Youngmok C. Jang

Age-associated increases in oxidative stress and antioxidant enzyme activities in cardiac interfibrillar mitochondria: implications for the mitochondrial theory of aging

Mitochondrial dysfunction and the accumulation of oxidative damage to macromolecules are believed to play key roles in the aging process. Characterization of age‐related changes to cardiac mitochondria has been complicated by the fact that two distinct populations of mitochondria exist in the myocardium: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). We investigated whether differences in hydrogen peroxide production (H2O2) and oxidative stress existed between cardiac SSM and IFM isolated from young (6 mo) and old (24 mo) male Fischer‐344 rats. There was a significant increase in oxidative stress levels (4‐hydroxy‐2‐nonenal‐modified proteins, protein carbonyls, and malondialdehyde) in IFM with age. In contrast, only protein carbonyls were elevated in SSM with age. Significant age‐related increases in MnSOD, GPX, and CAT activities were detected in IFM, while 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 and hydrogen peroxide. Indeed, SSM produced more H2O2 with age, while the increase in IFM was not significant, but this may be due to the higher antioxidant enzyme activity observed in IFM compared with SSM. Finally, reduced glutathione levels were significantly lower in IFM compared with SSM in both young and old rats, while glutathione reductase activity was not different with age or mitochondrial subpopulations, indicating increased consumption of glutathione. The accumulation of oxidant‐induced damage in IFM may be a major contributing factor to the age‐related alterations in myocardial function. Our results emphasize the importance of studying both mitochondrial populations when attempting to elucidate the contribution of mitochondrial dysfunction to myocardial aging.

Increased superoxide in vivo accelerates age-associated muscle atrophy through mitochondrial dysfunction and neuromuscular junction degeneration

Oxidative stress has been implicated in the etiology of age‐related muscle loss (sarcopenia). However, the underlying mechanisms by which oxidative stress contributes to sarcopenia have not been thoroughly investigated. To directly examine the role of chronic oxidative stress in vivo, we used a mouse model that lacks the antioxidant enzyme CuZnSOD (Sodl). Sod1−/− mice are characterized by high levels of oxidative damage and an acceleration of sarcopenia. In the present study, we demonstrate that muscle atrophy in Sod1−/− mice is accompanied by a progressive decline in mitochondrial bioenergetic function and an elevation of mitochondrial generation of reactive oxygen species. In addition, Sod1−/− muscle exhibits a more rapid induction of mitochondrial‐mediated apoptosis and loss of myonuclei. Furthermore, aged Sod1−/− mice show a striking increase in muscle mitochondrial content near the neuromuscular junctions (NMJs). Despite the increase in content, the function of mitochondria is significantly impaired, with increased denervated NMJs and fragmentation of acetylcholine receptors. As a consequence, contractile force in aged Sod1−/− muscles is greatly diminished. Collectively, we show that Sod1−/− mice display characteristics of normal aging muscle in an accelerated manner and propose that the superoxide‐induced NMJ degeneration and mitochondrial dysfunction are potential mechanisms of sarcopenia.—Jang, Y. C., Lustgarten, M. S., Liu, Y., Muller, F. L., Bhattacharya, A., Liang, H., Salmon, A. B., Brooks, S. V., Larkin, L., Hayworth, C. R., Richardson, A., and Van Remmen, H. Increased superoxide in vivo accelerates age‐associated muscle atrophy through mitochondrial dysfunction and neuro‐muscular junction degeneration. FASEB J. 24, 1376–1390 (2010). www.fasebj.org

Overexpression of Mn Superoxide Dismutase Does Not Increase Life Span in Mice

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.

The mitochondrial theory of aging: Insight from transgenic and knockout mouse models

A substantial body of evidence has accumulated over the past 35 years in support of a role for oxidative damage to the mitochondrial respiratory chain and mitochondrial DNA in the determination of mammalian lifespan. The goal of this review is to provide a concise summary of recent studies using transgenic and knockout mouse models with altered expression of mitochondrial antioxidant enzymes (MnSOD (Sod2Tg and Sod2(+/-)), thioredoxin 2 (Trx2(+/-)), mitochondrial targeted catalase (mCAT) and mutant mice models that have been genetically manipulated to increase mitochondrial deletions or mutations (Polgamma(D257A/D257A) mutant mice) to examine the role of mitochondrial oxidative stress in aging. The majority of studies using these strategies do not support a clear role for mitochondrial oxidative stress or a vicious cycle of oxidative damage in the determination of lifespan in mice and furthermore do not support the free radical theory of aging. However, several key questions remain to be addressed and clearly more studies are required to fully understand the role of mitochondria in age-related disease and aging.

Age-associated alterations of the neuromuscular junction

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 and 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.

High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates

Despite the considerable interest in superoxide as a potential cause of pathology, the mechanisms of its deleterious production by mitochondria remain poorly understood. Previous studies in purified mitochondria have found that the highest rates of superoxide production are observed with succinate-driven reverse-electron transfer through complex I, although the physiological importance of this pathway is disputed because it necessitates high concentrations of succinate and is thought not to occur when NAD is in the reduced state. However, very few studies have examined the rates of superoxide production with mitochondria respiring on both NADH-linked (e.g. glutamate) and complex II-linked substrates. In the present study, we find that the rates of superoxide production (measured indirectly as H2O2) with glutamate+succinate (approximately 1100 pmol of H2O2 x min(-1) x mg(-1)) were unexpectedly much higher than with succinate (approximately 400 pmol of H2O2 x min(-1) x mg(-1)) or glutamate (approximately 80 pmol of H2O2 x min(-1) x mg(-1)) alone. Superoxide production with glutamate+succinate remained high even at low substrate concentrations (<1 mM), was decreased by rotenone and was completely eliminated by FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), indicating that it must in large part originate from reverse-electron transfer through complex I. Similar results were obtained when glutamate was replaced with pyruvate, alpha-ketoglutarate or palmitoyl carnitine. In contrast, superoxide production was consistently lowered by the addition of malate (malate+succinate approximately 30 pmol of H2O2 x min(-1) x mg(-1)). We propose that the inhibitory action of malate on superoxide production can be explained by oxaloacetate inhibition of complex II. In summary, the present results indicate that reverse-electron transfer-mediated superoxide production can occur under physiologically realistic substrate conditions and suggest that oxaloacetate inhibition of complex II may be an adaptive mechanism to minimize this.

Doxorubicin treatment in vivo activates caspase‐12 mediated cardiac apoptosis in both male and female rats

We investigated in vivo the chemotherapeutic anthracycline agents doxorubicin and its ability to activate mitochondrial‐mediated, receptor‐mediated and endoplasmic/sarcoplasmic reticulum‐mediated apoptosis transduction pathways in cardiac tissue from male and female rats. We administered a single low dose of doxorubicin (10 mg/kg of body weight, i.p.) and then isolated mitochondrial and cytosolic proteins one and four days later from the heart. Caspase‐3 protein content and caspase‐3 activity were significantly increased after day four of doxorubicin treatment in both male and female rats. However, while males had DNA fragmentation at day one but not day four following doxorubicin administration, females showed no significant increase in DNA fragmentation at either time. Caspase‐12, localized in the SR, is considered a central caspase, and its activation by cleavage via calpain indicates activation of the SR‐mediated pathway of apoptosis. Cleaved caspase‐12 content and calpain activity significantly increased after day four of doxorubicin treatment in both sexes. In the mitochondrial‐mediated pathway, there were no significant treatment effects observed in cytosolic cytochrome c and cleaved (active) caspase‐9 in either sex. In control rats (saline injection), glutathione peroxidase (GPX) activity and hydrogen peroxide (H2O2) production were lower in females compared to males. Doxorubicin treatment did not significantly affect H2O2, GPX activity or ATP production in isolated mitochondria in either sex. Female rats produced significantly lower levels of H2O2 production one day after doxorubicin treatment, whereas male rats produced significantly less mitochondrial H2O2 four days after doxorubicin treatment. The receptor‐mediated pathway (caspase‐8 and c‐FLIP) showed no evidence of being significantly activated by doxorubicin treatment. Hence, doxorubicin‐induced apoptosis in vivo is mediated by the SR to a greater extent than other apoptotic pathways and should therefore be considered for targeted therapeutic interventions. Moreover, no major sex differences exist in apoptosis signaling transduction cascade due to doxorubicin treatment.

PGC-1α protects neurons and alters disease progression in an amyotrophic lateral sclerosis mouse model

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. We sought to determine whether peroxisome proliferator–activated receptor γ coactivator 1α (PGC‐1α) would have a beneficial effect on this disease. Methods: PGC‐1α transgenic mice were crossed with SOD1 mutant G93A DL mice. Results: We observed a moderate but non‐significant increase in average lifespan in PGC‐1α/G93A DL mice, as compared with G93A DL mice (292 ± 3 days vs. 274 ± 7 days). Although the onset of ALS was not altered, progression of the disease was significantly slower (∽34% increase in duration) in the PGC‐1α/G93A DL mice. These mice also exhibited markedly improved performance on the rotarod test, and the improved motor activity was associated with a decreased loss of motor neurons and less degeneration of neuromuscular junctions. Conclusion: A sustained level of excitatory amino acid transporter protein 2 (EAAT2) in astrocytes of the PGC‐1α/G93A DL mice may contribute to neuronal protection. Muscle Nerve 2011

Conditional knockout of Mn-SOD targeted to type IIB skeletal muscle fibers increases oxidative stress and is sufficient to alter aerobic exercise capacity.

In vitro studies of isolated skeletal muscle have shown that oxidative stress is limiting with respect to contractile function. Mitochondria are a potential source of muscle function-limiting oxidants. To test the hypothesis that skeletal muscle-specific mitochondrial oxidative stress is sufficient to limit muscle function, we bred mice expressing Cre recombinase driven by the promoter for the inhibitory subunit of troponin (TnIFast-iCre) with mice containing a floxed Sod2 (Sod2(fl/fl)) allele. Mn-SOD activity was reduced by 82% in glycolytic (mainly type II) muscle fiber homogenates from young TnIFastCreSod2(fl/fl) mice. Furthermore, Mn-SOD content was reduced by 70% only in type IIB muscle fibers. Aconitase activity was decreased by 56%, which suggests an increase in mitochondrial matrix superoxide. Mitochondrial superoxide release was elevated more than twofold by mitochondria isolated from glycolytic skeletal muscle in TnIFastCreSod2(fl/fl) mice. In contrast, the rate of mitochondrial H(2)O(2) production was reduced by 33%, and only during respiration with complex II substrate. F(2)-isoprostanes were increased by 36% in tibialis anterior muscles isolated from TnIFastCreSod2(fl/fl) mice. Elevated glycolytic muscle-specific mitochondrial oxidative stress and damage in TnIFastCreSod2(fl/fl) mice were associated with a decreased ability of the extensor digitorum longus and gastrocnemius muscles to produce contractile force as a function of time, whereas force production by the soleus muscle was unaffected. TnIFastCreSod2(fl/fl) mice ran 55% less distance on a treadmill than wild-type mice. Collectively, these data suggest that elevated mitochondrial oxidative stress and damage in glycolytic muscle fibers are sufficient to reduce contractile muscle function and aerobic exercise capacity.

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging.

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 this 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 approximately 90% in old TnIFastCreSod2fl/fl mice, and was found to be 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 Adenosine‐Tri‐Phosphate (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 > 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.