Muscle Protein Oxidation Research Articles

Pathway-oriented action of dietary essential oils to prevent muscle protein oxidation and texture deterioration of farmed rainbow trout

Recently, great attention has been directed towards the use of essential oils from aromatic plants as antimicrobials and antioxidant in food matrix. Fish is well known to be a high perishable food. Indeed, fish muscle is susceptible to suffer protein and lipid oxidation during frozen storage, which can lead to the development of softening and undesirable volatile molecules. However, the possible inclusion of essential oils in fish feed for preserving fish flesh quality during storage is still unclear. For this reason, the potential protective effects of the incorporation of a dietary essential oil constituted by eucalyptol, carvacrol and thymol, to rainbow trout’s (Oncorhynchus mykiss) feed were here investigated. Frozen fish fillets resulting from trout fed the essential oil showed a significant protection of specific muscle proteins against the oxidation produced during frozen storage at –10ºC for 6 months. Essential oil-enriched feed decreased carbonylation of specific myofibrillar (α-actinins-1 and -3, myosin heavy chain, myomesin-1, pyruvate kinase, tropomyosin, troponin-T and actin) and sarcoplasmic proteins (glycogen phosphorylase, creatine kinase, fructose-bisphosphate aldolase A and phosphoglycerate mutase 2). Essential oils also increased actin stability and preserved muscle protein solubility and water holding capacity. In addition, essential oils inhibited the onset of lipid oxidation and rancidity, resulting in frozen fish with superior textural quality and sensory scores. As a final conclusion, the inclusion of essential oils in farmed rainbow trout feed is largely efficient for increasing fish quality and shelf life during frozen storage, mainly through a selective-antioxidant effect on muscle proteins.

Osmolality Selectively Offsets the Impact of Hyperthermia on Mouse Skeletal Muscle in vitro.

Hyperthermia and dehydration can occur during exercise in hot environments. Nevertheless, whether elevations in extracellular osmolality contributes to the increased skeletal muscle tension, sarcolemmal injury, and oxidative stress reported in warm climates remains unknown. We simulated osmotic and heat stress, in vitro, in mouse limb muscles with different fiber compositions. Extensor digitorum longus (EDL) and soleus (SOL) were dissected from 36 male C57BL6J and mounted at optimal length in tissue baths containing oxygenated buffer. Muscles were stimulated with non-fatiguing twitches for 30 min. Four experimental conditions were tested: isotonic-normothermia (285 mOsm•kg-1 and 35°C), hypertonic-normothermia (300 mOsm•kg-1 and 35°C), isotonic-hyperthermia (285 mOsm•kg-1 and 41°C), and hypertonic-hyperthermia (300 mOsm•kg-1 and 41°C). Passive tension was recorded continuously. The integrity of the sarcolemma was determined using a cell-impermeable fluorescent dye and immunoblots were used for detection of protein carbonyls. In EDL muscles, isotonic and hypertonic-hyperthermia increased resting tension (P < 0.001). Whereas isotonic-hyperthermia increased sarcolemmal injury in EDL (P < 0.001), this effect was absent in hypertonic-hyperthermia. Similarly, isotonic-hyperthermia elevated protein carbonyls (P = 0.018), a response not observed with hypertonic-hyperthermia. In SOL muscles, isotonic-hyperthermia also increases resting tension (P < 0.001); however, these effects were eliminated in hypertonic-hyperthermia. Unlike EDL, there were no effects of hyperthermia and/or hyperosmolality on sarcolemmal injury or protein carbonyls. Osmolality selectively modifies skeletal muscle response to hyperthermia in this model. Fast-glycolytic muscle appears particularly vulnerable to isotonic-hyperthermia, resulting in elevated muscle tension, sarcolemmal injury and protein oxidation; whereas slow-oxidative muscle exhibits increased tension but no injury or protein oxidation under the conditions and duration tested.

Effect of oxidized lipids stored under different temperatures on muscle protein oxidation in Sichuan-style sausages during ripening

This study was conceived to research muscle protein oxidation under the influence of four different degrees of oxidized lipids during the ripening of Sichuan-style sausages. Lipids were stored at different temperatures to obtain different oxidation degrees. To elucidate the relationship between lipid oxidation and protein oxidation, the indicators of lipid oxidation, protein oxidation and protein degradation were analysed. During ripening, the carbonyl, SH, SS and free amino acid contents changed significantly. The carbonyl and SS contents increased first in all samples, then decreased, whereas the SH content showed the opposite results. These results showed a positive correlation between protein oxidation and lipid oxidation. Lipids with a higher oxidation degree induced a stronger oxidation reaction to protein. Meanwhile, the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the influence of lipid oxidation on myofibrillar proteins was much more intense than on sarcoplasmic proteins.

Effects of melatonin on low-dose lipopolysaccharide-induced oxidative stress in mouse liver, muscle, and kidney.

Lipopolysaccharide (LPS) administration in an in vivo experimental mice model causes oxidative damage in the liver, muscle, and kidney. We aimed to determine specific mechanisms underlying melatonin's antioxidant protective role. Assays were carried out in quadruplicate in the control, melatonin (10 mg/kg, 10 days), acute LPS administration (once 150 μg), and LPS + melatonin groups. LPS stimulated lipid peroxidation processes (dienes and malondialdehyde) and antioxidant enzyme concentrations (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) were assessed in all investigated tissues. Protein oxidation processes (measured as aldehyde and kenotic carbonyl protein derivatives) were enhanced by LPS in the kidney and liver but not in muscle. Melatonin reversed LPS-induced changes, with the exception of muscle protein oxidation. LPS-induced oxidative stress resulted in augmented early-stage diene conjugated and end-stage malondialdehyde lipid peroxidation processes and affected antioxidant activity in liver, kidney, and muscle tissues. LPS activated protein oxidation processes in the kidney and liver. Melatonin ameliorated oxidative damage in the liver, kidney, and partially in the muscle. Melatonin modulates oxidative stress-induced states. Potential synergism between melatonin and systemic inflammation in terms of oxidative modification of muscle proteins needs to be clarified in further studies.

Tucum-do-cerrado (Bactris setosa Mart.) may enhance hepatic glucose response by suppressing gluconeogenesis and upregulating Slc2a2 via AMPK pathway, even in a moderate iron supplementation condition

Dietary phytochemicals may improve glucose metabolism while iron excess seems to be associated to impaired glucose homeostasis and insulin responses. This study investigated the effect of tucum-do-cerrado (Bactris setosa Mart.) consumption on the carbohydrate metabolism and redox response in rats supplemented or not with dietary iron. Male wistar rats were treated with one of the following diets: CT: control diet (AIN- 93G); +Fe: iron-enriched diet; Tuc: control diet +15% tucum-do-cerrado or Tuc + Fe: iron-enriched diet +15% tucum-do-cerrado. Iron supplementation increased muscle lipid and protein oxidation, hepatic glucokinase (GK) and phosphofrutokinase 1 (PFK1) activities and decreased hepatic glucose-6-phosphatase (G6Pase), intestinal Scl2a2 and muscle Slc2a4 and Prkaa2α mRNA levels compared to CT group. Tucum-do-cerrado consumption (Tuc) increased hepatic Slc2a2, Prkaa1α, Prkaa2α and intestinal Slc5a1 mRNA levels, also decreased hepatic G6Pase activity, muscle Slc2a4 and Prkaa2α in relation to CT group. The association of tucum-do-cerrado with iron-enriched diet increased hepatic Prkaa1 and Pck1 compared to the CT and + Fe groups, intestinal Slc2a2 mRNA levels compared to the +Fe group, while decreased hepatic G6Pase activity in relation to the CT, +Fe and Tuc + Fe groups and muscle Slc2a4 and Prkaa2α compared to CT group. These results suggest that tucum-do-cerrado consumption might induce Prkaa1α and Prkaa2α expression, which may inhibit gluconeogenic rate limiting enzyme, G6Pase, and upregulates GLUT2 hepatic glucose uptake. In addition, moderate iron supplementation improves intracellular hepatic glucose response, stimulating the glycolytic rate limiting enzymes GK and PFK1 while inhibiting gluconeogenic enzyme G6Pase.

Novel Observations of Peroxiredoxin-2 Profile and Protein Oxidation in Skeletal Muscle from Pigs that Differ In Residual Feed Intake and Health Status

ObjectivesEfficiency of pork production is critical as demand for animal protein rises with increases in global populations and income. Feed efficiency and response to disease challenge are affected by stressors that increase reactive oxygen species in muscle. Reactive oxygen species are detrimental to growth, as they require the expenditure of energy for tissue repair that could be used for weight gain. Peroxiredoxin-2 (Prdx-2) is an antioxidant enzyme that converts hydrogen peroxide to water using reactive cysteines, thus mitigating oxidation. Prdx-2 can exist in multiple oxidation states (reduced, oxidized, and hyperoxidized) and quantenary structures (dimer and decamer). Prdx-2 concentration is greater in more color stable muscles and may be an indicator of tenderness. The objective of this study was to determine differences in Prdx-2 profile and protein oxidation between pigs that differed in feed efficiency and health status. Materials and MethodsPigs selected for differing feed efficiency based on residual feed intake (RFI) were used in a 2 × 2 factorial design for this study. At 50 ± 7 kg in body weight, high RFI (less efficient) and low RFI (more efficient) barrows were distributed between 2 rooms in the same building. One room was inoculated with a dual respiratory/enteric bacterial health challenge. At 21 d post-infection (projected peak of illness), pigs were necropsied (total = 24, n = 6 per group) and longissimus muscle samples were collected. Reduced and nonreduced sarcoplasmic protein samples (β-mercaptoethanol) were used for western blot analyses. Reduced samples were used to quantify total Prdx-2, while nonreduced samples were used to evaluate hyperoxidized peroxiredoxin, Prdx-2 decamer, and Prdx-2 profile in nonreducing gels (2 distinct bands analyzed). Carbonyl content and diagonal gel electrophoresis were performed on sarcoplasmic protein samples to determine protein oxidation. Statistical analysis was performed using SAS v. 9.4 (SAS Inst. Inc., Cary, NC) with fixed effects of RFI line, infection status, and RFI line × infection status interaction. Gel repetition and necropsy day were included as random effects. ResultsInfection status was significant for the Prdx-2 band comparison in nonreducing gels (P = 0.014) indicating potential differences in posttranslational modifications. Compared to low RFI pigs, high RFI pigs had greater total Prdx-2 (P = 0.035), greater Prdx-2 decamer (P = 0.0007), greater Prdx-2 in the second band of nonreducing gels (P = 0.0006), and less hyperoxidized peroxiredoxin relative to the total immunoreactive protein (P = 0.028). An interaction existed between RFI line and infection status for the Prdx-2 band comparison (P = 0.02), with high RFI challenged pigs having greater ratios, and high RFI control pigs having lesser ratios relative to low RFI pigs. Visual analysis of diagonal gels showed more proteins with intermolecular disulfide formation in low RFI muscle samples. No significant difference was seen for carbonyl content. ConclusionDifferences exist in Prdx-2 profile based on RFI line and infection status. High and low RFI pigs may respond differently to health challenges at the molecular level based on differences in antioxidant protein response. By defining Prdx-2 in livestock muscle, we may be better able to manage animals to meet demand for meat without increasing inputs.

Long-term PGC1\u03b2 overexpression leads to apoptosis, autophagy and muscle wasting

Skeletal muscle wasting is prevalent in many chronic diseases, necessitating inquiries into molecular regulation of muscle mass. Nuclear receptor co-activator peroxisome proliferator-activated receptor co-activator 1 alpha (PGC1α) and its splice variant PGC1α4 increase skeletal muscle mass. However, the effect of the other PGC1 sub-type, PGC1β, on muscle size is unclear. In transgenic mice selectively over-expressing PGC1β in the skeletal muscle, we have found that PGC1β progressively decreases skeletal muscle mass predominantly associated with loss of type 2b fast-twitch myofibers. Paradoxically, PGC1β represses the ubiquitin-proteolysis degradation pathway genes resulting in ubiquitinated protein accumulation in muscle. However, PGC1β overexpression triggers up-regulation of apoptosis and autophagy genes, resulting in robust activation of these cell degenerative processes, and a concomitant increase in muscle protein oxidation. Concurrently, PGC1β up-regulates apoptosis and/or autophagy transcriptional factors such as E2f1, Atf3, Stat1, and Stat3, which may be facilitating myopathy. Therefore, PGC1β activation negatively affects muscle mass over time, particularly fast-twitch muscles, which should be taken into consideration along with its known aerobic effects in the skeletal muscle.

Hyperthermia and extracellular hyperosmolality affect sarcolemmal damage and protein oxidation in mammalian skeletal muscle in vitro

In the US Armed Services, the incidence of rhabdomyolysis is 5–7 fold higher in peak summer months, suggesting that muscle membrane injury could be amplified by heat or to secondary dehydration. In this study we tested the extent each of these variables influences damage by evaluating sarcolemmal integrity and ROS‐induced protein oxidation. Extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected from 24 adult male C57BL6J mice and placed at optimal length in tissue baths containing 4 ml of Krebs (95%O2/5%CO2). Muscles were intermittently stimulated to perform non‐fatiguing twitches for 30 min. Buffer temperature and osmolality were manipulated to provide four experimental conditions: normothermia/isotonic (35°C and 285 mOsm/kg), normothermia/hypertonic (35°C and 300 mOsm/kg), hyperthermia/isotonic (41°C and 285 mOsm/kg), and hyperthermia/hypertonic (41°C and 300 mOsm/kg). We quantified the integrity of the sarcolemma using both procion orange leakage into the fibers and a fluorometric assay to detect leakage out of double‐stranded cell‐free DNA (cfDNA). To assess protein oxidation, we performed immunoblot assays for protein carbonyl derivatives. For SOL we did not observe a significant effect of hyperthermia and/or hyperosmolality on procion orange leakage, but we observed an increase in cfDNA (p = 0.03) and protein carbonyls with hyperthermia (p = 0.02). Interestingly, these effects were absent when hyperosmolality was superimposed on hyperthermia. For EDL, an increase in procion orange leakage was observed with hyperthermia alone (p = 0.04) and when hyperthermia and hyperosmolality were combined (p = 0.03), but the combination did not elicit changes in cfDNA in the buffer. EDL protein carbonyls were independently augmented with hyperthermia (p = 0.01) and hyperosmolality (p = 0.02), but also did not increase when hyperthermia was combined with hyperosmolality. In conclusion, hyperthermia caused sarcolemma damage in EDL muscle, both alone and when combined with hyperosmolality. SOL was more resistant to hyperthermia‐induced procion orange leakage, despite an increase release of extracellular cfDNA. Furthermore, physiological increases in extracellular osmolality prevented protein oxidation in hyperthermia in both muscle types. These observations suggest that different muscle types may respond differently to hyperthermic and osmotic challenges. The compensatory effect of hyperosmolality on hyperthermia‐induced oxidative stress is consistent with a role for ROS production in compensatory responses to cell swelling in the heat.Support or Funding InformationSupported by the BK and Betty Stevens Endowment and the Brazilian Council for the Development of Science and Technology (CNPq no. 249094/2013‐4).

Fatty acids, lipid and protein oxidation, metmyoglobin reducing activity and sensory attributes of &lt;i&gt;biceps femoris&lt;/i&gt; muscle in goats fed a canola and palm oil blend

The link between the consumption of saturated fatty acids of ruminant meat and the incidence of chronic diseases in humans substantiates efforts to modify its lipid profile. However, altering the lipid profile of ruminant meat could affect its quality attributes and shelf life. This study investigated the effect of a blend of 80% canola oil and 20% palm oil (BCPO) and post mortem ageing on lipid composition, antioxidant status, colour, metmyoglobin reducing activity (MRA) and lipid and protein oxidation in biceps femoris muscle in goats. Twenty-four bucks were randomly assigned to diets containing on a dry matter basis, 0%, 4% or 8% BCPO, fed for 100 d, and slaughtered. The biceps femoris was subjected to 7 d post mortem ageing (4 oC). Neither diet nor post mortem ageing altered catalase, superoxide dismutase and glutathione peroxidase activities or sensory attributes of the biceps femoris . Dietary BCPO improved α- and γ-tocopherol, but did not affect total carotenoids and δ-tocopherol, colour, metmyoglobin content, MRA, TBARS, carbonyl content and free thiol content. Goats fed 4% BCPO had higher cis-9 trans -11 CLA, while those fed 8% BCPO had higher C18:3n-3 and C20:5n-3 compared with other treatments. The 4% and 8% BCPO meat had lower C14:0, but higher C18:1n-9 and C22:5n-3 than the control goats. Post mortem ageing decreased muscle polyunsaturated fatty acids (PUFA), antioxidant compounds, redness, MRA and oxidative stability of lipid and proteins, but increased metmyoglobin content. Dietary BCPO enhanced beneficial muscle lipids without compromising the sensory quality, and lipid and protein stability of the goat meat. Keywords: Antioxidants, carbonyl, fatty acids, metmyoglobin, thiol

Oxidative fuel selection and shivering thermogenesis during a 12- and 24-h cold-survival simulation.

Because the majority of cold exposure studies are constrained to short-term durations of several hours, the long-term metabolic demands of cold exposure, such as during survival situations, remain largely unknown. The present study provides the first estimates of thermogenic rate, oxidative fuel selection, and muscle recruitment during a 24-h cold-survival simulation. Using combined indirect calorimetry and electrophysiological and isotopic methods, changes in muscle glycogen, total carbohydrate, lipid, protein oxidation, muscle recruitment, and whole body thermogenic rate were determined in underfed and noncold-acclimatized men during a simulated accidental exposure to 7.5 °C for 12 to 24 h. In noncold-acclimatized healthy men, cold exposure induced a decrease of ∼0.8 °C in core temperature and a decrease of ∼6.1 °C in mean skin temperature (range, 5.4-6.9 °C). Results showed that total heat production increased by approximately 1.3- to 1.5-fold in the cold and remained constant throughout cold exposure. Interestingly, this constant rise in Ḣprod and shivering intensity was accompanied by a large modification in fuel selection that occurred between 6 and 12 h; total carbohydrate oxidation decreased by 2.4-fold, and lipid oxidation doubled progressively from baseline to 24 h. Clearly, such changes in fuel selection dramatically reduces the utilization of limited muscle glycogen reserves, thus extending the predicted time to muscle glycogen depletion to as much as 15 days rather than the previous estimates of approximately 30-40 h. Further research is needed to determine whether this would also be the case under different nutritional and/or colder conditions.

Iron Deprivation May Enhance Insulin Receptor and Glut4 Transcription in Skeletal Muscle of Adult Rats.

Considering that phenotype related to iron overload associated with pathological conditions differs from that caused by dietary iron excess, our study set out to evaluate the impact of dietary iron restriction and dietary iron supplementation on oxidative stress and functional outcome in adult, healthy rats. adult rats were divided into the three groups and fed diets containing 10, 35 or 350 mg/kg iron (restricted-diet, control-diet and supplemented- diet groups, respectively) for 78 days. Hematological variables, fasting blood glucose, hepatic enzyme activity and C-reactive protein levels were analyzed. Iron and glycogen concentrations in liver and skeletal muscle were determined. The extent of tissue damage caused by either dietary iron restriction or iron supplementation was accessed by measuring malondialdehyde, carbonyl, NADPH oxidase, glutathione peroxidase, glutathione reductase and glutathione-s-transferase in various tissues. The mRNA expression levels of insulin receptor, glucose transporter 4 and p53 were also determined. Fasting blood glucose values trended toward a decrease by dietary iron restriction, moreover, hepatic glycogen content decreased with concomitant increases in skeletal muscle. In addition, dietary iron restriction resulted in a twofold increase in mRNA expression of Insr and fourfold increase in Glut4 expression in skeletal muscle. Although the dietary iron restriction did not affect body iron status, it caused hepatic low oxidative damages. However, high liver NADPH oxidase activity and increased levels of protein oxidation in muscle were observed. Chronic feeding of high iron diet induces iron overload and resulted in elevated levels of stress markers in tissues. Dietary iron deprivation may improve insulin receptor and glucose transporter transcription in muscle; however, our results show that dietary iron restriction can prevent and/or promote oxidative damage in a tissue-specific manner, emphasizing the importance of maintaining optimal iron intake.

Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress.

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male patients with advanced COPD and lung cancer

Muscle dysfunction and wasting are predictors of mortality in advanced COPD and malignancies. Redox imbalance and enhanced protein catabolism are underlying mechanisms in COPD. We hypothesized that the expression profile of several biological markers share similarities in patients with cachexia associated with either COPD or lung cancer (LC). In vastus lateralis of cachectic patients with either LC (n=10) or advanced COPD (n=16) and healthy controls (n=10), markers of redox balance, inflammation, proteolysis, autophagy, signaling pathways, mitochondrial function, muscle structure, and sarcomere damage were measured using laboratory and light and electron microscopy techniques. Systemic redox balance and inflammation were also determined. All subjects were clinically evaluated. Compared to controls, in both cachectic groups of patients, a similar expression profile of different biological markers was observed in their muscles: increased levels of muscle protein oxidation and ubiquitination (p<0.05, both), which positively correlated (r=0.888), redox-sensitive signaling pathways (NF-κB and FoxO) were activated (p<0.05, all), fast-twitch fiber sizes were atrophied, muscle structural abnormalities and sarcomere disruptions were significantly greater (p<0.05, both). Structural and functional protein levels were lower in muscles of both cachectic patient groups than in controls (p<0.05, all). However, levels of autophagy markers including ultrastructural autophagosome counts were increased only in muscles of cachectic COPD patients (p<0.05). Systemic oxidative stress and inflammation levels were also increased in both patient groups compared to controls (p<0.005, both). Oxidative stress and redox-sensitive signaling pathways are likely to contribute to the etiology of muscle wasting and sarcomere disruption in patients with respiratory cachexia: LC and COPD.

Pharmacological Strategies in Lung Cancer-Induced Cachexia: Effects on Muscle Proteolysis, Autophagy, Structure, and Weakness

Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome system, nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-κB, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF-κB (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO-1, NF-κB and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-κB and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF-κB signaling pathway effects on muscles is beneficial in cancer-induced cachexia.

Reactive oxygen species play a role in muscle wasting during thyrotoxicosis.

The role of reactive oxygen species (ROS) in muscle protein hydrolysis and protein oxidation in thyrotoxicosis has not been explored. This study indicates that ROS play a role in skeletal muscle wasting pathways in thyrotoxicosis. Two experimental groups (rats) were treated for 5days with either 3,3',5-triiodothyronine (HT) or HT with α-tocopherol (HT + αT). Two controls were used, vehicle (Control) and control treated with αT (Control + αT). Serum T3, peritoneal fat, serum glycerol, muscle and body weight, temperature, mitochondrial metabolism (cytochrome c oxidase activity), oxidative stress parameters and proteolytic activities were examined. High body temperature induced by HT returned to normal when animals were treated with αT, although total body and muscle weight did not. An increase in lipolysis was observed in the HT + αT group, as peritoneal fat decreased significantly together with an increase in serum glycerol. GSH, GSSG and total radical-trapping antioxidant parameter (TRAP) decreased and catalase activity increased in the HT group. The glutathione redox ratio was higher in HT + αT than in both HT and Control + αT groups. Carbonyl proteins, AOPP, mitochondrial and chymotrypsin-like proteolytic activities were higher in the HT group than in the Control. HT treatment with αT restored mitochondrial metabolism, TRAP, carbonyl protein, chymotrypsin-like activity and AOPP to the level as that of the Control + αT. Calpain activity was lower in the HT + αT group than in HT and Control + αT and superoxide dismutase (SOD) activity was higher in the HT + αT group than in the Control + αT. Although αT did not reverse muscle loss, ROS was involved in proteolysis to some degree.

Curcumin counteracts loss of force and atrophy of hindlimb unloaded rat soleus by hampering neuronal nitric oxide synthase untethering from sarcolemma

Antioxidant administration aimed to antagonize the development and progression of disuse muscle atrophy provided controversial results. Here we investigated the effects of curcumin, a vegetal polyphenol with pleiotropic biological activity, because of its ability to upregulate glucose-regulated protein 94 kDa (Grp94) expression in myogenic cells. Grp94 is a sarco-endoplasmic reticulum chaperone, the levels of which decrease significantly in unloaded muscle. Rats were injected intraperitoneally with curcumin and soleus muscle was analysed after 7 days of hindlimb unloading or standard caging. Curcumin administration increased Grp94 protein levels about twofold in muscles of ambulatory rats (P < 0.05) and antagonized its decrease in unloaded ones. Treatment countered loss of soleus mass and myofibre cross-sectional area by approximately 30% (P ≤ 0.02) and maintained a force-frequency relationship closer to ambulatory levels. Indexes of muscle protein and lipid oxidation, such as protein carbonylation, revealed by Oxyblot, and malondialdehyde, measured with HPLC, were significantly blunted in unloaded treated rats compared to untreated ones (P = 0.01). Mechanistic involvement of Grp94 was suggested by the disruption of curcumin-induced attenuation of myofibre atrophy after transfection with antisense grp94 cDNA and by the drug-positive effect on the maintenance of the subsarcolemmal localization of active neuronal nitric oxide synthase molecules, which were displaced to the sarcoplasm by unloading. The absence of additive effects after combined administration of a neuronal nitric oxide synthase inhibitor further supported curcumin interference with this pro-atrophic pathway. In conclusion, curcumin represents an effective and safe tool to upregulate Grp94 muscle levels and to maintain muscle function during unweighting.

Computational Simulations Reveal How Calmodulin Methionine Oxidation Triggers Large-Scale Changes in Structural Dynamics

We have examined the structural consequences of methionine (Met) oxidation in the calcium-sensing muscle regulatory protein calmodulin (CaM) using molecular dynamics simulations. Protein oxidation by reactive oxygen species (ROS), and subsequent reduction by the antioxidant enzyme methionine sulfoxide reductase, has emerged as a crucial cell regulatory mechanism. In the context of oxidative stress, protein oxidation is implicated in disease progression and biological aging. Our goal is to bridge our understanding of muscle dysfunction and protein oxidation with atomic-level insights into site-specific methionine oxidation and calmodulin structural dynamics.We have carried out multiple 50 ns molecular dynamics simulations of explicitly solvated calmodulin, starting from the calcium-bound (1cll) and apo (1cfd) structures. Simulations were performed using NAMD (University of Illinois) and the CHARMM27 force-field. Simulations suggest that Met oxidation alters the flexibility of calcium binding sites, the conformation of the linker helix connecting the lobes, and the relative orientation of the lobes. This work is a component of a larger study in which spectroscopic distance measurements and NMR experiments have been used to resolve the structural impact of site-specific CaM Met oxidation. We expect that our in silico results will bring atomic-level insight to spectroscopic measurements, and will be integral to creating a more complete model for oxidation-induced changes in CaM structural dynamics.This work is supported by a University of Wisconsin-La Crosse Faculty Research Grant to JC Klein, NIH grants to DD Thomas (R37AG026160), and the Minnesota Supercomputing Institute.

Time‐dependent muscle‐specific protein oxidation in a mouse model of chronic hypoxia

Respiratory muscle redox homeostasis is altered by chronic hypoxia (CH). Protein oxidation potentially contributes to respiratory muscle dysfunction in respiratory‐related diseases where hypoxia features. The objective of this study was to determine the type and extent of respiratory and limb muscle protein oxidation temporally in a mouse model of CH.C57/Bl6J mice were exposed to hypoxia (1, 3 and 6 weeks; 10% FiO2) or normoxia. Following treatment, excised respiratory and limb muscles were homogenised and incubated with carbonyl‐ or thiol‐reactive fluorophores before gel electrophoresis and fluorescence scanning. Optical density (OD) of fluorescence was normalised to total protein, determined by colloidal coomassie staining.Diaphragm free‐thiol content increased ~9‐fold after 1 week of CH. By 6 weeks, both diaphragm and sternohyoid free‐thiol content were significantly decreased (40% and 80% decrease from control respectively; n=6–8 per group; P=0.0046 and P&lt;0.0001, Student's unpaired t‐test) whereas there was a significant increase in the extensor digitorum longus (232%; P=0.0007) and in soleus (143%; P&lt;0.0001). Diaphragm carbonyl content was unchanged after 1 week of CH, but was increased significantly by 3 weeks and was increased further again after 6 weeks. The extent of carbonylation was different between muscle groups.We conclude that CH exposure causes differential time‐dependent protein oxidation in respiratory and limb muscles.

Structural Impact of Site-Specific Calmodulin Methionine Oxidation

We have examined the structural consequences of methionine (Met) oxidation in the calcium-sensing muscle regulatory protein calmodulin (CaM) using molecular dynamics simulations. Protein oxidation by reactive oxygen species (ROS), and subsequent reduction by the antioxidant enzyme methionine sulfoxide reductase, has emerged as a crucial cell regulatory mechanism. In the context of oxidative stress, protein oxidation is implicated in disease progression and biological aging. Our goal is to bridge our understanding of muscle dysfunction and protein oxidation with atomic-level insights into site-specific methionine oxidation and calmodulin structural dynamics. We have carried out multiple 500 ns molecular dynamics simulations of explicitly solvated calmodulin, both the calcium-bound (1cll) and apo (1cfc) crystal structures. Results from preliminary simulations suggest that the structure of calcium bound CaM is structurally insensitive to methionine oxidation, while methionine oxidation in apo CaM causes considerable changes the relative orientation of the N-ter and C-ter lobes. Our work is a component of a larger study in which spectroscopic distance measurements and nuclear magnetic resonance experiments are being carried out for site-specifically oxidized CaM in both the calcium bound and apo biochemical states. We expect that our in silico results will bring atomic-level insight to spectroscopic measurements, and will be integral to creating a more complete model for oxidation-induced changes in calmodulin structural dynamics. Further, we anticipate that our results will be applicable to the many biological and pharmaceutical contexts in which a detailed understanding of protein oxidation, function and structure relationships is sought. This work is supported by an NIH grant to Dave Thomas (2R37AG026160-06) and the Minnesota Supercomputing Institute.

Hemodynamic and oxidative mechanisms of tourniquet-induced muscle injury: near-infrared spectroscopy for the orthopedics setting

During orthopedic procedures, the tourniquets used to maintain bloodless surgical fields cause ischemia and then reperfusion (I/R), leading to oxidative muscle injury. Established methods exist neither for monitoring orthopedic I/R nor for predicting the extent of tourniquet-associated oxidative injury. To develop a predictive model for tourniquet-associated oxidative muscle injury, this study combined real-time near-infrared spectroscopy (NIRS) monitoring of I/R with Western blotting (WB) for oxidized proteins. We hypothesized strong correlations between NIRS-derived I/R indices and muscle protein oxidation. In 17 patients undergoing ankle fracture repair, a thigh tourniquet was inflated on the injured limb (300 mmHg). Using a continuous-wave (CW) NIRS setup, oxygenated (O2Hb), deoxygenated (HHb), and total (tHb) hemoglobin were monitored bilaterally (tourniquet versus control) in leg muscles. Leg muscle biopsies were collected unilaterally (tourniquet side) immediately after tourniquet inflation (pre) and before deflation (post). Average ischemia duration was 43.2 ± 14.6 min. In post-compared to pre-biopsies, muscle protein oxidation (quantified using WB) increased 172.3%± 145.7% (P<0.0005). Changes in O2Hb and tHb were negatively correlated with protein oxidation (respectively: P=0.040, R2=0.25 and P=0.003, R2=0.58). Reoxygenation rate was positively correlated with protein oxidation (P=0.041, R2=0.25). These data indicate that using CW NIRS, it is possible to predict orthopedic tourniquet-associated muscle oxidative injury noninvasively.