Telmisartan and captopril ameliorate pregabalin-induced heart failure in rats

Journal Pre-proof Telmisartan and Captopril Ameliorate Pregabalin-Induced Heart Failure in Rats Zeinab M. Awwad, Samar O. El-Ganainy, Ahmed I. ElMallah, Mahmoud M. Khattab, Aiman S. El-Khatib

PII:

S0300-483X(19)30267-7

DOI:

https://doi.org/10.1016/j.tox.2019.152310

Reference:

TOX 152310

To appear in:

Toxicology

Received Date:

14 July 2019

Revised Date:

19 September 2019

Accepted Date:

15 October 2019

Please cite this article as: Awwad ZM, El-Ganainy SO, ElMallah AI, Khattab MM, El-Khatib AS, Telmisartan and Captopril Ameliorate Pregabalin-Induced Heart Failure in Rats, Toxicology (2019), doi: https://doi.org/10.1016/j.tox.2019.152310

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Telmisartan and Captopril Ameliorate Pregabalin-Induced Heart Failure in Rats Zeinab M. Awwada,*, Samar O. El-Ganainya, Ahmed I. ElMallahb, Mahmoud M. Khattabc, Aiman S. El-Khatibc a

Department of Pharmacology and Therapeutics, Faculty of Pharmacy and Drug

Manufacturing, Pharos University in Alexandria, Alexandria, Egypt. b

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University,

c

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Alexandria, Egypt. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University,

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Cairo, Egypt.

*

Corresponding author, Department of Pharmacology and Therapeutics, Faculty of Pharmacy and Drug

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Manufacturing, Pharos University in Alexandria, Alexandria, Egypt.

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Abstract

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E-mail: [email protected]

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Phone: 002 01223322436

Pregabalin (PRG) is highly effective in the treatment of epilepsy, neuropathic pain

and anxiety disorders. Despite its potential benefits, PRG administration has been reported to induce or exacerbate heart failure (HF). It has been previously documented that overactivation of the renin angiotensin system (RAS) is involved in HF pathophysiological

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mechanism. The target of the current study was to examine the possible cardioprotective effect of telmisartan (Tel), an angiotensin II type 1 receptor (AT1R) blocker, compared with that of captopril (Cap), an angiotensin converting enzyme (ACE) inhibitor, in ameliorating PRG-induced

HF

in

rats

by

assessing

morphometric,

echocardiographic

and

histopathological parameters. Furthermore, to investigate the role of RAS blockade by the two drugs in guarding against PRG-induced changes in cardiac angiotensin 1-7 (Ang 1-7) and angiotensin II (Ang II) levels, in addition to myocardial expression of ACE2, ACE, Mas

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receptor (MasR) and AT1R. Results showed that PRG administration induced morphometric, echocardiographic and histopathological deleterious alterations and significantly elevated cardiac Ang II, ACE and AT1R levels, while reduced Ang 1-7, ACE2 and MasR cardiac

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levels. Concurrent treatment with either Tel or Cap reversed PRG-induced morphometric, echocardiographic and histopathological abnormalities and revealed prominent protection

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against PRG-induced HF via downregulation of ACE/Ang II/AT1R and upregulation of

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ACE2/Ang 1-7/MasR axes. These are the first findings to demonstrate that the potential benefits of Tel and Cap are mediated by counteracting the altered balance between the RAS axes induced by PRG. Hence; Tel and Cap may attenuate PRG-induced HF partially through

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stimulation of ACE2/Ang 1-7/MasR pathway.

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Keywords: Pregabalin; Heart failure; Telmisartan; Captopril

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1. Introduction

Pregabalin (PRG) is a gamma-aminobutyric acid analogue (Tassone et al. 2007) that

has been shown to be highly efficacious in the treatment of epilepsy (Ryvlin et al. 2008), neuropathic pain (Freynhagen et al. 2005), generalized anxiety disorder (Pohl et al. 2005) and fibromyalgia (Arnold et al. 2008). However, its use is hampered by the presence of deleterious cardiac side effects (Ho et al. 2013). Previous studies have reported the association 2

between PRG administration and incidence or exaggeration of cardiovascular diseases (CVDs) (Ho et al. 2017; Murphy et al. 2007; Robert Lee Page et al. 2008; Schiavo et al. 2017). In addition, a causal link between PRG treatment and worsening of heart failure (HF) was evident by the resolved cardiac dysfunction in PRG-administered patient within four days of PRG discontinuation (Aksakal et al. 2012).

L-type voltage-dependent calcium channel (L-VDCC) blockade has been reported to

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underly the deleterious cardiac effects exerted by PRG as a result of decreased calcium influx in cardiomyocytes (Murphy et al. 2007). However, none of the previous studies have investigated protective strategies to overcome such a major adverse effect that, despite its low

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prevalence, occasionally necessitates drug discontinuation (Aksakal et al. 2012).

Echocardiography is the gold standard for diagnosing HF (Sorrell and Stewart 2001).

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Additionally, testing of natriuretic peptides is a recommended tool in HF diagnosis (Maisel et

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al. 2008). Plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) is a diagnostic marker for HF and is associated with left ventricular (LV) dysfunction (Ndumele et al. 2016).

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Overactivation of renin angiotensin system (RAS) has been established as a major contributing factor in the pathophysiology of HF (Unger and Li 2004). Consequently, RAS

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modulation is a cornerstone in the treatment of HF (Werner and Böhm 2008). Both angiotensin converting enzyme (ACE) inhibitors (ACEIs) and angiotensin II type 1 receptor

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(AT1R) blockers (ARBs) have been shown to lower the risk of mortality and hospitalization in post-acute myocardial infarction (MI) patients with congestive HF and/or LV dysfunction (Demers et al. 2005; McMurray and Pfeffer 2002; Pfeffer et al. 2003). A growing body of evidence shows that telmisartan (Tel), an ARB, and captopril (Cap), an ACEI, possess a great therapeutic and protective value in CVDs. Tel has been reported to ameliorate cardiac remodeling in rats with HF after experimental autoimmune 3

myocarditis (Sukumaran et al. 2010), reduce mortality and LV hypertrophy in rats with hypertension and HF (Kishi et al. 2013), and attenuate vascular hypertrophy in spontaneously hypertensive rats (Zhong et al. 2011). Cap was shown to decrease mortality and MI in a rat model of ischemia-reperfusion (Zhu et al. 2000), and to reduce cardiovascular morbidity among patients with MI complicated by LV systolic dysfunction or HF (Pfeffer et al. 2003). Although many studies have investigated the cardioprotective effect of ACEIs and ARBs in rat hypertensive models (Nagai et al. 2004), diabetic models (Zhang et al. 2008) and

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chemotherapeutic-induced cardiac toxicity (Ibrahim et al. 2009), none of these studies have examined their effects in PRG-induced HF.

Considering the scarce experimental work, the present study aimed to investigate the

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potential cardioprotective effects of both Tel and Cap on cardiac abnormalities induced by

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PRG in rats. Alterations in echocardiographic, neurohumoral, and histopathological parameters were assessed. In addition, the protective effect of Tel was compared with that of

2.1. Drugs

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2. Materials and methods

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Cap on PRG-induced tissue RAS altered components.

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Pregabalin was purchased from Pfizer Pharmaceutical Company (Egypt). Tel and Cap were purchased from Sigma-Aldrich Company (USA). All drugs were freshly prepared in

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sterile isotonic saline before oral administration. All other chemicals or reagents were of the highest analytical grade and purity. 2.2. Animals The present study was carried on adult male Sprague-Dawley rats, weighing 200±20 g each. Rats were obtained from the animal house of Pharos University in Alexandria, Egypt. They were acclimatized for one week prior to the study with free access to food and water. 4

Throughout the experimental period, animals were housed under standardized conditions of well ventilation, room temperature (25±2°C), relative humidity (60±10%) and 12 h light/dark cycle. Unnecessary disturbance of animals was avoided. All procedures for the care and anesthesia of animals comply with the guide for care and use of laboratory animals published by the US National Institutes of Health (NIH Publications No. 8023, revised 1978) and were performed in accordance with the recommendations for the proper care and use of laboratory animals approved by the Ethics

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Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University, Egypt (Permit number: PT 1979).

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2.3. Experimental protocol

Rats were allocated into four groups. Group I (Control): rats served as controls and

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received saline. Group II (PRG): rats received PRG in a dose of 10 mg/kg (Singh et al. 2017). Group III (PRG+Tel): rats received PRG (10 mg/kg) and Tel in a dose of 10 mg/kg

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(Sukumaran et al. 2011). Group IV (PRG+Cap): rats received PRG (10 mg/kg) and Cap in a dose of 30 mg/kg (Castro-Moreno et al. 2012). All drugs were administered by oral gavage

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for 21 days. The chosen dose of PRG was selected based on pilot studies that were conducted with higher doses of PRG (20 and 30 mg/kg). However, these high doses were excluded due

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to exaggerated cardiotoxic effects as compared to the 10 mg/kg dose. Furthermore, the

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reported therapeutic dose of PRG in rats is 10 mg/kg (Seto et al. 2017; Singh et al. 2017). After 24 h of the last dose, animals were lightly anesthetized with an intraperitoneal

mixture of ketamine/xylazine (50/5 mg/kg) (Alfasan International (Netherlands) and Adwia Pharmaceutical Company (Egypt), respectively) for echocardiographic examination. Following echocardiography, blood samples were collected from the abdominal aorta and centrifuged at 4000 rpm for 15 min for plasma separation. Hearts of all animal groups were 5

quickly isolated, washed with ice-cold saline, dried and weighed for determining heart weight (HW) and heart weight index (HWI). Heart apices were isolated and fixed for one week in 10% formalin-saline solution for histopathological examination. The remaining parts of the ventricles were divided into two parts and immediately kept at -80 °C for further biochemical determinations. 2.4. Echocardiographic measurements On day 22, rats were anesthetized and echocardiographic measurements started after

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the stabilization of heart rate. Cardiac abnormalities were assessed using echocardiography (MyLab Touch; Esaote, Netherlands) equipped with an 18 MHz linear transducer.

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To evaluate cardiac function, LV end diastolic diameter (LVEDd), LV end systolic diameter (LVESd), ejection fraction (EF) and fractional shortening (FS) were recorded in all

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groups with M-mode tracings from the short axis parasternal view. All measurements were performed by the same expert who was blinded to the treatments, and each measurement was

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2.5. Cardiac morphometry

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averaged over three cardiac cycles.

All isolated rats' hearts were weighed and HWI (mg/g) was calculated by dividing HW

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(mg) by body weight (BW) (g), (HWI= HW/BW), as an index of cardiac hypertrophy. 2.6. Assay of plasma NT-proBNP level

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Plasma samples were assayed for NT-proBNP using rat-specific ELISA kit (Cusabio

Life science, Wuhan, Hubei, China) and expressed as picograms per milliliter. 2.7. Enzyme-linked immunosorbent assays Cardiac tissue homogenates were used for determination of the following parameters, using rat-specific ELISA kits according to the manufacturer’s instructions; angiotensin 1-7

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(Ang 1-7) (MyBioSource, SanDiego, CA, USA), angiotensin II (Ang II) (Cusabio Life science, Wuhan, Hubei, China), protein kinase A (PKA) (MyBioSource, SanDiego, CA, USA) and phosphoinositide 3-kinase (PI3K) (Cusabio Life science, Wuhan, Hubei, China). The results were expressed as picograms per gram tissue for PI3K and as nanograms per gram tissue for Ang 1-7, Ang II and PKA. 2.8. Western blot analysis Myocardial protein expression of ACE, angiotensin converting enzyme 2 (ACE2),

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AT1R and Mas receptor (MasR) was estimated using western blot methodology. Radioimmunoprecipitation assay lysis buffer (Bio-Basic Inc., Ontario, Canada) was used for cardiac tissue homogenization and protein extraction. Protein quantification was carried out

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with Bradford protein assay kit (Bio-Basic Inc., Ontario, Canada). Proteins were separated by

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and identified with the following antibodies according to the manufacturer’s

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instructions to quantify the cardiac levels of proteins: ACE (antibodies-online GmbH, Germany), ACE2 (Novus Biologicals, USA), AT1R (Thermo Fisher Scientific Inc., USA) and

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MasR (Abgent, CA, USA) diluted in tris-buffered saline with Tween 20 (TBST). Precision Plus Protein All Blue standards (Bio-Rad, Hercules, CA, USA) were used as molecular

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weight markers. The membranes were stained transiently with Ponceau S solution (SigmaAldrich, St. Louis, MO, USA) to verify protein transfer. Membranes were incubated with a

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blocking solution containing TBST buffer and 3% bovine serum albumin at 4 °C overnight. The membranes were then washed with TBST and incubated with each primary antibody solution, against the blotted target protein, followed by horseradish peroxidase-conjugated secondary antibody solution (Novus Biologicals, USA) for 1 h at room temperature. Finally, chemiluminescence detection was performed using the chemiluminescent substrate (Bio-Rad, CA, USA) according to the manufacturer’s procedures. Image analysis software was used to 7

read the band intensity of the target proteins against control sample after normalization by beta (β)-actin on the ChemiDoc MP imager. Results were expressed as arbitrary units after normalization for β-actin protein expression. 2.9. Histopathological examination Formalin-saline-fixed heart apices were processed to wax blocks for histopathological examination. Paraffin embedded tissue sections of 5μm thickness were stained with hematoxylin and eosin (H&E). Stained samples were blindly interpreted by a pathologist

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under light microscopy. 2.10. Statistical analysis

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Statistical analysis was performed using GraphPad Prism software (version 7.0). All results were expressed as mean ± SE (n= 8). One-way analysis of variance test (one-way

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ANOVA) was used to compare between groups, followed by Student-Newman-Keuls

3. Results

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statistically significant.

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multiple comparison test. A probability level of less than 0.05 (p < 0.05) was considered

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3.1. Effect of Tel and Cap on echocardiographic parameters in PRG-treated rats Echocardiographic examination of PRG-treated group revealed a significant increase

in LVEDd and LVESd reaching 145% and 207%, respectively of the control value. Concurrent administration of Tel significantly decreased PRG-induced elevation in LVEDd and LVESd by 27% and 45%, respectively. Co-treatment with Cap also prominently reduced PRG-induced elevation in LVESd and LVESd by 28% and 47%, respectively. 8

Additionally, PRG-treated rats exhibited a 41% and 53% reduction in EF and FS, respectively as compared to the control group. Co-treatment with Tel induced a significant elevation of reduced EF and FS observed in PRG group. Cap co-administration similarly elevated EF and FS with respect to PRG value. Noteworthy, the percentage recovery to control values of the EF amounted 95.2% and 96.6% for Tel and Cap, respectively, as shown in Figure 1 and Table 1. 3.2. Effect of Tel and Cap on cardiac morphometry in PRG-treated rats

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Rats treated with PRG showed a significant increase in HW (143%) and HWI (136%) compared to control value. Tel or Cap co-administration significantly ameliorated PRGinduced rise in HW by 16% and 20%, respectively. PRG-induced rise in HWI was also

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attenuated by Tel and Cap by 13% and 17%, respectively (Table 1).

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3.3. Effect of Tel and Cap on plasma NT-proBNP level in PRG-treated rats A prominent rise in plasma level of NT-proBNP (6-fold) was observed in PRG-treated

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group with respect to control value. Co-treatment with either Tel or Cap significantly reduced

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NT-proBNP plasma level by 69% and 73%, respectively compared to PRG value (Figure 2). 3.4. Effect of Tel and Cap on cardiac tissue level of Ang 1-7 and Ang II in PRG-treated

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rats

In PRG-treated group, a significant decline in cardiac Ang 1-7 level was observed

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compared to control value. Concurrent administration of either Tel or Cap significantly raised cardiac Ang 1-7 content by about 3 times compared to that in PRG group (Figure 3A). Furthermore, PRG treatment was accompanied by a prominent elevation of Ang II cardiac content (7-fold) with respect to control group. Co-treatment with either Tel or Cap induced a significant comparable reduction (67% and 70%, respectively) in the rise of Ang II noticed in PRG group (Figure 3B). 9

3.5. Effect of Tel and Cap on cardiac protein expression of ACE2, MasR, ACE and AT1R in PRG-treated rats Cardiac protein expression of ACE2 and MasR was dramatically downregulated in PRG-treated rats as compared to the control group. Co-treatment with either Tel or Cap resulted in a nearly equivalent upregulation of ACE2 (4-fold) as compared to PRG value (Figure 4A). In addition, MasR was comparably upregulated (5-fold) in both Tel co-treated group and Cap co-treated group with respect to PRG-treated rats (Figure 4B).

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On contrary, cardiac ACE and AT1R levels were significantly upregulated in PRGtreated group compared to control group. Co-administration of Tel alleviated the upregulated ACE (53%) and AT1R (63%) levels observed in PRG-treated group. Similarly, Cap co-

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treated group showed significantly decreased expression levels of both ACE (59%) and AT1R

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(67%) levels compared to PRG values (Figure 4C-D).

3.6. Effect of Tel and Cap on cardiac tissue level of PI3K in PRG-treated rats

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The PI3K cardiac level was sharply reduced in PRG-treated group with respect to control value. Concurrent administration of Tel resulted in a significant elevation of PI3K

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level about 3 times the PRG value. However, more significant elevation of PI3K level was observed with Cap co-treatment compared to Tel co-treated group (Figure 5). Cap co-treated

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group showed 5 times elevation of PI3K level compared to PRG-treated group.

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3.7. Effect of Tel and Cap on cardiac tissue level of PKA in PRG-treated rats The PKA level in PRG-treated rats exhibited a significant decrease as compared to the

control group. Tel co-administration significantly replenished cardiac PKA content compared to PRG value. Noteworthy, Cap co-treatment showed more significant replenishment of PRGinduced reduced PKA content (4 times) than Tel co-treated group (Figure 6). 3.8. Effect of Tel and Cap on histopathological cardiac alterations in PRG-treated rats 10

Cardiac sections of control rats showed normal histological structure of longitudinally arranged fibers with central oval nuclei and closely arrangement fibers with minimal interstitial connective tissue in between (Figure 7A). On contrary, PRG-treated rats revealed histopathological alterations illustrated by widely spaced edematous cardiomyocytes, thinning of fibers and areas of hyper-eosinophilia (Figure 7B). In addition, evident waviness of cardiomyocytes fibers, congested blood vessels (BV) and loss of cell structure delineation were noticed (Figure 7C). Concurrent administration of Tel resulted in little spacing,

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indicating less edema, (e) between cardiomyocyte fibers and waviness at focal areas (Figure 7D) as compared to PRG-treated group. Likewise, co-treatment with Cap showed fewer areas of edema and thinned fibers (Figure 7E) with respect to PRG-treated group.

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4. Discussion

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Current results showed that PRG induces HF-like manifestations in rats. Significant

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increase in HWI was observed in PRG-treated rats, indicating myocardial hypertrophy (Mao et al. 2018). In addition, dramatic decrease in EF and significant rise in plasma NT-proBNP was found following PRG treatment. In accordance with these results, several studies have

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reported decreased EF and increased plasma NT-proBNP upon diagnosing HF (Ciampi and Villari 2007; Don-Wauchope and McKelvie 2015; Langenickel et al. 2000; Omar et al. 2016).

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Histopathological examination revealed myocardial edema, thinning of fibers and hyper-

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eosinophilia. Similar histological alterations were reported accompanying myocardial injury (Albayrak et al. 2009; Romano et al. 2012). Excessive activation of RAS is one of the well-established pathophysiological

mechanisms of HF (Tai et al. 2017). The findings of the present study have shown an imbalance in the RAS in PRG-treated rats. Results showed an upregulation of ACE and ATIR expression as well as an upsurge of Ang II tissue level. These results were accompanied by a 11

downregulation of ACE2 and MasR as well as a prominent reduction of Ang 1-7 level in PRG-treated rats. These findings indicated that PRG induced an imbalance between the two major RAS axes which could play an essential role in the underlying cardiac manifestations. The mechanism of action of PRG was previously reported to be attributed to blocking L-VDCC via binding to α2-δ subunit (Verma et al. 2014). Consequently, the deleterious cardiac effects of PRG could be due to L-VDCC blockade in the myocardium and the vasculature leading to peripheral vasodilatation and negative inotropic effects (Fong and Lee

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2014). However, according to the results of the present work, it seems that PRG not only exerts its actions by decreasing calcium influx but also by acting on RAS. As a matter of fact, it is reported that calcium channel blockers (CCBs), such as amlodipine, can activate the RAS

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and alter the ratio of Ang II/Ang 1-7 (Aritomi et al. 2015; Aritomi et al. 2012; Konoshita et al.

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2010). This inevitable characteristic of CCBs is mediated via many mechanisms; lowering renal blood flow, activation of sympathetic drive and reduction of intracellular calcium

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(Konoshita et al. 2010).

It should be noted that the therapeutic dose of PRG in animal models that is equivalent

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to clinical dose results in higher plasma drug concentration in animals than in humans which is associated with undesired effects in experimental studies (Esteban et al. 2018; Vartanian et

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al. 2006). This may explain, at least in part, the reason why the chosen dose (10 mg/kg) in the

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current study produced such prominent cardiotoxic effects. In the present experimental work, co-administration of either Tel or Cap protected

against PRG-induced cardiac alterations. Both drugs significantly reduced PRG-induced elevation in HWI. Concurrent treatment with Tel or Cap also significantly recovered the reduced EF to almost near the control value and decreased the rise in plasma NT-proBNP induced by PRG. The ability of Tel and Cap to improve humoral and echocardiographic 12

alterations was previously reported in clinical (Jagodzinski et al. 2017; Zhang et al. 2019) and experimental models of cardiac damage (Lihua et al. 2016; Zong et al. 2011). However, none of them have investigated their effects in PRG-induced HF. Histologically, a notable reduction in edema and hyper-eosinophilia was also observed in Tel and Cap co-treated groups, suggesting their protective role against PRG-induced cardiomyocyte microscopical abnormalities. Co-treatment with Tel significantly ameliorated the progression of cardiac

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hypertrophy through modulation of the two arms of RAS. Cardiac ACE2 and MasR expressions, as well as, Ang 1-7 level were elevated, while cardiac ACE, AT1R and Ang II

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content were reduced following Tel concurrent use.

Following AT1R blockade, ARBs shifts Ang II activity to AT2R, leading to

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vasodilation and natriuresis through bradykinin, nitric oxide (NO) and cyclic guanosine monophosphate pathways, opposing AT1R actions (Kurisu et al. 2003; Padia and Carey 2013;

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Xu et al. 2013).

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Co-administration of Cap showed similar results to Tel co-treatment in modulating PRG-induced RAS imbalance, presented as higher, however insignificant, increase in cardiac

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level of Ang 1-7, ACE2 and MasR. Concurrent reduction in Ang II, ACE and AT1R cardiac tissue levels were also observed following Cap co-treatment. These results are consistent with

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previous experimental studies that documented the ability of Cap to amplify Ang 1-7 level and to suppress the level of Ang II, ACE and AT1R (Castro-Moreno et al. 2012; Hirsch et al. 1992; Nyby et al. 2007). The beneficial effects of ACEIs in guarding against HF have been previously reported to be attributed to the reduction of both Ang II formation and Ang1-7 metabolism (Tai et al. 2017), as ACE is the primary pathway for the metabolism of Ang 1-7 (Chappell et al. 1998; 13

Ferrario et al. 2005). Increased Ang 1-7 expression after ACE inhibition resulted in MasR activation, inducing cardioprotective effects and tissue repair, as well as, enhancing the vasodilator effects of increased bradykinin concentrations (Flores-Monroy et al. 2016; Loot et al. 2002; Sharp et al. 2015). Accumulation of bradykinin could contribute to the benefits of ACEIs through potentiation of fibrinolytic effects and inhibition of cellular growth (FloresMonroy et al. 2016; Su 2014). Noteworthy, the sulfhydryl group on Cap has an additional function of being a free radical scavenger, reducing the amount of oxidative injury to the

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myocardium (Bahk et al. 2008). The present results revealed a significant increase in cardiac PI3K level with Tel coadministration that could be attributed to the significant rise in Ang 1-7 and MasR levels. Ang

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1-7/MasR can activate PI3K/Akt pathway with subsequent NO synthase activation. This

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pathway plays a protective role against myocardial remodeling via generating NO which inhibit hypertrophic gene transcription activated by Ang II (Gomes et al. 2012).

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In addition, results showed that rats co-treated with Tel exhibited a profound rise in PKA tissue level, an effect that could be also attributed to Ang1-7 amplification (De Mello

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2015). PKA plays an essential role in regulating cardiac contractility and performance. It prevents cardiac hypertrophy and death through activation of contractile proteins and calcium

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regulating proteins (Dhalla and Müller 2010).

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Noteworthy, the present work showed that concurrent use of Cap resulted in more significant elevation of PI3K and PKA cardiac tissue levels than that of Tel co-administration. The superior effect of Cap on PI3K could be attributed to the ability of ACEIs to upregulate the level of bradykinin, independent of their action on Ang II (Abdi et al. 2002). Bradykinin via activating both PKI3 and PKA induce NO production which in turn exerts

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anti-apoptotic effects on cardiac tissues (Bell and Yellon 2003; Su 2017) and prevents cardiac hypertrophy in response to pathological stimulus (McMullen et al. 2007). It is worth mentioning that whether ARBs are comparable or not to ACEIs in attenuating HF remains controversial (Sukumaran et al. 2011). While ACEIs exert both Ang II dependent and non-dependent mechanisms, ARBs have recently shown additional properties. It has been reported that ARBs have different tissue penetration properties, different binding and dissociation times, as well as, ligand-directed signaling. In particular,

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Tel has a partial agonistic effect on peroxisome proliferator-activated receptor gamma (PPAR-γ) (Michel et al. 2016). Previous studies have shown that PPAR-γ activation inhibits Ang II-induced cardiac hypertrophy and downregulates AT1R suppressing Ang II

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intracellular pathways (Asakawa et al. 2002; Takano and Komuro 2009). The retrieved data

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offer an explanation to the comparable protective effect of both Cap and Tel on HF

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manifestations induced by PRG administration observed in this work.

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5. Conclusion

In conclusion, this study elucidated an association between PRG use in rats and HF,

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manifested by the dramatic decrease in EF and the prominent rise in plasma NT-proBNP following PRG administration. PRG-induced RAS imbalance was demonstrated by the

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upregulation of ACE/Ang II/AT1R and the downregulation of ACE2/Ang1-7/MasR axes. A novel finding of the present study is the protective effect of Tel and Cap against PRG-induced HF via modulating cardiac RAS components. To our knowledge, this is the first experimental work to investigate the effect of ACEIs and ARBs against PRG-induced HF. The current experimental work reported a nearly similar cardioprotective effect of Tel and Cap in

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attenuating PRG-induced HF, except for the superior effect of Cap over Tel on elevating cardiac PI3K and PKA levels.

Declaration-of-interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Conflict of interest

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None declared.

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Funding

This research did not receive any specific grant from funding agencies in the public,

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commercial, or not-for-profit sectors.

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Acknowledgment

The authors would like to express their gratitude to Prof. Dr. Maha A. El-Demellawy

(City of Scientific Research and Technological Applications, SRTA-City, Alexandria, Egypt) and Prof. Dr. Ghada M. Mourad (Center of Excellence for Research in Regenerative Medicine Applications, CERRMA, Faculty of Medicine in Alexandria, Alexandria, Egypt) for their help in echocardiographic and histopathological examination, respectively. 16

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Figure Legends Figure 1. Original M-mode echocardiographic tracings showing the effect of Tel and Cap on PRGinduced changes in echocardiography to evaluate the cardiac function of rats revealing left ventricular end diastolic diameter; LVEDd ( ) and left ventricular end systolic diameter; LVESd ( ). (A) Control group; (B) PRG group; (C) PRG+Tel group; (D) PRG+Cap group. PRG, pregabalin; Tel, telmisartan; Cap, captopril.

Figure 2. Effect of Tel and Cap on PRG-induced changes in plasma level of N-terminal pro-brain natriuretic peptide (NT-proBNP). PRG, pregabalin; Tel, telmisartan; Cap, captopril. Each column represents

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the mean ± SE of 8 rats per group. Statistical analysis was done using one-way ANOVA followed by StudentNewman-Keuls multiple comparison test; *p < 0.05 vs. control; #p < 0.05 vs. PRG.

Figure 3. Effect of Tel and Cap on PRG-induced changes in cardiac tissue level of (A) angiotensin 1-7 (Ang 1-7) and (B) angiotensin II (Ang II). PRG, pregabalin; Tel, telmisartan; Cap, captopril. Each column

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represents the mean ± SE of 8 rats per group. Statistical analysis was done using one-way ANOVA followed by

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Student-Newman-Keuls multiple comparison test; *p < 0.05 vs. control; #p < 0.05 vs. PRG.

Figure 4. Western Blot analysis bands and depicted bar graph of Tel and Cap effect on PRG-induced

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changes in protein expression of cardiac (A) angiotensin converting enzyme 2 (ACE2), (B) Mas receptor (MasR), (C) angiotensin converting enzyme (ACE) and (D) angiotensin II type 1 receptor (AT1R). PRG, pregabalin; Tel, telmisartan; Cap, captopril. Each column represents the mean ± SE of 8 rats per group.

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Statistical analysis was done using one-way ANOVA followed by Student-Newman-Keuls multiple comparison

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test; *p < 0.05 vs. control; #p < 0.05 vs. PRG.

Figure 5. Effect of Tel and Cap on PRG-induced changes in cardiac tissue level of phosphoinositide 3-

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kinase (PI3K). PRG, pregabalin; Tel, telmisartan; Cap, captopril. Each column represents the mean ± SE of 8 rats per group. Statistical analysis was done using one-way ANOVA followed by Student-Newman-Keuls multiple comparison test; *p < 0.05 vs. control; #p < 0.05 vs. PRG; $p < 0.05 vs. Tel.

Figure 6. Effect of Tel and Cap on PRG-induced changes in cardiac tissue level of protein kinase A (PKA). PRG, pregabalin; Tel, telmisartan; Cap, captopril. Each column represents the mean ± SE of 8 rats per group. Statistical analysis was done using one-way ANOVA followed by Student-Newman-Keuls multiple comparison test; *p < 0.05 vs. control; #p < 0.05 vs. PRG; $p < 0.05 vs. Tel.

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Figure 7. Representative light photomicrographs of rat myocardial sections showing the effect of Tel and Cap on PRG-induced changes in cardiac histological structure (H&E, x200). (A) Control rat demonstrating longitudinally arranged fibers with central oval nuclei ( ). The fibers show close arrangement with minimal interstitial connective tissue in between (curved arrow). (B, C) PRG-treated rats revealing (B) widely spaced cardiomyocytes by edema (e), thinning of fibers ( ) and areas of hyper-eosinophilia (arrow heads). In addition to, (C) evident waviness of cardiomyocytes fibers (*), noticed congested blood vessels (BV) and loss of cell structure delineation ( ). (D) PRG+Tel-treated rat illustrating little spacing by edema (e) between cardiomyocyte fibers and waviness at focal areas ( *). (E) PRG+Cap-treated rat illustrating longitudinally branching

( ).

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show few thinned fibers

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cardiomyocytes separated by minimal spaces of edema (e). Some cardiomyocytes appear normal, while others

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Table 1: Cardiac echocardiographic and morphometric parameters Parameter

HW (g) HWI (mg/g)

PRG

PRG+Tel

PRG+Cap

0.49±0.01

0.70±0.02*

0.59±0.01#

0.56±0.01#

2.20±0.07

3.00±0.09*

2.60±0.05#

2.50±0.05#

4.78±0.37

6.91±0.24*

5.04±0.34#

4.95±0.25#

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LVEDd (mm)

Control

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Group

2.65±0.21

5.48±0.20*

2.99±0.22#

2.88±0.25#

EF (%)

81.51±0.83

47.82±0.69*

77.63±0.49#

78.74±0.54#

44.54±0.81

20.77±0.36*

40.77±0.24#

41.78±0.25#

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LVESd (mm)

FS (%)

HW, heart weight; HWI, heart weight index; LVEDd, left ventricular end diastolic diameter; LVESd, left ventricular end systolic diameter; EF, ejection fraction; FS, fractional shortening; PRG, pregabalin; Tel, telmisartan; Cap, captopril. Each value represents the mean ± SE of 8 rats per group. Statistical analysis was done using one-way ANOVA followed by Student-Newman-Keuls multiple comparison test; *p < 0.05 vs. control; #p < 0.05 vs. PRG.

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