Lipid hydroperoxide modification of proteins during myocardial ischaemia

Objective: Lipid hydroperoxides (LOOH) are lipid peroxidation products form ed during oxidative stress. A component of their cytotoxicity is mediated b y the direct modification of proteins. Objectives: (i) To assess whether is chaemia and reperfusion in the isolated rat heart generates LOOH-protein (i i) to characterise the extent, time-course and subcellular localization of any protein adducts formed. Methods: Using a well-characterised antibody wh ich binds to LOOH-modified proteins and densitometry of Western blots, we q uantified the amounts of LOOH-protein in control aerobically perfused rat h earts and those subjected to ischaemia with and without repel-fusion. Resul ts: Hearts (n=3/4 group), analysed after various periods (0, 5, 10, 20 and 30 min) of zero-flow global ischaemia, exhibited a time-dependent increase in the LOOH-mediated modification of a number of proteins. In hearts subjec ted to 30 min of ischaemia and then reperfused for various times (0, 5, 10, 20, 30 or 60 min) no changes in LOOH-protein content achieved during the p roceeding ischaemic episode were detected. Reperfusion after short Periods of ischaemia (5 or 10 min) also did not result in reperfusion-induced LOOH- protein formation, Administration of mercaptopropionylglycine (I mM) to hea rts for 5 min before the onset of 30 min ischaemia efficiently attenuated t he formation of LOOH-protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in si tu immunofluorescent studies which showed marked LOOH-protein immunostainin g in ischaemic tissue around the sarcolemmal membrane. Conclusions: We conc lude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the patho physiology of ischaemic injury. In addition, these modifications may be ini tiators of oxidant-induced signal transduction pathways. These findings are consistent with an oxidant stress occurring during ischaemia which is not exacerbated or reduced during the first 60 min of reperfusion. (C) 2001 Els evier Science BY All rights reserved.