Michael S. Lustgarten

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.

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.

Diminished skeletal muscle microRNA expression with aging is associated with attenuated muscle plasticity and inhibition of IGF-1 signaling

Older individuals have a reduced capacity to induce muscle hypertrophy with resistance exercise (RE), which may contribute to the age‐induced loss of muscle mass and function, sarcopenia. We tested the novel hypothesis that dysregulation of microRNAs (miRNAs) may contribute to reduced muscle plasticity with aging. Skeletal muscle expression profiling of protein‐coding genes and miRNA was performed in younger (YNG) and older (OLD) men after an acute bout of RE. 21 miRNAs were altered by RE in YNG men, while no RE‐induced changes in miRNA expression were observed in OLD men. This striking absence in miRNA regulation in OLD men was associated with blunted transcription of mRNAs, with only 42 genes altered in OLD men vs. 175 in YNG men following RE, demonstrating a reduced adaptability of aging muscle to exercise. Integrated bioinformatics analysis identified miR‐126 as an important regulator of the transcriptional response to exercise and reduced lean mass in OLD men. Manipulation of miR‐126 levels in myocytes, in vitro, revealed its direct effects on the expression of regulators of skeletal muscle growth and activation of insulin growth factor 1 (IGF‐1) signaling. This work identifies a mechanistic role of miRNA in the adaptation of muscle to anabolic stimulation and reveals a significant impairment in exercise‐induced miRNA/mRNA regulation with aging.—Rivas, D. A., Lessard, S. J., Rice, N. P., Lustgarten, M. S., So, K., Goodyear, L. J., Parnell, L. D., Fielding, R. A. Diminished skeletal muscle microRNA expression with aging is associated with attenuated muscle plasticity and inhibition of IGF‐1 signaling. FASEB J. 28, 4133‐4147 (2014). www.fasebj.org

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.

Dietary restriction attenuates age-associated muscle atrophy by lowering oxidative stress in mice even in complete absence of CuZnSOD.

Age‐related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age‐related pathology in a variety of species, has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress–induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (60% of ad libitum fed diet). We have previously shown that the Sod1−/− mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1−/− mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation, and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca2+ regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F2‐isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress–induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.

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.

Complex I generated, mitochondrial matrix-directed superoxide is released from the mitochondria through voltage dependent anion channels.

Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2(fl/fl)). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ∼4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (∼75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.

Loss of manganese superoxide dismutase leads to abnormal growth and signal transduction in mouse embryonic fibroblasts.

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 (MEFs) from Sod2(-/-) mice. We found that the proliferation of Sod2(-/-) MEFs was significantly decreased compared with wild-type MEFs despite the absence of morphological differences. The Sod2(-/-) MEFs produced less cellular ATP, had lower O(2) 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 Ca(2+) signal transduction was also severely suppressed in Sod2(-/-) MEFs. Finally, the ratio of microtubule-associated protein light chain 3 (LC3)-II/LC3-I, an index of autophagic activity, was increased in Sod2(-/-) MEFs, consistent with a reduction in 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.

Serum Glycine Is Associated with Regional Body Fat and Insulin Resistance in Functionally-Limited Older Adults

Background Metabolic profiling may provide insight into biologic mechanisms related to age-related increases in regional adiposity and insulin resistance. Objectives The objectives of the current study were to characterize the association between mid-thigh intermuscular and subcutaneous adipose tissue (IMAT, SCAT, respectively) and, abdominal adiposity with the serum metabolite profile, to identify significant metabolites as further associated with the homeostasis model assessment of insulin resistance (HOMA-IR), and, to develop a HOMA-IR associated metabolite predictor set representative of regional adiposity, in 73 functionally-limited (short physical performance battery ≤10; SPPB) older adults (age range, 70–85 y). Methods Fasting levels of 181 total metabolites, including amino acids, fatty acids and acylcarnitines were measured with use of an untargeted mass spectrometry-based metabolomic approach. Multivariable-adjusted linear regression was used in all analyses. Results Thirty-two, seven and one metabolite(s) were found to be associated with IMAT, abdominal adiposity and, SCAT, respectively, including the amino acid glycine, which was positively associated with SCAT and, negatively associated with both IMAT and abdominal adiposity. Glycine and four metabolites found to be significantly associated with regional adiposity were additionally associated with HOMA-IR. Separate stepwise regression models identified glycine as a HOMA-IR associated marker of both IMAT (model R2 = 0.51, p<0.0001) and abdominal adiposity (model R2 = 0.41, p<0.0001). Conclusion Our findings for a positive association between glycine with SCAT but, a negative association between glycine with IMAT and abdominal adiposity supports the hypothesis that SCAT metabolic processes are different from that found in other fat depots. In addition, because of the significant associations found between glycine with HOMA-IR, IMAT, SCAT and abdominal adiposity, our results suggest glycine as a serum biomarker of both insulin sensitivity and regional fat mass in functionally-limited older adults.