Muscle RING-finger Protein-1 Research Articles

Small molecule adiponectin receptor agonist GTDF protects against skeletal muscle atrophy

Skeletal muscle atrophy is a debilitating response to several major diseases, muscle disuse and chronic steroid treatment for which currently no therapy is available. Since adiponectin signaling plays key roles in muscle energetics, we assessed if globular adiponectin (gAd) or the small molecule adiponectin mimetic 6-C-β-D-glucopyranosyl-(2S,3S)-(+)-5,7,3′,4′-tetrahydroxydihydroflavonol (GTDF) could ameliorate muscle atrophy. Both GTDF and gAd induced C2C12 myoblast differentiation. GTDF and gAd effectively prevented reduction in myotube area and suppressed the expressions of atrophy markers; atrogin-1 and muscle ring finger protein-1 (MuRF1) in models of steroid, cytokine and starvation –induced muscle atrophy. The protective effects of GTDF and gAd were routed through AMPK and AKT activation and thereby stimulation of PPAR gamma coactivator 1α and inhibition of forkhead box O transcription factors. Finally, GTDF and gAd mitigated dexamethasone-induced muscle atrophy in vivo. Together, our results demonstrate that activating adiponectin signaling may be an effective therapeutic strategy against skeletal muscle atrophy.

The study on mechanism of the modified Chinese herbal compound, jianpijiedu, on a mouse model of hepatic carcinoma cachexia

Various studies have investigated hepatic carcinoma cachexia, however, there is little published information regarding the effect of Chinese Medicine carcinoma cachexia. The present study was performed to investigate the effect of modified Chinese herbal compound jianpijiedu (MJPJD) on a mouse model of ascites-induced hepatic carcinoma cachexia. C57BL/6 mice were randomized to five groups: Control (Group A); xenograft tumor (Group B); low concentration of MJPJD (Group C); high concentration of MJPJD (Group D) and medroxyprogesterone (MPA) combined with indometacin (IND; Group E). The mouse model of ascites-induced hepatic carcinoma cachexia was established by abdominal injection of H22 hepatic carcinoma cells. Subsequently, the body weight, food intake and gastrocnemius weight were recorded, and the levels of interleukin (IL)-lα, IL-6, tumor necrosis factor-α (TNF-α) in ascites were detected by enzyme-linked immunosorbent assay. The protein expression levels of muscle RING-finger protein-1 (MU-RF1) and atrogin 1 were detected by western blotting and immunohistochemistry, and the mRNA levels in gastrocnemius were detected by reverse transcription-quantitative polymerase chain reaction. Compared with the xenograft tumor group, the administration of MJPJD inhibited the increase in body weight and the volume of ascites, the consumption of gastrocnemius was reduced, the net weight of ascites was maintained, the food intake was enhanced and the levels of the cytokines IL-lα, IL-6, TNF-α in ascites and the levels of MU-RF1 and atrogin 1 proteins were reduced. These results indicated that MJPJD delays the pathological process of ascites-induced hepatic carcinoma cachexia, and the mechanism of action may be correlated with a reduction in the levels of IL-lα, IL-6, TNF-α and inhibiting the activation of the ubiquitin proteosome pathway.

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy

Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.

Lung injury-induced skeletal muscle wasting in aged mice is linked to alterations in long chain fatty acid metabolism.

Older patients are more likely to acquire and die from acute respiratory distress syndrome (ARDS) and muscle weakness may be more clinically significant in older persons. Recent data implicate muscle ring finger protein 1 (MuRF1) in lung injury-induced skeletal muscle atrophy in young mice and identify an alternative role for MuRF1 in cardiac metabolism regulation through inhibition of fatty acid oxidation. To develop a model of lung injury-induced muscle wasting in old mice and to evaluate the skeletal muscle metabolomic profile of adult and old acute lung injury (ALI) mice. Young (2 month), adult (6 month) and old (20 month) male C57Bl6J mice underwent Sham (intratracheal H2O) or ALI [intratracheal E. coli lipopolysaccharide (i.t. LPS)] conditions and muscle functional testing. Metabolomic analysis on gastrocnemius muscle was performed using gas chromatography-mass spectrometry (GC-MS). Old ALI mice had increased mortality and failed to recover skeletal muscle function compared to adult ALI mice. Muscle MuRF1 expression was increased in old ALI mice at day 3. Non-targeted muscle metabolomics revealed alterations in amino acid biosynthesis and fatty acid metabolism in old ALI mice. Targeted metabolomics of fatty acid intermediates (acyl-carnitines) and amino acids revealed a reduction in long chain acyl-carnitines in old ALI mice. This study demonstrates age-associated susceptibility to ALI-induced muscle wasting which parallels a metabolomic profile suggestive of altered muscle fatty acid metabolism. MuRF1 activation may contribute to both atrophy and impaired fatty acid oxidation, which may synergistically impair muscle function in old ALI mice.

Vitamin D deficiency aggravates diabetes-induced muscle wasting in female mice.

We recently reported that vitamin D deficiency aggravates diabetic bone loss in mice. Although vitamin D affects both muscle and bone, the role of the vitamin D state in diabetic muscle loss and muscle-bone relationships remains unclear. In the present study, we examined the effects of vitamin D deficiency on muscle mass, muscle differentiation and muscle-derived humoral factors linking muscle to bone in diabetic female mice. Diabetes was induced in mice by streptozotocin (STZ) injection after feeding with a normal or vitamin D-deficient diet for 6weeks. Quantitative computed tomography analysis showed that tibial muscle mass was significantly decreased in diabetic mice compared with control mice 4weeks after induction of diabetes. Vitamin D deficiency accelerated muscle loss in diabetic mice. Vitamin D deficiency augmented the decreases in Pax7 mRNA levels and the increases in muscle RING-Finger Protein-1 and atrogin-1 mRNA levels induced by diabetes in the gastrocnemius muscle of mice. Moreover, vitamin D deficiency decreased the mRNA levels of insulin-like growth factor-1, fibroblast growth factor-2 and osteoglycin in muscle of diabetic mice. In conclusion, we demonstrated that vitamin D deficiency aggravates muscle loss induced by diabetes in female mice. Vitamin D may exert significant effects on the maintenance of the musculoskeletal system partly through the muscle-bone relationships in diabetic state.

Consequences of Late-Stage Non–Small-Cell Lung Cancer Cachexia on Muscle Metabolic Processes

IntroductionThe loss of muscle is common in patients with advanced non–small-cell lung cancer (NSCLC) and contributes to the high morbidity and mortality of this group. The exact mechanisms behind the muscle loss are unclear. Patients and MethodsTo investigate this, 4 patients with stage IV NSCLC who met the clinical definitions for sarcopenia and cachexia were recruited, along with 4 age-matched healthy volunteers. After an overnight fast, biopsy specimens were obtained from the vastus lateralis, and the key components associated with inflammation and the control of muscle protein, carbohydrate, and fat metabolism were assessed. ResultsCompared with the healthy volunteers, significant increases in mRNA levels for interleukin-6 and NF-κB signaling and lower intramyocellular lipid content in slow-twitch fibers were observed in NSCLC patients. Although a significant decrease in phosphorylation of the mechanistic target of rapamycin (mTOR) signaling protein 4E-BP1 (Ser65) was observed, along with a trend toward reduced p70 S6K (Thr389) phosphorylation (P = .06), no difference was found between groups for the mRNA levels of MAFbx (muscle atrophy F box) and MuRF1 (muscle ring finger protein 1), chymotrypsin-like activity of the proteasome, or protein levels of multiple proteasome subunits. Moreover, despite decreases in intramyocellular lipid content, no robust changes in mRNA levels for key proteins involved in insulin signaling, glycolysis, oxidative metabolism, or fat metabolism were observed. ConclusionThese findings suggest that examining the contribution of suppressed mTOR signaling in the loss of muscle mass in late-stage NSCLC patients is warranted and reinforces our need to understand the potential contribution of impaired fat metabolism and muscle protein synthesis in the etiology of cancer cachexia.

Insulin modulates energy and substrate sensing and protein catabolism induced by chronic peritonitis in skeletal muscle of neonatal pigs.

Acute infection promotes skeletal muscle wasting and insulin resistance, but the effect of insulin on energy and substrate sensing in skeletal muscle of chronically infected neonates has not been studied. Eighteen 2-d-old pigs underwent cecal ligation and puncture (CLP) or sham surgery (CON) to induce a chronic infection for 5 d. On d 5, pancreatic-substrate clamps were performed to attain fasting or fed insulin levels but to maintain glucose and amino acids in the fasting range. Total fractional protein synthesis rates (Ks), translational control mechanisms, and energy sensing and degradation signal activation were measured in longissimus dorsi muscle. In fasting conditions, CLP reduced Ks and sirtuin 1 (SIRT1) and increased AMP-activated protein kinase α (AMPKα) activation and muscle RING-finger protein-1 (MuRF1). Insulin treatment increased Ks and mitochondrial protein synthesis, enhanced translation activation, and reduced SIRT1 in CON. In contrast, in CLP, insulin treatment increased Ks, protein kinase B (PKB) and Forkhead box O1 phosphorylation, antagonized AMPK activation, and decreased peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), MuRF1, and SIRT1. Energy and substrate sensing in skeletal muscle by the PKB-AMPK-SIRT1-PGC-1α axis is impacted by chronic infection in neonatal pigs and can be modulated by insulin.

Glucocorticoids and 11β-HSD1 are major regulators of intramyocellular protein metabolism.

The adverse metabolic effects of prescribed and endogenous glucocorticoid excess, ‘Cushing’s syndrome’, create a significant health burden. While skeletal muscle atrophy and resultant myopathy is a clinical feature, the molecular mechanisms underpinning these changes are not fully defined. We have characterized the impact of glucocorticoids upon key metabolic pathways and processes regulating muscle size and mass including: protein synthesis, protein degradation, and myoblast proliferation in both murine C2C12 and human primary myotube cultures. Furthermore, we have investigated the role of pre-receptor modulation of glucocorticoid availability by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in these processes. Corticosterone (CORT) decreased myotube area, decreased protein synthesis, and increased protein degradation in murine myotubes. This was supported by decreased mRNA expression of insulin-like growth factor (IGF1), decreased activating phosphorylation of mammalian target of rapamycin (mTOR), decreased phosphorylation of 4E binding protein 1 (4E-BP1), and increased mRNA expression of key atrophy markers including: atrogin-1, forkhead box O3a (FOXO3a), myostatin (MSTN), and muscle-ring finger protein-1 (MuRF1). These findings were endorsed in human primary myotubes, where cortisol also decreased protein synthesis and increased protein degradation. The effects of 11-dehydrocorticosterone (11DHC) (in murine myotubes) and cortisone (in human myotubes) on protein metabolism were indistinguishable from that of CORT/cortisol treatments. Selective 11β-HSD1 inhibition blocked the decrease in protein synthesis, increase in protein degradation, and reduction in myotube area induced by 11DHC/cortisone. Furthermore, CORT/cortisol, but not 11DHC/cortisone, decreased murine and human myoblast proliferative capacity. Glucocorticoids are potent regulators of skeletal muscle protein homeostasis and myoblast proliferation. Our data underscores the potential use of selective 11β-HSD1 inhibitors to ameliorate muscle-wasting effects associated with glucocorticoid excess.

Effects of slow-release urea and rumen-protected methionine and histidine on mammalian target of rapamycin (mTOR) signaling and ubiquitin proteasome-related gene expression in skeletal muscle of dairy cows

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis, whereas the ubiquitin-proteasome system (UPS) is regarded as the main proteolytic pathway in skeletal muscle. The objective of the current study was to investigate the effects of slow-release urea and rumen-protected (RP) Met and His supplementation of a metabolizable protein (MP)-deficient diet on the abundance of key components of the mTOR pathway and of the UPS in skeletal muscle of dairy cows. Sixty Holstein cows were blocked based on days in milk and milk yield and were randomly assigned within block to 1 of 5 diets in a 10-wk experiment (including the first 2 wk as covariate period) as follows: (1) MP-adequate diet (AMP; 107% of MP requirements, based on the National Research Council requirements); (2) MP-deficient diet (DMP; 95% of MP requirements); (3) DMP supplemented with slow-release urea (DMPU); (4) DMPU supplemented with RPMet (DMPUM); and (5) DMPUM supplemented with RPHis (DMPUMH). Muscle biopsies were collected from longissimus dorsi during the last week of the experiment. The mRNA abundance of key mTOR signaling genes was not affected by the treatments. The phosphorylated (P)-mTOR protein was or tended to be greater for DMP compared with DMPU and AMP, respectively. The P-mTOR protein in DMPUMH was decreased when compared against DMPUM. The P-ribosomal protein S6 tended to be increased by DMPUM compared with DMPU. The abundance of total-S6 was or tended to be greater for DMP compared with AMP and DMPU, respectively. The mRNA abundance of ubiquitin activating and conjugating enzymes was not affected by the treatments, whereas that of muscle ring-finger protein 1 (MuRF-1) was greater in DMP than DMPU. The increased abundance of mTOR-associated signaling proteins and MuRF-1 mRNA abundance indicates a higher rate of protein turnover in muscle of DMP-fed cows. The reduced abundance of P-mTOR by supplementation of RPHis may suggest that His is likely partitioned to the mammary gland in favor of milk protein synthesis rather than to the skeletal muscle in dairy cows fed MP-deficient diets.

Amino acids, independent of insulin, attenuate skeletal muscle autophagy in neonatal pigs during endotoxemia.

BACKGROUNDSepsis induces loss of skeletal muscle mass by activating the ubiquitin proteasome (UPS) and autophagy systems. Although muscle protein synthesis in healthy neonatal piglets is responsive to amino acid (AA) stimulation, it is not known if amino acids (AA) can prevent the activation of muscle protein degradation induced by sepsis. We hypothesize that AA attenuate the sepsis-induced activation of UPS and autophagy in neonates.METHODSNewborn pigs were infused for 8 h with liposaccharide (LPS) (0 and 10 µg·kg−1·h−1), while circulating glucose and insulin were maintained at fasting levels; circulating AA were clamped at fasting or fed levels. Markers of protein degradation and AA transporters in longissimus dorsi (LD) were examined.RESULTSFasting AA increased muscle microtubule-associated protein light 1 chain 3 II (LC3-II) abundance in LPS compared to control, while fed AA levels decreased LC3-II abundance in both LPS and controls. There was no effect of AA supplementation on AMP-activated protein kinase, forkhead box O1 and O4 phosphorylation, nor on sodium-coupled neutral AA transporter 2 and light chain AA transporter 1, muscle RING-finger protein-1 and muscle Atrophy F-Box/Atrogin-1 abundance.CONCLUSIONSThese findings suggest that supplementation of AA antagonize autophagy signal activation in skeletal muscle of neonates during endotoxemia.

Retraction: Myostatin is a novel tumoral factor that induces cancer cachexia.

Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFβ (transforming growth factor β) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin–proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy–lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.

Muscle function recovery after acute lung injury is impaired in aged mice, linked in part, to metabolic alterations attributed to MuRF1 by quantitative targeted metabolomics analysis of acyl‐carnitines

Most survivors of acute lung injury suffer from substantial physical complications months after the initial lung injury resolves, and persistent muscle weakness is a very common sequelae of lung injury. Using a mouse model of LPS‐induced lung injury [acute lung injury (ALI) mice], we found that old (20 month) ALI mice failed to recover skeletal muscle function 10 days after injury compared to young (6 month) ALI control mice. Non‐targeted GC‐MS analysis of old ALI gastrocnemius muscle vs. vehicle treated controls identified 18 significantly different metabolites, including the long chain fatty acid linoleic acid (1.75 fold increased), indicative of altered linoleic acid metabolism. Our laboratory recently identified that muscle ring finger protein 1 (MuRF1) inhibits fatty acid oxidation in vivo via mono‐ubiquitination of peroxisome proliferator‐activated receptor alpha. Moreover, we've identified that MuRF1−/− mice are resistant to LPS‐induced muscle atrophy. Western analysis of old and young ALI muscles for MuRF1 found significantly greater expression of MuRF1 3 days following lung injury in old ALI mice, leading us to hypothesize that old ALI muscles had inhibited fatty acid oxidation. We next performed targeted metabolomics analysis of fatty acid intermediates (acyl‐carnitines) and amino acids of the affected muscles. At day 10, we identified 13 significantly altered acyl carnitines (by t‐test, p<0.05) compared to vehicle treated controls (0.27–1.14 fold). We also identified that total long chain acyl‐carnitines were almost decreased by half, paralleling the acyl‐carnitine profile recently published in MuRF1−/− hearts. Together, these studies demonstrate for the first time age‐associated susceptibility of ALI‐induced muscle wasting and characterize the metabolic alterations in fatty acid metabolism, suggesting MuRF1's role in regulating fatty acid metabolism, which may contribute to impaired skeletal muscle functional recovery.Support or Funding InformationThis work was supported by the National Institutes of Health (R01HL104129 to M.W.), the Leducq Foundation Transatlantic Networks of Excellence (to M.W.), the Claude D. Pepper Older Americans Independence Center (P30AG21332 to D.C.F and O.D.), and the American Thoracic Society Foundation (D.C.F.).

Titin, a Central Mediator for Hypertrophic Signaling, Exercise-Induced Mechanosignaling and Skeletal Muscle Remodeling.

Titin is a giant scaffold protein with multiple functions in striated muscle physiology. Due to the elastic I-band domains and the filament-like integration in the half-sarcomere titin is an important factor for sarcomere assembly and serves as an adaptable molecular spring that determines myofilament distensibility. Protein-interactions e.g., with muscle ankyrin repeat proteins or muscle LIM-protein link titin to hypertrophic signaling and via p62 and Muscle Ring Finger proteins to mechanisms that control protein quality control. This review summarizes our current knowledge on titin as a central node for exercise-induced mechanosignaling and remodeling and further highlights the pathophysiological implications.

A Soluble Activin Receptor IIB Fails to Prevent Muscle Atrophy in a Mouse Model of Spinal Cord Injury.

Myostatin (MST) is a potent regulator of muscle growth and size. Spinal cord injury (SCI) results in marked atrophy of muscle below the level of injury. Currently, there is no effective pharmaceutical treatment available to prevent sublesional muscle atrophy post-SCI. To determine whether inhibition of MST with a soluble activin IIB receptor (RAP-031) prevents sublesional SCI-induced muscle atrophy, mice were randomly assigned to the following groups: Sham-SCI; SCI+Vehicle group (SCI-VEH); and SCI+RAP-031 (SCI-RAP-031). SCI was induced by complete transection at thoracic level 10. Animals were euthanized at 56 days post-surgery. RAP-031 reduced, but did not prevent, body weight loss post-SCI. RAP-031 increased total lean tissue mass compared to SCI-VEH (14.8%). RAP-031 increased forelimb muscle mass post-SCI by 38% and 19% for biceps and triceps, respectively (p < 0.001). There were no differences in hindlimb muscle weights between the RAP-031 and SCI-VEH groups. In the gastrocnemius, messenger RNA (mRNA) expression was elevated for interleukin (IL)-6 (8-fold), IL-1β (3-fold), and tumor necrosis factor alpha (8-fold) in the SCI-VEH, compared to the Sham group. Muscle RING finger protein 1 mRNA was 2-fold greater in the RAP-031 group, compared to Sham-SCI. RAP-031 did not influence cytokine expression. Bone mineral density of the distal femur and proximal tibia were decreased post-SCI (-26% and -28%, respectively) and were not altered by RAP-031. In conclusion, MST inhibition increased supralesional muscle mass, but did not prevent sublesional muscle or bone loss, or the inflammation in paralyzed muscle.

F-BOX proteins in cancer cachexia and muscle wasting: Emerging regulators and therapeutic opportunities.

Cancer cachexia is a debilitating metabolic syndrome accounting for fatigue, an impairment of normal activities, loss of muscle mass associated with body weight loss eventually leading to death in majority of patients with advanced disease. Cachexia patients undergoing skeletal muscle atrophy show consistent activation of the SCF ubiquitin ligase (F-BOX) family member Atrogin-1 (also known as MAFBx/FBXO32) alongside the activation of the muscle ring finger protein1 (MuRF1). Other lesser known F-BOX family members are also emerging as key players supporting muscle wasting pathways. Recent work highlights a spectrum of different cancer signaling mechanisms impacting F-BOX family members that feed forward muscle atrophy related genes during cachexia. These novel players provide unique opportunities to block cachexia induced skeletal muscle atrophy by therapeutically targeting the SCF protein ligases. Conversely, strategies that induce the production of proteins may be helpful to counter the effects of these F-BOX proteins. Through this review, we bring forward some novel targets that promote atrogin-1 signaling in cachexia and muscle wasting and highlight newer therapeutic opportunities that can help in the better management of patients with this devastating and fatal disorder.

Dietary HMB and β-alanine co-supplementation does not improve in situ muscle function in sedentary, aged male rats.

This study evaluated the effects of dietary β-hydroxy-β-methylbutyrate (HMB) combined with β-alanine (β-Ala) in sedentary, aged male rats. It has been suggested that dietary HMB or β-Ala supplementation may mitigate age-related declines in muscle strength and fatigue resistance. A total of 20 aged Sprague-Dawley rats were studied. At age 20 months, 10 rats were administered a control, purified diet and 10 rats were administered a purified diet supplemented with both HMB and β-Ala (HMB+β-Ala) for 8 weeks (approximately equivalent to 3 and 2.4 g per day human dose). We measured medial gastrocnemius (MG) size, force, fatigability, and myosin composition. We also evaluated an array of protein markers related to muscle mitochondria, protein synthesis and breakdown, and autophagy. HMB+β-Ala had no significant effects on body weight, MG mass, force or fatigability, myosin composition, or muscle quality. Compared with control rats, those fed HMB+β-Ala exhibited a reduced (41%, P = 0.039) expression of muscle RING-finger protein 1 (MURF1), a common marker of protein degradation. Muscle from rats fed HMB+β-Ala also exhibited a 45% reduction (P = 0.023) in p70s6K phosphorylation following fatiguing stimulation. These data suggest that HMB+β-Ala at the dose studied may reduce muscle protein breakdown by reducing MURF1 expression, but has minimal effects on muscle function in this model of uncomplicated aging. They do not, however, rule out potential benefits of HMB+β-Ala co-supplementation at other doses or durations of supplementation in combination with exercise or in situations where extreme muscle protein breakdown and loss of mass occur (e.g., bedrest, cachexia, failure-to-thrive).

The melanocortin receptor type 3 agonist d-Trp(8)-γMSH decreases inflammation and muscle wasting in arthritic rats.

BackgroundChronic inflammatory diseases induce cachexia that increases mortality and morbidity of the illness. Adjuvant‐induced arthritis is an experimental model of rheumatoid arthritis that is associated with body weight loss and muscle wasting. Alpha‐melanocyte stimulating hormone has an anti‐inflammatory effect in arthritic rats and decreases muscle wasting. The aim of this work was to elucidate whether the anti‐cachectic action of alpha‐melanocyte stimulating hormone is mediated by the melanocortin receptor type 3 pathway.MethodsArthritis was induced in male Wistar rats by intradermal injection of Freund's adjuvant, and 6 days afterwards, arthritic rats were injected with the selective melanocortin receptor type 3 agonist d‐Trp(8)‐gammaMSH ( d‐Trp(8)‐γMSH) 500 µg/kg subcutaneously. or saline twice a day, for 10 days.Results d‐Trp(8)‐γMSH decreased the external signs of inflammation and body weight loss, but it was not able to modify the anorexigenic effect of arthritis or the increase in hypothalamic cyclooxygenase‐2 (COX‐2) expression. In contrast, d‐Trp(8)‐γMSH prevented arthritis‐induced increase in hypothalamic IL‐1β and serum corticosterone levels and the decrease in serum IGF‐I levels. d‐Trp(8)‐γMSH treatment also prevented arthritis‐induced NF‐kB(p65) phosphorylation and tumour necrosis factor‐α mRNA increase in the gastrocnemius. d‐Trp(8)‐γMSH administration to arthritic rats increased gastrocnemius mass, its cross‐sectional area, and mean fast fibre area. Those effects of d‐Trp(8)‐γMSH were associated with a decreased expression of atrogin‐1 and muscle ring‐finger protein‐1 in the gastrocnemius. In rats treated with saline, arthritis increased the expression of autophagy marker genes LC3b, Bnip‐3, and Gabarap1 as well as the conversion of LC3b I to LC3b II by lipidation in the gastrocnemius. d‐Trp(8)‐γMSH decreased gastrocnemius LC3b, Bnip‐3, and Gabarap1 mRNA expression and prevented the increase in LC3b II in arthritic rats.ConclusionThese data suggest that d‐Trp(8)‐γMSH administration prevents the effect of arthritis on corticosterone and insulin‐like growth factor‐I serum levels and decreases muscle wasting, by down‐regulating atrogenes and autophagy through modifying the NF‐kB(p65)/tumour necrosis factor‐α signalling transduction pathway.

Heat shock protein 70 overexpression does not attenuate atrophy in botulinum neurotoxin type A-treated skeletal muscle

Botulinum neurotoxin type A (BoNT/A) is used clinically to induce therapeutic chemical denervation of spastically contracted skeletal muscles. However, BoNT/A administration can also cause atrophy. We sought to determine whether a major proteolytic pathway contributing to atrophy in multiple models of muscle wasting, the ubiquitin proteasome system (UPS), is involved in BoNT/A-induced atrophy. Three and ten days following BoNT/A injection of rat hindlimb, soleus muscle fiber cross-sectional area was reduced 25 and 65%, respectively. The transcriptional activity of NF-κB and Foxo was significantly elevated at 3 days (2- to 4-fold) and 10 days (5- to 6-fold). Muscle RING-finger protein-1 (MuRF1) activity was elevated (2-fold) after 3 days but not 10 days, while atrogin-1 activity was not elevated at any time point. BoNT/A-induced polyubiquitination occurred after 3 days (3-fold increase) but was totally absent after 10 days. Proteasome activity was elevated (1.5- to 2-fold) after 3 and 10 days. We employed the use of heat shock protein 70 (Hsp70) to inhibit NF-κB and Foxo transcriptional activity. Electrotransfer of Hsp70 into rat soleus, before BoNT/A administration, was insufficient to attenuate atrophy. It was also insufficient to decrease BoNT/A-induced Foxo activity at 3 days, although NF-κB activity was abolished. By 10 days both NF-κB and Foxo activation were abolished by Hsp70. Hsp70-overexpression was unable to alter the levels of BoNT/A-induced effects on MuRF1/atrogin-1, polyubiquitination, or proteasome activity. In conclusion, Hsp70 overexpression is insufficient to attenuate BoNT/A-induced atrophy. It remains unclear what proteolytic mechanism/s are contributing to BoNT/A-induced atrophy, although a Foxo-MuRF1-ubiquitin-proteasome contribution may exist, at least in early BoNT/A-induced atrophy. Further clarification of UPS involvement in BoNT/A-induced atrophy is warranted.

The administration of Fructus Schisandrae attenuates dexamethasone-induced muscle atrophy in mice.

In the present study, we aimed to determine whether ethanol extracts of Fructus Schisandrae (FS), the dried fruit of Schizandra chinensis Baillon, mitigates the development of dexamethasone-induced muscle atrophy. Adult SPF/VAT outbred CrljOri:CD1 (ICR) mice were either treated with dexamethasone to induce muscle atrophy. Some mice were treated with various concentrations of FS or oxymetholone, a 17α-alkylated anabolic-androgenic steroid. Muscle thickness and weight, calf muscle strength, and serum creatine and creatine kinase (CK) levels were then measured. The administration of FS attenuated the decrease in calf thickness, gastrocnemius muscle thickness, muscle strength and weight, fiber diameter and serum lactate dehydrogenase levels in the gastrocnemius muscle bundles which was induced by dexamethasone in a dose-dependent manner. Treatment with FS also prevented the dexamethasone-induced increase in serum creatine and creatine kinase levels, histopathological muscle fiber microvacuolation and fibrosis, and the immunoreactivity of muscle fibers for nitrotyrosine, 4-hydroxynonenal, inducible nitric oxide synthase and myostatin. In addition, the destruction of the gastrocnemius antioxidant defense system was also inhibited by the administration of FS in a dose-dependent manner. FS downregulated the mRNA expression of atrogin-1 and muscle RING-finger protein-1 (involved in muscle protein degradation), myostatin (a potent negative regulator of muscle growth) and sirtuin 1 (a representative inhibitor of muscle regeneration), but upregulated the mRNA expression of phosphatidylinositol 3-kinase, Akt1, adenosine A1 receptor and transient receptor potential cation channel subfamily V member 4, involved in muscle growth and the activation of protein synthesis. The overall effects of treatment with 500 mg/kg FS were comparable to those observed following treatment with 50 mg/kg oxymetholone. The results from the present study support the hypothesis that FS has a favorable ameliorating effect on muscle atrophy induced by dexamethasone, by exerting anti-inflammatory and antioxidant effects on muscle fibers, which may be due to an increase in protein synthesis and a decrease in protein degradation.

The absence of MuRF1 protects against Calpain1‐induced systolic dysfunction in vivo

Protein degradation to maintain the cardiac contractile machinery is essential to the ongoing performance required of the heart. Multiple enzyme systems have been implicated in this process, including the ubiquitin‐proteasome system and Calpain(Capn) enzymes. In the heart, Capn1 is the primary calcium‐activated cysteine protease that physiologically degrades protein aggregates and sarcomere proteins upon activation. Activation of Capn1 results from pathological injury or stress, such as ischemia reperfusion injury and cardiac hypertrophy/heart failure. Recent evidence supports Capn1 involvement in regulating ubiquitination and subsequent degradation of sarcomeric proteins by an unidentified ubiquitin ligase. Muscle RING‐finger protein‐1(MuRF1) is an ubiquitin ligase expressed in striated muscle with many of the same muscle‐specific substrates as Capn1 (eg. troponin I). We hypothesized both Capn1 and MuRF1 are required for the pathological phenotypes associated with increased Capn1 activity. To test this, we crossed α‐MHC Capn1 transgenic mice (Capn1 Tg+) with MuRF1‐/‐ mice and followed their cardiac phenotype by conscious echocardiography. At 6 months, the Capn1 Tg+//MuRF1+/+ mice exhibited a significant reduction in fractional shortening associated with systolic dysfunction; in contrast, sibling Capn1 Tg+//MuRF1‐/‐ mice were largely protected. Capn1Tg+//MuRF1‐/‐ mice also did not exhibit the increased βMHC and ANF mRNA observed in the Capn1Tg+//MuRF1+/+ mice, which supports the absence of MuRF1 protects against pathological hypertrophy. Taken together, these data support a mechanism by which Capn protease and Murf1 ubiquitin‐proteasome pathways work in concert to degrade cardiac proteins in Capn1‐induced heart failure.