Secondary Logo

Endothelin receptor blocker bosentan inhibits hypertensive cardiac fibrosis in pressure overload-induced cardiac hypertrophy in rats

Visnagri, Asjad; Kandhare, Amit D.; Ghosh, Pinaki; Bodhankar, Subhash L.

Cardiovascular Endocrinology & Metabolism: December 2013 - Volume 2 - Issue 4 - p 85–97
doi: 10.1097/XCE.0000000000000010
Original articles
Free

Aim The aim of this study was to investigate the effect of bosentan (25, 50, 100 mg/kg) on left ventricular contractile function, cardiac fibrosis, and oxidative stress in pressure overload-induced cardiac hypertrophy in rats.

Methods Male rats (200–250 g) were assigned into various groups, namely, sham, aortic constriction, aortic constriction+bosentan (25, 50, and 100 mg/kg), and sham+bosentan (100 mg/kg). Myocardial hypertrophy was produced by constriction of the abdominal aorta. Four weeks after treatment with bosentan (25, 50, and 100 mg/kg perorally), the left ventricular contractile function was measured using a Millar catheter and ECG was carried out.

Results Treatment with bosentan (50 and 100 mg/kg perorally) significantly and dose-dependently (P<0.01 and <0.001) restored hemodynamic parameters including ECG, blood pressure, and left ventricular function. It also significantly elevated the levels of biochemical markers (P<0.001), that is, superoxide dismutase, reduced glutathione, and membrane-bound inorganic phosphate enzymes like Na+–K+-ATPase and Ca2+-ATPase. The elevated levels of creatine kinase-MB and lactate dehydrogenase enzymes, as well as the activities of malondialdehyde and tumor necrosis factor-α, were significantly attenuated (P<0.05 and <0.001) by bosentan (50 and 100 mg/kg perorally) treatment. An upregulation in the mRNA expression of endothelin-1 was significantly (P<0.05 and <0.001) attenuated by bosentan (50 and 100 mg/kg perorally) treatment. Histological aberration induced after pressure overload was restored by bosentan treatment.

Conclusion Bosentan attenuates the development of cardiac hypertrophy by blocking the endothelin-1 receptor and improving left ventricular function; it also ameliorates endogenous biomarkers in pressure overload-induced cardiac hypertrophy in rats.

Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India

Correspondence to Subhash L. Bodhankar, PhD, Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune 411 038, India Tel: +91 20 24537237x29; fax: +91 20 25439386; e-mail: sbodh@yahoo.com

Received March 8, 2013

Accepted July 18, 2013

Back to Top | Article Outline

Introduction

Hypertension and aortic stenosis (AS) are the cardiac diseases that result in left ventricular hypertrophy (LVH) in more than 3% of geriatric patients. Sustained LVH may result in arrhythmias, diastolic dysfunction progressing into congestive heart failure, and sudden death 1. It has been reported that the survival rate for LVH symptomatic patients is about 2–3 years, which is much less than that reported for age-matched and sex-matched asymptomatic patients. Various studies have been carried on cardiac hypertrophy as well as congestive heart failure (CHF). The mechanism for LVH has not been completely deciphered 2. Work carried out by a group of researchers has reported that a wide range of signaling pathways are involved in the induction of hypertension due to left ventricular pressure overload or AS 3. CHF is a complex syndrome that could result in metabolic and neurohumoral alterations 4,5. It has been reported that the release of proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β is responsible for the induction of hypertrophy in myocytes through nitric oxide release in vitro 6.

A wide range of animal studies have reported that an elevated level of oxidative stress leads to myocardial remodeling and dysfunction 7–9. Remodeling and hypertrophy of cardiomyocytes, apoptosis, and fetal gene expression-like changes may occur because of the increase in oxidative stress 10,11. Treatment with antioxidants like coenzyme Q10, eugenol, and N-2-mercaptopropionyl glycine has been shown to inhibit progression of CHF and to preserve LV function 11–13.

Besides the elevated levels of proinflammatory cytokines and oxidative stress, the release of endothelin-1 (ET-1), a 21 amino acid residue vasoconstrictor peptide, has been implicated in causing hypertrophy of cardiomyocytes 14–16. ET-1 is an isoform of the endothelin receptor and plays a vital role in cardiovascular regulation. Besides its cardiovascular effect it also possesses pulmonary vasoconstriction and vascular remodeling potential 17.

Abdominal aortic banding is a well-established, widely used, reproducible animal model to screen the potential of various therapeutic moieties for LVH 3,18. It has been reported that abdominal aortic banding-induced pressure overload on the ventricle results in concentric hypertrophy through parallel addition of myofibrils 19. The main treatment modalities for LVH include the use of diuretics, β-blocking agents, calcium channel antagonists, angiotensin converting enzyme inhibitors, as well as combination therapy 20,21. However, these therapies are associated with reversal of the condition and their side-effect profiles limit their use.

Complete replacement of the aortic valve is the only available effective alternative to drug treatment in the case of severe LVH 20,21. Aortic valve replacement results in better symptomatic relief with increased survival, which is nearly normal. However, aortic valve replacement patients still show high operative mortality with a poor long-term outcome.

Bosentan [4-tert-butyl-N-{6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl} benzene-1-sulfonamide] is an orally active nonselective endothelin receptor antagonist used in the treatment of ventricular remodeling and hemodynamic function (Fig. 1) 22–25. It inhibits the development of endothelial dysfunction as well as ischemia, thus resulting in improvement of myocardial function. Treatment with bosentan has resulted in a significant decrease in portal pressure without affecting renal circulation in rats with portal hypertension. Clinically, bosentan has been shown to be effective in the treatment of pulmonary hypertension through the improvement of hemodynamic workload 26,27. Concomitant administration of bosentan with clizapril effectively reduces the mean arterial pressure 28,29. It has also been shown to have a protective effect against streptozotocin-induced cardiomyopathy in rats 22 through the activation of protein kinase C isoforms. Cosenzi and colleagues 30,31 have reported the nephroprotective as well as retinoprotective potential of bosentan in diabetic rats through the modulation of transforming growth factor-β.

Fig. 1

Fig. 1

The aim of the present study was to evaluate the cardioprotective effect of bosentan on pressure overload-induced cardiac hypertrophy in laboratory rats by assessing various hemodynamic, biochemical, and molecular changes.

Back to Top | Article Outline

Materials and methods

Experimental animals and research protocol approval

Adult male Sprague–Dawley rats (150–200 g) were obtained from the National Institute of Biosciences, Pune, India. They were maintained at 24±1°C, with a relative humidity of 45–55% and a 12 : 12 h dark/light cycle. The animals had free access to standard pellet chow (Pranav Agro Industries Ltd., Sangli, India) and water throughout the experimental protocol. All experiments were carried out between 09:00 and 17:00 h. The experiment was approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Pune, and performed in accordance with the guidelines of the Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India.

Back to Top | Article Outline

Chemicals and sample

Bosentan was procured from Medicines Private Limited (Mumbai, India). Epinephrine hydrochloride, superoxide dismutase (SOD), and malondialdehyde were purchased from Sigma Chemical Co. (St Louis, Missouri, USA). Reduced glutathione (GSH), 5,5′-dithiobis (2-nitro-benzoic acid), and thiobarbituric acid were obtained from Hi media (Mumbai, India). The creatine kinase (CK)-MB kit was obtained from Randox Laboratories Ltd. (Antrim, UK) and the lactate dehydrogenase (LDH) and aspartate transaminase kits were purchased from Ecoline (Merck Ltd, Mumbai, India). Rat TNF-α ELISA kits were purchased from Thermo Scientific (Rockford, Illinois, USA). All chemicals used were of analytical grade.

Back to Top | Article Outline

Experimental design and surgical procedure

The animals were anesthetized using sodium thiopentone (35 mg/kg intraperitoneally). A midabdominal incision was made to expose the abdominal aorta. The aorta above the left renal artery was dissected and constricted at the suprarenal level using a cannula of size 0.9×40 mm, which was ligated with the aorta and withdrawn afterwards. In age-matched and body weight-matched sham-operated rats, the abdominal aorta was isolated and placed without ligation 32. One week after surgery, the rats with aortic constrictions (ACs) were randomly divided into the following groups:

Group 1: sham: the abdominal aorta of the animals was exposed but not constricted at the suprarenal level and 5% aqueous solution of gum acacia was administered perorally.

Group 2: AC: the abdominal aorta of the animals was exposed and constricted at the suprarenal level and 10 g/kg of a 5% aqueous solution of gum acacia (vehicle) was administered perorally.

Group 3: AC+bosentan (25): the abdominal aorta of the animals was exposed and constricted at the suprarenal level and 25 mg/kg of bosentan in a 5% aqueous solution of gum acacia was administered perorally.

Group 4: AC+bosentan (50): the abdominal aorta of the animals was exposed and constricted at the suprarenal level and 50 mg/kg of bosentan in a 5% aqueous solution of gum acacia was administered perorally.

Group 5: AC+bosentan (100): the abdominal aorta of the animals was exposed and constricted at the suprarenal level and 100 mg/kg of bosentan in a 5% aqueous solution of gum acacia was administered perorally.

Group 6: sham+bosentan (100): the abdominal aorta of the animals was exposed but not constricted at the suprarenal level and 100 mg/kg of bosentan in a 5% aqueous solution of gum acacia was administered perorally.

Bosentan was freshly prepared in a 5% aqueous solution of gum acacia in three different dosages (25, 50, and 100 mg/kg) and administered for 4 weeks 30,33. After 4 weeks, the rats were killed under deep anesthesia and their hearts were immediately isolated; tissue homogenates were prepared in 0.1 mol/l Tris-HCl buffer (pH 7.4) for the biochemical and molecular estimations.

Back to Top | Article Outline

Assessment of electrocardiographic abnormalities

To obtain an ECG, the leads were placed on the right foreleg (negative electrode), left foreleg (positive electrode), and right hind leg (neutral electrode) of the rats. Electrocardiographic changes were recorded using an eight-channel Power Lab System (LabChart 7.3; AD Instrument Pvt. Ltd) 13.

Back to Top | Article Outline

Invasive measurement of hemodynamic changes and LV contractile function

Blood pressure was measured using a polyethylene cannula (PE 50) filled with heparinized saline (100 IU/ml), inserted into the left femoral artery. The cannula was connected to a transducer and the signal was amplified by a bioamplifier. Left ventricular systolic pressure was measured using a Millar mikro-tip transducer catheter (Model SRP-320; Millar Instrument Inc., Houston, Texas, USA) inserted into the left ventricle through the right carotid artery and connected to a bioamplifier. Heart rate, dP/dt max, dP/dt min, and left ventricular end-diastolic pressure signals were obtained from primary signals (left ventricular systolic pressure and blood pressure) using a data acquisition system (LabChart 7.3; AD Instrument Pvt. Ltd).

Back to Top | Article Outline

Cardiac serum markers

Serum levels of the CK-MB isoenzyme, aspartate transaminase, and LDH were measured on an automated chemical analyzer (Micro Lab 300; Merck) using reagent kits.

Back to Top | Article Outline

Myocardial endogenous antioxidant enzymes

Another portion of myocardial tissue (n=6) was individually homogenized in 10% chilled tris-hydrochloride buffer (10 mmol/l, pH 7.4) in a tissue homogenizer (Remi Motors, Mumbai, India) and centrifuged at 7500 rpm for 15 min at 0°C in an Eppendorf 5810-R high-speed cooling centrifuge (Eppendorf India Limited, Chennai, India). The clear supernatant was collected after centrifugation and used for the assay of endogenous antioxidant enzymes. SOD levels were determined using the method developed by Misra and Fridovich 34. GSH levels were determined using the method developed by Moron et al. 35. Lipid peroxidation [malondialdehyde (MDA)] was estimated using the method developed by Slater and Sawyer 36. The sediment was resuspended in ice-cold Tris buffer (10 mmol/l, pH 7.4) and used for the estimation of membrane-bound enzymes. Na+–K+-ATPase and Ca2+-ATPase were assayed according to the methods used by Bonting 37 and Hjerten and Pan 38, respectively. Inorganic phosphorus content was estimated by the method used by Fiske and Subbarow 39. Total protein content was determined using the method developed by Lowry et al. 40.

Back to Top | Article Outline

Hydroxyproline content and NO content

Hydroxyproline content was measured in the remaining myocardial tissue (n=6) by a procedure similar to that adopted previously by Numan and Logan 41. Myocardial fibrosis was measured using a standard curve for hydroxyproline, and fibrosis level was expressed as μg/mg of protein. Nitrite level was estimated in the cardiac homogenate using the Greiss reagent and served as an indicator of nitric oxide production. A measure of 500 μl of Greiss reagent (1 : 1 solution of sulfanilamide in 5% phosphoric acid and 0.1% naphthylamine diamine dihydrochloric acid in water) was added to 100 μl of tissue homogenate and absorbance was measured at 546 nm using the method developed by Green et al. 42. Nitrite concentration was measured using a standard curve for sodium nitrite. Nitrite levels were expressed as μg/ml.

Back to Top | Article Outline

Estimation of TNF-α

Levels of TNF-α in the myocardial tissue homogenate (n=4) were determined using an enzyme-linked immunosorbent assay (Thermo scientific and Pierce Biotech Int., Rockford, Illinois, USA) according to the manufacturer’s instructions. ELISA is a 4.5-h-long solid-phase enzyme-linked immunosorbent rat TNF-α immunoassay designed to measure rat TNF-α levels. The assay is based on the sandwich enzyme immunoassay technique. A monoclonal antibody specific to rat TNF-α is precoated onto the microplate. Briefly, 50 μl of pretreated buffer was added to each well. Thereafter, 50 μl of the standard, control, and test samples (aliquot of tissue homogenate) were added into each well and the plate was incubated at room temperature for 1 h. If any rat TNF-α was present, it bound to the immobilized antibody. After washing away any unbound substance, 50 μl of biotinylated antibody reagent was added to each well and the plate was incubated at room temperature for 1 h. After washing away any unbound substance, 100 μl of streptavidin–horse radish peroxidase reagent was added to each well, which is an enzyme-linked polyclonal antibody specific to rat TNF-α. This was followed by washing to remove any unbound antibody–enzyme reagent. Thereafter, 100 μl of TMB, a substrate solution, was added, and an enzymatic reaction occurred, which yielded a yellow product. The intensity of the color was measured at 550 nm, which was proportional to the amount of rat TNF-α bound in the initial steps. The sample values were determined from a standard curve. Values are expressed as mean±SEM.

Back to Top | Article Outline

Reverse transcriptase PCR for endothelin receptor

RNA isolation

The levels of mRNA were estimated using the reverse transcription-PCR approach as described previously 43. The cardiac tissue was chopped and minced. The liquid nitrogen-cooled specimens were then ground using a mortar and pestle. The total cytoplasmic RNA was extracted from the tissue samples using the guanidinium thiocyanate/phenol chloroform/trizol extraction method (Thermo Fischer Pvt. Ltd, Mumbai, India). Following precipitation with isopropanol, RNA was washed with 70% ethanol and treated with RNase Inhibitor (Thermo Fischer Pvt. Ltd) for 45 min. It was then resuspended at 65°C for 15 min, further purified using the Qiagen RNA isolation kit, and treated with RNase-free DNase as directed by the manufacturer (Qiagen, Valencia, California, USA). Thereafter, RNA was precipitated and resuspended in RNase-free water, and its concentration was determined by measuring the absorbance at 260 nm. RNA samples were stored at −80°C until analysis.

Back to Top | Article Outline

cDNA preparation

Single-stranded complementary DNA (cDNA) was synthesized from 5 μg of total cellular RNA using reverse transcriptase (Takara Bio, Mountain View, California, USA) and oligo-(dT)-primers (Takara Bio) as described previously 43. Briefly, 5 μg of total RNA was uncoiled by heating (65°C for 5 min) and then reverse transcribed into cDNA in a 50 μl reaction mixture that contained 50 U Moloney murine leukemia virus reverse transcriptase, 0.3 μg oligo-(dT)-primer, 1 μl RNase block ribonuclease inhibitor (40 U/μl), 2 μl of a 100 mmol/l mixture of deoxyadenosine triphosphate (dATP), deoxyribothymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP), and 5 μl of 109 RT buffer (10 mmol/l Tris-HCl, pH=8.3, 50 mmol/l KCl, 5 mmol/l MgCl2). The resultant cDNA (2 μl) was amplified in a 50 μl reaction volume containing 2 U Taq polymerase, 200 μmol/l (each) dNTP (Vivantis, Oceanside, California, USA), 1.5 mmol/l MgCl2, 5 μl 10× PCR buffer (50 nmol/l KCl, 10 nmol/l Tris-HCl, pH=8.3), and specific primers used at a final concentration of 0.5 μmol/l. The PCR mixture was amplified in a DNA thermal cycler (Eppendorf India Ltd). The primer sequences for ET-1 and β-actin were obtained on the basis of previously described methodology and are in shown Table 1 44. Amplification of β-actin served as a control for sample loading and integrity. The primers were synthesized by Ocimum Biosolutions (Hyderabad, India). PCR products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The size of the amplicons was confirmed using a 100-bp ladder (Takara Bio) as a standard size marker. The amplicons were visualized, and images were captured using a gel documentation system (Alpha Innotech Inc., San Leandro, California, USA). The expression of all the genes was assessed by generating densitometric data for band intensities in different sets of experiments by analyzing the gel images on the Image J program (Version 1.33; National Institutes of Health, Bethesda, Maryland, USA) semiquantitatively. The band intensities were compared with that of constitutively expressed β-actin. The intensities of the mRNAs were standardized against that of β-actin mRNA from each sample, and the results were expressed as the PCR-product/β-actin mRNA ratio.

Table 1

Table 1

Back to Top | Article Outline

Histopathological evaluation

Hearts were obtained from the experimental group of rats, fixed for histopathological evaluation with 4% buffered paraformaldehyde solution, and embedded in paraffin. Three to four-micrometer-thick paraffin sections were dewaxed and placed in water through graded ethanol. Sections were stained with hematoxylin and eosin stain; they were then dehydrated through graded ethanol, cleared in xylene, and mounted with DPX (Sigma-Aldrich Chemie GmbH, Buchs SG, Switzerland). For staining of myocardial fibers, the Yuccafine Masson’s trichrome staining kit (Yucca Diagnostics, Kolhapur, India) was used. Sections of formalin-fixed material were incubated with the mordant, Bouins fluid, for 1 h at 56°C or overnight at room temperature. The sections were then deparafinized with xyline, followed by 95% ethanol. Thereafter, the sections were stained with Weigert’s hematoxylin for 10 min and washed in tap water for 5 min. They were then stained with Ponceau fuchsin solution for 5 min, washed in distilled water for 3 min, stained with phosphomolybdic acid reagent for 5 min, and then stained with aniline blue solution for 5 min. The section were washed in distilled water for 3 min, dehydrated through graded ethanol, cleared in xylene for 3 min, and mounted with DPX. The sections stained with Masson’s trichrome stain were assessed for the presence of interstitial fibrosis, and hematoxylin and eosin-stained sections were graded for the presence of medullary congestion, tubular cell necrosis and dilation, cytoplasmic vacuolization, nuclear pyknosis, and cytoplasmic eosinophilia.

Back to Top | Article Outline

Statistical analysis

Data are expressed as mean±SEM and statistical analysis was carried out by one-way analysis of variance followed by post-hoc Dunnett’s test, performed using GraphPad Prism 5.0 software (GraphPad software, San Diego, California, USA). A P-value of less than 0.05 was considered to be statistically significant.

Back to Top | Article Outline

Results

Effect of bosentan on ventricular remodeling and morphology

As shown in Table 2, the heart weight to body weight ratio and the left ventricle weight to body weight ratio in the AC group were significantly increased (P<0.001, <0.001) compared with those in the sham group. Four weeks of treatment with bosentan (100 mg/kg) resulted in a significant reduction in the heart weight to body weight ratio as compared with that in the AC control group, whereas the left ventricle weight to body weight ratio was significantly and dose-dependently decreased on treatment with bosentan (50 and 100 mg/kg, P<0.01, and <0.01, respectively) as compared with that in AC control rats. There was no significant difference in the heart weight to body weight ratio and the left ventricle weight to body weight ratio in the sham and bosentan (100 mg/kg)-treated group as compared with the sham group.

Table 2

Table 2

Back to Top | Article Outline

Effect of bosentan on electrocardiographic abnormalities

There was a significant reduction (P<0.001) in heart rate in the AC group as compared with the sham group. This reduction in heart rate was significantly inhibited (P<0.01) by treatment with bosentan (100 mg/kg) when compared with that in the AC group. However, rats treated with bosentan (25 and 50 mg/kg) failed to produce any significant increase in heart rate compared with that in AC control rats. Results in Table 3 indicate that treatment with bosentan significantly improves electrocardiographic abnormalities as compared with those seen in the AC group.

Table 3

Table 3

LVH in the AC group was associated with significant prolongation in the QRS, QT, QTc, RR, and PR intervals. Treatment with bosentan (50 and 100 mg/kg) resulted in significant and dose-dependent (P<0.01 and <0.001, respectively) attenuation of prolongation of the QT, QTc, RR, and PR intervals (P<0.01), as well as the QRS complex, when compared with that in the AC group. There was no significant difference in QRS, QT, QTc, RR, and PR intervals of sham and bosentan (100 mg/kg)-treated rats as compared with sham-treated rats (Table 3).

Back to Top | Article Outline

Effect of bosentan on hemodynamic changes and LV contractile function

A significant increase (P<0.001) in mean arterial blood pressure, systolic blood pressure, and diastolic blood pressure was recorded in AC control rats when compared with the sham group, whereas 4 weeks of treatment with bosentan (100 mg/kg) significantly decreased the mean arterial blood pressure (P<0.05), systolic blood pressure (P<0.01), and diastolic blood pressure (P<0.01) when compared with those in the AC group. Rats with cardiac hypertrophy in the AC group showed significant increases (P<0.001) in pulse pressure and systolic and diastolic durations as compared with those in the sham group. Treatment with bosentan led to significant reduction in pulse pressure (P<0.05), systolic duration (P<0.01), and diastolic duration (P<0.05). When compared with the sham group, the AC group showed severe LV contractile dysfunction. The maximal rates of rise in LV pressure (dP/dtmax) and fall in LV pressure (dP/dtmin) were significantly decreased (P<0.001 and <0.01, respectively) in the AC group after the fourth week. However, treatment with bosentan (100 mg/kg) significantly restored changes in dP/dtmax (P<0.01) and dP/dtmin (P<0.05). In addition, hypertrophy of the heart in the AC group led to a significant increase in the time course of relaxation, which is also called exponential tau (P<0.001), and the pressure time index (P<0.001), whereas it led to a significant decrease in the contractility index (P<0.001) after the fourth week of stenosis. Treatment with bosentan significantly improved the contractility index (P<0.01), exponential tau (P<0.01), and pressure time index (P<0.001) as compared with those in the AC group (Table 4). These data indicated that treatment with bosentan (100 mg/kg) considerably improves hemodynamics, as well as left ventricular alterations.

Table 4

Table 4

Back to Top | Article Outline

Effect of bosentan on the cardiac marker enzyme and hydroxyproline level

As shown in Table 5, there was a significant increase (P<0.001) in the levels of cardiac marker enzymes, that is, CK-MB and LDH, in the serum samples of rats in the AC group as compared with sham-treated rats. Hydroxyproline levels in AC control rats were significantly increased as compared with those in sham-treated rats. Treatment with bosentan (100 mg/kg) resulted in a significant reduction in CK-MB (P<0.05), LDH (P<0.05), and hydroxyproline (P<0.01) levels as compared with those in the AC group. However, there was no significant difference in CK-MB, LDH, and hydroxyproline levels of sham and bosentan (100 mg/kg)-treated rats as compared with sham-treated rats.

Table 5

Table 5

Back to Top | Article Outline

Effect of bosentan on myocardial endogenous antioxidant enzymes

As shown in Fig. 2, rats with cardiac hypertrophy from the AC group showed significantly increased (P<0.001) levels of MDA, whereas levels of GSH and SOD were decreased significantly (P<0.001) as compared with those in sham control rats. Treatment with bosentan (25, 50, and 100 mg/kg) significantly and dose-dependently (P<0.05, <0.05, and <0.001) restored the levels of MDA as compared with those in AC control rats. The decreased activities of GSH and SOD in the cardiac homogenate were significantly restored (P<0.001) by bosentan (50 and 100 mg/kg) treatment as compared with those in the AC group. However there was no significant difference in the levels of SOD, GSH, and MDA in sham and bosentan (100 mg/kg)-treated rats as compared with sham-treated rats.

Fig. 2

Fig. 2

Back to Top | Article Outline

Effect of bosentan on membrane-bound inorganic phosphate enzymes

The activities of membrane-bound phosphatase enzymes – that is Na+–K+-ATPase and Ca2+-ATPase – in the myocardium of hypertrophied rats from the AC group were decreased significantly (P<0.001) as compared with those in sham control rats. The activity of Na+–K+-ATPase in the bosentan (100 mg/kg) group was increased significantly (P<0.001) as compared with that in the AC group; bosentan (50 and 100 mg/kg)-treated rats showed significant elevation in Ca2+-ATPase levels when compared with AC control rats. The activities of Na+–K+-ATPase and Ca2+-ATPase differ nonsignificantly between sham and bosentan (100 mg/kg)-treated rats and sham-treated rats (Fig. 2).

Back to Top | Article Outline

Effect of bosentan on the TNF-α level

There was significant increase in the TNF-α level in AC control rats as compared with sham control rats. This elevated TNF-α level was significantly and dose-dependently attenuated by treatment with bosentan (50 and 100 mg/kg) when compared with that in AC control rats. Treatment with bosentan (25 mg/kg) failed to produce any significant reduction in elevated levels of TNF-α when compared with those in AC control rats. However there was no significant difference in the TNF-α levels between sham and bosentan (100 mg/kg)-treated rats and sham-treated rats (Fig. 2).

Back to Top | Article Outline

Effect of bosentan on ET-1 mRNA levels

As shown in Fig. 3, ET-1 mRNA levels were significantly up-regulated in the AC control rats as compared with sham control rats. This elevation in the level of ET-1 mRNA was significantly and dose-dependently downregulated on treatment with bosentan (50 and 100 mg/kg) as compared with that in AC control rats. However, treatment with bosentan (25 mg/kg) failed to produce any significant downregulation of ET-1 mRNA expression as compared with that in AC control rats. When compared with sham control rats, sham and bosentan (100 mg/kg)-treated rats did not show any significant change in the ET-1 mRNA level.

Fig. 3

Fig. 3

Back to Top | Article Outline

Effect of bosentan on histopathological changes and myocardial fibrosis

Histological analysis of heart tissue after hematoxylin and eosin staining revealed marked tissue injury with inflammation, cardiac hypertrophy, nuclear pyknosis, cytoplasmic vacuolization, and cytoplasmic eosinophilia (Fig. 4b); however, the sham group lacked tissue injury in myocardial fibers (Fig. 4a). Histological sections of hearts from bosentan (25 mg/kg perorally)-treated rats showed altered myocardial vasculature, reflected by presence of vascular congestion, myocardial inflammation, and nuclear pyknosis (Fig. 4c). Treatment with bosentan (50 and 100 mg/kg perorally) remarkably improved the histological changes in the heart tissue in the form of reduced myocardial damage, inflammation, and nuclear pyknosis (Fig. 4d and e, respectively). Light microscopic evaluation of the heart sections from the AC group after Masson’s trichrome staining showed severe interstitial and perivascular fibrosis (Fig. 5b). Treatment with 50 and 100 mg/kg (perorally) bosentan effectively improved the histological changes, that is, interstitial and perivascular fibrosis (Fig. 5d and e, respectively); however, treatment with 25 mg/kg bosentan (perorally) failed to do so (Fig. 5c).

Fig. 4

Fig. 4

Fig. 5

Fig. 5

Back to Top | Article Outline

Discussion

CHF is a chronic complex syndrome leading to both hemodynamic and electrographic abnormalities, along with alterations in metabolic and neurohormonal conditions. Along with hemodynamic stress factors other mechanisms also play a vital role in induction and maintenance of disease including inflammation, oxidonitrosative stress, dysfunction of the endothelium, and apoptosis 45. An array of neurohormonal markers, that is, TNF-α, IL-1β, and C-reactive protein have been reported to cause CHF 46.

Abdominal aortic banding caused pressure overload in the LV, inducing concentric hypertrophy and cell death. Various in-vitro and in-vitro studies carried out previously have reported the roles of the c-Jun N-terminal kinases, p38 kinases, and endothelin signaling pathways in the induction of cardiac hypertrophy 47,48. Various animal models have been used to study the effects of different potential drug candidates on LV hypertrophy, which include constriction of the thoracic aorta 49, abdominal AC 4,5, and induction of renovascular stenosis 50,51, as well as the development of genetically modified animals including salt-sensitive rats 52 and spontaneously hypertensive rats 53,54. In the present investigation, we studied the effect of bosentan on early events following the induction of LV pressure overload, as well as various changes in endogenous biomarkers and endothelin signaling in laboratory rats on aortic banding.

In the present investigation, elevated blood pressure was recorded after a few days of AS, reflecting the successful induction of pressure overload through stenosis. This was followed by a significant increase in the LV mass and development of thicker LV walls after 4 weeks of abdominal stenosis 32. Along with intense and sustained blood pressure, various hemodynamic as well as ECG alterations occurred, such as an increase in the QRS interval, an increase in the QRS duration, and changes in the QT, QTc, and RR intervals, which impair left ventricular functions such as left ventricular end systolic pressure, left ventricular end diastolic pressure, dP/dtmax, and the pressure time index. The finding of the present study is a typical outcome of concentric LVH associated with pressure overload, which is in accordance with the findings from previous studies 4,5. Treatment with bosentan for 4 weeks inhibits the increased left ventricular weight and restores the hemodynamic and ECG alternations.

Elevated levels of CK and LDH are the hallmarks of myocardial injury. Insufficient supply of oxygen or glucose increased cell permeability and thus caused efflux of these enzymes – that is, CK-MB and LDH – in heart tissue after cardiac hypertrophy 55. The elevation in the enzyme levels in serum is proportional to the number of necrotic cells 56. It has been reported that cardiac hypertrophy is associated with collagen strut dissolution, causing myocyte slippage with accumulation of collagen 57. Hydroxyproline plays a crucial role in stabilizing intramolecular and intermolecular crosslinks in the myocardium. In our study, AC control rats showed an increased collagen content, reflected by elevated levels of hydroxyproline in comparison with that in sham rats. This further increased fibrosis, evidenced by histological evaluation of heart tissue after staining with Masson’s trichrome, as compared with that in sham rats. Myocardial fibrosis is a hallmark of myocardial remodeling, which led to an increase in extracellular matrix content; it may contribute to contractile impairment 13. Rats treated with bosentan showed a decrease in these elevated levels of CK-MB, LDH, and hydroxyproline, reflecting its cardioprotective potential. Findings of present investigation are in accordance with the findings of previous studies, which showed that bosentan significantly inhibits elevated levels of collagen in diabetic rats 30.

It has been reported that oxidative stress plays important role in progressive development of LV dysfunction in cardiac hypertrophy induced by pressure overload, which leads to cardiomyocyte apoptosis 7,58. Aikawa and colleagues 59,60 also reported the occurrence of cardiac myocyte apoptosis due to the generation of oxidative stress in vitro. It has been reported that treatment with novel antioxidants can inhibit cardiac hypertrophy and thus improve ventricular function 58. Various enzymes are responsible for the generation of reactive oxygen species (ROS) including nitric oxide synthase, cyclooxygenase, NADH/NADPH oxidase, and xanthine oxidase; the mitochondrial electron transport chain is also involved in the generation of ROS 58,61–64. This elevated level of ROS downregulates copper–zinc–SOD enzyme activity along with GSH concentration in myocytes. It caused significant loss of myocardial integrity and necrosis 8,65–67. Superoxide anions and their derivatives including hydroxyl radicals are the most abundantly generated ROS in cardiac myocytes, which cause peroxidation of the lipid membrane 68. In this aspect, elevated levels of SOD appear to be effective in reducing ROS 69,70. In agreement with the results of previous work, aortic banding causes a decrease in the level of SOD as well as GSH, which induces cardiac hypertrophy 71. Lipid peroxidation is another important mechanism that causes oxidative damage in cardiomyocytes through modification of cellular proteins and DNA damage 72–74. Gupta and Singal, as well as Dhalla et al. 71, have reported that the level of MDA was significantly increased in the hearts of hypertrophied guinea pigs after 10 weeks of ascending aorta banding. Rats treated with bosentan significantly restored altered levels of SOD, GSH, and MDA. The present study provides evidence that bosentan may prevent the development of LVH through inhibition of the formation of ROS, thus improving cardiomyocyte fibrosis and apoptosis.

Reduced Ca2+ levels in the heart is a key feature of diminished contractility of hypertrophic cardiac myocytes, which in turn affects systolic and diastolic phases of the cardiac cycle 75,76. Na+–H+ exchange is a major pathway involved in Na+ entry as well as intracellular Ca2+ overload of myocardial tissue under the ischemic condition 77. The persistent inhibition of Na+–K+-ATPase and Ca2+-ATPase pumps causes intracellular excitation toxicity, mediated through a decrease in Ca2+ overload. This leads to a decrease in the resting potential and an increase in cell automatism, which leads to homeostasis loss and cell death 13,70,78–80. The activities of Na+–K+-ATPase and Ca2+-ATPase in the myocardial homogenate were decreased significantly in the AC control group as compared with those in sham rats. These results corroborate the findings of previous studies that have demonstrated the role of Na+–K+-ATPase and Ca2+-ATPase in LVH 81. Treatment with bosentan significantly restored diminished levels of membrane-bound inorganic phosphate enzymes, that is, Na+–K+-ATPase and Ca2+-ATPase.

In LVH, along with hemodynamic and ECG alterations with increased oxidative stress, AS is also associated with elevated levels of proinflammatory cytokines such as IL-1β and TNF-α. These proinflammatory cytokines act in an autocrine or paracrine manner to induced cardiac remodeling and ventricular dysfunction through the activation of p38 82. The activation of p38 may lead to an increase in inflammation, extracellular matrix remodeling, contractile and myocardial dysfunction, and apoptosis 83. Finkel et al. 84 and Schulz et al. 85 have reported that TNF-α produces a negative inotropic effect and NO-dependent activation in vivo, as well as in vitro. Rat treated with bosentan showed inhibition in the upregulation of TNF-α.

The known potent vasoconstrictor, ET-1, has the property of growth promotion in fibroblasts. The synthesis and release of ET-1 occurs in cardiomyocytes and fibroblasts of heart tissue. This hypertrophic factor acts through an autocrine or paracrine mechanism through the ETA receptor 86 Rats with LV overload produced by aortic banding showed upregulation of ET-1 expression as compared with sham rats. The results of the present investigation are consistent with those of previous studies 86,87. Moreover, in-vitro studies carried out by various researchers in cultured rat cardiomyocytes have shown that ET-1 plays a vital role in the induction and maintenance of cardiac hypertrophy, along with muscle-specific genes, as well as the c-fos proto-oncogene 88,89. It has been reported that ET-1 receptor blockers such as sitaxentan, ambrisentan, atrasentan, zibotentan, macitentan, and tezosentan have the potential to inhibit the development of cardiac hypertrophy and fibrosis in AC rats 90. Bosentan is a potent analogue of Ro 46-2005 with the potential for nonselective (ETA and ETB) endothelin receptor antagonism 91,92. It has been reported previously that 0.3 nmol/kg bosentan significantly inhibits the upregulation of ET-1 expression 92. It has also been reported that administration of bosentan reverts hypoxia-induced pulmonary hypertension, right-heart hypertrophy, and pulmonary vascular remodeling through its action on the endothelin receptor 25,28. In the present investigation, treatment with bosentan was seen to restore the elevated expression of ET-1. The results of the present investigation are in accordance with the findings of previous studies 25,92.

Recently bosentan was shown to lower blood pressure clinically and satisfactorily in essential hypertension compared with an angiotensin-converting enzyme inhibitor 26,27. Therefore, bosentan, a potent ET-1 receptor antagonist, may exhibit its cardioprotective effect through the inhibition of oxidonitrosative stress and the release of proinflammatory cytokines (TNF-α), along with the downregulation of ET-1 mRNA expression in rats with pressure overload-induced cardiac hypertrophy.

Back to Top | Article Outline

Acknowledgements

The authors thank Dr S. S. Kadam, vice chancellor, Bharati Vidyapeeth University, and Dr K. R. Mahadik, principal, Poona College of Pharmacy, for their keen interest and providing the necessary facilities to carry out the study.

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP.Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study.N Engl J Med1990;322:1561–1566.
2. Dole S, Kandhare AD, Ghosh P, Gosavi TP, Bodhankar SL.The health outcome after homeopathic treatment in cases of BPH and LUTS: a prospective clinical study.J Pharm Biomed Sci2012;22:1–6.
3. Imamura M, Schluchter M, Fouad-Tarazi FM.Remodelling of left ventricle after banding of ascending aorta in the rat.Cardiovasc Res1990;24:641–646.
4. Norton GR, Woodiwiss AJ, Gaasch WH, Mela T, Chung ES, Aurigemma GP, et al..Heart failure in pressure overload hypertrophy: the relative roles of ventricular remodeling and myocardial dysfunction.J Am Coll Cardiol2002;39:664–671.
5. Stoyanova V, Zhelev NZ, Yanev IB, Ghenev ED, Nachev CK.Time course and progression of pressure overload-induced cardiac hypertrophy in rats.Folia Med (Plovdiv)2005;47:52.
6. Thaik CM, Calderone A, Takahashi N, Colucci WS.Interleukin-1 beta modulates the growth and phenotype of neonatal rat cardiac myocytes.J Clin Invest1995;96:1093–1099.
7. Heymes C, Bendall JK, Ratajczak P, Cave AC, Samuel JL, Hasenfuss G, Shah AM.Increased myocardial NADPH oxidase activity in human heart failure.J Am Coll Cardiol2003;41:2164–2171.
8. Patil MVK, Kandhare AD, Ghosh P, Bhise SD.Determination of role of GABA and nitric oxide in anticonvulsant activity of Fragaria vesca L. ethanolic extract in chemically induced epilepsy in laboratory animals.Orient Pharm Exp Med2012;12:255–264.
9. Kandhare AD, Raygude KS, Ghosh P, Gosavi TP, Bodhankar SL.Patentability of animal models: India and the globe.Int J Pharma Biol Arch2011;2:1024–1032.
10. Oh H, Wang SC, Prahash A, Sano M, Moravec CS, Taffet GE, et al..Telomere attrition and Chk2 activation in human heart failure.Proc Natl Acad Sci USA2003;100:5378–5383.
11. Sia YT, Lapointe N, Parker TG, Tsoporis JN, Deschepper CF, Calderone A, et al..Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat.Circulation2002;105:2549–2555.
12. Choudhary R, Baker KM, Pan J.All-trans retinoic acid prevents angiotensin II- and mechanical stretch-induced reactive oxygen species generation and cardiomyocyte apoptosis.J Cell Physiol2008;215:172–181.
13. Ghule AE, Kulkarni CP, Bodhankar SL, Pandit VA.Effect of pretreatment with coenzyme Q10 on isoproterenol-induced cardiotoxicity and cardiac hypertrophy in rats.Curr Ther Res Clin Exp2009;70:460–471.
14. Hirata Y, Yoshimi H, Takaichi S, Yanagisawa M, Masaki T.Binding and receptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells.FEBS Lett1988;239:13–17.
15. Suzuki T, Hoshi H, Mitsui Y.Endothelin stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serum-free medium.FEBS Lett1990;268:149–151.
16. McDonough PM, Brown JH, Glembotski CC.Phenylephrine and endothelin differentially stimulate cardiac PI hydrolysis and ANF expression.Am J Physiol1993;264:H625–H630.
17. Abraham A, Kay J, Cole R, Pincock AC.Haemodynamic and pathological study of the effect of chronic hypoxia and subsequent recovery of the heart and pulmonary vasculature of the rat.Cardiovasc Res1971;5:95–102.
18. Yin F, Spurgeon H, Weisfeldt M, Lakatta EG.Mechanical properties of myocardium from hypertrophied rat hearts. A comparison between hypertrophy induced by senescence and by aortic banding.Circ Res1980;46:292–300.
19. Grossman W.Cardiac hypertrophy: useful adaptation or pathologic process?Am J Med1980;69:576–584.
20. Drayer JIM, Weber MA, Gardin JM, Lipson JL.Effect of long-term antihypertensive therapy on cardiac anatomy in patients with essential hypertension.Am J Med1983;75:116–120.
21. Wollam GL, Hall W, Porter VD, Douglas MB, Unger DJ, Blumenstein BA, et al..Time course of regression of left ventricular hypertrophy in treated hypertensive patients.Am J Med1983;75:100–110.
22. Jiang J, Yuen V, Xiang H, McNeill JH.Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms.Mol Cell Biochem2006;282:177–185.
23. Kaddoura S, Firth JD, Boheler KR, Sugden PH, Poole-Wilson PA.Endothelin-1 is involved in norepinephrine-induced ventricular hypertrophy in vivo: acute effects of bosentan, an orally active, mixed endothelin ETA and ETB receptor antagonist.Circulation1996;93:2068–2079.
24. Roux S, Breu V, Ertel SI, Clozel M.Endothelin antagonism with bosentan: a review of potential applications.J Mol Med1999;77:364–376.
25. Weber C, Schmitt R, Birnboeck H, Hopfgartner G, van Marle SP, Peeters PA, et al..Pharmacokinetics and pharmacodynamics of the endothelin-receptor antagonist bosentan in healthy human subjects.Clin Pharmacol Ther1996;60:124–137.
26. Krum H, McMurray JJ.Statins and chronic heart failure: do we need a large-scale outcome trial?J Am Coll Cardiol2002;39:1567–1573.
27. Williamson DJ, Wallman LL, Jones R, Keogh AM, Scroope F, Penny R, et al..Hemodynamic effects of bosentan, an endothelin receptor antagonist, in patients with pulmonary hypertension.Circulation2000;102:411–418.
28. Kiowski W, Sütsch G, Hunziker P, Müller P, Kim J, Oechslin E, et al..Evidence for endothelin-1-mediated vasoconstriction in severe chronic heart failure.Lancet1995;346:732–736.
29. Underwood DC, Bochnowicz S, Osborn RR, Luttmann MA, Hay DW.Nonpeptide endothelin receptor antagonists. X. Inhibition of endothelin-1-and hypoxia-induced pulmonary pressor responses in the guinea pig by the endothelin receptor antagonist, SB 217242.J Pharmacol Exp Ther1997;283:1130–1137.
30. Cosenzi A, Bernobich E, Trevisan R, Milutinovic N, Borri A, Bellini G.Nephroprotective effect of bosentan in diabetic rats.J Cardiovasc Pharmacol2003;42:752–756.
31. Evans T, Deng DX, Chen S, Chakrabarti S.Endothelin receptor blockade prevents augmented extracellular matrix component mRNA expression and capillary basement membrane thickening in the retina of diabetic and galactose-fed rats].Diabetes2000;49:662–666.
32. van Deel ED, de Boer M, Kuster DW, Boontje NM, Holemans P, Sipido KR, et al..Exercise training does not improve cardiac function in compensated or decompensated left ventricular hypertrophy induced by aortic stenosis.J Mol Cell Cardiol2011;50:1017–1025.
33. Said SA, Ammar el SM, Suddek GM.Effect of bosentan (ETA/ETB receptor antagonist) on metabolic changes during stress and diabetes.Pharmacol Res2005;51:107–115.
34. Misera H, Fridovich I.The role of superoxide anion in the auto-oxidation of epinephrine and a simple assay for SOD.J Biol Chem1972;247:3170–3175.
35. Moron MS, Depierre JW, Mannervik B.Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.Biochim Biophys Acta1979;582:67–78.
36. Slater T, Sawyer B.The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver fractions in vitro. General features of the systems used.Biochem J1971;123:805.
37. Bonting SBilter EE.Presence of enzyme system in mammalian tissues. Membrane and Ion Transport.Presence of enzyme systems in mammalian tissues1970.London:Wiley Interscience;257–263.
38. Hjertén S, Pan H.Purification and characterization of two forms of a low-affinity Ca2+-ATPase from erythrocyte membranes.Biochim Biophys Acta1983;728:281–288.
39. Fiske CH, Subbarow Y.The colorimetric determination of phosphorus.J Biol Chem1925;66:375–400.
40. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.Protein measurement with the Folin phenol reagent.J Biol Chem1951;193:265–275.
41. Neuman RE, Logan MA.The determination of collagen and elastin in tissues.J Biol Chem1950;186:549–556.
42. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids.Anal Biochem1982;126:131–138.
43. Konturek PC, Duda A, Brzozowski T, Konturek SJ, Kwiecien S, Drozdowicz D, et al..Activation of genes for superoxide dismutase, interleukin-1beta, tumor necrosis factor-alpha, and intercellular adhesion molecule-1 during healing of ischemia-reperfusion-induced gastric injury.Scand J Gastroenterol2000;35:452–463.
44. Brown LA, Nunez DJ, Brookes CI, Wilkins MR.Selective increase in endothelin-1 and endothelin A receptor subtype in the hypertrophied myocardium of the aorto-venacaval fistula rat.Cardiovasc Res1995;29:768–774.
45. Kang PM, Izumo S.Apoptosis and heart failure: a critical review of the literature.Circ Res2000;86:1107–1113.
46. Vasan RS, Sullivan LM, Roubenoff R, Dinarello CA, Harris T, Benjamin EJ, et al..Inflammatory markers and risk of heart failure in elderly subjects without prior myocardial infarction The Framingham Heart Study.Circulation2003;107:1486–1491.
47. Liang Q, Molkentin JD.Redefining the roles of p38 and JNK signaling in cardiac hypertrophy: dichotomy between cultured myocytes and animal models.J Mol Cell Cardiol2003;35:1385–1394.
48. Nishida K, Yamaguchi O, Hirotani S, Hikoso S, Higuchi Y, Watanabe T, et al..p38α mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload.Mol Cell Biol2004;24:10611–10620.
49. Boluyt MO, Robinson KG, Meredith AL, Sen S, Lakatta EG, Crow MT, et al..Heart failure after long-term supravalvular aortic constriction in rats.Am J Hypertens2005;18:202–212.
50. Martino RF, Davicino RC, Mattar MA, Casali YA, Correa SG, Micalizzi B.Larrea divaricata Cav. enhances the innate immune response during the systemic infection by Candida albicans.Afr J Microbiol Res2011;5:753–761.
51. Rahimkhani M, Khavari-Daneshvar H, Jamali S.Serologic evaluation of hepatitis B and D in patients with cirrhosis [J].Afr J Microbiol Res2011;5:568–571.
52. Dahl LK, Heine M, Tassinari L.Role of genetic factors in susceptibility to experimental hypertension due to chronic excess salt ingestion.Nature1962;194:480–482.
53. Bing O.Hypothesis: apoptosis may be a mechanism for the transition to heart failure with chronic pressure overload.J Mol Cell Cardiol1994;26:943–948.
54. Pfeffer JM, Pfeffer MA, Fishbein MC, Frohlich ED.Cardiac function and morphology with aging in the spontaneously hypertensive rat [J].Am J Physiol1979;237:H461–H468.
55. Basil B, Jordan R, Loveless A, Maxwell DR.A comparison of the experimental anti-arrhythmic properties of acebutolol:(M & B 17 803), propranolol and practolol.Br J Pharmacol2012;50:323–333.
56. Kandhare AD, Ghosh P, Ghule AE, et al..Elucidation of molecular mechanism involved in neuroprotective effect of Coenzyme Q10 in alcohol induced neuropathic pain.Fundam Clin Pharmacol2013doi: 10.1111/fcp.12003. [Epub ahead of print].
57. Swynghedauw B.Molecular mechanisms of myocardial remodeling.Physiol Rev1999;79:215–262.
58. Nakamura K, Kusano K, Nakamura Y, Kakishita M, Ohta K, Nagase S, et al..Carvedilol decreases elevated oxidative stress in human failing myocardium.Circulation2002;105:2867–2871.
59. Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M, et al..Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats.J Clin Invest1997;100:1813.
60. von Harsdorf R, Li PF, Dietz R.Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis.Circulation1999;99:2934–2941.
61. Farquharson CAJ, Butler R, Hill A, Belch JJ, Struthers AD.Allopurinol improves endothelial dysfunction in chronic heart failure [J].Circulation2002;106:221–226.
62. Kandhare AD, Ghosh P, Ghule AE, Zambare GN, Bodhankar SL.Protective effect of Phyllanthus amarus by modulation of endogenous biomarkers and DNA damage in acetic acid induced ulcerative colitis: role of phyllanthin and hypophyllanthin.Apollo Med2013;10:87–97.
63. Kandhare AD, Kumar VS, Adil M, Rajmane AR, Ghosh P, Bodhankar SL.Investigation of gastro protective activity of Xanthium strumarium L. by modulation of cellular and biochemical marker.Orient Pharm Exp Med2012;12:287–299.
64. Patil MVK, Kandhare AD, Bhise SD.Anti-arthritic and anti-inflammatory activity of Xanthium srtumarium L. ethanolic extract in Freund’s complete adjuvant induced arthritis.Biomed Aging Pathol2012;2:6–15.
65. Rathore N, John S, Kale M, Bhatnagar D.Lipid peroxidation and antioxidant enzymes in isoproterenol induced oxidative stress in rat tissues.Pharmacol Res1998;38:297–303.
66. Srivastava S, Chandrasekar B, Gu Y, Luo J, Hamid T, Hill BG, Prabhu SD.Downregulation of CuZn-superoxide dismutase contributes to β-adrenergic receptor-mediated oxidative stress in the heart.Cardiovasc Res2007;74:445–455.
67. Raygude KS, Kandhare AD, Ghosh P, Ghule AE, Bodhankar SL.Evaluation of ameliorative effect of quercetin in experimental model of alcoholic neuropathy in rats.Inflammopharmacology2012;20:331–341.
68. Hemnani T, Parihar M.Reactive oxygen species and oxidative DNA damage.Indian J Physiol Pharmacol1998;42:440–452.
69. Gosavi TP, Kandhare AD, Ghosh P, et al..Anticonvulsant activity of Argentum metallicum, a homeopathic preparation.Der Pharmacia Lettre2012;4:626–637.
70. Kandhare A, Raygude K, Ghosh P, Bodhankar SL.The ameliorative effect of fisetin, a bioflavonoid, on ethanol-induced and pylorus ligation-induced gastric ulcer in rats.Int J Green Pharm2011;5:236–243.
71. Dhalla AK, Hill MF, Singal PK.Role of oxidative stress in transition of hypertrophy to heart failure.J Am Coll Cardiol1996;28:506–514.
72. Kandhare AD, Raygude KS, Ghosh P, Ghule AE, Bodhankar SL.Neuroprotective effect of naringin by modulation of endogenous biomarkers in streptozotocin induced painful diabetic neuropathy.Fitoterapia2012;83:650–659.
73. Kandhare AD, Raygude KS, Ghosh P, Ghule AE, Bodhankar SL.Therapeutic role of curcumin in prevention of biochemical and behavioral aberration induced by alcoholic neuropathy in laboratory animals.Neurosci Lett2012;511:18–22.
74. Kandhare AD, Raygude KS, Shiva Kumar V, Rajmane AR, Visnagri A, Ghule AE, et al..Ameliorative effects quercetin against impaired motor nerve function, inflammatory mediators and apoptosis in neonatal streptozotocin-induced diabetic neuropathy in rats.Biomed Aging Pathol2012;2:173–186.
75. Bers DM, Despa S.Cardiac myocytes Ca2+ and Na+ regulation in normal and failing hearts.J Pharmacol Sci2006;100:315–322.
76. Yano M, Ikeda Y, Matsuzaki M.Altered intracellular Ca2+ handling in heart failure.J Clin Invest2005;115:556–564.
77. Wei CL, Wu Q, Vega VB, Chiu KP, Ng P, Zhang T, et al..A global map of p53 transcription-factor binding sites in the human genome.Cell2006;124:207–219.
78. Di Lisa F, Menabò R, Canton M, Petronilli V.The role of mitochondria in the salvage and the injury of the ischemic myocardium.Biochim Biophys Acta1998;1366:69–78.
79. Raygude KS, Kandhare AD, Ghosh P, Bodhankar SL.Anticonvulsant effect of fisetin by modulation of endogenous biomarkers.Biomed Prev Nutr2012;2:215–222.
80. Kandhare AD, Raygude KS, Ghosh P, Ghule AE, Gosavi TP, Badole SL, et al..Effect of hydroalcoholic extract of Hibiscus rosa sinensis Linn. leaves in experimental colitis in rats.Asian Pac J Trop Biomed2012;5:337–344.
81. Kennedy D, Omran E, Periyasamy SM, Nadoor J, Priyadarshi A, Willey JC, et al..Effect of chronic renal failure on cardiac contractile function, calcium cycling, and gene expression of proteins important for calcium homeostasis in the rat.J Am Soc Nephrol2003;14:90–97.
82. Sun M, Chen M, Dawood F, Zurawska U, Li JY, Parker T, et al..Tumor necrosis factor-α mediates cardiac remodeling and ventricular dysfunction after pressure overload state.Circulation2007;115:1398–1407.
83. Kerkela R, Force T.p38 mitogen-activated protein kinase: a future target for heart failure therapy?J Am Coll Cardiol2006;48:556–558.
84. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL.Negative inotropic effects of cytokines on the heart mediated by nitric oxide.Science1992;257:5387.
85. Schulz R, Panas DL, Catena R, Moncada S, Olley PM, Lopaschuk GD.The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha [J].Br J Pharmacol1995;114:27–34.
86. Sakurai T, Yanagisawa M, Inoue A, Ryan US, Kimura S, Mitsui Y, et al..cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA.Biochem Biophys Res Commun1991;175:44–47.
87. Ito H, Hirata Y, Adachi S, Tanaka M, Tsujino M, Koike A, et al..Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes.J Clin Invest1993;92:398–403.
88. Ito H, Hirata Y, Hiroe M, Tsujino M, Adachi S, Takamoto T, et al..Endothelin-1 induces hypertrophy with enhanced expression of muscle-specific genes in cultured neonatal rat cardiomyocytes.Circ Res1991;69:209–215.
89. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, et al..Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy.J Biol Chem1990;265:20555–20562.
90. Lüscher TF, Barton M.Endothelins and endothelin receptor antagonists: therapeutic considerations for a novel class of cardiovascular drugs.Circulation2000;102:2434–2440.
91. Clozel M, Breu V, Burri K, Cassal JM, Fischli W, Gray GA, et al..Pathophysiological role of endothelin revealed by the first orally active endothelin receptor antagonist.Nature1993;365:759–761.
92. Clozel M, Breu V, Gray GA, Kalina B, Loffler BM, Burri K, et al..Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist.J Pharmacol Exp Ther1994;270:228–235.
Keywords:

bosentan; cardiac hypertrophy; endothelin-1; hypertension; reactive oxygen species; tumor necrosis factor-α

© 2013Wolters Kluwer Health Lippincott Williams Wilkins