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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 10, 2005 as doi:10.1096/fj.04-2622fje. |
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* University of Florida, Biochemistry of Aging Laboratory, Gainesville, Florida, USA; and
Linus Pauling Institute and Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon, USA
1Correspondence: University of Florida, Biochemistry of Aging Laboratory, P.O. Box 118206, Gainesville FL 32611, USA. E-mail: cleeuwen{at}ufl.edu
SPECIFIC AIMS
Mitochondrial dysfunction and the accumulation of oxidative damage to macromolecules are believed to play key roles in the aging process. According to the mitochondrial theory of aging, reactive oxygen species, produced via mitochondrial respiration attack mitochondrial DNA (mtDNA). Mitochondrial respiratory complex function may be altered as a result of mtDNA mutations, leading to increased reactive oxygen species production and further damage to mtDNA, as well as other macromolecules. The age-related increase in oxidative damage to DNA, lipids, and proteins may be partly responsible for mitochondrial dysfunction with age. Characterization of age-related changes to cardiac mitochondria has been complicated by the fact that two distinct populations, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), exist in the myocardium. For example, there is continuous debate as to whether mitochondrial oxidant production increases with age. Numerous published studies have supported an increase in cardiac mitochondrial oxidant production with age, but others have reported no changes in H2O2 production with age. These discrepancies may be a result of the fact that most isolation procedures yield either SSM alone or a mixed population of SSM and IFM. We investigated whether differences in hydrogen peroxide production and oxidative stress existed between cardiac SSM and IFM isolated from young (6 mo) and old (24 mo) male Fischer-344 rats. The novelty of this paper is that for the first time we investigate free radical production and oxidative stress in these two very different mitochondrial populations and our results suggest that the mitochondrial theory of aging may need some modification.
PRINCIPAL FINDINGS
We found that there was a significant increase in oxidative stress levels (4-hydroxy-2-nonenal (HNE)-modified proteins, protein carbonyls, and malondialdehyde) in the IFM with age. In contrast, only protein carbonyls were elevated in SSM with age; levels of oxidative stress were much greater in IFM than in SSM. Significant age-related increases in MnSOD, GPX, and CAT activities were detected in IFM; in SSM, MnSOD and GPX activities increased with age and CAT activity declined. These increases in antioxidant enzyme activity likely occurred in response to increased mitochondrial production of superoxide (O2•–) and hydrogen peroxide (H2O2). SSM produced more H2O2 with age whereas the increase in IFM was not significant, but this may be due to the higher antioxidant enzyme activity observed in IFM vs. SSM. Reduced glutathione levels were significantly lower in IFM than SSM in both young and old rats whereas glutathione reductase activity did not differ with age or mitochondrial subpopulations, indicating increased consumption of essential antioxidants. Hence, IFM are under greater oxidative stress than are SSM.
1. Hydrogen peroxide production
Under normal conditions, mitochondria produce hydrogen peroxide as a result of inefficient reduction of O2 to H2O. O2•– produced during electron transport is released into the mitochondrial matrix and rapidly dismutated to H2O2 by manganese superoxide dismutase (MnSOD), which is abundant in the mitochondrial matrix. Since O2•– production is difficult to measure in intact mitochondria without disturbing the respiratory quotient, H2O2 production (a relatively stable oxidant) is often measured outside the mitochondria and partly reflects mitochondrial O2•– production. H2O2 production was measured in intact SSM and IFM immediately following the isolation procedure (Fig. 1
). There was an age-associated increase in H2O2 production from SSM whereas H2O2 production from IFM with age tended to be higher, but was not statistically significant (P=0.1603). No significant differences in H2O2 production were detected between SSM and IFM isolated from young rats. In contrast, IFM from old rats produced significantly less H2O2 than SSM from old rats. This may be a result of the increased GPX and CAT activity observed in old IFM compared with old SSM, an adaptation that would confer greater ability to scavenge H2O2 inside the mitochondria.
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2. 4-Hydroxy-2-nonenal content
4-Hydroxy-2-nonenal (HNE) is an end product of lipid peroxidation that is highly reactive with other biological molecules, including proteins. HNE exerts numerous effects, including inhibition of protein and DNA synthesis and enzyme inactivation, and is believed to play a major role in oxidative stress-induced cellular dysfunction. No differences in the amount of HNE-modified proteins in the cytosol and SSM were detected between young and old animals (Fig. 2
A, B). In striking contrast, IFM from old animals exhibited significantly greater amounts of HNE-modified proteins than did young animals (Fig. 2C
).
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CONCLUSIONS AND SIGNIFICANCE
Biochemical and functional differences in SSM and IFM have been reported. However, only one study has investigated whether these two populations exhibit differences in oxidant production and oxidative stress. We have expanded on these data to show convincingly that oxidative stress increases with age and that IFM are subjected to greater amounts of stress than are SSM.
We detected a significant increase in H2O2 production from SSM but not IFM, with age. On the surface, these results appear to be in contrast to the findings recently reported by Suh et al., that oxidant production increased with age in IFM, but not SSM, isolated from heart. However, Suh and colleagues did not directly measure H2O2 production from isolated mitochondria, but used the rate of oxidation of 2’7’dihydrodichlorofluorescein (DCFH) in isolated mitochondria as an indicator of total mitochondrial oxidant production. DCFH is able to cross the mitochondrial membranes, whereas our assay specifically measures the amount of H2O2 released from intact mitochondria. Our findings of increased antioxidant enzyme activity, reduced glutathione levels, and increased oxidative damage in IFM support the notion that oxidant production inside old IFM was probably higher than in young IFM.
Although some studies report that antioxidant enzyme activities decline in the aging heart, in light of the fact that exposure to oxidants acts as a signal to increase the activity and expression of antioxidant enzymes, it seems more likely to expect an increase in antioxidant enzyme activity with age, as this adaptation may help to protect tissues from oxidative stress. Our findings that MnSOD and mitochondrial GPX activities are increased with age agree with previous studies. The fact that we detected higher GPX and CAT activities in old IFM vs. old SSM indicates that IFM may have a greater ability to scavenge H2O2 inside the mitochondria and this may be a likely explanation as to why we did not detect a significant increase in H2O2 production from IFM with age.
Despite the increased antioxidant enzyme activity, IFM are subjected to greater oxidative stress than SSM, as indicated by the increased levels of protein and lipid oxidative damage. An accumulation of oxidized proteins is believed to play a key role in the loss of physiological function with age, since oxidized proteins can lose catalytic activity and are also prone to forming large, potentially cytotoxic, protein aggregates. Furthermore, it has been reported that lipid peroxidation is a major contributor to the age-related loss of membrane fluidity and that two aldehydic lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), are primarily responsible for the decrease in membrane fluidity. The increase in carbonyls and lipid peroxidation in IFM likely contributes to the selective age-related decline in function and protein yield observed in this population.
There is growing evidence that SSM and IFM are differently affected by aging, as seen by our data and work by others. Since many published studies have isolated SSM or a mixture of SSM and IFM, caution should be used when evaluating conclusions regarding age-related changes in mitochondrial oxidant production and oxidative stress. We showed clear increases in oxidative stress in the mitochondria, but not in the cytosol. Levels of HNE (Fig. 2A
) and GSSG and activities of SOD, CAT, and GR (data not shown) did not change in the cytosol with age, lending further support to the idea that mitochondria are centrally involved in the aging process.
Our data, along with that from other groups, suggests that reactive oxygen species produced via mitochondrial respiration cause oxidative stress and impaired function in IFM to a greater extent than in SSM with age. Due to their close proximity to myofibrils, IFM are probably the primary source of ATP for myosin ATPases. Therefore, the accumulation of oxidant-induced damage in IFM may be the underlying cause of the alterations observed in myocardial function with age. Additional studies are needed to determine whether IFM dysfunction triggers apoptotic and/or necrotic cell death and contributes to the loss of myocytes and compensatory hypertrophy observed with age. It will also be of interest to determine whether IFM accumulate more mitochondrial DNA mutations with age than SSM.
Cardiac IFM not only exhibit significant declines in function with age, but are subjected to greater oxidative stress than are SSM (Fig. 3
). These results emphasize the importance of studying both mitochondrial populations when attempting to elucidate the contribution of mitochondrial dysfunction to myocardial aging and suggest that anatomical location and specific metabolic demand may be critical to the rate of mitochondrial aging and should be taken into consideration when discussing the mitochondrial theory of aging.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2622fje;
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