Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-(alpha)-lipoic acid

Abstract

Oxidative stress has been implicated as a causal factor in the aging process of the heart and other tissues. To determine the extent of age-related myocardial oxidative stress, oxidant production, antioxidant status, and oxidative DNA damage were measured in hearts of young (2 months) and old (28 months) male Fischer 344 rats. Cardiac myocytes isolated from old rats showed a nearly threefold increase in the rate of oxidant production compared to young rats, as measured by the rates of 2,7-dichlorofluorescin diacetate oxidation. Determination of myocardial antioxidant status revealed a significant twofold decline in the levels of ascorbic acid (P = 0.03), but not alpha-tocopherol. A significant age-related increase (P = 0.05) in steady-state levels of oxidative DNA damage was observed, as monitored by 8-oxo-2'-deoxyguanosine levels. To investigate whether dietary supplementation with (R)-alpha-lipoic acid (LA) was effective at reducing oxidative stress, young and old rats were fed an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death. Cardiac myocytes from old, LA-supplemented rats exhibited a markedly lower rate of oxidant production that was no longer significantly different from that in cells from unsupplemented, young rats. Lipoic acid supplementation also restored myocardial ascorbic acid levels and reduced oxidative DNA damage. Our data indicate that the aging rat heart is under increased mitochondrial-induced oxidative stress, which is significantly attenuated by lipoic acid supplementation.

Figure 1

O₂ consumption in isolated cardiac myocyte declines with age. O₂ consumption in isolated cardiac myocytes from young and old rats was monitored using a Clark type oxygen electrode (Yellow Springs Instruments, Yellow Springs, Ohio). Cardiac myocytes (1×10⁶) were added to 3 ml of Krebs-Henseleit buffer supplemented with 20 mM glucose, pH 7. 4, that had been pre-equilibrated to 37°C, and oxygen consumption was monitored for at least 15 min (25). Results show that O₂ consumption, an indicator of cellular metabolic rate, declines sharply with age. Results are expressed as mean ± SE for cells from young rats (n=5) and the mean ± SE (n=5) for cells from old rats.

Figure 2

Cardiac myocytes produce significantly more oxidants with age. A) Cells from old rats (24 months) produce ~45% more oxidants, as measured by DCFH fluorescence, than cells from young (3 months) rats. B) The rate of oxidant production is even more marked when oxidant production is normalized to the cellular rate of O₂ consumption. Results are expressed as the mean ± SE of 3 experiments.

Figure 3

A 2 wk regimen of LA markedly lowers the age-related increase in cellular oxidant production. Myocardial oxidant production in both young (2–4 months; n=9) and old (24–28 months; n=8) rats treated with or without 0.2% (w/w) lipoic acid was measured. Results show that lipoic acid treatment significantly reversed the age-related increase in oxidant production back to levels not different from the young. Results are expressed as mean ± SE for heart isolated from young (n=4), young LA (n=5), old (n=4), and old LA (n=4). One-way ANOVA was used for statistical analysis with the level of significance defined as P < 0.05.

Figure 4

Dietary supplementation of LA leads to restoration of ascorbate back to the levels found in the young. Ascorbate levels in freshly isolated hearts from old and young animals were determined after a 2-wk supplementation of the AIN-93M diet with or without 0.2% (w/w) (R)-α–lipoic acid. Ascorbate levels in the old supplemented animals (n=12) were significantly higher (P=0.03) than old unsupplemented animals (n=10), to a level no longer significantly different from young unsupplemented animals. Results are expressed as mean ± SE for heart isolated from young (3), young LA (n=3), old (n=10), and old LA (n=12). One-way analysis of variance test was used for statistical analysis with the level of significance defined as P < 0.05.

Figure 5

LA supplementation leads to marked reduction in the steady-state levels of 8-oxo-dG. The steady-state levels of 8-oxo-dG in freshly isolated hearts from old and young rats after 2 wk supplementation with an AIN-93M diet with or without 0.2% (w/w) (R)-α-lipoic acid by using coulometric detection after separation with HPLC. The results show that LA supplementation significantly lowered the age-related accumulation of 8-oxo-dG in hearts from old rats. Results are expressed as mean ± SE for heart isolated from young (n=8), young LA (n=3), old (n=10), and old LA (n=9). One-way ANOVA was used for statistical analysis with the level of significance defined as P < 0.05.