J. Pineal Res.

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

2012 John Wiley & Sons A/S

Journal of Pineal Research

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Melatonin protects against isoproterenol-induced alterations in cardiac mitochondrial energy-metabolizing enzymes, apoptotic proteins, and assists in complete recovery from myocardial injury in rats
Abstract: The present study was undertaken to explore the protective eect 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and to test whether melatonin has a role in preventing myocardial injury and recovery when the ISO-induced stress is withdrawn.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-specic 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 ndings indicate melatonins ability to provide cardio protection at a low pharmacological dose and its role in the recovery process. Melatonin, a molecule with very low or 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
1 Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University College of Science and Technology, University of Calcutta, Kolkata, India; 2 Molecular Endocrinology Laboratory, Indian Institute of Chemical Biology, Kolkata, India; 3 Department 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: debasish63@gmail.com Received December 9, 2011; Accepted February 1, 2012.

Introduction
Ischemic heart disease (IHD), a health problem of global concern, is characterized by a reduced blood supply (ischemia) to the heart muscle, usually because of coronary artery disease (atherosclerosis of the coronary arteries) [1]. A disparity between the oxygen requirement of the myocardium 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, and is more common in men than in women [2]. Studies have indicated the involvement of reactive oxygen species (ROS) in myocardial ischemia [3]. In addition to their ability to directly inict damage upon cellular macromolecules, ROS play a signicant role in 166

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 catecholamine 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-5methoxytryptamine), is a highly evolutionarily conserved molecule, present in virtually all organisms including plants and animals [8, 9]. Melatonin has several important

Melatonin protection against myocardial injury physiological functions in mammals including seasonal reproductive regulation, immune enhancement, and regulation of lightdark signal transduction along with the capacity to inuence some aspects of aging [10]. Additionally, melatonin has widespread antioxidant actions [11]. The antioxidant properties of melatonin and its possible regulatory eects on ROS production and redox signaling have been proposed to play a key role in antagonizing the mitochondrial pathway of apoptosis [12, 13]. In the recent years, several ndings support the antioxidant eect as well as a direct role of melatonin in mitochondrial homeostasis [14]. This latter action of melatonin may contribute to melatonins protective eects in degenerative disorders such as Parkinsons disease, Alzheimer disease, epilepsy, aging, ischemia-reperfusion and sepsis, all of which involve mitochondrial dysfunction as a primary or secondary cause of the disease [15, 16]. Melatonins ability to provide protection to the heart has been shown in dierent models of oxidative stress [1720] and is an emerging area of research. Earlier, we found that pretreatment for rats with melatonin at a dose of 10 mg/kg BW, administered intraperitoneally, protected against ISO-induced myocardial ischemic injury [5]. Although the protection aorded by melatonin was signicant 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 eects through antioxidant mechanisms were clearly shown, it remained to be deciphered whether ISO-induced myocardial 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 of myocardial 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 its antioxidant eects. 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 current 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 ISOdamaged heart by its direct and indirect antioxidant mechanism(s) as well as via the control of mitochondrial ROS generation and stress-activated signaling pathways, thereby raising the possibility of it being used as a therapeutic against IHD. 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), glutaraldehyde, 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 ferricyanide (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), Mumbai, 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-jB p65 antibody was obtained from Cell Signaling Technology Inc., Danvers, MA, USA. Donkey anti-goat and goat anti-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 the vehicle-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 was discontinued. 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.

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

167

Mukherjee et al. The measurement of the activities of the mitochondrial Krebs cycle enzymes Cardiac tissue was homogenized (10%) in ice-cold 50 mm phosphate buer, 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 buer and used for assaying the mitochondrial enzymes. Pyruvate dehydrogenase activity was measured spectrophotometrically according to the method of Chretien et al. [21] with some modications by following the reduction of NAD+ to NADH at 340 nm using 50 mm phosphate buer, 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. One milliliter assay volume contained 50 mm phosphate buer, 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 measured 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 buer, pH 7.4 as assay buer, 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 reduction of potassium ferricyanide (K3FeCN6) at 420 nm according to the method of Veeger et al. [23] with some modications. One ml assay mixture contained 50 mm phosphate buer, 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 measured spectrophotometrically by following the reduction in oxidized 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 buer, 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 spectrophotometrically 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 buer, 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 modications. Briey, the LV was homogenized in a buer 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 homogenate was centrifuged to separate the nuclear, mitochondrial, and cytosolic fractions. The dierent 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 from the 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 buer. After transfer, the membranes were blocked using 10% nonfat dried milk in Tris-buered 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-buered 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 further washing twice with TBS-T for 15 min. The immunoreactive bands were detected with alkaline phosphatase buer (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 quantied 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 enzyme activity 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 modications. The enzyme activity was expressed as Units/mL. The cardiac-specic Type 1 isoform of lactate dehydrogenase (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.

168

Melatonin protection against myocardial injury 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 and Aust [29] with some modication as adopted by Bandyopadhyay et al. [25]. Briey, the homogenate 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 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 and Lindsey [30] with some modications [25]. Cardiac tissue was homogenized (10%) in 2 mm ice-cold ethylenediaminetetraacetic acid (EDTA). The homogenate was mixed with TrisHCl buer, pH 9.0, followed by DTNB for color development. The absorbance was measured at 412 nm using a UVVIS spectrophotometer to determine GSH content. Values were expressed as nmoles/mg protein. Estimation of proteins Proteins of the dierent 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 identied and ligated cranially. A miniaturized conductance 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 parameters were then calculated (Millar analysis software PVAN 3.4). Each experiment was repeated at least with three animals. Histological studies The extirpated hearts were xed immediately in 10% formalin and embedded in paran 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, and images 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 measured and expressed as the % collagen volume [33]. The cardiac tissue sections were processed for scanning electron microscopy according to standard procedures with some modications [34]. Briey, LV sections were xed with 2.5% glutaraldehyde in 50 mm phosphate buer pH 7.2 and kept overnight. The sections were washed with several changes of 50 mm phosphate buer pH 7.2 and then dehydrated rst 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 evaluation Each experiment was repeated at least three times with dierent rats. Data are presented as means S.E.M. The level of signicance was calculated using one-tailed Students t-test.

Results
Fig. 1(A) depicts a signicant decrease in the activity of pyruvate dehydrogenase (PDH) (P < 0.001 versus control), the enzyme that couples glycolysis to tricarboxylic acid (TCA) cycle. Pretreatment for the rats with melatonin signicantly ameliorated the ISO-induced eects (31% increase versus I P < 0.01). Fig. 1(BD) show the ISOinduced decrease (P < 0.001 versus control) in the activity of the TCA cycle enzymes ICDH, a-ketoglutarate dehydrogenase (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 signicantly (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 rats with melatonin (P < 0.001 versus I). ISO-induced inhibition of mitochondrial TCA cycle and respiratory chain enzymes leads to the leakage of cytochrome 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 dierence 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 < 0.001 versus control) of the apoptotic protease activating factor 1 169

Mukherjee et al.
8

(A)

12

(B)

Pyruvate dehydrogenase activity (Units/mg protein)

7 6 5 4 3 2 1 0

** *

Isocitrate dehydrogenase activity (Units/mg protein)

10
8 6 4 2 0

** *

CON

I+m

CON

I+m

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

5.0

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

(C)
80

(D)
Succinate dehydrogenase activity (Units/mg protein)

Alpha-ketoglutarate dehydrogenase activity (Units/mg protein)

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

#
70 60 50 40 30 20 10 0

*
CON I I+m

CON

I+m

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

12

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

0.9

Fig. 1. Protective eect of melatonin against ISO-induced decrease in the activities of (A) pyruvate dehydrogenase, (B) isocitrate dehydrogenase, (C) a-ketoglutarate dehydrogenase, and (D) succinate 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 < 0.01 versus I; #P < 0.001 versus I.

(A)

(B)

NADH-Cytochrome c reductase (Units/mg protein)

Cytochrome c oxidase activity (Units/mg protein)

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

CON I I+m

10 8 6 4 2 0
CON I I+m

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

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

Fig. 2. Protective eect of melatonin against ISO-induced decrease in the activities of (A) NADH-cytochrome c oxidoreductase and (B) cytochrome oxidase 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.

(A) Con

I+m

Con

I+m

Cytochrome c (cytoplasm) Con I

Cytochrome c (mitochondria) I+m Actin

(B)
Cytochrome c (cytoplasm) Pixel density (arbitrary unit)

90 80 70 60 50 40 30 20 10 0 CON

**

(C)
Cytochrome c (mitochondria) Pixel density (arbitrary unit)
180 160 140 120 100 80 60 40 20 0 CON I I+m

I+m

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

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

Fig. 3. (A) Representative results of Western blot analysis for determining the level of cytoplasmic and mitochondrial cytochrome c (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 [(B) for cytoplasmic and (C) for mitochondrial] obtained through Western blotting was quantied with ImageJ software (NIH), and the values (means S.E.M.) were presented below in the form of a bar graph. **P < 0.01 versus CON.

170

Melatonin protection against myocardial injury

(A)
Con I I+m Apaf-1 Actin
60 40

(B)

Con

I+m Caspase9 Actin

Caspase 9 Pixel density (arbitrary unit)

Apaf-1 Pixel density (arbitrary unit)

35 30 25 20 15 10 5 0
CON I

50 40 30 20 10 0

*
**

I+m

CON

I+m

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

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

Fig. 4. (A) Representative result of Western blot analysis for determining the level of apoptotic protease activating factor (Apaf)-1 (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 quantied 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 < 0.001 versus I. (B) Representative result of Western blot analysis for determining the level of caspase9. Actin served as loading control. The pixel density of bands obtained through Western blotting was quantied 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 < 0.01 versus I.

(A)

Con

I+m ERK2 Actin

(B)

Con

I+m pP38 Actin

Fig. 5. Western blot analysis of levels of (A) ERK 2 and (B) phosphorylated P 38 of heart tissue in control (CON), ISOtreated (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 quantied 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 < 0.01 versus I.

160

*
pP38 Pixel density (arbitrary unit)

70 60 50 40 30 20 10 0

ERK 2 Pixel density (arbitrary unit)

**

140 120 100 80 60 40 20 0 CON I I+m

CON

I+m

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

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

(Apaf-1), the protein involved in the formation of apoptosome complex with cytochrome c that cleaves Procaspase 9 to its activated form. This ISO-induced elevation of Apaf-1 level is decreased signicantly by melatonin (P < 0.001 versus I). Fig. 4(B) shows the signicant 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 eect of ISO on the stress-activated proteins ERK2, P38, HSP70, and P53 as well as transcription factors cJUN and NFjB. Figs 5(A,B), 6(A) and (B) demonstrate that ISO signicantly 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 signicant 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 control values. Fig. 7(A) shows the ISO-induced elevation in the level of the transcription factor cJUN, which was found to be signicantly lowered when the rats were pretreated with melatonin. The level of NFjB, another important transcription 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 myocardial injury at the dose of 25 mg/kg BW s.c., as is evident from a signicant increase in SGOT activity (P < 0.001 171

Mukherjee et al.
(A)
Con I I+m HSP-70 Actin
50

(B)

Con

I+m pP53 Actin

HSP-70 Pixel density (arbitrary unit)

**
80 60 40 20 0 CON I I+m

pP 53 Pixel density (arbitrary unit)

100

*
40

#
30

20

10

0 CON I I+m

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

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

Fig. 6. Representative result of Western blot analysis for determining the level of (A) HSP 70 and (B) phosphorylated P 53 of heart tissue (lanes from left) 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 quantied 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 < 0.01 versus I; #P < 0.001 versus I.

(A)

Con

I+m c-Jun (nucleus) Actin

(B)

Con

I+m NF (nucleus) Actin

80

c-Jun Pixel density (arbitrary unit)

70 60 50 40 30 20 10 0 CON

NFkB (nucleus) Pixel density (arbitrary unit)

* **

60 50 40 30 20 10 0

**

I+m

CON

I+m

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

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

Fig. 7. Western blot analysis of levels of (A) cJUN and (B) NFjB 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 quantied 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 < 0.01 versus I.

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 signicantly (P < 0.001 versus 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 the withdrawal of ISO, there was a gradual reduction in the activity of SGOT, which was found to be statistically signicant 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 were continued 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 conrmed 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-specic Type 1 isoform (LDH1) (Fig. 8C). Both the gures show a signicant increase in enzyme activity following ISO treatment (P < 0.001 versus control), which was found to be signicantly decreased when the rats were pretreated with melatonin. However, the 172

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 ISOtreated rat hearts at 20 magnication (Fig. 9A) showed myo-degeneration as characterized by a loss of cardiac myobers and a mononuclear cell inltration. However, when the rats were pretreated with melatonin, the ISOinduced degenerative changes in the myocardial tissue were found to be signicantly lower. The recovery from the tissue 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 signicant reduction in the level of a-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 of a-actinin to almost control levels.

Melatonin protection against myocardial injury

(B)
7 6.5 6

Serum lactate dehydrogenase (LDH) activity

#I
Iw 2D

IU/L

(A)
14

*
Iw 4D

Serum glutamate oxaloacetate transaminase activity

#
I Iw 2D Iw 4D

5.5 5 4.5 4
CON

*
I+m

**
Iw + m 2D

12

## ^
Iw + m 4D

IU/L

*
10
CON I+m

* **
Iw + m 2D

0 Day

2 Day

4 Day

6 Day

##
Iw + m 4D

Duration (days) of isoproterenol bitartrate & melatonin

(C) 0.3
0.25

Serum LDH1 activity

#
I Iw 2D

0 Day

2 Day

4 Day

6 Day

IU/L

Duration (days) of isoproterenol bitartrate & melatonin

*
Iw 4D

0.20 0.15 0.1

I* m +

**
Iw + m 2D

## ^
Iw + m 4D

CON

0 Day

2 Day

4 Day

6 Day

Duration (days) of isoproterenol bitartrate & melatonin

Fig. 8. (A) Protective eect of melatonin against ISO induced alterations of SGOT activity. The rats were treated with ISO (I) at a dose of 25 mg/kg body weight s.c. for 2 days. ISO treatment was then discontinued, and the rats were killed after 2days (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 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). 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 < 0.01 versus Iw 2D; ^P < 0.01 versus Iw 4D; ##P < 0.001 versus I + m. (B) Protective eect of melatonin against 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 eect of melatonin against ISO induced elevation of cardiac-specic LDH 1 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.

(A)

CON

ISO

Iw 2D

Iw 4D

200

Fig. 9. (A) Representative images (200 magnication) of hematoxylin/eosinstained left ventricular longitudinal sections 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) Representative result of Western blot analysis for 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 at least three times. Actin served as loading control. (C) The pixel density of bands obtained through Western blotting and quantied with ImageJ software (NIH), and the values (means S.E.M.) presented in the form of a bar graph. *P < 0.001 versus CON; #P < 0.001 versus I.

MEL

I+m

Iw + m 2D

Iw + m 4D

200

(C) (B)
Con I I + m Iw + m Iw + m (2D) (4D)

140 120 100 80 60 40 20 0

Alpha-actinin Pixel density (arbitrary unit)

Alpha-actinin Actin

Con

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

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

173

Mukherjee et al. The cytoarchitectural damage to the cardiac tissue because of ISO treatment was further conrmed 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 timedependent manner when the animals were initially pretreated with melatonin, and then the melatonin treatment was 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 pronounced 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 signicantly prevented when the rats were pretreated 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 signicant increase in the level of lipid peroxidation (LPO) measured as TBARS in the cardiac tissue (Fig. 12A, P < 0.001 versus control). Only slight dierence to the elevated LPO levels was found even when the rats were left untreated for a further 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 signicant 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 timedependant 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-ISO treatment period.

(A)

CON

I+m

Iw + m (2D)

Iw + m (4D)

600

(B)
Fig. 10. (A) Representative images (600 magnication) of Sirius red-stained left ventricular longitudinal sections 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. Red color stretches are collagen depositions. (B) The similar images captured by confocal laser scanning microscope for quantication of brosis. Arrow heads indicate collagen bers. (C) Histogram showing % collagen volume in the ventricular tissues. Values are means S.E.M. % collagen from three images from each of three rats of each group; *P < 0.001 versus CON; ##P < 0.001 versus I; #P < 0.001 versus I + m.
I+m Iw + m (2D)
Iw + m (4D)

600

(C)
Collagen volume (%)

6 5 4 3 2 1 0
CON I I +m Iw + m (2 D)

# ##

Iw + m (4 D)

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

CON

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 membrane blebbings.

174

Melatonin protection against myocardial injury

0.07

(A) Lipid peroxidation level

nmoles GSH/mg protein
#I

35

(B)
CON

GSH level
**
Iw + m 2D

nmoles TBARS/mg protein

##
Iw + m 4D

30

0.06

*
I+m

Iw 2D

*
25
I+m Iw 2D

* **
Iw + m 2D Iw 4D

*
Iw 4D

0.05
CON

##
Iw + m ^ 4D

20

#
I

0.04 0 Day

2 Day

4 Day

6 Day

15 0 Day

2 Day

4 Day

6 Day

Duration (days) of isoproterenol bitartrate & melatonin

Duration (days) of isoproterenol bitartrate & melatonin

Fig. 12. (A) Protective eect of melatonin against ISO induced increase in lipid peroxidation (LPO) level of rat heart tissue. The rats were treated 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 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). 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 eect of melatonin against ISO induced decrease in reduced glutathione (GSH) level of rat heart tissue 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. 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 Heart rate (bpm) Pmax (mmHg) Pmin (mmHg) CO (uL/min) dP/dtmax (mmHg/s) dP/dtmin (mmHg/s) Control 367 116.60 20.10 45,971 6975 )6019 3.00 1.60 1.50 744 225 211.10 ISO 348 72.10 18.745 18,416 1913 )1510 1.00a 0.52a 0.23 211a 20a 14.10a ISO + MEL 393 116 10.00 26,775 4158 )4248 2.00b 1.50b 0.40 470b 91b 64.45b ISOwth + MEL 2 days 368 116.7 14.51 37,728 6226 )5211 2.00 1.50 0.40 402 154 137 ISOwth + MEL 4 days 371 128.4 13.0 44,594.5 7044 )5077 1.0c 2.0c 0.6 467c 95c 100c

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

As shown in Table 1, the systolic blood pressure was signicantly (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 signicantly (P < 0.001, n = 6) reduced in ISO-treated rats. Pretreatment for rats with melatonin for 2 days signicantly (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 signicantly reduced by ISO compared to control, which were restored signicantly by melatonin. These data indicate that melatonin restores the ISOinduced 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 myocytes [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 amplication of the initial ROS-mediated cardiomyocyte dysfunction and/or cytotoxicity. In our earlier study [6], we demonstrated that ISO, 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 inhibiting the 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 ISOinduced stress. However, the details of the cellular mechanisms involved in the induction of oxidative stress by ISO and 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 eect 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 signicantly inhibited in ISO-treated rats. Pretreatment for rats with melatonin 175

Mukherjee et al. signicantly 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 stressinduced dysfunction has been implicated in the pathogenesis of aging, cancer, diabetes, ischemia/reperfusion injury, neurodegenerative disorders, and other diseases [37]. A reduction in the activity of NADH-cytochrome c reductase (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 following ISO treatment. Inhibition of mitochondrial TCA cycle and respiratory chain enzymes leads to a leakage of electrons, producing a reducing environment within the mitochondria, thereby generating free radicals [38, 39]. These free radicals, particularly the free OH radical, can damage the mitochondrial 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 ISO treatment for rats cause a highly signicant 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 signicant 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 in the 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 membrane 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 our experiments prompted us to investigate whether ISO treatment for rats induces any apoptotic and/or stress signaling protein. We found a signicant 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 signicantly 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 the mitochondrial level into the so-called intrinsic (or dam176 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 eects 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 administrations 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 protein are linked directly to intracellular redox status, while NFjB activation is involved in hypoxia-reoxygenation injury, especially in the vascular endothelium, where NFjB activation 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 stress induced because of ISO. Melatonin and its metabolites have been proposed to regulate ROS uxes and provide mitochondrial protection [42]. The current work also indicates toward melatonins capability of inuencing the activation of fundamental signaling pathways. Even though our current study explored the role of melatonin in protecting ISO-induced mitochondrial dysfunction and activation of stress-activated apoptotic and signaling pathways, it was found that, in case of many of the parameters studied, the restoration by melatonin, at the present dose and duration, although signicant, 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 pretreatment 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) days after the withdrawal of ISO treatment (after the 2nd dose) could lead to complete recovery of the cardiac tissue after ISO-induced damage. To conrm 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 corresponding 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-specic Type 1 isoform of LDH (LDH1). A signicant increase in the activity of all these biomarker

Melatonin protection against myocardial injury enzymes (Fig. 8) indicated the induction of myocardial injury. Pretreatment for the rats with melatonin partially, although signicantly, ameliorated these eects. 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 from the histopathological studies of the myocardial tissue. Our studies on hematoxylin/eosin-stained cardiac tissue sections showed signicant cellular damage and degeneration following 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 conrmed by the expression level of one of the important structural proteins of cardiac tissue of rat, the a-actinin, which was signicantly 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 observed in the control rats after 2 days of treatment as was also evident in our earlier work [6]. The reason for this may be that for complete restoration, the dose of melatonin was insucient 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 the fact that on continuing melatonin treatment for 2 and 4 days, respectively, after discontinuation of ISO treatment, a complete restoration of a-actinin was found in the cardiac tissue samples from the rats. Thus, the pattern of restoration was found to be time-dependant. Additionally, the depletion of collagen in the cardiac tissue following ISO treatment for rats was found to be partially but signicantly 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 oxidative 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 signicant increase in LPO level in the cardiac tissue following ISO treatment. This may be due to the oxidation 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 catecholamines [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 and deamination catalysis among other deleterious eects. 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 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 [49]. Our studies further indicate that the continuation 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 signicant reduction in the GSH content of cardiac tissue. Pretreatment for rats with melatonin signicantly restored the GSH levels of the cardiac tissue indicating that melatonin 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 Mukherjee et al. [6]. Melatonin has been shown to restore the GSH levels of tissues in various models of oxidative stress, perhaps, through its stimulatory eect on GSH synthesis [50]. However, continuation of melatonin treatment for two and 4 days after the withdrawal of ISO helped the tissue to regain its GSH content as observed in the control tissues indicating again melatonins ability to promote cellular physiological 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 parameters of cardiac function studied, particularly heart rate, CO, and systolic and diastolic pressure to that observed in control animals, indicating melatonins ecacy in improving heart function even in postinfarction period. These observations suggest that melatonin could have a potential clinical application in the treatment for myocardial 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 decreases melatonin synthesis remains to be determined [54]. Many 177

Mukherjee et al. of the drugs used in the treatment for dierent cardiac diseases do possess various side eects, 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 antioxidants [25, 5759]. They also reduce the toxicity of dierent drugs [60, 61]. Moreover, pharmacological doses of melatonin possess very low or no toxicity [62]. Therefore, it will be 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.
and binding to melatonin receptors in vertebrates. Biochem Mol Biol Int 1999; 35:327334. Tan DX, Manchester LC, Terron MP, Flores LJ,Reiter RJ. One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? J Pineal Res 2007; 42:2842. Tan DX, Hardeland R, Manchester LC et al. The changing biological roles of melatonin during evolution: from an antioxidant to signals of darkness, sexual selection and tness. Biol Rev 2010; 85:607623. 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. 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. 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. Leon J, Acuna-Castroviejo D, Escames G, Tan DX, Reiter RJ. Melatonin mitigates mitochondrial malfunction. J Pineal Res 2005; 38:19. 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. 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. 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. Ozturk M, Ozler M, Kurt YG et al. Ecacy of melatonin in mercaptoethyl guanidine and 1800W in doxorubicin-and trastuzumab-induced cardiotoxicity. J Pineal Res 2011; 50: 8996. Forman K, Vara E, Garcia C et al. Benecial eects of melatonin on cardiological alterations in a murine model of accelerated aging. J Pineal Res 2010; 49:312320. Dominguez-Rodriguez A, Abreu-Gonzalez P, SanchezSanchez JJ et al. Melatonin and circadian biology in human cardiovascular disease. J Pineal Res 2010; 49:1422. Chretien D, Pourrier M, Bourgeron T et al. An improved spectrophotometric assay of pyruvate dehydrogenase in lactate deheydrogenase contaminated mitochondrial preparations from human skeletal muscles. Clin Chem Acta 1995; 240:129136. Duncan MJ, Fraenkel DG. Alpha-ketoglutarate dehydrogenase mutant of Rhizobium meliloti. J Bacteriol 1979; 37:415419. Veeger C, Vartanian DV, Zeylemaker WP. Succinate dehydrogenase. Meth Enzymol 1969; 13:8190. Goyal N, Srivastava VML. Oxidation and reduction of cytochrome c by mitochondrial enzymes of Setaria cervi. J Helminth 1995; 69:1317. BandyopadhyayD,GhoshG,BandyopadhyayA, ReiterRJ. Melatonin protects against piroxicam-induced gastric ulceration. J Pineal Res 2004; 36:195203. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 27:680685.

9.

10.

11.

12.

13.

Acknowledgements
Debasri Mukherjee gratefully acknowledges the receipt of a Senior 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 is partially 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 gratefully acknowledged. The help from Bose Institute, Kolkata, is also gratefully acknowledged.

14.

15.

16.

17.

18.

References
1. Tengattini S, Reiter RJ, Tan DX et al. Cardiovascular diseases: protective eects 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 disease: protection by vitamin E. Pharmacol Rep 2006; 58:179 187. 4. Peyrot F, Ducrocq C. Potential role of tryptophan derivatives in stress responses characterized by the generation of reactive oxygen and nitrogen species. J Pineal Res 2008; 45:235244. 5. BandyopadhyayD,ChattopadhyayA,GhoshG,DattaAG. Oxidative stress-induced ischemic heart disease: protection by antioxidants. Curr Med Chem 2004; 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. Eect of isoproterenol on lipid peroxidation and antioxidant enzymes of myocardial tissue of mice and protection by quinidine. Mol Cell Biochem 2003; 245:4349. 8. Hattori A, Migitaka H, Iigo M et al. Identication of melatonin in plants and its eects on plasma melatonin levels 19.

20.

21.

22. 23. 24.

25.

26.

178

Melatonin protection against myocardial injury

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 Instrumentation A Practical Approach, 1st edn. World Scientic 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 reagent. Anal Biochem 1968; 25:192205. 31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein 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 glucocorticoid induces cardiac dysfunction via activating angiotensin II pathway. Cell Physiol Biochem 2009; 24:110. 34. Noronha-Dutra AA, Steen EM, Woolf N. The early changes 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. Melatonin 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 Dier 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 activation 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. Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial 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. Melatonin role in the mitochondrial function. Front Biosci 2007; 12:947963. 46. Zhang GX, Kimura S, Nishiyama A. Cardiac oxidative stress in acute and chronic isoproterenol-infused rats. Cardiovasc 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 cardiomyopathy. 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 quininederived 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 protects 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, GarciaGonzalez 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 ischemiareperfusion 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, metabolites 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 ecacy of drugs. J Pharm Pharmacol 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 an adjunct in patients with acute myocardial infarction undergoing 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