The development of metabolic dysfunctions like diabetes and insulin resistance in mammals is regulated by a myriad of factors. Oxidative stress seems to play a central role in this process as recent evidence shows a general increase in oxidative damage and a decrease in oxidative defense associated with several metabolic diseases. These changes in oxidative stress can be directly correlated with increased fat accumulation, obesity and consumption of high calorie/high fat diets. Modulation of oxidant protection through either genetic mutation or treatment with antioxidants can significantly alter oxidative stress resistance and accumulation of oxidative damage in laboratory rodents. Antioxidant mutant mice have previously been utilized to examine the role of oxidative stress in other disease models, but have been relatively unexplored as models to study the regulation of glucose metabolism. In this review, we will discuss the evidence for oxidative stress as a primary mechanism linking obesity and metabolic disorders and whether alteration of antioxidant status in laboratory rodents can significantly alter the development of insulin resistance or diabetes.

We examined the effects of increased levels of thioredoxin 1 (Trx1) on resistance to oxidative stress and aging in transgenic mice overexpressing Trx1 [Tg(TRX1)+/0]. The Tg(TRX1)+/0 mice showed significantly higher Trx1 protein levels in all the tissues examined compared with the wild-type littermates. Oxidative damage to proteins and levels of lipid peroxidation were significantly lower in the livers of Tg(TRX1)+/0 mice compared with wild-type littermates. The survival study demonstrated that male Tg(TRX1)+/0 mice significantly extended the earlier part of life span compared with wild-type littermates, but no significant life extension was observed in females. Neither male nor female Tg(TRX1)+/0 mice showed changes in maximum life span. Our findings suggested that the increased levels of Trx1 in the Tg(TRX1)+/0 mice were correlated to increased resistance to oxidative stress, which could be beneficial in the earlier part of life span but not the maximum life span in the C57BL/6 mice.

Manganese superoxide dismutase (MnSOD) in the mitochondria plays an important role in cellular defense against oxidative damage. Homozygous MnSOD knockout (Sod2−/−) mice are neonatal lethal, indicating the essential role of MnSOD in early development. To investigate the potential cellular abnormalities underlying the aborted development of Sod2−/− mice, we examined the growth of isolated mouse embryonic fibroblasts (MEF) from Sod2−/− mice. We found that the proliferation of Sod2−/− MEFs was significantly decreased when compared with wild type MEFs despite the absence of morphological differences. The Sod2−/− MEFs produced less cellular ATP, had lower O2 consumption, generated more superoxide, and expressed less Prdx3 protein. Furthermore, the loss of MnSOD dramatically altered several markers involved in cell proliferation and growth, including decreased growth stimulatory function of mTOR signaling and enhanced growth inhibitory function of GSK-3β signaling. Interestingly, the G protein coupled receptor-mediated intracellular Ca2+ ([Ca2+]i) signal transduction was also severely suppressed in Sod2−/− MEFs. Finally, the ratio of LC3-II/LC3-I, an index of autophagic activity, was increased in Sod2−/− MEFs, consistent with a reduction of mTOR signal transduction. These data demonstrate that MnSOD deficiency results in alterations in several key signaling pathways, which may contribute to the lethal phenotype of Sod2−/− mice.

OBJECTIVE

Aging increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes. It has been proposed that increased reactive oxygen species (ROS) generation by dysfunctional mitochondria could play a role in the pathogenesis of these metabolic abnormalities. We examined whether aging per se (in subjects with normal glucose tolerance [NGT]) impairs mitochondrial function and how this relates to ROS generation, whether older subjects with IGT have a further worsening of mitochondrial function (lower ATP production and elevated ROS generation), and whether exercise reverses age-related changes in mitochondrial function.

RESEARCH DESIGN AND METHODS

Mitochondrial ATP and ROS production were measured in muscle from younger individuals with NGT, older individuals with NGT, and older individuals with IGT. Measurements were performed before and after 16 weeks of aerobic exercise.

RESULTS

ATP synthesis was lower in older subjects with NGT and older subjects with IGT versus younger subjects. Notably, mitochondria from older subjects (with NGT and IGT) displayed reduced ROS production versus the younger group. ATP and ROS production were similar between older groups. Exercise increased ATP synthesis in the three groups. Mitochondrial ROS production also increased after training. Proteomic analysis revealed downregulation of several electron transport chain proteins with aging, and this was reversed by exercise.

CONCLUSIONS

Old mitochondria from subjects with NGT and IGT display mitochondrial dysfunction as manifested by reduced ATP production but not with respect to increased ROS production. When adjusted to age, the development of IGT in elderly individuals does not involve changes in mitochondrial ATP and ROS production. Lastly, exercise reverses the mitochondrial phenotype (proteome and function) of old mitochondria.

Background

Protective effects of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1) against endotoxic shock have not been demonstrated in animal models. We used a murine model to investigate whether overexpression of SOD1 protects against endotoxic shock, and whether the genetic background of SOD1 affects its effective protective effects and susceptibility to endotoxic shock.

Methods

Transgenic (tg) mice overexpressing human SOD1 and control mice were divided into four groups based on their genetic background: (1) tg mice with mixed genetic background (tg-JAX); (2) wild-type (WT) littermates of tg-JAX strain (WT-JAX); (3) tg mice with C57BL/6J background (tg-TX); (4) WT littermates of tg-TX strain (WT-TX). Activity of SOD1 in the intestine, heart, and liver of tg and control mice was confirmed using a polyacrylamide activity gel. Endotoxic shock was induced by intraperitoneal injection of lipopolysaccharide. Survival rates over 120 hours (mean, 95% confidence interval) were analyzed using Kaplan–Meier survival curves.

Results

Human SOD1 enzymatic activities were significantly higher in the intestine, heart, and liver of both tg strains (tg-JAX and tg-TX) compared with their WT littermates (WT-JAX and WT-TX, respectively). Interestingly, the endogenous SOD1 activities in tg-JAX mice were decreased compared with their WT littermates (WT-JAX), but such aberrant changes were not observed in tg-TX mice. There was no difference in the survival time between tg-JAX and WT-JAX groups after endotoxic shock (P > 0.05). However, the survival time in the tg-TX group was more than twofold longer than that in the WT-TX group (P < 0.05). In addition, WT-JAX mice survived significantly longer than WT-TX mice (P < 0.05).

Conclusion

Aberrant decrease of endogenous SOD1 activities may have overshadowed the effect of overexpression of SOD1 in tg mice (tg-JAX). Mice with C57BL/6J background (tg-TX) are more susceptible to lipopolysaccharide-induced endotoxic shock than those with mixed genetic background (tg-JAX). Overexpression of SOD1 is protective only in mice with C57BL/6J background (tg-TX).

Summary

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2fl/fl mice). In the present study, we used TnIFastCreSod2fl/fl mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2fl/fl mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2fl/fl mice, when compared with control mice. Complex II activity is reduced by 47% in young and by ~90% in old TnIFastCreSod2fl/fl mice, associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2fl/fl mice. Complex II-linked mitochondrial ATP production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2fl/fl mice. Furthermore, in old TnIFastCreSod2fl/fl mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains greater than 2-fold elevated; and oxidative damage (measured as F2 isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2fl/fl mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.

The p53 DNA damage response attenuated with age and we have evaluated downstream factors in the DNA damage response. In old animals p21 protein accumulates in the whole cell fraction but significantly declines in the nucleus, which may alter cell cycle and apoptotic programs in response to DNA damage. We evaluated the transcriptional response to DNA damage in young and old and find 2692 genes are differentially regulated in old compared to young in response to oxidative stress (p<0.005). As anticipated, the transcriptional profile of young mice is consistent with DNA damage induced cell cycle arrest while the profile of old mice is consistent with cell cycle progression in the presence of DNA damage, suggesting the potential for catastrophic accumulation of DNA damage at the replication fork. Unique sets of DNA repair genes are induced in response to damage in old and young, suggesting the types of damage accumulating differs between young and old. The DNA repair genes upregulated in old animals point to accumulation of replication-dependent DNA double strand breaks (DSB). Expression data is consistent with loss of apoptosis following DNA damage in old animals. These data suggest DNA damage responses differ greatly in young and old animals.

Age-related loss of muscle mass and function greatly affects quality of life in the elderly population. Several hypotheses have been proposed but accumulating evidence point to alterations in neuromuscular system during aging as a key event that leads to functional denervation, muscle wasting, and weakness. Over the past few decades, age-associated degeneration of the neuromuscular junction (NMJ) and its components have been well documented. With advancing age, pre-terminal portions of motor axons exhibit regions of abnormal thinning, distension, and sprouting whereas postsynaptic endplates decrease in size, reduce in number, length, and density of postsynaptic folds. Although the exact underlying mechanisms are still lacking, recent studies provided direct evidence that age-associated increase in oxidative stress plays a crucial role in NMJ degeneration and progression of sarcopenia. Homozygous deletion of an important antioxidant enzyme, Cu,Zn superoxide dismutase (CuZnSOD, SOD1) leads to acceleration of age-dependent muscle atrophy, with a significant NMJ degeneration similar to that seen in old wild type sarcopenic animals. In this short review, we briefly summarize the current understanding of some of the cellular and molecular changes in the NMJ during aging and suggest a role for oxidative stress and mitochondrial dysfunction in age-related changes in the maintenance of neuromuscular innervation.

Aging is associated with reduced ability to maintain normal glucose homeostasis. It has been suggested that an age-associated increase in chronic pro-inflammatory state could drive this reduction in glucoregulatory function. Thioredoxins (Trx) are oxido-reductase enzymes that play an important role in the regulation of oxidative stress and inflammation. In this study, we tested whether overexpression of Trx1 in mice [Tg(TRX1)+/0] could protect from glucose metabolism dysfunction caused by high fat diet feeding. Body weight and fat mass gains with high fat feeding were similar in Tg(TRX1)+/0 and wild-type mice; however, high fat diet induced glucose intolerance was reduced in Tg(TRX1)+/0 mice relative to wild-type mice. In addition, expression of the pro-inflammatory cytokine TNF-α was reduced in adipose tissue of Tg(TRX1)+/0 mice compared to wild-type mice. These findings suggest that activation of thioredoxins may be a potential therapeutic target for maintenance of glucose metabolism with obesity or aging.

Loss of skeletal muscle mass and function is observed in many insulin-resistant disease states such as diabetes, cancer cachexia, renal failure and ageing although the mechanisms for this remain unclear. We hypothesised that impaired insulin signalling results in reduced muscle mass and function and that this decrease in muscle mass and function is due to both increased production of atrogenes and aberrant reactive oxygen species (ROS) generation. Maximum tetanic force of the extensor digitorum longus of muscle insulin receptor knockout (MIRKO) and lox/lox control mice was measured in situ. Muscles were removed for the measurement of mass, histological examination and ROS production. Activation of insulin signalling pathways, markers of muscle atrophy and indices of protein synthesis were determined in a separate group of MIRKO and lox/lox mice 15 min following treatment with insulin. Muscles from MIRKO mice had 36% lower maximum tetanic force generation compared with muscles of lox/lox mice. Muscle fibres of MIRKO mice were significantly smaller than those of lox/lox mice with no apparent structural abnormalities. Muscles from MIRKO mice demonstrated absent phosphorylation of AKT in response to exogenous insulin along with a failure to phosphorylate ribosomal S6 compared with lox/lox mice. Atrogin-1 and MuRF1 relative mRNA expression in muscles from MIRKO mice were decreased compared with muscles from lox/lox mice following insulin treatment. There were no differences in markers of reactive oxygen species damage between muscles from MIRKO mice and lox/lox mice. These data support the hypothesis that the absence of insulin signalling contributes to reduced muscle mass and function though decreased protein synthesis rather than proteasomal atrophic pathways.

To test the impact of increased mitochondrial oxidative stress as a mechanism underlying aging and age-related pathologies, we generated mice with a combined deficiency in two mitochondrial-localized antioxidant enzymes, Mn superoxide dismutase (MnSOD) and glutathione peroxidase-1 (Gpx-1). We compared life span, pathology, and oxidative damage in Gpx1−/−, Sod2+/−Gpx1+/−, Sod2+/−Gpx1−/−, and wild-type control mice. Oxidative damage was elevated in Sod2+/−Gpx1−/− mice, as shown by increased DNA oxidation in liver and skeletal muscle and increased protein oxidation in brain. Surprisingly, Sod2+/−Gpx1−/− mice showed no reduction in life span, despite increased levels of oxidative damage. Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2+/−Gpx1−/− mice (28–30 months). Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.

Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4–6 months) and old (26–28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.

Currently, the Oxidative Stress (or Free Radical) Theory of Aging is the most popular explanation of how aging occurs at the molecular level. While data from studies in invertebrates (e.g., C. elegans and Drosophila) and rodents show a correlation between increased lifespan and resistance to oxidative stress (and in some cases reduced oxidative damage to macromolecules), direct evidence showing that alterations in oxidative damage/stress play a role in aging are limited to a few studies with transgenic Drosophila that overexpress antioxidant enzymes. Over the past eight years, our laboratory has conducted an exhaustive study on the effect of under- or overexpressing a large number and wide variety of genes coding for antioxidant enzymes. In this review, we present the survival data from these studies together. Because only one (the deletion of the Sod1 gene) of the 18 genetic manipulations we studied had an effect on lifespan, our data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice.

Low levels of reactive oxygen species (ROS) production are necessary to optimize muscle force production in unfatigued muscle. In contrast, sustained high levels of ROS production have been linked to impaired muscle force production and contraction-induced skeletal muscle fatigue. Using genetically engineered mice, we tested the hypothesis that the independent transgenic overexpression of catalase (CAT), copper/zinc superoxide dismutase (CuZnSOD; SOD1) or manganese superoxide dismutase (MnSOD; SOD2) antioxidant enzymes would negatively affect force production in unfatigued diaphragm muscle but would delay the development of muscle fatigue and enhance force recovery after fatiguing contractions. Diaphragm muscle from wild-type littermates (WT) and from CAT, SOD1 and SOD2 overexpressing mice were subjected to an in vitro contractile protocol to investigate the force–frequency characteristics, the fatigue properties and the time course of recovery from fatigue. The CAT, SOD1 and SOD2 overexpressors produced less specific force (in N cm−2) at stimulation frequencies of 20–300 Hz and produced lower maximal tetanic force than WT littermates. The relative development of muscle fatigue and recovery from fatigue were not influenced by transgenic overexpression of any antioxidant enzyme. Morphologically, the mean cross-sectional area (in μm2) of diaphragm myofibres expressing myosin heavy chain type IIA was decreased in both CAT and SOD2 transgenic animals, and the percentage of non-contractile tissue increased in diaphragms from all transgenic mice. In conclusion, our results do not support the hypothesis that overexpression of independent antioxidant enzymes protects diaphragm muscle from contraction-induced fatigue or improves recovery from fatigue. Moreover, our data are consistent with the concept that a basal level of ROS is important to optimize muscle force production, since transgenic overexpression of major cellular antioxidants is associated with contractile dysfunction. Finally, the transgenic overexpression of independent endogenous antioxidants alters diaphragm skeletal muscle morphology, and these changes may also contribute to the diminished specific force production observed in these animals.

Many age-associated degenerative diseases commonly involve degradation of the extracellular matrix and aberrant matrix metalloproteinase-1 (MMP-1) expression. In diverse cell lines MMP-1 or interstitial collagenase (CL) expression is tightly regulated through a network of signals involving reactive oxygen species (ROS). However, whether the in vivo age-associated increase in CL expression is also sensitive to ROS-mediated signaling has not been established. To evaluate the contribution of ROS to the age-dependent increase in CL we monitored the levels of murine CL in two well-established models of oxidant stress. Analysis of murine CL levels in mice deficient in either of the intracellular superoxide dismutases (Sod2+/− or Sod1−/−) revealed its age- and redox-dependent expression relative to WT controls. Both age- and redox-dependent increases in murine CL expression were associated with elevations in phosphorylation of the MAP Kinases, Erk, JNK and p38. CL expression was highest in renal and skeletal muscle tissue from the aged Sod1−/− mice and was associated with a decrease in collagen staining. These findings suggest that MAPK signaling and CL production are both age- and redox-responsive. The redox sensitivity of age-associated CL expression suggests that degenerative disease associated with aberrant matrix remodeling and oxidant stress may be amenable to antioxidant-based therapies.

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes, CuZn-Superoxide Dismutase (Sod1) and Glutathione Peroxidase-1 (Gpx1).

Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3 to 6 hours in wild type mice without any lethality. In contrast, treating Sod1−/− or Gpx1−/− mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classical antioxidant genes, though long-term oxidative stress in the Sod1−/− mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor, Nrf2. The main finding of our study is that the common response to elevated oxidative stress, with diquat treatment in wild type, Gpx1−/−, Sod1−/− mice and in untreated Sod1−/− mice, is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53-target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

We evaluated the effect of overexpressing antioxidant enzymes on the lifespans of transgenic mice that overexpress copper zinc superoxide dismutase (CuZnSOD), catalase, or combinations of either CuZnSOD and catalase or CuZnSOD and manganese superoxide dismutase (MnSOD). Our results show that the overexpression of these major antioxidant enzymes, which are known to scavenge superoxide and hydrogen peroxide in the cytosolic and mitochondrial compartments, is insufficient to extend lifespan in mice.

Age-related skeletal muscle sarcopenia has been extensively studied and smooth muscle sarcopenia has been recently described, but age-related cardiac sarcopenia has not been previously examined. Therefore, we evaluated adult (7.5±0.5 months; n=27) and senescent (31.8±0.4 months; n=26) C57BL/6J mice for cardiac sarcopenia using physiological, histological, and biochemical assessments. Mice do not develop hypertension, even into senescence, which allowed us to decouple vascular effects and monitor cardiac-dependent variables. We then developed a mathematical model to describe the relationship between age-related changes in cardiac muscle structure and function. Our results showed that, compared to adult mice, senescent mice demonstrated increased left ventricular (LV) end diastolic dimension, decreased wall thickness, and decreased ejection fraction, indicating dilation and reduced contractile performance. Myocyte numbers decreased, and interstitial fibrosis was punctate but doubled in the senescent mice, indicating reparative fibrosis. Electrocardiogram analysis showed that PR interval and QRS interval increased and R amplitude decreased in the senescent mice, indicating prolonged conduction times consistent with increased fibrosis. Intracellular lipid accumulation was accompanied by a decrease in glycogen stores in the senescent mice. Mathematical simulation indicated that changes in LV dimension, collagen deposition, wall stress, and wall stiffness precede LV dysfunction. We conclude that age-related cardiac sarcopenia occurs in mice and that LV remodeling due to increased end diastolic pressure could be an underlying mechanism for age-related LV dysfunction.

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/105 deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 ± 0.002 for liver to 0.015 ± 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of γ-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after γ-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.

Oxidative damage caused by free radicals in vivo is believed to play an important role in the etiology of aging and age-associated degenerative diseases. The most direct evidence supporting this theory is the recent finding that the transgenic Drosophila that overexpress the antioxidant enzymes catalase and superoxide dismutase exhibit an increase in life span. Although the increase in life span in Drosophila by these enzymes is certainly important, the next logical direction is to demonstrate whether increased antioxidant protection occurs similarly in mammals. Several transgenic mouse models that overexpress antioxidant enzymes are currently available. However, one major shortcoming in using these transgenic mice is the difficulty of producing antioxidant overexpression in more than a few tissues. Despite the potential shortcomings of using transgenic mice, these animals provide a unique system in which individual components of a complex system, such as the antioxidant defense system, can be modulated and examined independently. Transgenic mice are therefore potentially powerful tools to study the role of various components of the antioxidant system in the aging process.

A parallel direction in the study of free radical roles in aging is to investigate the modulation of transcription factors by oxidative stress. Among these, the transcription factors, NF-κB and AP-1 are implicated in oxidative stress. The activities of these oxidative stress-response transcription factors are regulated by upstream signaling molecules, which involve a cascade of phosphorylation and dephosphorylation events leading to their activation. In this article, we review recent studies that use molecular approaches to investigate the biological role of oxidant stress. Each of these studies potentially provide new insights into the roles of free radicals and free radical damage in the aging process.

In muscle, aging is associated with a failure of adaptive responses to contractile activity, and this is hypothesized to play an important role in age-related loss of muscle mass and function. Mice lacking the Cu,Zn superoxide dismutase (Cu,ZnSOD, SOD1) show an accelerated, age-related loss of muscle mass and function. This work determined whether adult mice lacking Cu,ZnSOD (Sod1−/− mice) show a premature failure of adaptive responses to contractions in a similar manner to old wild-type (WT) mice. Adult Sod1−/− mice (6–8 months of age) had a ∼ 30% reduction in gastrocnemius muscle mass compared with age-matched WT mice. This lower muscle mass was associated with an activation of DNA binding by NFκB and AP-1 at rest. Measurements of the activity of reactive oxygen species (ROS) in single fibres from the muscles of Sod1−/− mice at rest indicated an elevation in activity compared with fibres from WT mice. Following 15 min of isometric contractions, muscle fibres from WT mice showed an increase in the intracellular ROS activities and activation of NFκB and AP-1, but no changes in either ROS activity or NFκB and AP-1 activation were seen in the muscles of Sod1−/− mice following contractions. This pattern of changes mimics that seen in the muscles of old WT mice, suggesting that the attenuated responses to contractile activity seen in old mice result from chronic exposure to increased oxidant activity. Data support the use of the Sod1−/− mouse model to evaluate potential mechanisms that contribute to the loss of muscle mass and function in the elderly.

Summary

Mice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age-related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild-type (WT) and Sod1−/− mice were loaded with NO-sensitive (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, DAF-FM) and superoxide-sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3-nitrotyrosine (3-NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from Sod1−/− mice compared with those from WT mice. Fibers from Sod1−/− mice had decreased NO availability (decreased DAF-FM fluorescence), increased 3-NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1−/− mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1−/− mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1−/− mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.

Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ) treatment of wild type mice) and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1−/−)) models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O) at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax) was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain.

The side effects of cancer therapy on normal tissues limit the success of therapy. Generation of reactive oxygen species (ROS) has been implicated for numerous chemotherapeutic agents including doxorubicin (DOX), a potent cancer chemotherapeutic drug. The production of ROS by DOX has been linked to DNA damage, nuclear translocation of p53, and mitochondrial injury; however, the causal relationship and molecular mechanisms underlying these events are unknown. The present study used wild-type (WT) and p53 homozygous knock-out (p53−/−) mice to investigate the role of p53 in the crosstalk between mitochondria and nucleus. Injecting mice with DOX (20 mg/kg) causes oxidative stress in cardiac tissue as demonstrated by immunogold analysis of the levels of 4-hydroxy-2′-nonenal (4HNE)-adducted protein, a lipid peroxidation product bound to proteins. 4HNE levels increased in both nuclei and mitochondria of WT DOX-treated mice but only in nuclei of DOX-treated p53(−/−) mice, implicating a critical role for p53 in causing DOX-induced oxidative stress in mitochondria. The stress-activated protein c-Jun amino-terminal kinase (JNKs) was activated in response to increased 4HNE in WT mice but not p53(−/−) mice receiving DOX treatment, as determined by co-immunoprecipitation of HNE and pJNK. The activation of JNK in DOX treated WT mice was accompanied by Bcl-2 dissociation from Beclin in mitochondria and induction of type II cell death (autophagic cell death), as evidenced by an increase in LC3-I/LC-3-II ratio and γ-H2AX, a biomarker for DNA damage. The absence of p53 significantly reduces mitochondrial injury, assessed by quantitative morphology, and decline in cardiac function, assessed by left ventricular ejection fraction and fraction shortening. These results demonstrate that p53 plays a critical role in DOX-induced cardiac toxicity, in part, by the induction of oxidative stress mediated retrograde signaling.