MuRF1 Protein Research Articles

Unexpected regulation pattern of the IKKβ/NF-κB/MuRF1 pathway with remarkable muscle plasticity in the Daurian ground squirrel (Spermophilus dauricus).

As a typical hibernator, the Daurian ground squirrel (Spermophilus dauricus) spends considerable time in a state of reduced activity with prolonged fasting. Despite this, they experience little muscle atrophy and have thus become an interesting anti-disuse muscle atrophy model. The IKKβ/NF-κB signaling pathway is significant to muscle atrophy due to the protein degradation resulting from the upregulation of the E3 ubiquitin ligase MuRF1. The current study showed that the IKKβ/NF-κB signaling pathway and MuRF1 maintained relatively steady mRNA and protein expression levels, with little muscle atrophy observed in the soleus (slow-twitch, SOL) or extensor digitorum longus (fast-twitch, EDL) during hibernation (HIB); however, mRNA expression significantly increased in the SOL and EDL muscle during interbout arousal (IBA), as did the MuRF1 mRNA level in the SOL and MuRF1 protein level in the EDL. Interestingly, the expressions of p50 and MuRF1 significantly increased during HIB in the gastrocnemius (mixed muscle, GAS) and showed moderate atrophy, but dramatically decreased during IBA. Elevated IKKβ and p50 mRNA and protein expression in the cardiac muscle (CM) during HIB did not accompany increased MuRF1 expression or muscle wasting. Importantly, almost all increased or decreased indicators in the tested tissues recovered to pre-hibernation levels after HIB. This is the first study to report on the unexpected regulation of the IKKβ/NF-κB/MuRF1 pathway with remarkable muscle plasticity in Daurian ground squirrels during hibernation. Furthermore, we found that different types of muscles exhibited different strategies to cope with prolonged hibernation-induced disuse muscle atrophy.

Resistance Training-induced Decrease In Circulating C1q Is Associated With Attenuated Muscle Degradation In Senescent Mice

Our recent study has showed that reduction of aging-induced elevation of serum C1q level by resistance training was involved in muscle hypertrophy in old adults. C1q activates Wnt/β-catenin in aged muscle, resulting in enhancement of muscle protein degradation-related genes, such as MuRF-1 and Atrogin-1. Although resistance training attenuates muscle protein degradation, it is still unclear that resistance training-induced decrease in serum C1q level is associated with attenuation of muscle protein degradation. PURPOSE: The purpose of this study was to investigate whether resistance training-induced change in circulating C1q level affects muscle protein degradation in senescent mice. METHODS: Male 13-week-old SAMP1 mice (Young) and 38-week-old SAMP1 mice (Aged) were randomly divided into three groups; young-sedentary control (Young-Con), aged-sedentary control (Aged-Con) and aged-resistance training (Aged-RT) groups (n=10 each group). Resistance training was performed 3 days a week for 12 weeks using a climbing ladder with 70% of 1 repetition maximum weight. RESULTS: Muscle strength and mass and cross-sectional area (CSA) of tibialis anterior muscle in the Aged-Con group significantly decreased as compared with the Young-Con group (p<0.05), whereas those in the Aged-RT group significantly increased as compared with the Aged-Con group (p<0.05). Serum C1q level and expression levels of muscle β-catenin, MuRF-1 and Atrogin-1 proteins significantly increased in the Aged-Con group as compared with the Young-Con group (p<0.05), whereas those expression levels in the Aged-RT group significantly decreased as compared with the Aged-Con group (p<0.05). Additionally, serum C1q level was positively correlated with protein expression levels of muscle MuRF-1 (r = 0.713, p<0.05) and Atrogin-1 (r = 0.584, p<0.05), but was negatively correlated with muscle mass (r = -0.469, p<0.05) and CSA (r = -0.595, p<0.01). CONCLUSIONS: These results suggest that resistance training-induced decrease in serum C1q level is associated with attenuation of muscle protein degradation thereby leading to muscle hypertrophy in senescent mice. Supported by Grants-in-Aid for Scientific Research (#17H02182 and #16K13059, M. Iemitsu) and Sasakawa Scientific Research Grant (#29-628 for N. Horii).

Skeletal myofiber VEGF‐A‐targeted gene deletion induces mitochondrial, structural and contractile alterations in mouse diaphragm

Respiratory muscle weakness is a hallmark of COPD, heart failure, and critical illness. Vascular endothelial growth factor (VEGF) may be an important therapeutic target in chronic cardiopulmonary disease as it is essential for neovascularization and vessel repair following trauma. We hypothesized that respiratory muscle, which is required to persistently function, would exhibit impaired mitochondrial, structural, and contractile function in skeletal myofiber VEGF gene deleted mice.We measured diaphragm biochemistry, structure, and contractile function in adult mice with conditional deletion of the VEGF‐A gene in skeletal myofibers (SkmVEGF−/−). Male SkmVEGF−/− mice (n=12) were compared to wild type (WT) controls (n=13) – both on a C57BL/6J background. Fiber bundles were isolated and electrically stimulated in vitro to evaluate force‐frequency and fatigue profiles. Mitochondrial O2 consumption and H2O2 flux (index of mitochondrial ROS) were simultaneously measured in saponin‐permeabilized fiber bundles (Oroboros, Innsbruck, AT). Fiber structure and biochemistry were measured using standard techniques (IHC, Western, ELISA).Diaphragm VEGF protein was lower in SkmVEGF−/− than WT mice (1.5±0.2 vs. 3.0±0.4 pg/total protein, p&lt;0.05). Compared to WT mice, the force‐frequency relationship was depressed in SkmVEGF−/− (F[7,161]=15.0, p&lt;0.05), with maximal specific force reduced by ~10 % (24±1 vs. 21±1 N/cm2; p&lt;0.05). However, fatigue resistance tended to be greater following SkmVEGF−/− (F[2,115]=3.3, p=0.06). Fiber type proportions were unchanged, but type I fiber cross‐sectional area was reduced in SkmVEGF−/− (653±45 vs 493±53 μm2; p&lt;0.05). Compared to WT, sarcomeric actin protein expression was reduced by ~30% in SkmVEGF−/− (p&lt;0.05) while myosin heavy chain, MAFbx, MuRF1, PGC1α, and HIF1α protein remained unchanged (p&gt;0.05). Mitochondrial respiration was not different between SkmVEGF−/− and WT (F[1,80]=0.9, p&gt;0.05) in each of the respiratory states. However, H2O2 flux was reduced in SkmVEGF−/− (F[1,75]=14.6, p&lt;0.05).Skeletal myofiber‐specific VEGF deletion contributed to diaphragm weakness, but tended to improve fatigue resistance in this highly oxidative muscle. Reductions in oxidative fiber size, sarcomeric actin protein content, and ROS generation following VEGF gene deletion may contribute to protecting mitochondrial respiratory function in the diaphragm. The adaptations may be part of compensatory mechanisms mitigating larger deficits in force generation and fatigue resistance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct signal transductions in fast- and slow- twitch muscles upon denervation.

Denervation induces skeletal muscle atrophy, which primarily impairs oxidative slow twitch fibers. The underlying mechanism of this phenomenon, however, remains to be addressed. We hypothesize that denervation‐induced fiber‐specific atrophy may result from the distinct activities of different signaling pathways that are involved in protein synthesis and degradation in fast‐ and slow‐twitch fibers. In this study, 1‐month‐old male mice were subjected to unilateral sciatic denervation for 4 days. Fast‐twitch muscle extensor digitorum longus (EDL) and slow‐twitch muscle soleus were collected from the denervated side and the control side of hind limbs. Total and phosphorylated protein levels of key factors of major signaling pathways in these tissues were determined using western blot assay. Our data showed that total AKT and FoxO3 protein levels were upregulated in denervated muscles as compared with control sides. Phosphorylation of AKT and FoxO3 were proportionally enhanced in denervated EDL but not soleus, indicating AKT activation drives phosphorylation of FoxO3 in EDL but not in soleus upon denervation. As a result, FoxO3‐targeted atrogenes MurF1 and Atrogin1 protein abundances were reduced in denervated EDL but not altered in soleus. In consistent with this change, polyubiquitination were significantly increased in denervated soleus, but only a slight increase in ubiquitination was found in denervated EDL. Autophagy marker LC3 protein level was significantly increased in both muscle types, but in greater extent in EDL after denervation. IRS1 protein level and active ERK were reduced in both muscles upon denervation, which might contribute to the upregulation of total AKT protein level and FoxO3 abundance in EDL and soleus. Total and phosphorylated AMPK protein levels were increased in denervated soleus but not in EDL. Overall, these data reveal that the key signaling pathways that regulate protein synthesis and degradation are more sensitive in soleus than EDL in response to denervation.

Decreased grip strength, muscle pain, and atrophy occur in rats following long-term exposure to excessive repetitive motion.

Work‐related musculoskeletal disorders (WMSD) are caused by the overuse of muscles in the workplace. Performing repetitive tasks is a primary risk factor for the development of WMSD. Many workers in highly repetitive jobs exhibit muscle pain and decline in handgrip strength, yet the mechanisms underlying these dysfunctions are poorly understood. In our study, rats performed voluntary repetitive reaching and grasping tasks (Task group), while Control group rats did not perform these activities. In the Task group, grip strength and forearm flexor withdrawal threshold declined significantly from week 2 to week 6, compared with these values at week 0 (P < 0.05). Relative muscle weight and muscle fiber cross‐sectional area of flexor digitorum superficialis (FDS) muscles decreased significantly in the Task group, compared with the Control group, at 6 weeks (P < 0.05 and P < 0.01, respectively). Nerve growth factor, glial cell line‐derived neurotrophic factor, and tumor necrosis factor α‐expression in FDS muscles were not significantly different in Control and Task groups at 3 and 6 weeks. At 6 weeks, the Task group had elevated MuRF1 protein levels (P = 0.065) and significant overexpression of the autophagy‐related (Atg) proteins, Beclin1 and Atg5–Atg12, compared with in the Control group (both P < 0.05). These data suggested that long‐term exposure to excessive repetitive motion causes loss of grip strength, muscle pain, and skeletal muscle atrophy. Furthermore, this exposure may enhance protein degradation through both the ubiquitin‐proteasome and autophagy‐lysosome systems, thereby decreasing skeletal muscle mass.

The role of calpains in ventilator-induced diaphragm atrophy

BackgroundControlled mechanical ventilation (CMV) is associated with diaphragm dysfunction. Dysfunction results from muscle atrophy and injury of diaphragm muscle fibers. Enhanced proteolysis and reduced protein synthesis play an important role in the development of atrophy. The current study is to evaluate the effects of the calpains inhibitor calpeptin on the development of diaphragm atrophy and activation of key enzymes of the ubiquitin-proteasome pathway in rats under CMV.MethodsThree groups of rats were studied: control animals (CON, n = 8), rats subjected to 24 h of MV (CMV, n = 8), and rats subjected to 24 h of MV after administration of the calpain inhibitor calpeptin (CMVC, n = 8). The diaphragm was analyzed for calpain activity, myosin heavy chain (MHC) content, and cross-sectional area (CSA) of diaphragmatic muscle fibers as a marker for muscle atrophy. In addition, key enzymes of the ubiquitin-proteasome pathway (MAFbx and MuRF1) were also studied.ResultsCMV resulted in loss of both MHCfast and MHCslow. Furthermore, the CSA of diaphragmatic muscle fibers was significantly decreased after 24 h of CMV. However, calpain inhibitor calpeptin prevented loss of MHC and CSA after CMV. In addition, calpeptin prevented the increase in protein expression of calpain1 and calpain2 and reduced calpain activity as indicated by reduced generation of the calpain cleavage product αII-spectrin in the diaphragm. CMV-induced upregulation of both MAFbx and MuRF1 protein levels was attenuated by treatment with calpeptin.ConclusionsThe calpain inhibitor calpeptin prevents MV-induced muscle atrophy. In addition, calpeptin attenuated the expression of key proteolytic enzymes known to be involved in ventilator-induced diaphragm atrophy, including MAFbx and MuRF1.

Chinese Herbal Medicine Baoyuan Jiedu Decoction Inhibited Muscle Atrophy of Cancer Cachexia through Atrogin-l and MuRF-1.

The aim of this study is to investigate the mechanisms of Chinese herbal medicine called “Baoyuan Jiedu” (BYJD for short) decoction, improving life quality and preventing muscle atrophy of cancer cachexia model mice. We showed that the effect of BYJD decoction increased body weights of mice and reduced tumor volume and tumor mass. Furthermore, BYJD decoction increased the gastrocnemii mass and the transverse diameter of muscle fiber morphology. Moreover, BYJD reduced the expression of Atrogin-1 and MuRF-1 protein. Collectively, our results show that BYJD decoction improves the life quality of cancer cachexia mice and prevents muscle atrophy by downregulating expression of Atrogin-1 and MuRF-1.

Preventive Effects of the Dietary Intake of Medium-chain Triacylglycerols on Immobilization-induced Muscle Atrophy in Rats.

Previous studies have shown that medium-chain triacylglycerols (MCTs) exert favorable effects on protein metabolism. This study evaluated the effects of the dietary intake of MCTs on rat skeletal muscle mass and total protein content during casting-induced hindlimb immobilization, which causes substantial protein degradation and muscle atrophy. Rats were fed a standard diet containing long-chain triacylglycerols (LCTs) or MCTs for 3 days and then a unilateral hindlimb was immobilized while they received the same diet. After immobilization for 3, 7, and 14 days, muscle mass and total protein content in immobilized soleus muscle in the LCT-fed rats had markedly decreased compared to the contralateral muscle; however, these losses were partially suppressed in MCT-fed rats. Autophagosomal membrane proteins (LC-I and -II), which are biomarkers of autophagy-lysosome activity, did not differ significantly between the LCT- and MCT-fed rats. In contrast, the immobilization-induced increase in muscle-specific E3 ubiquitin ligase MuRF-1 protein expression in immobilized soleus muscle relative to contralateral muscle was completely blocked in the MCT-fed rats and was significantly lower than that observed in the LCT-fed rats. Collectively, these results indicate that the dietary intake of MCTs at least partly alleviates immobilization-induced muscle atrophy by inhibiting the ubiquitin-proteasome pathway.

Exercise Training Prevents Diaphragm Contractile Dysfunction in Heart Failure.

Patient studies have demonstrated the efficacy of exercise training in attenuating respiratory muscle weakness in chronic heart failure (HF), yet direct assessment of muscle fiber contractile function together with data on the underlying intracellular mechanisms remains elusive. The present study, therefore, used a mouse model of HF to assess whether exercise training could prevent diaphragm contractile fiber dysfunction by potentially mediating the complex interplay between intracellular oxidative stress and proteolysis. Mice underwent sham operation (n = 10) or a ligation of the left coronary artery and were randomized to sedentary HF (n = 10) or HF with aerobic exercise training (HF + AET; n = 10). Ten weeks later, echocardiography and histological analyses confirmed HF. In vitro diaphragm fiber bundles demonstrated contractile dysfunction in sedentary HF compared with sham mice that was prevented by AET, with maximal force 21.0 ± 0.7 versus 26.7 ± 1.4 and 25.4 ± 1.4 N·cm, respectively (P < 0.05). Xanthine oxidase enzyme activity and MuRF1 protein expression, markers of oxidative stress and protein degradation, were ~20% and ~70% higher in sedentary HF compared with sham mice (P < 0.05) but were not different when compared with the HF + AET group. Oxidative modifications to numerous contractile proteins (i.e., actin and creatine kinase) and markers of proteolysis (i.e., proteasome and calpain activity) were elevated in sedentary HF compared with HF + AET mice (P < 0.05); however, these indices were not significantly different between sedentary HF and sham mice. Antioxidative enzyme activities were also not different between groups. Our findings demonstrate that AET can protect against diaphragm contractile fiber dysfunction induced by HF, but it remains unclear whether alterations in oxidative stress and/or protein degradation are primarily responsible.

Angiotensin-(1-7) Prevents Skeletal Muscle Atrophy Induced by Transforming Growth Factor Type Beta (TGF-β) via Mas Receptor Activation

Background: Transforming growth factor type beta 1 (TGF-β1) produces skeletal muscle atrophy. Angiotensin-(1-7) (Ang-(1-7)), through the Mas receptor, prevents the skeletal muscle atrophy induced by sepsis, immobilization, or angiotensin II (Ang-II). However, the effect of Ang-(1-7) on muscle wasting induced by TGF-β1 is unknown. Aim: To evaluate whether Ang-(1-7)/Mas receptor axis could prevent the skeletal muscle atrophy induced by TGF-β1. Methods: This study assessed the atrophic effect of TGF-β1 in C₂C₁₂ myotubes and mice in absence or presence of Ang-(1-7), and the receptor participation using A779, an antagonist of the Mas receptor. The levels of myosin heavy chain (MHC), polyubiquitination, and MuRF-1 were detected by western blot. Myotube diameter was also evaluated. In vivo analysis included the muscle strength, fibre diameter, MHC and MuRF-1 levels by western blot, and ROS levels by DCF probe detection. Results: The results showed that Ang-(1-7) prevented the increase in MuRF-1 and polyubiquitined protein levels, the decrease of MHC levels, the myotubes/fibre diameter diminution, and the increased production of reactive oxygen species (ROS) induced by TGF-β1. Utilizing A779 inhibited the anti-atrophic effect of Ang-(1-7). Conclusion: The preventive effect of Ang-(1-7) on skeletal muscle atrophy induced by TGF-β1 is produced through inhibition of ROS production and proteasomal degradation of MHC.

Six-week anaerobic training improves proteolytic profile of diabetic rats.

To evaluate the effect of six-week anaerobic training on the mRNA expression of genes related to proteolysis Ubb (Ubiquitin), E2-14kDa, Trim63 (MuRF1 protein) and Nfkb1 in the skeletal muscle of diabetic rats. Four groups were established: DE (DiabetesExercised), DS (Diabetes Sedentary), CE (Control Exercised) and CS (Control Sedentary). The training consisted of 3 sets of 12 jumps in the liquid mean with load equivalent to 50% of BW for 6 weeks. Euthanasia occurred under ip anesthesia, and blood, adipose tissue and skeletal muscles were collected. Gene expression was quantified by RT-PCR in the gastrocnemius muscle. ANOVA one-way was used for comparison among groups, with post-hoc (Tukey) when necessary, considering p < 0.05. We observed reduction in the body weight and adipose tissue in the diabetic groups. The muscle mass was reduced in DS, which could be reversed by training (DE). Although DS and DE have presented similar body weight, the training protocol in DE promoted reduction in the adipose tissue, and increase of muscle mass. Anaerobic training was efficient to reduce glycaemia only in the diabetic animals until 6 hours after the end of training. The Trim63 gene expression was increased in DS; decreased Ubb gene level was observed in trained rats (CE and DE) compared to sedentary (CS and DS), and DE presented the lowest level of E2-14kDa gene expression. Six-week anaerobic training promoted muscle mass gain, improved glycemic control, and exerted inhibitory effect on the proteolysis of gastrocnemius muscle of diabetic rats.

Growth Hormone Administration Influence on Soleus Muscle of Heart Failure Rats

Objective: To evaluate the effects of growth hormone (GH) on satellite cells activation and expression of trophism-related proteins in soleus muscle of rats with aortic stenosis (AS)-induced heart failure. Methods: Male Wistar rats were subjected to thoracotomy for ascending AS induction. After developing tachypnea, rats were assigned into three groups (n=8): Sham, AS and AS treated with GH (AS-GH, 2 mg/kg/day for 14 days). Echocardiogram was performed before and after treatment. Muscle trophism was evaluated by histologic analysis. Protein expression was analyzed by Western-blot and satellite cells activation by immunofluorescence. Statistical analysis: Anova and Tukey or Kruskal-Wallis and Student-Newman-Keuls. Results: Before treatment, cardiac variables did not differ between AS and AS-GH groups. GH administration preserved body weight (Sham 492±32; AS 420±32; AS-GH 486±69 g; p<0.05 AS vs Sham and AS-GH). Left ventricular posterior wall thickness and right ventricle weight/body weight ratio were higher in AS and AS-GH than Sham. Soleus cross-sectional area was smaller in both AS groups than Sham (Sham 3,556±447; AS 2,882±422; AS-GH 2,868±591 µm2; p=0.02). IGF-1 expression was higher in AS-GH (Sham 1.0±0.3; AS 1.0±0.5; AS-GH 1.7±0.5 arbitrary units; p=0.004). p-PI3K/GAPDH was higher in AS and AS-GH than Sham and total PI3K [Sham 1.0 (0.6-1.3); AS 1.3 (1.0-2.5); AS-GH 2.5 (2.0-6.0) arbitrary units; p=0.04] was higher in AS-GH than Sham. MyoD, MRF4, myogenin, myostatin, follistatin, akt, atrogin-1, MuRF-1 and Pax-7 protein levels did not differ between groups. Satellite cells activation was increased in both AS groups. Conclusion Growth hormone administration increases IGF-1 and PI3K intracellular signaling in the soleus muscle of heart failure rats.

Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease

Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems.

P22 - De novo ceramides synthesis is not involved in skeletal muscle atrophy induced by short-term mechanical unloading

Patients admitted to the intensive care unit commonly develop skeletal muscle weakness that can exacerbate illness and complicate their recovery. Beyond the primary disease or aging, weakness is promoted by a variety of prolonged hospitalization-associated conditions. These include altered nutritional status, pharmacologic side effects, physical inactivity, and prolonged bed rest. The two latter conditions (i.e. inactivity and bed rest) are the most ubiquitous, affecting all patients during a prolonged hospitalization. In both cases, skeletal muscle utilization is decreased with a concomitant reduction in fatty acid oxidation. Subsequent fatty acids accumulation converted to ceramides could be a cellular mechanism leading to muscle wasting. Indeed these sphingolipids act as second messengers in several of molecular signaling pathways involved in muscle atrophy. Consequently, the aim of this work is to determine the effects of immobilization on muscle ceramides accumulation, and identify the role of these ectopic lipids in molecular mechanisms involved in skeletal muscle atrophy. For this purpose, male Wistar rats were treated with an inhibitor of de novo synthesis of ceramides (i.e. myriocin) and subjected to hindlimb unloading for 7 days. We found that hindlimb unloading induced skeletal muscle atrophy, in part through proteolysis (i.e. decrease in AKT activation, increase in MuRF1 and polyubiquinated proteins content) and apoptosis activations (i.e. increase in Bax/Bcl-2 ratio and cleaved caspase-3). Myriocin treatment did not prevent skeletal muscle atrophy and concomitant induction of apoptosis and proteolysis. Data concerning muscle ceramides content are being analyzed. Together, these results suggest that de novo synthesis of ceramides is not involved in muscle atrophy induced by a short period of hindlimb unloading.

Regulation of ubiquitin proteasome pathway molecular markers in response to endurance and resistance exercise and training.

Knowledge on the effects of divergent exercise on ostensibly protein degradation pathways may be valuable for counteracting muscle wasting and for understanding muscle remodelling. This study examined mRNA and/or protein levels of molecular markers of the ubiquitin proteasome pathway (UPP), including FBXO32 (atrogin-1), MURF-1, FBXO40, FOXO1 and FOXO3. Protein substrates of atrogin-1-including EIF3F, MYOG and MYOD1-and of MURF-1-including PKM and MHC-were also measured. Subjects completed 10 weeks of endurance training (ET) or resistance training (RT) followed by a single-bout of endurance exercise (EE) or resistance exercise (RE). Following training, atrogin-1, FBXO40, FOXO1 and FOXO3 mRNA increased independently of exercise mode, whereas MURF-1 mRNA and FOXO3 protein increased following ET only. No change in other target proteins occurred post-training. In the trained state, single-bout EE, but not RE, increased atrogin-1, MURF-1, FBXO40, FOXO1, FOXO3 mRNA and FOXO3 protein. In contrast to EE, FBXO40 mRNA and protein decreased following single-bout RE. MURF-1 and FOXO1 protein levels as well as the protein substrates of atrogin-1 and MURF-1 were unchanged following training and single-bout exercise. This study demonstrates that the intracellular signals elicited by ET and RT result in an upregulation of UPP molecular markers, with a greater increase following ET. However, in the trained state, the expression levels of UPP molecular markers are increased following single-bout EE, but are less responsive to single-bout RE. This suggests that adaptations following endurance exercise training are more reliant on protein UPP degradation processes than adaptations following resistance exercise training.

β-Hydroxy β-methylbutyrate improves dexamethasone-induced muscle atrophy by modulating the muscle degradation pathway in SD rat.

Skeletal muscle atrophy results from various conditions including high levels of glucocorticoids, and β–hydroxy β–methylbutyrate (HMB; a metabolite of leucine) is a potent therapeutical supplement used to treat various muscle disorders. Recent studies have demonstrated that HMB inhibits dexamethasone-induced atrophy in cultured myotubes, but its effect on dexamethasone-induced muscle atrophy has not been determined in vivo. In the present study, we investigated the effect of HMB on dexamethasone-induced muscle atrophy in rats. Treatment with dexamethasone weakened grip strengths and increased muscle damage as determined by increased serum creatine kinase levels and by histological analysis. Dexamethasone treatment also reduced both soleus and gastrocnemius muscle masses. However, HMB supplementation significantly prevented reductions in grip strengths, reduced muscle damage, and prevented muscle mass and protein concentration decrease in soleus muscle. Biochemical analysis demonstrated that dexamethasone markedly increased levels of MuRF1 protein, which causes the ubiquitination and degradation of MyHC. Indeed, dexamethasone treatment decreased MyHC protein expression and increased the ubiquitinated-MyHC to MyHC ratio. However, HMB supplementation caused the down-regulations of MuRF1 protein and of ubiquitinated-MyHC. Furthermore, additional experiments provided evidence that HMB supplementation inhibited the nuclear translocation of FOXO1 induced by dexamethasone, and showed increased MyoD expression in the nuclear fractions of soleus muscles. These findings suggest that HMB supplementation attenuates dexamethasone-induced muscle wasting by regulating FOXO1 transcription factor and subsequent MuRF1 expression. Accordingly, our results suggest that HMB supplementation could be used to prevent steroid myopathy.

Autophagy and Protein Turnover Signaling in Slow-Twitch Muscle during Exercise

The aim of this study was to characterize skeletal muscle protein breakdown and mitochondrial dynamics markers at different points of endurance exercise. Mice run at 10 m·min(-1) during 1 h, and running speed was increased by 0.5 m·min(-1) every minute during 40 min and then by 1 m·min(-1) until exhaustion. Animals were killed by cervical dislocation at 30, 60, 90, and 120 min; at time to exhaustion (Te); and at 3 and 24 h during recovery. The soleus and the deep red regions of the quadriceps muscles were pooled. AMPK phosphorylation (Thr172) increased from 30 min to Te, and FoxO3a phosphorylation (Thr32 and Ser253) decreased from 120 min to 3 h after exercise. FoxO3a-dependent E3 ligases Mul1 and MuRF1 proteins increased from 30 min to Te and at Te and 3 h after exercise, respectively, whereas MAFbx/atrogin-1 protein expression did not change significantly. The autophagic markers LC3B-II increased at 120 min and Te, and p62 significantly decreased at Te. The AMPK-dependent phosphorylation of Ulk1 at Ser317 and Ser555 increased from 60 min to Te and at 30 and 60 min, respectively. Akt (Ser473), MTOR (Ser2448), and 4E-BP1 (Thr37/46) phosphorylation decreased from 90 min to Te, and the MTOR-dependent phosphorylation of Ulk1 (Ser757) decreased from 120 min to Te. Ser616 phosphorylation of the mitochondrial fission marker DRP1 increased from 60 min to Te, but protein expression of the fusion markers mitofusin-2, a substrate of Mul1, and OPA1 did not significantly change. These results fit with a regulation of protein breakdown triggered by FoxO3a and Ulk1 pathways after AMPK activation and Akt/MTOR inhibition. Furthermore, our data suggest that mitochondrial fission is quickly increased, and mitochondrial fusion is unchanged during exercise.

Low-intensity resistance training attenuates dexamethasone-induced atrophy in the flexor hallucis longus muscle

This study investigated the potential protective effect of low-intensity resistance training (RT) against dexamethasone (DEX) treatment induced muscle atrophy. Rats underwent either an 8 week period of ladder climbing RT or remained sedentary. During the last 10 days of the exercise protocol, animals were submitted to a DEX treatment or a control saline injection. Muscle weights were assessed and levels of AKT, mTOR, FOXO3a, Atrogin-1 and MuRF-1 proteins were analyzed in flexor hallucis longus (FHL), tibialis anterior (TA), and soleus muscles. DEX induced blood glucose increase (+46%), body weight reduction (−19%) and atrophy in FHL (−28%) and TA (−21%) muscles, which was associated with a decrease in AKT and an increase in MuRF-1 proteins levels. Low-intensity RT prevented the blood glucose increase, attenuated the FHL atrophy effects of DEX, and was associated with increased mTOR and reductions in Atrogin-1 and MuRF-1 in FHL. In contrast, TA muscle atrophy and signaling proteins were not affected by RT. These are the first data to demonstrate that low-intensity ladder-climbing RT specifically mitigates the FHL atrophy, which is the main muscle recruited during the training activity, while not preventing atrophy in other limb muscle not as heavily recruited. The recruitment-dependent prevention of atrophy by low intensity RT likely occurs by a combination of attenuated muscle protein degradation signals and enhanced muscle protein synthesis signals including mTOR, Atrogin-1 and MuRF-1.

Muscle atrophy, ubiquitin–proteasome, and autophagic pathways in dysferlinopathy

Muscle fiber atrophy and the molecular pathways underlying this process have not been investigated in dysferlinopathy patients. In 22 muscles from dysferlinopathy patients we investigated fiber atrophy by morphometry and ubiquitin-proteasome and autophagic pathways using protein and/or transcriptional analysis of atrophy- and autophagy-related genes (MuRF1, atrogin1, LC3, p62, Bnip3). Dysferlinopathy showed significant fiber atrophy and higher MuRF-1 protein and mRNA levels, which correlated with fiber size, suggesting activation of the atrophy program by proteasome induction. Some of the MuRF-1 upregulation and proteasome induction may be attributed to the prominent regeneration found. A potential role of impaired autophagy was suggested by p62-positive protein aggregates in atrophic fibers and significantly higher levels of LC3-II and p62 proteins and overexpression of p62 and Bnip3 mRNA. Damaged muscle fibers and prominent inflammatory changes may also enhance autophagy due to the insufficient level of proteasomal degradation of mutant dysferlin.

Molecular analyses provide insight into mechanisms underlying sarcopenia and myofibre denervation in old skeletal muscles of mice

Molecular mechanisms that are associated with age-related denervation and loss of skeletal muscle mass and function (sarcopenia) are described for female C57Bl/6J mice aged 3, 15, 24, 27 and 29 months (m). Changes in mRNAs and proteins associated with myofibre denervation and protein metabolism in ageing muscles are reported, across the transition from healthy adult myofibres to sarcopenia that occurs between 15 and 24m. This onset of sarcopenia at 24m, corresponded with increased expression of genes associated with neuromuscular junction denervation including Chnrg, Chrnd, Ncam1, Runx1, Gadd45a and Myog. Sarcopenia in quadriceps muscles also coincided with increased protein levels for Igf1 receptor, Akt and ribosomal protein S6 (Rps6) with increased phosphorylation of Rps6 (Ser235/236) and elevated Murf1 mRNA and protein, but not Fbxo32: many of these changes are also linked to denervation. Global transcription profiling via microarray analysis confirmed these functional themes and highlighted additional themes that may be a consequence of pathology associated with sarcopenia, including changes in fatty acid metabolism, extracellular matrix structure and protein catabolism. Ageing was also associated with increased global gene expression variance, consistent with decreased control of gene regulation.