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SALBUTAMOL: A SUBSTITUENT OF ISOPROTERENOL TO ESTABLISH AN EXPERIMENTAL ANIMAL MODEL TO INDUCE MYOCARDIAL INFARCTION.

Byline: N. Afsheen, Khalil-ur-Rehman, N. Jahan, K. M. Khan and M. A. Zia

ABSTRACT

Isoproterenol, a catecholamine acting as a [beta] adrenergic agonist, is commonly used to establish myocardial infarction (MI) in animal models for drug development. Although cheaper than isoproterenol, salbutamol is another type of catecholamine, may be potentially and equally used for the onset of MI in animal model. However the concentrations of salbutamol for inducing MI need to be optimized and standardized. In current work, following the treatment with different concentrations of salbutamol, the blood samples of rats were taken at different time intervals according to "Central Composite Design" (CCD) for measuring the level of cardiac markers, CK-MB, LDH and SGOT. Response Surface Methodology (RSM) indicated an optimal salbutamol dosage of 80 mg/kg for inducing MI, which resulted in elevated level of SGOT after 20 hr with CK-MB and LDH level changes in 23 hr following salbutamol (80 mg/kg) administration.

The level changes of cardiac marker Trop I and ECG also confirmed the onset of myocardial infarction at the optimal concentration of salbutamol. Current work obtained the optimal dose and the feasibility of salbutamol as a substituent of isoproterenol for inducing MI.

Key words: Salbutamol, CCD, MI, RSM.

INTRODUCTION

The cardiovascular disease (CVD) is a global life threat (Srivastav et al., 2013). 17.3 million People died from CVDs in 2008, this number will rise up to 23.6 million in 2030 (Cao et al., 2013). Among CVDs, myocardial infarction (MI) is increasing with frequent proportion all over the world (Clarke et al., 2014). As the number of cardiac patients is increasing therefore to combat heart diseases new drugs with new concepts is needed. The animal models impart crucial role to discover new drugs for management and treatment of various diseases in human being (Ahsan et al., 2014). The chemically provoked MI in animals is a well recognized typical model to examine the valuable outcomes of medicinal plants on cardiac dysfunction (Sahreen et al., 2014).

Catecholamine is potent inducer of myocardial contractility in animal models at its high dose and may lead to ischemia, hypoxia, myocardial hyperactivity and coronary hypertension (Jahan et al., 2012; Yousefi et al., 2014; Beaulah et al., 2014). Among various catecholamine, the isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropylamino ethanol hydrochloride] is documented to cause severe oxidative stress in the myocardium (Beaulah et al., 2014; Kumar and Gurusamy 2014; Prabha et al., 2014). The necrosis of cardiomyocytes causes the alteration in membrane permeability and leads to the loss of functions and integrity of myocardial membranes (Ramadoss et al., 2012).

Salbutamol is also synthetic catecholamine that brings about severe myocardium stress and necrosis due to its structural similarity and mode of action with isoproterenol (Aslam et al., 2015). Salbutamol elevates intracellular concentration of cyclic AMP (cAMP) by activating adenylate cyclase. The elevated level of cAMP also inhibits the release of inflammatory mediators from mast cells and eosinophils (Rahman et al., 2012). High dose of salbutamol resulted in tachycardia which may lead to myocardial infarction (Hina et al., 2010). As the salbutamol is economical, easily available as compared to isoproterenol but very few literature citations with varying concentrations of salbutamol to induce myocardial infarction are available.

A variety of cardiotoxic agents are available for preclinical trials and the most commonly reported toxin used in experimental animals is isoproterenol. The salbutamol may be used as an alternative drug to induce MI because of its accessibility and low price. In this research the animal model was established to optimize the concentration of salbutamol to induce MI. statistically the animal model was designed by applying Central Composite Design (CCD) of Response Surface Methodology (RSM) to get the optimal concentration that may induce myocardial infarction.

MATERIALS AND METHODS

Selection of animals: The rats having 150-250 g weight were kept in animal house, Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad to acclimatize for one week under laboratory conditions. They were fed with standard rats' feed throughout the experimental period. The husk in the cages was renewed three times a week to ensure hygiene and maximum comfort for rats.

Experimental design for dose optimization: The rats were orally administered with varying concentrations of salbutamol as suggested by "Central Composite Design" of Response Surface Methodology (RSM-CCD) for two consecutive days. The blood sampling was also performed at different time intervals from 0 to 116 hr, also proposed by RSM-CCD (Table. 1).

Table 1. Experimental design suggested by Central Composite Design to optimize the dose of salbutamol.

Sr. No.###Time of blood###Conc. of salbutamol

###sampling (hr) X1###(mg/kg) X2

1###0###50

2###96###50

3###0###125

4###96###125

5###0###88

6###116###88

7###48###34

8###48###141

9###48###88

10###48###88

11###48###88

12###48###88

13###48###88

Biochemical analysis: The rats were anesthetized and the blood sample was drawn by aortic puncture. The serum was used to analyze the effect of different concentrations of salbutamol on cardiac markers (CK-MB, SGOT and LDH) by using Biomed kits through chemistry analyzer. After biochemical analysis the statistical tool "RSM" was applied to predict the optimal dose of salbutamol at which it may induce MI.

Endorsement of optimized dose: To confirm the optimal dose of salbutamol, rats were administered with optimized concentration of salbutamol orally for two consecutive days. The blood sample was taken at optimal time to confirm the indication of MI by performing Trop I test. The ECG was also performed for the corroboration of myocardial infarction. Standard lead II was recorded by power Lab and needle electrodes were placed subcutaneously in the extended limbs of the supine animals.

Statistical analysis: The dose optimization of salbutamol was executed by applying Central Composite Design (CCD) of Response Surface Methodology (RSM).

RESULTS AND DISCUSSION

The response of cardiac markers was predicted by applying multiple regression analysis on the experimental data. The quadratic models obtained from regression analysis for Y1 (CK-MB), Y2 (SGOT) and Y3 (LDH) were as follows.

Y1 = 85.70810+1.39235 X1+1.81761 X2+6.11111E-003 X1X2-0.018873 X12-8.87704E-003 X22

Y2 = -48.13573+1.43497 X1+2.42125 X2+5.46484E-003 X1X2-0.017539 X12-0.012166 X22

Y3 = 201.45349+1.50376 X1+2.10444 X2+0.010833 X1X2-0.022602 X12-0.011076 X22

Where the X1 and X2 are the coded variables for time of blood sampling (hr) and concentrations of salbutamol (mg/kg) respectively.

The relationship between dependant (cardiac markers) and independent variables (concentrations of salbutamol and time intervals) was graphically presented by 3D Response surface plots in Fig. 1-3.

The interaction of different concentrations of salbutamol and time intervals of blood sampling on the level of CK-MB has been given in Fig. 1. The increased in concentration of salbutamol from 34 to 88 mg/kg b.wt. after 48 hr of salbutamol administration resulted in elevation in CK-MB level from 152 to 297 IU/L. Further increase in concentration from 88 to 141 mg/kg of salbutamol showed comparatively low elevation in the CK-MB level. However after 116 hr of salbutamol administration (88 mg/kg.b.wt), there was considerable decline in enzyme level from 293 to 184 IU/L. The possible explanation could be that the CK-MB level fall to normal with passage of time intervals after 48 to 72 hr of MI development (Dianita et al., 2015). In case of the rats administered the salbutamol at the concentrations of 125 and 50 mg/kg did not show the considerable elevation in the level of CK-MB after intervals of 96 hr. Thus the quantity of enzymes released, depends upon the degree of cellular damage (Khan et al., 2014).

The Fig. 2 described the relation between dependent (SGOT) and independent variables (conc. and time intervals). The salbutamol at the concentrations of 34, 88 and 141 mg/kg b.wt depicted the inclined in the level of SGOT after 48 hr of salbutamol administration. This might be due to the reason that salbutamol causes leakage of SGOT from cardiomyocytes into the blood stream. It occurs as a result of collapse of cellular and subcellular compartments that reflect pathological alterations in myocardium (Jaffe et al., 2006). After 96 hr of salbutamol administration at the concentration of 50 and 125 mg/kg, the SGOT level was comparatively low.

This decline in SGOT level might be due to the increase in time intervals as the SGOT level shifted towards normal with passage of time (Dianita et al., 2015).

In case of LDH, the increase in concentrations (34 to 88 mg/kg b.wt) resulted in increase in level of LDH after 48 hr interval of salbutamol administration as the LDH is detectable from 8 to12 hours of post MI and reached at its peak at 24-72 hours (Rosenblat et al., 2012). The rats treated with salbutamol at the concentration of 88 mg/kg depicted the considerable fall in the level of LDH with further increase in time interval of 116 hr after salbutamol administration (Fig. 3). This decline in LDH level supported the fact that serum levels of LDH approaches to normal value after 96 hours after MI (Wang et al., 2006). The level of LDH was 278 and 357 IU/L in response to the corresponding concentrations of 50 and 125 mg/kg b.wt. after 96 hr intervals of salbutamol administration. This increase in the level of cardiac markers in serum indicated the altered membrane permeability and leakage of these enzymes into blood stream.

The salbutamol increased the enzyme level by inducing oxidative stress and myocardial cell necrosis (Dianita et al., 2015; Zafar et al., 2015). The extent of leakage of cardiac markers indicated the onset of myocardial infarction and act as sensitive markers of myocyte injury (Nandave et al., 2007). Previous researchers also indicated the cardiotoxicity due to administration of salbutamol which resulted in increased cardiac marker, lipid profile and alleviated antioxidant enzymes as compared to normal control group (Zafar et al., 2015). The histopathological examination of heart after salbutamol administration also indicated the development of myocardial infarction in another animal trial (Aslam et al., 2015).

The analysis of variance (ANOVA) presented the statistical significance of the fitted quadratic polynomial model (Table. 2). The F values of quadratic model were, 48.18, 27.27 and 122.30 for CK-MB, SGOT and LDH respectively which suggested the significance of this model. The model is adequate to present the relationship between the response of dependent (cardiac markers) and independent variables (concentrations of salbutamol and time intervals). The value of determination coefficient (R2) for studied cardiac markers with non significant lack of fit at P>0.05 depicted that the calculated model was able to explain more than 97.18 % of the results. Meanwhile, a relatively lower value of coefficient of variation showed a better precision and reliability of the experiment.

The Table. 3. presented the predicted optimal concentration and time at which salbutamol elevated the level of cardiac markers. The cardioprotective potential of Coriandrum sativum against salbutamol induced cardiac injury in another experimental model also favored the increased in cardiac markers after administration of two doses of salbutamol (Kousar et al., 2012).

Depletion in myocardial LDH and CK-MB isoenzymes levels due to salbutamol administration indicated the altered membrane permeability and leakage of these enzymes into blood stream (Nandave et al., 2007). Increase in cardiac enzymes was due to unnecessary formation of free radicals which activate membrane permeability variations, causing the failure of functions and integrity of myocardial membranes (Barman et al., 2013).

Endorsement of optimized dose: The onset of MI was confirmed by administrating the optimized dose (80 mg/kg) of salbutamol for two consecutive days and blood sample was taken after 23 hr of salbutamol administration to perform the Trop I test. The positive indication of Trop-I confirmed the onset of myocardial infarction (Fig. 4). Troponin is a protein found in cardiac tissue and locates in the thin filament of striated muscles consisting of the three subunits Trop T, Trop I and Trop C. Out of the three troponins; Troponin T and I are being used as the biochemical markers for the diagnosis of myocardial injury (Subashini et al., 2011).

The significant increased level of Trop I might be due to the leakage of Trop I from the damaged heart tissues into the blood stream as a result of necrosis induced by optimized dose of salbutamol in rats. Electrocardiograph abnormalities are the main criteria generally used for the definite diagnosis of MI. The ECG showed the significant elevation of ST-segment in rats to which the salbutamol was administered.

The characteristic findings were reductions in the P wave intensity, increase in QRS amplitude, QT interval and prolongation of cardiac cycle along with ST segment elevation. These alterations could be due to the consecutive loss of cell membrane in injured myocardium (Thippeswamy et al., 2009). These alterations depicted the onset of MI by optimized dose of salbutamol (80 mg/kg).

Table 2. Analysis of variance (ANOVA) for the fitted quadratic model of CK-MB, LDH and SGOT activity as a function of independent variables.

Parameter###Source###SS###Df###MS###F Value###P>F

###Model###54546.23###5###10909.25###48.18###<0.0001

###A-Time###1188.22###1###1188.22###5.25###0.0557

###B-Conc###4585.08###1###4585.08###20.25###0.0028

CK-MB###AB###400.00###1###400.00###1.77###<0.0001

R2=0.9718###A2###34111.31###1###34111.31###150.65###<0.0001

CV=6.86###B2###20304.00###1###20304.00###89.67###<0.0001

###Residual###1585.00###7###226.43

###Lack of Fit###1564.20###3###521.40###100.27###<0.0521

###Pure Error###20.80###4###5.20

###Model###17167.89###5###3433.58###27.27###0.0002

###A-Time###969.95###1###969.95###7.7###0.0275

###B-Con###3459.58###1###3459.58###27.48###0.0012

SGOT###AB###387.04###1###387.04###3.07###0.123

R2=0.9512###A2###11359.67###1###11359.67###90.22###<0.0001

CV=12.96###B2###2036.11###1###2036.11###16.17###0.0051

###Lack of Fit###731.36###3###243.79###6.5###0.0541

###Residual###881.36###7###125.91

###Pure Error###150###4###37.5

###Model###1.571E+005###5###31423.93###122.30###<0.0001

###A-Time###2680.09###1###2680.09###10.43###<0.0145

###B-Conc###7444.98###1###7444.98###28.97###<0.0010

LDH###AB###1521.00###1###1521.00###5.92

R2=0.9887###A2###99216.21###1###99216.21###386.13

CV=4.20###B2###64680.21###1###64680.21###251.72

###Residual###1798.64###7###256.95

###Lack of Fit###1777.44###3###592.48###111.79###0.0613

###Pure Error###21.20###4###5.30

Table 3. Effects of optimized dose of salbutamol on different cardiac markers suggested by Response Surface Methodology.

Parameter###Time (hr)###Concentration(mg/Kg)###Optimized dose (IU/L)###Desirability

CK-MB###23###80###265###0.907

SGOT###20###80###99###0.786

LDH###23###80###467###0.912

Conclusions: The research concluded that salbutamol being economic and effective agent might be used as an alternate of isoproterenol to induce MI successfully with an optimal concentration of 80 mg/kg.

Author's contribution: Nadia Afsheen (NA), Ph.D research scholar performed the optimization of salbutamol as a substituent of isoproterenol to standardize an experimental animal model to induce MI. All the research work was planned under the kind supervision of Prof. Khalil-ur-Rehhman (KR). Nazish Jahan (NJ) and Anjum Zia (AZ) also interpreted the data. All the authors read and approved the manuscripts carefully.

Acknowledgement: Author is highly thankful to Higher Education Commission (HEC), Islamabad, Pakistan who financially supported the research work under "HEC Indigenous PhD Fellowship Scheme Batch-2012".

Compliance with ethical guidelines:

Competing interests: The authors declare that they have no competing interests.

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