MuRF1 Protein Expression Research Articles

Therapeutic ultrasound treatment for the prevention of chronic kidney disease-associated muscle wasting in mice

Low-intensity pulsed ultrasound (LIPUS) is a kind of therapeutic ultrasound. It can help improve bone fracture repair and soft tissue healing. Our previous study found that LIPUS treatment could halt the chronic kidney disease (CKD) progression in mice; unexpectedly, we observed the improvement of CKD-reduced muscle weights by LIPUS treatment. Here, we further tested the protective potential of LIPUS on CKD-associated muscle wasting/sarcopenia using the CKD mouse models. Mouse models of both unilateral renal ischemia/reperfusion injury (IRI) with nephrectomy and adenine administration were used to induce CKD. LIPUS with condition of 3 MHz, 100 mW/cm2, 20 min/day was applied to the kidney of CKD mice. LIPUS treatment significantly reversed the increased serum BUN/creatinine levels in CKD mice. LIPUS effectively prevented the decrease in grip strength, muscle weight (soleus, tibialis anterior, and gastrocnemius muscles), cross-section areas of muscle fibres, and muscular phosphorylated Akt protein expression by immunohistochemistry, and the increase in muscular atrogenes Atrogin1 and MuRF1 protein expression by immunohistochemistry in CKD mice. These results indicated that LIPUS could help improve weak muscle strength, muscle mass loss, muscle atrophy-related protein expression, and Akt inactivation. LIPUS application may be an alternative non-invasive therapeutic intervention on the management of CKD-associated muscle wasting.

Suppressive Effects of Turmeric Extract on Muscle Atrophy in Dexamethasone-Treated Mice and Myotubes.

Sarcopenia is the decline in skeletal muscle mass, strength, and functions, which decreases the quality of life in elderly people. This study investigated the suppressive effect of turmeric (Curcuma longa) extract (TE) on muscle atrophy in dexamethasone (DEX)-treated mice and C2C12 myotubes. DEX treatment significantly decreased the muscle weight and significantly increased Fbxo32 and Murf1 expression in mice, and these changes were suppressed by the supplementation of an AIN-93 based diet with 2% TE. A similar pattern was observed in FBXO32 and MuRF1 protein expression. In C2C12 myotubes, DEX treatment significantly increased FBXO32 and MuRF1 gene and protein expression, and these increases were significantly suppressed by TE supplementation at a concentration of 200 µg/mL. Furthermore, one of the five TE fractions, which were separated by high-performance liquid chromatography had a similar effect with TE supplementation. The present study proposes the suppressive effect of turmeric on sarcopenia.

Lactate Administration Increases Skeletal Muscle Synthesis in Mice

PurposeLactate, secreted by skeletal muscle during exercise, had been known as fatigue substance. However, several physiological roles of lactate have been revealed in the last decades, and previous studies reported that lactate treatment increased protein synthesis factors in skeletal muscle. Nevertheless, underlying molecular mechanisms of exogenous lactate on protein synthesis and degradation remain unknown, and there was no study that confirm long‐term lactate administration with exercise training. Therefore, the purpose of study is to investigate the effect of long‐term exogenous lactate administration and lactate administration with exercise training on the skeletal muscle synthesis and degradation.Methods32 mice were randomly divided into four groups: sedentary + saline (Sed/Con), sedentary + lactate (Sed/Lac), exercise training + saline (Ex/Con), and Exercise training + lactate (Ex/Lac). Exercise training (VO2max 70%) and lactate administration (3 g/kg sodium lactate) performed 5 days a week. The timing of lactate administration was immediately after every exercise training, and saline administration groups were given same volume of saline as lactate. After 5 weeks, mice were sacrificed and immediately obtained skeletal muscle and blood. To confirm the effect of lactate on protein synthesis (IGF receptor, Akt and mTOR phosphate/total) and degradation (MuRF1, MAFbx1, FOXO1), mRNA and protein expression analyzed respectively plantaris and extensor digitorum longus. Blood parameters of lactate, insulin, IGF1, and glucose were measured.ResultsSkeletal muscle weight tended to higher Sed/Lac than Sed/Con, but Sed/Lac had significantly supplement effects compared to Sed/Con in only gastrocnemius weight (p = .000). Blood IGF1 showed that Sed/Lac was significantly lower than Sed/Con (p = .039) and Ex/Lac (p = .007). mRNA of IGF receptor had only exercise effects (p = .000). mRNA of Akt and mTOR showed that Sed/Lac was significantly higher than Sed/Con (respectively, p = .000 and p = .043). Protein expression of MuRF1, Sed/Lac was significantly lower than Sed/Con (p = .046), but exercise training effects was found saline groups (Sed/Con vs Ex/Con; p = .001).ConclusionThis study showed that long‐term lactate administration had positive effects on Akt / mTOR and MuRF1. Especially, it was confirmed that long‐term lactate administration increased skeletal muscle mass in mice. Hence, we suggest that long‐term lactate administration increases skeletal muscle synthesis and that lactate administration can be used as supplement for sarcopenia. Nevertheless, this study did not confirm synergistic effects of lactate administration with exercise training. It is suggested that post‐exercise lactate administration, unlike lactate administration at rest state, affects energy metabolism rather skeletal muscle synthesis. Therefore, future study would be necessary to investigate lactate administration timing with exercise training.

L-carnitine ameliorates the muscle wasting of cancer cachexia through the AKT/FOXO3a/MaFbx axis

BackgroundRecent studies suggest potential benefits of applying L-carnitine in the treatment of cancer cachexia, but the precise mechanisms underlying these benefits remain unknown. This study was conducted to determine the mechanism by which L-carnitine reduces cancer cachexia.MethodsC2C12 cells were differentiated into myotubes by growing them in DMEM for 24 h (hrs) and then changing the media to DMEM supplemented with 2% horse serum. Differentiated myotubes were treated for 2 h with TNF-α to establish a muscle atrophy cell model. After treated with L-carnitine, protein expression of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K was determined by Western blotting. Then siRNA-Akt was used to determine that L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx. In vivo, the cancer cachexia model was established by subcutaneously transplanting CT26 cells into the left flanks of the BALB/c nude mice. After treated with L-carnitine, serum levels of IL-1, IL-6 and TNF-α, and the skeletal muscle content of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K were measured.ResultsL-carnitine increased the gastrocnemius muscle (GM) weight in the CT26-bearing cachexia mouse model and the cross-sectional fiber area of the GM and myotube diameters of C2C12 cells treated with TNF-α. Additionally, L-carnitine reduced the protein expression of MuRF1, MaFbx and FOXO3a, and increased the p-FOXO3a level in vivo and in vitro. Inhibition of Akt, upstream of FOXO3a, reversed the effects of L-carnitine on the FOXO3a/MaFbx pathway and myotube diameters, without affecting FOXO3a/MuRF-1. In addition to regulating the ubiquitination of muscle proteins, L-carnitine also increased the levels of p-p70S6K and p70S6K, which are involved in protein synthesis. Akt inhibition did not reverse the effects of L-carnitine on p70S6K and p-p70S6K. Hence, L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx and p70S6K pathways. Moreover, L-carnitine reduced the serum levels of IL-1 and IL-6, factors known to induce cancer cachexia. However, there were minimal effects on TNF-α, another inducer of cachexia, in the in vivo model.ConclusionThese results revealed a novel mechanism by which L-carnitine protects muscle cells and reduces inflammation related to cancer cachexia.

Effects of Geniposide and Geniposidic Acid on Fluoxetine-Induced Muscle Atrophy in C2C12 Cells

Fluoxetine, an antidepressant known as a selective 5-hydroxytryptamine reuptake inhibitor (SSRI), can cause side effects such as muscle atrophy with long-term use, but the mechanism is not fully understood. Geniposide (GPS) and geniposidic acid (GPSA), the main components of Gardenia jasminoides fruit, have been shown to have biological activity in disease prevention, but their role in preventing FXT-related side effects such as muscle atrophy remains unclear. The process of muscle atrophy is a complex physiological mechanism involving the balance of protein synthesis and catabolism. In this study, we hypothesized that FXT may suppress hypertrophy signaling and activate the atrophy mechanisms, resulting in proteolysis and reduced protein synthesis, while geniposide (GPS) and geniposide acid (GPSA) may be beneficial in improving muscle weakness caused by FXT. The C2C12 cell model was used to examine the expression of hypertrophy signaling (PI3K, Akt, and mTOR) and protein break signals (FOXO, MuRF-1, and MyHC). Our data indicated that FXT inhibited MyHC and promoted MuRF-1 protein expression by downregulating the signaling pathways of p-ERK1/2, p-Akt, p-mTOR, and p-FOXO, resulting in a decrease in differentiation and myotube formation in C2C12 muscle cells, which further resulted in muscle atrophy. However, GPS and GPSA can positively regulate the atrophy mechanism induced by FXT in muscle cells, thereby ameliorating the imbalance in muscle synthesis. In conclusion, GPS and GPSA have the potential to attenuate the muscle loss caused by long-term FXT administration, diseases, or the aging process.

Wheel running improves fasting-induced AMPK signaling in skeletal muscle from tumor-bearing mice.

Disruptions to muscle protein turnover and metabolic regulation contribute to muscle wasting during the progression of cancer cachexia. The initiation of cachexia is also associated with decreased physical activity. While chronic muscle AMPK activation occurs during cachexia progression in Apc Min /+ (MIN) mice, a preclinical cachexia model, the understanding of muscle AMPK’s role during cachexia initiation is incomplete. Therefore, we examined if voluntary wheel exercise could improve skeletal muscle AMPK signaling in pre‐cachectic MIN mice. Next, we examined muscle AMPK’s role in aberrant catabolic signaling in response to a 12‐h fast in mice initiating cachexia. Male C57BL/6 (B6: N = 26) and MIN (N = 29) mice were subjected to ad libitum feeding, 12‐h fast, or 4 wks. of wheel access and then a 12‐h fast during the initiation of cachexia. Male tamoxifen‐inducible skeletal muscle AMPKα 1 α 2 (KO) knockout mice crossed with Apc Min /+ and floxed controls were examined (WT: N = 8, KO: N = 8, MIN: N = 10, MIN KO: N = 6). Male mice underwent a 12‐h fast and the gastrocnemius muscle was analyzed. MIN gastrocnemius mass was reduced compared to B6 mice. A 12‐h fast induced MIN muscle AMPKT172, FOXOS413, and ULK‐1S555 phosphorylation compared to B6. Wheel running attenuated these inductions. A 12‐h fast induced MIN muscle MuRF‐1 protein expression compared to B6 and was suppressed by wheel running. Additionally, fasting induced muscle autophagy signaling and disrupted mitochondrial quality protein expression in the MIN, which was prevented in the MIN KO. We provide evidence that increased skeletal muscle AMPK sensitivity to a 12‐h fast is an adverse event in pre‐cachectic MIN mice, and exercise can improve this regulation.

Ubiquitin-proteasome-system and enzymes of energy metabolism in skeletal muscle of patients with HFpEF and HFrEF.

BackgroundSkeletal muscle (SM) alterations contribute to exercise intolerance in heart failure patients with preserved (HFpEF) or reduced (HFrEF) left ventricular ejection fraction (LVEF). Protein degradation via the ubiquitin‐proteasome‐system (UPS), nuclear apoptosis, and reduced mitochondrial energy supply is associated with SM weakness in HFrEF. These mechanisms are incompletely studied in HFpEF, and a direct comparison between these groups is missing.Methods and resultsPatients with HFpEF (LVEF ≥ 50%, septal E/e′ > 15 or >8 and NT‐proBNP > 220 pg/mL, n = 20), HFrEF (LVEF ≤ 35%, n = 20) and sedentary control subjects (Con, n = 12) were studied. Inflammatory markers were measured in serum, and markers of the UPS, nuclear apoptosis, and energy metabolism were determined in percutaneous SM biopsies. Both HFpEF and HFrEF showed increased proteolysis (MuRF‐1 protein expression, ubiquitination, and proteasome activity) with proteasome activity significantly related to interleukin‐6. Proteolysis was more pronounced in patients with lower exercise capacity as indicated by peak oxygen uptake in per cent predicted below the median. Markers of apoptosis did not differ between groups. Mitochondrial energy supply was reduced in HFpEF and HFrEF (complex‐I activity: −31% and −53%; malate dehydrogenase activity: −20% and −29%; both P < 0.05 vs. Con). In contrast, short‐term energy supply via creatine kinase was increased in HFpEF but decreased in HFrEF (47% and −45%; P < 0.05 vs. Con).ConclusionsSimilarly to HFrEF, skeletal muscle in HFpEF is characterized by increased proteolysis linked to systemic inflammation and reduced exercise capacity. Energy metabolism is disturbed in both groups; however, its regulation seems to be severity‐dependent.

A New Muscle Disuse Model Using A Removable Tissue-engineering Muscle

PURPOSE: Physical inactivity and subsequent skeletal muscle disuse may cause muscle atrophy, which is associated with a reduction in muscle function, physical performance, quality of life, and loss of independence, particularly in elderly. Traditionally, 2D muscle cell cultures (e.g. C2C12) have been used to study biological processes and identify and validate pharmacological and/or nutritional compounds to treat skeletal muscle disease including muscle atrophy. However, cell culture studies basically use a pharmacological method to induce muscle atrophy, and not an actual muscle disuse induced by a reduction of mechanical stress (i.e., mechanical unloading). Previously Nakamura et al. (2017) developed 3D engineered muscle (OITem: Osaka institute technology engineered muscle) with artificial tendons at both ends of the muscle, which is removable, thereby allowing us to mechanically release muscle tension by taking off (removing) one side of the muscle. Therefore, the purpose of the present study was to investigate whether removable tissue-engineering muscle effectively induce muscle atrophy, and thus is useful for muscle disuse model. METHODS: OITem was made from C2C12 skeletal muscle cells and a cold type-I collagen gel and placed between two artificial tendons (Nakamura et al., 2017). OITem was differentiated for 2-weeks and then divided into two groups: 1-week stretched on control group (CON: n = 6) in which the both ends of the muscle fixed with tendons, and 1-week stretched off group (OFF: n = 6) in which the one side of the muscle was removed. We analyzed expressions of muscle atrophy-related proteins MuRF-1 and Atrogin-1, and myogenesis-related protein myogenin using Western blotting. As well, we analyzed expression of MHC-fast or -slow (fast- or slow-twitch muscle fiber specific myosin heavy chain (MHC), respectively). RESULTS: The protein expression of MuRF-1 and myogenin was significantly lower in the OFF than those in the CON (P < 0.05). Moreover, the protein expression of MHC-fast and -slow was significantly lower in the OFF than those in the CON (P < 0.01). However, the protein expression of Atrogin-1 did not differ between two groups. CONCLUSIONS: The present findings suggest that OITem may be useful model for muscle disuse.

840 Myostatin Deficiency Inhibits Muscle Wasting and Improves Bacterial Clearance and Survival in Septic Mice

Abstract Introduction Sepsis is a leading cause of the mortality of burn patients. Muscle wasting is a major complication of sepsis and burn injury and negatively affects clinical outcomes of patients with sepsis and burn injury. Myostatin (MSTN) is a myokine that causes muscle atrophy by activating the activin type 2 receptor (ActRII) pathway and myostatin deficiency increases skeletal muscle mass. However, a role of myostatin in critical illness (e.g., sepsis, burn injury)-induced muscle wasting has not yet been investigated. Moreover, a role of muscle wasting in immune suppression and mortality in sepsis is unknown. Methods Sepsis was induced by cecum ligation and puncture (CLP) in male MSTN-deficient mice at 8 weeks of age and age- and body weight (BW)-matched wild type (WT) mice. Survival was monitored for 14 days. Bacterial clearance was evaluated at 16 h after CLP by measuring bacterial load in the peritoneal cavity and circulation. The weight of gastrocnemius (GC), tibialis anterior (TA) and soleus (SOL) muscle and cross-sectional areas of GC were measured before and at 14 days after CLP. To evaluate neutrophil organ infiltration, myeloperoxidase (MPO) activity was measured in the liver and kidney at 16 h after CLP. To evaluate liver dysfunction, acute kidney injury and inflammatory response, plasma levels of AST, ALT, NGAL and high mobility group box 1 (HMGB1) were measured at 16 h after CLP. Protein expression of Murf-1 and Atrogin-1, key players in muscle wasting, and ActRIIB was evaluated in GC muscle by immunoblotting at 3 days and 16 h after CLP, respectively. Results MSTN deficiency increased skeletal muscle mass and inhibited sepsis-induced muscle wasting compared with BW-matched WT mice. Sepsis-induced increase in Murf-1, but not Atrogin-1, expression in muscle was attenuated by MSTN deficiency. CLP increased protein expression of ActRIIB in muscle. Moreover, MSTN deficiency reduced mortality of septic mice compared with age- and BW-matched WT mice. MSTN deficiency improved bacterial clearance and ameliorated increases in MPO activity in the liver and kidney and plasma concentrations of AST, ALT, NGAL and HMGB1 in septic mice. Conclusions Our data showed that MSTN deficiency inhibited muscle wasting and improved bacterial clearance and survival in septic mice. These data indicate that MSTN plays an important role in sepsis-induced muscle wasting. Moreover, our findings suggest that muscle wasting may not be just a complication of sepsis but a driver of sepsis development contributing to mortality of septic mice. This study also raises the possibility that muscle wasting and/or activation of the MSTN-ActRII pathway may exacerbate sepsis-induced immune dysfunction. Applicability of Research to Practice Our study identifies the MSTN-ActRII pathway as a novel, potential molecular target to ameliorate muscle wasting and improve survival of septic burned patients.

The effect of vitamin D on sarcopenia depends on the level of physical activity in older adults.

ObjectiveSarcopenia in older adults is closely related to vitamin D deficiency and reduced levels of physical activity, but little has been reported on the interaction between physical activity and the positive effects of vitamin D. The purpose of this study was to explore the interactive effect of vitamin D and physical activity on muscle mass and function through animal experiments and population surveys.MethodsMale 4‐week‐old C57BL/6J mice were fed different purified diets: a vitamin D‐deficient diet (with increased calcium and phosphorus to prevent the effects of abnormal mineral levels on muscle) or a 1,25‐dihydroxyvitamin D3 (1,25D)‐supplemented diet. After 24 weeks on the assigned diets, the mice were immobilized. The level of skeletal muscle atrophy in the mice was determined by grip strength, gastrocnemius (GA) muscle mass and muscle fiber cross‐sectional area (CSA); additionally, the protein expression levels of FOXO3a and the E3 ubiquitin ligases MuRF1 and MAFbx were detected. A cross‐sectional study included data from 4139 older adults (64.9% women, 67.9 ± 6.7 years) as part of a survey in Shenyang, Northeast China. The associations of serum 25(OH)D3 and physical activity with timed up and go test (TUG) performance, handgrip strength, calf circumference, and body muscle mass were assessed by a linear regression analysis that was adjusted for covariates.ResultsIn activity‐limited mice, vitamin D deficiency accelerated the decrease in GA muscle weight, muscle fiber CSA, and grip strength and increased the protein expression of MuRF1, MAFbx, and FOXO3a (all P < 0.05). In addition, 1,25D supplementation may inhibit the grip‐strength reduction induced by limited activity (P = 0.069). Serum 25(OH)D3 and physical activity were linearly related to TUG time (P < 0.001) and handgrip strength (P < 0.05) after adjustment for sex, age, body mass index (BMI), education level, smoking status, and serum calcium level. Serum 25(OH)D3 and physical activity had interactive effects on TUG (P < 0.001) and handgrip strength (P < 0.05) but not calf circumference or body muscle mass in older adults.ConclusionsThe effect of vitamin D on muscle strength and physical performance depends on physical activity level in the elderly. It is recommended that older adults strive to avoid both physical inactivity and vitamin D deficiency. Because physical inactivity and vitamin D deficiency may exacerbate muscle atrophy, the biological mechanism may involve synergistic effects of vitamin D and physical activity on the promotion of muscle protein ubiquitination and degradation.

Immobilization Decreases FOXO3a Phosphorylation and Increases Autophagy-Related Gene and Protein Expression in Human Skeletal Muscle

Immobilization of the lower limbs promotes a catabolic state that reduces muscle mass, whereas physical training promotes an anabolic state that increases muscle mass. Understanding the molecular mechanisms underlying this is of clinical interest, as loss of muscle mass is a major complication to critical illness in humans. To determine the molecular regulation of protein synthesis and degradation during muscle loss and hypertrophy, we examined skeletal muscle biopsies from healthy human subjects after 2 weeks unilateral immobilization of a lower limb and during 6 weeks of physical rehabilitation. We have previously shown that cross-sectional area of the knee muscle-extensors decreased by ∼10% during immobilization and was completely restored during rehabilitation. Here we provide novel data to suggest that autophagy is an important underlying mechanism involved in regulation of muscle mass. Protein expression of MuRF1 and ATROGIN-1 did not change during the study, indicating that the recruitment of substrates to the proteasomes was unaltered. Phosphorylation of mTORat Ser2448 did not change during the study, and neither did phosphorylation of the mTORC1 substrates 4EBP1 Thr37/46 and p70S6K Thr389, suggesting that this pathway does not suppress protein synthesis during muscle wasting. Protein levels of p62 and ULK1 increased during immobilization and returned to baseline levels during rehabilitation. Same pattern was observed for FOXO3a phosphorylation at Ser318/321, suggesting transcriptional activation during immobilization and inactivation during rehabilitation. To investigate this further, we analyzed mRNA expression of seven autophagy-related genes controlled by FOXO3a. Five of these (p62, LC3B, BECLIN-1, ATG12, and BNIP3) increased during immobilization and returned to baseline during rehabilitation. In conclusion, immobilization of a lower limb increases autophagy-related gene and protein expression in human skeletal muscle in a pattern that mirrors FOXO3a phosphorylation. These findings could imply that FOXO3a dependent transcriptional regulation of autophagy is involved in the regulation of muscle mass in humans.Clinical Trial RegistrationThe study was approved by the Ethics Committee of Copenhagen (j.no. H-1-2010-016).

AHCC Supplementation Attenuates Muscle Atrophy via Akt Activation in Hindlimb-suspended Rat

We investigated the inhibitory effect of active hexose correlated compound (AHCC) on muscle atrophy in hindlimb-suspended rats. Twenty-four six-week-old male Sprague-Dawley rats were randomly divided into three groups: the control sedentary group (CS, n = 8), the hindlimb-suspended group (HS, n = 8), and the hindlimb-suspended and AHCC-supplemented group (HSA, n = 8). Hindlimb suspension and AHCC supplementation were performed for two weeks. The HSA group was treated with AHCC (1 g/1 kg of body weight (BW)) orally in 0.3 mL of PBS solution, while the HS group received the vehicle (PBS solution) only. After two weeks, the cross-sectional area (CSA) of the HS and HSA groups decreased by approximately 36% (p < 0.05) and 19%, respectively, compared to the CS group. In addition, myonuclear numbers of the HS and HSA groups and the extensor digitorum longus (EDL) muscle weight of the HS group decreased 30% (p < 0.05) and 18%, respectively, compared to the CS group. AHCC supplementation increased the phosphorylation of pAkt/Akt in the HSA group compared to the HS group (p < 0.05). Furthermore, Fbx32 and MuRF1 protein expression in the HSA group recovered to the level of the CS group. Based on these results, AHCC supplementation may have a positive role in the prevention of muscle atrophy via Akt activation in hindlimb-suspended rats.

PO-242 Effects of tail suspension on the expression of FNDC5/Irisin protein in rat skeletal muscle

Objective Irisin is a myokine secreted by skeletal muscle,and it is a type I membrane protein factor encoded by the protein 5(FNDC5) gene after cleavage and modification of the type III fibronectin component.Dependence of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α).In this study, the potential association between skeletal muscle atrophy and irisin was explored by detecting changes in rat soleus and gastrocnemius irisin-related proteins during unloading.&#x0D; Methods Twenty male 8-week rats were randomly divided into control group C (n=10) and suspension group T (n=10). The tail suspension system (TSS) was used to perform a 2-week tail suspension experiment on the T group. Two weeks after the tail suspension test, the weights of the rats and the wet weights of soleus and gastrocnemius muscles were measured. HE staining was performed under light microscope to observe the changes of muscle fiber area of skeletal muscle in each group. Western-blot was used to detect the protein expression of MURF1, PGC-1α and FNDC5 in soleus muscle and gastrocnemius muscle of each group.&#x0D; Results (1) The soleus muscle and gastrocnemius muscle mass in T group decreased by 28.6% (P&lt;0.05) and 25.8% (P&lt;0.01), respectively. (2) The cross-sectional area of soleus muscle and gastrocnemius muscle fiber in T group decreased by 20.5% (P&lt;0.01) and 25.2% (P&lt;0.05), respectively. (3) The MURF1 protein expression in the gastrocnemius muscle and soleus muscle in the T group was significantly higher than that in the C group (P&lt;0.01). (4) The expression of PGC-1α protein in gastrocnemius muscle and soleus muscle of T group was significantly lower than that in group C (P&lt;0.05). (5) The expression of FNDC5 protein in gastrocnemius muscle and soleus muscle in T group was significantly lower than that in group C (P&lt;0.05).&#x0D; Conclusions After sole tail suspension for two weeks, the soleus and gastrocnemius muscles of the rats were obviously atrophied, and soleus muscle atrophy was more obvious. Skeletal muscle atrophy may be related to increased expression of MURF1. The decrease of FNDC5/Irisin content may be related to the occurrence of skeletal muscle atrophy, and PGC-1α also may be involved in this process.

PL - 027 Up-regulation of NRG1 improves cardiac repair in zebra fish and involved in the cardioprotective effects of exercise training in rats of myocardial infarction

Objective Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. Exercise training could improve cardiac function following MI. However, the mechanisms are still not well-known. Neuregulin 1 (NRG1)plays an important role in heart development and regeneration.In this study, we investigated the effect of NRG1 on cardiac regeneration in a zebrafish model, detected whether exercise could improve cardiac function through regulating NRG1 expression in infarcted heart and explore the possible role of up-regulation of NRG1 in skeletal muscle play in the cardioprotective effects in rats with MI.&#x0D; Methods Transgenic zebrafish line, cmlc2:CreERandβ-act2:BSNrg1,wereusedto study the effect of NRG1 on heart growth and regeneration after injury. PCNA was detected by immunofluorescence staining andmRNAexpression of gata4, nkx2.5, tbx5, smyd1b, hsp90α and murf were tested by RT-PCR.Sprague-Dawley rats were used to establish MI model and underwent fourweeks of exercise training (ET) or pAAV-{dMCK promoter}rNRG1-eGFP intervention.AG1478 was used asan inhibitor of NRG1/ErbBs signaling pathway. Cardiac function and structure,cardiomyocyte proliferation and NRG1 expression were detected in the heart or skeletal.&#x0D; Results Cardiac-specific overexpression of NRG1 induced cardiac hypertrophy and cardiomyocyte proliferation, regulated the mRNA expression of gata4, nkx2.5, tbx5, smyd1b, hsp90α andmurf in uninjuriedzebrafish, and promote cardiac repair and regeneration after injury in the zebrafish.Exercise activated NRG1/ErbBs signaling pathway, improved cardiac remodeling and heart function, enhanced cardiomyocyte proliferation, reduced cardiomyocyte apoptosis, ROS level and MuRF1 protein expression in rats with MI. BlockingErbB signaling attenuated the ET-induced cardioprotection effects in rat with MI.up-regulation of NRG1 expression in skeletal muscle could increase the protein level of NRG1 in serum and infarcted heart, improve cardiomyocyte proliferation and reduce the level of cardiac fibrosis, finally promote cardiac function.&#x0D; Conclusions Up-regulation of NRG1 expression in the heart or skeletal musclemay be one of the underlying mechanisms of thebeneficial effects of exercise training following MI.

Muscle-specific activation of calpain system in hindlimb unloading rats and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse.

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.

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.

Resistance exercise-induced S6K1 kinase activity is not inhibited in human skeletal muscle despite prior activation of AMPK by high-intensity interval cycling.

Combining endurance and strength training in the same session has been reported to reduce the anabolic response to the latter form of exercise. The underlying mechanism, based primarily on results from rodent muscle, is proposed to involve AMPK-dependent inhibition of mTORC1 signaling. This hypothesis was tested in eight trained male subjects who in randomized order performed either resistance exercise only (R) or interval cycling followed by resistance exercise (ER). Biopsies taken from the vastus lateralis before and after endurance exercise and repeatedly after resistance exercise were assessed for glycogen content, kinase activity, protein phosphorylation, and gene expression. Mixed muscle fractional synthetic rate was measured at rest and during 3 h of recovery using the stable isotope technique. In ER, AMPK activity was elevated immediately after both endurance and resistance exercise (∼90%, P < 0.05) but was unchanged in R. Thr(389) phosphorylation of S6K1 was increased severalfold immediately after exercise (P < 0.05) in both trials and increased further throughout recovery. After 90 and 180 min recovery, S6K1 activity was elevated (∼55 and ∼110%, respectively, P < 0.05) and eukaryotic elongation factor 2 phosphorylation was reduced (∼55%, P < 0.05) with no difference between trials. In contrast, markers for protein catabolism were differently influenced by the two modes of exercise; ER induced a significant increase in gene and protein expression of MuRF1 (P < 0.05), which was not observed following R exercise only. In conclusion, cycling-induced elevation in AMPK activity does not inhibit mTOR complex 1 signaling after subsequent resistance exercise but may instead interfere with the hypertrophic response by influencing key components in protein breakdown.

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets x 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNFalpha protein expression, and IKK(Ser180/181) and p38MAPK(Thr180/Tyr182) phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNFalpha and IKK(Ser180/181). There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). Akt(Ser473) and mTOR(Ser2448) phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k(Thr389) increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6(Ser235/236) increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.

Catabolic Changes In Skeletal Muscle In Chronic Heart Failure And Aging - Effects Of Aerobic Exercise Training

BACKGROUND: After the initial event leading to left ventricular dysfunction symptom progression and prognosis of chronic heart failure (CHF) are also related to secondary peripheral alterations such as skeletal muscle atrophy. However, the molecular basis of muscle wasting is still incompletely understood and pharmacological interventions are not yet available. We therefore analysed the activation of the proteolytic ubiquitin-proteasome pathway in the skeletal muscle of CHF patients and healthy subjects (HS) by measuring expression of a muscle specific E3-ligase (MuRF-1) and compared the response of MuRF-1 expression to a 4 week training program in old (>65 years) and young (<55 years) subjects. METHODS: A total of 53 CHF-patients and 57 HS were randomized to 4 weeks of ergometer training at 60-70% of the heart rate reserve 4 times 20 min/day or to control. Before and after training a cardiopulmonary exercise test, and an echocardiogram were performed, and a skeletal muscle biopsy from the vastus lateralis muscle was obtained. We quantified MuRF-1 m-RNA expression by real-time PCR (standardized for 18S-rRNA) and protein expression by Western blot. RESULTS: After training in CHF patients (age 60.8±2.7 years, LV-EF 27.8±1.2%) VO2 max increased from 14.7±3.0 to 18.2±4.5 ml/min/kg (p<0.01 versus control), and LV-EF from 27.3±4.2 to 32.1±5.0% (p=0.001 versus control). At baseline skeletal muscle MuRF-1 expression in CHF was significantly higher versus HS. After training MuRF-1 mRNA expression decreased by 26±8% and Murf-1 protein expression by 23±5% (both p<0.05 versus control). The relative reduction of MuRF-1 mRNA expression was more pronounced in patients >65 years (-46.2±5.3%) as compared to those <55 years (-13.5±9.1%, p<0.05). CONCLUSIONS: CHF is associated with activation of the catabolic ubiquitin-proteasome system in the skeletal muscle. Four weeks of ergometer training significantly reduced the elevated MuRF-1 expression by 29%. Surprisingly, the anticatabolic training effects were augmented among older CHF patients. These findings extend our current understanding of how physical activity readjusts the catabolic-anabolic balance in CHF in different age groups.