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International Journal of Arrhythmia 2014;15(1): 6-19.
ORIGINAL ARTICLE
Losartan attenuates atrial structural
and electrical remodeling in rat ischemic
heart failure model: Implications of
endothelin for atrial fibrillation

Kyoung-Im Cho, MD, Soo-Jeong Lee, MD, Tae-Joon Cha, MD, PhD, Sang-Ho Koo, MS, Jung-Ho Heo, MD, PhD, Hyun-Su Kim, MD, and Jae-Woo Lee, MD, PhD
Division of Cardiology, Department of Internal Medicine, Kosin University College of Medicine, Busan, Korea




Introduction

   Atrial fibrillation (AF) is the most common form of sustained arrhythmia. It is associated with cardiac dysfunction and thrombus formation, two major complications resulting in an increased risk of morbidity and mortality from congestive heart failure (CHF) and stroke.1,2 AF is commonly associated with CHF, because CHF creates the conditions necessary for AF development via structural remodeling and neurohormonal remodeling.3 Therefore, elucidating the exact role that CHF plays in the development AF is of paramount importance.
   There are two mechanisms of arrhythmia development in CHF. One mechanism is reentry, which is associated with increased tissue fibrosis4 due to activation of the renin-angiotensin system (RAS).5 In a nonclinical study, it was demonstrated that angiotensin II receptor blockers (ARB) prevent AF by reducing atrial structural remodeling.6 The other important mechanism of arrhythmia development in CHF is triggered activity.7 By performing biatrial mapping experiments in dogs with CHF, Fenelon et al. demonstrated that the majority of AF episodes involved a focal mechanism. Another anti-AF mechanism of ARBs may be the prevention of focal electrical discharge.8
   Multiple neurohormonal factors, including endothelin-1 (ET-1), are altered in patients with CHF. ET-1 concentration is increased in cardiac tissues during pathological conditions such as CHF9 and myocardial infarction (MI).10 ET-1 can provide short-term inotropic support for failing hearts, however, this benefit comes with the potential burden of arrhythmogenesis and remodeling.11 The arrhythmogenic effect of ET-1 is mediated by activation of inositol 1,4,5-triphosphate.12 ET receptor antagonists have shown promising results in animal models of CHF,13,14 but have failed to improve morbidity and mortality in clinical trials in humans.15 Recent reports have suggested that angiotensin II is a powerful stimulator of ET synthesis and release in vascular smooth muscle and endothelial cells.16,17 Therefore, in this study, we investigated the role of the ET system in the development of AF in a model of CHF. We also evaluated the potential AF-suppressing effect of ARBs to determine whether blocking ET-1 synthesis is of significance.


Materials and Methods

Animal preparation

   All animal experiments were performed in accordance with Korean Council on Animal Care guidelines, and under the authority of the Animal Research Ethics Committee of the Kosin University School of Medicine. Male Sprague-Dawley (SD) rats were purchased from Daehan Biolink Inc. (Korea). Rats were 7-8 weeks old and weighed between 250 and 300 g. Rats were fed a normal sodium diet and offered tap water ad libitum. MI was induced by ligation of the left anterior descending coronary artery (LAD) as described in detail previously.18 Rats were allowed to recover for 2 days and then randomized into 3 groups: sham (n=20), MI (n=19), and MI + losartan (n=20). Losartan (10 mg/kg/day) was added to the drinking water and administered for a period of 10 weeks. Water consumption and body weight were carefully monitored. Sham operated rats served as the control group.

MI procedure

   Rats were anesthetized by i.p. administration of 50 mg/kg ketamine and 10 mg/kg xylazine. Rats were ventilated with room air at 60 strokes per min and a stroke volume of 1 mL/100 g body weight (Harvard, Rodent Ventilator, Model 683). After opening the chest by mid-sternotomy, the LAD was ligated with a 5.0 polypropylene suture (Ethicon, Belgium) approximately 2 to 3 mm from its origin. Control rats were subjected to a sham operation using a similar procedure, but without coronary ligation. Perioperative mortality was around 40% in rats subjected to coronary artery ligation.

Echocardiography

   At the end of the study, rats were lightly anesthetized by i.p. administration of 25 mg/kg ketamine with 5 mg/kg xylazine (n=10/group). Transthoracic echocardiography was performed using an Acuson Sequoia C512 12-MHz phased-array probe (Mountain View, CA, USA). Wall thickness and left ventricular (LV) diameters during diastole and systole were measured from M-mode recordings according to the leading-edge method. Two-dimensional, short-axis images at the mid-papillary muscle level were obtained for measurement of LV end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD), fractional shortening (FS), and wall thickness. Ejection fraction (EF) was calculated as follows: EF = (LVEDD2- LVESD2)/LVEDD2×100. Left atrial (LA) and aortic diameters were measured from M-mode recordings in a modified, parasternal, long-axis view.

Electrophysiological study

   On the open-chest study days, rats were anesthetized with ketamine (90 mg/kg) and xylazine (13 mg/ kg) and ventilated mechanically. A sternotomy was performed, and bipolar electrodes were hooked into the right-atrial appendage for recording and stimulation. AF was induced by burst pacing (10 Hz, 4 times threshold, 30 seconds). Mean AF duration was estimated by the average of 10 inductions if AF duration was ≤20 min, and from 5 inductions if AF duration lasted between 20 and 30 min. AF lasting longer than 30 minutes was considered persistent, and in this case, mean AF duration was recorded as 30 minutes.

Quantification of fibrosis

   After completion of the electrophysiological study, hearts were rapidly excised and weighed in order to calculate heart:body weight ratio. Hearts were immersed in a 10% buffered formalin solution and then embedded in paraffin. Sections were stained with Masson’s trichrome staining to characterize the collagen fibers. Fibrous tissue content of the LA was analyzed with Image-Pro Plus 5.1 (Media Cybernetics, GA, USA), and quantified as percent surface area, excluding blood vessel-containing regions.

RNA purification

   Total RNA was isolated from sham, MI, and MI + losartan LA tissues (10-20 mg) with Reboex reagent (Genall, Korea), followed by chloroform extraction and isopropanol precipitation. Genomic DNA was eliminated by incubation in rDNase I (2 U/μL, 37℃, Ambion, USA) for 30 minutes. RNA was quantified by spectrophotometric absorbency at a wavelength of 260 nm; purity was confirmed by calculating the A260/A280 ratio.

Real-time RT-PCR

   Gene-specific primers for real-time reverse transcription (RT)-polymerase chain reaction (PCR) were designed based on published cDNA sequences for rat ET-1 (Table 1). First-strand cDNA synthesized by RT of rat atrial mRNA samples was used as a template for subsequent RT-PCR experiments. Two-step RT PCR was conducted with LightCycler® Real-Time RT-PCR system (Roche Applied Science). Primers used for the detection of ET-1 and GAPDH are shown in Table 1. RT PCR was run in the presence of a double-stranded DNA binding dye (SYBR). GAPDH was used as an internal standard, and all ET-1 results were normalized to GAPDH data obtained from the same samples at the same time. Total RNA was run in duplicate for each experiment.



Western blot

   LA tissue (~25 mg) was homogenized in a modified tonic sucrose solution (0.3 mol/L sucrose, 10 mmol/L imidazole, 10 mmol/L sodium metabisulfite, 1 mmol/L DTT, 0.3 mmol/L PMSF), and centrifuged at 1300 xg for 15 min at 4°C. Extracted protein was quantified by Bradford assay. Aliquots containing 50 μg ETaR and ETbR were separated with 13%-polyacrylamide gel-SDS electrophoresis (90 min), then transferred to PVDF membrane (Pierce, USA). Immunoblotting was performed with ETa and ETb polyclonal antibodies (Alamone, USA), at a 1:500 dilution in 5% milk/ TBST. GAPDH (1:5000, Santa Cruz, USA) was used as control for protein loading. Immunoreactive bands were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce, USA) and exposed to X-ray film. Densitometric analysis of western blots was performed, and band intensities are expressed relative to GAPDH intensity from the same sample.

Immunohistochemistry for ET-1, ETa, and ETb

   Additional hearts were used for the immunohistochemical visualization of ET-1, ETaR, and ETbR. Hearts were dissected, fixed for 3 h in 4% phosphatebuffered paraformaldehyde solution (pH 7.4), and processed for embedding in paraffin. Sections (6-μm thickness) were cut, mounted on silane-coated slides, and heated to 60°C for 35 min. Slides were then deparaffinized in xylene, and rehydrated in graded immunoglobulin-free bovine serum albumin (BSA, Sigma, USA) in PBS for 20 min at room temperature. Slides were subsequently incubated for 2 h at room temperature with rabbit anti-ET-1 (1:50, Peninsula laboratories), rabbit anti-rat-ETa (1:50, Alamone, USA), and rabbit anti-rat-ETb (1:50, Alamone, USA) polyclonal antibodies diluted in 3% BSA. Sections were washed once in 0.1% Triton X-100, and twice in PBS. Antibody binding was detected using anti-rabbit and anti-mouse UltraVision LP Detection System (Lab Vision Corporation, Suffolk, UK), according to the manufacturer’s instructions.

Drugs

   Lorsartan (a gift from Merck Pharmaceuticals, Seoul, Korea) was dissolved at strength in vehicle.

Statistical Analysis

   Data were expressed as mean ± SEM unless otherwise indicated. Kruskal-Wallis tests were used for statistical comparisons. If Kruskal-Wallis tests were significant, group-to-group comparisons were performed using the Mann-Whitney test. Differences were considered statistically significant when p<0.05. Analyses were performed with SPSS 12.0 (SPSS Inc., Chicago, IL, USA).


RESULTS

Echocardiographic indices

   Echocardiographic parameter changes are presented in Figure 1 and Table 2. LV EF was significantly lower in the MI group compared with the sham group (32.3 ± 4.3 vs. 84.8 ± 2.7%, p<0.01). However, LV EF was significantly higher in the MI + losartan group compared with the MI group (47.8 ± 4.4 vs. 32.3 ± 4.3%, p<0.05).



AF induction study

   AF inducibility was significantly higher in the MI group compared with sham controls (22.6 ± 5.3 vs. 5.5 ± 2.2%, p=0.001). Treatment with losartan significantly lowered AF inducibility compared with the MI group (13.7 ± 5.3% vs MI, p=0.038). Mean AF duration significantly increased in MI rats compared with sham controls (293.2 ± 153.8 vs. 9.8 ± 7.4 seconds, p=0.001), and was moderately reduced after losartan treatment (182.1 ± 123.7 seconds, p=0.022 vs. MI) (Figure 2 and 3).




Preventive effects of losartan on LA fibrosis

   Interstitial fibrosis in the LA was significantly increased in the MI group compared with the sham group (n=5/group, 2.37 ± 0.30 vs. 0.24 ± 0.03%, p=0.008). After treatment with losartan, the level of LA fibrosis was significantly decreased compared with the MI group (0.99 ± 0.12% in MI + losartan group, p=0.008 vs. MI group) (Figure 4 and 5).



Change in transcription level of ET-1

   The expression of ET-1 mRNA in LA tissue of rats from the MI group was ~3.5 times upregulated compared with the sham group (p=0.002). This change recovered slightly in rats that received losartan treatment (p=0.023), as shown in Figure 6.



Expression of ETa receptor and ETb receptor proteins

   Protein level of ETaR in LA tissue was significantly increased in the MI group as compared to the sham group (p=0.002), and it was almost abolished in the losartan-treated group (p=0.026). In contrast, protein expression of ETbR in LA tissue significantly decreased in the MI group, but recovered to 80% of the sham group level following treatment with losartan (p=0.004) (Figure 7).



Immunohistochemistry results

   Immunoreactive ET-1 and ETaR were rarely detected in cardiomyocytes obtained from sham controls. However, expression of ET-1 and ETaR were increased in the MI group, which was prevented with losartan treatment. Immunohistochemical staining for ETbR showed some degree of ETbR activity in the sham group, but myocardial ETbR activity was lower in the MI group (Figure 8). In general, immunohistochemical results correlated with those of western blot analysis.





DISCUSSION

   This study demonstrated that expression of ET-1 and ETaR significantly increased and expression of ETbR significantly decreased in the LA tissue of MI-induced CHF rats. These observations were accompanied by increased AF inducibility and increased mean AF duration in the same model; thus implicating the ET system in the promotion of AF. After treatment with losartan, these changes observed in the ET system and AF development were reversed. Together, these findings suggest a possible mechanism to explain the anti-AF effects of ARBs in an animal model of CHF.

The endothelin system in CHF

   The ET system, a vascular regulatory system, has received considerable attention in recent years.9-17 Of the 4 active ETs, ET-1 is the predominant isoform in the cardiovascular system and exerts its effects through activation of 2 distinct G-protein-coupled receptors, ETaR and ETbR.19,20 ETaRs are found in the medial smooth muscle layers of blood vessels, and the myocardium of the atria and ventricles. When stimulated, ETaRs induce vasoconstriction and cellular proliferation by increasing intracellular calcium.21 ETbRs are localized in endothelial cells and, to some extent, smooth muscle cells and macrophages.22 Activation of ETbRs stimulates the release of nitric oxide and prostacyclin, and prevents apoptosis.23 ETbRs are down-regulated in CHF and contribute to neurohormonal activation, hemodynamic deterioration, and cardiovascular remodeling.24 Elevated plasma ET-1 and big ET-1 levels have been reported after spontaneous and triggered non-sustained supraventricular and ventricular tachycardias. Dezsi et al. reported significantly decreased plasma ET-1 levels after catheter ablation of tachyarrhythmias.25 In the present study, we demonstrated that ET-1 and ETaR density increased in LA tissues of rats with MI-induced CHF, which might be linked with the induction of AF and increased duration of AF.

Relationship between angiotensin II, AF, and ET-1

   Increased atrial interstitial fibrosis has been demonstrated in patients with AF.26,27 Furthermore, elevated quantities of interstitial fibrosis have been shown to increase AF vulnerability in animal models of CHF4,26-29 and in a transgenic model for selective atrial fibrosis.30 ARBs prevent the promotion of AF by reducing atrial fibrosis,5 and the LIFE study has shown that ARBs prevent subsequent AF development in patients with hypertension.31 Based on these studies, the preventive effects of ARBs on AF are thought to be due to an attenuation of structural remodeling that can lead to atrial interstitial fibrosis. CHF is known to facilitate the release of angiotensin II, which activates the secretion of ET-1 from various tissues. We demonstrated increased mRNA and immunoreactivity of ET-1 in MI-induced CHF rats (Figure 6 and 8), and conclude that this could be due to activation of angiotensin II and/or toxicity of CHF. Along with increased ETaR expression, ET-1 binding to ETaR could cause vasoconstriction of heart vessels, proliferation of cardiomyocytes and/ or non-cardiomyocytes in atrial tissue, and activate intracellular inositol 1,4,5-trisphosphate receptors. These receptors trigger spontaneous Ca2+ increase,12 resulting in increased AF inducibility and atrial fibrosis. Losartan treatment in MI-induced CHF rats partially reversed the expression of ET-1 and EtaR, and lessened subsequent AF development and LA fibrosis. Therefore, it is postulated that the effects of ET-1 on MI-induced CHF are most likely mediated through angiotensin II. The role of ETbR in the heart is less well known than that of EtaR. However, decreased ETbR was consistent with decreased tissue nitric oxide level in MI-induced CHF rats, which was subsequently reversed with losartan treatment.

Limitations of study

   In the present study, we did not directly measure angiotensin II levels in LA tissue; however, increased angiotensin II levels have already been documented in CHF. Furthermore, the direct relationship between elevation of ET-1 and arrhythmogenesis was not evaluated using an ET receptor blocker. Bosentan, an ET receptor blocker, failed to demonstrate efficacy in a CHF clinical trial, and is currently only used for the treatment of primary pulmonary hypertension. Since bosentan is not clinically relevant to CHF, we did not evaluate the antiarrhythmic effects of such direct ET-1 blockade in our CHF model. Moreover, ET 1-induced arrhythmogenesis has been reported several times in the literature,32-34 therefore we did not investigate ET-1-induced arrhythmogensis in our study.

Acknowledgements

   We thank Merck Pharmaceuticals for providing losartan.

Disclosure Statement

   The authors state no conflict of interest.


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