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USE OF ELECTROPHORETICALLY SEPARATED SERUM PROTEIN FRACTIONS FOR THE DIAGNOSIS OF CARDIOMYOPATHY.

Byline: Zahid Hussain Siddiqui and Abdul Majeed Cheema

ABSTRACT: In an investigation of molecular pathogenesis in cardiovascular diseases, the blood samples of the patients diagnosed for cardiomyopathy (CMP) were obtained from the Punjab Institute of Cardiology, Lahore. Blood samples of the healthy subjects of comparable age group without any history of cardiac ailment were also collected for the control comparisons. The sera of CMP were separated and used for the study of the protein profiles with sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in first dimension. Quantification of various protein fractions done by Gene Genius Bio-imaging Gel Documentation System that provide the data of molecular weights and the percent raw volume covered by each of the fractions. The protein fractions that showed significant variation were separated by using the technique of electroblotting and electroelution and run on isoelectric focusing (IEF) in second dimension to determine their isoelectric points.

The most pertinent results in the comparison were the significant increase in apolipoprotein B, Ceruloplasmin, apolipoprotein A-I and transthyretin in the sera of patients of CMP compared to healthy subjects. These results show that level of apolipoprotein B, Ceruloplasmin, apolipoprotein A-I and transthyretin are strong predictor of CMP and can also be used for the diagnosis of CMP.

Key words: Cardiomyopathy, Protein fractions, Electrophoresis.

INTRODUCTION

The term cardiomyopathy (CMP) could be applied to describe the primary myocardial diseases of undetermined cause [1]. Cardiomyopathy is one of the leading causes of sudden cardiac death in young and still carries a high risk for mortality and morbidity. It is associated with viral myocarditis, alcohol abuse and immune mediated pathogenesis [2]. Genetic defects have also been documented in some cases [3, 4].

Cardiomyopathy is diagnosed on the basis of chest X-ray, ECG changes, echocardiography and cardiac biopsy. During the last few years interest has focused on serum proteins for the diagnosis of cardiovascular disease [5]. Some authors demonstrated variations in proteins in CMP patients. A case of low alpha-1-antitrypsin serum level was demonstrated in cardiomyopathy patient [6]. Marked decrease in haptoglobin and elevated lactate dehydrogenase were observed in a patient of hypertrophic cardiomyopathy [7]. Elevated level of cathepsin L [8] and cytokin [9] whereas lower levels of albumin and prealbumin [10] were observed in cardiomyopathy group. Another study showed increase in heat shock protein 70 (HSP70) in CMP patient which may hold diagnostic potential in clinical practice [11].

Electrophoresis is a powerful technique for the analysis of proteins including serum proteins which can be used to study the variations in the protein profiles in CMP patients. By the use of SDS gel electrophoresis, a decrease in molecular weight of titin in dilated cardiomyopathy compared with normal was revealed which is correlated with the stage of disease [12].

Presently two-dimensional gel electrophoresis (2-DE), with one dimension on isoelectric focusing (IEF) and other on sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and vice versa, is mostly employed in such analysis. SDS-PAGE is likely to yield protein distribution profile consistent with the patient's clinical status and may have an important role in diagnostic investigation [13].

The comparison of two-dimensional gel electrophoresis pattern from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified [14]. Protein composition of human myocardium studied by two-dimensional gel electrophoresis (2-DE) in dilated cardiomyopathy is characterized by transferrin accumulation in myocardium and by enhanced expression of protein with molecular weight of 3 kDa and isoelectric point 5 [15]. Two-dimensional gel electrophoresis also showed 28% mutant actin in heart muscles of CMP patient [16]. These studies demonstrated that two-dimensional gel electrophoresis appears to be a powerful method for the detection of disease associated alterations in the myocardial protein pattern.

The aim of the present study was to investigate the protein patterns in the sera of CMP patients and their comparison with those of healthy subjects through SDS-PAGE and 2-DE and characterization of the proteins specifically found in the serum of CMP patient in our local population for prediction and diagnosis of CMP.

MATERIALS AND METHODS

Blood samples from twenty patients from the Punjab Institute of Cardiology, Lahore, with a diagnosis of CMP (based upon Chest X-ray, echocardiography and ECG) were collected. Five ml blood was collected from each patient with the help of syringe. Blood samples of same number of healthy subjects with negative family history of CVD were also collected for use as controls. Serum was separated by centrifugation and stored at -70 degC until used for analysis. For SDS-PAGE, the serum samples were diluted in phosphate buffer (pH 7.2) and proteins were denatured by heating with loading dye (1.54 g dithiothreitol, 2 g sodiumdodocyle sulfate, 8 mL of 1.0 M Tris HCl; pH 6.8, 10 mL of glycerol and 20 mg of bromophenol blue dye) in boiling water bath for two minutes before loading on the gel. Lypholized mixture of proteins SDS-6H for high (205-45 kDa) and SDS VII-L for low (66-14.2 kDa) molecular weight proteins (Sigma Chemicals) were used as molecular weight markers.

It was reconstituted, separately, in 1.5 mL of sample buffer (0.0625M Tris HCl pH 6.75, 2% SDS, 5% mercaptoethanol, 10% glycerol and 0.001% bromophenol blue). Heated in a boiling water bath for 2 minutes and stored in aliquots at -70degC. Polyacrylamide gels, 5 % for high and 12 % for low molecular weight proteins, were prepared [17]. Protein size marker and each of the samples were loaded in separate wells and gels were electrophoresed at 20 mA and 200 volts in a cooling chamber maintained at 4degC. Electrophoresis was stopped, immediately, after dye seemed to diffuse in the buffer in the lower chamber. Following electrophoresis, the 5% gel was stained with coomassie brillient blue for 30 minutes and 12 % gel for two hours. After staining the gels were destained until the clearance of blue background. Protein fractions of different molecular weights were visible in the form of blue bands on a transparent background.

Gels were photographed and their images were saved for protein quantification by Gene Genius Bio-imaging Gel Documentation System provides the data of molecular weights against protein markers and the total area covered by each of the protein fractions. The data was employed in finding the enhancement or reduction and the appearance and disappearance of particular protein fractions for comparison of the healthy individuals and CMP patients.

Table-1: Average raw volumes (%) exhibited by electrophoretically separated serum protein fractions of control and cardiomyopathy (CMP) groups and their percentage differences.

Molecular###Average rawof###Average raw###Percentage###

weight###volume (%)###volume (%)###difference in

of###protein fractions###of protein###protein fractions

proteins###in control group###fractions###in CMP group

(kDa)###in CMP group

270###2.45 +- 0.21###3.88 +- 0.36###58

190###7.37 +- 0.30###7.65 +- 0.29###04

186###3.50 +- 0.26###3.55 +- 0.23###01

135###4.25 +- 0.23###5.31 +- 0.16###25

115###5.23 +- 0.37###5.92 +- 0.41###13

100###3.25 +- 0.24###3.33 +- 0.10###03

77###10.21 +- 0.23###10.46 +- 0.21###03

66###26.52 +- 0.18###27.49 +- 0.16###04

54###14.55 +- 0.31###14.65 +- 0.15###01

45###10.64 +- 0.41###10.57 +- 0.27###01

36###2.22 +- 0.12###2.48 +- 0.17###12

28###9.48 +- 0.48###10.60 +- 0.37###12

24###12.42 +- 0.29###12.94 +- 0.22###04

23###3.38 +- 0.25###4.32 +- 0.24###28

17###1.34 +- 0.05###1.52 +- 0.11###13

14###0.96 +- 0.11###1.71 +- 0.26###78

Samples containing significant quantities of desired protein fractions were run on SDS-PAGE and the unstained gel was electroblotted on polyvinylidene diflouride (PVDF) membrane [18]. The required protein band from PVDF membrane was excised and electroeluted [19]. Each of the eluted protein was freeze dried and reconstituted in the buffer when used for isoelectric focusing.10 uL solution D (10 % w/v sodiumdodecyle sulfate in 2.3 % w/v dithioerythreitol) was added to 60 uL eluted protein solution, mixed and heated at 95degC for 5 minutes. Brought to room temperature and added 5 uL solution E (8 M urea, 4 % CHAPS (3-[(3cholamidopropyl)dimethylammonio]-1-Propanesulfonate), 40 mM Tris HCl and 65 mM dithioerythretol, traces of bromophenol blue). The eluted protein was subjected, afterwards, to isoelectric focusing [20] in order to determine its isoelectric point/s against isoelectric focusing markers.

RESULTS

Variations regarding enhancement of some of the protein fractions but reduction of few others in CMP patients compared to healthy subjects were notable. No new fractions were, however, detected in CMP group when compared to healthy group. Variations in the protein profile in CMP were studied separately.

SDS-PAGE of serum of control subjects. (M indicates protein markers; MW indicates molecular weight of protein markers on left side and serum protein fractions on right side; 5% gel above and 12% gel below).

The comparison of the protein fractions in CMP group with that of control group indicated that the prominent high as well as low molecular weight fractions did not show any significant difference in CMP group compared to the control group except the fractions of 270, 135, 23 and 14 kDa. The fraction of 270 kDa and 135 kDa exhibited 58 % and 25 % (P less than 0.01) greater expression when compared with normal subjects. These two fractions exhibited raw volume of 2.45 +- 0.21 % and 4.25 +- 0.23 % respectively in control subjects, however, in cardiomyopathy patients the raw volumes of 3.88 +- 0.36 % and 5.31 +- 0.16 % were determined respectively. Amongst low molecular weight protein fractions, the fractions of 23 and 14 kDa expressed significantly 28 % and 78 % greater intensity respectively (P less than 0.01) in cardiomyopathy patients compared to control group.

The values of percent raw volumes were 3.38 +- 0.25 % and 0.96 +- 0.11 % respectively in the control group and the comparable values were estimated as 4.32 +- 0.24 % and 1.71 +- 0.26 % respectively in the cardiomyopathy group. (Table-1; Fig. 1-2).

The protein fractions showing considerable variations were subjected to isoelectric focusing. High molecular weight protein fraction of 270 kDa was resolved into two bands corresponding to isoelectric points 5.9 and 6.1. The fraction of 135 kDa was resolved into two bands whose isoelectric points were determined as 4.8 and 5.2 Amongst low molecular weight proteins the fraction of 23 kDa was resolved into three fractions corresponding to isoelectric points 5.1, 5.3 and 5.5. Lastly the fraction of 14 kDa resolved into two bands whose isoelectric points were determined as 5.3 and 5.5 (Fig. 3).

From the above data regarding the molecular weight and the isoelectric points, each of the proteins was then identified using human plasma protein map [21]. Protein fractions of 270, 135, 23 and 14 kDa were found to be apolipoprotein B, ceruloplasmin, apolipoprotein A-I and transthyretin respectively.

DISCUSSION

The present study was undertaken to find the variations in the serum protein profiles in the patients of CMP in local population because in the recent years the proteonomics is a rapidly growing research area. It has increased the understanding of many diseases and protein composition represents the functional status of biological compartment. Due to resolution and sensitivity the technique of 2-DE is a powerful tool for the analysis and detection of protein from complex biological sources [20]. Variations in the serum protein profile in CMP were therefore detected by SDS-PAGE in first dimension. IEF was performed, in second dimension electrophoresis (2-DE), of those proteins which exhibited significant variations and could be diagnostically significant in identifying the different categories of cardiovascular disease.

IEF gels showing distinct bands of 270, 135, 23 and 14 kDa protein fraction in Fig. A, B, C and D respectively. (M indicates protein markers and SPF indicates serum protein fractions. pI indicates isoelectric points of protein markers on left side and serum protein fractions on right side).

In present study apoplipoprotein B (270 kDa) have been found to be elevated by 58 % in patients of cardiomyopathy compared to the control subjects. The excess circulatory levels of any lipoprotein can be caused by one or two factors, either excess production or decreased catabolism [22]. It is caused by increased apolipoprotein B synthesis by the liver [23, 24] and also due to defect in low density lipoprotein (LDL) receptor, leading to inadequate hepatic uptake of LDL and markedly increasing circulatory LDL or apolipoprotein B, a component of LDL [25]. An important reason of increased LDL level is defective apolipoprotein B in which a substitution of glutamine for arginine at position 3500 results in a form of apolipoprotein B that binds poorly to the receptor and result in reduced LDL clearance and increase LDL or apolipoprotein B concentration in blood [26].

Another mutation of apolipoprotein B in which a substitution of cystine for arginine at position 3531 impairs binding of apolipoprotein B to the LDL receptor [27]. It is speculated that number of LDL receptor is not fixed and modified by genetic defects, dietary intake of saturated fat, cholesterol and calories and certain pharmacological agents. Thus the interaction of genetic and environmental factors control the number of lipoprotein receptors. These interactions may explain different responses within populations to similar dietary constituents [22, 28]. High use of alcohol [29] and hypertension [30] also the cause of increased level of apolipoprotein B. Smoking increase concentration of LDL [31, 32] and testosterone increase LDL-cholesterol in blood [33]. From the above discussion it is suggested that the higher level of apolipoprotein B may be used as diagnostic marker for cardiovascular disease.

It is also indicated that the factors responsible for the enhancement of apolipoprotein B are genetic, dietary and environmental. Management of these factors may be helpful in reducing the chances of the disease.

Ceruloplasmin (135 kDa) was found to be elevated by 25% in patients of cardiomyopathy. These results are in agreement with observations of many authors. A higher level of ceruloplasmin was found in patients of hypertrophic cardiomyopathy than in healthy volunteers [34]. Elevated level of copper was observed in cardiomyopathy group compared to normal subjects [10]. This elevated level of copper may suggest the increase in the concentration of ceruloplasmin which is a copper carrying protein. Ceruloplasmin level also rises in disorders producing inflammation or tissue injury. Ceruloplasmin is copper carrying protein which functions as a ferroxidase and superoxidase scavenger. It is an acute phase protein. The term "acute phase response" encompasses a complex range of physiological changes that occur following infection, inflammation and related conditions. Increase occurs in plasma concentration of ceruloplasmin as a result of increased synthesis, mediated primarily by interleukin-6 and other cytokines.

Cytokines secreted by cells involved in inflammation and immunity [35]. It is suggested that ceruloplasmin may prevent lipid peroxidation and free radical production in inflammatory state. This is perhaps its role in an acute phase reaction [36].

Apolipoprotein A-I (23 kDa) expressed 28 % higher values in the patients of cardiomyopathy. In some previous reports increased amount of apolipoprotein A-I was found in the urine of cardiomyopathy affected cattles analyzed by sodium dodecyle sulfate polyacrylamide gel electrophoresis [37]. It is suggested that altered metabolism caused by the mutation may be a significant factor in apolipoprotein A-I fibrillogenesis. It has been found that Apolipoprotein A-I in an amyloid protein which may

be the cause of amyloidotic neuropathy [38].

Transthyretin (14kDa) exhibited 78 % increase in patients of cardiomyopathy. Transthyretin is transport protein and binds thyroxin and triiodothyronine. There is abundant evidence that thyroid hormone may alter cardiac function directly. The available data suggest that the direct effect of thyroid hormone on the heart is mediated via a change in protein synthesis. Patients with hyperthyroidism are at increased risk of developing atherosclerosis. There is increased frequency of hyperthyroidism in patients with familial hypertrophic cardiomyopathy. There is also evidence that thyroid hormone both increase the synthesis of myosin and alter its structure, increasing its contractile properties [39]. Transthyretin, also known as prealbumin or thyroxin binding protein, is synthesized in liver, from where it is secreted into plasma. Transthyretin is the main constituent of amyloid that deposit preferentially in the heart leading to familial amyloid cardiomyopathy [40].

Some studies revealed that cause of some forms of cardiomyopathy is the deposition of normal and/or mutant transthyretin molecules in the form of amyloid fibrils [41]. In another report a new transthyretin gene mutation was also identified in CMP patient [42].

The results of present study clearly indicated that in CMP patients alteration in protein fractions occur as a result of displaced molecular homeostasis. This pattern of variations in CMP patients is similar to most of the populations of the world. The technique of electrophoresis, due to its resolution and sensitivity, is being used for the diagnosis of CMP in these different populations of the world. Therefore the technique of electrophoresis, particularly two-dimensional gel electrophoresis, is also very useful for the diagnosis of CMP in our Pakistani population where the work on the diagnosis of cardiovascular disease is merely conventional and highly underdeveloped.

ACKNOWLEDGEMENT

This project was funded by University of the Punjab, Lahore, Pakistan.

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Publication:Science International
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