cardioprotective effect of melatonin against isoproterenol induced myocardial infarction in rats: a...

European Journal of Pharmacology 644 (2010) 160–168

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Cardiovascular Pharmacology

Cardioprotective effect of melatonin against isoproterenol induced myocardialinfarction in rats: A biochemical, electrocardiographic andhistoarchitectural evaluation

Vaibhav Patel ⁎, Aman Upaganlawar, Rishit Zalawadia, R. BalaramanPharmacy Department, Faculty of Technology and Engineering, The M.S. University of Baroda, Vadodara-390001, India

⁎ Corresponding author. Pharmacy Department, Facneering, The M.S. University of Baroda, Kalabhavan, VaTel.: +91 265 2434187; fax: +91 265 2418927.

E-mail address: [email protected] (V. Patel).

0014-2999/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ejphar.2010.06.065

a b s t r a c t
a r t i c l e i n f o
Article history:Received 13 March 2010Received in revised form 10 June 2010Accepted 24 June 2010Available online 13 July 2010

Keywords:MelatoninIsoproterenolMyocardial infarctionAntioxidantElectrocardiography

The present study was designed to investigate the cardioprotective effect of melatonin against isoproterenolinduced myocardial infarction in rats by studying myocyte injury markers, antioxidant defense system,serum and heart lipid profile, inflammatory markers, electrocardiographic and histopathological changes.Male Sprague Dawley (SD) rats were randomly divided into four groups, namely control, melatonin,isoproterenol and melatonin+isoproterenol treated group. Melatonin treatment group received melatonin(10 mg/kg/day, i.p.) for 7 days. Myocardial infarction in rats was induced by isoproterenol administration(150 mg/kg, s.c.) at an interval of 24 h on 6th and 7th day. On 8th day ECG, gravimetric, biochemical andhistopathological parameters were assessed. Isoproterenol administration showed changes in ECG pattern,including ST-segment elevation (diagnostic of myocardial infarction) increase in the serum levels of cardiacinjury markers (creatine kinase-MB, lactate dehydrogenase, aspartate transaminase and alanine transami-nase), decreased antioxidant defense system in the heart and altered lipid profile in the serum and heart.Isoproterenol administration also resulted in release of inflammatory markers and neutrophil infiltrationalong with histopathological changes. Melatonin pre-co-treatment prevented almost all the parameters ofisoproterenol induced myocardial infarction in rats. The above finding was confirmed by the histopatho-logical examination. In the baseline group (melatonin alone treated group) no significant change wasobserved. Results of the present study suggest that melatonin has a significant effect on the protection of theheart against isoproterenol induced myocardial infarction through maintaining endogenous antioxidantenzyme activities.

ulty of Technology and Engi-dodara-390001, Gujarat, India.

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Myocardial infarction is the common presentation of the ischemicheart disease. It occurs when myocardial ischemia surpasses the criticalthreshold level for an extended time resulting in irreversible myocardialcell damage. Although clinical care is improved, public awareness israised and health innovations are widely used, myocardial infarction stillremains the leading cause of death worldwide (Aronow, 2006).According to the World Health Organization it will be the major causeof death in theworldby theyear2020 (Lopez andMurrau, 1998). In India,the number of patients being hospitalized for myocardial infarction,commonly knownasheart attack, is increasing over thepast 35 years andmale patients have shown a more striking increase (Krishnaswami,1998).

Isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanolhydrochloride] is a synthetic catecholamine and β-adrenergic agonist,

whichhas been documented to produce severe stress in themyocardiumresulting in the myocardial infarction, if administered in supramaximaldoses (Rona, 1985; Rona et al., 1959). It produces myocardial necrosiswhich caused cardiac dysfunction, increased lipid peroxidation alongwith an increase in the level of myocardial lipids, altered activities of thecardiac enzymes and antioxidants (Karthikeyan et al., 2007; Rajaduraiand Prince, 2006). Thepathophysiological andmorphological aberrationsproduced in the heart of this myocardial necrotic rat model arecomparable with those taking place in human myocardial infarction(Rona, 1985). Among the various mechanisms proposed to explain theisoproterenol induced cardiotoxicity, generation of highly cytotoxic freeradicals throughauto-oxidationof catecholamineshasbeen implicatedasone of the important causative factors.

Free radicals and reactive oxygen species have been implicated inlarge number of diseases and have a deleterious effect on heartfunctioning. Various experimental and clinical studies have shown thatenormous amount of reactive oxygen species such as, superoxide,hydrogen peroxide and hydrogen radicals are generated in failingmyocardium (Rajadurai and Prince, 2006). Therefore, therapeuticinterventions having antioxidants or free radical scavenging activity

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161V. Patel et al. / European Journal of Pharmacology 644 (2010) 160–168

may be useful against oxidative stress associated with various cardio-vascular diseases including myocardial infarction.

Melatonin, the chief indoleamine produced by the pineal gland, isknown to influence a variety of biological processes including circadianrhythms, neuroendocrine, cardiovascular and immune functions as wellas thermoregulation. Recently, there was an increased interest inmelatonin as a good antioxidant and potent scavenger of toxic freeradicals, such as peroxynitrites (ONOO•), hydroxyl (•OH) and peroxylradicals (ROO•). The scavenging ability of melatonin is more effectivethan that of the well known intracellular antioxidant, reduced glutathi-one (GSH) (Tan et al., 1993).

Therefore the present study was designed to study the effect ofmelatonin pretreatment on the myocardial infarction induced bysupramaximal doses of isoproterenol. The present study also attemptedto demonstrate the possible mechanism of its therapeutic efficacy bystudying the biochemical markers, antioxidant defense system, lipidprofile, electrocardiographic and histopathological changes.

2. Materials and methods

2.1. Chemicals

Isoproterenol hydrochloride was purchased from Sigma chemicalcompany, St. Louis, MO, USA. Melatonin was purchased from Hi-mediaChemical Company, Vadodara, India. All other biochemical reagents andchemicals were of analytical grade.

2.2. Animals

All experiments and protocols described in present study wereapproved by the Institutional Animal Ethics Committee (IAEC) ofPharmacy Department, The M.S. University of Baroda and withpermission from Committee for the Purpose of Control and Supervisionof Experiments on Animals (CPCSEA), Ministry of Social Justice andEmpowerment,Governmentof India. Tenweekoldmale SpragueDawleyrats (200±25 g) were housed in-group of 3 animals and maintainedunder standardized conditions (12-h light/dark cycle, 24 ± 2 °C, 35 to60% humidity) and provided free access to palleted CHAKKAN diet (NavMaharashtra Oil Mills Pvt. Ltd., Pune) and purified drinking water adlibitum.

2.3. Induction of experimental myocardial infarction

Isoproterenol was dissolved in normal saline and injected to rats(150 mg/kg, s.c.) at an interval of 24 h for 2 days to induce experimentalmyocardial infarction (Ribeiro et al., 2009). Animals were sacrificed 48 hafter the first dose of isoproterenol.

2.4. Experimental design

After acclimatization, the animals were randomly divided into thefollowing groups consisting of 6 rats each:

Group 1 (control): Animals received standard laboratory diet anddrinking water ad libitum and served as a controlgroup.

Group 2 (melatonin): Animals receivedmelatonin (10 mg/kg/day, i.p.)treatment for 7 days (Acikel et al., 2003). Animalswere not injected with isoproterenol.

Group 3 (isoproterenol): Animals were injected with isoproterenol
(150 mg/kg/day, s.c.) at an interval of 24 h for2 days and served as isoproterenol group.
Group 4 (melatonin+isoproterenol): Animals received melatonin
treatment (10 mg/kg/day, i.p.) for 7 days andwere injected with isoproterenol (150 mg/kg/day, s.c.) on 6th and 7th day.
2.5. Electrocardiography

At the end of the experimental period, needle electrodes wereinserted under the skin of the animals under light ether anesthesia inlead II position. ECG recordings were made using computerized MP30data acquisition system (BIOPAC, Santa Barbara, California) andchanges in ECG pattern were considered.

2.6. Biochemical analysis

After recording the ECG, the animals were sacrificed and bloodsamples were collected. Serum was separated from each sample andused for the biochemical analysis. Immediately after sacrifice, hearttissues were excised in ice cold condition. They were blotted free ofblood and tissue fluids. Then they were weighed and stored at−80 °Ctill further use for the analysis (CryoScientific, India). Heart weight tobodyweight ratio was calculated by dividing heart weight (g) by bodyweight (g) and multiplying it with 100 (Ramesh et al., 1998).

2.6.1. Biochemical parameters in serumThe collected serumwas used for the estimation of cardiac marker

enzymes creatinine kinase-MB (CK-MB), lactate dehydrogenase(LDH), alkaline phosphatase (ALP), aspartate transaminase (AST)and alanine transaminase (ALT) using commercially available stan-dard enzymatic kits (Span Diagnostics Pvt. Ltd., India). Serumwas alsoestimated for the lipid profile including total cholesterol, HDL-cholesterol, triglycerides using commercially available standardenzymatic kits (Span Diagnostics Pvt. Ltd., India) and phospholipidsby the method described by Fiske and Subbarow (1925). Serum LDL-cholesterol was calculated by the Friedewald formula (Friedewaldet al., 1972).

LDL = Total cholesterol– HDL � cholesterol + Triglycerides= 5ð Þf g:

2.6.1.1. TNF-α quantitation by ELISA. Serum TNF-α levels weredetermined using an enzyme-linked immunosorbent assay (ELISA)(Rat TNF-α kit, Endogen, USA) according to the manufacturer'sinstructions.

2.6.2. Biochemical parameters in heart tissue

2.6.2.1. Lipid peroxidation (LPO), reduced glutathione and antioxidantenzymes. The excised heart tissue was homogenized in chilled Tris–HCl buffer (0.1 M) pH 7.4. The homogenate was then centrifuged at10,000×g at 0 °C using the Remi C-24 high speed cooling centrifuge.The clear supernatant obtained was used for the assay of lipidperoxidation (MDA content), endogenous antiperoxidative enzymes(superoxide dismutase (SOD), catalase) GSH, glutathione-S-transfer-ase (GST) and glutathione peroxidase (GPx). SOD was determined bythe method of Mishra and Fridovich (1972). Catalase was estimatedby the method given by Aebi (1984). GSH was determined by themethod of Moron and Depierre (1979). Lipid peroxidation ormalondialdehyde (MDA) formation was estimated by the method ofSlater and Sawyer (1971). GST and GPx were estimated by themethods described by Habig and Jakoby (1981) and Rotruck et al.(1979), respectively.

2.6.2.2. Membrane-bound ATPases. The sediment after centrifugationwas resuspended in ice cold Tris–HCl buffer (0.1 M)pH7.4. Thiswas usedfor the estimations of membrane-bound enzymes and protein content.The membrane-bound enzymes such as Na+/K+-ATPase, Ca++-ATPase,and Mg++-ATPase activity were assayed by estimating the amount ofphosphorus liberated from the incubation mixture containing tissuehomogenate, ATP, and the respective chloride salt of the electrolytes(Bonting, 1970;Hjerten andPan, 1983;Ohnishi et al., 1982). Total protein

Table 1Effect of melatonin pretreatment on heart weight, body weight and heart weight/bodyweight ratio in isoproterenol induced myocardial infarction in rats.

Control Melatonin Isoproterenol Melatonin+isoproterenol

Heart weight (g) 0.54±0.01 0.54±0.02 0.85±0.03c 0.69±0.03f

Body weight (g) 237.5±5.79 234.0±4.94 223.7±6.12ns 226.3±2.28ns

Heart weight/body weightratio

0.226±0.002 0.231±0.003 0.379±0.005c 0.303±0.012f

All values are presented as mean±S.E.M. for six rats in each group.aPb0.05, bPb0.01, cPb0.001 as compared to control group.dPb0.05, ePb0.01, fPb0.001 as compared to isoproterenol group.ns — non significant.

162 V. Patel et al. / European Journal of Pharmacology 644 (2010) 160–168

content was estimated by themethod described previously (Lowry et al.,1951).

2.6.2.3. Tissue nitrite content. Nitrite was estimated colorimetricallywith the Griess reagent (Guevara et al., 1998) in protein freesupernatant of heart homogenate. Briefly equal volumes of proteinfree supernatant of heart homogenate and Griess reagent (sulfanil-amide 1% w/v, naphthylethylenediamine dihydrochloride 0.1% w/v,and orthophosphoric acid 2.5% v/v) were mixed and incubated atroom temperature for 10 min and the absorbance was determined at540 nm wavelength and compared to those of known concentrationsof sodium nitrite.

2.6.2.4. Myeloperoxidase assay. To quantify myocardial neutrophilinfiltration the cardiac activity of myeloperoxidase (MPO), anabundant enzyme of neutrophils, was assessed using a modifiedmethod (Mullane et al., 1985). The myocardial tissue was homoge-nized in 50 mM K2HPO4 buffer (pH 6) containing 0.5% hexadecyl-trimethylammonium bromide using a Polytron tissue homogenizer.After freeze-thawing three times, the samples were centrifuged at11,000×g for 30 min at 40 °C, and the resulting supernatant wasassayed spectrophotometrically for myeloperoxidase determination.In brief, 40 μL of sample was mixed with 960 μL of 50 mM phosphatebuffer (pH 6), containing 0.167 mg/mL O-dianisidine dihydrochlorideand 0.0005% H2O2. The change in absorbance at 460 nm wasmeasured with the spectrophotometer. One unit of enzyme activitywas defined as the amount of myeloperoxidase present that caused achange in absorbance measured at 460 nm for 3 min. Myeloperox-idase activity data are presented as U/gm tissue.

2.6.2.5. Extraction and estimation of heart tissue lipids. Extraction oftotal lipids from heart tissue was performed by the method of Folch etal. (1957). The total lipid extract was estimated for total cholesterol,triglycerides and phospholipids by the methods described earlier(Section 2.6.1).

2.7. Histopathology

After sacrifice, the heart was rapidly dissected out and washedimmediately with saline and fixed in 10% buffered formalin. The fixedtissues were embedded in paraffin and serial sections (5 μm thick)were cut. Each section was stained with hematoxylin and eosin(H&E). The sections were examined under the light microscope(Olympus BX10, Tokyo, Japan) for histopathological changes andphotomicrographs (Olympus DP12 camera, Japan) were taken. Thepathologist performing histopathological evaluation was blinded tothe treatment assignment of different study groups.

2.8. Statistical analysis

All the values are expressed as mean±S.E.M. Statistical signifi-cance between more than two groups was tested using one-wayANOVA followed by the Bonferroni multiple comparisons test orunpaired two-tailed Student's t-test as appropriate using computerbased fitting program (Prism, Graphpad). Differences were consid-ered to be statistically significant when Pb0.05.

3. Results

The effects of isoproterenol andmelatonin treatment on heartweight,body weight and heart weight to body weight ratio are depicted inTable 1. There was no significant difference in the body weight betweenthe groups observed, although isoproterenol treated animals showed aslight reduction in body weight which was not significant. The heartweight and the heart weight to body weight ratio were increasedsignificantly (Pb0.001) in isoproterenol-administered rats when com-

pared with normal control rats. In rats pretreated with melatonin andthen treated with isoproterenol, there was a significant (Pb0.001)reduction in the heart weight and the ratio as compared to isoproterenoltreated rats. No significant difference was observed in rats treated withmelatonin alone when compared to normal control rats.

Electrocardiographic pattern of normal and experimental animalsis shown in Fig. 1 and Table 2. Normal control and melatonin alonetreated rats showed normal pattern of ECG, whereas rats treated withisoproterenol showed a significant (Pb0.01) increase in ST-segmentand significant (Pb0.01) decrease in R-amplitude as compared tocontrol rats, indicative of infarctedmyocardium (Fig. 1). Isoproterenoltreated rats also exhibited the pathological Q wave, indicating theinduction of transmural myocardial infarction. Moreover, a significant(Pb0.01 for P wave and R–R interval and Pb0.001 for QRS complex)decrease in P wave, QRS complex, R–R interval and a significant(Pb0.001) increase in heart rate was observed in rats injected withisoproterenol (Table 2) as compared to control rats. Melatoninpretreatment in isoproterenol treated rats showed significant(Pb0.05) decrease in ST-segment and increase in R-amplitude ascompared to isoproterenol alone treated rats (Fig. 1). Melatoninpretreatment also resulted in a significant increase (Pb0.01 for QRScomplex and Pb0.05 for R–R interval) in QRS complex and R–Rinterval along with a significantly (Pb0.05) decreased heart ratewhen compared to isoproterenol intoxicated rats (Table 2).

Table 3 represents the effects of isoproterenol and melatonintreatment on cardiac marker enzymes including CK-MB, LDH, AST,ALT and ALP. The activities of these enzymes were increasedsignificantly (Pb0.001) in isoproterenol treated rats as compared tonormal control group rats. Melatonin pretreatment in isoproterenoltreated animals significantly (Pb0.001 for CK-MB and LDH, Pb0.01 forAST and ALT) decreased the CK-MB, LDH, AST and ALT activities.Melatonin pretreatment, however, did not show any significant effecton the activity of ALP enzyme. No significant difference was observedin rats treated with melatonin alone when compared to normalcontrol rats.

The levels of LPO and GSH along with the activities of theantioxidant enzymes GST, GPx, SOD and catalase in normal andexperimental rats are listed in Table 4. Isoproterenol treated ratsshowed significantly (Pb0.001) elevated levels of MDA, end productof lipid peroxidation andmarker for oxidative stress, in heart tissue ascompared to control rats. Isoproterenol treatment also resulted in thesignificant reduction (Pb0.001) in the levels of GSH, an endogenousantioxidant in comparison to normal control rats. Activities ofglutathione-dependent antioxidant enzymes (GST and GPx) andantiperoxidative enzymes (catalase and SOD) were significantly(Pb0.001) lowered in the heart tissue of isoproterenol injected ratsas compared to normal control rats. Pre-co-treatment with melatoninin isoproterenol intoxicated rats significantly (Pb0.001) reduced thelevels of LPO as compared to isoproterenol alone treated rats. Theprior administration of melatonin for 7 days along with isoproterenoladministration on 6th and 7th day resulted in a significant (Pb0.05 for

Fig. 1. Effect of melatonin pretreatment on electrocardiographic pattern {(A) control, (B) melatonin, (C) isoproterenol and (D) melatonin+isoproterenol}, ST-segment (E) and R-amplitude (F) in isoproterenol induced myocardial infarction in rats [**Pb0.01 as compared to control group, #Pb0.05 as compared to isoproterenol group].


SOD, Pb0.01 for catalase, GSH and GST, Pb0.001 for GPx) increase inlevels of GSH and activities of GST, GPx, SOD and catalase. The normalrats receiving melatonin alone did not show any significant changewhen compared with normal rats, indicating that it does not per sehave any adverse effects.

The activities of the membrane-bound phosphatases in the heart ofnormal and experimental animals are shown in Table 5. Membrane-bound phosphatases such as Na+–K+-ATPase and Mg++-ATPase weresignificantly (Pb0.001 for Na+–K+-ATPase and Pb0.01 for Mg++-ATPase)decreased in isoproterenol alonegroupwhencomparedwith thenormal group. However, isoproterenol alone treated group showed asignificantly (Pb0.001) elevated activity of Ca++-ATPase as comparedwith the normal group. Melatonin pretreatment in isoproterenolintoxicated rats showed the significant (Pb0.001 for Na+–K+-ATPaseand Pb0.05 for Mg++-ATPase) increase in activities of Na+–K+-ATPaseand Mg++-ATPase and the activity of Ca++-ATPase was significantly(Pb0.001) decreased in comparison to isoproterenol alone treated rats.Treatment with melatonin in normal rats produced no marked changes.

Isoproterenol administration showed a significant (Pb0.001)increase in inflammatory markers such as serum TNF-α level

Table 2Effect of melatonin pretreatment on electrocardiographic parameters in isoproterenol indu

Control Melatonin

P wave (s) 0.027±0.0019 0.025±0.0010QRS complex (s) 0.040±0.0010 0.040±0.0015QT interval (s) 0.074±0.0028 0.075±0.0034R–R interval (s) 0.162±0.0043 0.167±0.0071Heart Rate (bpm) 372.1±9.87 362.1±15.04

See demonstration of Table 1.

(Fig. 2) and myocardial MPO activity (Fig. 3) when compared tonormal control rats. Isoproterenol intoxicated rats also showedsignificantly (Pb0.01) decreased levels of nitrite, a metabolite ofnitric oxide as compared to normal control rats (Fig. 4). Melatoninpretreatment in isoproterenol intoxicated rats showed a significantly(Pb0.001) decreased level of TNF-α and myocardial MPO activity ascompared to isoproterenol alone treated rats. However, myocardialnitrite content was found to be increasing significantly (Pb0.05) inmelatonin pretreated isoproterenol injected rats as compared toisoproterenol alone treated rats.

Table 6 lists the levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides and phospholipids in serum of normal andexperimental animals. Rats treated with isoproterenol alone showed asignificant (Pb0.001) increase in these levels with the levels of HDL-cholesterol being exception where there was a significant (Pb0.001)decrease. The prior administration of melatonin for 7 days along withisoproterenol administration on 6th and 7th day significantly (Pb0.05)decreased the levels of lipoproteins, triglycerides and phospholipidswitha subsequent nonsignificant increase in the levels of HDL-cholesterol ascompared to isoproterenol alone treated rats. No significant change in

ced myocardial infarction in rats.

Isoproterenol Melatonin+isoproterenol

0.019±0.0013b 0.023±0.0015ns

0.029±0.0012c 0.036±0.0015e

0.084±0.0039ns 0.081±0.0026ns

0.131±0.0037b 0.154±0.0055d

460.5±13.50c 393.3±13.55d

Table 3Effect of melatonin pretreatment on cardiac marker enzymes in isoproterenol inducedmyocardial infarction in rats.


CK-MB (IU/L) 86.15±3.23 87.65±1.88 227.4±14.96c 162.7±11.66f

LDH (IU/L) 186.2±24.0 187.4±5.10 556.5±27.54c 316.7±28.92f

AST (U/L) 47.54±3.36 47.79±1.74 106.7±7.93c 73.68±6.73e

ALT (U/L) 35.03±2.10 36.68±1.43 86.15±5.73c 57.80±6.81e

ALP (IU/L) 125.5±8.42 131.0±2.02 284.6±24.03c 245.8±11.99ns



total cholesterol was observed when compared to isoproterenol alonetreated group. In the baseline group (melatonin alone treated), therewasno significant change observed in the serum lipoproteins, triglyceridesand phospholipid levels in comparison to those of control rats.

The effects of melatonin treatment on total cholesterol, triglycer-ides and phospholipids in the heart of normal and isoproterenoltreated animals are listed in Table 7. Isoproterenol treated ratsshowed a significant (Pb0.001) increase in myocardial lipid levelsexcept phospholipids as compared to control rats. There was asignificant (Pb0.001) decrease in the myocardial phospholipid levelsin isoproterenol intoxicated rats as compared to control rats.Melatonin pretreatment in isoproterenol intoxicated rats showed asignificant (Pb0.01) reduction in myocardial total cholesterol levelsand also resulted in a significant (Pb0.05) increase in the myocardialphospholipid levels in comparison to isoproterenol alone treated rats.Melatonin pretreatment did not show significant effect on myocardialtriglyceride levels as compared to isoproterenol alone treated rats.There was no significant difference in myocardial lipid levels inmelatonin alone treated rats as compared to control rats.

Fig. 5 illustrates the histopathological photographs of heart tissuesof normal and experimental rats. Histopathological examination ofmyocardial tissue obtained from normal control animals exhibitedclear integrity of myocardial membrane. Normal untreated ratsshowed normal cardiac fibers without any infarction and infiltrationof inflammatory cells was not seen in this group. Histopathologicalfindings confirmed the induction of myocardial infarction byisoproterenol. Heart tissues from isoproterenol treated rats showedwidespread myocardial structure disorder and subendocardial necro-sis with capillary dilatation and leukocyte infiltration (Fig. 5) ascompared to control group. Pretreatment with melatonin depicteddecreased degree infiltration of inflammatory cells and the morphol-ogy of cardiac muscle fibers was relatively well preserved with noevidence of focal necrosis. Baseline group (melatonin alone treated)rats showed no change in histo-architecture of heart tissue ascompared to normal control rats.

4. Discussion

Catecholamines are important regulators of myocardial contractilityandmetabolism. However, it has been known for a long time that excesscatecholamines are responsible for cellular damage, observed in clinicalconditions like angina, transient myocardial hypoxia, acute coronary

Table 4Effect of melatonin pretreatment on lipid peroxidation, endogenous antioxidant and antiox

Control

SOD (U/mg protein) 11.50±0.66CAT (μmol of H2O2 consumed/mg protein) 14.59±0.60GSH (μg/mg protein) 9.30±0.55LPO (nmol of MDA/mg protein) 1.36±0.12GST (μmol of CDNB conjugated/mg protein) 773.5±14.99GPx (μmol ofglutathione oxidized/mg protein)

1.87±0.07


insufficiency and subendocardial infarct. Animals develop infarct likelesions when injected with isoproterenol, a potent synthetic catechol-amine. These lesions are morphologically similar to those of ‘coagulativemyocytolysis’ or myofibrillar degeneration, one of the finding describedin acute myocardial infarction and sudden death in man (Baroldi, 1974).

Several mechanisms of isoproterenol induced myocardial infarc-tion have been reported. Isoproterenol acts both on β1 and β2

adrenoceptors, activation of which leads to positive inotropic andchronotropic effects. Thus, isoproterenol produces relative ischemiadue to myocardial hyperactivity and coronary hypotension (Yeagerand Iams, 1981). Other probable mechanisms include increased cyclicadenosine monophosphate (Bhagat et al., 1978), increased intracel-lular Ca++ overload (Bloom and Davis, 1972), depletion of high-energy phosphate stores and oxidative stress (Rajadurai and Prince,2006; Singal et al., 1982). Increased generation of cytotoxic freeradicals, due to the auto-oxidation metabolic products of isoproter-enol, is one of the well recognized mechanisms of isoproterenolinducedmyocardial necrosis (Rajadurai and Prince, 2006; Singal et al.,1982). Isoproterenol, upon auto-oxidation produces quinones whichreact with oxygen to produce superoxide anion (O2

−) and H2O2. Theproduction of superoxide radical results in secondary formation ofH2O2 and hydroxyl radical (•OH) (Singal et al., 1982).

Following isoproterenol administration, the heart weight increasedsignificantly, with relatively unchanged body weight resulting in theincreaseof theheartweight tobodyweight ratio. Increase inheartweightmightbeattributed to increasedwater content, edematous intramuscularspace (Upaganlawar et al., 2009) and increased protein content. Theseresults are in consistent with the previous report (Judd and Wexler,1974), which has observed extensive edematous intramuscular space,accumulation ofmucopolysaccharides and cellular infiltration after 4 h ofinduction of myocardial infarction. It has been proposed that a 1%increase in myocardial water content could be expected to result inpossibly a 10% reduction in myocardial function (Laine and Allen, 1991).Pre-co-treatment of melatonin brings down the heart weight to bodyweight ratio indicativeof itsprotectionofmyocardiumagainst infiltrationand it also could be due to the decrease in water content of themyocardium.

The ECG is considered the singlemost important initial clinical test fordiagnosis ofmyocardial ischemia and infarction. Its correct interpretationis usually the basis for immediate therapeutic interventions and/orsubsequent diagnosis tests. Isoproterenol administration in rats showedpathological Qwave. The appearance of pathological Qwaves is themostcharacteristic ECG finding of transmural myocardial infarction of the leftventricle. The Q wave appears when the infarcted muscle is electricallyinert and the loss of forces normally generated by the infarcted arealeaves unbalanced forces of variable magnitude in the opposite directionfrom the remote region, for example, an opposite wall. Isoproterenoladministration in rats also showed a decrease in P wave intensity, QRScomplex, R–R interval and increase in the heart rate. These changes couldbe due to the consecutive loss of cell membrane in injured myocardium(Holland and Brooks, 1977). It has been demonstrated that an increase inheart rate is responsible for increased oxygen consumption leading toaccelerated myocardial necrosis (Rona, 1985). Isoproterenol administra-tion also resulted in increased ‘ST-segment’ and decreased ‘R-amplitude’.

idant enzymes in isoproterenol induced myocardial infarction in rats.

Melatonin Isoproterenol Melatonin+isoproterenol

10.63±0.56 5.89±0.39c 8.48±0.38d

13.85±0.28 9.55±0.33c 11.91±0.32e

9.29±0.07 5.83±0.60c 8.22±0.21e

1.50±0.07 4.34±0.17c 3.23±0.23f

764.6±4.67 525.1±18.55c 589.4±5.63e

1.78±0.06 1.04±0.03c 1.53±0.09f

Table 5Effect of melatonin pretreatment on membrane-bound ATPase enzymes in isoproterenol induced myocardial infarction in rats.


Na+–K+ ATPase (μmol of Pi liberated/min/mg protein) 7.96±0.17 7.72±0.15 3.15±0.09c 5.02±0.32f

Ca++ ATPase (μmol of Pi liberated/min/mg protein) 5.21±0.11 5.30±0.11 12.07±0.27c 8.43±1.19e

Mg++ ATPase (μmol of Pi liberated/min/mg protein) 5.31±0.61 5.18±0.07 3.12±0.17b 4.77±0.31d



This is in consistent with the observations of the earlier reports. ST-segment elevation reflects the potential difference in the boundarybetween ischemic and non-ischemic zones and consequent loss of cellmembrane function,whereas decreasedR-amplitudemight bedue to theonset of myocardial edema following isoproterenol administration(Ramesh et al., 1998). Melatonin pretreatment in isoproterenol treatedrats prevented thepathological alterations in theECG suggestiveof its cellmembrane protective effect.

Myocardium contains plentiful concentrations of diagnosticmarkers of myocardial infarction and once metabolically damaged,it releases its contents into the extracellular fluid (Upaganlawar et al.,2009). Of all the macromolecules that leak from the damaged tissue,enzymes because of their tissue specificity and catalytic activity arethe best markers of tissue damage. Assay of the activity of CK-MB inserum is an important diagnosis, because of the marked abundance ofthis enzyme in myocardial tissue and virtual absence from most ofother tissues and its consequent sensitivity. CK-MB isoenzyme activityis useful not only as an index of early diagnosis of myocardialinfarction, but also any type of myocardial injury. Cytosolic enzymesviz. CK-MB, LDH, AST, ALT and ALP which serve as the diagnosticmarkers, leak out from the damaged tissue to blood stream when cellmembrane becomes permeable or rupture (Farvin et al., 2004). Theamount of these cellular enzymes in serum reflects the alterations inplasma membrane integrity and/or permeability (Farvin et al., 2004).In the present study isoproterenol injected rats showed significantelevation in the levels of these marker enzymes in serum, which werein line with the previous reports and indication of isoproterenolinduced necrotic damage of the myocardium and leakiness of theplasma membrane. Melatonin pre-co-treatment resulted in thelowered activity of the marker enzymes in serum. It demonstratedthat melatonin could maintain membrane integrity thereby restrict-ing the leakage of these enzymes.

Free radical scavenging enzymes such as SOD, catalase and GPx arethe first line of cellular defense system against oxidative stress,eliminating reactive oxygen radicals such as superoxide and hydrogen

Fig. 2. Effect of melatonin pretreatment on serum TNF-α levels in isoproterenol inducedmyocardial infarction in rats [***Pb0.001 as compared to control group, ###Pb0.001 ascompared to isoproterenol group].

peroxide and preventing the formation of more deterioratinghydroxyl radicals. The second line of defense comprises of non-enzymatic scavengers viz. ascorbic acid, α-tocopherol, ceruloplasmin,and sulphydryl containing compounds, which scavenge residual freeradicals escaping decomposition by the antioxidant enzymes (Sharmaet al., 2001). The equilibrium between antioxidants and free radicals isan important process for the effective removal of oxidative stress inintracellular organelles. However, in pathological conditions likemyocardial infarction, the generation of reactive oxygen species candramatically disturb this balance with an increased demand of theantioxidant defense system (Singal et al., 1982).

As discussed earlier, isoproterenol auto-oxidation leads to gener-ation of enormous amounts of reactive oxygen species. These reactiveoxygen species may attack any type of molecules, but their maintarget appears to be polyunsaturated fatty acids (PUFAs) withinmembranes forming peroxyl radicals. These radicals then attackadjacent fatty acids within membranes causing a chain reaction oflipid peroxidation (Priscilla and Prince, 2009). Lipid peroxidation is animportant pathogenic event in myocardial necrosis and accumulationof lipid hydroperoxides reflects damage of the cardiac constituents(Rajadurai and Prince, 2006). MDA is a major lipid peroxidation endproduct; increased MDA content may contribute to increasedgeneration of free radicals and/or decreased activities of antioxidantdefense system (Zhou et al., 2006). In the present study isoproterenoladministration resulted in marked elevation in LPO, expressed asMDA content, which is in line with the previous reports (Priscilla andPrince, 2009; Rajadurai and Prince, 2006; Upaganlawar et al., 2009).Melatonin pre-co-treatment showed a significant decrease in the levelof myocardial MDA content which can be attributed to potentantioxidant activity of melatonin against isoproterenol auto-oxidationgenerated free radicals (Allegra et al., 2003).

Activities of antiperoxidative enzymes (SOD and catalase) weredecreased significantly in the heart tissue of isoproterenol injectedanimals. SOD is a class of enzymes, which catalyses the dismutation oftwo superoxide radicals to form hydrogen peroxide and molecular

Fig. 3. Effect of melatonin pretreatment on MPO activity in the heart in isoproterenolinduced myocardial infarction in rats [***Pb0.001 as compared to control group,###Pb0.001 as compared to isoproterenol group].

Fig. 4. Effect of melatonin pretreatment on nitrite content in the heart in isoproterenolinduced myocardial infarction in rats [**Pb0.01 as compared to control group, #Pb0.05as compared to isoproterenol group.]

Table 7Effect of melatonin pretreatment on lipid profile in the heart in isoproterenol inducedmyocardial infarction in rats.


Total cholesterol(mg/g tissue)

4.56±0.18 4.49±0.09 9.15±0.30c 6.49±0.74e

Triglycerides(mg/g tissue)

4.27±0.27 4.32±0.11 6.31±0.39c 5.26±0.20ns

Phospholipids(mg/g tissue)

27.67±0.57 26.92±0.32 17.74±1.95c 23.42±1.25d



oxygen. Thus, generated H2O2 is inactivated by either catalase or bythe GSH redox system consisting of reduced glutathione as thecofactor for GPx and glutathione reductase (Jayalakshmi et al., 2006).Decrease in the activities of these enzymes in isoproterenol inducedmyocardial infarction may be due to increased generation of reactiveoxygen species, such as superoxide and hydrogen peroxide, which inturn leads to inhibition of these enzymes. Furthermore decreasedactivities of these antiperoxidative enzymes result in decreasedremoval of superoxide radical and hydrogen peroxide radicals. It iswell known that superoxide anion can participate in metal ion-mediated Haber Weiss reaction that converts hydrogen peroxide tovery toxic hydroxyl radicals (Zhongyi et al., 1998). Melatonintreatment restored the decreased levels of SOD and catalase in hearttissue. This could be due to direct free radical scavenging effect ofmelatonin (Allegra et al., 2003), indirect effect through its ability toaugment the activities of antioxidant enzymes (Reiter et al., 2000) orits ability to protect antioxidant enzymes from oxidative damage(Mayo et al., 2003).

In the present studywe observed a decreased concentration of GSH inthe heart and decreased activities of glutathione dependant enzymessuch as GPx and GST in the heart of isoproterenol injected rats. GSH, atripeptide, is one of the most abundant non-enzymatic antioxidant bio-molecules present in the body. GSH has a direct antioxidant function byreacting with superoxide radicals, peroxy radicals and singlet oxygenfollowed by the formation of oxidized GSH and other disulfides (Meister,1988). It forms an important substrate for GPx, GST and several otherenzymes, which are involved in free radical scavenging (Meister, 1988).Decreased GSH levels in isoproterenol induced rats might be due to itsincreased utilization in protecting –SH group containing proteins from

Table 6Effect of melatonin pretreatment on lipid profile in serum in isoproterenol inducedmyocardial infarction in rats.


Total cholesterol(mg/dl)

78.17±1.30 79.17±0.87 110.7±5.26c 102.2±1.76ns

Triglycerides (mg/dl) 40.83±1.56 41.23±1.89 64.00±2.07c 54.00±2.28d

HDL-cholesterol(mg/dl)

21.83±1.35 20.60±0.54 14.83±1.42c 16.50±0.43ns

LDL-cholesterol(mg/dl)

48.29±0.69 50.48±1.19 83.05±3.43c 72.53±2.04d

Phospholipids(mg/dl)

101.4±3.58 103.4±1.90 152.4±8.72c 130.4±2.66d


free radicals (Lil et al., 1988). Lowered activities of GPx and GST in theheart in isoproterenol treated rats may be due to reduced availability ofGSH (Priscilla and Prince, 2009; Upaganlawar et al., 2009). Decreasedactivity of these enzymes lead to accumulation of oxidants and makemyocardial cell membranes more susceptible to oxidative damage.Furthermore, inactivation of glutathione reductase, an enzyme respon-sible for conversion of oxidized glutathione (GSSG) to GSH, leads toaccumulation of GSSGwhich in turn inactivates enzymes containing –SHgroup and inhibits protein synthesis (Lil et al., 1988). The present studyobserved a significant rise in the concentration of GSH and activities ofGPx and GST in the heart of melatonin pretreated rats. This can beattributed to the activity of melatonin in stimulating the γ-glutamylcys-teine synthase (Urata et al., 1999), a rate limiting enzyme in GSHsynthesis or stimulatory effect of melatonin on antioxidant enzymesincluding GPx and glutathione reductase (Reiter et al., 2003).

ATPases are intimately associated with the plasma membrane andparticipates in the energy requiring translocation of sodium, potassium,calcium and magnesium (Upasani and Balaraman, 2001). The presentstudy showeddecreased activities ofNa+–K+-ATPase andMg++-ATPaseand increased activity of Ca++-ATPase in isoproterenol injected rats,which is in linewith theprevious report (Upaganlawar et al., 2009).Na+–K+-ATPase is a lipid-dependant enzyme containing –SH group andincreased lipid peroxidation leads to oxidation of the protein and in turninactivates the enzymes (Jayachandran et al., 2009). The inhibition ofNa+–K+-ATPase activates the Na+ and Ca++ ion exchange mechanismin themyocardium. The increased activity of Ca++-ATPasemaybedue toactivation of adenylate cyclase. Melatonin pretreatment showedincreased activities of Na+–K+-ATPase andMg++-ATPase anddecreasedactivity of Ca++-ATPase, which may be attributed to the directantioxidant activity (Allegra et al., 2003) and antioxidant enzymestimulatory effect (Reiter et al., 2000) of melatonin which therebyprotects the –SH group from oxidative damage.

Inflammatory cytokines such as TNF-α are upregulated inmyocardial injury states, ischemia–reperfusion, chronic ischemiapost-myocardial infarction, and heart failure (Ramani et al., 2004).TNF-α may contribute to myocardial injury because it has directeffects on myocardial contractile function by altering calciumhomeostasis, excitation–contraction coupling, nitric oxide metabo-lism, and signaling through ceramide/sphingosine secondmessengers(Ramani et al., 2004). Myeloperoxidase, an enzyme that is associatedwith the azurophilic granules of neutrophils, has been measured toform an index of the infiltration of neutrophils into inflamed tissue.Myeloperoxidase, a green hemoprotein enzyme can use H2O2

generated by NADPH oxidase to oxidize halides (Cl−, Br− and I−) totheir corresponding hypohalous acids (an additional class of activeoxygen metabolite) (Kettle et al., 1997). In the present studyisoproterenol administration showed a significant increase in serumTNF-α and myocardial MPO levels indicative of necrosis inducedinflammation of myocardial tissue and neutrophil infiltration. Inmelatonin pretreated group, significantly decreased levels of inflam-matory markers indicated that the melatonin pretreatment sup-pressed the neutrophil infiltration to the injured myocardium andrelease of inflammatory cytokine, viz. TNF-α. Nitric oxide (NO) is an

Fig. 5. Effect of melatonin pretreatment on histopathological changes {(A) control, (B) melatonin, (C) isoproterenol and (D) melatonin+isoproterenol} in heart tissue inisoproterenol induced myocardial infarction in rats (Heart tissues were stained with hematoxylin and eosin and visualized under light microscope at 40× magnification).


important signaling messenger known to play important roles inmany physiological (such as host defense and homeostasis) andpathological (such as inflammation) conditions. Some studies havepostulated that isoproterenol exerts complex effects on the myocar-dium and that the generation of NO through increased expression ofiNOS could be an important factor involved into the pathogenesis ofmyocyte injury, such as cardiac dysfunction (Zhang et al., 2008). In thepresent study, isoproterenol administration showed decreased levelof nitrite, an oxidized end product of nitric oxide, which may beattributed to formation of peroxynitrite by reaction of NO withgenerated superoxide radicals (Gandhi et al., 2009). Restored levels ofmyocardial nitrite with melatonin pretreatment may be attributed tothe antioxidant activity of melatonin, which decreased the formationof peroxynitrite through inhibition of formation of superoxideradicals.

Lipids play an important role in cardiovascular diseases, not onlyby way of hyperlipidemia and the development of atherosclerosis, butalso by modifying the composition, structure and stability of thecellular membranes. A significant elevation in the total cholesteroland triglycerides was observed in serum and heart of isoproterenoltreated rats. Isoproterenol treated rats also showed an increase in LDLfraction alongwith a decrease in HDL-cholesterol. Phospholipids werefound to be decreasing in the heart and increasing in the serum ofisoproterenol treated rats. These changes could be attributed toenhanced lipid biosynthesis by cardiac cyclic adenosine monopho-sphate (Paritha and Devi, 1997). A strong positive correlation hasbeen documented between the risk of developing ischemic heartdisease and serum LDL level, whereas a negative correlation has beenreported with HDL-cholesterol (Karthikeyan et al., 2007). Theincrease in serum phospholipids may be due to an increaseperoxidation of membrane phospholipids released via phospholipaseA2 (Karthikeyan et al., 2007) which in turn decreased the phospho-lipid levels in heart tissue. The pretreatment with melatoninsuccessfully restored the elevated triglycerides, LDL-cholesterol andphospholipid levels in serum and also restored the total cholesteroland phospholipid levels in the heart in the treatment group.

Histopathological examination of myocardial tissue in controlillustrated clear integrity of the myocardial cell membrane and noinflammatory cell infiltration was observed. Isoproterenol injectedrats showed coagulative necrosis, separation of cardiac muscle fibers

and infiltration of inflammatory cells. The reduced inflammatory cellinfiltration and normal cardiac muscle fiber architecture furtherconfirmed the cardioprotective effect of melatonin.

5. Conclusion

In conclusion, present study demonstrated that subcutaneousinjections of isoproterenol produced myocardial infarction in rats asevident by the release of myocyte injury markers in serum.Myocardial lesions were associated with decreased antioxidantdefense status in the heart electrocardiographic, histopathologicalchanges and release of inflammatorymarkers. In addition, the presentstudy provided experimental evidence that melatonin maintained theantioxidant enzyme levels and improved cardiac performancefollowing high-dose isoproterenol administration. This findingmight be a scientific support to understand the beneficial effects ofmelatonin on cardioprotection against myocardial injury, in whichoxidative stress has long been known to contribute to thepathogenesis.

Acknowledgement

The authors are thankful to All India Council for TechnicalEducation (AICTE) for providing financial assistance in the form ofNational Doctoral Fellowship (NDF).

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