Debasri Paper 2010

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Melatonin protects against isoproterenol-induced myocardial

injury in the rat: antioxidative mechanisms

Introduction

The pineal secretory product melatonin (N-acetyl-5-

methoxytryptamine) is a highly evolutionarily conserved

molecule present virtually in all organisms, in both plants

and animals. Melatonin has several important physiological

functions in mammals including seasonal reproductiveregulation, immune enhancement and regulation of light

dark signal transduction along with the capacity to influ-

ence some aspects of aging. Additionally, melatonin has

widespread antioxidant actions [13].

The well-documented effects of melatonin and its metab-

olites as antioxidants have shown that they protect cells,

tissues and organs from oxidative damage induced by

reactive oxygen species (ROS) as well as from nitrogen-

based reactants [4, 5]. Melatonin is particularly effective in

neutralizing the hydroxyl radical (OH) which attacks

DNA, proteins and lipids leading to a variety of disorders

[6, 7]. Melatonin also detoxifies superoxide anion

radical(O2-) [8], nitric oxide (NO), peroxynitrite anion

(ONOO-) [9], hypochlorous acid (HOCl) [10], the hemo-

globin oxoferryl radical [11], ABTS+ cation radical and

possibly the peroxyl radical (LOO) [12] all of which cause

cell damage [13]. In addition, melatonin inhibits inducible

nitric oxide synthetase (iNOS) [14] and stimulates several

antioxidant enzymes [15]. Additionally, it increases theefficiency of the electron transport chain and, as a conse-

quence, likely reduces electron leakage and the generation

of free radicals [16].

Reactive oxygen species play a critical role in the

pathogenesis of various diseases including cardiovascular

injury associated with circulatory disturbance. Recent

studies have indicated the involvement of ROS in myocar-

dial ischemia. Myocardial infarction is associated with

ischemic necrosis of cardiac muscles due to a decrease in the

supply of blood to a portion of the myocardium below a

critical level necessary for viability and proper physiological

function [4]. A disparity between the oxygen requirement of

Abstract: The present study was undertaken to explore the protective effect

of melatonin against isoproterenol bitartrate (ISO)-induced myocardial

injury in rat. Treatment of rats with ISO increased the level of lipid

peroxidation products and decreased the reduced glutathione levels in

cardiac tissue indicating that this synthetic catecholamine induces oxidative

damage following oxidative stress. Pretreatment of ISO-injected rats with

melatonin at a dose of 10 mg/kg body weight, i.p. prevented these changes.

Additionally, melatonin also restored the activities and the levels of

antioxidant enzymes which were found to be altered by ISO treatment.

Treatment of rats with ISO resulted into an increased generation of hydroxyl

radicals with melatonin pretreatment significantly reducing their production.

Finally, treatment of rats with ISO caused a lowering of systolic pressure

with reduced cardiac output and diastolic dysfunction whereas melatoninpretreatment significantly restored many of these parameters to normal.

The findings document melatonins ability to provide cardio protection at

a low pharmacological dose. Melatonin has virtually no toxicity which

raises the possibility of this indole being a therapeutic treatment for ischemic

heart disease.

Debasri Mukherjee1,

Sreerupa Ghose Roy2,

Arun Bandyopadhyay2,

Aindrila Chattopadhyay3,

Anjali Basu1,

Elina Mitra1, Arnab Kr. Ghosh1,

Russel J. Reiter4 and Debasish

Bandyopadhyay1

1Oxidative Stress and Free Radical Biology

Laboratory, Department of Physiology,

University of Calcutta, University College of

Science and Technology, Kolkata, India;2Molecular Endocrinology Laboratory, Indian

Institute of Chemical Biology, Kolkata, India;3Department of Physiology, Vidyasagar

College, Kolkata, India; 4Department of

Cellular and Structural Biology, University of

Texas Health Science Center at San Antonio,

TX, USA

Key words: antioxidant, hydroxyl radical,

isoproterenol, melatonin, myocardial injury

Address reprint requests to Debasish

Bandyopadhyay, Oxidative stress and Free

Radical Biology Laboratory, Department of

Physiology, University of Calcutta, UniversityCollege of Science and Technology, 92 APC

Road, Kolkata 700009, India.

E-mail: [email protected]

Received October 11, 2009;

accepted December 23, 2009.

J. Pineal Res. 2010; 48:251262Doi:10.1111/j.1600-079X.2010.00749.x

2010 The AuthorsJournal compilation 2010 John Wiley & Sons A/S

Journal of Pineal Research

251

M

olecular,Biological,Physiolog

icalandClinicalAspectsofMelatonin


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the myocardium and the ability of the coronary artery to

meet the oxygen needs, results in the ischemic apoptosis and

necrosis of the heart muscle [17, 18].

The administration of isoproterenol, a synthetic cate-

cholamine as well as a b-adrenergic receptor agonist,

produces gross and microscopic infarcts in the rat heart

[19]. Studies have shown that the pathophysiological

changes that take place in heart following myocardialinfarction induced by isoproterenol administration are

comparable with the changes taking place after myocardial

infarction in humans [20].

Isoproterenol (ISO), upon oxidation, produces quinon-

es which react with oxygen to produce O2- and hydrogen

peroxide (H2O2). The production of O2- results in the

liberation and reduction of iron from tissue ferritin [21] as

well as the secondary formation of H2O2 and the OH

[22]. Because iron and OH are both initiators of lipid

peroxidation (LPO) [23] one might expect LPO to be an

important determinant of myocardial injury. Melatonins

ability to provide protection to the heart has been shown

in different models of oxidative stress [2426] and is an

emerging area of research. In these studies, melatoninprovided cardio protection likely through its antioxidant

mechanisms. Here, we provide additional evidence that

ISO-induced myocardial injury is ameliorated by pre-

treatment of the experimental rats with a low pharmaco-

logical dose of melatonin [27]. The current studies further

reveal that this low molecular weight natural indole

provides protection to the rat heart because of ISO

administration through its indirect antioxidant mecha-

nism(s) as well as by directly scavenging the endogenously

generated OH.

Materials and methodsAnimals

Male SpragueDawley rats, weighing 180220 g, were

obtained from the animal facility of the Indian Institute

of Chemical Biology. The animals were handled as per the

guidelines of the Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), Min-

istry of Social Justice and Empowerment, Government of

India.

Drugs, reagents and antibodies

Melatonin, isoproterenol bitartrate, thiobarbituric acid,

eosin, NAD+, Direct Red-80, 2,2-dithiobis-nitro benzoicacid (DTNB), xanthine, xanthine oxidase, cytochrome

c, fast blue BB salt, nitro blue tetrazolium (NBT),

5-bromo-4-chloro-3-indolyl phosphate (BCIP) and gluta-

thione Peroxidase kit were obtained from Sigma, St. Louis,

MO, USA. Hematoxylin, H2O2 and dimethyl sulfoxide

(DMSO) were obtained from Merck Limited, Delhi, India.

The superoxide dismutase (SOD) 1(C-17), SOD 2(G-20),

glutathione-S-transferase (GST) (Z-5), glutathione reduc-

tase (GR) (H-300) and actin (I-19) antibodies were obtained

from Santa Cruz Biotechnology Inc., Santa Cruz, CA,

USA. Monoclonal anti-a-actinin and anti-catalase were

obtained from Sigma (MO, USA).

Donkey anti-goat and goat anti-mouse immunoglobulin

G (IgG) conjugated with alkaline phosphatase were pur-

chased from Santa Cruz Biotechnology Inc., and anti-

rabbit IgG-AP was purchased from Sigma.

Induction of myocardial infarction with isoproterenol

Myocardial infarction was induced in rats by s.c. injectionof Isoproterenol bitartrate (ISO). Briefly, male Sprague

Dawley rats (food and water ad libitum) weighing 180

220 g were divided into two groups. The rats of the first

group constituted the vehicle-treated controls. The rats of

the second group were injected s.c. with different doses of

Isoproterenol bitartrate (12.5, 25.0, 50.0 mg/kg body

weight) twice at an interval of 24 hr. The animals were

kept at room temperature and were sacrificed 24 hr after

the second injection by cervical dislocation and the hearts

collected and stored at )80C for further biochemical

analyses. Prior to sacrifice, the blood was collected by

cardiac puncture for the preparation of the serum. Devel-

opment of myocardial infarction was confirmed by observ-

ing the ischemic area and measurement of serum glutamateoxaloacetate transaminase (SGOT) levels.

Isoproterenol-induced myocardial ischemia and

protection by melatonin

Male SpragueDawley rats (food and water ad libitum)

weighing 180220 g were divided into three groups. The

rats of the first group constituted the vehicle-treated

controls. The rats of the second group were injected s.c.

with isoproterenol bitartrate (25 mg/kg body weight) twice

at an interval of 24 hr. Rats of the third group were injected

i.p. with different doses of melatonin (5, 10, 20, 40 mg/kg

body weight) 30 min prior to ISO injection. The animalswere kept at room temperature and were sacrificed 24 hr

after the second ISO injection by cervical dislocation and

the heart was collected and stored at )80C for further

biochemical analyses. Prior to sacrifice blood was collected

from the animals by cardiac puncture for preparation of

serum.

Measurement of SGOT level

Serum glutamate oxaloacetate transaminase was measured

by standard routine methods. Values are expressed as IU/L.

Measurement of lipid peroxidation and reduced

GSH level

Cardiac tissue was homogenized (10%) in ice-cold 0.9%

saline (pH 7.0) with a Potter Elvenjem glass homogenizer

(Belco Glass Inc., Vineland, NJ, USA) for 30 s and lipid

peroxides in the homogenate were determined as thiobar-

bituric acid reactive substances (TBARS) according to the

method of Buege and Aust [28] with some modification as

adopted by Bandyopadhyay et al. [29]. Briefly, the homog-

enate was mixed with thiobarbituric acidtrichloro acetic

acid (TBATCA) reagent with thorough shaking and heated

for 20 min at 80C. The samples were then cooled to room

temperature. The absorbance of the pink chromogen

Mukherjee et al.

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present in the clear supernatant after centrifugation at

1200 g for 10 min at room temperature was measured

at 532 nm using a UVvis spectrophotometer (Bio-Rad,

Hercules, CA, USA). Tetrahydroxypropane was used as

standard. Values were expressed as nmoles of TBARS/mg

protein.

Reduced GSH content (as acid soluble sulfhydryl) was

estimated by its reaction with DTNB (Ellmans reagent)

following the method of Sedlac and Lindsey [30] with some

modifications [29]. Cardiac tissue was homogenized (10%)

in 2 mm ice-cold ethylenediaminetetraacetic acid (EDTA).

The homogenate was mixed with TrisHCl buffer, pH 9.0,

followed by DTNB for color development. The absorbance

was measured at 412 nm using a UVvis spectrophoto-

meter to determine GSH content. Values were expressed

as nmoles/mg protein.

Assays of superoxide dismutase and catalase

Copper-zinc superoxide dismutase (SOD1) activity was

measured by hematoxylin autooxidation method of Martin

et al. [31]. Briefly, cardiac tissue was homogenized (10%) inice-cold 50 mm phosphate buffer containing 0.1 mm EDTA

pH 7.4. The homogenate was centrifuged at 12,000 g for

15 min and supernatant collected. Inhibition of hematox-

ylin autooxidation by the cell free supernatant was

measured at 560 nm using a UVvis spectrophotometer.

The enzyme activity was expressed as U/min/mg of tissue

protein.

Manganese superoxide dismutase (SOD2) activity was

measured in the mitochondrial fraction by the xanthine

oxidasecytochrome c method as described by McCord and

Fridovich [32] with some modifications as adopted by

Bandyopadhyay et al. [33]. In brief, cardiac tissue was

homogenized (10%) in ice-cold 50 mm

phosphate buffer,pH 7.8. The homogenate was then centrifuged at 500 g for

10 min and the supernatant was again centrifuged at

12,000 g for 15 min to obtain the mitochondrial fraction.

The supernatant was discarded and the pellet was

re-suspended in the buffer and used for assay carried out

spectrophotometrically at 550 nm with a O2- generating

system (xanthine/xanthine oxidase) in the presence of

cytochrome c. The enzyme activity was expressed as

U/mg protein.

Catalase was assayed by the method of Beers and Sizer

[34] with some modifications as adopted by Chattopadhy-

ay et al. [20]. Cardiac tissue was homogenized (5%) in

ice-cold 50 mm phosphate buffer pH 7.0. The homogenate

was centrifuged in cold at 12,000 g for 12 min. Thesupernatant was then collected and incubated with

0.01 mL of absolute ethanol at 4C for 30 min, after

which 10% Triton X-100 was added to have a final

concentration of 1%. The sample thus obtained was used

to determine catalase activity by measuring the breakdown

of H2O2 spectrophotometrically at 240 nm. Values were

expressed as lm H2O2/min/mg protein.

Assay of glutathione peroxidase

Cardiac tissue was homogenized (10%) in ice-cold 50 mm

TrisHCl buffer containing 0.5 mm EDTA pH 8.0. The

homogenate was centrifuged at 3000 g for 10 min and

supernatant collected. The supernatant was assayed for

GPx activity spectrophotometrically at 340 nm using com-

mercially available GPx kit (Sigma, St. Louis, MO, USA).

The enzyme activity was expressed as Units/mg of tissue

protein.

Measurement of tissue free hydroxyl radical (OH)

The OH generated in cardiac tissue was measured by using

DMSO as a specific OH radical scavenger following the

method of Bandyopadhyay et al. [29]. DMSO forms a

stable product (methane sulfonic acid [MSA]) on reaction

with OH. Accumulation of MSA was measured to

estimate the OH generated after forming a colored

complex with Fast blue BB salt. Three groups of rats

containing four animals each were used for each experi-

ment. The animals of the first group were injected i.p. with

0.4ml of 25% DMSO per 100 g body weight 30 min before

s.c. injection of Isoproterenol (25 mg/kg body weight). The

second group was injected with melatonin (10 mg/kg body

weight, i.p.) 15 min after DMSO injection which wasfollowed by isoproterenol injection (25 mg/kg body weight,

s.c.) 30 min after melatonin injection. The third group of

rats was the control group and was treated only with

DMSO (i.p. injection). The animals of each group were

kept at room temperature for 48 hr and then sacrificed by

cervical dislocation, the chest cavity opened and the hearts

were collected. The cardiac tissue was then processed in

cold for MSA which was allowed to react with Fast blue BB

salt to yield a yellow product. This was measured spectro-

photometrically at 425 nm using benzenesulfinic acid as

standard. The values obtained were expressed as nm of

OH/g tissue.

Measurement of superoxide anion radical (O2-)

generation by the xanthine oxidase/xanthine

dehydrogenase system

Xanthine oxidase was assayed by measuring the conversion

of xanthine to uric acid following the method of Greenlee

and Handler [35]. Briefly, cardiac tissues were homogenized

in cold (10%) in 50 mm Phosphate buffer pH 7.8. The

homogenates were centrifuged at 500 g for 10 min. The

supernatant obtained was further centrifuged at 12,000 g

for 20 min. The supernatant, thus obtained, was collected

and used for spectrophotometric assay at 295 nm using

0.1 mm xanthine in 50 mm phosphate buffer pH 7.8 as

the substrate. The enzyme activity was expressed as milliUnits/mg protein. Xanthine dehydrogenase was assayed by

following the reduction of NAD+ to NADH according to

the method of Strittmatter [36] with some modifications. In

brief, cardiac tissues were homogenized in cold (10%) in

50 mm phosphate buffer with 1 mm EDTA pH 7.2. The

homogenates were centrifuged in cold at 500 g for 10 min.

The supernatant, thus obtained, was further centrifuged in

cold at 12,000 g for 20 min. The supernatant was used for

enzyme assay at 340 nm with 0.3 mm xanthine as the

substrate (in 50 mm phosphate buffer pH 7.5) and 0.7 mm

NAD+ as an electron donor. The enzyme activity was

expressed as milli Units/mg protein.

Melatonin protection against myocardial injury

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Western blot analysis

Western blot analysis was performed with LV homogenates

which were prepared as described earlier by Bandyopadhyay

et al. [29] with minor modifications. Briefly, the LV was

homogenized in a buffer containing 50 mm TrisHCl (pH

7.4), 150 mm NaCl, 1 mm PMSF, 1 mm sodium orthovana-

date, 1 lg/mL each of pepstatin A, leupeptin, and aprotinin.The homogenate was centrifuged at 800 g for 10 min. The

supernatant was again centrifuged at 12,000 g for 15 min to

obtain mitochondrial fraction. The supernatant was col-

lected and the pellet (containing the mitochondrial fraction)

was resuspended in the buffer. The supernatant was resolved

by 10% SDSPAGE according to Laemmlis method [37]

using Mini Protean II apparatus (Bio-Rad Laboratories,

Hercules, CA, USA). Protein (25 lg) for SOD1 (Cu-Zn

SOD), 35 lg protein for GST, catalase and a-actinin and

50 lg protein for GR and actin were loaded for immun-

odetection. Protein (30 lg) from the mitochondrial fraction

was loaded for the detection of SOD2 (Mn-SOD).

After SDSPAGE, the proteins were transferred to

nitrocellulose membranes in an electroblotting apparatus(Mini Trans-Blot, Bio-Rad) at 85 V for 60 min using

193 mm glycine, 25 mm Tris and 20% methanol as transfer

buffer. After transfer the membranes were blocked using

10% nonfat dried milk in Tris-buffered saline containing

0.05% Na-azide (blocking solution, pH 7.6), and incubated

at room temperature for 2 hr. The membranes were then

rinsed twice with Tris-buffered saline containing 0.1%

Tween-20 (TBS-T) and then incubated with the respective

primary antibody (1:2000 dilutions for all in 5% blocking

solution) overnight. After washing thrice with TBS-T, the

membranes were incubated with secondary antibody for

2 hr at room temperature, followed by a further washing

with TBS-T for 15 min twice. The immunoreactive bandswere detected with alkaline phosphatase buffer (100 mm

NaCl, 5 mm MgCl2, and 100 mm TrisHCl; pH 9.5) in

presence of nitro blue tetrazolium (NBT) and BCIP in the

ratio of 2:1. The pixel density of bands obtained through

Western blotting was quantified using ImageJ software

(NIH, Bethesda, MD, USA).

Estimation of proteins

Proteins of the different samples were determined by the

method of Lowry et al. [38].

Hemodynamic study

Hemodynamic studies were conducted as described earlier

[39]. The rats were anaesthetized with sodium pentobarbital

(50 mg/kg, body weight) and heparin (500 units/kg, body

weight). The right internal carotid artery was identified and

ligated cranially. A miniaturized conductance catheter

(SPR-838 Millar instruments, Houston, TX, USA) wasinserted into the carotid artery and then advanced into the

left ventricle until stable pressurevolume (PV) loops were

obtained [40]. Data were then acquired under steady state

conditions. Using the pressure conductance data a range of

functional parameters were then calculated (Millar analysis

software PVAN 3.4). Each experiment was repeated at least

with three animals.

Statistical evaluation

Each experiment was repeated at least three times with

different rats. Data are presented as means S.E.M.

Significance was calculated using one-tailed Students t-test.

Results

Figure 1A reveals a dose-dependent increase in the activity

of SGOT following treatment of rats with ISO which

indicates myocardial tissue damage. At 50 mg/kg body

weight, s.c., the serum level of SGOT increased to a

maximal value (P < 0.001 versus control). Figure 1B

documents that pretreatment of rats with melatonin dose-

dependently prevented the rise in serum SGOT level

following ISO treatment at a dose of 25 mg/ kg body

weight, s.c.

To examine whether administration of ISO induces

oxidative stress, we measured two important biomarkers,namely, LPO and reduced glutathione content of rat heart.

Treatment of rats with different doses of ISO elicited a

dose-dependant increase in the level of LPO measured as

TBARS in the cardiac tissue (Fig. 2A, P < 0.001 versus

control at the dose 50 mg/kg body weight s.c.). However, as

there was no mortality of rats at 25 mg/kg body weight, s.c.

the rest of the experiments were carried out with this dose

of ISO. Figure 2B reveals that pretreatment of rats with

melatonin dose-dependently prevented the ISO-induced

elevation in the level of LPO of the cardiac tissue

(P < 0.001 versus control).

12

14

*

12 *

8

10

12

6

8

10 **

4

6(IU/L)

2

4

6

0

2

I-50I-25I-12.5CON

Serumglutamateoxaloacetate

transaminaseactivity

(IU/L)

Serumglutamateoxaloacetate

transaminaseactivity

0

2

I-25

+m-40

I-25

+m-20

I-25

+m-10

I-25

+m-5

I-25CON

Isoproterenol (mg/kg) Isoproterenol(mg/kg)+melatonin (mg/kg)

(A) (B)Fig. 1. (A) Effect of ISO on serum gluta-

mate oxaloacetate transaminase activity.

Rats were given increasing doses of ISO

(I) s.c. Control (CON) animals were trea-

ted similarly with vehicle only. Values are

means S.E.M. of eight rats in each

group; *P < 0.001 versus CON. (B) Pro-

tective effect of melatonin against ISO-

induced alterations in SGOT activity.

Rats were treated with ISO and increasing

doses of melatonin (m). Values are


group; *P < 0. 001 ve rsu s CON.

**P < 0.001 versus I.

Mukherjee et al.

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Treatment of rats with ISO caused a highly significant

decrease in the reduced glutathione (GSH) content of therat heart tissue (Fig. 3A). However, a dose-dependant

restoration of the GSH content by melatonin pretreatment

of the rats is also evident from the data presented in

Fig. 3B.

To determine the effect of ISO on the activities of the

antioxidant enzymes, we measured the activities of Cu-Zn

SOD, Mn-SOD, catalase, and GPx. The results presented in

Fig. 4A reveals that ISO at the doses of 12.5, 25.0 and

50.0 mg/kg body weight, s.c. significantly increased dose-

dependently the activity of Cu-Zn SOD in cardiac tissue.

There was no mortality with this dose (25 mg/kg body

weight) of ISO. Thus, subsequent experiments were carried

out with this dose of ISO. Figure 4B further reveals that theenhancement of Cu-Zn SOD activity of the cardiac tissue

was restored to control levels by pretreatment of rats with

melatonin, also in a dose-dependent manner. Figure 4C

demonstrates a significant elevation in the level of Cu-Zn

SOD following treatment of rats with ISO. This elevation

was restored to normal level when these rats were

pretreated with melatonin.

Figure 5A reveals a highly significant increase in the

activity of Mn-SOD in therats treated with the same dose (25

mg/kg body weight) of ISO. The activity of Mn-SOD comes

back to near control values when the rats were pretreatedwith melatonin. That this enhancement of Mn-SOD activity

is due to elevation in the level of Mn-SOD protein is evident

from the results presented in Fig. 5B. Mn-SOD levels are

significantly elevated following treatment of rats with ISO.

However, this increment is significantly reduced when the

rats were pretreated with melatonin.

Figure 6A,B demonstrates that ISO also reduces catalase

activity, another important antioxidant enzyme, in a dose-

dependent manner with the maximum inhibition at 50 mg/

kg body weight, s.c. (P < 0.001 versus control). However,

in a separate experiment, a highly significant decrease of

catalase activity of rat cardiac tissue following treatment of

the animals with ISO at a dose of 25 mg/kg body weight,s.c. was restored to near normal by pretreatment of rats

with melatonin in a dose-dependant manner. This inhibited

activity of catalase following ISO treatment of rats is

supported by a reduced level of the enzyme protein as is

evident from Western blot analysis which was restored to

near normal level in the rats pretreated with 10 mg/kg body

weight melatonin, i.p. (Fig. 6C).

Treatment of rats with ISO significantly reduced the

activity of GPx in cardiac tissue (Fig. 7). However, the rats

0.07

(A) (B)

* *

0.04

0.05

0.06**

0.02

0.03

L

0.00

0.01

0.07

0.04

0.05

0.06

0.02

0.03

0.00

0.01

I-50I-25I-12.5CON

Lipidperoxidation

(nmolT

BARS/mgprotein)

Lipidperoxidation

(nmolT

BARS/mgprotein)

I-25I-25I-25I-25I-25CON

Isoproterenol (mg/kg)+m-40+m-20+m-10+m-5

Isoproterenol (mg/kg)+melatonin (mg/kg)

Fig. 2. (A) Effect of ISO on lipid peroxidation level measured as thiobarbituric acid reactive substances (TBARS). Rats were treated with

increasing doses of ISO (I). Control (CON) rats were treated with vehicle only. Values are means S.E.M. of eight rats in each group;

*P < 0.001 versus CON. (B) Protective effect of melatonin against ISO-induced increase in lipid peroxidation level. Rats were treated with

ISO (I) and increasing doses of melatonin (m). Control (CON) animals were treated with vehicle only. Values are means S.E.M. of eight

rats in each group; *P < 0.001 versus CON. **P < 0.001 versus I.

30

35(A) (B)

30

35

**

20

25

*

15

20

25

*

5

10

15

5

10

15

0

5

I-50I-25I-12.5CON

nmoleGSH/mgprotein

nmoleGSH/mgprotein

0

5

I-25

+m-40

I-25

+m-20

I-25

+m-10

I-25

+m-5

I-25CONIsoproterenol (mg/kg)

Isoproterenol (mg/kg) + melatonin (mg/kg)

Fig. 3. (A) Effect of ISO on glutathione

levels of rat heart. The rats were treated

with increasing doses of ISO (I). Control

(CON) rats were treated with vehicle only.

Values are means S.E.M. of eight rats

in each group; *P < 0.001 versus CON.

(B) Protective effect of melatonin against

ISO-induced decrease in the levels of glu-

ta thio ne o f rat h ear t. Va lue s a re


group; *P < 0.00 1 ve rsu s CON.

**P < 0.001 versus I.


255


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when pretreated with melatonin exhibited a near normal

activity of GPx of cardiac tissue.

Figure 8A,B reveals that ISO-induced myocardial oxida-

tive stress is associated with a reduction in the level of the

enzymes GR and GST which play an essential role in the

metabolism of GSH in the cardiac tissue. Pretreatment of

the rats with melatonin at a dose of 10 mg/kg i.p. restored

the level of these enzymes to those observed in the control

rats.

We also examined whether ISO administration to rats

induced the generation of ROS. The results presented in

Fig. 9AE clearly indicate that there was an enhancement

in the generation of O2- in vivo following treatment of rats

with ISO. The activities of xanthine oxidase (XO), xanthinedehydrogenase (XD), total enzyme activity, that is, XO plus

XD, XO - XD ratio and XO/XO + XD ratio all increased

significantly following ISO treatment of rats. All these

parameters were restored to normal levels when the rats

were pretreated with melatonin indicating melatonins

ability to neutralize free radicals in vivo.

Figure 10 illustrates the effect of melatonin on the

scavenging of OH generated in vivo following treatment

of rats with ISO. Treatment of rats with ISO caused nearly

a six-fold increase of endogenous generation of OH.

Pretreatment of rats with melatonin decreased the ISO-

induced OH formation to near basal levels.

Figure 11 reveals that treatment of rats with ISO caused

a significant reduction in the level of a-actinin, an impor-

tant structural protein of myocardial tissue. However, this

protein was not restored to the levels observed in control

rats when they were pretreated with melatonin.

As shown in Table 1, the systolic blood pressure was

significantly (P < 0.01, n = 5) decreased in ISO (25 mg/

kg, body weight) treated rat (Pmax, 76 3 mm Hg) com-

pared with those of control (Pmax, 109 2 mm Hg). The

cardiac output (CO) was significantly (P < 0.01, n = 5)

reduced in ISO-treated rat. The parameters of systolic

(dP/dt max) and diastolic function (dP/dt min) were

significantly reduced by ISO compared with control.

Melatonin significantly restored the ISO-induced altera-tions of hemodynamic parameters.

Discussion

The therapeutic effect of melatonin has been well docu-

mented in various pathophysiological conditions including

cardiovascular diseases [4, 41]. Here we demonstrate that

melatonin not only protects the heart from myocardial

injury but also improves ventricular function in the ISO-

induced ischemic rat. We provide evidence that melatonin

improves cardiac physiology of ISO-treated rat mainly

because of its antioxidant ability.

2.0

2.5

(A)

(C)

(B)

*

1.0

1.5

-ZnSOD

CON I-25

0.0

0.5

I-50I-25I-12.5CON

Cu-

ZnSODactivity

(Units/min/mgprotein)

Cu-ZnSODactivity

(Units/min/mgprotein)

Cu Zn

actin

1.8

2.0 *

Isoproterenol(mg/kg)

60

70

80

**

*

1.0

1.2

1.4

1.6

**

30

40

50

0.0

0.2

0.4

0.6

0.8

CON0

10

20

Cu-Znsuperoxidedismutase

pixeldensity(arbitraryunit)

I-25

+m-40

I-25

+m-20

I-25

+m-10

I-25

+m-5

I-25CON

Isoproterenol (mg/kg)+melatonin(mg/kg)

I-25+m-10

I-25 I-25+m-10

Fig. 4. (A) Effect of ISO on Cu-Zn SOD activity of rat heart tissue. The rats were treated with increasing doses of ISO (I). The control rats

were treated with vehicle only. Values are means S.E.M. of eight rats in each group; * P < 0.001 versus CON. (B) Protective effect of

melatonin against ISO-induced increase in Cu-Zn SOD activity of rat heart tissue. The rats were treated with ISO (I) and increasing doses of

melatonin (m). The control rats were treated with vehicle only. Values are means S.E.M. of eight rats in each group; * P < 0.001 versus

CON. **P < 0.001 versus I. (C) Representative result of Western blot analysis for determining the level of Cu-Zn SOD (lanes from left) of

heart tissue in control (CON), ISO-treated (I) and melatonin (m) protected rats. The Western blot analysis was repeated at least three times.

Actin served as loading control. The pixel density of bands obtained through Western blotting was quantified with ImageJ software (NIH,

Bethesda, MD, USA) and the values (means S.E.M.) were presented below in the form of a bar graph. *P < 0.001 versus CON;

**P < 0.001 versus I.

Mukherjee et al.

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The cellular mechanisms involved in the pathogenesis ofmyocardial ischemia/reperfusion (I/R) injury are complex

and involve the interaction of a number of cell types,

including coronary endothelial cells, circulating blood cells

(e.g., leukocytes, platelets), and cardiac myocytes [17, 42],

all of which are capable of generating ROS. ROS have the

potential to injure vascular cells and cardiac myocytes

directly, and can initiate a series of local chemical reactions

and genetic alterations that ultimately results in an ampli-

fication of the initial ROS-mediated cardiomyocyte

dysfunction and/or cytotoxicity.

Isoproterenol bitartrate, when administered at high

doses, causes myocardial ischemia and infarction via

b-adrenergic pathway [43]. In this study, the dose at which

ISO was administered to induce myocardial ischemia in rats

was 25 mg/kg body weight, s.c. twice at an interval of 24 hr

with no mortality of animals during the treatment period. A

significant increase of SGOT level in the ISO-treated rats

indicated the development of myocardial ischemia in rat

heart. The activity of this enzyme was restored to control

level when the ISO-treated animals were pretreatedwith melatonin. SGOT is one of the diagnostic enzymes

of clinical importance for the detection of myocardial

infarction.

The treatment of rats with ISO induced LPO in the

cardiac tissue. LPO may be due to the oxidation of ISO to

semiquinones which react with oxygen to produce O2- and

H2O2 [44]. Catecholamines readily form chelate complexes

with metal ions such as iron, copper, and manganese, which

strongly catalyze oxidation of catecholamines [44]. Copper

and iron are mobilized in the coronary flow following

myocardial ischemia [45]. Both these ions are present in the

coronary flow fraction in a redox active form that supports

free radical-mediated deleterious reactions [45]. Another

study revealed that catecholamines undergo cyclization toaminochromes. This process can occur enzymatically or

through autooxidation and involves the formation of free

radicals. Aminochromes are highly reactive molecules that

can cause oxidation of protein sulfhydryl groups and

deamination catalysis among other deleterious effects.

Melatonin may reduce LPO levels by interfering with any

of the steps in catecholamine metabolism or by scavenging

the free radicals generated due to redox-active transition

metals such as copper or iron. Melatonin may also reduce

the level of LPO by detoxifying the transition metals that

are reported to be mobilized following myocardial ischemia

[45].

That ISO treatment of rats induces oxidative stress isevident from a highly significant reduction in the GSH

content of cardiac tissue. Melatonin pretreatment, however,

dose-dependently restored the GSH levels of the cardiac

tissue indicating that melatonin is able to mitigate the

oxidative stress induced due to ISO. The decreased tissue

GSH content may be the outcome of an alteration in the

glutathione metabolizing pathway as we observed a reduc-

tion in the protein level of the two key enzymes, GR and

GST following ISO treatment. Both the enzymes were

found to be restored to control levels when the ISO-treated

animals were pretreated with melatonin. This indicates that

melatonin raises the GSH level in vivo in the face of

oxidative challenge. Melatonin has also been shown to

restore the cellular GSH levels of tissues in various modelsof oxidative stress, perhaps, through its stimulatory effect

on GSH synthesis [46]. ISO-induced myocardial ischemia

has been earlier shown to cause cardiac damage although

no doseresponse studies were performed.

We also studied the expression level of one of the

important structural proteins of cardiac tissue of rat, the

a-actinin, by Western blot analysis. Treatment of rats with

ISO significantly reduced the level of a-actinin when

compared with control. However, melatonin did not restore

the level of this protein to that observed in the control rats.

The reason for this may be that for complete restoration,

the dose of melatonin may be insufficient or the time

60

70

80

90(A)

(B)

*

10

20

30

40

50**

Mn

-SODactivity

(Un

its/mgprotein)

0

10

I-25+m-10I-25CON


Mn-SOD

CON I-25 I-25+m-10

50

60

70

**

*

actin

20

30

40

50

CON I-25 I-25+m-100

10Mn-superoxidedismutase


Fig. 5. (A) Effect of ISO on Mn-SOD activity of rat heart tissue.

The rats were treated with ISO (I) at a dose of 25 mg/kg. Melatonin(m) protected rats were treated with 10 mg/kg 30 min before ISO

treatment. The control (CON) rats were treated with vehicle only.

Values are means S.E.M. of eight rats in each group;

*P < 0.001 versus CON; **P < 0.001 versus I. (B) Representative

result of Western blot analysis for determining the level of Mn-

SOD (lanes from left) of heart tissue in control (CON), ISO-treated

(I) and melatonin (m) protected rats. The Western blot analysis was

repeated at least three times. Actin served as loading control. The

pixel density of bands obtained through Western blotting was

quantified with ImageJ software (NIH, Bethesda, MD, USA) and

the values (means S.E.M.) were presented below in the form of a

bar graph. *P < 0.001 versus CON; **P < 0.001 versus I treated.


257


8/12

required for restoration of this protein may be longer than

the period for which the experiments were carried out.

The increase in SOD activity (both cytosolic and mito-

chondrial) in ISO-treated animals may probably be an

adaptive response towards oxidative stress. Many studies

indicate over expression of various SODs which confers

significant protection against ischemia-reperfusion injury

[47]. However, when O2- levels are high, several enzymes

vital to cardiac function is vulnerable to inactivation by thisradical. The decrease in catalase activity after ISO admin-

istration may be due to excessive generation of O2- leading

to the inactivation of the enzyme. O2- is small enough to

gain access to the hemes of catalase and might convert the

resting enzyme to ferro-oxy state (compound III) which is

known to be inactive [48]. A decreased activity of GPx

following ISO treatment of rats as observed is expected to

further aggravate the situation of oxidative stress. Interest-

ingly, melatonin at the low pharmacological dose of 10 mg/

kg restored the activities of the key antioxidant enzymes to

normal. The increased SOD and a decreased catalase

protein level as evident from the Western blot analysis

demonstrate that increased and decreased activity of the

key antioxidant enzyme are the result of altered proteinexpression following treatment of rats with ISO. Once

again, melatonin restored the antioxidant enzyme protein

level to near normal. These observations support the notion

that melatonin protects tissues and organs against oxidative

stress through its indirect antioxidant mechanism(s).

The current studies clearly reveal that following ISO

treatment, the activities of XO and XD are highly signif-

icantly increased compared with control with a concomi-

tant increase in the XO plus XD, XO/XD ratio, XO/

XO + XD ratio. This strongly indicates that metabolic

reactions involving these two enzymes do serve as the

source of this ROS. Earlier workers have also indicated the

25(A)

(C)

(B)

10

15

20

*CON

Catalase

0

5

I-50I-25I-12.5CON

C

atalaseactivity

(M

icromolarH2O2

consu

med/min/mgprotein.)

Catalaseactivity

(MicromolarH2O2

consumed/min/mgprotein.)

120

actin

25

80

100 **

*

15

20

*

**

20

40

60

Catalase

5

10

CON I-25 I-25+m-100


0

I-25+m-40

I-25+m-20

I-25+m-10

I-25+m-5

I-25CON

Isoproterenol(mg/kg)+melatonin(mg/kg)

Isproterenol(mg/kg)

I-25 I-25+m-10

Fig. 6. (A) Effect of ISO on catalase activity of rat heart tissue. The rats were treated with increasing doses of ISO (I). The control rats were

treated with vehicle only. Values are means S.E.M. of eight rats in each group; * P < 0.001 versus CON. (B) Protective effect of

melatonin against ISO-induced increase in Cu-Zn SOD activity of rat heart tissue. The rats were treated with ISO (I) and increasing doses of

melatonin (m). The control rats were treated with vehicle only. Values are means S.E.M. of eight rats in each group; * P < 0.001 versus

CON. **P < 0.001 versus I. (C) Representative result of Western blot analysis for determining the level of catalase (lanes from left) of heart

tissue in control (CON), ISO-treated (I) and melatonin (m) protected rats. The Western blot analysis was repeated at least three times. Actin

served as loading control. The pixel density of bands obtained through Western blotting was quantified with ImageJ software (NIH,

Bethesda, MD, USA) and the values (means S.E.M.) were presented below in the form of a bar graph. *P < 0.001 versus CON;

**P < 0.001 versus I.

1.8

2.0

**

1.2

1.4

1.6*

0.4

0.6

0.8

1.0

0.0

0.2

I-25+m-10I-25CONG

(Units/mgprotein)

Glutathioneperoxidaseactiv

ity


Fig. 7. Protective effect of melatonin against ISO-induced reduc-

tion in glutathione peroxidase activity of rat heart tissue. The rats

were treated with ISO (I) at a dose of 25 mg/kg. Melatonin (m)protected rats were treated with 10 mg/kg 30 min before ISO



*P < 0.001 versus CON; **P < 0.001 versus I.

Mukherjee et al.

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involvement of XO in free radical production [49]. More-

over, our studies clearly demonstrate nearly a six-fold rise

in the endogenous generation of OH following treatment

of rats with ISO. The formation of OH following ISO

treatment in rats pretreated with melatonin was reduced to

basal levels. This clearly documents melatonins ability to

directly neutralize OH. Melatonins ability to scavenge free

hydroxyl radical in vivo has also been shown by earlier

workers [6, 29, 33].

Melatonin protects the isolated rat heart from I/R injury

by scavenging OH, significantly improving left ventricular

function and duration of ventricular tachycardia or

ventricular fibrillation. The result of another study has

shown a spectacular protection against I/R injuries (on

arrhythmias as well as on infarct size) in rats pretreated

with melatonin [25]. This observation suggests that mela-

tonin could have a potential clinical application in the

treatment of myocardial ischemia, even if the mechanism(s)

CON I-25 I-25+m-10

Glutathione reductase

60

(A)

(B)

actin

40

50

** Glutathione-S

CON I-25 I-25+m-10

20

30*

Glutathionereduc

tase

-transferase

actin

80

CON I-25 I-25+m-10

0

10

pixeldensity(arbitraryunits)

50

60

70

**

*

Glutathione-S-tranferase

20

30

40


CON I+25 I-25+m-100

10

Fig. 8. Western blot analysis of levels of

glutathione reductase and glutathione-S-

transferase of heart tissue in control

(CON), ISO-treated (I) and melatonin (m)

protected rats. The Western blot analysis

was repeated at least three times. Actin

served as loading control. The pixel den-

sity of bands obtained through Western

blotting was quantified with ImageJ soft-

ware (NIH, Bethesda, MD, USA) and the

values (means S.E.M.) were presented

b elo w in t he for m of a b ar gr aph.

*P < 0.001 versus CON; **P < 0.001

versus I.

5

(A) (B) (C)

(D) (E)

*10 *

16*

2

3

4

4

6

8

**6

8

10

12

14

**

0

1

2

I-25+m-10I-25CON

**

Xanthineoxidaseactivity

mUnits/mgprotein

0

2

4

I-25+m-10I-25CON

Xanthinedehydrogenaseactivity

mUnits/mgprotein

Isoproterenol (mg/kg)+melatonin (mg/kg)Isoproterenol (mg/kg)+melatonin (mg/kg)

0

2

4

I-25+m-10I-25CON

mUnits/mgprotein

Isoproterenol (mg/kg)+melatonin (mg/kg)Totalenzymeactivitylevel(XO+XDH)

0.7

0.8*

0.35

0.40 *

0.2

0.3

0.4

0.5

0.6

**

XO/XDHratio

0.15

0.20

0.25

0.30**

0.0

0.1

I-25+m-10I-25CONIsoproterenol (mg/kg)+melatonin (mg/kg)

0.00

0.05

0.10

I-25+m-10I-25CON

XO/XO+XDHratio


Fig. 9. Protective effect of melatonin against ISO-induced increase in the activities of (A) xanthine oxidase and (B) xanthine dehydrogenase

in control (CON), ISO-treated (I), and melatonin (m) protected rats. Values are means S.E.M. of eight rats in each group. * P < 0.001

versus CON, **P < 0.001 versus I. (C) Total enzyme activity (XO + XDH), (D) xanthine oxidase/xanthine dehydrogenase (XO/XDH)

ratio, (E) xanthine oxidase/xanthine oxidase + xanthine dehydrogenase (XO/XO + XDH) ratio.


259


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underlying this protection remain to be determined [26, 50].

Night-time melatonin synthesis is reduced in patients with

coronary artery disease [51]. Whether a decreased melato-

nin level may be a predisposing factor for coronary artery

disease, or whether the occurrence of coronary artery

disease decreases melatonin synthesis remains to be deter-

mined [52].

Oxidative mutilation of essential bio-macromolecules

involved in cardiac metabolism and cardiac contractility

leads to diminished cardiac function [17]. Our results also

clearly provide evidence of a diminished cardiac function in

the rats treated with ISO. However, pretreatment of the

ISO-treated rats with melatonin restored cardiac function

to that observed in the control rats. This improvement of

cardiac function in ISO-treated rats by melatonin may be of

future therapeutic importance.

Many of the drugs used in the treatment of differentcardiac diseases do possess various side effects which limits

their use by clinicians. Recently, attention has been focused

on the cardio-protective ability of melatonin [4, 53, 54].

This small indole and several of its metabolites are excellent

antioxidants [29, 55, 56]. They also reduce the toxicity of

different drugs [57, 58]. Moreover, pharmacological doses

of melatonin do posses very low or no toxicity [59].

Therefore, it will be worth investigating whether melatonin

can be used along with other cardio-protective drugs as a

co-therapeutic in the treatment of ischemic heart disease.

The available information to date suggests that melatonin

may be an ideal candidate for thorough investigation with

respect of its cardio-protective activity.

Acknowledgements

Debasri Mukherjee gratefully acknowledges the receipt of a

project fellowship from UPE Scheme of UGC, Govt. of

India, under University of Calcutta. Sreerupa Ghose Roy is

a recipient of a Senior Research Fellowship from CSIR,

New Delhi, Govt. of India. Elina Mitra is a recipient of a

project fellowship from UPE Scheme of UGC, Govt. of

India, under University of Calcutta. The technical help of

Swapan Mandal, Prabir Das and Sumanta Ghoshal is also

acknowledged. This work is partially supported by CSIR

grant to Arun Bandyopadhyay (SIC 007).

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300

*

150

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250

50

100

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**

nm

olhydroxylradical

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Heart rate 341 7.0 346 1.0 388 2.0

Pmax (mmHg) 109 2.0 76 3.0* 133 7.0

Pmin (mmHg) 21.3 2.0 18.4 0.3 9.0 0.5

CO (lL/min) 22494 1070 10837 342* 15442 403#

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