1 Department of Cardiothoracic Surgery, Heart Science Centre, National Heart and Lung Institute, Imperial College School of Medicine, Harefield, UK and
2 Arizona Prevention Center, College of Medicine, University of Arizona, Tucson, Arizona, USA
Received 8 July 2002; in revised form 11 September 2002; accepted 28 September 2002
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ABSTRACT |
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INTRODUCTION |
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It is becoming increasingly apparent that HIV infection and the development of AIDS are often associated with pathological heart conditions, such as cardiomyopathy (Lewis, 2000). Indeed, as patient survival increases with more effective antiretroviral therapy, cardiomyopathy in AIDS has become more prevalent. The interactions of cellular and viral factors in AIDS and their relationships to the development of cardiomyopathy are still unclear. The consequences to the heart of excessive consumption of ethanol are well known and include dilation of the left ventricle, cardiomegaly and contractile abnormalities (Richardson et al., 1986
; Preedy and Richardson, 1994
). It has also been suggested that excessive alcohol intake can aggravate the symptoms of AIDS and possibly contribute towards the development of AIDS cardiomyopathy. This study was designed to utilize a proteomic approach to investigate global alterations in protein expression in hearts from MuLV-infected mice. An experimental group of infected mice was also given dietary ethanol in order to determine the consequences of excessive alcohol consumption on the patterns of cardiac protein expression.
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MATERIALS AND METHODS |
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Preparation of tissue samples
All tissue samples were stored in liquid nitrogen prior to processing. The frozen tissue specimens (typically 0.2 g) were ground to a fine powder under liquid nitrogen using a pestle and mortar. The resulting powder was collected into 1.5 ml microcentrifuge tubes and homogenized using a handheld homogenizer for 1 min in 1 ml of lysis buffer, containing 9.5 M urea, 1% dithiothreitol (DTT), 2% CHAPS and 0.8% Pharmalyte pH 310 (Amersham Biosciences, Amersham, UK). We have found that this method of sample preparation is optimal for cardiac tissue and that reagents such as thiourea, alternative zwitterionic detergents and reducing agents do not give improved solubility or better separation of heart proteins by two-dimensional polyacrylamide gel electrophoresis (2-DE) (M. J. Dunn and J. Weekes, unpublished data). After vortexing for 30 s, samples were centrifuged at 15 000 r.p.m. (20 000 g) for 1 h and the resulting supernatants were collected. Total homogenate protein concentration was measured in duplicate using a modification of the method described by Bradford (1976). Briefly, the BSA used for preparation of standard curves and the protein samples to be measured were made up to 10 µl with lysis buffer prior to the addition of 10 µl of 0.1 M HCl and 80 µl of H2O. Bradford reagent diluted 1 in 4 with H2O was added to a final volume of 3.5 ml and the protein concentration was then determined spectrophotometrically.
Two-dimensional polyacrylamide gel electrophoresis
First-dimension isoelectric focusing (IEF) was performed using 18 cm immobilized pH gradient (IPG) strips (Amersham Biosciences), with pH ranges 47 L (linear) and 310 NL (non-linear), using an in-gel rehydration method. The samples were diluted with rehydration solution containing 8 M urea, 0.5% CHAPS, 0.2% DTT and 0.2% Pharmalyte pH 310 prior to rehydration overnight in a re-swelling tray. For analytical gels, total protein loaded was 100 µg in a total volume of 450 µl, and for preparative gels, 400 µg in a total volume of 450 µl. The strips were focused at 0.05 mA/IPG strip for 60 kVh at 20°C. After IEF, the strips were equilibrated in 1.5 M Tris pH 8.8 buffer containing 6 M urea, 30% glycerol, 2% SDS and 0.01% bromophenol blue, with the addition of 1% DTT for 15 min, followed by the same buffer with the addition of 4.8% iodoacetamide for 15 min (Weekes et al., 1999). Second-dimension SDSPAGE was performed using 22 cm 12% T, 2.6% C separating gels without a stacking gel, using a Hoefer DALT system. The separation was carried out overnight at 20 mA/gel at 8°C and was stopped just as the dye front left the bottom of the gels.
Protein visualization, densitometry and computer analysis
Analytical gels were fixed and stained using the Daiichi 2-D silver staining kit (Insight Biotechnology, Wembley, UK). Micro-preparative gels were stained using a modified version of the PlusOne silver staining kit (Amersham Biosciences) that is compatible with mass spectrometry (Yan et al., 2000). All silver-stained gels were scanned at 100 µm resolution using a Molecular Dynamics Personal SI laser densitometer. Gels were analysed using Melanie II image analysis software (BioRad, Hemel Hempstead, UK) running on a Sun Sparc Ultra1 workstation. After detection of spots, the gels were aligned, landmarked and matched. Gels were then placed into the appropriate experimental classes and differential analysis was performed. All spots that differed between the classes by 50% or more and passed a Students t-test (P < 0.05) were accepted as being significant. All gel spots detected as significantly different between the groups were then highlighted and checked manually to eliminate any artefactual differences due to gel pattern distortions, abnormal silver staining and inappropriately matched or badly detected spots. For simplicity, the optical density values for each spot were averaged across each group.
Peptide mass finger-printing by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
Protein spots were excised from preparative-scale 2-DE gels stained with modified PlusOne silver stain, cut into 1 mm cubes, destained by washing in 20 µl aliquots of 50 mM ammonium bicarbonate in 30% (v/v) acetonitrile for 1 h repeatedly until colourless. The samples were then dried in a centrifugal evaporator and treated as described by Yan et al.(2000). Modified (methylated) porcine trypsin (Promega, Southampton, UK) was prepared as a stock solution in water (0.1 µg/µl). For digestion, 4 µl of trypsin solution were added to 21 µl of Tris buffer (5 mM pH 8.8, prepared fresh for each use) and added to the gel pieces before incubation overnight at room temperature. Digestion was stopped by addition of 15 µl of 50% acetonitrile, 0.1% TFA. MALDI-MS was performed using a TofSpec 2E mass spectrometer (Micromass, Manchester, UK) operated in the positive ion reflection mode at 20 kV accelerating voltage with time-lag focusing enabled. The matrix was
-cyanohydroxycinnamic acid (4 mg/ml) as described by Yan et al. (2000)
. Peptide mass maps were searched against SWISS-PROT/TrEMBL release 35, using Protein Probe (Micromass), or against a non-redundant database maintained by the National Center for Biotechnology Information (NCBI) using the Mascot search engine.
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RESULTS |
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(2) Comparison of the cardiac proteins from uninfected and infected, ethanol-treated mice indicated spots altered due to infection or ethanol or a combined effect of ethanol and infection 53 proteins were reduced and 15 were increased in the infected, ethanol-treated group.
(3) Comparing cardiac protein profiles of the uninfected and infected groups detected spots altered due to infection. Any spots that were detected in this comparison and also appeared altered in comparisons (1) and (2) above were subtracted from this group, leaving the proteins that were altered due to infection alone. A total of 15 protein spots were reduced in expression and nine were increased.
Of the 24 spots altered due to infection alone, eight were altered by ethanol treatment. For six of these spots, the effect of ethanol was to bring these altered proteins back towards the levels seen in the uninfected hearts. Alterations of two proteins were exacerbated by ethanol. Examples of such spots are shown in the histogram plots from quantitative computer analysis in Fig. 2. An example of a region of the 2-D gel protein profile showing a protein spot that is modulated by viral infection and ethanol consumption is shown in Fig. 3
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DISCUSSION |
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As might be expected, the comparison of the uninfected and infected, ethanol-treated mice (which indicated spots altered due to infection or ethanol or a combined effect of ethanol and infection) produced the largest number of protein expression changes 53 proteins were reduced and 15 were increased in the infected, ethanol-treated group. In a number of cases, the effect of infection mirrored that of alcohol. In most cases, the effect of the combination of alcohol and infection did not increase or decrease the effect of either treatment in isolation. Such examples are spots 536 and 482 shown in Fig. 2. It is not clear why this should be the case, but it is possible that these proteins may be part of the stress response, which may be activated in a similar fashion by both infection and alcohol feeding.
It is interesting to note that, of the 24 proteins shown to be altered in expression in the heart due to LP-BM5 infection alone, eight appeared to be further altered by ethanol feeding. However, of these eight proteins, the majority (six) were ameliorated by ethanol and for only two was the effect of infection exacerbated by ethanol. This may appear to indicate that alcohol could produce a level of cardiac protection from the virus, which would perhaps contradict the tenet that ethanol consumption aggravates the effects of AIDS. However, if these proteins represent part of a protective response to viral infection it is possible that the effect of alcohol consumption may lead to a de-protection of the cardiac myocyte and a consequent aggravation of the effects of infection. Indeed, one of the proteins altered in response to ethanol feeding was identified as the 27 kDa HSP27. It is possible, therefore, that ethanol consumption may accentuate the effects of retroviral infection in the heart by down-regulating the antiviral response and thereby compromising the ability of the cardiac myocyte to protect itself against the effect of viral damage. The identification of the remaining differentially expressed proteins will contribute to the understanding of the biochemical alterations in murine heart after LP-BM5 infection and the role of ethanol in the disease.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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* Author to whom correspondence should be addressed at: Department of Neuroscience, Box P045, Institute of Psychiatry, Kings College London, De Crespigny Park, London SE5 8AF, UK.
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