Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories—a regimen that increases life span by 30–40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.     

Possible differences in the mechanism of malignant transformation of HaCaT cells by arsenite and its dimethyl metabolites,particularly dimethylthioarsenics

Background: As a confirmed human carcinogen, arsenic can cause skin cancer, lung cancer, etc. However, its carcinogenic mechanism is still unclear. In recent years, the oxidative stress hypothesis has become widely accepted. In mammals it has been found that arsenic can be converted to dimethylarsinous acid (DMA^III) and dimethylmonothioarsinic acid (DMMTA^V) through a series of methylation and redox reactions. DMA^III and DMMTA^V are highly toxic.Methods: Human keratinocytes (HaCaT) were exposed to different concentrations of NaAsO₂ (IAs^III), DMMTA^V and DMA^III for 24 h. Reactive oxygen species (hydrogen peroxide and superoxide), oxidative damage markers (8-hydroxydeoxyguanosine and malondialdehyde), and antioxidant markers (glutathione and superoxide dismutase) were measured. In addition, sulfane sulfurs were measured in HaCaT cells and a cell-free system.Results: In the DMMTA^V and DMA^III treatment groups, the levels of hydrogen peroxide and superoxide in HaCaT cells were higher than in the IAs^III treatment groups at the same dose. Levels of 8−OHdG and MDA in the DMMTA^V and DMA^III treatment groups were also higher than those in the IAs^III treatment groups at the same dose. However, in the DMMTA^V and DMA^III treatment groups, the levels of GSH and SOD activity were lower than that in the IAs^III treatment groups. In DMMTA^V-treated HaCaT cells, sulfane sulfurs were produced. Further, it was found that DMMTA^V could react with DMDTA^V to form persulfide in the cell-free system, which may explain the mechanism of the formation of sulfane sulfurs in DMMTA^V-treated HaCaT cells.Conclusions: DMMTA^V and DMA^III more readily induce reactive oxygen species (ROS) and cause oxidative damage in HaCaT cells than inorganic arsenic. Further, the persulfide formed by the reaction of DMMTA^V and DMDTA^V produced from the metabolism of DMMTA^V may induce a stronger reductive defense mechanism than GSH against the intracellular oxidative stress of DMMTA^V. However, the cells exposed to arsenite are transformed by the continuous nuclear translocation of Nrf2 due to oxidative stress, and the persulfide from dimethylthioarsenics may promote Nrf2 by the combination with thiol groups, especially redox control key protein, Keap1, eventually cause nuclear translocation of sustained Nrf2.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

Effects of statin treatment and withdrawal on angiotensin II-induced phosphorylation of p38 MAPK and ERK1/2 in cultured vascular smooth muscle cells

Abrupt discontinuation of 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors (statins) is associated with increased cardiovascular risk. To investigate the molecular mechanisms determining the increased cardiovascular risk after statin withdrawal, we studied the effects of statin treatment and withdrawal on angiotensin II (AII) actions in rat aortic vascular smooth muscle cells (VSMC) in culture. In VSMC, AII stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and of p38 mitogen-activated protein kinase (p38 MAPK), with an EC50% of 0.86 and 3 nM, respectively. Maximal stimulation was observed after 5-10 min of exposure to AII. Pretreatment with 1-3 microM simvastatin for 24h inhibited AII-mediated stimulation of ERK1/2 and p38 MAPK phosphorylation; without affecting the levels on non-phosphorylated MAPK. Washout of simvastatin produced a rebound increase above control levels of AII-mediated phosphorylation of ERK1/2 and p38 MAPK. As previously reported for other agonists, the rebound increase of AII effects was observed from 1 to 3h after statin withdrawal, and was lost at later times. The basal levels of phosphorylation and the amount of non-phosphorylated kinases were unaffected by statin withdrawal. Similar effects were observed with lovastatin. Our results suggest that statins modulate AII effects in VSMC, and that transient increases in AII effects mediated via the MAPK pathway may play a role in the vascular dysfunction associated with statin withdrawal.     

MicroRNAs in skeletal muscle biology and exercise adaptation

MicroRNAs (miRNAs) have emerged as important players in the regulation of gene expression, being involved in most biological processes examined to date. The proposal that miRNAs are primarily involved in the stress response of the cell makes miRNAs ideally suited to mediate the response of skeletal muscle to changes in contractile activity. Although the field is still in its infancy, the studies presented in this review highlight the promise that miRNAs will have an important role in mediating the response and adaptation of skeletal muscle to various modes of exercise. The roles of miRNAs in satellite cell biology, muscle regeneration, and various myopathies are also discussed.     

Peroxiredoxin 3 has a crucial role in the contractile function of skeletal muscle by regulating mitochondrial homeostasis

Antioxidant systems against reactive oxygen species (ROS) are important factors in regulating homeostasis in various cells, tissues, and organs. Although ROS are known to cause to muscular disorders, the effects of mitochondrial ROS in muscle physiology have not been fully understood. Here, we investigated the effects of ROS on muscle mass and function using mice deficient in peroxiredoxin 3 (Prx3), which is a mitochondrial antioxidant protein. Ablation of Prx3 deregulated the mitochondrial network and membrane potential of myotubes, in which ROS levels were increased. We showed that the DNA content of mitochondria and ATP production were also reduced in Prx3-KO muscle. Of note, the mitofusin 1 and 2 protein levels decreased in Prx3-KO muscle, a biochemical evidence of impaired mitochondrial fusion. Contractile dysfunction was examined by measuring isometric forces of isolated extensor digitorum longus (EDL) and soleus muscles. Maximum absolute forces in both the EDL and the soleus muscles were not significantly affected in Prx3-KO mice. However, fatigue trials revealed that the decrease in relative force was greater and more rapid in soleus from Prx3-KO compared to wild-type mice. Taken together, these results suggest that Prx3 plays a crucial role in mitochondrial homeostasis and thereby controls the contractile functions of skeletal muscle.     

Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats

Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer-induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4-dinitrophenylhydrazine assay), both malondialdehyde- (MDA-) and 2-hydroxy-4-nonenal- (HNE-) protein adducts, Mn-superoxide dismutase (Mn-SOD), catalase, heme oxygenase-1 (HO-1) and 3-nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH-130 hepatoma. In the muscles of the tumour-bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA-protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn-SOD, catalase, and HO-1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer-induced cachexia.     

Omega-3 fatty acids differentially modulate enzymatic anti-oxidant systems in skeletal muscle cells

During physical activity, increased reactive oxygen species production occurs, which can lead to cell damage and in a decline of individual’s performance and health. The use of omega-3 polyunsaturated fatty acids as a supplement to protect the immune system has been increasing; however, their possible benefit to the anti-oxidant system is not well described. Thus, the aim of this study was to evaluate whether the omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) can be beneficial to the anti-oxidant system in cultured skeletal muscle cells. C2C12 myocytes were differentiated and treated with either eicosapentaenoic acid or docosahexaenoic acid for 24 h. Superoxide content was quantified using the dihydroethidine oxidation method and superoxide dismutase, catalase, and glutathione peroxidase activity, and expression was quantified. We observed that the docosahexaenoic fatty acids caused an increase in superoxide production. Eicosapentaenoic acid induced catalase activity, while docosahexaenoic acid suppressed superoxide dismutase activity. In addition, we found an increased protein expression of the total manganese superoxide dismutase and catalase enzymes when cells were treated with eicosapentaenoic acid. Taken together, these data indicate that the use of eicosapentaenoic acid may present both acute and chronic benefits; however, the treatment with DHA may not be beneficial to muscle cells.     

Lysosomal activation in mouse skeletal muscle induced by protamine in vitro

Summary Incubation of mouse skeletal muscle in a physiological Ringer solution containing protamine (60 g/ml) at +37° C for 1 h induced ultrastructural changes including proliferation of tubular profiles and vesicles at the I-band level close to the A-I junction, formation of numerous acid phosphatase positive lysosomes in the longitudinal sarcoplasmic reticulum and autophagic vacuolation starting at the level of the A-I junction.Biochemical determination of acid phosphatase in the incubated muscles showed that protamine caused an increase in acid phosphatase activity of about 25 % compared to enzyme activities obtained from muscles incubated without protamine at +37°C or with protamine at +4°C.The morphological findings suggest that the vesicles arising adjacent to the A-I junction originate from transverse tubules. Such vesicles, designated as endocytic, may acquire acid phosphatase activity in the longitudinal SR and be active in an autophagic process resulting in large vacuoles. A causal relationship between endocytosis and lysosomal activation is suggested.We wish to thank Miss Britt-Marie Svensson, Miss Ann-Christin Nilsson and Mrs. Lena Kvist for their able technical assistance. This work was supported by grants from the Medical Faculty, University of Lund, Sweden, and the Swedish Medical Research Council, Stockholm, Sweden (04P-4289, 14X-4286, 14X-3112) and from Muscular Dystrophy Association of America, Inc.     

Indices of free-radical-mediated damage following maximum voluntary eccentric and concentric muscular work

This study monitored plasma and skeletal muscle markers of free-radical-mediated damage following maximum eccentric and concentric exercise, to examine the potential role of free radicals in exercise-induced muscle damage. Fourteen male volunteers performed either (1) a bout of 70 maximum eccentric and a bout of 70 maximum concentric muscle actions of the forearm flexors (the bouts being separated by 4 weeks; n = 8) or (2) a bout of 80 maximum eccentric and a bout of 80 maximum concentric muscle actions of the knee extensors (the bouts being separated by 1 week; n=6). Plasma markers of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and diene-conjugated compounds (DCC) were monitored in the arm protocol and skeletal muscle markers of oxidative lipid and protein damage, malondialdehyde (MDA) and protein carbonyl derivatives (PCD) respectively, were monitored in the leg protocol. In both protocols, the contralateral limb was used for the second bout and the order of the bouts was randomised between limbs. Repeated measures ANOVA indicated significant changes from baseline following eccentric arm work on the measures of serum creatine kinase activity (P < 0.05), maximum voluntary torque production (P < 0.01) and relaxed arm angle (P < 0.01). Subjective muscle soreness peaked 2 days after eccentric arm work (P < 0.05, Wilcoxon test). However, there were no changes in the plasma levels of TBARS or DCC following the eccentric or concentric arm exercise. Immediately after concentric leg exercise, skeletal muscle PCD concentrations was significantly higher than that observed immediately after eccentric work (P < 0.05). However, no significant difference between the eccentric and concentric knee extensor bouts was observed on the measure of skeletal muscle MDA concentration. The results of this study offer no support for the involvement of oxygen free radicals in exercise-induced muscle damage.     

The antioxidant requirement for plasma membrane repair in skeletal muscle

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of the pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.     

Piceatannol markedly upregulates heme oxygenase-1 expression and alleviates oxidative stress in skeletal muscle cells

Piceatannol (PIC), a phytochemical, is abundant in passion fruit (Passiflora edulis) seeds. In this study, we investigated the effects of PIC on the expression levels of antioxidant enzymes in C2C12 skeletal muscle cells and compared its effects with those of PIC analogues and polyphenols. We also evaluated its effects on hydrogen peroxide–induced accumulation of reactive oxygen species in C2C12 myotubes. Treatment with PIC led to dose-dependent upregulation of heme oxygenase-1 (Ho-1) and superoxide dismutase 1 (Sod1) mRNA expression in C2C12 myotubes. PIC was the most potent inducer of Ho-1 among the PIC analogues and major polyphenols tested. In addition, treatment with PIC suppressed the hydrogen peroxide–induced increase in intracellular reactive oxygen species levels. Our results suggest that PIC protects skeletal muscles from oxidative stress by activating antioxidant enzymes such as HO-1 and SOD1 and can therefore help prevent oxidative stress–induced muscle dysfunction such as muscle fatigue and sarcopenia.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.     

Diazeniumdiolation of protamine sulfate reverses mitogenic effects on smooth muscle cells and fibroblasts

After vascular interventions, endothelial cells are typically injured or lacking, resulting in decreased NO synthesis to maintain vascular health. Moreover, inflammation as a result of the tissue injury and/or the presence of an implanted foreign polymer such as a vascular graft causes excessive generation of reactive oxygen species (ROS) (e.g., superoxide), which can react with NO. The combination of the above creates a general decline in NO bioavailability, as well as oxidative stress due to less available NO to scavenge ROS. Localized NO delivery is an attractive solution to alleviate these issues; however, NO donors typically exhibit unpredictable NO payload release when using nitrosothiols or the risk of nitrosamine formation for synthetic diazeniumdiolates. The objective of this study was therefore to synthesize an NO donor from a biological peptide that could revert to its native form upon NO release. To this effect, protamine sulfate (PS), an FDA-approved peptide with reported vasodilator and anticoagulant properties, was diazeniumdiolated to form PS/NO. PS/NO showed diazeniumdiolate-characteristic UV peaks and NO release in physiological solutions and was capable of scavenging radicals to decrease oxidative stress. Furthermore, PS/NO selectively inhibits the proliferation of smooth muscle cells and adventitial fibroblasts, thereby reversing reported mitogenic properties of PS. Endothelial cell growth, on the other hand, was promoted by PS/NO. Finally, PS retained its anticoagulant properties upon diazeniumdiolation at clinically relevant concentrations. In conclusion, we have synthesized an NO prodrug from a biological peptide, PS/NO, that selectively inhibits proliferation of smooth muscle cells and fibroblasts, retains anticoagulant properties, and reverts back to its native PS form upon NO payload release.     

血管紧张素-(1-7)对大鼠血管平滑肌细胞PKC和ERK1/2的抑制作用

采用Westernblot、氚 胸腺嘧啶 ( 3H TdR)和氚 亮氨酸 ( 3H Leu )掺入等技术和方法 ,用血管紧张素Ⅱ(AngⅡ )和血管紧张素 ( 1 7) [Ang ( 1 7) ]刺激大鼠血管平滑肌细胞 (VSMCs) ,观察和分析Ang ( 1 7)对VSMCs增殖及蛋白激酶C (PKC)和胞外调节蛋白激酶 (ERK)表达的影响。Ang ( 1 7)能明显抑制基础和AngⅡ刺激下的VSMCsPKC ζ和ERK1/ 2蛋白表达 (P <0 0 1或P <0 0 5 ) ,减少3H TdR和3H Leu掺入量 (P <0 0 1或P <0 0 5 )。结果提示 ,Ang ( 1 7)对VSMCs增殖有抑制作用 ,这可能与影响PKC ζ和ERK1/ 2蛋白表达有关。     

Glutaredoxin 2 knockout increases sensitivity to oxidative stress in mouse lens epithelial cells

Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.     

Mitochondrial respiration of complex II is not lower than that of complex I in mouse skeletal muscle

Skeletal muscle (SKM) requires a large amount of energy, which is produced mainly by mitochondria, for their daily functioning. Of the several mitochondrial complexes, it has been reported that the dysfunction of complex II is associated with several diseases, including myopathy. However, the degree to which complex II contributes to ATP production by mitochondria remains unknown. As complex II is not included in supercomplexes, which are formed to produce ATP efficiently, we hypothesized that complex II-linked respiration was lower than that of complex I. In addition, differences in the characteristics of complex I and II activity suggest that different factors might regulate their function. The isolated mitochondria from gastrocnemius muscle was used for mitochondrial respiration measurement and immunoblotting in male C57BL/6J mice. Student paired t-tests were performed to compare means between two groups. A univariate linear regression model was used to determine the correlation between mitochondrial respiration and proteins. Contrary to our hypothesis, complex II-linked respiration was not significantly less than complex I-linked respiration in SKM mitochondria (complex I vs complex II, 3402 vs 2840 pmol/[s × mg]). Complex I-linked respiration correlated with the amount of complex I incorporated in supercomplexes (r = 0.727, p < 0.05), but not with the total amount of complex I subunits. In contrast, complex II-linked respiration correlated with the total amount of complex II (r = 0.883, p < 0.05), but not with the amount of each complex II subunit. We conclude that both complex I and II play important roles in mitochondrial respiration and that the assembly of both supercomplexes and complex II is essential for the normal functioning of complex I and II in mouse SKM mitochondria.