Debasri paper 2012

7/31/2019 Debasri paper 2012

1/14

Melatonin protects against isoproterenol-induced alterations in

cardiac mitochondrial energy-metabolizing enzymes, apoptotic

proteins, and assists in complete recovery from myocardial injuryin rats

Introduction

Ischemic heart disease (IHD), a health problem of global

concern, is characterized by a reduced blood supply (ische-mia) to the heart muscle, usually because of coronary artery

disease (atherosclerosis of the coronary arteries) [1].

A disparity between the oxygen requirement of the myocar-

dium and the ability of the coronary artery to meet the

oxygen need results in the ischemic apoptosis and necrosis of

the heart muscle (myocardial infarction). Its risk increases

with age, smoking, hypercholesterolemia, diabetes, and

hypertension, andis more commonin menthanin women [2].

Studies have indicated the involvement of reactive

oxygen species (ROS) in myocardial ischemia [3]. In

addition to their ability to directly inflict damage upon

cellular macromolecules, ROS play a significant role in

activating stress-sensitive signaling pathways that regulate

gene expression leading to cellular damage [4]. Antioxidants

have been evaluated for both primary and secondary

prevention of IHD [5].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

[6]. Studies have shown that the pathophysiological changes

that take place in rat heart following myocardial infarction

induced by isoproterenol administration are comparable by

the changes taking place after myocardial infarction in

humans [7].

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

methoxytryptamine), is a highly evolutionarily conserved

molecule, present in virtually all organisms including

plants and animals [8, 9]. Melatonin has several important

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

of melatonin against isoproterenol bitartrate (ISO)-induced rat myocardial

injury and to test whether melatonin has a role in preventing myocardial

injury and recovery when the ISO-induced stress is withdrawn. Treatment for

rats with ISO altered the activities of some of the key mitochondrial enzymes

related to energy metabolism, the levels of some stress proteins, and the

proteins related to apoptosis. These changes were found to be ameliorated

when the animals were pretreated with melatonin at a dose of 10 mg/kg BW,

i.p. In addition to its ability to reduce ISO-induced mitochondrial

dysfunction, we also studied the role of melatonin in the recovery of the

cardiac tissue after ISO-induced damage. Continuation of melatonin

treatment in rats after the withdrawal of ISO treatment was found to reduce

the activities of cardiac injury biomarkers including serum glutamate

oxaloacetate transaminase (SGOT), lactate dehydrogenase (LDH), and

cardio-specific LDH1 to control levels. The levels of tissue lipid peroxidation

and reduced glutathione were also brought back to that seen in control

animals by continued melatonin treatment. Continuation of melatonin

treatment in post-ISO treatment period was also found to improve cardiac

tissue morphology and heart function. Thus, the findings indicate

melatonins ability to provide cardio protection at a low pharmacological

dose and its role in the recovery process. Melatonin, a molecule with very lowor no toxicity may be considered as a therapeutic for the treatment for

ischemic heart disease.

Debasri Mukherjee1, Arnab K.

Ghosh1, Arun Bandyopadhyay2,

Anjali Basu1, Santanu Datta3,

Sanjib K. Pattari4, Russel J.

Reiter5 and Debasish

Bandyopadhyay1

1Oxidative Stress and Free Radical Biology

Laboratory, Department of Physiology,

University College of Science and Technology,University of Calcutta, Kolkata, India;2Molecular Endocrinology Laboratory, Indian

Institute of Chemical Biology, Kolkata, India;3Department of Cardiothoracic Surgery, SSKM

Hospital, Kolkata, India; 4RN Tagore

International Institute of Cardiac Sciences,

Kolkata, India; 5Department of Cellular and

Structural Biology, University of Texas Health

Science Center at San Antonio, San Antonio,

TX, USA

Key words: antioxidant, heart function,

isoproterenol, melatonin, myocardial injury,

oxidative stress, tissue recovery

Address reprint requests to Debasish

Bandyopadhyay, Oxidative Stress and Free

Radical Biology Laboratory, Department of

Physiology, University College of Science and

Technology, University of Calcutta, 92, APC

Road, Kolkata 700 009, India.

E-mail: [email protected]

Received December 9, 2011;

Accepted February 1, 2012.

J. Pineal Res. 2012; 53:166179Doi:10.1111/j.1600-079X.2012.00984.x

2012 John Wiley & Sons A/S

Journal of Pineal Research

166

M

olecular,Biological,Physiolog

icalandClinicalAspectsofMelatonin


2/14

physiological functions in mammals including seasonal

reproductive regulation, immune enhancement, and regula-

tion of lightdark signal transduction along with the

capacity to influence some aspects of aging [10]. Addition-

ally, melatonin has widespread antioxidant actions [11]. The

antioxidant properties of melatonin and its possible regu-

latory effects on ROS production and redox signaling have

been proposed to play a key role in antagonizing themitochondrial pathway of apoptosis [12, 13]. In the recent

years, several findings support the antioxidant effect as well

as a direct role of melatonin in mitochondrial homeostasis

[14]. This latter action of melatonin may contribute to

melatonins protective effects in degenerative disorders such

as Parkinsons disease, Alzheimer disease, epilepsy, aging,

ischemia-reperfusion and sepsis, all of which involve mito-

chondrial dysfunction as a primary or secondary cause of

the disease [15, 16]. Melatonins ability to provide protection

to the heart has been shown in different models of oxidative

stress [1720] and is an emerging area of research.

Earlier, we found that pretreatment for rats with mela-

tonin at a dose of 10 mg/kg BW, administered intraperito-

neally, protected against ISO-induced myocardial ischemicinjury [5]. Although the protection afforded by melatonin

was significant with respect to biomarkers of organ damage,oxidative stress and antioxidant enzyme activities, and

protein levels, the results indicated that the protection was

never complete. Moreover, although melatonins protective

effects through antioxidant mechanisms were clearly shown,it remained to be deciphered whether ISO-induced myocar-

dial injury was owing to disturbances in the mitochondrial

energy metabolism and whether induction of apoptosis in

the myocardial tissue was one of the causative factors ofmyocardial tissue injury. It also remains to be seen whether

pretreatment of rats with melatonin is capable of providing

protection to the heart through mechanisms other than itsantioxidant effects.

Herein, we provide evidence that pretreatment for rats

with melatonin provides protection against ISO-induced

myocardial injury by ameliorating the disturbances

observed in the activities of the enzymes related to the

substrate metabolism in mitochondria and protecting the

myocardial cells against apoptosis. Additionally, the cur-

rent work demonstrates that continuation of melatonin

treatment results in complete recovery of the myocardial

tissue from ISO-induced ischemic changes.

Thus, the current studies reveal that this low-molecular

weight natural indole provides protection to the ISO-

damaged heart by its direct and indirect antioxidant

mechanism(s) as well as via the control of mitochondrialROS generation and stress-activated signaling pathways,

thereby raising the possibility of it being used as a

therapeutic against IHD.

Materials and methods

Animals

Male SpragueDawley rats, weighing 180220 g were

handled as per the guidelines of the Committee for the

Purpose of Control and Supervision of Experiments on

Animals (CPCSEA), Ministry of Social Justice and

Empowerment, Government of India, with the approval

of the Institutional Animal Ethics Committee (IAEC) of

the Department of Physiology, University of Calcutta.

Chemicals and reagents

Melatonin, ISO, thiobarbituric acid (TBA), eosin, NAD

+

,NADH, 2,2-dithiobis-nitro benzoic acid (DTNB), glutar-

aldehyde, cytochrome c, nitro blue tetrazolium (NBT),

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

obtained from Sigma, St Louis, MO, USA. Hematoxylin,

trichloroacetic acid (TCA), MnSO4, and potassium ferri-

cyanide (K3FeCN6) were obtained from Merck Limited,

Delhi, India. Sodium pyruvate, isocitrate, succinate,

a-ketoglutarate, and bovine serum albumin (BSA) were

obtained from Sisco Research Laboratories (SRL), Mum-

bai, India. The cytochrome c (7H8), Apaf-1 (H 324),

caspase 9 (H 83), ERK 2 (C 14), pP38 (Tyr 182), HSP 70

(K 20), c-Jun (H 79), and actin (I-19) polyclonal

antibodies were obtained from Santa Cruz Biotechnology,

Inc., Santa Cruz, CA, USA. Polyclonal phospho-NF-jBp65 antibody was obtained from Cell Signaling Technol-

ogy Inc., Danvers, MA, USA. Donkey anti-goat and goatanti-mouse immunoglobulin G (IgG) conjugated with

alkaline phosphatase were purchased from Santa Cruz

Biotechnology Inc. The anti-rabbit IgG-AP was purchased

from Sigma.

Isoproterenol-induced myocardial ischemia and

protection by melatonin

Male SpragueDawley rats, weighing 180220 g, kept at

room temperature (food and water ad libitum) were divided

into seven groups. The rats of the 1st group constituted thevehicle-treated control. The rats of the 2nd7th groups

were injected s.c. with ISO (25 mg/kg body weight) twice at

an interval of 24 hr. Rats of the 3rd, 4th, and 5th groups

were injected i.p. with melatonin (10 mg/kg body weight)

30 min prior to ISO injection (25 mg/kg body weight s.c.).

The animals of the 1st, 2nd, and 3rd groups were killed

24 hr after the second ISO injection by cervical dislocation,

and the hearts were collected and stored at )80C for

further biochemical analyses. Prior to killing, blood was

collected from the animals by cardiac puncture for the

preparation of serum. The animals of the 4th group were

injected i.p. with melatonin (10 mg/kg body weight) for two

more days after discontinuation of ISO treatment, while in

case of those in the 6th group, only ISO treatment wasdiscontinued. The animals of these two groups were killed

on the 5th day (third day after second ISO injection), and

cardiac tissue and serum were collected as before. The

animals of the 5th group were treated with melatonin

(10 mg/kg BW, i.p.) for a further 4 days after second ISO

injection, while those of the 7th group were left undisturbed

for four more days after discontinuation of ISO treatment.

The animals of the 5th and 7th groups were killed on the

7th day from the start of experiment. Cardiac tissue and

serum were collected as before and stored at )80C for

further analyses.

Melatonin protection against myocardial injury

167


3/14

The measurement of the activities of the

mitochondrial Krebs cycle enzymes

Cardiac tissue was homogenized (10%) in ice-cold 50 mmphosphate buffer, pH 7.4 with a Potter Elvenjem glass

homogenizer (Belco Glass, Inc., Vineland, NJ, USA) for

30 s. 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 pellet thus obtained was resuspended in the buffer and

used for assaying the mitochondrial enzymes.Pyruvate dehydrogenase activity was measured spectro-

photometrically according to the method of Chretien et al.

[21] with some modifications by following the reduction of

NAD+ to NADH at 340 nm using 50 mm phosphate

buffer, pH 7.4, 0.5 mm sodium pyruvate as substrate, and

0.5 mm NAD+ in addition to enzyme. The enzyme activity

was expressed as Units/mg protein.

Mitochondrial isocitrate dehydrogenase (ICDH) activity

was measured according to the method of Duncan et al.

[22] by measuring the reduction of NAD+ to NADH at

340 nm with the help of a UVVIS spectrophotometer. Onemilliliter assay volume contained 50 mm phosphate buffer,

pH 7.4, 0.5 mm isocitrate, 0.1 mm MnSO4, 0.1 mm NAD+,

and enzyme. The enzyme activity was expressed as Units/

mg protein.

Alpha-ketoglutarate dehydrogenase activity was mea-

sured spectrophotometrically according to the method of

Duncan et al. [22] by measuring the reduction of 0.35 mm

NAD+ to NADH at 340 nm using 50 mm phosphate

buffer, pH 7.4 as assay buffer, and 0.1 mm a-ketoglutarate

as substrate. The enzyme activity was expressed as Units/

mg protein.

Mitochondrial succinate dehydrogenase activity was

measured spectrophotometrically by following the reduc-

tion of potassium ferricyanide (K3FeCN6) at 420 nmaccording to the method of Veeger et al. [23] with some

modifications. One ml assay mixture contained 50 mm

phosphate buffer, pH 7.4, 2% (w/v) BSA, 4 mm succinate,2.5 mm K3FeCN6, and enzyme. The enzyme activity was

expressed as Units/mg protein.

The measurement of mitochondrial respiratory chain

enzymes

NADH-Cytochrome c oxidoreductase activity was mea-

sured spectrophotometrically by following the reduction inoxidized cytochrome c at 565 nm according to the method

of Goyal and Srivastava [24].One millilitre assay mixture

contained in addition to enzyme 50 mm phosphate buffer,

0.1 mg BSA, 20 mm oxidized cytochrome c, and 0.5 lM

NADH. The enzyme activity was expressed as Units/mg

protein.

Cytochrome c oxidase activity was determined spectro-

photometrically by following the oxidation of reduced

cytochrome c at 550 nm according to the method of Goyal

and Srivastava [24]. One ml assay mixture contained 50 mm

phosphate buffer, pH 7.4, 40 mm reduced cytochrome c,

and enzyme. The enzyme activity was expressed as Units/

mg protein.

Western blot analysis

Western blot analysis was performed with left ventricular

(LV) homogenates that were prepared as described

earlier by Bandyopadhyay et al. [25] with minor modifi-

cations. Briefly, the LV was homogenized in a buffer

containing 50 mm TrisHCl (pH 7.4), 150 mm NaCl,

1 mm PMSF, 1 mm sodium orthovanadate, 1 lg/mL each

of pepstatin A, leupeptin, and aprotinin. The homogenatewas centrifuged to separate the nuclear, mitochondrial,and cytosolic fractions. The different fractions were

resolved by 10% SDSPAGE according to Laemmlis

method [26] using Mini Protean II apparatus (Bio-Rad

Laboratories, Hercules, CA, USA). Protein (60 lg) for

a-Actinin, 35 lg protein for pP38, HSP-70, and ERK-2,

50 lg protein of cytochrome c, Apaf-1, Caspase-9,

and 65 lg protein of cJUN and actin were loaded

for immunodetection. Seventy micrograms protein fromthe nuclear fraction was loaded for the detection of

NFjB.

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 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, fol-

lowed by further washing twice with TBS-T for 15 min.

The immunoreactive bands were detected with alkalinephosphatase buffer (pH 9.5) in the 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).

Measurement of SGOT and serum LDH levels

Serum glutamate oxaloacetate transaminase (SGOT) was

measured by standard routine methods. The enzymeactivity was expressed as IU/L.

Total serum lactate dehydrogenase (LDH) activity was

obtained by measuring the oxidation of NADH (0.1 mm) to

NAD+ at 340 nm using 1.0 mm sodium pyruvate as

substrate according to the method of Strittmatter [27] with

some modifications. The enzyme activity was expressed as

Units/mL.

The cardiac-specific Type 1 isoform of lactate dehydro-

genase (LDH1) activity was obtained according to the

method of Varcoe et al. [28] by incubating the serum

samples at 65C for 30 mins, which destroys all isoforms

except LDH1, then assaying the enzyme as before by

measuring NADH oxidation. The enzyme activity was

expressed as Units/mL.

Mukherjee et al.

168


4/14

Measurement of lipid peroxidation and reduced

glutathione level

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

saline (pH 7.0) with a Potter Elvenjem glass homogenizer

(Belco Glass, Inc.) for 30 s, and lipid peroxides in the

homogenate were determined as thiobarbituric acid reactive

substances (TBARS) according to the method of Buege andAust [29] with some modification as adopted by Bandyo-

padhyay et al. [25]. Briefly, the homogenate was mixed with

thiobarbituric acidtrichloro acetic acid (TBATCA) re-

agent with thorough shaking and heated for 20 min at

80C. The samples were then cooled to room temperature.

The absorbance of the pink chromogen present in the clear

supernatant after centrifugation at 1200 g for 10 min at

room temperature was measured at 532 nm using a UV

VIS spectrophotometer (Bio-Rad). Tetraethoxypropane

(TEP) was used as standard. Values were expressed as

nmoles of TBARS/mg protein.

Reduced glutathione (GSH) content (as acid-soluble

sulfhydryl) was estimated by its reaction with DTNB

(Ellmans reagent) following the method of Sedlac andLindsey [30] with some modifications [25]. Cardiac tissue

was homogenized (10%) in 2 mm ice-cold ethylenediamine-tetraacetic 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 spectrophotometer to determine GSHcontent. Values were expressed as nmoles/mg protein.

Estimation of proteins

Proteins of the different samples were determined by the

method of Lowry et al. [31].

Hemodynamic study

Hemodynamic studies were conducted as described earlier

by Connelley et al. [32].The rats were anesthetized with

sodium pentobarbital (50 mg/kg BW) and heparin

(500 units/kg BW). The right internal carotid artery was

identified and ligated cranially. A miniaturized conduc-

tance catheter (SPR-838, Millar Instruments, Houston,

TX, USA) was inserted into the carotid artery and then

advanced into the left ventricle until stable pressure

volume (PV) loops were obtained [33]. Data were then

acquired under steady state conditions. Using the

pressure conductance data, a range of functional param-

eters were then calculated (Millar analysis softwarePVAN 3.4). Each experiment was repeated at least with

three animals.

Histological studies

The extirpated hearts were fixed immediately in 10%

formalin and embedded in paraffin following routine

procedure [33]. Left ventricular sections (5 lm thick) were

prepared and stained with hematoxylineosin (H/E). The

tissue sections were examined under an Olympus BX51

(Olympus Corporation, Tokyo, Japan) microscope, andimages were captured with a digital camera attached to it.

Left ventricular sections (5 lm thick) were stained with

Sirius red (Direct Red 80; Sigma Chemical Co) and imaged

with laser scanning confocal system (Zeiss LSM 510

META, Carl Zeiss MicroImaging GmbH, Jena, Germany),

and the stacked images through multiple slices were

captured. The digitized images were then analyzed using

image analysis system (Image J, NIH Software), and the

total collagen area fraction of each image was measuredand expressed as the % collagen volume [33].

The cardiac tissue sections were processed for scanning

electron microscopy according to standard procedures with

some modifications [34]. Briefly, LV sections were fixed

with 2.5% glutaraldehyde in 50 mm phosphate buffer pH

7.2 and kept overnight. The sections were washed with

several changes of 50 mm phosphate buffer pH 7.2 and then

dehydrated first with graded alcohol then with graded amyl

acetate in alcohol. The tissue was then critical-point-dried,

mounted on aluminium stubs, coated with a gold-palladium

mixture, and examined in a Quanta 200 FEI microscope.

Statistical evaluationEach experiment was repeated at least three times with

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

level of significance was calculated using one-tailed Stu-

dents t-test.

Results

Fig. 1(A) depicts a significant decrease in the activity of

pyruvate dehydrogenase (PDH) (P < 0.001 versus con-

trol), the enzyme that couples glycolysis to tricarboxylic

acid (TCA) cycle. Pretreatment for the rats with melatonin

significantly ameliorated the ISO-induced effects (31%

increase versus I P < 0.01). Fig. 1(BD) show the ISO-induced decrease (P < 0.001 versus control) in the activity

of the TCA cycle enzymes ICDH, a-ketoglutarate dehy-

drogenase (a-KGDH), and succinate dehydrogenase

(SDH), respectively. The activities of all the three enzymes

were improved back to near control levels on pretreatment

for the rats with melatonin [P < 0.01 versus I for ICDH

(31%); P < 0.001 versus I for a-KGDH (70%) and SDH

(38.5%)].

The activity of mitochondrial respiratory chain enzymes,

like NADH-cytochrome c oxidoreductase and cytochrome

c oxidase also decreased significantly (P < 0.001 versus

control) following ISO treatment for rats as is evident from

the data presented in Fig. 2(A) and (B). Both these enzymes

were brought back to control level by pretreatment for ratswith melatonin (P < 0.001 versus I).

ISO-induced inhibition of mitochondrial TCA cycle and

respiratory chain enzymes leads to the leakage of cyto-

chrome c from the mitochondria into the cytoplasm as is

evident from Fig. 3(A) and (B) (P < 0.01 versus control).

Pretreatment for rats with melatonin is unable to prevent

the leakage of cytochrome c into the cytoplasm (Fig. 3A

and B). This leakage, however, causes very slight difference

in the mitochondrial cytochrome c pool, which is evident

from the results presented in Fig. 3(A) and (C).

Fig. 4(A) shows an ISO-induced elevation (P < 0.001versus control) of the apoptotic protease activating factor 1


169


5/14

10

12

**6

7

8

*

**

4

6

8

*

3

4

5*

0

2

0

1

2

Pyruvatedehydrogenaseactivity

(U

nits/mgprotein)

Isocitra

tedehydrogenaseactivity

(Units/mgprotein)

I I + m

4.5

5.0

70

80

#

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

CON

I I + m


CON I I + m


CON

I I + m


CON

2.5

3.0

3.5

4.0

#40

50

60

*

0.5

1.0

1.5

2.0

* 1020

30

Succin

atedehydrogenaseactivity

(Units/mgprotein)

0.0

Alpha-ketoglutaratedehydrogenase

activity(Units/mgprotein)

0

(A) (B)

(C) (D)

Fig. 1. Protective effect of melatoninagainst ISO-induced decrease in theactivities of (A) pyruvate dehydrogenase,(B) isocitrate dehydrogenase, (C) a-keto-glutarate dehydrogenase, and (D) succi-

nate dehydrogenase in control (CON),ISO-treated (I), and melatonin (m)-pro-tected rats. Values are means S.E.M. ofeight rats in each group. *P < 0.001 ver-sus CON; **P < 0.01 versus I;#P < 0.001 versus I.

12 #0.9 #

8

10

0.6

0.7

0.8

*

4

6

*

0.2

0.3

0.4

0.5 *

0

2

NADH-Cytochromecr

eductase

(Units/mgprotein)

CON I I + m

0.0

0.1Cyto

chromecoxidaseactivity

(Units/mg

protein)


CON I I + m


(A) (B)

Fig. 2. Protective effect of melatoninagainst ISO-induced decrease in theactivities of (A) NADH-cytochrome coxidoreductase and (B) cytochrome oxi-dase in control (CON), ISO-treated (I),and melatonin (m) protected rats. Valuesare means S.E.M. of eight rats in eachgroup. *P < 0.001 versus CON;#P < 0.001 versus I.

Cytochrome c

(cytoplasm)Cytochrome c

(mitochondria)

Actin

**

Con I I + m

Con I I + m Con I I + m

60

70

80

90 **

140

160

180

30

40

50

60

80

100

120

0

10

20Cytochromec(

cytoplasm)

Pixeldensity(arbitraryunit)

Cytochromec(

mitochondria)


CON I I + m

0

20

40


CON I I + m


(A)

(B) (C)

Fig. 3. (A) Representative results ofWestern blot analysis for determining thelevel of cytoplasmic and mitochondrialcytochrome c (lanes from left) of hearttissue in control (CON), ISO-treated (I),and melatonin (m)-protected rats. TheWestern blot analysis was repeated at leastthree times. Actin served as loading con-trol. The pixel density of bands [(B) forcytoplasmic and (C) for mitochondrial]obtained through Western blotting wasquantified with ImageJ software (NIH),and the values (means S.E.M.) werepresented below in the form of a bargraph. **P < 0.01 versus CON.

Mukherjee et al.

170


6/14

(Apaf-1), the protein involved in the formation of apopto-

some complex with cytochrome c that cleaves Procaspase 9

to its activated form. This ISO-induced elevation of Apaf-1level is decreased significantly by melatonin (P < 0.001

versus I). Fig. 4(B) shows the significant elevation of the

level of activated Caspase 9, the protein involved in cellular

apoptosis, by ISO (P < 0.001 versus control) and its

amelioration by melatonin (P < 0.01 versus I).

We also studied the effect of ISO on the stress-activated

proteins ERK2, P38, HSP70, and P53 as well as transcrip-

tion factors cJUN and NFjB. Figs 5(A,B), 6(A) and (B)

demonstrate that ISO significantly increased the levels of

the stress proteins, ERK2, phosphorylated P38, HSP70,

and phosphorylated P53 (P < 0.001 versus control).

Although pretreatment for rats with melatonin did not

cause any significant change in ERK 2 levels, it was,

however, able to lower the levels of pP38, HSP70 (P < 0.01

versus I), and pP53 (P < 0.001 versus I) to near controlvalues.

Fig. 7(A) shows the ISO-induced elevation in the level of

the transcription factor cJUN, which was found to be

significantly lowered when the rats were pretreated with

melatonin. The level of NFjB, another important tran-

scription factor, (Fig. 7B) was also found to be elevated

following ISO treatment (P < 0.001 versus control). This

elevation was also found to be ameliorated by pretreatment

for rats with melatonin (P < 0.01 versus I).

Fig. 8(A) reveals that isoproterenol (ISO) causes myo-

cardial injury at the dose of 25 mg/kg BW s.c., as is evident

from a significant increase in SGOT activity (P < 0.001

Con I I + m

Actin

Caspase9

40

50

60

*

**30

35

40 *#

20

30

Caspase9


15

20

25

Apaf-1


10

0

5

10

0


CON I I + m


CON I I + m

Con I I + m

Apaf-1

Actin

(A) (B)

Fig. 4. (A) Representative result of Western blot analysis for determining the level of apoptotic protease activating factor (Apaf)-1 (lanesfrom left) of heart tissue in control (CON), ISO-treated (I), and melatonin (m)-protected rats. The Western blot analysis was repeated atleast three times. Actin served as loading control. The pixel density of bands obtained through Western blotting was quantified with ImageJ

software (NIH), and the values (means S.E.M.) were presented below in the form of a bar graph. * P < 0.001 versus CON; #P < 0.001versus I. (B) Representative result of Western blot analysis for determining the level of caspase9. Actin served as loading control. The pixeldensity of bands obtained through Western blotting was quantified with ImageJ software (NIH), and the values (means S.E.M.) werepresented below in the form of a bar graph. *P < 0.001 versus CON; **P < 0.01 versus I.

pP38ERK2


70 *

ActinActin

50

60

***

120

140

160 *

20

30

40

pP38

Pixeldensity(arbitraryu

nit)

60

80

100

ERK2


0

10

0

20

40

CON I I + m

Isoproterenol (mg/kg) + melatonin (mg/kg)CON I I + m


(A) (B)

Fig. 5. Western blot analysis of levels of(A) ERK 2 and (B) phosphorylated P 38of heart tissue in control (CON), ISO-treated (I), and melatonin (m)-protectedrats. The Western blot analysis was re-peated at least three times. Actin served asloading control. The pixel density ofbands obtained through Western blottingwas quantified with ImageJ software(NIH), and the values (means S.E.M.)were presented below in the form of a bargraph. *P < 0.001 versus CON;**P < 0.01 versus I.


171


7/14

versus control) in ISO-treated rats. However, when the rats

were pretreated with melatonin at a dose of 10 mg/kg, BW

i.p. the SGOT activity decreased significantly (P < 0.001versus I) indicating the ability of melatonin in protecting

against ISO-induced injury to cardiac tissue. Fig. 8(A) also

reveals that when the rats were left undisturbed for a

further period of 2 (Iw 2D) and 4 (Iw 4D) days after thewithdrawal of ISO, there was a gradual reduction in the

activity of SGOT, which was found to be statistically

significant at the 6th day (Iw 4D, P < 0.001 versus I) from

the start of the experiment. However, this reduction in the

SGOT activity was found to be more when the rats werecontinued to be treated with melatonin (10 mg/kg BW, i.p.)

for a further period of 2 (Iw + m 2D) and 4 (Iw + m 4D)

days after ISO withdrawal.

The ISO-induced myocardial injury and its protection by

melatonin in a time-dependant manner were further con-

firmed by the results presented in Fig. 8(B) and (C), which

show the serum activity levels of the enzyme LDH (Fig. 8B)

and its cardiac-specific Type 1 isoform (LDH1) (Fig. 8C).

Both the figures show a significant increase in enzyme

activity following ISO treatment (P < 0.001 versus con-

trol), which was found to be significantly decreased when

the rats were pretreated with melatonin. However, the

enzyme activity reached almost control level when the rats

were continued to be treated with melatonin up to 4 days

after the withdrawal of ISO treatment (P < 0.001 versus I).

Fig. 9(AC) reveal the tissue morphological changes in

the myocardium following ISO treatment. Hematoxylin

and eosin staining of the LV tissue sections of the ISO-

treated rat hearts at 20 magnification (Fig. 9A) showedmyo-degeneration as characterized by a loss of cardiac

myofibers and a mononuclear cell infiltration. However,

when the rats were pretreated with melatonin, the ISO-

induced degenerative changes in the myocardial tissue were

found to be significantly lower. The recovery from thetissue injury was found to be complete when melatonin

treatment was continued for a further 2 and 4 days after the

withdrawal of ISO treatment. The ISO-induced damage to

the cardiac cytoarchitecture was further evident from a

significant reduction in the level ofa-actinin, an important

structural protein of the myocardial tissue (Fig. 9B and

9C). Pretreatment for rats with melatonin for 2 days

(I + m) was unable to restore the levels of this structural

protein. However, continuation of melatonin treatment for

a further period of 2 (Iw + m 2D) and 4 (Iw + m 4D)

days after the withdrawal of ISO caused a gradual

restoration ofa-actinin to almost control levels.

NFc-Jun


Actin

(nucleus)(nucleus)

Actin

40

50

60

***

50

60

70

80*

**

20

30

20

30

40c-Jun


NFkB(nucleus)


**

0

10

0

10

Isoproterenol(mg/kg) + melatonin(mg/kg)CON I I + m

Isoproterenol(mg/kg) + melatonin(mg/kg)CON I I + m

(A) (B)

Fig. 7. Western blot analysis of levels of(A) cJUN and (B) NFjB of heart tissue incontrol (CON), ISO-treated (I), and mel-atonin (m)-protected rats. The Westernblot analysis was repeated at least threetimes. Actin served as loading control.The pixel density of bands obtainedthrough Western blotting was quantifiedwith ImageJ software (NIH), and thevalues (means S.E.M.) were presentedbelow in the form of a bar graph.*P < 0.001 versus CON; **P < 0.01

versus I.

pP53


50

ActinActin

HSP-70

40

*

#80

100 *

**

20

30

pP53

Pixeldensity(arbitraryun

it)

40

60

HSP-70

Pixeldensity(arbitraryun

it)

0

10

0

20

CON I I + m

Isoproterenol (mg/kg) + melatonin (mg/kg)CON I I + m


(A) (B)

Fig. 6. Representative result of Westernblot analysis for determining the level of(A) HSP 70 and (B) phosphorylated P 53of heart tissue (lanes from left) in control(CON), ISO-treated (I), and melatonin(m)-protected rats. The Western blotanalysis was repeated at least three times.Actin served as loading control. The pixeldensity of bands obtained through Wes-tern blotting was quantified with ImageJsoftware (NIH), and the values (mean-s S.E.M.) were presented below in theform of a bar graph. *P < 0.001 versusCON; **P < 0.01 versus I; #P < 0.001versus I.

Mukherjee et al.

172


8/14

7

Serum lactate dehydrogenase

(LDH) activity

Serum glutamate oxaloacetate

transaminase activity

5.5

6

6.5

IU/L

IU/L

*

*

Iw4DIw2D

#I

IIw2D Iw4D

#

*4

4.5

5

CON

**

Iw + m 2D

I + m

^##

Iw + m 4D

IU/L

*

CON

I + mIw + m 2D

Iw + m 4D

**

^

##

0.3 Serum LDH1 activity

14

12

10

8

Duration (days) of isoproterenol bitartrate

& melatonin


& melatonin

0 Day 2 Day 4 Day 6 Day



& melatonin


0.20

0.25

Iw4D

*Iw2D

I

#

0.15Iw + m4D

##^

Iw + m 2D

***I + m

CON

0.1

(A)

(B)

(C)

Fig. 8. (A) Protective effect of melatonin against ISO induced alterations of SGOT activity. The rats were treated with ISO (I) at a dose of25 mg/kg body weight s.c. for 2 days. ISO treatment was then discontinued, and the rats were killed after 2days (I w 2D) and 4 days (Iw 4D)post-ISO, respectively. Melatonin (m)-protected rats were treated with 10 mg/kg body weight i.p. 30 min before ISO treatment for 2 days(I + m). ISO was then discontinued and melatonin treatment continued post-ISO for 2days (Iw + m 2D) and 4 days (Iw + m 4D). Thecontrol (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; **P < 0.01 versus Iw 2D; ^P < 0.01 versus Iw 4D; ##P < 0.001 versus I + m. (B) Protective effect of melatoninagainst ISO induced increase in serum LDH activity. The rats were treated with ISO (I) at a dose of 25 mg/kg body weight s.c. Melatonin(m)-protected rats were treated with 10 mg/kg body weight i.p. The CON rats were treated with vehicle only. Values are means S.E.M.(C) Protective effect of melatonin against ISO induced elevation of cardiac-specific LDH 1 activity. The rats were treated with ISO (I) at adose of 25 mg/kg body weight s.c. Melatonin (m)-protected rats were treated with 10 mg/kg body weight i.p. The CON rats were treatedwith vehicle only. Values are means S.E.M.

CON ISO Iw 2D Iw 4D

200

I + m Iw + m 2D Iw + m 4DMEL

200

140

Alpha-actinin

Con I I + m Iw + m

(2D)

Iw + m

(4D)

Con I I + m I w + m

(2D)

Iw + m

(4D)

80

100

120*

#

Actin

20

40

60

Alpha-actinin


0


(A)

(B)(C)

Fig. 9. (A) Representative images (200magnification) of hematoxylin/eosin-stained left ventricular longitudinal sec-tions of rat hearts of control (CON), ISO(I)-treated, and melatonin (m)-protected,pre (I + m) and post (Iw + m 2D,Iw + m 4D) ISO treatment. (B) Repre-

sentative result of Western blot analysisfor determining the level of a-Actinin(lanes from left) of heart tissue in CON,ISO-treated (I), and melatonin (I + m)-protected rats, pre (I + m) and post(Iw + m 2D, Iw + m 4D) ISO treatment.The Western blot analysis was repeated atleast three times. Actin served as loadingcontrol. (C) The pixel density of bandsobtained through Western blotting andquantified with ImageJ software (NIH),and the values (means S.E.M.) pre-sented in the form of a bar graph.*P < 0.001 versus CON; #P < 0.001versus I.


173


9/14

The cytoarchitectural damage to the cardiac tissue

because of ISO treatment was further confirmed by the

observation that there was a loss of collagen from the

intercellular space compared to control, and the results are

presented in the Fig. 10(A) and (B). This loss of collagen

was found to be almost completely prevented in a time-

dependent manner when the animals were initially pre-

treated with melatonin, and then the melatonin treatmentwas also continued for a period of 2 and 4 days after the

withdrawal of ISO. The results indicate that melatonin has

the ability to provide protection to the myocardial tissue

against ISO-induced damage.

Fig. 11 shows the changes brought about to the cardiac

endo and myocardium following ISO treatment and studied

through scanning electron microscopy. The cardiac tissue

sections of the ISO-treated rats showed a perforated

endocardium having cells with convoluted cell membranes.

These cells, which were markedly contracted, with pro-

nounced nuclear bulges, also had large membrane blebs

covering the cell surface. A few cells appeared to be

separating from each other, and a few polymorphonuclear

neutrophils were present adhering to the endocardial cells.These ISO-induced changes in the rat heart endocardium

were found to be significantly prevented when the rats werepretreated with melatonin, and the recovery from the

damage was almost complete when melatonin treatment

was continued for a further period of 2 and 4 days post-ISO

treatment.

Treatment for rats with ISO elicited a significant increase

in the level of lipid peroxidation (LPO) measured as

TBARS in the cardiac tissue (Fig. 12A, P < 0.001 versus

control). Only slight difference to the elevated LPO levels

was found even when the rats were left untreated for afurther 2 (Iw 2D) and 4 (Iw 4D) days after the withdrawal

of ISO treatment. Pretreatment for rats with melatonin

prevented the ISO-induced elevation in the level of LPO of

the cardiac tissue (P < 0.001 versus I), and the LPO level

was further lowered to control levels on continuation of

melatonin treatment after the withdrawal of ISO

(P < 0.001 versus I and P < 0.001 versus I + m).

Treatment for rats with ISO caused a significant decrease

(P < 0.001 versus control) in the reduced GSH content of

the rat heart tissue.

(Fig. 12B). This reduction in tissue GSH was found to be

only slightly elevated on its withdrawal. However, a time-

dependant restoration of the cardiac GSH content was

observed when the rats were pretreated with melatonin(P < 0.001 versus I) as well as when melatonin treatment

was continued for two and four more days in the post-ISOtreatment period.

CON I I + m Iw + m (2D) Iw + m (4D)

600

600

6#

3

4

5

#

##

1

2*

Collagenvolume(%)

0


CON I I + m Iw + m(2 D)

Iw + m(4 D)

(A)

(B)

(C)

Fig. 10. (A) Representative images (600magnification) of Sirius red-stained leftventricular longitudinal sections of rathearts of control (CON), ISO (I)-treated,and melatonin (m)-protected, pre (I + m)and post (Iw + m 2D, Iw + m 4D) ISOtreatment. Red color stretches are colla-gen depositions. (B) The similar imagescaptured by confocal laser scanningmicroscope for quantification of fibrosis.Arrow heads indicate collagen fibers. (C)Histogram showing % collagen volume inthe ventricular tissues. Values are mean-s S.E.M. % collagen from three images

from each of three rats of each group;*P < 0.001 versus CON; ##P < 0.001versus I; #P < 0.001 versus I + m.

CON I I + m Iw + m (2D) Iw + m (4D)

6000

Fig. 11. Scanning electron micrograph (6000) of endocardial cells of rat hearts of control (CON), ISO (I)-treated, and melatonin (m)-protected, pre (I + m) and post (Iw + m 2D, Iw + m 4D) ISO treatment. Arrow heads indicate perforated tissue structure and membraneblebbings.

Mukherjee et al.

174


10/14

As shown in Table 1, the systolic blood pressure wassignificantly (P < 0.001, n = 6) decreased in ISO-treated

rats (Pmax, 72.10 0.52 mm Hg) compared to those of

control (Pmax, 116.60 1.60 mm Hg). The cardiac output

(CO) was also significantly (P < 0.001, n = 6) reduced inISO-treated rats. Pretreatment for rats with melatonin for

2 days significantly (P < 0.001, n = 6) increased CO. The

CO was further increased when the animals were continued

to be treated with melatonin for 2 (Iw + m 2D) and 4(Iw + m 4D) days after ISO withdrawal. The parameters

of systolic (dP/dt max) as well as diastolic function (dP/dt

min) were significantly reduced by ISO compared to

control, which were restored significantly by melatonin.

These data indicate that melatonin restores the ISO-induced alterations of hemodynamic parameters in a

time-dependent manner.

Discussion

Generation of oxidative stress plays a major role in the

pathogenesis of myocardial ischemia/reperfusion (I/R)

injury, which involve the interaction of a number of cell

types, including coronary endothelial cells, circulating

blood cells (e.g., leukocytes, platelets), and cardiac myo-

cytes [5, 35] all of which are capable of generating ROS.

Reactive oxygen species 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 result in an amplification of the

initial ROS-mediated cardiomyocyte dysfunction and/or

cytotoxicity. In our earlier study [6], we demonstrated thatISO, a synthetic b-adrenergic agonist, caused myocardial

ischemia (at a dose of 25 mg/kg BW s.c.) via the induction

of oxidative stress. Isoproterenol caused oxidative stress

both by direct generation of ROS as well as by inhibitingthe antioxidant defense mechanisms of the myocardial cells.

We also demonstrated that melatonin (at the dose of

10 mg/kg BW i.p.) was capable of ameliorating the ISO-

induced stress. However, the details of the cellular mech-

anisms involved in the induction of oxidative stress by ISOand protection by melatonin remained to be explored.

Because mitochondria are the seat as well as the principal

target of oxidative stress, herein, we have investigated the

effect of ISO on the mitochondrial enzymes related to

energy metabolism. The current studies have investigated

the status of activity of pyruvate dehydrogenase and some

of the mitochondrial Krebs cycle enzymes, particularly,

ICDH, alpha-ketoglutarate dehydrogenase, and succinate

dehydrogenase related to ATP production in mitochondria

following treatment of rats with ISO. In each case, the

activities of the enzymes are highly significantly inhibited in

ISO-treated rats. Pretreatment for rats with melatonin

0.06

0.07Lipid peroxidation level

I# 30

35 GSH level

CON

*

Iw + m2D

**

Iw + m4D^

##

0.05

Iw2D

Iw + m2D

Iw4DI + m

*

**

*

##20

25I + m

*

Iw4D

*

Iw2D

#

0.04nmolesTBARS/mgprotein

nm

olesGSH/mgprotein

CONIw + m4D^

15

I


& melatonin



& melatonin


(A) (B)

Fig. 12. (A) Protective effect of melatonin against ISO induced increase in lipid peroxidation (LPO) level of rat heart tissue. The rats weretreated with ISO (I) at a dose of 25 mg/kg body weight s.c. for 2 days. ISO treatment was discontinued, and the rats were killed after 2 days(Iw 2D) and 4 days (Iw 4D) post-ISO, respectively. Melatonin (m)-protected rats were treated with 10 mg/kg body weight i.p. 30 min beforeISO treatment for 2 days (I + m). ISO was then discontinued and melatonin treatment continued post-ISO for 2days (Iw + m 2D) and4 days (Iw + m 4D). 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; **P < 0.001 versus Iw 2D; ^P < 0.001 versus Iw 4D; ##P < 0.001 versus I + m.(B) Protective effect of melatonin against ISO induced decrease in reduced glutathione (GSH) level of rat heart tissue The rats were treatedwith ISO (I) at a dose of 25 mg/kg body weight s.c. Melatonin (m)-protected rats were treated with 10 mg/kg body weight i.p. pre (I + m)and post (Iw + m 2D, Iw + m 4D) ISO treatment. The CON rats were treated with vehicle only. Values are means S.E.M. of eight rats

in each group.

Table 1. Hemodynamic parameters in control hearts and those treated with isoproterenol (ISO) with or without melatonin (MEL)

Experiment Control ISO ISO + MEL ISOwth + MEL 2 days ISOwth + MEL 4 days

Heart rate (bpm) 367 3.00 348 1.00a 393 2.00b 368 2.00 371 1.0c

Pmax (mmHg) 116.60 1.60 72.10 0.52a 116 1.50b 116.7 1.50 128.4 2.0c

Pmin (mmHg) 20.10 1.50 18.745 0.23 10.00 0.40 14.51 0.40 13.0 0.6CO (uL/min) 45,971 744 18,416 211a 26,775 470b 37,728 402 44,594.5 467c

dP/dtmax (mmHg/s) 6975 225 1913 20a 4158 91b 6226 154 7044 95c

dP/dtmin (mmHg/s) )6019 211.10 )1510 14.10a )4248 64.45b )5211 137 )5077 100c

Values are means S.E.M. of 8 rats in each group.aP < 0.001 versus CON; bP < 0.001 versus I; cP < 0.001 versus I+m.


175


11/14

significantly elevated the activities of these crucial enzymes

toward normal indicating melatonins ability to protect

these enzymes either through scavenging the toxic reactants

produced within the mitochondria in ISO-treated rats or

protecting the substrate-binding sites of these enzymes

through some hitherto unknown mechanism(s). Inhibition

of mitochondrial Krebs cycle enzymes enhances free

radical formation [36]. Mitochondrial oxidative stress-induced dysfunction has been implicated in the pathogen-

esis of aging, cancer, diabetes, ischemia/reperfusion injury,

neurodegenerative disorders, and other diseases [37].

A reduction in the activity of NADH-cytochrome creductase (Complex I) and cytochrome c oxidase (Complex

IV) of the respiratory chain following ISO treatment for

rats is clearly indicative of an elevated state of oxidative

stress in cardiac mitochondria. Pretreatment for rats with

melatonin, however, completely restored the activity of

these enzymes indicating that melatonin is capable of

mitigating mitochondrial oxidative stress generated follow-

ing ISO treatment.

Inhibition of mitochondrial TCA cycle and respiratory

chain enzymes leads to a leakage of electrons, producing areducing environment within the mitochondria, thereby

generating free radicals [38, 39]. These free radicals,particularly the free OH radical, can damage the mito-

chondrial membrane causing a leakage of cytochrome c

from the mitochondria into the cytoplasm [39, 40]. This is

evident from our current study which shows that ISOtreatment for rats cause a highly significant increase in the

release of cytochrome c from the mitochondria. However,

in our experimental setup, pretreatment for rats with

melatonin could not decrease cytochrome c leakage to

any significant extent as evident from our Western blot

analysis for the measurement of cytochrome c level both in

the cytosol and mitochondria. The reason for this may lie inthe fact that within 48 hr, melatonin may not be able to

repair the mitochondrial membrane damage and need more

time for complete repair, although it improves activity of

many of the mitochondrial enzymes related to energy

metabolism within the same time period. However, the

requirement of a higher dose of melatonin or longer

duration of treatment for complete mitochondrial mem-

brane repair may not be ruled out and needs further

investigation. Thus, alterations in mitochondrial redox

metabolism and respiratory functions may lead to the

increased production of ROS in cells [40]. Melatonins

ability to protect and improve mitochondrial functions has

also been reported earlier [41].

The leakage of mitochondrial cytochrome c in ourexperiments prompted us to investigate whether ISO

treatment for rats induces any apoptotic and/or stress

signaling protein. We found a significant elevation in the

levels of apaf-1 and caspase 9 proteins because of ISO

treatment. Apaf-1 is known to form a complex with

cytochrome c, which triggers caspase 9 and eventually

apoptosis. Melatonin pretreatment significantly reduced the

levels of both proteins indicating a protective role of this

small indole against stress-induced cellular apoptosis.

Earlier workers have shown that the apoptotic signaling

activated during UVB stress mainly converges at themitochondrial level into the so-called intrinsic (or dam-

age-induced) pathway [42]. Recent convincing evidence

suggests that this pathway might represent the main target

of melatonin to antagonize apoptosis in human leukocytes

[43] and in other tumor cell lines and in vivo models [42,

43]. The antioxidant properties of melatonin and its

possible regulatory effects on ROS production and redox

signaling have been proposed to play a key role in

antagonizing the mitochondrial pathway of apoptosis [44,45].

Our studies also demonstrated ISO-induced elevation in

the levels of key stress proteins like ERK-2, phosphorylated

p38, HSP-70, phosphorylated p53, cJUN, and NFjB.

Earlier workers have also demonstrated that acute admin-

istrations of ISO to conscious rats induced dose-dependent

increases in cardiac LPO and ERK1/2, p38, and JNK MAP

kinase phosphorylation via b-adrenoceptor [46]. Reactive

oxygen species act as important mediators for intracellular

signaling in a variety of cells leading to changes in gene

expression. Phosphorylation and activation of c-Jun pro-

tein are linked directly to intracellular redox status, while

NFjB activation is involved in hypoxia-reoxygenation

injury, especially in the vascular endothelium, where NFjBactivation leads to neutrophil adhesion in vivo [47]. In our

studies, most of these stress-related proteins were brought

back to near control levels by melatonin pretreatment

indicating that melatonin is capable of providing protection

to the cardiac tissue by reducing the level of oxidative stressinduced because of ISO. Melatonin and its metabolites have

been proposed to regulate ROS fluxes and provide mito-

chondrial protection [42]. The current work also indicates

toward melatonins capability of influencing the activation

of fundamental signaling pathways.Even though our current study explored the role of

melatonin in protecting ISO-induced mitochondrial dys-

function and activation of stress-activated apoptotic andsignaling pathways, it was found that, in case of many of

the parameters studied, the restoration by melatonin, at the

present dose and duration, although significant, was not

completely back to the levels seen in control animals.

A similar question was raised in our earlier study [6] where

some of the parameters studied, particularly those relating

to the cardiac histopathology and heart function, were not

restored to the levels seen in control animals by pretreat-

ment of rats with melatonin at the dose of 10 mg/kg BW

i.p. for 2 days, raising the possibility of testing a higher

dose of melatonin or a longer duration of treatment with

melatonin. So, in the second part of our present study, we

explored the fact whether continuation of melatonin

treatment for 2 (Iw + m 2D) and 4 (Iw + m 4D) daysafter the withdrawal of ISO treatment (after the 2nd dose)

could lead to complete recovery of the cardiac tissue after

ISO-induced damage. To confirm that the recovery of the

cardiac tissue after the withdrawal of ISO treatment was

truly because of melatonin and not because of a natural

healing of the myocardial tissue, we also left two corre-

sponding groups of animals untreated post-ISO treatment

for 2 (Iw 2D) and 4 (Iw 4D) days, respectively.

We studied the diagnostic enzymes for myocardial

infarction, that is, SGOT and LDH as well as the

cardiac-specific Type 1 isoform of LDH (LDH1).

A significant increase in the activity of all these biomarker

Mukherjee et al.

176


12/14

enzymes (Fig. 8) indicated the induction of myocardial

injury. Pretreatment for the rats with melatonin partially,

although significantly, ameliorated these effects. However,

continuation of melatonin treatment for 2 (Iw + m 2D)

and 4 (Iw + m 4D) days, respectively, after the withdrawal

of ISO (after the 2nd dose) brought back the activities of

these enzymes completely to that seen in control animals.

The improvement of cardiac status was also evident fromthe histopathological studies of the myocardial tissue. Our

studies on hematoxylin/eosin-stained cardiac tissue sections

showed significant cellular damage and degeneration fol-

lowing ISO treatment. Pretreatment for rats with melatonin

for only 2 days was unable to restore the cardiac tissue

architecture. However, continued melatonin treatment for

another 2 and 4 days after discontinuation of the ISO

treatment was found to restore the architecture of the

damaged cardiac tissue in a time-dependant manner.

This was further confirmed by the expression level of one

of the important structural proteins of cardiac tissue of rat,

the a-actinin, which was significantly reduced following ISO

treatment. Melatonin, at the dose of 10 mg/kg body weight

i.p. did not restore the level of this protein to that observedin the control rats after 2 days of treatment as was also

evident in our earlier work [6]. The reason for this may bethat for complete restoration, the dose of melatonin was

insufficient or the time required for restoration of this

protein needed to be longer than the period for which the

experiments were carried out. This was evident from thefact that on continuing melatonin treatment for 2 and

4 days, respectively, after discontinuation of ISO treatment,

a complete restoration ofa-actinin was found in the cardiac

tissue samples from the rats. Thus, the pattern of restora-tion was found to be time-dependant.

Additionally, the depletion of collagen in the cardiac

tissue following ISO treatment for rats was found to bepartially but significantly ameliorated by pretreatment for

the animals with melatonin for 2 days. But the tissue

collagen was found to be completely restored to that

observed in the control cardiac tissues when treatment of

rats with melatonin was continued for another 2 and 4 days

after discontinuation of ISO treatment. This observation

was further supported from our studies of the cardiac tissue

sections with confocal microscopy.

Besides, the LV tissue morphological studies through

scanning electron microscopy also revealed severe tissue

injury at the infarcted site following ISO treatment for rats.

This tissue injury was also found to be partially restored

when the rats were pretreated with melatonin. However, the

cardiac tissue recovered almost completely from the oxida-tive injury because of ISO when the melatonin treatment

was continued for 2 and 4 days post-ISO treatment period.

The results indicate the ability of melatonin to serve not

only as a protective antioxidant but also point toward its

role in promoting recovery of tissue injury resulting from

oxidative onslaught. This seems to be an area of intense

research in the future days.

That ISO-induced myocardial damage was caused

because of the induction of oxidative stress was evident

from the significant increase in LPO level in the cardiac

tissue following ISO treatment. This may be due to theoxidation of ISO to semiquinones that react with oxygen to

produce O2 and H2O2 [46]. Catecholamines readily form

chelate complexes with metal ions such as iron, copper, and

manganese, which strongly catalyze oxidation of catechol-

amines [48]. Catecholamines may also undergo cyclization

to aminochromes. 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 anddeamination 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 because of redox-active transi-

tion 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 [49]. Our studies further indicate that the contin-

uation of the melatonin treatment for rats for 2 and 4 days

after the withdrawal of ISO decreased the LPO level to

almost control value indicating that this indole also plays a

role in tissue recovery.

Induction of oxidative stress by ISO is also evident from

a highly significant reduction in the GSH content of cardiactissue. Pretreatment for rats with melatonin significantly

restored the GSH levels of the cardiac tissue indicating thatmelatonin is able to mitigate the oxidative stress induced

because of ISO. The decreased tissue GSH content may be

the outcome of an alteration in the GSH-metabolizing

pathway that has been demonstrated earlier by Mukherjeeet al. [6]. Melatonin has been shown to restore the GSH

levels of tissues in various models of oxidative stress,

perhaps, through its stimulatory effect on GSH synthesis

[50]. However, continuation of melatonin treatment for twoand 4 days after the withdrawal of ISO helped the tissue to

regain its GSH content as observed in the control tissues

indicating again melatonin

s ability to promote cellularphysiological processes that were otherwise compromised

following oxidative insult to cardiac tissue following ISO

treatment.

Oxidative mutilation of essential bio-macromolecules

involved in cardiac metabolism and cardiac contractility

leads to diminished cardiac function [6]. Our results also

clearly provide evidence of a diminished cardiac function in

the rats treated with ISO. In our previous work also, we

have demonstrated improvement of heart function by

melatonin pretreatment in ISO-injected rats [6] but the

restoration was not complete. However, continuation of

melatonin treatment in rats for another 2 and 4 days after

the withdrawal of ISO treatment improved all the param-

eters of cardiac function studied, particularly heart rate,CO, and systolic and diastolic pressure to that observed in

control animals, indicating melatonins efficacy in improv-

ing heart function even in postinfarction period.

These observations suggest that melatonin could have a

potential clinical application in the treatment for myocar-

dial ischemia, even if the mechanisms(s) underlying this

protection remains to be determined [51, 52]. Night-time

melatonin synthesis is reduced in patients with coronary

artery disease [53]. Whether a decreased melatonin level

may be a predisposing factor for coronary artery disease, or

whether the occurrence of coronary disease decreasesmelatonin synthesis remains to be determined [54]. Many


177


13/14

of the drugs used in the treatment for different cardiac

diseases do possess various side effects, which limit their use

by clinicians. Recently, attention has been focused on the

cardio-protective ability of melatonin [1, 55, 56]. This small

indole and several of its metabolites are excellent antiox-

idants [25, 5759]. They also reduce the toxicity of different

drugs [60, 61]. Moreover, pharmacological doses of mela-

tonin possess very low or no toxicity [62]. Therefore, it willbe worth investigating whether melatonin can be used along

with other cardio-protective drugs as a co-therapeutic in the

treatment for IHD. The results of the current studies clearly

indicate that melatonin not only has the ability to protect

the heart against ischemic stress but may also play a critical

role in the improvement and maintenance of normal

cardiac function even after ischemic episode. The available

information to date suggests that melatonin may be an ideal

candidate for thorough investigation with respect of its

cardio-protective ability.

Acknowledgements

Debasri Mukherjee gratefully acknowledges the receipt of aSenior Research Fellowship (SRF) from CSIR, Govt. of

India, New Delhi. AKG is a Senior Research Fellow (SRF)under RFSMS Fellowship Program, Govt. of India.

A. Basu is supported from the funds available to Dr. DB

under BI 92 (7). Dr. SD is supported from the funds

available to him from Govt. of West Bengal. This work ispartially supported by UGC Major Research Project Grant

to Dr. DB [F. No. 37-396/2009 (SR)] and also by CSIR

Grant to Dr. AB (SIP 007). Technical help from Swapan

Mandal, Prabir Das, and Sumanta Ghoshal is also grate-

fully acknowledged. The help from Bose Institute, Kolkata,

is also gratefully acknowledged.

References

1. Tengattini S, Reiter RJ, Tan DX et al. Cardiovascular

diseases: protective effects of melatonin. J Pineal Res 2008;

44:1625.

2. Reiter RJ, Tan DX. Melatonin: a novel protective agent

against oxidative injury of the ischemic/reperfused heart.

Cardiovasc Res 2003; 58:1019.

3. Chattopadhyay A, Bandyopadhyay D. Ischemic heart dis-

ease: protection by vitamin E. Pharmacol Rep 2006; 58:179

187.

4. Peyrot F, Ducrocq C. Potential role of tryptophan deriva-

tives in stress responses characterized by the generation of

reactive oxygen and nitrogen species. J Pineal Res 2008;

45:235244.

5. Bandyopadhyay D,Chattopadhyay A,GhoshG,DattaAG.

Oxidative stress-induced ischemic heart disease: protection by

antioxidants. Curr Med Chem2004; 11:369387.

6. Mukherjee D, Roy SG, Bandyopadhyay A et al. Melatonin

protects against isoproterenol-induced myocardial injury in the

rat: antioxidative mechanisms. J Pineal Res 2010; 48:251262.

7. Chattopadhyay A, Biswas S, Bandyopadhyay D, Sarkar C,

Datta AG. Effect of isoproterenol on lipid peroxidation and

antioxidant enzymes of myocardialtissue of miceand protection

by quinidine. Mol Cell Biochem 2003; 245:4349.

8. Hattori A, Migitaka H, Iigo M et al. Identification of

melatonin in plants and its effects on plasma melatonin levels

and binding to melatonin receptors in vertebrates. Biochem

Mol Biol Int 1999; 35:327334.

9. TanDX,Manchester LC,TerronMP,Flores LJ,ReiterRJ.

One molecule, many derivatives: a never-ending interaction of

melatonin with reactive oxygen and nitrogen species?

J Pineal Res2007; 42:2842.

10. Tan DX, Hardeland R, Manchester LC et al. The chang-

ing biological roles of melatonin during evolution: from an

antioxidant to signals of darkness, sexual selection and fitness.

Biol Rev 2010; 85:607623.

11. Reiter RJ, Paradies SO, Manchester LC, Tan DX.

Reducing oxidative/nitrosative stress: a newly discovered genre

for melatonin. Crit Rev Biochem Mol Biol 2009; 44:175200.

12. Jou MJ, Reng TI, Hsu LF et al. Visualization of melatonins

multiple levels of protection against mitochondrial Ca2+-

mediated permeability transition and beyond in rat brain

astrocytes. J Pineal Res 2010; 48:2038.

13. Paradies G, Petnosillo G, Paradies V, Reiter RJ,

Ruggiero FM. Melatonin, cardiolipin and mitochondrial

bioenergetics in health and disease. J Pineal Res 2010; 48:297

319.

14. Leon J, Acuna-Castroviejo D, Escames G, Tan DX,

Reiter RJ. Melatonin mitigates mitochondrial malfunction.J Pineal Res 2005; 38:19.

15. Reiter RJ, Acuna-Castroviejo D, Tan DX, Burkhardt S.

Free radical-mediated molecular damage. Mechanisms for the

protective actions of melatonin in the central nervous system.

Ann N Y Acad Sci 2001; 939:200215.

16. Oragicevi N, Copes N, Oneal-Moffit G et al. Melatonin

treatment restores mitochondrial function in Alzheimers mice:

a mitochondrial protective role of melatonin membrane

receptor signaling. J Pineal Res 2011; 51:7586.

17. Tan DX, Manchester LC, Reiter RJ, Qi W, Kim SJ,

El-Sokkary GH. Ischemia/reperfusion-induced arrhythmias

in an isolated rat heart: prevention by melatonin. J Pineal Res

1998; 25:184191.

18. Ozturk M, Ozler M, Kurt YG et al. Efficacy of melatoninin mercaptoethyl guanidine and 1800W in doxorubicin-and

trastuzumab-induced cardiotoxicity. J Pineal Res 2011; 50:

8996.

19. Forman K, Vara E, Garcia C et al. Beneficial effects of

melatonin on cardiological alterations in a murine model of

accelerated aging. J Pineal Res 2010; 49:312320.

20. Dominguez-Rodriguez A, Abreu-Gonzalez P, Sanchez-

Sanchez JJ et al. Melatonin and circadian biology in human

cardiovascular disease. J Pineal Res 2010; 49:1422.

21. Chretien D, Pourrier M, Bourgeron T et al. An improved

spectrophotometric assay of pyruvate dehydrogenase in lactate

deheydrogenase contaminatedmitochondrial preparationsfrom

human skeletal muscles. Clin Chem Acta 1995; 240:129136.

22. Duncan MJ, Fraenkel DG. Alpha-ketoglutarate dehydroge-nase mutantofRhizobiummeliloti. J Bacteriol1979;37:415419.

23. Veeger C, Vartanian DV, Zeylemaker WP. Succinate

dehydrogenase. Meth Enzymol 1969; 13:8190.

24. Goyal N, Srivastava VML. Oxidation and reduction of

cytochrome c by mitochondrial enzymes of Setaria cervi.

J Helminth 1995; 69:1317.

25. Bandyopadhyay D,GhoshG,Bandyopadhyay A,ReiterRJ.

Melatonin protects against piroxicam-induced gastric ulcera-

tion. J Pineal Res2004; 36:195203.

26. Laemmli UK. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 1970;

27:680685.

Mukherjee et al.

178


14/14

27. Strittmatter CF. Studies on avian xanthine dehydrogenases:

properties and patterns of appearance during development.

J Biol Chem 1965; 240:25572564.

28. Varcoe JS. Clinical Biochemistry: Techniques and Instru-

mentation A Practical Approach, 1st edn. World Scientific

Publishing Company, Singapore, 2001.

29. Buege JA, Aust SD. Microsomal lipid peroxidation. Meth

Enzymol 1978; 52:302310.

30. Sedlak J, Lindsay RH. Estimation of total protein bound

and non-protein sulfhydryl groups in tissue with Ellmans re-

agent. Anal Biochem 1968; 25:192205.

31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Pro-

tein measurement with Folin phenol reagent. J Biol Chem

1970; 193:265275.

32. Connelly KA, Prior DL, Kelly DJ, Feneley MP, Krum H,

Gilbert RE. Load-sensitive measures may overestimate global

systolic function in the presence of left ventricular hypertrophy:

a comparison with load-insensitive measures. Am J Physiol

Heart Circ Physiol 2006; 290:H1699H1705.

33. Ghose Roy S, De P, Mukherjee D et al. Excess of gluco-

corticoid induces cardiac dysfunction via activating angioten-

sin II pathway. Cell Physiol Biochem 2009; 24:110.

34. Noronha-Dutra AA, Steen EM, Woolf N. The earlychanges induced by isoproterenol in the endocardium and

adjacent myocardium. Am J Pathol 1984; 114:231239.

35. Lucchesi BR. Modulation of leukocyte-mediated myocardial

reperfusion injury. Annu Rev Physiol 1990; 52:561.

36. Fariss MW, Chan CB, Patel M et al. Role of mitochondria

in toxic oxidative stress. Mol Interv 2005; 5:94111.

37. Wallace DC. A mitochondrial paradigm of metabolic and

degenerative diseases, and aging: a dawn for evolutionary

medicine. Annu Rev Genet 2005; 39:359407.

38. Martin M, Macias M, Leon J, Escames G, Khaldy H,

Acuna-Castroviejo D. Melatonin increases the activity of the

oxidative phosphorylation enzymes and the production of

ATP in rat brain and liver mitochondria. Int J Biochem Cell

Biol 2002; 34:348357.39. Reiter RJ, Tan DX, Manchester LC, El-Sawi MR. Mela-

tonin reduces oxidant damage and promotes mitochondrial

respiration: implications for aging. Ann N Y Acad Sci 2002;

959:238250.

40. Zamzami N, Larochetti N, Kroemer G. Mitochondrial

permeability transition in apoptosis and necrosis. Cell Death

Differ 2005; 12:14781480.

41. Leon J, Acuna-Castroviejo D, Sainz RM et al. Melatonin

and mitochondrial function. Life Sci 2004; 75:765790.

42. Luchetti F, Betti M, Canonico B et al. ERK MAPK acti-

vation mediates the antiapoptotic signaling of melatonin

inUVB-stressed U937 cells. Free Radic Biol Med 2009; 46:339

351.

43. Radogna F, Cristofanon S, Paternoster L et al. Melato-nin antagonizes the intrinsic pathway of apoptosis via mito-

chondrial targeting of Bcl-2. J Pineal Res 2008; 44:316325.

44. Galano A, Tan DX, Reiter RJ. Melatonin as a natural ally

against oxidative stress: a physicochemical examination.

J Pineal Res 2011; 51:116.

45. Acuna-CastroviejoD,EscamesG,RodriguezMI,LopezLC.

Melatoninrole in themitochondrialfunction. FrontBiosci2007;

12:947963.

46. Zhang GX, Kimura S, Nishiyama A. Cardiac oxidative

stress in acute and chronic isoproterenol-infused rats. Car-

diovasc Res 2005; 65:230238.

47. Li C, Jackson RM. Reactive species mechanisms of cellular

hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002;

282:227241.

48. Singal PK, Kapur N, Dhillon KS, Beamish RE, Dhalla

NS. Role of free radicals in catecholamine-induced cardio-

myopathy. Can J Physiol Pharmacol 1982; 60:13901397.

49. Segura-Aguilar J, Lind C. On the mechanism of the Mn3+

induced neurotoxicity of dopamine. prevention of quinine-

derived oxygen toxicity of DT diaphorase and superoxide

dismutase. Chem Biol Interact 1989; 72:309324.

50. Winiarska K, Fraczy KT, Malinska O, Drozak J, Bryla

J. Melatonin attenuates diabetes-induced oxidative stress in

rabbits. J Pineal Res 2006; 40:168176.

51. Reiter RJ, Tan DX, Sainz RM, Mayo JC. Melatonin pro-

tects the heart against both ischemia/reperfusion injury and

chemotherapeutic drugs. Cardiovasc Drugs Ther 2002; 16:56.

52. Kaneko S, Okumura K, Numaguchi Y et al. Melatonin

scavenges hydroxyl radical and protects isolated rat hearts

from ischemic reperfusion injury. Life Sci 2000; 67:101112.

53. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, Reiter RJ. Relation of nocturnal melatonin

levels to serum matrix metalloproteinase-9 concentrations in

patients with myocardial infarction. Thromb Res 2007;

120:361366.

54. Bertuglia S, Reiter RJ. Melatonin reduces ventricular

arrhythmias and preserves capillary perfusion during ischemia-

reperfusion events in cardiomyopathic hamsters. J Pineal Res

2007; 42:5563.

55. Korkmaz AA, Reiter RJ, Topal T et al. Melatonin: an

established antioxidant worthy of use in clinical trials. Mol

Med 2009; 15:4350.

56. Reiter RJ, Tan DX. Melatonin and cardiac pathophysiology.

Heart Metab 2009; 44:3134.

57. Tan DX, Chen LD, Poeggeler B et al. Melatonin: a potent,endogenous hydroxyl radical scavenger. Endocr J 1993; 1:

5760.

58. Hardeland R, Tan DX, Reiter RJ. Kynuramines, metabo-

lites of melatonin and other indoles: the resurrection of an

almost forgotten class of biogenic amines. J Pineal Res 2009;

47:109126.

59. Reiter RJ, Korkmaz A. Clinical aspects of melatonin. Saudi

Med J 2008; 29:15371547.

60. Reiter RJ, Tan DX, Sainz RM et al. Melatonin: reducing the

toxicity and increasing the efficacy of drugs. J Pharm Phar-

macol 2002; 54:12991321.

61. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia

Gonzalez MJ et al. A unicenter, randomized, double-blind,

parallel-group, placebo-controlled study of melatonin as anadjunct in patients with acute myocardial infarction undergo-

ing primary angioplasty the melatonin adjunct in the acute

myocardial infarction treated with angioplasty (MARIA) trial:

study design and rationale. Contemp Clin Trials 2007; 28:532

539.

62. Bandyopadhyay D, Chattopadhyay A. Reactive oxygen

species-induced gastric ulceration: protection by melatonin.

Curr Med Chem 2006; 13:11871202.


179

Documents

Debasri paper 2012