Alterations in levels of O-linked N-acetylglucosamine modified proteins in the uraemic heart

Myocardial insulin resistance is a characteristic of patients with chronic kidney disease and may contribute to the high cardiac mortality in this patient group. However, the mechanisms underlying this abnormality remain unclear. The aim of this study was to characterise the relative level of O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins in control and uraemic cardiac tissue. Experimental uraemia was induced in male Sprague–Dawley rats via a two stage sub-total nephrectomy. Left ventricular tissue was harvested after 8 weeks, homogenised and O-GlcNAc modified proteins isolated by binding and precipitation with immobilised wheat germ agglutinin (a lectin specific for glucosamine). Bound proteins were recovered and separated by 1D- and 2D-PAGE. Gels were probed with an O-GlcNAc-specific antibody (1D) or stained with Sypro Red (2D). 1D-PAGE demonstrated separation of different populations of O-GlcNAc modified proteins. Image analysis of 2D gels gave >1500 matched spots in control and uraemic samples. Of these 78 showed ≥2.5-fold upregulation and 72 showed ≥2.5-fold reduction in the uraemic heart relative to control. Identification of O-GlcNAc modified proteins was evaluated by excising spots from a control gel, digesting with trypsin and analysing by MALDI-TOF/TOF mass spectrometry. Of 12 spots selected, 10 were identified including stress related proteins (e.g. HSP-70 and αBcrystallin), cytoskeletal proteins (actin and desmin) and key mitochondrial enzyme subunits (dihydrolipoamide transacylase E2).