Expression Of Atrogin-1 Research Articles

Ellagic acid attenuates muscle atrophy in STZ-induced diabetic mice.

Diabetes is closely connected with skeletal muscle dysfunction. Ellagic acid (EA) possesses a variety of bio-effects and is applied to the improvement of diabetes. The purpose of this study was to explore the potential improvement effect and mechanisms of EA in streptozotocin (STZ)-induced diabetic muscle atrophy. The model of diabetic mice was established by intra-peritoneal STZ to evaluate treatment effect of EA (100 mg/kg/d for 8 weeks) on muscle atrophy. Our data exhibited that EA enhanced fiber size and weight of gastrocnemius, and promoted grip strength to relieve STZ-induced muscle lesions. In serum, the levels of Creatine kinase (CK), lactate dehydrogenase (LDH), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL) were inhibited, while high-density lipoprotein cholesterol (HDL) level was enhanced by EA treatment in diabetic mice. In gastrocnemius, EA decreased Atrogin-1 and MuRF-1 expressions to relieve STZ-induced muscle atrophy. Moreover, EA increased NRF-1 and PGC-1alpha expressions to alleviate mitochondrial disorder. Meanwhile, EA suppressed CHOP and GRP-87 levels to relieve ER stress. Lastly, EA inhibited BAX expressions and enhanced Bcl-2 expressions to mitigate apoptosis. In conclusion, EA is preventing the event of STZ-induced gastrocnemia by amelioration of mitochondrial dysfunction, ER stress and apoptosis, and could be used in the protection and therapeutic of muscle atrophy in diabetes.

Muscle contractile exercise through a belt electrode device prevents myofiber atrophy, muscle contracture, and muscular pain in immobilized rat gastrocnemius muscle.

Immobilization of skeletal muscles causes muscle atrophy, muscle contracture, and muscle pain, the mechanisms of which are related to macrophage accumulation. However, muscle contractile exercise through a belt electrode device may mitigate macrophage accumulation. We hypothesized that such exercise would be effective in preventing myofiber atrophy, muscle contracture, and muscular pain. This study tested this hypothesis in immobilized rat gastrocnemius muscle. A total of 32 rats were divided into the following control and experimental groups: immobilization (immobilized treatment only), low-frequency (LF; immobilized treatment and muscle contractile exercise with a 2 s (do) /6 s (rest) duty cycle), and high-frequency (HF; immobilized treatment and muscle contractile exercise with a 2 s (do)/2 s (rest) duty cycle). Electrical stimulation was performed at 50 Hz and 4.7 mA, and muscle contractile exercise was applied to the lower limb muscles for 15 or 20 min/session (once daily) for 2 weeks (6 times/week). After the behavioral tests, the bilateral gastrocnemius muscles were collected for analysis. The number of macrophages, the Atrogin-1 and MuRF-1 mRNA expression, and the hydroxyproline content in the HF group were lower than those in the immobilization and LF groups. The cross-sectional area (CSA) of type IIb myofibers in the superficial region, the PGC-1α mRNA expression, and the range of motion of dorsiflexion in the HF group were significantly higher than those in the immobilization and LF groups. The pressure pain thresholds in the LF and HF groups were significantly higher than that in the immobilization group, and the nerve growth factor (NGF) content in the LF and HF groups was significantly lower than that in the immobilization group. Muscle contractile exercise through the belt electrode device may be effective in preventing immobilization-induced myofiber atrophy, muscle contracture, and muscular pain in the immobilized rat gastrocnemius muscle.

Research Note: Creatine monohydrate alleviates protein breakdown induced by corticosterone via inhibiting ubiquitin proteasome pathway in chicken myotubes

Stress is a common problem diminishing the muscle development of broilers. Creatine (Cr), an energy buffer in skeletal muscle, plays a fundamental role in muscle physiology. This study aimed to evaluate the effect of Cr monohydrate (CMH) on protein breakdown in chicken myotubes challenged by corticosterone (CORT) in vitro. The morphology of myotube was measured and the activation of ubiquitin proteasome (UP) pathway was determined. The result showed that CORT treatment decreased myotube diameter (P < 0.05), increased 3-methyl-histidine (3M-His) content in medium, enhanced the mRNA expression levels of muscle ring finger1(MuRF1) and Atrogin1 (P < 0.001), and Atrogin1 protein level (P < 0.05) compared with control. By contrast, CMH increased myotube diameter (P < 0.05) and myosin heavy chain (MHC) expression (P < 0.001), whereas decreased 3M-His and the mRNA and protein levels of Atrogin1 (P < 0.05), compared to control. In the present of CMH, the decreased myotube diameter and increased 3M-His, mRNA levels of MuRF1 and Atrogin1, and Atrogin1 protein level by CORT were partially relieved (P < 0.05). Hence, the result suggests that CMH alleviates CORT-induced protein breakdown by suppressing Atrogin1 expression in chicken myotubes. The result highlights the potential application of CMH in regulating muscle protein catabolism in chickens under stress.

Pathophysiological levels of GDF11 activate Smad2/Smad3 signaling and induce muscle atrophy in human iPSC-derived myocytes.

Skeletal muscle mass is negatively regulated by several TGF-β superfamily members. Myostatin (MSTN) is the most prominent negative regulator of muscle mass. Recent studies show that in addition to MSTN, GDF11, which shares a high sequence identity with MSTN, induces muscle atrophy in vitro and in vivo at supraphysiological levels, whereas controversy regarding its roles exists. Furthermore, higher circulating GDF11 levels associate with frailty in humans. On the other hand, little is known about the effect of pathophysiological levels of GDF11 on muscle atrophy. Here we seek to determine whether pathophysiological levels of GDF11 are sufficient to activate Smad2/Smad3 signaling and induce muscle atrophy using human iPSC-derived myocytes (hiPSC myocytes). We first show that incubating hiPSC myocytes with pathophysiological concentrations of GDF11 significantly reduces myocyte diameters. We next demonstrate that pathophysiological levels of GDF11 are sufficient to activate Smad2/3 signaling. Finally, we show that pathophysiological levels of GDF11 are capable of inducing the expression of Atrogin-1, an atrophy-promoting E3 ubiquitin ligase and that FOXO1 blockage reverses the GDF11-induced Atrogin-1 expression and atrophic phenotype. Collectively, our results suggest that GDF11 induces skeletal muscle atrophy at the pathophysiological levels through the GDF11-FOXO1 axis.

Ubiquitin E3 ligase Atrogin-1 protein is regulated via the rapamycin-sensitive mTOR-S6K1 signaling pathway in C2C12 muscle cells

Atrogin-1 and Muscle-specific RING finger protein 1 (MuRF1) are highly expressed in multiple conditions of skeletal muscle atrophy. The phosphoinositide 3-kinase (PI3K)/Akt/forkhead box (FoxO) signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mechanistic target of rapamycin complex 1 (mTORC1), which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 with a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 μM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased by PF-4708671 treatment for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by the rapamycin-sensitive mTOR-S6K1-dependent signaling pathway.

Protein Hydrolysate from Spirulina platensis Prevents Dexamethasone-Induced Muscle Atrophy via Akt/Foxo3 Signaling in C2C12 Myotubes.

Loss of muscle mass is the primary symptom of sarcopenia. Protein intake is recommended to prevent muscle mass loss, and Spirulina platensis, a microalga with high protein content, is a potential protein supplement. Here, we evaluated the differentiation ability of C2C12 cells and the inhibitory effect of Spirulina hydrolysates (SPH) prepared by Collupulin on dexamethasone (DEX)-treated C2C12 cells. SPH contained 578.27 mg/g protein and 92.30 mg/g branched-chain amino acids. SPH increased C2C12 myotube length and diameter, likely owing to increased MyoD1 and Myf5 expression. Inhibition of increased Atrogin-1, MuRF-1, and FoxO3 expression by SPH in DEX-treated C2C12 cells suppressed DEX-induced muscle atrophy. Moreover, SPH inhibited the DEX-induced increase in cytosolic p-Akt protein expression and suppressed the increase in nuclear FoxO3a protein expression, thereby suppressing the increase in the protein expression of the ubiquitin-proteasome-related factors Atrogin-1 and MuRF-1, which are involved in muscle atrophy. SPH suppressed DEX-induced muscle atrophy by activating the Akt/FoxO3a pathway. SPH promoted C2C12 myoblast differentiation into myotubes and inhibited DEX-induced myotube atrophy by suppressing Atrogin-1 and MuRF-1 expression and regulating the FoxO3a transcription factor. Collectively, SPH can be used as a functional food to inhibit muscle atrophy and promote muscle regeneration.

AB0064 PHARMACOLOGICAL INHIBITION OF IL-6/JAK/STAT AXIS INCREASES MUSCLE MASS IN AN EXPERIMENTAL MODEL OF SARCOPENIA ASSOCIATED TO RHEUMATOID ARTHRITIS

BackgroundSarcopenia is a frequent comorbidity of rheumatoid arthritis (RA) due to an imbalance in muscle remodelling with dominance of catabolism over anabolism. IL-6/JAK/STAT axis is of particular importance in this regulation because of its dual activity. According to local and acute factors, it promotes muscle healing and hypertrophy, whereas in chronic inflammation, this signalling substantially increases muscle catabolism activating pro-atrophic pathways involving atrogene expression and ubiquitin proteasome system (UPS). JAK/STAT blockade suppresses muscle wasting induced by IL-6, through STAT3 activation. Interestingly, different clinical trials have shown that JAK inhibitors produce an asymptomatic increase in serum creatine kinase (CK) and creatinine levels in RA patients, suggesting an impact on muscle.ObjectivesTo evaluate the effect of JAK inhibition and its effect in muscle remodelling in an experimental model that accurately mimics human rheumatoid sarcopenia.MethodsAn experimental model of antigen-induced arthritis (AIA) was carried out in 14 rabbits by immunization against ovalbumin followed by 4 intra-articular (i.a) injections of this protein. One week after the first i.a injection, 7 of these rabbits received tofacitinib (TOFA, orally 10mg/kg/day) for 2 wk. Animals were euthanized one day after the last i.a injection, when tibialis anterior (TA), extensor digitorum longus (EDL) and gastrocnemius (GN) were isolated from both hind limbs. Histological changes and ATPase staining were analysed in TA, while the number of myonuclei was evaluated in EDL fibres by immunofluorescence studies. C-reactive protein (CRP) and myostatin (MSTN) serum concentration were determined by ELISA. The gene expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α, MCP-1), atrogenes (Atrogin-1, MuRF-1) and MSTN was measured by quantitative PCR, while proliferative muscle marker (PAX-7), differentiation muscle markers (MyoD, Myogenin), pSTAT3, pSTAT1, MSTN and CK protein expression was analysed by western blot in GN. Creatine presence in GN was analysed by a colorimetric assay.ResultsA significant increase in body weight was observed in the AIA+TOFA group vs. AIA at the end of the study. A significant increase in TA cross-sectional area and diameter in the AIA+TOFA, in comparison to AIA, was found, while the decrease in the area of type II fibres observed in the AIA animals was not noticed in AIA+TOFA. Moreover, the number of myonuclei in the EDL that was increased in the AIA group when compared to healthy animals, was significantly decreased in AIA+TOFA. However, systemic inflammation measured by CRP was not modified by TOFA treatment at the end of the study, in comparison to AIA, and so did serum MSTN concentration. TOFA evoked a significant reduction in the gene expression of IL-6, MCP-1, atrogin-1 and MuRF-1 in GN in comparison to untreated AIA rabbits. Notably, AIA+TOFA showed higher protein levels of CK and lower creatine compared to AIA in muscle. Simultaneously, no differences in the proliferative marker PAX-7 were found between groups, while AIA rabbits showed an increase in the differentiation markers MyoD and Myogenin in the muscle that was prevented in AIA+TOFA.ConclusionThese data provide novel insights into the effects of JAK inhibitors in muscle remodelling during rheumatoid cachexia. Despite an elevated systemic inflammatory status, JAK inhibition was able to rapidly increase muscle mass through attenuating IL-6/JAK/STAT activation, decreasing atrogene expression, and restoring to baseline the muscle cell differentiation markers in the tissue. The increase in muscle mass was accompanied by an increase in CK presence, supporting the role of CK as a valuable marker of muscle gain following treatment with JAK inhibitors. However, the low levels of MCP-1 and the disorganization of muscle fibre myonuclei raise the question of whether these treatments could trigger a complete regeneration of muscle fibre cell or just hypertrophy of cells already present.Disclosure of InterestsIsmael Bermejo: None declared, Sandra Pérez-Baos: None declared, Juan Pablo Medina: None declared, Paula Gratal: None declared, Ivan Prieto-Potín: None declared, Raquel Largo: None declared, Gabriel Herrero-Beaumont Grant/research support from: The Bone and Joint Research Unit has received funding from several pharmaceutical laboratories (Pfizer, LILLY...).

A High-Fat Diet Induces Muscle Mitochondrial Dysfunction and Impairs Swimming Capacity in Zebrafish: A New Model of Sarcopenic Obesity.

Obesity is a highly prevalent disease that can induce metabolic syndrome and is associated with a greater risk of muscular atrophy. Mitochondria play central roles in regulating the physiological metabolism of skeletal muscle; however, whether a decreased mitochondrial function is associated with impaired muscle function is unclear. In this study, we evaluated the effects of a high-fat diet on muscle mitochondrial function in a zebrafish model of sarcopenic obesity (SOB). In SOB zebrafish, a significant decrease in exercise capacity and skeletal muscle fiber cross-sectional area was detected, accompanied by high expression of the atrophy-related markers Atrogin-1 and muscle RING-finger protein-1. Zebrafish with SOB exhibited inhibition of mitochondrial biogenesis and fatty acid oxidation as well as disruption of mitochondrial fusion and fission in atrophic muscle. Thus, our findings showed that muscle atrophy was associated with SOB-induced mitochondrial dysfunction. Overall, these results showed that the SOB zebrafish model established in this study may provide new insights into the development of therapeutic strategies to manage mitochondria-related muscular atrophy.

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Linoleic Acid Attenuates Denervation-Induced Skeletal Muscle Atrophy in Mice through Regulation of Reactive Oxygen Species-Dependent Signaling.

Muscle atrophy is a major muscle disease, the symptoms of which include decreased muscle volume leading to insufficient muscular support during exercise. One cause of muscle atrophy is the induction of oxidative stress by reactive oxygen species (ROS). This study aimed to identify the antioxidant mechanism of linoleic acid (LA) in muscle atrophy caused by oxidative stress. H2O2 has been used to induce oxidative stress in myoblasts in vitro. C2C12 myoblasts treated with H2O2 exhibited decreased viability and increased ROS synthesis. However, with LA treatment, the cells tended to recover from oxidative effects similar to those of the control groups. At the molecular level, the expression of superoxide dismutase 1 (SOD1), Bax, heat shock protein 70 (HSP70), and phosphorylated forkhead box protein O1 was increased by oxidative stress, causing apoptosis. LA treatment suppressed these changes. In addition, the expression of MuRF1 and Atrogin-1/MAFbx mRNA increased under oxidative stress but not in the LA-treated group. Sciatic denervation of C57BL/6 mice manifested as atrophy of the skeletal muscle in micro-computed tomography (micro-CT). The protein expression levels of SOD1, HSP70, and MuRF1 did not differ between the atrophied muscle tissues and C2C12 myoblasts under oxidative stress. With LA treatment, muscle atrophy recovered and protein expression was restored to levels similar to those in the control. Therefore, this study suggests that LA may be a candidate substance for preventing muscle atrophy.

N-Chlorotaurine Reduces the Lung and Systemic Inflammation in LPS-Induced Pneumonia in High Fat Diet-Induced Obese Mice.

Lung infection can evoke pulmonary and systemic inflammation, which is associated with systemic severe symptoms, such as skeletal muscle wasting. While N-chlorotaurine (also known as taurine chloramine; TauCl) has anti-inflammatory effects in cells, its effects against pulmonary and systemic inflammation after lung infection has not been elucidated. In the present study, we evaluated the anti-inflammatory effect of the taurine derivative, TauCl against Escherichia coli-derived lipopolysaccharide (LPS)-induced pneumonia in obese mice maintained on a high fat diet. In this study, TauCl was injected intraperitoneally 1 h before intratracheal LPS administration. While body weight was decreased by 7.5% after LPS administration, TauCl treatment suppressed body weight loss. TauCl also attenuated the increase in lung weight due to lung edema. While LPS-induced acute pneumonia caused an increase in cytokine/chemokine mRNA expression, including that of IL-1β, -6, TNF-α, MCP-1, TauCl treatment attenuated IL-6, and TNF-alpha expression, but not IL-1β and MCP-1. TauCl treatment partly attenuated the elevation of the serum cytokines. Furthermore, TauCl treatment alleviated skeletal muscle wasting. Importantly, LPS-induced expression of Atrogin-1, MuRF1 and IκB, direct or indirect targets for NFκB, were suppressed by TauCl treatment. These findings suggest that intraperitoneal TauCl treatment attenuates acute pneumonia-related pulmonary and systemic inflammation, including muscle wasting, in vivo.

The regulating pathway of creatine on muscular protein metabolism depends on the energy state.

Creatine (Cr) is beneficial for increasing muscle mass and preventing muscle atrophy via involving in energy metabolism through the Cr and phosphocreatine (PCr) system. This study aimed to evaluate the supplemental effect of Cr on protein metabolism under normal and starvation conditions. The primary myoblasts were obtained from the breast muscle of chicks. The mammalian target of rapamycin (mTOR)/P70S6 kinase (P70S6K), ubiquitin-proteasome (UP) pathways, and mitochondrial function of myotubes were evaluated at normal or starvation state and with or without glucose supplementation. Under normal condition, Cr supplementation enhanced protein synthesis rate as well as upregulated the total and phosphorylated P70S6K expressions. Cr had little influence on protein catabolism and mitochondrial function. In a starvation state, however, Cr alleviated myotube atrophy and enhanced protein accretion by inhibiting Atrogin1 and myostatin (MSTN) expression. Furthermore, Cr treatment upregulated the transcriptional coactivators peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression and decreased reactive oxygen species (ROS) accumulation under starvation condition. In the presence of glucose, however, the favorable effect of Cr on protein content and myotube diameter did not occur under starvation condition. The present result indicates that at a normal state, Cr stimulated protein synthesis via the mTOR/P70S6K pathway. In a starvation state, Cr mainly takes a favorable effect on protein accumulation via suppression of the UP pathway and mediated mitochondrial function mainly by serving as an energy supplier. The result highlights the potential clinical application for the modulation of muscle mass under different nutritional conditions.

Assessment of the Preventive Effect of L-carnitine on Post-statin Muscle Damage in a Zebrafish Model

Statins, such as lovastatin, are lipid-lowering drugs (LLDs) that have been used to treat hypercholesterolaemia, defined as abnormally elevated cholesterol levels in the patient’s blood. Although statins are considered relatively safe and well tolerated, recipients may suffer from adverse effects, including post-statin myopathies. Many studies have shown that supplementation with various compounds may be beneficial for the prevention or treatment of side effects in patients undergoing statin therapy. In our study, we investigated whether L-carnitine administered to zebrafish larvae treated with lovastatin alleviates post-statin muscle damage. We found that exposure of zebrafish larvae to lovastatin caused skeletal muscle disruption observed as a reduction of birefringence, changes in muscle ultrastructure, and an increase in atrogin-1. Lovastatin also affected heart performance and swimming behaviour of larvae. Our data indicated that the muscle-protective effect of L-carnitine is partial. Some observed myotoxic effects, such as disruption of skeletal muscle and increase in atrogin-1 expression, heart contraction could be rescued by the addition of L-carnitine. Others, such as slowed heart rate and reduced locomotion, could not be mitigated by L-carnitine supplementation.

The Mitochondria-Targeting Agent MitoQ Improves Muscle Atrophy, Weakness and Oxidative Metabolism in C26 Tumor-Bearing Mice.

Cancer cachexia is a debilitating syndrome characterized by skeletal muscle wasting, weakness and fatigue. Several pathogenetic mechanisms can contribute to these muscle derangements. Mitochondrial alterations, altered metabolism and increased oxidative stress are known to promote muscle weakness and muscle catabolism. To the extent of improving cachexia, several drugs have been tested to stimulate mitochondrial function and normalize the redox balance. The aim of this study was to test the potential beneficial anti-cachectic effects of Mitoquinone Q (MitoQ), one of the most widely-used mitochondria-targeting antioxidant. Here we show that MitoQ administration (25 mg/kg in drinking water, daily) in vivo was able to improve body weight loss in Colon-26 (C26) bearers, without affecting tumor size. Consistently, the C26 hosts displayed ameliorated skeletal muscle and strength upon treatment with MitoQ. In line with improved skeletal muscle mass, the treatment with MitoQ was able to partially correct the expression of the E3 ubiquitin ligases Atrogin-1 and Murf1. Contrarily, the anabolic signaling was not improved by the treatment, as showed by unchanged AKT, mTOR and 4EBP1 phosphorylation. Assessment of gene expression showed altered levels of markers of mitochondrial biogenesis and homeostasis in the tumor hosts, although only Mitofusin-2 levels were significantly affected by the treatment. Interestingly, the levels of Pdk4 and CytB, genes involved in the regulation of mitochondrial function and metabolism, were also partially increased by MitoQ, in line with the modulation of hexokinase (HK), pyruvate dehydrogenase (PDH) and succinate dehydrogenase (SDH) enzymatic activities. The improvement of the oxidative metabolism was associated with reduced myosteatosis (i.e., intramuscular fat infiltration) in the C26 bearers receiving MitoQ, despite unchanged muscle LDL receptor expression, therefore suggesting that MitoQ could boost β-oxidation in the muscle tissue and promote a glycolytic-to-oxidative shift in muscle metabolism and fiber composition. Overall, our data identify MitoQ as an effective treatment to improve skeletal muscle mass and function in tumor hosts and further support studies aimed at testing the anti-cachectic properties of mitochondria-targeting antioxidants also in combination with routinely administered chemotherapy agents.

Codonopsis lanceolata and its active component Tangshenoside I ameliorate skeletal muscle atrophy via regulating the PI3K/Akt and SIRT1/PGC-1α pathways

Background: Skeletal muscle atrophy is caused by aging, disuse, malnutrition, and several diseases. However, there are still no effective drugs or treatments for muscle atrophy. Codonopsis lanceolata (CL), a traditional medicinal plant and food, has been reported to have anti-oxidative, anti-inflammatory, anti-tumor, and anti-obesity effects.Purpose: This study aimed to investigate the efficacy and active component of CL on muscle atrophy in vitro and to confirm the effect of CL and its active component on muscle atrophy and the underlying molecular mechanisms in vivo.Study: design/Methods This study used the dexamethasone (Dex)-induced muscle atrophy C2C12 myotube model and immobilization (IM)-induced muscle atrophy C57BL/6 mice model. In vitro study, the myotube diameter was measured. In vivo study, the grip strength, muscle mass (quadriceps, gastrocnemius, and soleus) and muscle fiber cross-sectional area (CSA) was measured. Western blot analysis and qRT-PCR were performed to confirm the underlying molecular mechanismsResults:In vitro study, CL and its main component, Tangshenoside I (TSI), effectively restored C2C12 myotube diameters decreased by Dex. Surprisingly, TSI was identified as the active component responsible for the overall efficacy of CL on muscle atrophy. In vivo study, CL and TSI, dose-dependently increased grip strength, mass muscle, and muscle fiber CSA reduced by IM. In the molecular mechanism studies, CL and TSI increased muscle protein synthesis via activating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin complex 1 (mTORC1) pathway and decreased muscle protein degradation via inhibiting the muscle ring finger-1 (MuRF1) and muscle atrophy F-box protein (Atrogin-1) expressions. It also upregulated mitochondrial biogenesis via the silent information regulator 1 (SIRT1)/ peroxisome proliferator-activated receptor gamma and coactivator-1 alpha (PGC-1α) pathway.Conclusion: This study suggests that CL and its active component, TSI, can be potential drug candidates for the prevention and treatment of muscle atrophy.

Long-Acting Thioredoxin Ameliorates Doxorubicin-Induced Cardiomyopathy via Its Anti-Oxidative and Anti-Inflammatory Action.

Although the number of patients with heart failure is increasing, a sufficient treatment agent has not been established. Oxidative stress and inflammation play important roles in the development of myocardial remodeling. When thioredoxin (Trx), an endogenous anti-oxidative and inflammatory modulator with a molecular weight of 12 kDa, is exogenously administered, it disappears rapidly from the blood circulation. In this study, we prepared a long-acting Trx, by fusing human Trx (HSA-Trx) with human serum albumin (HSA) and evaluated its efficacy in treating drug-induced heart failure. Drug-induced cardiomyopathy was created by intraperitoneally administering doxorubicin (Dox) to mice three times per week. A decrease in heart weight, increased myocardial fibrosis and markers for myocardial damage that were observed in the Dox group were suppressed by HSA-Trx administration. HSA-Trx also suppressed the expression of atrogin-1 and myostatin, myocardial atrophy factors in addition to suppressing oxidative stress and inflammation. In the Dox group, a decreased expression of endogenous Trx in cardiac tissue and an increased expression of macrophage migration inhibitory factor were observed, but these changes were restored to normal levels by HSA-Trx administration. These findings suggest that HSA-Trx improves the pathological condition associated with Dox-induced cardiomyopathy by its anti-oxidative/anti-inflammatory and myocardial atrophy inhibitory action.

MiR-27b-3p Attenuates Muscle Atrophy by Targeting Cbl-b in Skeletal Muscles

As it is well known, muscle atrophy is a process in which protein degradation increases and protein synthesis decreases. This process is regulated by a variety of links. Among them, microRNAs play an essential role in this process, which has attracted widespread attention. In this paper, we find that miR-27b-3p and Cbl-b genes are significantly differentially expressed in the induced atrophy model. The dual-luciferase experiment and Western blot analysis confirmed that miR-27b-3p could regulate the expression of Cbl-b. In C2C12-differentiated myotubes, the overexpression of the Cbl-b gene showed that Cbl-b could upregulate the expression of MuRF-1 and Atrogin-1, which are related marker genes of muscle atrophy, at both the mRNA and protein levels, indicating that the Cbl-b gene can specifically affect muscle atrophy. The knockdown of the Cbl-b gene after C2C12-differentiated myotubes induced atrophy treatment can downregulate the expression of muscle-atrophy-related genes, indicating that manual intervention to downregulate the expression of Cbl-b has a certain alleviating effect on muscle atrophy. These data suggest that miR-27b-3p can regulate the expression of the Cbl-b gene and then exert a particular influence on muscle atrophy through the Cbl-b gene.

Clinical-Like Cryotherapy in Acute Knee Arthritis Protects Neuromuscular Junctions of Quadriceps and Reduces Joint Inflammation in Mice.

Rheumatoid arthritis is an autoimmune and inflammatory disease that affects synovial joint tissues and skeletal muscle. Clinical-like cryotherapy benefits signs of joint inflammation in knee osteoarthritis after 60 days of anterior cruciate ligament transection surgery. However, it is unknown whether it also benefits acute knee arthritis (e.g., reduces inflammatory process and protects neuromuscular junction [NMJ] and muscle fibers). We aimed to analyze the effects of clinical-like cryotherapy on NMJ and quadriceps muscle fibers in a model of acute knee arthritis. Twenty-four male C57BL/6 mice (20 to 25 g) were randomly allocated into three groups: control (mice with no intervention), antigen-induced arthritis (AIA; mice sensitized and immunized with intra-articular [i.a.] injection of methylated bovine serum albumin [mBSA]), and AIA+cryotherapy (mice sensitized, immunized with i.a. injection of mBSA, and submitted to a clinical-like cryotherapy protocol). Twenty-one days after sensitization, arthritis was induced in immunized mice via i.a. injection of mBSA (100 μg/joint). Two clinical-like cryotherapy sessions (crushed ice pack for 20 min) were applied two hours apart. The first session was applied immediately after i.a. injection of mBSA. The quadriceps was removed two hours after the second clinical-like cryotherapy session for morphological analysis of muscle fibers (cross-sectional area), frequency distribution of muscle fiber area (%), and NMJ (area, perimeter, and maximum diameter). Gene expressions of mRNA involved in NMJ signaling (γ-nAChR, α1-nAChR, ε-nAChR, Agrin-MusK-Rapsyn, α-dystrobrevin, and utrophin) and atrophy (muscle RING-finger protein-1 and Atrogin-1) pathways were analyzed. Inflammatory signs were assessed in knee joint (swelling, articular surface temperature, and neutrophil migration in synovial fluid). Regarding morphological analysis of muscle fibers, 180 to 270 and >270 μm2 classes were higher in the AIA+cryotherapy than the AIA group. Area, perimeter, and maximum diameter of NMJ also increased in the AIA+cryotherapy compared with the control group. Agrin mRNA expression increased in the AIA+cryotherapy compared with the control and AIA groups. In the atrophy pathway, Atrogin-1 increased compared with the control and AIA groups. The AIA+cryotherapy group reduced knee swelling and neutrophil migration compared with the AIA group. In conclusion, clinical-like cryotherapy increased Agrin expression, contributing to NMJ maintenance and increased Atrogin-1 expression, thus protecting NMJ and muscle fiber. Furthermore, clinical-like cryotherapy reduced inflammatory signs (swelling and neutrophil migration) of acute knee arthritis.

Neuron-derived neurotrophic factor protects against dexamethasone-induced skeletal muscle atrophy

Skeletal muscle atrophy caused by various conditions including aging, nerve damage, and steroid administration, is a serious health problem worldwide. We recently reported that neuron-derived neurotrophic factor (NDNF) functions as a muscle-derived secreted factor, also known as myokine, which exerts protective actions on endothelial cell and cardiomyocyte function. Here, we investigated whether NDNF regulates skeletal muscle atrophy induced by steroid administration and sciatic denervation.NDNF-knockout (KO) mice and age-matched wild-type (WT) mice were subjected to continuous dexamethasone (DEX) treatment or sciatic denervation. NDNF-KO mice exhibited decreased gastrocnemius muscle weight and reduced cross sectional area of myocyte fiber after DEX treatment or sciatic denervation compared with WT mice. Administration of an adenoviral vector expressing NDNF (Ad-NDNF) or recombinant NDNF protein to gastrocnemius muscle of WT mice increased gastrocnemius muscle weight after DEX treatment. NDNF-KO mice showed increased expression of ubiquitin E3-ligases, including atrogin-1 and MuRF-1, in gastrocnemius muscle after DEX treatment, whereas Ad-NDNF reduced expression of atrogin-1 and MuRF-1 in gastrocnemius muscle of WT mice after DEX treatment. Pretreatment of cultured C2C12 myocytes with NDNF protein reversed reduced myotube diameter and increased expression of atrogin-1 and MuRF-1 after DEX stimulation. Treatment of C2C12 myocytes increased Akt phosphorylation. Pretreatment of C2C12 myotubes with the PI3-kinase/Akt inhibitor reversed NDNF-induced increase in myotube fiber diameter after DEX treatment. In conclusion, our findings indicated that NDNF prevents skeletal muscle atrophy in vivo and in vitro through reduction of ubiquitin E3-ligases expression, suggesting that NDNF could be a novel therapeutic target of muscle atrophy.

A new therapeutic effect of fenofibrate in Duchenne muscular dystrophy: The promotion of myostatin degradation.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease with no effective drug treatment. This study investigated the positive effects of fenofibrate on dystrophic muscles. Myostatin expression in serum and muscle tissue from patients with Duchenne muscular dystrophy and mdx mice were tested. Primary myoblasts isolated from mdx mice were challenged with an inflammatory stimulus and treated with fenofibrate. In animal experiments, 6-week-old male mdx mice were treated with fenofibrate (100 mg kg-1 ) administered orally once per day for 6weeks. Effects of fenofibrate were evaluated by tests of muscle function plus histology and biochemical analyses of serum. Expression of myostatin, MuRF1, and atrogin-1 in skeletal muscle was evaluated by western blotting and real-time PCR. Total and oxidative myosin heavy chain (MHC) were assessed via immunofluorescence. Expression of myostatin protein was increased in dystrophic muscle of patients with Duchenne muscular dystrophy and mdx mice. Fenofibrate enhanced myofibre differentiation by down-regulating the expression of myostatin protein but not mRNA in primary myoblasts of mdx mice. Fenofibrate significantly improved muscle function while ameliorating muscle damage in mdx mice. These benefits were accompanied by an anti-inflammatory effect. Fenofibrate treatment returned myofibre function by inhibiting the expressions of myostatin, MuRF1, and atrogin-1 protein in the gastrocnemius muscle and diaphragm, while leaving the mRNA level of myostatin unaffected. Fenofibrate substantially slows muscle dystrophy by promoting the degradation of myostatin protein, which may indicate a new therapeutic focus for patients with Duchenne muscular dystrophy.