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Anti-apoptotic potential of rosuvastatin pretreatment in murine modelof cardiomyopathy

Himanshu Sharma a, Rahila Ahmad Pathan a, Vinay Kumar a, Saleem Javed b, Uma Bhandari a,⁎a Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi-110062, Indiab Department of Biochemistry, Faculty of Science, Hamdard University, New Delhi-110062, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 24 October 2009Received in revised form 5 January 2010Accepted 2 April 2010Available online 7 May 2010

Keywords:ApoptosisStatinsBiomarkersDoxorubicin

Background: Apoptosis is a key pathologic feature in myocardial infarction and heart failure. Recent evidencesuggests that statins may have beneficial effects on cardiovascular outcomes in patients with heart failure.The present study was planned to investigate the anti-apoptotic potential of rosuvastatin pretreatment indoxorubicin-induced cardiomyopathy.Methods: Sixty male Wistar rats were randomly divided into six groups: Group-I (vehicle control group),Group-II (pathological Control group), Group-III (rosuvastatin 0.5 mg/kg), Group IV (rosuvastatin 2 mg/kg),Group-V (rosuvastatin 2 mg/kg per se), and Group-VI (carvedilol 1 mg/kg). Myocardial apoptosis wasdetected by caspase-3 assay, DNA gel electrophoresis and Na+/K+ ATPase estimation. The animals wereevaluated for various biochemical parameters in serum followed by histopathological studies of heart tissue.Results: Doxorubicin treated rats exhibited cardiac dysfunctions as indicated by an increase in systolic,diastolic, mean BP, heart rate and tail blood flow and volume and increased serum LDH, TC, TGs, LDL-C, VLDL-C levels and atherogenic indexes. A marked induction in caspase-3 and Na+-K+ ATPase levels and DNAladdering as revealed by agarose gel electrophoresis was observed in rat myocardium of pathological group.Pretreatment with the test drug, rosuvastatin significantly reduced the increase in hemodynamicparameters, serum LDH, lipid profile and myocardial caspase-3, Na+-K+ ATPase activity as compared tothe pathogenic control group. Further, DNA ladder formation was attenuated by rosuvastatin treatment.Histopathological studies further confirm its myocardial salvaging effects. The results were comparable withcarvedilol.Conclusions: The study demonstrates the cardioprotective potential of rosuvastatin against doxorubicin-induced myocardial apoptosis.

© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Cardiovascular disease is a leading cause of death worldwide; itremains to be one of themajor killers inmodern society. It is believed toaccount for approximately twelve million deaths annually [1]. In recentyears, it has emerged that loss of myocardial cells may be a majorpathogenic factor. Cell death can occur in a destructive, uncontrolledmanner via necrosis or by a highly regulated, energy dependent,programmed cell suicidemechanism termed ‘apoptosis’. As cell death inconditions such as heart failure and myocardial infarction does notalways followa typically apoptotic pathway, it remains to be establishedwhether it occurs by apoptosis, necrosis, or a novel uncharacterizedmechanism combining aspects of both types of cell death.

Apoptosis has been shown to contribute to loss of cardiomyocytes incardiomyopathy, progressive decline in left ventricular (LV) function,myocardial infarction, ischemia/reperfusion injury and congestive heart

failure. More recent research on the underlying biochemical eventsleading to these distinct features of apoptosis has revealed the pivotalrole of a family of proteases termed ‘caspases’ (caspase3). Na+-K+

ATPase (NKA) deficiency has been identified as a contributor toapoptosis and pathogenesis [2]. A central role for the NKA inpathogenesis has beenwidely implicated, particularly in heart ischemia[3] and congestive heart failure (CHF) [4]. Lactate dehydrogenase (LDH)test is used to detect tissue alterations and as an aid in the diagnosis ofheart attack, anemia, and liver disease. Massive necrosis of cardiac cellsin heart damage may cause a large rise in serum LDH levels.

Statins are highly effective and widely used lipid-lowering agentsin clinical practice that also display a number of pleiotropic propertiesbeyond cholesterol lowering, such as anti-inflammatory, antioxidant,atrial remodeling attenuation, ion channel stabilization, and auto-nomic nervous system regulation. Statins may therefore play animportant role in the treatment of heart failure a physiologic statecharacterized by endothelial dysfunction, sympathetic upregulation,and inflammation [5]. Rosuvastatin, a relatively new molecule, hasemerged as an effective 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase inhibitor with respect to lipid-lowering activity

International Journal of Cardiology 150 (2011) 193–200

⁎ Corresponding author. Dept. of Pharmacology, Faculty of Pharmacy, HamdardUniversity, New Delhi-110062, India. Tel.: +91 11 26059688; fax: +91 11 26059663.

E-mail address: uma_bora@hotmail.com (U. Bhandari).

0167-5273/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ijcard.2010.04.008

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in low- to high-risk patients, showing a safety and tolerability profilesimilar to commonly used doses of other statins [6]. Rosuvastatin hasbeen reported to decrease blood pressure and triglyceride levels alongwith prevention of increase in capillary filtration and lymphaticdysfunction in diabetic rats [7]. In one study, Zacà et al. (2007)reported that early, long-term monotherapy with high dose rosuva-satin (3.0 mg/kg once daily, n=7), prevents the progressive leftventricular (LV) dysfunction and remodeling in dogs with moderateheart failure (HF). The conclusion was supported by the findings that3 months of therapy with high dose rosuvasatin increased LV ejectionfraction (LV EF) and prevented progressive LV enlargement thuspreventing progressive LV global remodeling. Furthermore, thebenefits observed with high dose rosuvasatin on LV function andremodeling were associated, at the cellular level, with a lower volumefraction of interstitial fibrosis, increased capillary density, improvedoxygen diffusion distance, and reduced cardiomyocyte hypertrophy[8]. Recently, few large randomized clinical trials examined the effectsof rosuvastatin on the mortality, morbidity, and functional status ofpatients with established symptomatic HF of both ischemic andnonischemic etiology [9–12].

Doxorubicin (trade name Adriamycin) or hydroxyl-daunorubicin is adrug widely used in cancer chemotherapy. Due to its known cardiotoxi-city, it is one of the major research tools to induce cardiotoxicity in theexperimental animals [13]. To date, no such studies have beenperformedon cardioprotective potential of rosuvastatin in doxorubicin-inducedcardiomyopathy.

Carvedilol, a vasodilating β-adrenoceptor antagonist and a potentantioxidant, is known to produce a high degree of cardioprotection in avariety of experimental models of ischemic cardiac injury [14].Carvedilol also prevented cardiomyocyte apoptosis in an experimentalmodel of ischemia/reperfusion [15]. Carvedilol is already indicated inthe treatment of clinically documented doxorubicin-induced cardio-myopathy to halt the further decline in LV function, to amelioratesymptoms, and to improve prognosis [16]. Therefore it is selected as astandard drug in present study.

Pretreatment with drugs in experimental animals is a prophylacticmeasure to prevent the occurrence and progression of diseasecomplications. Hence, in the present study, pretreatment with testdrugswas planned for 30 days, followed by doxorubicin (30 mg/kg/i.p.)injection on the 31st day, which is clinically significant as pretreatmentwith the drugs under investigation, if found beneficial will prevent theprogression of cardiomyopathy and apoptotic changes induced bydoxorubicin. This study might suggest that statins may be therapeuti-cally useful for the treatment of heart failure. Similar treatmentstrategies were followed in number of research studies [17,18].

The present studywas planned to investigate themodulatory role ofrosuvastatin pretreatment on various cardiovascular biomarkers asso-ciatedwithdoxorubicin-induced cardiotoxicity and apoptosis inmurinemodel of cardiomyopathy and explore its anti-apoptotic potential.Further, the results were compared with carvedilol, a standardcardioprotective drug.

2. Materials and methods

2.1. Animals and study protocol

Male albino rats (Wistar strain), 150–200 g, were procured from Central AnimalHouse Facility, Hamdard University, New Delhi. The animals were kept in polypropyl-ene cages (8 in each cage) under standard laboratory conditions (12 h light and 12hdark:day:night cycle) and had free access to commercial pellet diet (Amrut rat feed,Mfd by Nav Maharashtra Chakan Oil Mills Ltd, Delhi, India) and water ad libitum. Theanimal house temperature was maintained at 25±2 °C and relative humidity was alsomaintained at 50±15%.

They were divided into 6 groups, each group consisting of 8 animals: (a) Group-I(vehicle control rats), rats were treated with 0.5% carboxy methyl cellulose (CMC) innormal saline (2 ml/kg/day, single i.p. injection) for 30 days (b) Group-II (PathologicalControl rats), rats were treated with 0.5%CMC in normal saline (2 ml/kg/day, single i.p.injection) for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day (c) Group-III (Test Drug, Dose 1 treated), ratswere treatedwith rosuvastatin (0.5 mg/kg/day, single i.p.

injection) for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day (d) GroupIV (Test Drug, Dose 2 treated), rats were treated with rosuvastatin (2 mg/kg/day, single i.p.injection) for 30 days+doxorubicin (30 mg/kg, single i.p. injection)on31stday (e)Group-V(rosuvastatin per se), rats were treatedwith rosuvastatin (2 mg/kg/day, single i.p. injection)for 30 days (f) Group-VI (Standard Drug), rats were treated with carvedilol (1 mg/kg/day,single i.p. injection) for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day.

The dose of carvedilol (1 mg/kg) was selected based on earlier studies in whichcarvedilol has shown cardioprotective effect against doxorubicin-induced cardiomy-opathy in rats (15, 16). The doses of rosuvastain taken were based on our earlierreported research work in rats [19].

Rats were kept on overnight fasting with water ad libitum. Hemodynamicmeasurements were carried out using tail cuff method on CODA non invasive bloodpressure measurement instrument (Kent Scientific, USA) on 32nd day and after that theblood was collected from the retro-orbital plexus of overnight fasted rats usingmicrocapillary tube and serum was separated using sterile pipette after centrifugation at3000 rpm for 15 min. The rats were then killed by an overdose of anesthetic ether vaporsand hearts were removed by opening the thoracic cavity. The hearts were weighed andkept at−80 °C, except one heart in each group for histopathological examination.

2.2. Hemodynamic measurements

Every rat in eachgroupwas given training in the restrainer for a period of 15min everyday at least 10–15 days prior to the day of measurement of the hemodynamic parameters(systolic, diastolic,meanbloodpressures, heart rate, tail bloodflowand tail bloodvolume).Restless or stressedout rats, whichwas seen by their tendency to defecate or urinate,weregiven repeated trainings every time they showed the symptoms. Hemodynamicmeasurements were carried out using tail cuff method on CODA Non Invasive BloodPressure Recorder Instrument (Kent Scientific, USA).

2.3. Biochemical estimations in serum

Biochemical parameters in serum were estimated using commercially available kitsviz. lactate dehydrogenase (LDH) [20], total cholesterol (TC) [21], triglycerides (TGs) [22],high density lipoprotein-cholesterol (HDL-C) [23] (Span Diagnostics, Surat, Gujrat, India).Low density lipoprotein-cholesterol (LDL-C) and very low density lipoprotein-cholesterol(VLDL-C) were calculated using formula given by Friedward et al. (1972) [24] andatherogenic index was calculated by formula TC/HDL-C and LDL-C/HDL-C.

2.4. Biochemical estimation in cardiac tissue

Na+/K+-ATPase activity was assayed in the heart homogenates. Activity wasdetermined in reaction mixtures A and B, in absence and presence of Ouabain. Reactionmixture A for total ATPase activity contained 0.2 ml of 200 mM KCl, 0.2 ml of 1 M NaCl,0.1 ml of 100 mM MgCl2, 1 ml of 200 mM Tris buffer pH 7.4, 0.2 ml of distilled water,and 0.1 ml of heart homogenate. The mixture was allowed to pre-incubate at roomtemperature for 5 min and then incubated for 15 min at 37 °C after adding 0.2 ml of25 mM ATP disodium salt. For Mg2+ ATPase activity reaction mixture B contained0.1 ml of 100 mM MgCl2, 1 ml of 200 mM Tris buffer pH 7.4, 0.1 ml of hearthomogenate, 0.16 ml water, 0.2 ml of 10 mM Ouabain preincubated for 5 min. Thereactionwas initiated by adding 0.24 ml of 1 MNaCl and 0.2 ml of 25 mMATP disodiumsalt and incubating for 15 min at 37 °C. The reaction in both reaction mixtures wasterminated by adding 1 ml of 10% trichloroacetic acid and centrifuged at 3000 rpm for5 min. The supernatant was used for inorganic phosphate estimation (Pi) by themethod of Fiske and Subba Row [25]. To 0.5 ml of supernatant, 3.3 ml of water, 0.5 ml of2.5% ammonium molybdate in 5NH2SO4 and 0.2 ml of 1,2,4,aminonaphthol sulphonicacid (ANSA) were added. The mixture was vortexed and absorbance was read at600 μm after 10 min. The difference of the Pi liberated in the two reaction mixturesgave the activity of Na+-K+-ATPase and was expressed as µmol Pi liberated/h/mgprotein [26].

Caspase-3 activity was measured using Caspase-3/CPP32 Colorimetric Assay Kit(BioVision, USA) [27]. 50 µl supernatant from homogenized tissue with cooled lysisbuffer was taken from each sample and 50 μl of 2× Reaction Buffer (containing 10 mMDTT) was added to each sample. Then, 5 μl of the 4 mM DEVD-pNA substrate (200 μMfinal conc.) added and incubated at 37 °C for 1-2 h to allow a dissociation of p-nitroanilide (pNA) from the conjugate DEVD-pNA. CPP-32 activity was measuredspectrophotometrically at 405 nm using a 100-μl micro quartz cuvette (Sigma), ordilute sample to 1 ml with Dilution Buffer. Caspase-3 activity was calculated as units/mg/protein/h.

2.5. Agarose gel electrophoresis [28]

DNA isolation from the cardiac tissue of rat: 1% heart tissue homogenate wasprepared in solution containing 50 mmol/L Tris–HCl (pH 8.0), 100 mmol/L EDTA,100 mmol/L NaCl, and 1%SDS. Then, the tissue was digested with 5 µl of proteinase K(stock soln 20 mg/ml) at 56 °C for 2 h and incubated with RNase A (1 µl/ml) at 37 °C for1 h. After that phenol/choloroform extraction was performed twice. The upper clearlayer was transferred into another fresh tube. To this clear layer, 600 µl isopropanol wasadded and kept at room temperature for 1 h and later centrifuged at 10,000 rpm for10 min. Then the isopropanol layer was discarded by decantation and 100 µl 75%chilled ethanol was added and again centrifuged at 10,000 rpm for 10 min. The upper

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layer was discarded and DNA pellets were kept at room temperature overnight to dry.To the above test tubes, 100 µl TE solution (10 mmol/L Tris-Hcl [pH 8.0], 1 mmol/LEDTA) was added and kept for 1 h dissolution.

2.6. Loading of the DNA and running the gel for final analysis

DNA samples (5 µl DNA+1 µl gel loadingdye)were subjected to electrophoresis on 2%agarose gel, stainedwith ethidium bromide. DNA laddering, an indicator of tissue apoptoticnucleosomal DNA fragmentation was visualized and photographed under ultraviolettransilluminator.

2.7. Histopathological study of heart tissue

Cardiac tissue was fixed in 10% formalin, routinely processed and embedded inparaffin wax. Paraffin section (5 µm) were cut on glass slides and stained withhematoxylin (H) and eosin (E) and examined under a light microscope by a pathologistblinded to the groups studied.

2.8. Statistical analysis

Statistical analysiswas carried out usingGraphpadPrism3.0 (Graphpad software; SanDiego, CA). All results were expressed as mean±standard error of mean (S.E.M.). Groupsof data were compared with the analysis of variance (ANOVA) followed by Dunnett's t-test. Values were considered statistically significant when pb0.01.

3. Results

3.1. Effect on hemodynamic parameters

The systolic, diastolic and mean BP were significantly increased indoxorubicin-induced group (i.e. pathogenic control, group II) ascompared to the normal control healthy rats (i.e. group I). Therewere insignificant (pN0.05) changes in systolic, diastolic and mean BPwith rosuvastatin (0.5 mg/kg/day, i.p., i.e. group III) treatment ascompared to the pathogenic control group (i.e. group II) whiletreatment with both rosuvastatin (2 mg/kg/day i.p., i.e. group IV)and carvedilol (1 mg/kg/day i.p. i.e. group VI) showed reduction in theelevated systolic, diastolic and mean BP (pb0.01) as compared to thepathogenic control group (i.e. group II). There were no significant(pN0.05) changes in the systolic, diastolic, mean BP in rosuvastatin perse (2 mg/kg/day i.p.) treated rats (i.e. group V) as compared to thevehicle control group (i.e. group I) rats.

An increase in the mean heart rate was observed in doxorubicin-induced group (i.e. pathogenic control, group II) as compared to thenormal control healthy rats (i.e. group I) (pb0.01). There was aninsignificant (pN0.05) decrease in mean heart rate with rosuvastatin(2 mg/kg/day i.p., i.e. group IV) treatment as compared to thepathogenic control group (i.e. group II) while treatment with bothrosuvastatin (0.5 mg/kg/day, i.p., i.e. group III) and carvedilol (1 mg/kg/day i.p., i.e. group VI) showed significant (pb0.01) reduction in theelevated heart rate as compared to the pathogenic control group(i.e. group II). There was no significant (pN0.05) change in the meanheart rate in rosuvastatin per se (2 mg/kg/day i.p.) treated rats (i.e.group V) as compared to the vehicle control group (i.e. group I) rats.

Therewasa significant increase inmean tail bloodflowandmean tailblood volume in doxorubicin-induced group (i.e. pathogenic control,group II) as compared to the normal control healthy rats (i.e. group I)while no significant (pN0.05) change in the mean blood flow was seenwith rosuvastatin (0.5 mg/kg/day, i.p., i.e. group III and 2 mg/kg/day i.p.,i.e. group IV). But a less significant (pb0.05) decrease in themean bloodflowwith carvedilol (1 mg/kg/day i.p., i.e. group VI) as compared to thepathogenic control group (i.e. group II). A significant (pb0.01) decreasein the elevated mean blood volume level was further observed withrosuvastatin (0.5 mg/kg/day, i.p., i.e. group III and 2 mg/kg/day i.p., i.e.group IV) and carvedilol (1 mg/kg/day i.p., i.e. group VI) as compared tothe pathogenic control group (i.e. group II). There were no significant(pN0.05) changes in the mean blood volume in rosuvastatin per se(2 mg/kg/day i.p.) treated rats (i.e. group V) as compared to the vehiclecontrol group (i.e. group I) rats (Table 1).

3.2. Effect on serum LDH levels (IU/L)

Themean serumLDH levelswere significantly (pb0.01) increased indoxorubicin-induced group (i.e. pathogenic control, group II) ascompared to the normal control healthy rats (i.e. group I). Rosuvastatin(0.5 mg/kg/day, i.p. and 2 mg/kg/day, i.p., i.e. group III and IVrespectively) as well as carvedilol (1 mg/kg/day i.p., i.e. group VI)treatment for 30 days significantly (pb0.01) decreased the increase inthe serum LDH levels as compared to the pathogenic control group (i.e.group II). There was no significant (pN0.05) change in the serum LDHlevel in rosuvastatin per se (2 mg/kg/day i.p.) treated rats (i.e. group V)as compared to the vehicle control group (i.e. group I) rats (Table 2).

3.3. Effect on serum lipid profile levels

The mean serum TC, TGs, LDL-C, VLDL-C levels and atherogenicindexes (TC/HDL-C & LDL-C/HDL-C) were significantly increased in thedoxorubicin-induced group (i.e. pathogenic control, group II) ascompared to the levels in normal control healthy rats (i.e. group I).Doxorubicin-induced pathogenic rats, when treated with rosuvastatin(0.5 mg/kg/day i.p. and 2 mg/kg/day i.p. i.e. group III and IV respective-ly), as well as carvedilol (1 mg/kg/day, i.e. group VI), it was observedthat there were significant (pb0.01) decreases in serum TC, TGs, LDL-C,VLDL-C levels and atherogenic indexes as compared to pathogeniccontrol group (i.e. group II) rats. There were no significant (pN0.05)changes in the serum TC, TG, LDL-C, VLDL-C levels and atherogenicindexes in rosuvastatinper se (2 mg/kg/day i.p.) treated rats (i.e. groupV)as compared to the vehicle control group (i.e. group I) rats.

The mean serum HDL-C was significantly (pb0.01) decreased indoxorubicin-induced group (i.e. pathogenic control, group II) ascompared to the normal control healthy rats (i.e. group I).Rosuvastatin (0.5 mg/kg/day i.p. and 2 mg/kg/day i.p. i.e. group IIIand IV respectively) as well as carvedilol (1 mg/kg/day i.p., i.e. groupVI) treatment for 30 days, significantly (pb0.01) elevated the reducedHDL-C levels in serum as compared to the pathogenic control group(i.e. group II). There was a nonsignificant (pN0.05) change in theserum HDL-C level in rosuvastatin per se (2 mg/kg/day i.p.) treatedrats (i.e. group V) as compared to the vehicle control group (i.e. groupI) rats (Table 2 and Table 3).

3.4. Effect on Caspase-3 activity (units/mg/protein/h)

The mean Caspase-3 levels were significantly (pb0.01) increasedby 3.5 folds in doxorubicin-induced group (i.e. pathogenic control,group II) as compared to the caspase-3 levels in hearts of the normalcontrol healthy rats (i.e. group I). Rosuvastatin (0.5 mg/kg/day, i.p., i.e.group III) showed no significant (pN0.05) change in the caspase-3levels but rosuvastatin (2 mg/kg/day, i.p., i.e. group IV) as well ascarvedilol (1 mg/kg/day i.p., i.e. group VI) treatment for 30 daysshowed significant (pb0.05) decrease in the caspase-3 levels in thecardiac tissue as compared to the pathogenic control group (i.e. groupII) rats. There was no significant (pN0.05) change in the cardiaccaspase-3 levels in rosuvastatin per se (2 mg/kg/day i.p.) treated rats(i.e. group V) as compared to the vehicle control group (i.e. group I)rats (Table 4).

3.5. Effect on Na+-K+ ATPase activity (Pi/min/mg tissue)

The mean Na+-K+ ATPase levels were significantly (pb0.01)decreased in the hearts of doxorubicin-induced group (i.e. pathogeniccontrol, group II) as compared to the hearts of the normal controlhealthy rats (i.e. group I). Rosuvastatin (0.5 mg/kg/day, i.p., i.e. groupIII) showed slightly significant (pb0.05) while rosuvastatin (2 mg/kg/day, i.p., i.e. group IV) as well as carvedilol (1 mg/kg/day i.p., i.e. groupVI) treatment showed significant increase in the decreased Na+-K+

ATPase levels in the cardiac tissue as compared to the pathogenic

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control group (i.e. group II) hearts. There was no significant (pN0.05)change in the cardiac Na+-K+ ATPase levels in rosuvastatin per se(2 mg/kg/day i.p.) treated rats (i.e. group V) as compared to thevehicle control group (i.e. group I) rats (Table 4).

3.6. Effect on DNA gel electrophoresis

Electrophoresis of DNA extracted from the left ventricle region ofthe heart of doxorubicin treated rats (i.e. group II) showed DNAladdering with the lowest band below 200 bps, indicating apoptoticinter-nucleosomal DNA fragmentation. Ladders were not detected innormal control group (i.e. group I), rosuvastatin (2 mg/kg) treatedgroup (i.e. group IV), cardvedilol treated group (i.e. group VI) androsuvastatin per se treated group (i.e. group V), where genomic DNAband was preserved. Little DNA laddering was shown in rosuvaststin(0.5 mg/kg) treated group (i.e. group III) as compared to pathogeniccontrol group (i.e. group II) (Fig. 1).

3.7. Histopathological studies in cardiac tissue

Photomicrograph of vehicle control group (i.e. group I) revealedmyocardium fibres with normal architecture and regular morphologyof myocardial cell membrane (Fig. 2A). Photomicrograph of patho-genic control group (i.e. group II) showed cardiotoxic myocardialfibres with small and large cytoplasmic vacuoles (vacuolar myopathy)with no evidence of myocardial necrosis (Fig. 2B). Photomicrograph of

rosuvastatin (0.5 mg/kg/day) treated group (i.e. group III) showedmyocardium with few vacuolated fibres (moderate vacuolar myop-athy) and no evidence of necrosis, inflammation or fibrosis ascompared to pathogenic control group (i.e. group II) (Fig. 2C).

Photomicrograph of rosuvastatin (2 mg/kg/day) treated group (i.e.group IV) showed myocardium with no evidence of vacuolated fibresor vacuolar myopathy and no evidence of necrosis, inflammation orfibrosis as compared to pathogenic control group (i.e. group II)(Fig. 2D). Photomicrograph of rosuvastatin per se (2 mg/kg/day)treated group (i.e. group V) showed normal myocardium with novacuolated fibres or evidence of vacuolar myopathy and no evidenceof necrosis, inflammation or fibrosis (Fig. 2E). Photomicrograph ofcarvedilol (1 mg/kg/day) treated group (i.e. group VI) showedmyocardium with no vacuolated fibres or evidence of vacuolarmyopathy and no evidence of necrosis, inflammation or fibrosis ascompared to pathogenic control group (i.e. group II) (Fig. 2F).

4. Discussion

Present research work was planned to investigate the role ofrosuvastatin pre-treatment in doxorubicin-induced cardiomyopathyand apoptotic changes in murine model. Doxorubicin-induced cardio-toxicity is oneof themajor research tools toprovide cardiotoxicity in theexperimental animals [13,29]. Acute toxic effects after 24 h ofdoxorubicin 30 mg/kg i.p. single injectionwere observed in the presentstudy which was in correlation to that of Chakrabartia et al. [30].

Table 2Effect of rosuvastatin on serum LDH, TG, TC in doxorubicin-induced cardiotoxicity in rats.

Groups Serum LDH (IU/L) Serum TC (mg/dl) Serum TG (mg/dl)

I Vehicle Control [0.5%CMC in normal saline (2 ml/kg/day, single i.p. injection) for 30 days] 59.92±6.68 117.32±3.64 71.42±3.21II Pathological Control [0.5%CMC in normal saline (2 ml/kg/day, single i.p. injection) for30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

177.66±11.06⁎⁎ 189.87±4.86⁎⁎ 197.7±5.93⁎⁎

III Test Drug (Dose1) treated [rosuvastatin (0.5 mg/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

128.49±5.63## 131.82±2.07## 104.02±3.64##

IV Test Drug (Dose2) treated [rosuvastatin (2 mg/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

118.15±11.21## 135.19±2.87## 115.65±4.37##

V Rosuvastatin per se treated [rosuvastatin (2 mg/kg/day, single i.p. injection)for 30 days]

66.67±7.29ns1 110.09±2.82ns1 53.19±2.83ns1

VI Standard Drug treated [carvedilol (1 mg/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

85.99±6.06## 141.31±4.32## 102.63±2.14##

n=10 animals per group; Mean±SEM., ns1(nonsignificant), ⁎⁎pb0.01, as compared with Vehicle Control group (i.e. group I), one-way ANOVA followed by Dunnett's t-test##pb0.01, as compared with Pathological Control group (i.e. group II), one-way ANOVA followed by Dunnett's t-test.

Table 1Effect of rosuvastatin on hemodynamic changes using tail cuff method in doxorubicin-induced cardiotoxicity in rats.

Groups Systolic BP(mm Hg)

Diastolic BP(mm Hg)

Mean BP(mm Hg)

Heart rate(beats/min)

Flow(µL/cycle)

Volume(µL/cycle)

I Vehicle Control [0.5%CMC in normal saline(2 ml/kg/day, single i.p. injection) for 30 days]

127.83±6.86 96±4.65 106.16±5 399±8.39 12.25±1.65 31.5±4.05

II Pathological Control [0.5%CMC in normal saline(2 ml/kg/day, single i.p. injection) for 30 days+doxorubicin(30mg/kg, single i.p. injection) on 31st day]

192.83±7.65⁎⁎ 151.16±7.45⁎ 164.83±6.96⁎ 702.75±19.24⁎⁎ 42.75±1.87⁎ 93±5.22⁎⁎

III Test Drug (Dose1) treated [rosuvastatin(0.5 mg/kg/day, single i.p. injection) for 30 days+doxorubicin(30mg/kg, single i.p. injection) on 31st day]

167.33±6.70ns2 109 s±9.52ns2 128.16±8.4ns2 475.25±12.25## 22.25±1.7ns2 30.25±4.76##

IV Test Drug (Dose2) treated [rosuvastatin(2 mg/kg/day, single i.p. injection) for 30 days+doxorubicin(30mg/kg, single i.p. injection) on 31st day]

126.66±10.28## 70.66±4.78## 89±2.45## 547.75±18.13# 18.25±2.05ns2 61.5±5.04##

V Rosuvastatin per se treated [rosuvastatin(2 mg/kg/day, single i.p. injection) for 30 days]

120.16±6.30ns1 64.5±9.68ns1 82.66±10.37ns1 433±20.83ns1 10.5±1.84ns1 28±4.6ns1

VI Standard Drug treated [carvedilol(1 mg/kg/day, single i.p. injection) for 30 days+doxorubicin(30mg/kg, single i.p. injection) on 31st day]

137.66±2.50# 83.66±2.53## 101.33±8.22## 443.25±13.28## 16±2.12# 48.25±3.63##

n=10 animals per group; Mean±SEM., ns1(nonsignificant), ⁎pb0.05; ⁎⁎pb0.01, as compared with Vehicle Control group (i.e. group I), one-way ANOVA followed by Dunnett's t-testns2(nonsignificant), #pb0.05;##pb0.01, as compared with Pathological Control group (i.e. group II), one-way ANOVA followed by Dunnett's t-test.

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Most of the studies support the view that an increase in oxidativestress evidenced by an increase in free radicals and lipid peroxidationas well as a decrease in antioxidants and sulfahydryl groups play animportant role in the pathogenesis of doxorubicin-induced cardio-myopathy [31,32]. Our study demonstrated the presence of apoptoticmachinery in terms of caspase-3, to be involved in the cardiotoxicityof doxorubicin.

Doxorubicin, which was used to induce cardiotoxicity in rats,produced a significant increase (pb0.01) in the blood pressure;systolic, diastolic and mean, as well as heart rate, tail blood flow andtail flow volume in the pathogenic control rats (i.e. group II) ascompared to vehicle control group (i.e. group I), as recorded by thevolume pressure recording using the rat tail cuff method. The increasein heart rate in pathogenic rats as compared to normal healthy controlrats could be the result of increased oxygen consumption due toaccelerated myocardial apoptosis and necrosis following doxorubicintreatment and might be because of catecholamines release. Indifferent study, Yagmurca et al., [33] found an increase in systolicBP, diastolic BP and heart rate after doxorubicin administration.Findings of Steven et al., [34] shows that doxorubicin affects thedevelopment of dilated cardiomyopathy, afterload excess and latereduction in systolic and diastolic blood pressure. However, the latterstudied the affect of cumulative dose, while our study considered theeffect of acute toxicity of doxorubicin.

In the present study, marked elevation (3-folds increase) in theactivity of LDH in the serum of doxorubicin intoxicated rats (i.e. group II)wasobserved(pb0.01), as compared tovehicle control group (i.e. group I)(Table 2)which is consistentwith previous studieswhereGnanapragasanet al., [35] reported 1.5-folds increase in serum LDH after cumulative dose

of15 mg/kg. This increase in cardiacenzymecouldbedue toan increase intheir release during doxorubicin-induced damage of cardiac membrane.

Elevation in the apoptosis specific enzyme caspase-3 in thepathogenic control, confirms the postulation that doxorubicin-induced cardiomyopathy is mediated via apoptosis mediated by thepathway involving caspase-3 (Table 4). Our results corroborates wellwith the findings of Chularojmontri et al., [36] which reported 2.27-folds increase in caspase-3 level in doxorubicin-induced cardiotoxi-city. Doxorubicin damages cell membrane as evident from significantdecrease (1.6-folds) in levels of membrane bound enzyme i.e. NKA ascompared to normal control (pb0.01) (Table 4). Our results are inagreement with the findings of Shan et al., [2], who also reported a 1.5folds decrease in NKA levels in doxorubicin (10 mg/kg i.v. single dose)treated rats as compared to normal rats. The decrease of ATPase ondoxorubicin administration could be due to enhanced lipid peroxida-tion by free radicals [37].

Histopathological effects of doxorubicin show severe degenerationof themyofibrils, vacuolated cytoplasmwere clearly seen indoxorubicintreated group. Thus the result of the study has confirmed thatadministration of a single dose of doxorubicin (30 mg/kg, i.p.) producedsignificant cardiomyopathy in rats alongwithbloodpressure changes asa result of apoptosis (marked by increase cardiac tissue caspase-3 level,NKA activity) which was further supported by histopathologicalfindings and agarose gel electrophoresis.

Treatment with the test drug, rosuvastatin (2 mg/kg/day, single i.p.injection, for 30 days) (i.e. group IV) and carvedilol (1 mg/kg/day, singlei.p. injection for 30 days) (i.e. group VI) significantly (pb0.01) reducedthe increase in hemodynamic parameters, namely, systolic BP, diastolicBP, mean BP, heart rate and blood volume as compared to the

Table 4Effect of rosuvastatin on Caspase-3 activity and Na+-K+ ATPase activity in heart tissue in doxorubicin-induced cardiotoxicity in rats.

Groups Caspase-3 (units/mg/protein/h)in cardiac tissue

Na+-K+ ATPase (Pi/min/mg tissue)in cardiac tissue

I Vehicle Control [0.5%CMC in normal saline (2 ml/kg/day, single i.p. injection) for 30 days] 62.34±9.28 0.297±0.012II Pathological Control [0.5%CMC in normal saline (2 ml/kg/day, single i.p. injection) for 30 days+

doxorubicin (30 mg/kg, single i.p. injection) on 31st day]218.98±9.56⁎⁎ 0.177±0.007⁎⁎

III Test Drug (Dose1) treated [rosuvastatin (0.5 mg/kg/day, single i.p. injection) for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

184.96±14.27ns2 0.227±0.016#

IV Test Drug (Dose2) treated [rosuvastatin (2 mg/kg/day, single i.p. injection) for 30 days+doxorubicin(30 mg/kg, single i.p. injection) on 31st day]

103.35±13.77# 0.241±0.008##

V Rosuvastatin per se treated [rosuvastatin (2 mg/kg/day, single i.p. injection) for 30 days] 63.44±8.75ns1 0.291±0.011ns1

VI Standard Drug treated [carvedilol (1 mg/kg/day, single i.p. injection) for 30 days+doxorubicin(30 mg/kg, single i.p. injection) on 31st day]

109.52±4.45# 0.276±0.008##

n=10 animals per group; Mean±SEM., ns1(nonsignificant),⁎⁎pb0.01, as compared with Vehicle Control group (i.e. group I), one-way ANOVA followed by Dunnett's t-test ns2

(nonsignificant), #pb0.05;##pb0.01, as compared with Pathological Control group (i.e. group II), one-way ANOVA followed by Dunnett's t-test.

Table 3Effect of rosuvastatin on serum HDL-C, LDL-C, VLDL-C and atherogenic index in doxorubicin-induced cardiotoxicity in rats.

Groups Serum HDL-C(mg/dl)

Serum LDL-C(mg/dl)

Serum VLDL-C(mg/dl)

Atherogenic index

TC/HDL-C LDL-C/HDL-C

I Vehicle Control [0.5%CMC in normal saline (2 ml/kg/day, single i.p. injection) for 30 days] 43.15±2.73 59.94±1 14.28±0.64 2.78±0.16 1.49±0.13II Pathological Control [0.5%CMC in normal saline (2 ml/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

17.77±1.53⁎⁎ 132.55±5.71⁎⁎ 39.53±1.18⁎⁎ 10.66±0.77⁎⁎ 8.42±0.97⁎⁎

III Test Drug (Dose1) treated [rosuvastatin (0.5 mg/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

34.74±1.68## 76.25±3.08## 20.79±0.73## 3.85±0.19## 2.25±0.19##

IV Test Drug (Dose2) treated [rosuvastatin (2 mg/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

32.99±1.65## 79.04±3.46## 23.12±0.87## 4.17±0.23## 2.43±0.20##

V Rosuvastatin per se treated [rosuvastatin (2 mg/kg/day, single i.p. injection)for 30 days]

51.30±2.31ns1 48.15±2.27ns1 10.63±0.56ns1 2.16±0.07ns1 0.92±0.05ns1

VI Standard Drug treated [carvedilol (1 mg/kg/day, single i.p. injection)for 30 days+doxorubicin (30 mg/kg, single i.p. injection) on 31st day]

42.16±1.63## 78.58±4.68## 20.52±0.42## 3.69±0.13## 2.47±0.28##

n=10 animals per group; Mean±SEM., ns1(nonsignificant), ⁎⁎pb0.01, as compared with Vehicle Control group (i.e. group I), one-way ANOVA followed by Dunnett's t-test##pb0.01, as compared with Pathological Control group (i.e. group II), one-way ANOVA followed by Dunnett's t-test.

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pathogenic control group (i.e. group II) after administering doxorubicin(30 mg/kg, single i.p. injection) on 31st day.

Rosuvastatin (0.5 mg/kg/day, i.p. and 2 mg/kg/day, i.p., i.e. groupIII and IV respectively) as well as carvedilol (1 mg/kg/day i.p., i.e.group VI) treatment for 30 days significantly (pb0.01) reduced theincrease in the serum LDH, serum TC, serum TG, serum LDL-C, serumVLDL-C & atherogenic index as compared to the pathogenic controlgroup (i.e. group II). Further, the test drug rosuvastatin the higherdose (2 mg/kg/day for 30 days) and standard drug, carvedilol (1 mg/kg/day for 30 days) (i.e. group IV, VI) showed significant (pb0.01)reduction in the elevated levels of pro-apoptotic enzyme, caspase-3 inthe heart tissue as compared to the pathogenic control group (i.e.group II). The smaller dose (0.5 mg/kg/day for 30 days) also reducedthe elevated caspase-3 levels but the decrease was not significant.

Our findings are in conformity to the studies carried out byKotamraju et al. [38]. According to this study, doxorubicin-inducedtoxicity, which is mediated via increase in the toxic iron content, leadsto cardiomyocyte apoptosis via free radical dependent activation ofCaspase-3.

According to a latest study, Ipseeta et al., [39] the cell death inischemia-reperfusion is mediated via both apoptotic and necroticpathways. At first due to the ischemic insult, the myocardial cellsundergo apoptosis. Since apoptosis is an energy requiring process, theATP remains of the cells are utilized for the same. Later due toreperfusion, the apoptotic cells get access tomore ATP and the processof apoptosis continues. As the cell's ATP stores deplete, the mode ofcell death also shifts from apoptotic to necrotic pathway. This type ofbifocal cell death mechanism is known as ‘hybrid cell death’.

Doxorubicin-induced pathogenic rats when treated with test drugrosuvastatin (0.5 mg/kg/day and 2 mg/kg/day) and standard drug,carvedilol (1 mg/kg/day for 30 days) (i.e. group III, IV and VI)significantly (pb0.01) elevated the decreased NKA levels in theheart as compared to the pathogenic control group (i.e. group II).

Internucleosomal DNA fragmentation in doxorubicin-treatedrats (i.e. group II) was confirmed by the consistent observation ofcharacteristic DNA ladders on agarose gels by AGE. Treatmentwith carvedilol (1 mg/kg) (i.e. group VI) and rosuvastatin (2 mg/kg)(i.e. group IV) significantly decreased the appearance of DNAladdering in the left ventricular region of rat heart, howeverrosuvaststin (0.5 mg/kg) (i.e. group III) showed mild DNA laddering.Our findings are in agreement with the previous report of Nakamuraet al., [40] where doxorubicin (2 mg/kg/i.v.) administration for 8 weeksshowed a nucleosomal breakdown of genomic DNA, indicating DNAfragmentation.

Histopathological evidences suggest that rosuvastatin (2 mg/kg/day for 30 days) pre-treatment significantly protects the myocardiumfrom doxorubicin-induced vacuolar myopathy as compared topathogenic control group (i.e. group II) while rosuvastatin (0.5 mg/kg/day for 30 days) partially protects the myocardium from doxoru-bicin-induced toxicity, as evident by few vacuolated fibres (moderatevacuolar myopathy). Further, standard carvedilol (1 mg/kg/day for30 days) protected the myocardium against doxorubicin-inducedcardiotoxicity as shown by myocardium with no vacuolated fibres orevidence of vacuolar myopathy as compared to pathogenic controlgroup (i.e. group II).

In conclusion, our results suggest that rosuvastatin pretreatmentfor 30 days significantly decreases the cardiac tissue caspase-3activity, DNA fragmentation as well as decreasing serum LDH andlipid levels, along with reversal of hemodynamic changes induced bydoxorubicin. Rosuvastatin treatment was also shown to recover theNKA activity lost due to doxorubicin-induced cardiotoxicity. Thepresent study reports for the first time the anti-apoptotic potential ofrosuvastatin pre-treatment in doxorubicin-induced cardiotoxic rats asis evident from the significant decrease in myocardial caspase-3 levelsand DNA damage along with an increase in myocardial Na+-K+-ATPase levels.

Statins may be very promising pharmacological molecules forreducing the cardiovascular risks in both primary and secondaryprevention of cardiovascular disease. Statinsmay offer beneficial effectsin patients with HF. In the present study restoring hemodynamicparameters, inhibition of apoptosis and improving lipid profile alteredby doxorubicin-induced cardiomyopathy are the mechanisms involvedfor the cardioprotective effects of rosuvastatin. However, evidence ofpleiotropic activities for statins is emerging. Statins have also beenshown to possess a host of other non cholesterol-lowering propertiesincluding ability to decrease LV mass, decrease LV fibrosis, decreaseinflammation, decrease immune activation, alter metalloproteinaseactivity, decreaseoxidative stress, increase arterial compliance, decreasethrombosis, all of which can contribute to the improvement of LVfunction and prevention or attenuation of progressive LV remodeling inheart failure. There appear to bemultiple plausible biologicmechanismsfor the observed benefits,many ofwhich are independent of cholesterollowering. These benefits are distinct from other clinically usedcardioprotective drugs, suggesting that statin therapy may have aunique and additive role. If true, this conclusion has far-reachingimplications, suggesting that statins may be therapeutically useful forthe treatment of HF, even in patients for whom themedicationmay nototherwise be indicated.

Acknowledgements

The authors of this manuscript have certified that they complywith the Principles of Ethical Publishing in the International Journal ofCardiology [41].

Fig. 1. Effect of rosuvastatin on DNA fragmentation detected by agarose gelelectrophoresis. Lane M=100 bps ladder, L1=vehicle control group, L2=pathologicalcontrol group, L3=carvedilol (1 mg/kg, standard drug) treated group, L4=rosuvas-tatin (0.5 mg/kg) treated group, L5=rosuvastatin (2 mg/kg) treated, L6=rosuvastatin(2 mg/kg) per se treated group, L7=vehicle control group.

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Fig. 2. Photomicrographs of heart tissue (H&E 40×). A) Photomicrograph of vehicle control group showing myocardiumwith normal architecture (H&E 40×). B) Photomicrograph ofpathogenic control group showing cardiotoxic myocardium (H&E 40×). C) Photomicrograph of rosuvastatin [0.5 mg/kg single i.p.+DOX] showing myocardium with no evidence ofnecrosis, inflammation or fibrosis (H&E 40×). D) Photomicrograph of rosuvastatin [2 mg/kg single i.p.+DOX] showing myocardium with no evidence of necrosis, inflammation orfibrosis (H&E 40×). E) Photomicrograph of rosuvastatin per se [2 mg/kg, single i.p.] showing normal myocardium with no evidence of necrosis, inflammation or fibrosis (H&E 40×).F) Photomicrograph of carvedilol [1 mg/kg, single i.p.+DOX] showing myocardium with no evidence of necrosis, inflammation or fibrosis (H&E 40×).

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