Activated Receptor- Draft Corresponding author: Dr. Ola Ahmed El-Gohary ... proliferator-activated receptor gamma ... 2.3. ECG monitoring:

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Effect of vitamin D on isoprenaline induced myocardial

infarction in rats; possible role of Peroxisome Proliferator Activated Receptor- ɣ (PPAR-ɣ)

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0150.R2

Manuscript Type: Article

Date Submitted by the Author: 20-Jul-2016

Complete List of Authors: El-Gohary, Ola; Benha University, physiology allam, mona; B

Keyword: vitamin D, isoprenaline, myocardial infarction, Peroxisome Proliferator Activated Receptor- ɣ, oxidative stress.

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Title page

Effect of vitamin D on isoprenaline induced myocardial infarction in rats; possible

role of Peroxisome Proliferator Activated Receptor- ɣ (PPAR-ɣ)

Ola Ahmed El-Gohary and Mona Maher Allam

Physiology department, Faculty of Medicine, Benha University, Egypt.

Running title: Effect of vitamin D on isoprenaline induced myocardial infarction in rats and

role of PPAR-ɣ.

Key words: vitamin D, isoprenaline, myocardial infarction, Peroxisome Proliferator

Activated Receptor- ɣ, oxidative stress.

Name of Authors:

1. Ola Ahmed El-Gohary ([email protected],

[email protected] , [email protected]) – Benha Faculty of

Medicine – Benha, Egypt

2. Mona Maher Allam ([email protected]) – Benha Faculty of

Medicine – Benha, Egypt

The Corresponding author: Dr. Ola Ahmed El-Gohary

E.Mail: [email protected] ,

[email protected],

[email protected]

Mobile: 00201284499665.

Address: Physiology department, Benha Faculty of Medicine, Benha, Qualubia, Egypt.

(www.fmed.bu.edu.eg)

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Effect of vitamin D on isoprenaline induced myocardial infarction in rats; possible

role of Peroxisome Proliferator Activated Receptor- ɣ (PPAR-ɣ)

Ola A. EL-Gohary and Mona M. Allam

Department of Physiology

Faculty of Medicine, Benha University, Benha, Egypt.

Abstract:

Infarct-like lesion induced by isoprenaline is a well-known model to study myocardial

infarction (MI). Vitamin D has been shown to have anti-inflammatory and antioxidant

effects. Recent studies highlighted cross talk between vitamin D and peroxisome

proliferator-activated receptor gamma (PPAR- ɣ). The present study was designed to

investigate the effect of pretreatment with vitamin D on the isoprenaline-induced infarct-

like lesion in rats and the role of PPAR- ɣ as a novel mechanism in vitamin D-mediated

cardio protective effect. Markers chosen to assess cardiac damage included serum level

of creatine kinase (CPK), lactate dehydrogenase (LDH), tumor necrosis factor-alpha

(TNF-α), and interleukin-6 (IL-6). Cardiac contents of malondialdehyde (MDA),

superoxide dismutase (SOD) and glutathione peroxidase (GSH) have been also assessed.

Furthermore, ECG monitoring and measurement of injury extension were carried out.

Isoprenaline increased the level of cardiac enzymes as well as inflammatory and

oxidative stress biomarkers. In addition, it produced ST-segment elevation. Pretreatment

with vitamin D significantly improved previous parameters. The prior treatment with

PPAR- ɣ antagonist; bisphenol A diglycidyl ether; (BADGE) significantly attenuated the

protective effect of vitamin D. In conclusion, vitamin D can be demonstrated as a

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promising cardio-protective agent in MI and PPAR- ɣ significantly contributes toward

vitamin D-mediated protection.

Key words: vitamin D, isoprenaline, myocardial infarction, Peroxisome Proliferator

Activated Receptor- ɣ, oxidative stress.

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1. Introduction:

Despite great efforts during the last decades, cardiovascular diseases (CVDs) remain

the major cause of death worldwide, increasing their prevalence every year (Basak et al.

2015). Myocardial infarction (MI) is a cardiovascular disease that occurs when the blood

supply to a part of the heart is interrupted, causing death to the heart tissue. It is a

complex phenomenon affecting the mechanical, electrical, structural, and biochemical

properties of the heart (Bharti et al. 2010; Upaganlawar et al. 2010).

Isoprenaline (ISO), a β adrenergic agonist, has been reported to induce infarct-like lesions

in rats and other animal species (Zhang et al. 2008). It has been shown to exhibit many

metabolic and morphological aberrations in the heart tissue of experimental animals

similar to those seen in humans with myocardial infarction (Panda and Naik 2009).

Injected isoprenaline undergoes auto-oxidation resulting in the generation of free radicals

that stimulate lipid peroxidation which causes destruction and damage to the myocardial

cell membrane (Loh et al. 2007; Upaganlawar et al. 2010). Inflammation is a key process

involved in mediating myocardial tissue damage through the release of proteolytic

enzymes (Jordan et al. 1999).

Vitamin D plays a classical hormonal role in skeletal structures by regulating calcium and

phosphorus metabolism. Recently, much research has focused on the cardio protective

effects of vitamin D (Brandenburg et al. 2012; Mancuso et al. 2008) and on its anti-

inflammatory and anti-oxidant actions (Shab-Bidar et al. 2014; Zittermann and Koerfer

2008), which may play a role during acute infarction. It has been shown that insufficient

vitamin D levels are linked to nonskeletal major chronic diseases, especially

cardiovascular diseases (Wang et al. 2013). Vitamin D deficiency has been found in a

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high proportion of patients with myocardial infarction (Goleniewska et al. 2014; Hlaing

et al. 2014) and has been associated with coronary heart diseases and myocardial

infarctions (Lee et al. 2011).

Vitamin D receptors have been found in all major cardiovascular cell types including

cardiomyocytes, arterial wall cells, and immune cells. It has been established that they are

closely related to the Peroxisome Proliferator Activated Receptor-ɣ (PPAR-ɣ) (Al Mheid

et al. 2013). Both vitamin D receptors and PPAR-ɣ are ligand-activated nuclear receptors.

Recently, a few in vitro studies suggested cross talk between these two nuclear receptors

with involvement of PPAR-ɣ in vitamin D mediated biological responses (Dai et al.

2008).

In spite of the accumulating data about the cardiovascular effect of vitamin D, the role of

vitamin D supplementation in the management of cardiovascular diseases remains to be

established. Hence, the present study was designed to investigate the effect of vitamin D

on the outcome of ISO- induced myocardial infarct-like lesions in rats, and also to outline

the role of (PPAR-ɣ) as a novel mechanism for this effect if found. Parameters chosen to

assess the myocardial damage and the protective effect of vitamin D included; serum

cardiac enzymes, inflammatory markers and oxidative stress parameters in addition to

ECG monitoring and measurement of injury extension.

2. Material and Methods

2.1. Animals:

This study was conducted on 50 adult Wistar albino male rats, 6-8 weeks old and

weighing between 200 and 250 g. Animals were housed in the animal laboratory at the

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Medical Research Center of Benha Faculty of Medicine. They were housed at room

temperature (25°C) and 12h/12h light/dark cycle. All Rats were fed a standard diet and

water. The study was carried out according to the guidelines of the Ethics Committee,

Faculty of Medicine, Benha University.

2.2. Experimental design:

The rats were randomly divided into five groups (n:10). Group I (Control group):

Received no medication and was given free access to food and water. Group II (Vit. D

group): Received Vitamin D ( 0.5 µg/kg; i.p.) once daily for 7 days (Akanksha et al.

2013). Group III (ISO group): Infarct-like lesion was induced in the rats by

subcutaneous injection of 100mg/ kg isoprenaline hydrochloride dissolved in saline once

daily for two successive days (Kumaran and Prince 2010). Group IV (ISO+Vit. D

group): Received Vitamin D ( 0.5 µg/kg; i.p.) once daily for 7 days. For induction of

infarct-like lesion, rats received isoprenaline (100mg/kg; s.c.) after 1 h of vitamin D

administration on the last 2 days of the treatment period. Group V (ISO+Vit. D+ BADGE

group): This group was treated with PPAR-γ antagonist; Bisphenol A diglycidyl ether

(BADGE); It was dissolved in minimal volume of ethanol, diluted with saline and

injected intraperitoneally at a dose of (30 mg/kg) given 30 min before vitamin D injection

( 0.5 µg/kg; i.p.) once daily for 7 days (Akanksha et al. 2013). For induction of infarct-

like lesion, rats received isoprenaline (100mg/kg; s.c.) after 1 h of vitamin D

administration on the last 2 days of the treatment period. Isoprenaline hydrochloride,

vitamin D and Bisphenol A diglycidyl ether (BADGE) were purchased from Sigma–

Aldrich (USA).

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24 hours after the last treatment, overnight- fasted rats were anaesthetized with urethane

(1.5 g/kg; i.p.) for ECG monitoring (Abood and Elshal 2015; Zaafan et al. 2013).

Thereafter, blood samples were collected via cardiac puncture for serum separation and

estimation of cardiac marker enzymes such as serum level of creatine kinase (CPK) and

lactate dehydrogenase (LDH) as well as inflammatory markers such as tumor necrosis

factor- α (TNF-α) and interleukin-6 (IL-6). The rats were then sacrificed by decapitation

and their hearts were rapidly isolated. The injury extension was measured in the excised

hearts. Portions of the heart tissues were used to determine the oxidative stress as

malondialdehyde (MDA) and antioxidant enzymes such as superoxide dismutase (SOD)

and glutathione peroxidase (GSH).

2.3. ECG monitoring:

The anesthetized rats were placed in the supine position on a board and ECG was

recorded continuously with standard artifact free lead II (right forelimb to left hind limb).

Needle electrodes were inserted subcutaneously into the paw pads of each rat, and

connected to Biocare ECG 101 (Shenzhen Biocare Electronics Co., Ltd., China). The

ECG was measured to determine the duration and amplitude of the P wave, the QRS

complex, and the ST segment alterations.

2.4. Measurement of extension of cardiac injury:

The excised beating heart was submerged in cold (8ºC) 30 mmol KCl to achieve diastolic

arrest. The right ventricle and both atria were excised to isolate the left ventricle (the

septum and free wall). The left ventricle was then sectioned by a sharp surgical scissor

into transverse slices, each of about 1.5mm thick. The slices were stained in a 1.5%

triphenyltetrazolium chloride (TTC) (MP biomedical, France) in phosphate buffer, PH

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7.4, for 10-15 minutes at 37ºC. The TTC stain formed red color precipitates in the

presence of an intact dehydrogenase enzyme system. Areas of necrosis lost

dehydrogenase activity and therefore failed to stain (Sharma and Singh 2000; Vivaldi et

al. 1985). The slices were washed with saline and then clear glass plates were placed over

both sides of each slice. Epicardial and endocardial outlines as well as the TTC stained

and non- stained areas were traced on clear plastic sheets. The plastic sheets were then

fixed on an E.C.G paper and the small squares occupying the stained and non-stained

areas were counted giving each in mm2. The sum of the stained and the non-stained areas

give the surface area of the whole heart slices. The surface area of the whole left ventricle

was calculated by adding the surface areas of all cardiac sections measured on E.C.G

paper. The surface area of the injured tissue of the whole heart was obtained by adding

the surface area of the injured tissue in all cardiac slices and the injury extension was

calculated as percentage of the sum of the injured areas to the sum of surface areas of all

the slices (Evans et al. 1985).

2.5. Biochemical analysis:

The serum was separated by centrifugation (5000 rpm for 5 min) and used for

biochemical analysis. Cardiac marker enzymes such as CPK and LDH were detected

using Stanbio CK-MB diagnostic kit (USA). In addition inflammatory markers such as

TNF-α and IL-6 were determined by the ELISA technique using standard kits (Ray

Biotech, Inc., USA). The sensitivities of the methods as stated in the instructions of

enzyme immunoassay kits are 12 pg/ml for IL-6 and 11.2 pg/ml for TNFα. The intra-

assay coefficient of variation for the measurements was <5%.

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Portions of the heart tissue were homogenized in a saline solution (0.9%) and centrifuged

at 3000 rpm for 15 min; the supernatant was kept at – 20 C and used to determine the

oxidative stress as level of MDA (Uchiyama and Mihara 1978) and antioxidant enzymes

such as SOD (Das et al. 2000) and GSH (Moron et al. 1979).

2.6. Statistical analysis:

All analyses were performed using the program "Statistical Package for Social Sciences

(SPSS) version 16" (SPSS Inc, Chicago, IL, USA). The data are presented as the mean ±

standard deviation (SD). Comparisons among groups, in all studied parameters, were

analyzed by using one-way analysis of variance (ANOVA) test and Post-Hoc multiple

comparisons (LSD). Probability of chance (P value) < 0.05 was considered statistically

significant.

3. Results

3.1. Effect of vitamin D on ECG parameters and extension of cardiac injury in

isoprenaline-induced infarct-like lesion in rats and the role of PPAR-ɣ (Fig. 1, Table

2):

Isoprenaline injection induced infarct-like lesion represented by ST segment elevation,

and a decrease in R wave amplitude as compared to the normal group. Pretreatment with

vitamin D resulted in a reduction in the ST segment elevation with an increase in R wave

amplitude as compared to the isoprenaline group. The BADGE pretreatment abolished

the protective effect of vitamin D in rats subjected to infarct-like lesion (Fig. 1).

As regards the extension of cardiac injury, vitamin D treatment significantly decreased

the injury extension (P<0.05) as compared to the isoprenaline group. Moreover, BADGE

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pretreatment in rats receiving vitamin D and undergone infarct-like lesion significantly

increased the injury extension (P<0.05) that was still reduced (P<0.05) as compared to

isoprenaline group. (Table 2).

3.2. Effect of vitamin D on the biochemical changes associated with isoprenaline-

induced infarct-like lesion in rats and the role of PPAR-ɣ (Table1, 2):

The levels of cardiac enzymes (CPK and LDH) in the plasma showed a significant rise

(P˂0.05) in the ISO group, ISO + Vit.D group and ISO + Vit. D + BADGE group

compared to the control group. Pretreatment with vitamin D significantly reduced the

cardiac enzymes to near normal values. On the other hand, BADGE pretreatment in the

rats that received vitamin D and subjected to infarct-like lesion significantly increased

(P<0.05) the plasma levels of cardiac enzymes that still showed significant reduction

(P<0.05) as compared to the isoprenaline group. (Table 1).

As regards pro-inflammatory cytokines, TNF-α and IL-6 increased significantly (P˂0.05)

in the ISO group, ISO + Vit.D group and ISO + Vit. D + BADGE group as compared

with the control group. However, these cytokines showed significant reduction in the

ISO-injected rats pretreated with vitamin D (P˂0.05) compared to the ISO group.

Furthermore, the pretreatment with BADGE in the rats that received vitamin D and

subjected to infarct-like lesion produced significant elevation (P˂0.05) of pro-

inflammatory cytokines that still revealed significant reduction (P<0.05) as compared to

isoprenaline group. (Table 1).

Similarly, cardiac MDA significantly increased (P˂0.05) with a parallel significant

decrease in SOD and GSH content (P˂0.05) in the ISO group, ISO + Vit.D group and

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ISO + Vit. D + BADGE group as compared to the normal rats. Pretreatment with vitamin

D resulted in a significant decrease in MDA (P˂0.05) and a significant increase in SOD

and GSH content (P˂0.05) as compared to the isoprenaline group. Similar to other

parameters, the BADGE pretreatment abolished the protective effect of vitamin D in rats

subjected to infarct-like lesion. There was a significant decrease (P<0.05) in cardiac

MDA while there was a significant increase (P<0.05) in SOD content and a non-

significant increase (P>0.05) in GSH content comparing the ISO + Vit. D + BADGE

group to the ISO group. (Table 2).

4. Discussion

MI continues to be a major health problem worldwide and contributes significantly to

mortality statistics (Gao et al. 2001). The term MI is thought to reflect death of cardiac

myocytes due to prolonged ischemia (Patel et al. 2011). In the current study, infarct-like

lesion was induced in rats by intraperitoneal injection of ISO. It has been reported that

isoprenaline administration in high doses to animals produces ‘infarct like’ lesions in the

heart (Vennila et al. 2010). The mechanisms proposed to explain isoprenaline-induced

cardiac damage include increases in heart rate, myocardial contractility, hypoxia due to

myocardial hyperactivity, depletion in energy reserve, calcium overload, excessive

production of highly cytotoxic free radicals, and mitochondrial injury or dysfunction

(Mohanty et al. 2004; Rathore et al. 2000).

In the present study, isoproterenol injection was seen to result in significant alterations in

ECG patterns such as ST segment elevation coupled with marked decrease in R wave

amplitude that reflect isoprenaline-induced infarct-like lesion. ECG pattern alterations by

isoprenaline were previously demonstrated by other investigators (Prince and Sathya

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2010; Tawfik et al. 2010). In addition to the measurement of injury extension, ECG

abnormalities were the main criteria used for the diagnosis of infarct-like lesion. These

alterations could be due to the consecutive loss of cell membrane potential in the injured

myocardium as a result of oxidative stress (Peacock et al. 2007). It was reported that ST

elevation correlates well with the leak of creatine kinase from the myocardium (Mohan et

al. 2010).

Cardiac enzymes, as a marker for acute myocardial infarction were all significantly

elevated in our model. Since theses enzymes are released from necrotic cells to the

extracellular fluid upon the incidence of infarction, their high levels give another

evidence for necrosis and infarction (Wang et al. 2009). Our findings are in agreement

with those of Kurian et al. (2005) and Rajadurai et al. (2007), who reported that these

cardio specific marker enzymes were released from the heart into the blood during

myocardial damage.

Inflammation is a key process involved in mediating myocardial tissue damage after an

ischemic event. In the current study, isoprenaline produced an increase in the serum level

of pro-inflammatory cytokine TNF-α and IL-6, an effect that is in accordance with the

work of other investigators (Cusack et al. 2002; Tawfik et al. 2010). The injured region

undergoes local necrosis and myocyte apoptosis resulting in complement activation, free

radicals generation, and accumulation of cellular debris. Phagocytosis of the resultant

cellular debris by macrophages and neutrophils triggers the inflammatory cytokines as

TNF-α (Frangogiannis et al. 2002).

The increase in the cardiac MDA content (an indicator of lipid peroxidation) and the

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decrease in the cardiac content of SOD and GSH (cardiac antioxidants) in isoprenaline

group could be expected. Isoprenaline causes oxidative stress, which results from a

serious imbalance between the generation of reactive oxygen species (ROS) and their

clearance by the body’s endogenous antioxidative defenses. Our findings are in

agreement with those of Shikalgar and Naikwade (2010) and Upaganlawar et al. (2010).

Administration of vitamin D for 7 days before induction of infarct-like lesion resulted in

amelioration of cardiac injury. Specifically, there was improvement in ECG parameters,

cardiac injury extension and cardiac enzymes of ISO-injected rats pretreated with vitamin

D. These findings are in line with a recent study of Abood and Elshal (2015), who

reported a cardio-protective effect against MI through vitamin D receptor (VDR)

stimulation based on improvement of ECG criteria in addition to return of cardiac

enzyme parameters to near normal.

To explain the protective effect of vitamin D on isoprenaline-induced infarct-like lesion

we measured plasma TNF-α and IL-6, as markers for inflammation, together with cardiac

MDA, SOD and GSH content, as markers for the oxidative state. The down regulation of

inflammatory cytokines demonstrated by vitamin D administration in ISO-injected rats

denotes that vitamin D exerts its protective effect, at least in part, by an anti-

inflammatory action. The reduction of inflammatory cytokines as evidenced in our study

has previously been reported by Arnson et al. (2013). Contrarily, Witham et al (2013)

showed no reduction of inflammatory cytokines after 8 weeks of vitamin D

supplementation. This contradiction is probably due to genetic polymorphism that may

modulate the response to vitamin D supplementation (Gagnon et al. 2014). In addition,

pretreatment with vitamin D in ISO-injected rats was found to be associated with a

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significant reduction in lipid peroxidation products resulting in amelioration of the

oxidative stress. This conforms to the recent data published by Cavalcante and colleagues

(2015) regarding the antioxidant effect of vitamin D.

We revealed that vitamin D treatment reduced ISO-induced infarct-like lesion through

PPAR- ɣ activation. The PPAR- ɣ agonists are well documented to protect against IRI in

various tissues through down-regulation of molecular pathways like nuclear factor kappa

beta, thromboxane synthase, monocyte chemoattractant protein-1, inducible nitric oxide

synthase, and fibronectin (Chatterjee et al. 2004; Panchapakesan et al. 2005). Moreover,

the PPAR- ɣ agonists are proposed to inhibit Jun N-terminal kinase phosphorylation that

belongs to mitogen-activated protein kinase and production of endothelin-1 that are

produced in response to stress stimuli including cytokines and are responsible for cellular

apoptosis and damage (Delerive et al. 1999). There are previous studies that reported the

protective action of vitamin D on the heart (Abood and Elshal 2015; Creighton et al.

2012; Schleithoff et al. 2006). To the best of our knowledge, our study reports for the

first time that vitamin D essentially involves PPAR- ɣ activation for its cardio protective

effect, as pharmacologic inhibition of PPAR- ɣ using BADGE abolished vitamin D-

mediated cardio protection, supporting the involvement of PPAR- ɣ in protective action

of vitamin D.

Conclusion:

In conclusion, the present study revealed that vitamin D pretreatment could improve the

outcome of isoprenaline-induced infarct-like lesion in rats. This protective effect could be

attributed to the strong anti-inflammatory and antioxidant effects of vitamin D. The study

also demonstrated for the first time that PPAR- ɣ significantly contributes toward vitamin

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D mediated cardio protection.

Declaration of interest statement:

The authors declare that they have no conflict of interest.

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Table 1: Changes in serum cardiac enzymes and inflammatory markers in different

experimental groups.

control

group

Vit. D

group

ISO group ISO + Vit. D

group

ISO + Vit. D +

BADGE group

CPK (U/l) 681.4 ± 2.9 679.5 ± 3.2 1011.3 ±7.3*

743.3 ± 9.3*†

983.1 ± 4.0*† ‡

LDH (U/l) 2156 ± 5.2 2160 ± 7.1 8234 ± 3.9*

3642 ± 4.6*†

7454 ± 1.9*† ‡

TNF-α (pg/ml) 80.4 ± 0.43

79.6 ± 0.52

132.0 ± 0.12*

91.1 ± 0.90*†

124.5 ± 0.41*† ‡

IL-6 (pg/ml) 41.5 ± 0.57 39.8 ± 0.78 126.4 ± 0.61* 57.0 ± 0.24*† 119.2 ± 0.74*† ‡

CPK, creatine kinase; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor- α; IL-6, interleukin-6.

Data is expressed as mean ± standard deviation (n = 10 per group). P < 0.05 is significant

tested by using One-way analysis of variance (ANOVA) and Post Hoc multiple

comparisons (LSD). * P< 0.05 vs. control group; †P < 0.05 vs. ISO group; ‡ P < 0.05 vs.

ISO+Vit. D group.

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Table 2: Changes in cardiac oxidative stress, cardiac antioxidant status and size of

infarction in different experimental groups.

control

group

Vit. D

group

ISO group ISO + Vit. D

group

ISO + Vit. D +

BADGE group

MDA (nmol/g tissue) 115.1 ± 1.3 113.8 ± 3.9 157.8 ± 6.5*

126.5 ± 7.1*†

149.9 ± 5.2*† ‡

SOD (IU/mg protein) 7.4 ± 0.93 7.0 ± 0.49 3.1 ± 0.12* 6.2 ± 0.61*† 3.6 ± 0.40*† ‡

GSH (IU/mg protein) 4.6 ± 0.82 4.8 ± 0.63 1.2 ± 0.60* 3.5 ± 0.34*† 1.7 ± 0.57*

‡

Infarction size (% LV) 0.0 0.0 36.4 ± 2.7* 19.3 ± 3.1*† 28.8 ± 4.3*† ‡

MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione peroxidase.

Data is expressed as mean ± standard deviation (n = 10 per group). P < 0.05 is significant

tested by using One-way analysis of variance (ANOVA) and Post Hoc multiple

comparisons (LSD). * P < 0.05 vs. control group; † P < 0.05 vs. ISO group; ‡ P < 0.05 vs.

ISO+Vit. D group.

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Figure Caption:

Fig. 1 (A,B,C,D,E): Lead II trace showing ECG changes in different experimental groups

(A) Normal control group

(B) Vitamin D-treated group

(C) Isoprenaline group

(D) Isoprenaline group pretreated with vitamin D

(E) isoprenaline group pretreated with vitamin D received BADGE injection

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40x49mm (300 x 300 DPI)

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Activated Receptor- Draft Corresponding author: Dr. Ola Ahmed El-Gohary ... proliferator-activated receptor gamma ... 2.3. ECG monitoring: