Evidence for oxidative and not reductive stress in the aged rabbit heart

Reductive stress, which is due to a paradoxical excess of antioxidants such as reduced glutathione (GSH) and GSH-related enzymes associated with decreased oxidant levels, has emerged as a pathogenetic mechanism of myocardial damage in pathological conditions such as protein aggregation cardiomyopathy. Notably, in the aged heart a cardiomyopathy-like pathology occurs leading to myocardial dysfunction. Whether reductive stress, or instead its counterpart oxidative stress, is operative in the aged mammalian heart needs to be elucidated also for the different therapeutic implications of such redox stress conditions. In the present investigation, we assessed GSH and the specific enzymatic activities of γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GSSG-Red) and selenium-dependent glutathione peroxidase (GSH-Px) as endogenous antioxidants, together with oxidized glutathione (GSSG) and the glutathione redox ratio (GSH/GSSG), in the aerobically perfused hearts of aged rabbits (about 4.5 years old) and young adult control rabbits (3-4 months old). We also assessed in the aged and control hearts H₂O₂ and catalytically active low molecular weight iron (LMWI) as oxidant forces, as well as fluorescent damage products of lipid peroxidation (FDPL) and protein carbonyls (PC) as biomarkers of lipid and protein oxidation. Moreover, the effects of 4.5 mM N-acetylcysteine (NAC) as reducing thiol antioxidant were studied on hemodynamic parameters and lipid peroxidation in the perfused hearts of the aged and control rabbits. The levels of GSH and of the GSH/GSSG ratio were lower, and those of GSSG higher, in the aged than in the control hearts. The aged hearts were also characterized by decreased activities of the antioxidant enzymes γ-GCS, GSSG-Red and GSH-Px, as well as by heightened levels of H₂O₂, LMWI, FDPL and PC, highlighting the occurrence of aging-dependent oxidative stress. Associated with such biochemical alterations, hemodynamic dysfunction occurred in the aged rabbit hearts, as evidenced by lowered developed pressure (DP) and enhanced end-diastolic pressure (EDP) with decreased coronary flow (CF). Remarkably, NAC administration significantly improved DP and EDP, and lowered lipid peroxidation, electively in the aged hearts. In conclusion, oxidative and not reductive stress is operative in the aged rabbit heart, whose hemodynamic dysfunction is improved by NAC together with reduction in myocardial lipid peroxidation.