Lipid hydroperoxide modification of proteins during myocardial ischaemia.
OBJECTIVE
Lipid hydroperoxides (LOOH) are lipid peroxidation products formed during oxidative stress. A component of their cytotoxicity is mediated by the direct modification of proteins.


OBJECTIVE
(i) To assess whether ischaemia and reperfusion in the isolated rat heart generates LOOH-protein (ii) to characterise the extent, time-course and subcellular localization of any protein adducts formed.


METHODS
Using a well-characterised antibody which binds to LOOH-modified proteins and densitometry of Western blots, we quantified the amounts of LOOH-protein in control aerobically perfused rat hearts and those subjected to ischaemia with and without reperfusion.


RESULTS
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 subjected 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 proceeding 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 (1 mM) to hearts for 5 min before the onset of 30 min ischaemia efficiently attenuated the formation of LOOH-protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in situ immunofluorescent studies which showed marked LOOH-protein immunostaining in ischaemic tissue around the sarcolemmal membrane.


CONCLUSIONS
We conclude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the pathophysiology of ischaemic injury. In addition, these modifications may be initiators 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.
Eaton
P
P
Cardiovascular Research, The Centre for Cardiovascular Biology and Medicine, King's College London, The Rayne Institute, St. Thomas' Hospital, SE1 7EH, London, UK. philip.eaton@kcl.ac.uk
Hearse
D J
DJ
Shattock
M J
MJ
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D007106:Immune Sera; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000378:metabolism*; D008297:Male; D017202:Myocardial Ischemia / Q000378:metabolism*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar
D007106:Immune Sera; D008054:Lipid Peroxides; D011506:Proteins