Muscle RING-finger Protein-1 Research Articles

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.

Characterization of RING Finger/SPRY Domain Containing 1 (Rspry1) and NEFA‐interacting Nuclear Protein 30 (Nip30) Transcriptional Regulation

Muscle atrophy results from a range of conditions, including immobilization, spinal cord damage, inflammation and aging. An investigation designed to identify changes in gene expression in skeletal muscle following denervation revealed a set of genes that show differential expression patterns in response to neurogenic atrophy. Nip30 and Rspry1, two genes not previously characterized in muscle, were found to be transcriptionally induced during muscle wasting in mice and protein expression was confirmed in C2C12 cells by western blot. In order to characterize the transcriptional regulation of Nip30 and Rspry1, a fragment of the promoter region of each gene was cloned, fused to a reporter gene, and transfected into muscle cells in combination with expression plasmids for Muscle RING Finger 1 (MuRF1) and myogenic regulatory factors (MRFs). The MuRF1 protein is an E3 ubiquitin ligase that is induced under most atrophic conditions and is believed to promote protein degradation; however, data presented in this study suggests that MuRF1 may also regulate the transcriptional activity of genes that are differentially expressed following neurogenic atrophy. Myogenic regulatory factors (MRFs) are a class of E-box binding proteins that regulate muscle-specific gene expression. The results of this study demonstrate that MuRF1 and MRFs cooperatively repress Rspry1 and Nip30 reporter activity. Furthermore, mutation of a conserved E-box element in the shared regulatory region of Nip30 and Rspry1 eliminated the MuRF1 and MRF cooperative repression of these genes. Identifying novel atrophy-induced genes and characterizing their role in the atrophy cascade may help further our understanding of the molecular mechanisms of muscle wasting.

Pathophysiological Mechanism of Bone Loss in Type 2 Diabetes Involves Inverse Regulation of Osteoblast Function by PGC-1α and Skeletal Muscle Atrogenes: AdipoR1 as a Potential Target for Reversing Diabetes-Induced Osteopenia.

Type 2 diabetes is associated with increased fracture risk and delayed fracture healing; the underlying mechanism, however, remains poorly understood. We systematically investigated skeletal pathology in leptin receptor-deficient diabetic mice on a C57BLKS background (db). Compared with wild type (wt), db mice displayed reduced peak bone mass and age-related trabecular and cortical bone loss. Poor skeletal outcome in db mice contributed high-glucose- and nonesterified fatty acid-induced osteoblast apoptosis that was associated with peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) downregulation and upregulation of skeletal muscle atrogenes in osteoblasts. Osteoblast depletion of the atrogene muscle ring finger protein-1 (MuRF1) protected against gluco- and lipotoxicity-induced apoptosis. Osteoblast-specific PGC-1α upregulation by 6-C-β-d-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF), an adiponectin receptor 1 (AdipoR1) agonist, as well as metformin in db mice that lacked AdipoR1 expression in muscle but not bone restored osteopenia to wt levels without improving diabetes. Both GTDF and metformin protected against gluco- and lipotoxicity-induced osteoblast apoptosis, and depletion of PGC-1α abolished this protection. Although AdipoR1 but not AdipoR2 depletion abolished protection by GTDF, metformin action was not blocked by AdipoR depletion. We conclude that PGC-1α upregulation in osteoblasts could reverse type 2 diabetes-associated deterioration in skeletal health.

Possible Involvement of Mitochondrial Reactive Oxygen Species Production in Protein Degradation Induced by Heat Stress in Avian Muscle Cells

Heat stress (HS) stimulates mitochondrial reactive oxygen species (ROS) production and protein degradation in skeletal muscle. The present study investigated the stimulatory effects of HS-induced mitochondrial ROS production on the ubiquitin-proteasome protein degradation system in primary cultured avian muscle cells. Cells were isolated from the breast muscle of neonatal chicks, and then grown for 48 h. Thereafter, the cells were subjected to 37°C or 41°C (HS). Exposure to 6 h of HS treatment significantly decreased the cellular protein content compared to that of normal cells, an effect was completely suppressed by the addition of a proteasome-specific inhibitor. Whereas the mRNA levels of the 20S proteasome C2 subunit, which is one of the subunits of the 26S proteasome, did not change at any time during HS treatment (1, 3, 6 h), the mRNA levels of atrogin-1 and muscle ring-finger protein 1, both of which are muscle-specific ubiquitin ligases, increased after 1 h of HS but then decreased to near-normal values with time. Intracellular ROS production (the sum of H2O2, hydroxyl radicals, peroxyl radicals, peroxynitrite) did not change in the 1 h HS-exposed cells, but was significantly increased after 3 h and 6 h of HS. Mitochondrial superoxide production was significantly increased after 1 h of HS, which might increase the mRNA expression of ubiquitin ligase in muscle cells. In cells pretreated with 4-hydroxy TEMPO, which is able to decrease mitochondrial superoxide production, the increases in mitochondrial superoxide production and ubiquitin ligase mRNA levels observed after 1 h of HS were suppressed. The protein content of these cells was not decreased, which was observed after the longest period of HS (6 h). These findings suggest that mitochondrial superoxide production may play an important role in activating the ubiquitin-proteasome system, probably via the induction of ubiquitin ligases, in HS-exposed muscle cells.

Influence of exercise intensity on atrophied quadriceps muscle in the rat

[Purpose] The aim of this study was to determine the effect of resistance training on atrophied skeletal muscle in rats based on evidence derived from physical therapy. [Subjects and Methods] Rats were forced to undergo squats as resistance training for 3 weeks after atrophying the rectus femoris muscle by hindlimb suspension for 2 weeks. The intensity of resistance training was adjusted to 50% and 70% of the maximum lifted weight, i.e., 50% of the one-repetition maximum and 70% of the one-repetition maximum, respectively. [Results] Three weeks of training did not alter the one-repetition maximum, and muscle fibers were injured while measuring the one-repetition maximum and reloading. The decrease in cross-sectional area in the rectus femoris muscle induced by unloading for 2 weeks was significantly recovered after training at 70% of the one-repetition maximum. The levels of muscle RING-finger protein-1 mRNA expression were significantly lower in muscles trained at 70% of the one-repetition maximum than in untrained muscles. [Conclusion] These results suggest that high-intensity resistance training can promote atrophic muscle recovery, which provides a scientific basis for therapeutic exercise methods for treatment of atrophic muscle in physical therapy.

Atomoxetine prevents dexamethasone-induced skeletal muscle atrophy in mice.

Skeletal muscle atrophy remains a clinical problem in numerous pathologic conditions. β2-Adrenergic receptor agonists, such as formoterol, can induce mitochondrial biogenesis (MB) to prevent such atrophy. Additionally, atomoxetine, an FDA-approved norepinephrine reuptake inhibitor, was positive in a cellular assay for MB. We used a mouse model of dexamethasone-induced skeletal muscle atrophy to investigate the potential role of atomoxetine and formoterol to prevent muscle mass loss. Mice were administered dexamethasone once daily in the presence or absence of formoterol (0.3 mg/kg), atomoxetine (0.1 mg/kg), or sterile saline. Animals were euthanized at 8, 16, and 24 hours or 8 days later. Gastrocnemius muscle weights, changes in mRNA and protein expression of peroxisome proliferator-activated receptor-γ coactivator-1 α (PGC-1α) isoforms, ATP synthase β, cytochrome c oxidase subunit I, NADH dehydrogenase (ubiquinone) 1 β subcomplex, 8, ND1, insulin-like growth factor 1 (IGF-1), myostatin, muscle Ring-finger protein-1 (muscle atrophy), phosphorylated forkhead box protein O 3a (p-FoxO3a), Akt, mammalian target of rapamycin (mTOR), and ribosomal protein S6 (rp-S6; muscle hypertrophy) in naive and muscle-atrophied mice were measured. Atomoxetine increased p-mTOR 24 hours after treatment in naïve mice, but did not change any other biomarkers. Formoterol robustly activated the PGC-1α-4-IGF1-Akt-mTOR-rp-S6 pathway and increased p-FoxO3a as early as 8 hours and repressed myostatin at 16 hours. In contrast to what was observed with acute treatment, chronic treatment (7 days) with atomoxetine increased p-Akt and p-FoxO3a, and sustained PGC-1α expression and skeletal muscle mass in dexamethasone-treated mice, in a manner comparable to formoterol. In conclusion, chronic treatment with a low dose of atomoxetine prevented dexamethasone-induced skeletal muscle wasting and supports a potential role in preventing muscle atrophy.

Single muscle fiber gene expression with run taper.

This study evaluated gene expression changes in gastrocnemius slow-twitch myosin heavy chain I (MHC I) and fast-twitch (MHC IIa) muscle fibers of collegiate cross-country runners (n = 6, 20±1 y, VO2max = 70±1 ml•kg−1•min−1) during two distinct training phases. In a controlled environment, runners performed identical 8 kilometer runs (30∶18±0∶30 min:s, 89±1% HRmax) while in heavy training (∼72 km/wk) and following a 3 wk taper. Training volume during the taper leading into peak competition was reduced ∼50% which resulted in improved race times and greater cross-section and improved function of MHC IIa fibers. Single muscle fibers were isolated from pre and 4 hour post run biopsies in heavily trained and tapered states to examine the dynamic acute exercise response of the growth-related genes Fibroblast growth factor-inducible 14 (FN14), Myostatin (MSTN), Heat shock protein 72 (HSP72), Muscle ring-finger protein-1 (MURF1), Myogenic factor 6 (MRF4), and Insulin-like growth factor 1 (IGF1) via qPCR. FN14 increased 4.3-fold in MHC IIa fibers with exercise in the tapered state (P<0.05). MSTN was suppressed with exercise in both fiber types and training states (P<0.05) while MURF1 and HSP72 responded to running in MHC IIa and I fibers, respectively, regardless of training state (P<0.05). Robust induction of FN14 (previously shown to strongly correlate with hypertrophy) and greater overall transcriptional flexibility with exercise in the tapered state provides an initial molecular basis for fast-twitch muscle fiber performance gains previously observed after taper in competitive endurance athletes.

Forkhead box O1 and muscle RING finger 1 protein expression in atrophic and hypertrophic denervated mouse skeletal muscle.

BackgroundForkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass. The function of FoxO transcription factors is regulated by post-translational modifications such as phosphorylation and acetylation. In the present study FoxO1 protein expression, phosphorylation and acetylation as well as MuRF1 protein expression, were examined in atrophic and hypertrophic denervated skeletal muscle.MethodsProtein expression, phosphorylation and acetylation were studied semi-quantitatively using Western blots. Muscles studied were 6-days denervated mouse hind-limb muscles (anterior tibial as well as pooled gastrocnemius and soleus muscles, all atrophic), 6-days denervated mouse hemidiaphragm muscles (hypertrophic) and innervated control muscles. Total muscle homogenates were used as well as separated nuclear and cytosolic fractions of innervated and 6-days denervated anterior tibial and hemidiaphragm muscles.ResultsExpression of FoxO1 and MuRF1 proteins increased 0.3-3.7-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic. Phosphorylation of FoxO1 at S256 increased about 0.8-1-fold after denervation in pooled gastrocnemius and soleus muscles and in hemidiaphragm but not in unfractionated anterior tibial muscle. A small (0.2-fold) but statistically significant increase in FoxO1 phosphorylation was, however, observed in cytosolic fractions of denervated anterior tibial muscle. A statistically significant increase in FoxO1 acetylation (0.8-fold) was observed only in denervated anterior tibial muscle. Increases in total FoxO1 and in phosphorylated FoxO1 were only seen in cytosolic fractions of denervated atrophic anterior tibial muscle whereas in denervated hypertrophic hemidiaphragm both total FoxO1 and phosphorylated FoxO1 increased in cytosolic as well as in nuclear fractions. MuRF1 protein expression increased in cytosolic as well as in nuclear fractions of both denervated atrophic anterior tibial muscle and denervated hypertrophic hemidiaphragm muscle.ConclusionsIncreased expression of FoxO1 and MuRF1 in denervated muscles (atrophic as well as hypertrophic) suggests that these proteins participate in the tissue remodelling occurring after denervation. The effect of denervation on the level of phosphorylated and acetylated FoxO1 differed in the muscles studied and may be related to differences in fiber type composition of the muscles.

Ghrelin relieves cancer cachexia associated with the development of lung adenocarcinoma in mice

Cancer cachexia is a multifactorial, critical illness syndrome characterized by an ongoing loss of skeletal muscle and adipose tissue. The reductions in body weight and skeletal muscle mass are important prognostic indicators for cancer patients that are refractory to current therapies. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced in the stomach, stimulates food intake and growth hormone secretion, suppresses inflammation, and prevents muscle catabolism. We investigated the pharmacological potential of ghrelin in the treatment of cancer cachexia by using urethane-treated, bronchioalveolar epithelium-specific Pten-deficient mice that developed lung adenocarcinomas. Ghrelin or phosphate-buffered saline was given to mice daily for four weeks beginning at five months after urethane injection, which corresponded to the time point of lung adenocarcinoma formation. Ghrelin inhibited the inductions of C-reactive protein, tumor necrosis factor-α, interleukin-1β, and interleukin-6, mitigated the reduction of food intake and fat mass, and consequently ameliorated body weight loss in the mouse model of lung adenocarcinoma. We also demonstrated that skeletal muscle mass and muscle contraction force in both fast-twitch muscle and slow-twitch muscle were retained in ghrelin-treated mice in conjunction with an upregulation of local insulin-like growth factor 1/Akt signaling. In addition, ghrelin administration reduced the expressions of phosphorylated-p38 mitogen-activated protein kinase, phosphorylated-nuclear factor-kappa B, Forkhead box protein O1, muscle RING-finger protein-1, and F-Box protein 32 in the lysates of skeletal muscle in the tumor-bearing state. Our results indicate that ghrelin administration exerts a protective effect against cancer cachexia by ameliorating skeletal muscle wasting and regulating systemic inflammation.

Skeletal Muscle Disuse Atrophy Is Not Attenuated by Dietary Protein Supplementation in Healthy Older Men

Short successive periods of muscle disuse, due to injury or illness, can contribute significantly to the loss of muscle mass with aging (sarcopenia). It has been suggested that increasing the protein content of the diet may be an effective dietary strategy to attenuate muscle disuse atrophy. We hypothesized that protein supplementation twice daily would preserve muscle mass during a short period of limb immobilization. Twenty-three healthy older (69 ± 1 y) men were subjected to 5 d of one-legged knee immobilization by means of a full-leg cast with (PRO group; n = 11) or without (CON group; n = 12) administration of a dietary protein supplement (20.7 g of protein, 9.3 g of carbohydrate, and 3.0 g of fat) twice daily. Two d prior to and immediately after the immobilization period, single-slice computed tomography scans of the quadriceps and single-leg 1 repetition maximum strength tests were performed to assess muscle cross-sectional area (CSA) and leg muscle strength, respectively. Additionally, muscle biopsies were collected to assess muscle fiber characteristics as well as mRNA and protein expression of selected genes. Immobilization decreased quadriceps' CSAs by 1.5 ± 0.7% (P < 0.05) and 2.0 ± 0.6% (P < 0.05), and muscle strength by 8.3 ± 3.3% (P < 0.05) and 9.3 ± 1.6% (P < 0.05) in the CON and PRO groups, respectively, without differences between groups. Skeletal muscle myostatin, myogenin, and muscle RING-finger protein-1 (MuRF1) mRNA expression increased following immobilization in both groups (P < 0.05), whereas muscle atrophy F-box/atrogen-1 (MAFBx) mRNA expression increased in the PRO group only (P < 0.05). In conclusion, dietary protein supplementation (∼20 g twice daily) does not attenuate muscle loss during short-term muscle disuse in healthy older men. This trial was registered at clinicaltrials.gov as NCT01588808.

Constant heat stress reduces skeletal muscle protein deposition in broilers

This experiment was conducted to evaluate the effects of constant heat stress on growth performance and protein metabolism in skeletal muscle of Arbor Acres broilers. Two hundred and seventy 21-day-old Arbor Acres broilers with similar body weight (1298 ± 28 g) were selected for a 3-week trial (29-49 days of age). The broilers were randomly assigned to three groups including the control group, constant heat stress group and pair-fed group. Up-regulation of the rectal temperature and the mRNA expression of heat shock protein 70 in liver indicate that the model for constant heat stress was success. The average daily gain, feed conversion ratio, breast and thigh muscle weight, percentage of breast muscle, crude protein content in breast and thigh muscle in constant heat stress group were significantly lower than in control group and pair-fed group. Serum uric acid content and the glutamic-oxaloacetic transaminase activity were significantly higher, while protein content and glutamic-pyruvate transaminase activity were significantly lower in liver of heat stress group than of the control and pair-fed groups. The expression of insulin-like growth factor 1, phosphatidylinositol 3-kinase and p70S6 kinase associated with protein synthesis were lower in breast muscle but higher in thigh muscle in heat stress group compared to the control or fed-pair groups. In thigh muscles, the expression of muscle ring-finger protein-1 and MAFbx associated with protein degradation were higher in the heat stress group than in the control and pair-fed groups. Poor performance of the birds under heat stress may be due to lower synthesis and increased degradation of proteins.

Muscle-specific inhibition of the classical nuclear factor-κB pathway is protective against diaphragmatic weakness in murine endotoxemia.

Diaphragmatic weakness and acute respiratory failure are common in sepsis. Nuclear factor-κB acts as a general coordinator of the systemic inflammatory response, but its role within the diaphragm itself during sepsis is unknown. We investigated the potential protective effect upon the diaphragm of inhibiting nuclear factor-κB only within muscle fibers during acute endotoxemia. Prospective study in experimental animals. University research laboratory. Wild-type and transgenic (muscle-specific IκBα super-repressor) mice with skeletal muscle-specific inhibition of the classical nuclear factor-κB pathway were subjected to acute endotoxemia. Muscle-specific ubiquitin ligases (muscle RING-finger protein 1 and atrogin-1), caspase-3 activity, inhibitor of apoptosis proteins, proinflammatory cytokines (interleukin-1β, monocyte chemoattractant protein-1, and tumor necrosis factor-α), and diaphragmatic contractility were evaluated after 24 hours. In wild-type mice, endotoxemia significantly increased proinflammatory cytokines (fold-change messenger RNA: interleukin-1β = 7.6, monocyte chemoattractant protein-1 = 15.3, and tumor necrosis factor-α = 2.2) and proteolysis effectors (fold-change messenger RNA: muscle RING-finger protein 1 = 5.7, atrogin-1 = 2.8; caspase-3 activity elevated by 28%) in the diaphragm, while reducing its force-generating capacity by 38%. In nonendotoxemic muscle-specific IκBα super-repressor diaphragms, caspase-3 activity was unexpectedly increased by 40% above basal wild-type levels and inhibitors of apoptosis proteins were down-regulated, but force production remained normal. In muscle-specific IκBα super-repressor mice subjected to endotoxemia, proinflammatory cytokines, muscle RING-finger protein 1, and atrogin-1 were not significantly increased above their basal levels, and diaphragmatic weakness and further increases in caspase-3 activity were completely prevented. These results suggest that nuclear factor-κB signaling within skeletal muscle fibers is a key pathway leading to diaphragmatic weakness during acute endotoxemia, most likely via effects on multiple inflammatory mediators. In addition, inhibition of nuclear factor-κB signaling within diaphragm muscle fibers has complex effects on caspase-3 activation, which could have implications for the treatment of sepsis-induced diaphragmatic dysfunction.

Transcriptional regulation of myotrophic actions by testosterone and trenbolone on androgen-responsive muscle

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth.

β-Hydroxy-β-methylbutyrate improves bone properties and attenuates the depression of protein synthesis during a simulated sustained operation.

Soldiers lose muscle and bone density during sustained operations. We investigated the impact of β-hydroxy-β-methylbutyrate (HMB) on bone properties, muscle mass, and markers of skeletal muscle regeneration under simulated military sustained operations. Male mice were divided into four groups (10/group): (1) ALT = ad libitum + trained (1h/d for 3 d/wk); (2) ALTH = ALT + HMB (0.5 g/kg BW/d); (3) C = caloric restricted (-30%) + trained (6h/d, 6d/wk); and (4) CH = C + HMB. Assessments included bone mineral density/content by dual-energy X-ray absorptiometry, muscle wet weight (quadriceps) and expression of selected genes regulating muscle mass and protein turnover. Analysis of variances were used with significance set at p < 0.05. Bone mineral content increased in the ALT group (+16%) and decreased in the C group (-32%). Quadriceps muscle mass was lower in C (-27%) and CH (-19%) compared to ALT and ALTH. Myogenin mRNA expression was higher in C than ALT, ALTH and CH. Protein kinase B (Akt) mRNA expression was higher in both C and CH than ALT and ALTH. Mammalian target of rapamycin expression was higher in CH than ALT and ALTH. Muscle RING-finger protein-1 expression was higher in both C and CH than ALT and ALTH. HMB intake improved bone properties and attenuated the depression of protein synthesis during a simulated sustained military operation.

Rare variants in genes encoding MuRF1 and MuRF2 are modifiers of hypertrophic cardiomyopathy.

Modifier genes contribute to the diverse clinical manifestations of hypertrophic cardiomyopathy (HCM), but are still largely unknown. Muscle ring finger (MuRF) proteins are a class of muscle-specific ubiquitin E3-ligases that appear to modulate cardiac mass and function by regulating the ubiquitin-proteasome system. In this study we screened all the three members of the MuRF family, MuRF1, MuRF2 and MuRF3, in 594 unrelated HCM patients and 307 healthy controls by targeted resequencing. Identified rare variants were confirmed by capillary Sanger sequencing. The prevalence of rare variants in both MuRF1 and MuRF2 in HCM patients was higher than that in control subjects (MuRF1 13/594 (2.2%) vs. 1/307 (0.3%), p = 0.04; MuRF2 22/594 (3.7%) vs. 2/307 (0.7%); p = 0.007). Patients with rare variants in MuRF1 or MuRF2 were younger (p = 0.04) and had greater maximum left ventricular wall thickness (p = 0.006) than those without such variants. Mutations in genes encoding sarcomere proteins were present in 19 (55.9%) of the 34 HCM patients with rare variants in MuRF1 and MuRF2. These data strongly supported that rare variants in MuRF1 and MuRF2 are associated with higher penetrance and more severe clinical manifestations of HCM. The findings suggest that dysregulation of the ubiquitin-proteasome system contributes to the pathogenesis of HCM.

Proteomic and bioinformatic analysis of differentially expressed proteins in denervated skeletal muscle

The aim of this study was to improve our understanding and the current treatment of denervation-induced skeletal muscle atrophy. We used isobaric tags for relative and absolute quantification (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) to identify the differentially expressed proteins in the tibialis anterior (TA) muscle of rats at 1 and 4 weeks following sciatic nerve transection. A total of 110 proteins was differentially expressed and was further classified using terms from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to unravel their molecular functions. Among the differentially expressed metabolic enzymes involved in glycolysis, Krebs cycle and oxidative phosphorylation, α- and β-enolase displayed an increased and decreased expression, respectively, which was further validated by western blot analysis and immunohistochemistry. These findings suggest that the enolase isozymic switch during denervation-induced muscle atrophy is the reverse of that occurring during muscle maturation. Notably, protein‑protein interaction analysis using the STRING database indicated that the protein expression of tumor necrosis factor receptor-associated factor-6 (TRAF6), muscle ring-finger protein 1 (MuRF1) and muscle atrophy F-box (MAFBx) was also upregulated during denervation‑induced skeletal muscle atrophy, which was confirmed by western blot analysis. TRAF6 knockdown experiments in L6 myotubes suggested that the decreased expression of TRAF6 attenuated glucocorticoid‑induced myotube atrophy. Therefore, we hypothesized that the upregulation of TRAF6 may be involved in the development of denervation‑induced muscle atrophy, at least in part, by regulating the expression of MAFBx and MuRF1 proteins. The data from the present study provide valuable insight into the molecular mechanisms regulating denervation-induced muscle atrophy.

The kinin B1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

Regulation of Large Conductance Ca2+-activated K+ (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels

The large conductance Ca(2+)-activated K(+) (BK) channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK channelopathy and vasculopathy in diabetes.

Regulation of the protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs (137.1)

The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin (Ins) and amino acids (AA), especially leucine (leu), modulate protein synthesis in skeletal muscle and how this changes with development. These studies aimed to determine whether Ins, AA or Leu alter the abundance and activation of ubiquitin‐proteasome, i.e., atrogin‐1 (MAFbx) and muscle RING‐finger protein‐1 (MuRF1), and autophagy‐lysosome, i.e., microtubule‐associated protein light chain 3 (LC3) and lysosomal‐associated membrane protein 2 (Lamp‐2), protein degradation systems and whether the responses change with age. In study 1, overnight fasted 6‐ and 26‐d‐old pigs were infused with saline or with insulin or AA to achieve fed levels. In study 2, overnight fasted 6‐d‐old pigs were infused with Leu while Ins and AA were kept at fasting levels. Abundance of atrogin‐1, but not MuRF1, was greater in 26‐ than in 6‐day‐old pigs (P&lt;0.05) and was not affected by Ins, AA, or Leu. Abundance of LC3 was higher in younger pigs and not affected by treatment (P&lt;0.05). LC3‐II/LC3‐I ratio was reduced by Ins, AA, and Leu (P&lt;0.05). This response was more profound in younger pigs. Abundance of Lamp‐2 was not affected by treatment or development. In conclusion, Ins, AA, and, in particular, Leu act as signals to modulate muscle protein degradation in neonates.Grant Funding Source: Supported by NIH AR444474 and USDA/ARS 6250‐51000‐055