Muscle-specific RING Finger-1 Research Articles

MicroRNA-140 is not involved in sepsis-induced muscle atrophy.

Sepsis is a life-threatening inflammatory response to infection, often accompanied by skeletal muscle atrophy. A previous study demonstrated that the administration of microRNA-140 (miR-140) attenuated lipopolysaccharides (LPS)-induced muscle atrophy, whereas miR-140 knockdown with siRNA promoted atrophy. Therefore, we investigated whether miR-140 is involved in LPS-induced muscle atrophy using a genetic model, miR-140-/- mice. We found that a single injection of LPS induced atrophy both in slow-twitch and fast-twitch muscles. The muscle weights and fiber cross-sectional areas were significantly reduced in both the wild-type (WT) and miR-140-/- mice, with no difference between genotypes. The expression of several proteolysis markers, muscle-specific RING-finger 1 (MuRF1) and MAFbx/atrogin-1, increased in both groups after LPS injection. The ubiquitinated proteins in the miR-140-/- mice were similar to those in the WT mice. Therefore, the deletion of miR-140 did not affect sepsis-induced muscle atrophy.

Exercise attenuates angiotensinⅡ-induced muscle atrophy by targeting PPARγ/miR-29b

BackgroundExercise is beneficial for muscle atrophy. Peroxisome proliferator-activated receptor gamma (PPARγ) and microRNA-29b (miR-29b) have been reported to be responsible for angiotensinⅡ (AngⅡ)-induced muscle atrophy. However, it is unclear whether exercise can protect AngⅡ-induced muscle atrophy by targeting PPARγ/miR-29b. MethodsSkeletal muscle atrophy in both the control group and the run group was established by AngⅡ infusion; after 1 week of exercise training, the mice were sacrificed, and muscle weight was determined. Myofiber size was measured by hematoxylin-eosin and wheat-germ agglutinin staining. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression level of muscle atrogenes, including F-box only protein 32 (FBXO32, also called Atrogin-1) and muscle-specific RING-finger 1 (MuRF-1), the phosphorylation level of protein kinase B (PKB, also called AKT)/forkhead box O3A (FOXO3A)/mammalian target of rapamycin (mTOR) pathway proteins, the expression level of PPARγ and apoptosis-related proteins, including B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), cysteine-aspartic acid protease 3 (caspase-3), and cleaved-caspase-3, were determined by western blot. The expression level of miR-29b was checked by reverse-transcription quantitative polymerase chain reaction. A PPARγ inhibitor (T0070907) or adeno-associated virus serotype-8 (AAV8)-mediated miR-29b overexpression was used to demonstrate whether PPARγ activation or miR-29b inhibition mediates the beneficial effects of exercise in AngⅡ-induced muscle atrophy. ResultsExercise can significantly attenuate AngⅡ-induced muscle atrophy, which is demonstrated by increased skeletal muscle weight, cross-sectional area of myofiber, and activation of AKT/mTOR signaling and by decreased atrogenes expressions and apoptosis. In AngⅡ-induced muscle atrophy mice models, PPARγ was elevated whereas miR-29b was decreased by exercise. The protective effects of exercise in AngⅡ-induced muscle atrophy were inhibited by a PPARγ inhibitor (T0070907) or adeno-associated virus serotype-8 (AAV8)-mediated miR-29b overexpression. ConclusionExercise attenuates AngⅡ-induced muscle atrophy by activation of PPARγ and suppression of miR-29b.

PINK1/Parkin-mediated mitophagy in mechanical ventilation-induced diaphragmatic dysfunction.

Background:Mechanical ventilation (MV) often leads to ventilation-induced diaphragm dysfunction (VIDD). Although the development of this disorder had been linked to oxidative stress, mitochondrial energy deficiency, autophagy activation, and apoptosis in the diaphragm, it remains unclear whether the activation of mitophagy can induce VIDD. With our research, our endeavor is to uncover whether PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy affects the MV-caused diaphragmatic dysfunctionMethods:Sprague-Dawley rats were subjected to MV treatment for 6 h (MV-6h), 12 h (MV-12h), or 24 h (MV-24h). Post MV, the diaphragm muscle compound action potential (CMAP) and cross-sectional areas (CSAs) of the diaphragm of these rats were measured. The levels of proteins of interest were examined to assess muscle health, mitochondrial dynamics, and mitophagy in the diaphragm. The co-localization of PINK1 with the mitochondrial protein marker tom20 was examined, as well as transmission electron microscopy analysis to detect changes in diaphragm mitochondrial ultrastructure.Results:MV-12h and MV-24h treatments resulted in a decrease in CSA of diaphragm and CMAP amplitude. In addition, the expressions of F-box (MFAbx), muscle-specific ring finger 1 (MURF1), PINK1, and p62 were elevated in rats treated with MV for 12 h and 24 h, while mfn2 expression was reduced. Rats following MV-24h treatment displayed an increase in mitochondrial dynamic protein (Drp1) and Parkin expression and microtubule-associated protein 1 light chain 3/1 (LC3II/I) ratio. Moreover, decreased SOD and GSH activity and membrane potential were observed after MV-12h and MV-24h treatment, while H2O2 activity increased after MV-24h treatment. In addition, a strong co-localization between PINK1 and tom20 was identified.Conclusion:These results reveal that MV leads to various changes in mitochondrial dynamics and significantly increases the mitophagy levels, which subsequently cause the variation in diaphragmatic function and muscle atrophy, indicating that mitophagy could be one of the possible mechanisms by which MV induces diaphragmatic dysfunction. The reviews of this paper are available via the supplemental material section.

Aspirin alleviates denervation-induced muscle atrophy via regulating the Sirt1/PGC-1α axis and STAT3 signaling.

BackgroundOur prior studies have shown that inflammation may play an important triggering role during the process of denervated muscle atrophy. The nonsteroidal anti-inflammatory drug aspirin exhibits the effect of anti-inflammatory factors. This study will investigate the protective effect of aspirin on denervated muscle atrophy and the underlying mechanism.MethodsMouse models of denervated muscle atrophy were established. The protective effect of aspirin (20 mg/kg/d, i.p.) on denervated muscle atrophy was analyzed using the wet weight ratio of tibialis anterior (TA) muscle and muscle fiber cross-sectional area (CSA). The levels of inflammatory factors were detected using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Sirtuins1 (SIRT1)/Peroxisome Proliferator-Activated Receptor γ Co-Activator 1α (PGC-1α) and Signal transducer and activator of transcription 3 (STAT3) signaling pathway and the muscle fiber type related proteins in TA muscle after denervation were analyzed by western blot assay.ResultsIntraperitoneal injection of aspirin (20 mg/kg/d) effectively alleviated denervation-induced muscle atrophy. This mainly manifested as follows: The wet weight ratio of TA muscle and muscle fiber CSA of mice treated with aspirin were significantly greater compared with mice treated with normal saline. The level of myosin heavy chain (MHC) increased, and the levels of muscle specific E3 ubiquitin ligase Muscle-specific RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx) were decreased. Mitochondrial vacuolation and autophagy were inhibited, as evidenced by reduced level of autophagy related proteins PINK1, BNIP3, LC3B and Atg7 in mice treated with aspirin compared with mice treated with saline. In addition, aspirin treatment inhibited the slow-to-fast twitch muscle fiber conversion, which were related with triggering the expression of Sirt1 and PGC-1α. Moreover, aspirin reduced the levels of inflammatory factors interleukin-6, interleukin-1β and tumor necrosis factor-α and decreased the activation of STAT3 signaling pathway.ConclusionsThis is the first study to find that aspirin can alleviate denervation-induced muscle atrophy and inhibit the type I-to-type II muscle fiber conversion and mitophagy possibly through regulating the STAT3 inflammatory signaling pathway and Sirt1/PGC-1α signal axis. This study expands our knowledge regarding the pharmacological function of aspirin and provides a novel strategy for prevention and treatment of denervated muscle atrophy.

Dietary supplementation with arginine and glutamic acid alters the expression of amino acid transporters in skeletal muscle of growing pigs

Sixty Duroc × Large White × Landrace pigs with an average initial body weight (BW) of 77.1 ± 1.3kg were selected to investigate the effects of dietary supplementation with arginine (Arg) and/or glutamic acid (Glu) on free amino acid (FAA) profiles, expression of AA transporters, and growth-related genes in skeletal muscle. The animals were randomly assigned to one of five treatment groups (basic diet, iso-nitrogenous, Arg, Glu, and Arg + Glu groups). The results showed that plasma Glu concentration was lowest in the Arg + Glu group and highest in the Glu group (P < 0.05). In the longissimus dorsi (LD) muscle, the concentrations of histidine, Arg, and taurine in the Arg + Glu group were higher, and the concentrations of 3-methylhistidinewas lower, than in the basic diet group (P < 0.05). The mRNA levels of ASC amino acid transporter-2 (ASCT2), L-type AA transporter 1, and sodium-coupled neutral amino acid transporter 2 in the LD muscle, as well as the mRNA levels of ASCT2 and proton-assisted amino acid transporter in the biceps femoris (BF) muscle, were higher in the Arg + Glu group compared to the basic diet group (P < 0.05). The mRNA levels of the muscle-specific RING finger-1 and muscle atrophy F-box genes in the LD muscle were downregulated in the Glu and Arg + Glu groups compared to the basic diet group (P < 0.05). Collectively, these findings suggest that dietary supplementation with both Arg and Glu increases intramuscular FAA concentrations and decreases the mRNA levels of genes involved in protein degradation in skeletal muscle.

Role of Akirin1 in the regulation of skeletal muscle fiber-type switch.

Akirin1 is a highly conserved ubiquitously expressed nuclear protein. Owing to its strong nuclear localization signal and protein-protein interaction properties, Akirin1 has been speculated to regulate transcription of target genes as a cofactor. Previous studies have reported Akirin1 as a downstream target of myostatin, a potent negative regulator of myogenesis. Mice lacking myostatin displayed enhanced Akirin1 gene expression. Further, in vitro evidence has shown Akirin1 overexpression leads to hypertrophy in C2 C 12 myotubes. In this study, we used Akirin1 knockout mice as a model system to further elucidate the function of Akirin1 in fully differentiated skeletal muscle. Akirin1 knockout mice did not show any obvious phenotypic difference when compared with wild type. However, promoter-reporter assay suggested that Akirin1 regulated the transcription of muscle-specific RING finger 1 (MuRF-1), an important E3 ubiquitin ligase in skeletal muscle. Furthermore, ablation of Akirin1 resulted in increased type IIa and decreased type I muscle fibers, which was further supported by an increase in Myh2 and decrease in Myh7 gene expression. Also, histochemical studies for succinate dehydrogenase activity revealed a less oxidative muscle in the absence of Akirin1. Together, our study suggests a novel role of Akirin1 in maintaining the muscle fiber type and regulation of the metabolic activity of the skeletal muscle.

Mechanistic Role of Reactive Oxygen Species and Therapeutic Potential of Antioxidants in Denervation- or Fasting-Induced Skeletal Muscle Atrophy.

Skeletal muscle atrophy occurs under various conditions, such as disuse, denervation, fasting, aging, and various diseases. Although the underlying molecular mechanisms are still not fully understood, skeletal muscle atrophy is closely associated with reactive oxygen species (ROS) overproduction. In this study, we aimed to investigate the involvement of ROS in skeletal muscle atrophy from the perspective of gene regulation, and further examine therapeutic effects of antioxidants on skeletal muscle atrophy. Microarray data showed that the gene expression of many positive regulators for ROS production were up-regulated and the gene expression of many negative regulators for ROS production were down-regulated in mouse soleus muscle atrophied by denervation (sciatic nerve injury). The ROS level was significantly increased in denervated mouse soleus muscle or fasted C2C12 myotubes that had suffered from fasting (nutrient deprivation). These two muscle samples were then treated with N-acetyl-L-cysteine (NAC, a clinically used antioxidant) or pyrroloquinoline quinone (PQQ, a naturally occurring antioxidant), respectively. As compared to non-treatment, both NAC and PQQ treatment (1) reversed the increase in the ROS level in two muscle samples; (2) attenuated the reduction in the cross-sectional area (CSA) of denervated mouse muscle or in the diameter of fasted C2C12 myotube; (3) increased the myosin heavy chain (MHC) level and decreased the muscle atrophy F-box (MAFbx) and muscle-specific RING finger-1 (MuRF-1) levels in two muscle samples. Collectively, these results suggested that an increased ROS level was, at least partly, responsible for denervation- or fasting-induced skeletal muscle atrophy, and antioxidants might resist the atrophic effect via ROS-related mechanisms.

Iron limitation promotes the atrophy of skeletal myocytes, whereas iron supplementation prevents this process in the hypoxic conditions.

There is clinical evidence that patients with heart failure and concomitant iron deficiency have increased skeletal muscle fatigability and impaired exercise tolerance. It was expected that a skeletal muscle cell line subjected to different degrees of iron availability and/or concomitant hypoxia would demonstrate changes in cell morphology and in the expression of atrophy markers. L6G8C5 rat skeletal myocytes were cultured in normoxia or hypoxia at optimal, reduced or increased iron concentrations. Experiments were performed to evaluate the iron content in cells, cell morphology, and the expression of muscle specific atrophy markers [Atrogin1 and muscle-specific RING-finger 1 (MuRF1)], a gene associated with the atrophy/hypertrophy balance [mothers against decapentaplegic homolog 4 (SMAD4)] and a muscle class-III intermediate filament protein (Desmin) at the mRNA and protein level. Hypoxic treatment caused, as compared to normoxic conditions, an increase in the expression of Atrogin-1 (P<0.001). Iron-deficient cells exhibited morphological abnormalities and demonstrated a significant increase in the expression of Atrogin-1 (P<0.05) and MuRF1 (P<0.05) both in normoxia and hypoxia, which indicated activation of the ubiquitin proteasome pathway associated with protein degradation during muscle atrophy. Depleted iron in cell culture combined with hypoxia also induced a decrease in SMAD4 expression (P<0.001) suggesting modifications leading to atrophy. In contrast, cells cultured in a medium enriched with iron during hypoxia exhibited inverse changes in the expression of atrophy markers (both P<0.05). Desmin was upregulated in cells subjected to both iron depletion and iron excess in normoxia and hypoxia (all P<0.05), but the greatest augmentation of mRNA expression occurred when iron depletion was combined with hypoxia. Notably, in hypoxia, an increased expression of Atrogin-1 and MuRF1 was associated with an increased expression of transferrin receptor 1, reflecting intracellular iron demand (R=0.76, P<0.01; R=0.86, P<0.01). Hypoxia and iron deficiency when combined exhibited the most detrimental impact on skeletal myocytes, especially in the context of muscle atrophy markers. Conversely, iron supplementation in in vitro conditions acted in a protective manner on these cells.

Characterizing the Role of Muscle‐Specific RING Finger 1 (MuRF1) as a Regulator of Atrophy‐Induced Gene Expression

Skeletal muscle wasting occurs as a consequence of numerous physiological conditions, including denervation, immobilization, and aging. The E3 ubiquitin ligases, MuRF1 and MAFbx, are induced under nearly all atrophy conditions and are believed to play a key role in protein degradation. Preliminary data presented here provides intriguing evidence that MuRF1 may also act as a transcriptional modulator of muscle atrophy‐induced gene expression. The transcriptional regulation of MuRF1 and MAFbx were analyzed using a reporter gene system and exhibited repression in C2C12 cells overexpressing MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRFs), MyoD and myogenin, caused significant activation of the MuRF1 and MAFbx reporter gene constructs, while co‐overexpression of MuRF1 with MRFs resulted in reversal of MRF induction of reporter gene activity. Ectopic expression of a catalytically dead MuRF1 RING mutant failed to reverse MRF activation of the reporters, suggesting that ubiquitin ligase activity may be important for MuRF1 transcriptional regulation. In order to further investigate a potential mechanism for MuRF1 modulation of MRF activity, Western blot analysis was performed to analyze MRF protein expression levels in C2C12 cells overexpressing MuRF1 or MuRF1 RING mutant. Cells with ectopic MuRF1 or MuRF1 RING mutant showed repressed levels of myogenin. Additionally, cells overexpressing MuRF1 and MuRF1 RING mutant treated with MG132 showed a modest rescue of myogenin, but myogenin protein levels were still reduced. Finally, chromatin immunoprecipitation (ChIP) was performed in order to analyze MRF occupancy of the MuRF1 promoter. As expected, MRF overexpression resulted in increased MuRF1 promoter immunoprecipitation. Interestingly, co‐overexpression of MuRF1 with MRFs resulted in a reversal of promoter occupancy. These findings suggest that MuRF1 may regulate MRF transcriptional activity by interfering with promoter binding, which provides additional insight into the potential mechanism by which MuRF1 may act to transcriptionally regulate atrophy‐induced gene expression.Support or Funding InformationThis work was supported by University of North Florida Transformational Learning Opportunity Grants to D.W.

Cancer cachexia-when proteasomal inhibition is not enough.

Cachexia is a life threatening syndrome associated with several diseases, such as end‐stage heart failure, end‐stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1, 2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2, 4, 5, 6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non‐Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small‐cell and non‐small‐cell lung cancer patients develop cachexia.7, 8, 9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi‐organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7, 12, 13, 14, 15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18, 19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.

Transcriptional Regulation of Skeletal Muscle Atrophy‐induced Gene Expression by Muscle‐Specific RING Finger 1 (MuRF1)

Skeletal muscle wasting is a consequence of numerous physiological conditions, including denervation, corticosteroid treatment, immobilization, and aging. The E3 ubiquitin ligases, MuRF1 and MAFbx, are induced under nearly all atrophy conditions and are believed to play a key role in protein degradation in atrophying muscle. However, the preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of neurogenic atrophy‐induced gene activity, including the regulation of MAFbx and MuRF1 expression. To characterize the transcriptional regulation of MuRF1 and MAFbx, reporter gene constructs containing fragments of the proximal promoter regions of these genes were developed, transfected into C2C12 cells with or without a MuRF1 expression plasmid and monitored for differences in reporter gene activity. The MuRF1 and MAFbx reporters each showed repressed activity in cells ectopically expressing MuRF1 compared to cells that did not overexpress MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRF), MyoD1 and myogenin, caused significant activation of the MuRF1 and MAFbx reporter constructs. However, co‐overexpression of MuRF1 with MyoD1 or myogenin resulted in reversal of MRF induction of reporter gene activity. To further characterize the role of the MuRF1 gene product in repression of MuRF1 expression, a MuRF1 RING domain mutant was created. The RING mutant construct was then co‐transfected along with MRF expression plasmids and the MuRF1 or MAFbx reporter gene into C2C12 cells and reporter gene activity was assessed. The MuRF1 RING mutant failed to reverse MRF activation of the reporter gene, suggesting that ubiquitin ligase activity is required for MuRF1 transcriptional regulatory effects. To further investigate the potential regulation of MRFs by MuRF1, Western blot analysis was performed in order to determine the impact on MRF protein levels in C2C12 cells in response to overexpression of MuRF1. Cells with ectopic expression of MuRF1 showed reduced levels of myogenin protein. In addition, cells overexpressing MuRF1 were treated with MG132, a general proteasome inhibitor, followed by Western blot analysis. The results of the Western blot revealed that cells treated with MG132 alone had increased myogenin protein levels, while cells ectopically expressing MuRF1 still exhibited reduced myogenin levels. MuRF1 repression of myogenin expression, with or without 26S proteasome inhibition, suggests that MuRF1 may be acting by a previously uncharacterized mechanism to regulate MRF expression, providing additional support that MuRF1 may act as a transcriptional regulator of atrophy‐induced gene expression. These data offer exciting evidence of a potential new function for MuRF1 as a transcriptional modulator of atrophy‐mediated changes in gene expression.Support or Funding InformationUniversity of North Florida Transformational Learning Opportunity Grant

High‐intensity resistance training attenuates dexamethasone‐induced muscle atrophy

In this study we investigated the effects of high-intensity resistance training (RT) on dexamethasone (DEX)-induced muscle atrophy in flexor hallucis longus (FHL), tibialis anterior (TA), and soleus (SOL) muscles. Rats underwent either high-intensity RT or were kept sedentary. In the last 10 days they received either DEX (0.5 mg/kg/day, intraperitoneally) or saline. DEX reduced body weight (-21%), food intake (-28%), FHL and TA muscle mass (-20% and -18%, respectively), and increased muscle-specific ring finger 1 (MuRF-1) protein level (+37% and +45.5%). RT attenuated FHL muscle atrophy through a combination of low increase in MuRF-1 protein level (-3.5%) and significant increases in mammalian target of rapamycin (mTOR) (+63%) and p70S6K (+46% and +49% for control and DEX, respectively) protein levels. RT attenuated DEX-induced muscle atrophy through a combination of increases in mTOR and p70S6K protein levels and a low increase in MuRF-1 protein level.

DJ-1 protects against undernutrition-induced atrophy through inhibition of the MAPK-ubiquitin ligase pathway in myoblasts.

The purpose of this study is to explore whether antioxidant DJ-1 protein affects the atrophy of skeletal muscle cell induced by undernutrition. To determine cell atrophic responses, L6 cell line and skeletal primary cells from mouse hind limbs were cultivated under condition of FBS-free and low glucose. Changes of protein expression were analyzed using Western blot. Overexpression and knockdown of DJ-1 was performed in cells to assess its influence on cell atrophic responses. Undernutrition decreased cell size and increased the abundance of oxidized form and total form of DJ-1 protein in L6 myoblasts. The undernourished cells revealed an elevation in the expression of muscle-specific RING finger-1 (MuRF-1) and atrogin-1, and in the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase compared with control groups. Moreover, DJ-1-knockout mice showed a decrease in cell size and an enhancement in the expression of MuRF-1 and atrogin-1, as well as in the phosphorylation of MAPKs in gastrocnemius muscles; these changes were also observed in L6 cells transfected with siRNA of DJ-1. On the other hand, L6 cells overexpressing full-length DJ-1 did not exhibit the alterations in cell size and ubiquitin ligases seen after undernourished states of control cells. Myotubes differentiated from L6 cells also showed elevated expression of MuRF-1 and atrogin-1 in response to undernutrition. These results suggest that DJ-1 protein may contribute to undernutrition-induced atrophy via MAPKs/ubiquitin ligase pathway in skeletal muscle cells.

Leucine improves growth performance of intrauterine growth retardation piglets by modifying gene and protein expression related to protein synthesis

ObjectiveLeucine has been reported to alter the gene expression of proteins, the activation of signaling components, and the fractional rates of protein synthesis in multiple organs of piglets. The aim of this study was to investigate the effects of leucine on molecular mechanisms regulating protein synthesis and degradation in skeletal muscle and determine how these adaptations affect body weight gain in intrauterine growth retardation (IUGR) pigs. MethodsThirty-two weaned piglets were randomly assigned to the following four experimental groups (n = 8 per group): normal birth weight, normal birth weight supplemented with leucine, IUGR, and IUGR supplemented with leucine. Piglets were fed from 14 to 35 d of age. Growth performance, major serum biochemical parameters, and enzyme activities were evaluated. The messenger RNA expression of muscle mammalian target of rapamycin (mTOR), muscle atrophy F-box (MAFbx), and muscle-specific ring finger-1 were investigated. Additionally, total and phosphorylated levels of mTOR and ribosomal protein S6 kinase 1 were measured in longissimus muscle. ResultsAverage daily weight gain and average daily feed intake were increased by leucine in IUGR pigs. At the end of the experiment, IUGR pigs had lower liver and kidney weight compared with the normal piglets. However, IUGR supplemented with leucine decreased serum concentration of urea. Leucine supplementation affected the concentrations of isoleucine, valine, lysine, and phenylalanine in serum. There was no significant difference in the expression of mTOR and muscle-specific ring finger-1 in IUGR piglets, whereas muscle atrophy F-box was reduced only by IUGR dependent of leucine. Compared with the IC group, dietary supplementation with 0.35% l-leucine increased the phosphorylated levels of mTOR and ribosomal S6 kinase 1 in IUGR piglets. ConclusionsThe present study identified a major role for leucine in the activation of the mTOR signaling pathway and reducing muscle atrophy in IUGR piglets, which contributed significantly to differences in body weight gain.

SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner.

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.

Transcriptional regulation of dual‐specificity phosphatase 4 (Dusp4) by muscle‐specific RING finger 1 (MuRF1) (LB822)

Skeletal muscle wasting is a consequence of numerous physiological conditions, including denervation, corticosteroid treatment, and aging. The E3 ubiquitin ligase, MuRF1, is induced under most atrophy conditions and is believed to play a key role in protein degradation in atrophying muscle. However, the preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of atrophy‐induced gene activity, including the regulation of Dusp4 expression. In order to characterize the transcriptional regulation of Dusp4, reporter gene constructs containing fragments of the proximal promoter region of this gene were developed, transfected into C2C12 cells with or without a MuRF1 expression plasmid and analyzed for differences in reporter gene activity.The Dusp4 reporters showed repressed activity in cells ectopically expressing MuRF1 compared to cells that did not overexpress MuRF1. Furthermore, ectopic expression of the myogenic regulatory factors (MRFs), MyoD1 and myogenin, also caused repression of the Dusp4 reporter constructs, while co‐overexpression of MuRF1 with MyoD1 or Myogenin resulted in additional cooperative repression of reporter gene activity. To further characterize the role of MuRF1 in the repression of Dusp4 expression, a MuRF1 RING domain mutant was created. The MuRf1 RING mutant was overexpressed in combination with MyoD1 or Myogenin and Dusp4 reporter gene activity was assessed. The MuRF1 RING mutant failed to cooperate with the MRFs to repress the Dusp4 reporter gene, suggesting that ubiquitin ligase activity is required for MuRF1 transcriptional regulatory effects. These data offer evidence that MuRF1 may act as a transcriptional modulator of atrophy‐induced gene expression.

Tiny transporters: how exosomes and calcineurin signaling regulate miR-23a levels during muscle atrophy. Focus on “miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export”

skeletal muscle atrophy is characterized by an increase in protein degradation that is accomplished through a variety of cellular mechanisms. Significant attention has been given to the ubiquitin-proteasome pathway and to expression of the E3 ubiquitin ligases muscle-specific RING finger-1 (MuRF1)

Whey protein modifies gene expression related to protein metabolism affecting muscle weight in resistance-exercised rats

ObjectiveThe aim of this study was to evaluate the effects of resistance exercise on the mRNA expression of muscle mammalian target of rapamycin (mTOR), muscle-specific RING finger-1 (MuRF-1), and muscle atrophy F-box (MAFbx) in the presence or absence of whey protein ingestion. We hypothesized that resistance exercise in combination with whey protein ingestion alters the gene expression of proteins related to muscle protein synthesis (mTOR) and/or degradation (MuRF-1 and MAFbx), thus affecting muscle weight gain in rats. MethodsThirty-two male Fischer rats were randomly assigned to the following four experimental groups (n = 8/group): Control sedentary, control exercised, whey protein sedentary, and whey protein exercised. Exercise consisted of inducing the animals to perform sets of jumps for 8 wk. Body weight gain, muscle weights, food intake, and feeding efficiency were evaluated. Gene expressions were analyzed by quantitative real-time reverse transcription polymerase chain reaction. Statistical evaluation was performed using a two-way analysis of variance with a Tukey post hoc test. ResultsWhey protein exercised rats exhibited higher body and muscle weight gain compared with control-exercised rats (P = 0.032). The expression of mTOR was reduced by exercise but increased when whey protein was consumed as a dietary protein (P = 0.005). MuRF-1 expression was reduced by exercise (P < 0.001), whereas MAFbx was reduced only by whey protein ingestion (P = 0.008) independent of exercise. ConclusionsA reduction in MAFbx gene transcription induced by whey protein and the interaction between exercise and whey protein ingestion on mTOR gene expression contributed significantly to differences in body and muscle weight gain.

Altered gene expression patterns in muscle ring finger 1 null mice during denervation- and dexamethasone-induced muscle atrophy

Muscle atrophy can result from inactivity or unloading on one hand or the induction of a catabolic state on the other. Muscle-specific ring finger 1 (MuRF1), a member of the tripartite motif family of E3 ubiquitin ligases, is an essential mediator of multiple conditions inducing muscle atrophy. While most studies have focused on the role of MuRF1 in protein degradation, the protein may have other roles in regulating skeletal muscle mass and metabolism. We therefore systematically evaluated the effect of MuRF1 on gene expression during denervation and dexamethasone-induced atrophy. We find that the lack of MuRF1 leads to few differences in control animals, but there were several significant differences in specific sets of genes upon denervation- and dexamethasone-induced atrophy. For example, during denervation, MuRF1 knockout mice showed delayed repression of metabolic and structural genes and blunted induction of genes associated with the neuromuscular junction. In the latter case, this pattern correlates with blunted HDAC4 and myogenin upregulation. Lack of MuRF1 caused fewer changes in the dexamethasone-induced atrophy program, but certain genes involved in fat metabolism and intracellular signaling were affected. Our results demonstrate a new role for MuRF1 in influencing gene expression in two important models of muscle atrophy.

Role of ubiquitin–proteasome proteolysis in muscle fiber destruction in experimental chloroquine‐induced myopathy

Previous studies have documented the presence of rimmed vacuoles, atrophic fibers, and increased lysosomal cathepsin activity in skeletal muscle from animal models of chloroquine-induced myopathy, suggesting that muscle fibers in this type of myopathy may be degraded via the lysosomal-proteolysis pathway. Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, in this study we chose to examine the significance of the ubiquitin-proteasome proteolytic system in the process of muscle fiber destruction in experimental chloroquine myopathy. Expression of ubiquitin, 26S proteasome proteins, and ubiquitin ligases, such as muscle-specific RING finger-1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (MAFbx), was analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Abnormal accumulation of rimmed vacuoles was observed only in chloroquine-treated denervated muscles. Ubiquitin and proteasome immunostaining, and ubiquitin, MuRF-1, and atrogin-1/MAFbx mRNAs were significantly increased in denervated soleus muscles from saline- and chloroquine-treated rats when compared with contralateral innervated muscles. Further, ubiquitin and ubiquitin ligase mRNA levels were higher in denervated muscles from chloroquine-treated rats when compared with saline-treated rats. These data demonstrate increased proteasomes and ubiquitin in denervated muscles from chloroquine-treated rats and suggest that the ubiquitin-proteasome proteolysis pathway as well as the lysosomal-proteolysis pathway mediate muscle fiber destruction in experimental chloroquine myopathy.