced ventricular arrhythmias.

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Conflicts of interest: none.

References

Rabinovitch M. Investigational approaches to pulmonary hypertension. Toxicol Pathol 1991;19:458–69.

Campiani ME, Hardziyenka M, Michel MC, Tan HL. How valid are animal models to

CE

[2]

PT

[1]

AC

evaluate treatments for pulmonary hypertension? Naunyn-Schmiedeberg`s Arch Pharmacol 2006;373:391–400. [3]

Li XQ, Hong Y, Wang Y, Zhang XH, Wang HL. Sertraline pritects against monocrotaline-induced pulmonary hypertension in rats. Clin Exp Pharmacol Physiol 2006;33:1047–51.

[4]

Umar S, Hessel M, Steendijk P, Bax W, Schutte C, Schalij M, et al. Activation of signaling molecules and matrix metalloproteinases in right ventricular myocardium of rats with pulmonary hypertension. Pathol Res Pract 2007;203:863–72.

ACCEPTED MANUSCRIPT [5]

14

Leichsenring-Silva F, Tavares A, Mosele F, Berger B, Llesuy S, Belló-Klein A. Association of time course of pulmonary arterial hypertension with changes in

Thenappan T, Prins KW, Pritzker MR, Scandurra J, Volmers K, Weir EK. The critical

RI P

[6]

T

oxidative stress in the left ventricle. Clin Exp Pharmacol Physiol 2011;38:804–10.

role of pulmonary arterial compliance in pulmonary hypertension. Ann Am Thorac

[7]

SC

Soc 2016;13:276–84.

Antzelevitch C. Role of spatial dispersion of repolarization in inherited and acquired

MA NU

sudden cardiac death syndromes. Am J Physiol Heart Circ Physiol 2007;293:H2024– 38. [8]

Killeen MJ, Sabir IN, Grace AA, Huang CL. Dispersions of repolarization and

ED

ventricular arrhythmogenesis: Lessons from animal models. Prog Biophys Mol Biol 2008;98:219–29.

Mam V, Tanbe AF, Vitali SH, Arons E, Christou HA, Khalil RA. Impaired

PT

[9]

vasoconstriction and nitric oxide-mediated relaxation in pulmonary arteries of

CE

hypoxia- and monocrotaline-induced pulmonary hypertensive rats. J Pharmacol Exp

AC

Ther 2010;332:455–62. [10] Dell RB, Holleran S, Ramakrishnan R. Sample size determination. ILAR J 2002;43:207–13. [11] Millar CK, Kralios FA, Lux RL. Correlation between refractory periods and activation–recovery intervals from electrograms: Effects of rate and adrenergic interventions. Circulation 1985;72:1372–9. [12] Naeije R, Dewachter L. Modèles animaux d’hypertension artérielle pulmonaire [Animal models of pulmonary arterial hypertension]. Rev Mal Respir 2007;24:481–96.

ACCEPTED MANUSCRIPT

15

[13] Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary

arterial

hypertension:

the

hope

for

etiological

discovery

and

T

pharmacological cure. Am J Physiol Lung Cell Mol Physiol 2009;297:L1013–32.

RI P

[14] Li X, Jiang W. Electrical remodeling of membrane ionic channels of hypertrophied ventricular myocytes from spontaneously hypertensive rats. Chin Med J (Engl)

SC

2000;113:584–7.

[15] Naqvi RU, Tweedie D, MacLeod KT. Evidence for the action potential mediating the

MA NU

changes to contraction observed in cardiac hypertrophy in the rabbit. Int J Cardiol 2001;77:189–206.

[16] Volk T, Nguyen TH-D, Schultz J-H, Faulhaber J, Ehmke H. Regional alterations of

ED

repolarizing K+ currents among the left ventricular free wall of rats with ascending aortic stenosis. J Physiol 2001;530:443–55.

PT

[17] Kharin SN, Krandycheva VV, Strelkova MV, Tsvetkova AS, Shmakov DN. Doxorubicin-induced changes of ventricular repolarization heterogeneity: results of a

CE

chronic rat study. Cardiovasc Toxicol 2012;12:312–7.

AC

[18] Kharin S, Krandycheva V, Tsvetkova A, Strelkova M, Shmakov D. Remodeling of ventricular repolarization in a chronic doxorubicin cardiotoxicity rat model. Fundam Clin Pharmacol 2013;27:364–72. [19] Krandycheva V, Kharin S, Strelkova M, Shumikhin K, Sobolev A, Shmakov D. Ventricular repolarization in rat model of global heart failure. Clin Exp Pharmacol Physiol 2013;40:431–7. [20] Clark RB, Bouchard RA, Salinas-Stefanon E, Sanchez-Chapula J, Giles WR. Heterogeneity of action potential waveforms and potassium currents in rat ventricle. Cardiovasc Res 1993;27:1795–9.

ACCEPTED MANUSCRIPT

16

[21] Volk T, Noble PJ, Wagner M, Noble D, Ehmke H. Ascending aortic stenosis selectively increases action potential-induced Са2+ influx in epicardial myocytes of the

T

rat left ventricle. Exp Physiol 2004;90:111–21.

RI P

[22] Bryant SM, Shipsey SJ, Hart G. Normal regional distribution of membrane current density in rat left ventricle is altered in catecholamine-induced hypertrophy.

SC

Cardiovasc Res 1999;42:391–401.

[23] Tomaselli GF, Marbán E. Electrophysiological remodeling in hypertrophy and heart

MA NU

failure. Cardiovasc Res 1999;42:270–83.

[24] Lee JK, Kodama I, Honjo H, Anno T, Kamiya K, Toyama J. Stage-dependent changes in membrane currents in rats with monocrotaline-induced right ventricular

ED

hypertrophy. Am J Physiol 1997;272:H2833–42.

[25] Furukawa T., Kurokawa J. Potassium channel remodeling in cardiac hypertrophy. J

PT

Mol Cell Cardiol 2006;41:753–61.

[26] Benoist D, Stones R, Drinkhill M, Bernus O, White E. Arrhythmogenic substrate in

CE

hearts of rats with monocrotaline-induced pulmonary hypertension and right

AC

ventricular hypertrophy. Am J Physiol Heart Circ Physiol 2011;300:H2230–7. [27] Benoist D, Stones R, Drinkhill MJ, Benson AP, Yang Z, Cassan C, et al. Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension. Am J Physiol Heart Circ Physiol 2012;302:H2381–95. [28] Rich JD, Thenappan T, Freed B, Patel AR, Thisted RA, Childers R, et al. QTc prolongation is associated with impaired right ventricular function and predicts mortality in pulmonary hypertension. Int J Cardiol 2013;167:669–76. [29] Rocchetti M, Sala L, Rizzetto R, Staszewsky LI, Alemanni M, Zambelli V, et al. Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension. Cardiovasc Res 2014;104:37–48.

ACCEPTED MANUSCRIPT

17

[30] Brunner F, Wölkart G, Haleen S. Defective intracellular calcium handling in monocrotaline-induced right ventricular hypertrophy: protective effect of long-term

(PD155080).

J

Pharmacol

Exp

Ther

RI P

phenyl-)-4-oxobut-2-enoate-sodium

T

endotelin-A receptor blockade with 2-Benzo[1,3]dioxol-5-yl-3-benzil-4-(4-methoxy-

2002;300:442–9.

SC

[31] Krandycheva VV, Kharin SN, Azarov JE, Shmakov DN. The effects of renovascular hypertension on repolarization of ventricular epicardium. Exp Clin Cardiol

MA NU

2009;14:E51–6.

[32] Campian ME, Hardziyenka M, de Bruin K, van Eck-Smit BL, de Bakker JM, Verberne HJ, et al. Early inflammatory response during the development of right ventricular

ED

heart failure in a rat model. Eur J Heart Fail 2010;12:653–8. [33] Fontoura D, Oliveira-Pinto J, Tavares-Silva M, Leite S, Vasques-Nóvoa F, Mendes-

PT

Ferreira P, et al. Myocardial and anti-inflammatory effects of chronic bosentan therapy in monocrotaline-induced pulmonary hypertension. Rev Port Cardiol 2014;33:213–22.

CE

[34] Tang B, Chen G, Liang M, Yao J, Wu Z. Ellagic acid prevents monocrotaline-induced

AC

pulmonary artery hypertension via inhibiting NLRP3 inflammasome activation in rats. Int J Cardiol 2015;180:134–41. [35] Stones R., Benoist D., Peckham M., White E. Microtubule proliferation in right ventricular myocytes of rats with monocrotaline-induced pulmonary hypertension. J Mol Cell Cardiol 2013;56:91–6. [36] Kay JM, Harris P, Heath D. Pulmonary hypertension produced in rats by ingestion of Crotalaria spectabilis seeds. Thorax 1967;22:176–9. [37] Bristow MR, Zisman LS, Lowes BD, Abraham WT, Badesch DB, Groves BM, et al. The

pressure-overloaded

1998;114:101S–6S.

right

ventricle

in

pulmonary hypertension.

Chest

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[38] Hessel MN, Steendijk P, den Adel B, Schutte CI, van der Laarse A. Characterization of right ventricular function after monocrotaline-ibduced pulmonary hypertension in

HH,

Haddad

F.

Pulmonary

Hypertension.

A

stage

for

ventricular

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[39] Hsia

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the intact rat. Am J Physiol Heart Circ Physiol 2006;291:2424–30.

interdependence? J Am Coll Cardiol 2012;59:2203–05.

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[40] Hardziyenka M, Campian ME, Verkerk AO, Surie S, van Ginneken AC, Hakim S, et al. Electrophysiologic remodeling of the left ventricle in pressure overload-induced

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right ventricular failure. J Am Coll Cardiol 2012;59:2193–202. [41] Sedova KA, Goshka SL, Vityazev VA, Shmakov DN, Azarov JE. Load-induced changes in ventricular repolarization: evidence of autonomic modulation. Can J Pysiol

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CE

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Pharmacol 2011;89:935–44.

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Table 1 Body weight, RV/LV weight ratio and hemodynamic parameters in rats treated with

T

monocrotaline. TG3

(n = 12)

(n = 8)

209 ± 9

208 ± 11

206 ± 12

-10 ± 17.2*

-21 ± 25*

-45 ± 30*†‡

TG1

(n = 19)

(n = 8)

at baseline

206 ± 22

gain

23 ± 13

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Bodyweight (g)

TG2

RI P

Control group Parameter

0.27 ± 0.02

0.49 ± 0.10*

0.56 ± 0.05*†

0.56 ± 0.08*†

Cardiac output (ml/min)

36 ± 10

38 ± 5

19 ± 5*†

12 ± 2*†‡

LV systolic pressure (mmHg)

106 ± 16

98 ± 15

83 ± 16*†

51 ± 11*†‡

3251 ± 1109

3135 ± 1009

2284 ± 680*†

1222 ± 249*†‡

2670 ± 982

2413 ± 778

1839 ± 653 *†

807 ± 302 *†‡

27 ± 4

60 ± 19*

50 ± 9*†

26 ± 4†‡

852 ± 247

1694 ± 346*

1194 ± 257*†

585 ± 140*†‡

623 ± 141

1477 ± 419 *

1103 ± 262 *†

478 ± 143 *†‡

RV/LV weight ratio

ED

LV dP/dt max (mmHg/sec)

RV systolic pressure (mmHg/sec)

CE

RV dP/dt max (mmHg/sec)

PT

LV dP/dt min (mmHg/sec)

AC

RV dP/dt min (mmHg/sec )

Data are the mean ± SD. * P < 0.05 compared with the control group, † P < 0.05 compared with TG1, ‡ P < 0.05 compared with TG2 (the Mann–Whitney test). RV, right ventricle; LV, left

ventricle;

TG,

test

group.

ACCEPTED MANUSCRIPT Table 2 Structural damage in pulmonary and myocardial tissues in rats treated with monocrotaline. Lungs

Ventricular myocardium

TG1

interstitial infiltration

tortuosity of myocytes

(n = 8)

lymphoid hyperplasia

RI P

T

Group

hyperplasia of myocytes

emphysematous foci

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foci of atelectasis vasculitis

bronchitis TG2

alveolar edema

(n = 12)

lymphoid hyperplasia

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small/focal hemorrhages

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desquamation of alveolar macrophages emphysema vasculitis

tortuosity of myocytes collagenation inflammation perivascularis infiltration interstitial edema turgent epithelium

zones of carnification

paretic vasodilatation

small/focal hemorrhages

capillary hyperemia

serous exudates in bronchi

hemorrhages

TG3

interstitial inflammation

tortuosity of myocytes

(n = 8)

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hypertrophy of arterial walls

perivascularis infiltration

perivascular fibrosis

sparse foci of emphysema with alveolar edema

interstitial infiltration

organized atelectasis

interstitial edema

hypertrophy of arterial walls

hypertrophy of myocytes

carnification

paretic vasodilatation

hemorrhages

hemorrhages

bronchial spasm TG, test group.

20

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Table 3 Electrophysiological parameters in rats treated with monocrotaline. Control group

TG1

TG2

of the heart ventricles

(n = 19)

(n = 8)

(n = 12)

RI P

ARIs (ms)

T

Area

TG3 (n = 8)

30.9 ± 6.5 *†

36.5 ± 15.3 *†

25.2 ± 7.2 *

35.1 ± 13.7 *†

40.0 ± 30.4 *

14.8 ± 4.3

24.6 ± 4.1 *

27.7 ± 6.7 *

33.6 ± 18.1 *

RV apex

10.9 ± 2.8

25.2 ± 7.8 *

35.9 ± 12.7 *†

42.3 ± 31.5 *

RV base

9.8 ± 2.0

24.4 ± 6.7 *

31.5 ± 17.3 *

37.4 ± 30.2 *

LV apex

15.6 ± 5.0

25.4 ± 4.6 *

28.9 ± 7.9 *

34.0 ± 20.1 *

LV base

14.0 ± 4.1

23.8 ± 4.4 *

26.6 ± 6.0 *

33.1 ± 15.8 *

12.7 ± 3.1

24.2 ± 3.3 *

Right ventricle

10.4 ± 2.2

Left ventricle

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Both ventricles

PT

ARI dispersion (ms)

15.6±4.2

18.8 ± 4.5 *

34.8 ± 13.9 *†

48.0 ± 28.0 *†

9.1 ± 3.8

13.8 ± 3.7 *

28.7 ± 11.5 *†

19.3 ± 4.8 *†‡

12.1 ± 4.4

11.9 ± 3.9

22.4 ± 5.7 *†

26.9 ± 11.2 *†

6.5 ± 3.3

7.8 ± 4.3

19.6 ± 13.2 *†

13.1 ± 4.7 *†

6.2 ± 3.1

10.1 ± 4.7 *

15.0 ± 7.0 *(†)

10.2 ± 4.4 *

LV apex

10.1 ± 4.0

8.3 ± 3.4

19.3 ± 7.4 *†

16.6 ± 10.2 *†

LV base

8.2 ± 4.6

8.5 ± 4.01

13.6 ± 7.0 *†

20.0 ± 15.6 *†

Right ventricle

RV apex RV base

AC

Left ventricle

CE

Both ventricles

Data are the mean ± SD. * P < 0.05 compared with the control group, † P < 0.05 compared with TG1, ‡ P < 0.05 compared with TG2 (the Mann–Whitney test). ARI, activation–recovery interval; RV, right ventricle; LV, left ventricle; TG, test group.

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Table 4 The threshold dose of aconitine producing sustained (> 3 s) ventricular tachycardia in rats

Control group

8

TG1

6

TG2 + TG3

7

Aconitine dose (µg/kg)

RI P

n

74 ± 9

SC

Group

T

treated with monocrotaline.

65 ± 15

50 ± 14 *†

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Data are the mean ± SD. * P < 0.05 compared with the control group, † P < 0.05 compared

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PT

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with TG1 (the Mann–Whitney test). RV, right ventricle; LV, left ventricle; TG, test group.

19

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SC

RI P

T

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Fig. 1. The representative epicardial electrogram (solid line) and its first time derivative (dotted line) with the markers indicating activation time (AT), repolarization time (RT), and

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activation-recovery interval (ARI).

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B

SC

RI P

T

Control

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TG1

AC

TG3

CE

PT

ED

TG2

Fig. 2. Representative images of hematoxylin-eosin-stained lung (A) and myocardial (B) sections obtained from a control rat (Control) and MCT-treated rats (TG1, TG2, TG3). Cont, A and B: a typical image showing normal tissues. A, TG1: a typical image showing atelectasis, emphysema, vasculitis, and bronchitis; A, TG2: a typical image showing emphysema and edema; A, TG3: a typical image showing edema and hemorrhages. B, TG1: a typical image showing tortuosity of myocytes; B, TG2: a typical image showing

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inflammation and fibrosis; B, TG3: a typical image showing tortuosity of myocytes and

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RI P

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hemorrhages.

22

RI P

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Fig. 3. Typical ECG traces showing normal sinus rhythm (left panel) and aconitine-induced

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ventricular tachycardia (right panel) in a MCT-treated rat.

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Highlights 1. Ventricular repolarization is prolonged and repolarization heterogeneity is increased in

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RVH.

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2. The degree of these alterations depends on hemodynamic changes and myocardial injuries.

SC

3. Vulnerability of the failing heart but not the compensated heart to fatal ventricular

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arrhythmias is increased.