Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart

Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, p articularly metabolites and proteins containing thiol groups, HNE is cytoto xic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reper fusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and dens itometry of Western blots, we quantified the amount of HNE-protein adduct i n the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemi a, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In stud ies with individual hearts (n = 4/group), control aerobic perfusion (70 min ) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), admin istration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by similar to 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that se en with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. West ern immunoblot results were confirmed in studies using in situ immunofluore scent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to ref lect a toxic sequence of events that might act to compromise tissue surviva l during ischemia and recovery on reperfusion.