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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
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olecular,Biological,Physiolog
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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
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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.
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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
means S.E.M. of eight rats in each
group; *P < 0. 001 ve rsu s CON.
**P < 0.001 versus I.
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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
means S.E.M. of eight rats in each
group; *P < 0.00 1 ve rsu s CON.
**P < 0.001 versus I.
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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.
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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
Isoproterenol (mg/kg)+melatonin (mg/kg)
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
pixeldensity(arbitraryunit)
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.
Melatonin protection against myocardial injury
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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
pixeldensity(arbitraryunit)
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
Isoproterenol (mg/kg)+melatonin (mg/kg)
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
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.
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
pixeldensity(arbitraryunit)
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
Isoproterenol (mg/kg)+melatonin (mg/kg)
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.
Melatonin protection against myocardial injury
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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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*
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50
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**
nm
olhydroxylradical
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treated with isoproterenol (ISO) with or without melatonin
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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#
dP/dt max(mmHg/s)
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dP/dt min
(mmHg/s)
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