Antioxidant vitamins prevent cardiomyocyte apoptosis produced by norepinephrine infusion in ferrets

Background: Norepinephrine (NE) induces apoptosis in cultured neonatal rat myocytes. To determine whether this change occurred in intact animals after chronic subhypertensive doses of NE, and whether the effect was mediated via oxidative stress produced by NE, we measured myocyte apoptosis and apoptotic gene proteins in ferrets receiving chronic NE with and without antioxidant vitamin treatment. Methods: Ferrets were administered either subcutaneous NE or vehicle and simultaneously assigned to receive antioxidant vitamins (beta -carotene, ascorbic acid and alpha -tocopherol) or vehicle for 4 weeks. Resting hemodynamics and plasma NE were measured at 4 weeks. Animals were then sacrificed for measuring cardiac myocyte size by electron microscopy, and oxidative stress by reduced to oxidized glutathione (GSH/GSSG) ratio and mitochrondrial DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Cardiomyocyte apoptosis was detected by both terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and monoclonal antibody to single-stranded DNA (Mab) staining. Western blot analysis was used to measure the expression of the antiapoptotic protein Bcl-2 and apoptotic protein Bax. Results: NE administration produced a 4-fold increase in plasma NE, but had no effect on resting hem rate, heart weight, arterial pressure, left ventricular systolic function or cardiac cell size. NE infusion decreased tissue GSH/GSSG ratio, and increased mtDNA 8-oxo-dG, and TUNEL- and Mab-positive apoptotic cells. These changes were associated with a 27% decrease in Bcl-2 protein, a 42% increase in Bax and a 57% reduction in the ratio of Bcl-2/Bax. All of the changes were prevented by co-administration of antioxidant vitamins. Conclusion: NE administration at a dose which produced no significant increase in blood pressure or myocyte hypertrophy caused cardiomyocyte apoptosis in intact animals. This effect was associated with an increase in oxidative stress, up-regulation of Bax protein and down-regulation of Bcl-2 protein. Antioxidant vitamins prevented the changes produced by NE. The findings suggest that NT-induced myocyte apoptosis is mediated by oxidative stress, and that antioxidant vitamins may be beneficial in heart failure in which cardiac NE release is increased. (C) 2001 Elsevier Science B.V. All rights reserved.