Dataset on transcriptome signature of skeletal muscle of young, adult and aged mice.

This data article reports the level of expression of messenger RNA (mRNA) obtained from a set of 18 skeletal muscle samples using Affymetrix Genechips Exon arrays. Data were obtained from Gastrocnemius muscle of C57BL6 male mice at 3 distinct age groups, 2, 11 and 25 months old representing young, mature adult and aged groups. The data submitted to GEO constitute a large dataset of 15,300 mRNA levels. The data include eighteen .CEL files obtained after scanning mouse exon arrays and one .xls file obtained after processing with Genespring GX 14.9. Three distinct files containing affymetrix data processed using Genespring and analyzed for differences between stages 2 per 2 are provided as supplementary data.

A Single Bout of Ultra-Endurance Exercise Reveals Early Signs of Muscle Aging in Master Athletes.

Middle-aged and master endurance athletes exhibit similar physical performance and long-term muscle adaptation to aerobic exercise. Nevertheless, we hypothesized that the short-term plasticity of the skeletal muscle might be distinctly altered for master athletes when they are challenged by a single bout of prolonged moderate-intensity exercise. Six middle-aged (37Y) and five older (50Y) master highly-trained athletes performed a 24-h treadmill run (24TR). Vastus lateralis muscle biopsies were collected before and after the run and assessed for proteomics, fiber morphometry, intramyocellular lipid droplets (LD), mitochondrial oxidative activity, extracellular matrix (ECM), and micro-vascularisation. Before 24TR, muscle fiber type morphometry, intramyocellular LD, oxidative activity, ECM and micro-vascularisation were similar between master and middle-aged runners. For 37Y runners, 24TR was associated with ECM thickening, increased capillary-to-fiber interface, and an 89% depletion of LD in type-I fibers. In contrast, for 50Y runners, 24TR did not alter ECM and capillarization and poorly depleted LDs. Moreover, an impaired succinate dehydrogenase activity and functional class scoring of proteomes suggested reduced oxidative phosphorylation post-24TR exclusively in 50Y muscle. Collectively, our data support that middle-aged and master endurance athletes exhibit distinct transient plasticity in response to a single bout of ultra-endurance exercise, which may constitute early signs of muscle aging for master athletes.

Magnesium Homeostasis in Myogenic Differentiation-A Focus on the Regulation of TRPM7, MagT1 and SLC41A1 Transporters.

Magnesium (Mg) is essential for skeletal muscle health, but little is known about the modulation of Mg and its transporters in myogenic differentiation. Here, we show in C2C12 murine myoblasts that Mg concentration fluctuates during their differentiation to myotubes, declining early in the process and reverting to basal levels once the cells are differentiated. The level of the Mg transporter MagT1 decreases at early time points and is restored at the end of the process, suggesting a possible role in the regulation of intracellular Mg concentration. In contrast, TRPM7 is rapidly downregulated and remains undetectable in myotubes. The reduced amounts of TRPM7 and MagT1 are due to autophagy, one of the proteolytic systems activated during myogenesis and essential for the membrane fusion process. Moreover, we investigated the levels of SLC41A1, which increase once cells are differentiated, mainly through transcriptional regulation. In conclusion, myogenesis is associated with alterations of Mg homeostasis finely tuned through the modulation of MagT1, TRPM7 and SLC41A1.

Association Between Physical Activity, Quadriceps Muscle Performance, and Biological Characteristics of Very Old Men and Women.

The aim of the study was to evaluate the association between physical activity, knee extensors (KE) performance (ie, isometric strength and fatigability), and biological parameters (ie, muscle structural, microvascular, and metabolic properties) in healthy very old men and women. Thirty very old adults (82 ± 1 years, 15 women) performed an isometric Quadriceps Intermittent Fatigue (QIF) test for the assessment of KE maximal force, total work (index of absolute performance), and fatigability. Muscle biopsies from the vastus lateralis muscle were collected to assess muscle fibers type and morphology, microvasculature, and enzymes activity. Correlation analyses were used to investigate the relationships between physical activity (steps/day, actimetry), KE performance, and biological data for each sex separately. Men, compared to women, showed greater total work at the QIF test (44 497 ± 8 629 Ns vs 26 946 ± 4 707 Ns; p < .001). Steps per day were correlated with total work only for women (r = 0.73, p = .011). In men, steps per day were correlated with the percentage (r = 0.57, p = .033), shape factor (r = 0.75, p = .002), and capillary tortuosity of type IIX fibers (r = 0.59, p = .035). No other relevant correlations were observed for men or women between steps per day and biological parameters. Physical activity level was positively associated with the capacity of very old women to perform a fatiguing test, but not maximal force production capacity of the KE. Physical activity of very old men was not correlated with muscle performance. We suggest that very old women could be at higher risk of autonomy loss and increasing the steps per day count could provide a sufficient stimulus for adaptations in less active women.

A Mix of Dietary Fibres Changes Interorgan Nutrients Exchanges and Muscle-Adipose Energy Handling in Overfed Mini-Pigs.

This study evaluates the capacity of a bread enriched with fermentable dietary fibres to modulate the metabolism and nutrients handling between tissues, gut and peripheral, in a context of overfeeding. Net fluxes of glucose, lactate, urea, short chain fatty acids (SCFA), and amino acids were recorded in control and overfed female mini-pigs supplemented or not with fibre-enriched bread. SCFA in fecal water and gene expressions, but not protein levels or metabolic fluxes, were measured in muscle, adipose tissue, and intestine. Fibre supplementation increased the potential for fatty acid oxidation and mitochondrial activity in muscle (acox, ucp2, sdha and cpt1-m, p < 0.05) as well as main regulatory transcription factors of metabolic activity such as pparα, pgc-1α and nrf2. All these features were associated with a reduced muscle fibre cross sectional area, resembling to controls (i.e., lean phenotype). SCFA may be direct inducers of these cross-talk alterations, as their feces content (+52%, p = 0.05) was increased in fibre-supplemented mini-pigs. The SCFA effects could be mediated at the gut level by an increased production of incretins (increased gcg mRNA, p < 0.05) and an up-regulation of SCFA receptors (increased gpr41 mRNA, p < 0.01). Hence, consumption of supplemented bread with fermentable fibres can be an appropriate strategy to activate muscle energy catabolism and limit the establishment of an obese phenotype.

Concurrent BMP Signaling Maintenance and TGF-ß Signaling Inhibition Is a Hallmark of Natural Resistance to Muscle Atrophy in the Hibernating Bear.

Muscle atrophy arises from a multiplicity of physio-pathological situations and has very detrimental consequences for the whole body. Although knowledge of muscle atrophy mechanisms keeps growing, there is still no proven treatment to date. This study aimed at identifying new drivers for muscle atrophy resistance. We selected an innovative approach that compares muscle transcriptome between an original model of natural resistance to muscle atrophy, the hibernating brown bear, and a classical model of induced atrophy, the unloaded mouse. Using RNA sequencing, we identified 4415 differentially expressed genes, including 1746 up- and 2369 down-regulated genes, in bear muscles between the active versus hibernating period. We focused on the Transforming Growth Factor (TGF)-ß and the Bone Morphogenetic Protein (BMP) pathways, respectively, involved in muscle mass loss and maintenance. TGF-ß- and BMP-related genes were overall down- and up-regulated in the non-atrophied muscles of the hibernating bear, respectively, and the opposite occurred for the atrophied muscles of the unloaded mouse. This was further substantiated at the protein level. Our data suggest TGF-ß/BMP balance is crucial for muscle mass maintenance during long-term physical inactivity in the hibernating bear. Thus, concurrent activation of the BMP pathway may potentiate TGF-ß inhibiting therapies already targeted to prevent muscle atrophy.

UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin.

The ubiquitin proteasome system (UPS) is the main player of skeletal muscle wasting, a common characteristic of many diseases (cancer, etc.) that negatively impacts treatment and life prognosis. Within the UPS, the E3 ligase MuRF1/TRIM63 targets for degradation several myofibrillar proteins, including the main contractile proteins alpha-actin and myosin heavy chain (MHC). We previously identified five E2 ubiquitin-conjugating enzymes interacting with MuRF1, including UBE2L3/UbcH7, that exhibited a high affinity for MuRF1 (KD = 50 nM). Here, we report a main effect of UBE2L3 on alpha-actin and MHC degradation in catabolic C2C12 myotubes. Consistently UBE2L3 knockdown in Tibialis anterior induced hypertrophy in dexamethasone (Dex)-treated mice, whereas overexpression worsened the muscle atrophy of Dex-treated mice. Using combined interactomic approaches, we also characterized the interactions between MuRF1 and its substrates alpha-actin and MHC and found that MuRF1 preferentially binds to filamentous F-actin (KD = 46.7 nM) over monomeric G-actin (KD = 450 nM). By contrast with actin that did not alter MuRF1-UBE2L3 affinity, binding of MHC to MuRF1 (KD = 8 nM) impeded UBE2L3 binding, suggesting that differential interactions prevail with MuRF1 depending on both the substrate and the E2. Our data suggest that UBE2L3 regulates contractile proteins levels and skeletal muscle atrophy.

Magnesium Deficiency Alters Expression of Genes Critical for Muscle Magnesium Homeostasis and Physiology in Mice.

Chronic Mg2+ deficiency is the underlying cause of a broad range of health dysfunctions. As 25% of body Mg2+ is located in the skeletal muscle, Mg2+ transport and homeostasis systems (MgTHs) in the muscle are critical for whole-body Mg2+ homeostasis. In the present study, we assessed whether Mg2+ deficiency alters muscle fiber characteristics and major pathways regulating muscle physiology. C57BL/6J mice received either a control, mildly, or severely Mg2+-deficient diet (0.1%; 0.01%; and 0.003% Mg2+ wt/wt, respectively) for 14 days. Mg2+ deficiency slightly decreased body weight gain and muscle Mg2+ concentrations but was not associated with detectable variations in gastrocnemius muscle weight, fiber morphometry, and capillarization. Nonetheless, muscles exhibited decreased expression of several MgTHs (MagT1, CNNM2, CNNM4, and TRPM6). Moreover, TaqMan low-density array (TLDA) analyses further revealed that, before the emergence of major muscle dysfunctions, even a mild Mg2+ deficiency was sufficient to alter the expression of genes critical for muscle physiology, including energy metabolism, muscle regeneration, proteostasis, mitochondrial dynamics, and excitation-contraction coupling.

Magnesium Influences Membrane Fusion during Myogenesis by Modulating Oxidative Stress in C2C12 Myoblasts.

Magnesium (Mg) is essential to skeletal muscle where it plays a key role in myofiber relaxation. Although the importance of Mg in the mature skeletal muscle is well established, little is known about the role of Mg in myogenesis. We studied the effects of low and high extracellular Mg in C2C12 myogenic differentiation. Non-physiological Mg concentrations induce oxidative stress in myoblasts. The increase of reactive oxygen species, which occurs during the early phase of the differentiation process, inhibits myoblast membrane fusion, thus impairing myogenesis. Therefore, correct Mg homeostasis, also maintained through a correct dietary intake, is essential to assure the regenerative capacity of skeletal muscle fibers.

Muscle Proteomic and Transcriptomic Profiling of Healthy Aging and Metabolic Syndrome in Men.

(1) Background: Aging is associated with a progressive decline in muscle mass and function. Aging is also a primary risk factor for metabolic syndrome, which further alters muscle metabolism. However, the molecular mechanisms involved remain to be clarified. Herein we performed omic profiling to decipher in muscle which dominating processes are associated with healthy aging and metabolic syndrome in old men. (2) Methods: This study included 15 healthy young, 15 healthy old, and 9 old men with metabolic syndrome. Old men were selected from a well-characterized cohort, and each vastus lateralis biopsy was used to combine global transcriptomic and proteomic analyses. (3) Results: Over-representation analysis of differentially expressed genes (ORA) and functional class scoring of pathways (FCS) indicated that healthy aging was mainly associated with upregulations of apoptosis and immune function and downregulations of glycolysis and protein catabolism. ORA and FCS indicated that with metabolic syndrome the dominating biological processes were upregulation of proteolysis and downregulation of oxidative phosphorylation. Proteomic profiling matched 586 muscle proteins between individuals. The proteome of healthy aging revealed modifications consistent with a fast-to-slow transition and downregulation of glycolysis. These transitions were reduced with metabolic syndrome, which was more associated with alterations in NADH/NAD+ shuttle and ß-oxidation. Proteomic profiling further showed that all old muscles overexpressed protein chaperones to preserve proteostasis and myofiber integrity. There was also evidence of aging-related increases in reactive oxygen species but better detoxifications of cytotoxic aldehydes and membrane protection in healthy than in metabolic syndrome muscles. (4) Conclusions: Most candidate proteins and mRNAs identified herein constitute putative muscle biomarkers of healthy aging and metabolic syndrome in old men.

Mitophagy and Mitochondria Biogenesis Are Differentially Induced in Rat Skeletal Muscles during Immobilization and/or Remobilization.

Mitochondria alterations are a classical feature of muscle immobilization, and autophagy is required for the elimination of deficient mitochondria (mitophagy) and the maintenance of muscle mass. We focused on the regulation of mitochondrial quality control during immobilization and remobilization in rat gastrocnemius (GA) and tibialis anterior (TA) muscles, which have very different atrophy and recovery kinetics. We studied mitochondrial biogenesis, dynamic, movement along microtubules, and addressing to autophagy. Our data indicated that mitochondria quality control adapted differently to immobilization and remobilization in GA and TA muscles. Data showed i) a disruption of mitochondria dynamic that occurred earlier in the immobilized TA, ii) an overriding role of mitophagy that involved Parkin-dependent and/or independent processes during immobilization in the GA and during remobilization in the TA, and iii) increased mitochondria biogenesis during remobilization in both muscles. This strongly emphasized the need to consider several muscle groups to study the mechanisms involved in muscle atrophy and their ability to recover, in order to provide broad and/or specific clues for the development of strategies to maintain muscle mass and improve the health and quality of life of patients.

Lipid accumulation and mitochondrial abnormalities are associated with fiber atrophy in the skeletal muscle of rats with collagen-induced arthritis.

Rheumatoid arthritis (RA) has a negative impact on muscle mass, and reduces patient's mobility and autonomy. Furthermore, RA is associated with metabolic comorbidities, notably in lipid homeostasis by unknown mechanisms. To understand the links between the loss in muscle mass and the metabolic abnormalities, arthritis was induced in male Sprague Dawley rats (n = 11) using the collagen-induced arthritis model. Rats immunized with bovine type II collagen were compared to a control group of animals (n = 11) injected with acetic acid and complete Freund's adjuvant. The clinical severity of the ensuing arthritis was evaluated weekly by a semi-quantitative score. Skeletal muscles from the hind limb were used for the histological analysis and exploration of mitochondrial activity, lipid accumulation, metabolism and regenerative capacities. A significant atrophy in tibialis anterior muscle fibers was observed in the arthritic rats despite a non-significant decrease in the weight of the muscles. Despite moderate inflammation, accumulation of triglycerides (P < 0.05), reduced mitochondrial DNA copy number (P < 0.05) and non-significant dysfunction in mitochondrial cytochrome c oxidase activity were found in the gastrocnemius muscle. Concomitantly, our results suggested an activation of the muscle specific E3 ubiquitin ligases MuRF-1 and MAFbx. Finally, the adipose tissue from the arthritic rats exhibited decreased PPARγ mRNA suggesting reduced adipogenic capacities. In conclusion, the reduced adipose tissue adipogenic capacity and skeletal muscle mitochondrial capacity are probably involved in the activation of protein catabolism, inhibition of myogenesis, accumulation of lipids and fiber atrophy in the skeletal muscle during RA.

Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients.

Sickle cell disease (SCD) is a genetic hemoglobinopathy leading to 2 major clinical manifestations: severe chronic hemolytic anemia and iterative vaso-occlusive crises. SCD is also accompanied by profound muscle microvascular remodeling. The beneficial effects of endurance training on microvasculature are widely known. The aim of this study was to evaluate the effects of an endurance training program on microvasculature of skeletal muscle in SCD patients. A biopsy of the vastus lateralis muscle and submaximal incremental exercise was performed before and after the training period. Of the 40 randomized SCD patients, complete data sets from 32 patients were obtained. The training group (n = 15) followed a personalized moderate-intensity endurance training program, while the nontraining (n = 17) group maintained a normal lifestyle. Training consisted of three 40-minute cycle ergometer exercise sessions per week for 8 weeks. Histological analysis highlighted microvascular benefits in the training SCD patients compared with nontraining patients, including increases in capillary density (P = .003), number of capillaries around a fiber (P = .015), and functional exchange surface (P < .0001). Conversely, no significant between-group difference was found in the morphology of capillaries. Indexes of physical ability also improved in the training patients. The moderate-intensity endurance exercise training program improved the muscle capillary network and partly reversed the microvascular defects commonly observed in skeletal muscle of SCD patients. This trial was registered at www.clinicaltrials.gov as #NCT02571088.

Muscle wasting in patients with end-stage renal disease or early-stage lung cancer: common mechanisms at work.

BACKGROUND: Loss of muscle mass worsens many diseases such as cancer and renal failure, contributes to the frailty syndrome, and is associated with an increased risk of death. Studies conducted on animal models have revealed the preponderant role of muscle proteolysis and in particular the activation of the ubiquitin proteasome system (UPS). Studies conducted in humans remain scarce, especially within renal deficiency. Whether a shared atrophying programme exists independently of the nature of the disease remains to be established. The aim of this work was to identify common modifications at the transcriptomic level or the proteomic level in atrophying skeletal muscles from cancer and renal failure patients. METHODS: Muscle biopsies were performed during scheduled interventions in early-stage (no treatment and no detectable muscle loss) lung cancer (LC), chronic haemodialysis (HD), or healthy (CT) patients (n = 7 per group; 86% male; 69.6 ± 11.4, 67.9 ± 8.6, and 70.2 ± 7.9 years P > 0.9 for the CT, LC, and HD groups, respectively). Gene expression of members of the UPS, autophagy, and apoptotic systems was measured by quantitative real-time PCR. A global analysis of the soluble muscle proteome was conducted by shotgun proteomics for investigating the processes altered. RESULTS: We found an increased expression of several UPS and autophagy-related enzymes in both LC and HD patients. The E3 ligases MuRF1 (+56 to 78%, P < 0.01), MAFbx (+68 to 84%, P = 0.02), Hdm2 (+37 to 59%, P = 0.02), and MUSA1/Fbxo30 (+47 to 106%, P = 0.01) and the autophagy-related genes CTPL (+33 to 47%, P = 0.03) and SQSTM1 (+47 to 137%, P < 0.01) were overexpressed. Mass spectrometry identified >1700 proteins, and principal component analysis revealed three differential proteomes that matched to the three groups of patients. Orthogonal partial least square discriminant analysis created a model, which distinguished the muscles of diseased patients (LC or HD) from those of CT subjects. Proteins that most contributed to the model were selected. Functional analysis revealed up to 238 proteins belonging to nine metabolic processes (inflammatory response, proteolysis, cytoskeleton organization, glucose metabolism, muscle contraction, oxidant detoxification, energy metabolism, fatty acid metabolism, and extracellular matrix) involved in and/or altered by the atrophying programme in both LC and HD patients. This was confirmed by a co-expression network analysis. CONCLUSIONS: We were able to identify highly similar modifications of several metabolic pathways in patients exhibiting diseases with different aetiologies (early-stage LC vs. long-term renal failure). This strongly suggests that a common atrophying programme exists independently of the disease in human.

A mix of dietary fermentable fibers improves lipids handling by the liver of overfed minipigs.

Obesity induced by overfeeding ultimately can lead to nonalcoholic fatty liver disease, whereas dietary fiber consumption is known to have a beneficial effect. We aimed to determine if a supplementation of a mix of fibers (inulin, resistant starch and pectin) could limit or alleviate overfeeding-induced metabolic perturbations. Twenty female minipigs were fed with a control diet (C) or an enriched fat/sucrose diet supplemented (O + F) or not (O) with fibers. Between 0 and 56 days of overfeeding, insulin (+88%), HOMA (+102%), cholesterol (+45%) and lactate (+63%) were increased, without any beneficial effect of fibers supplementation. However, fibers supplementation limited body weight gain (vs. O, -15% at D56) and the accumulation of hepatic lipids droplets induced by overfeeding. This could be explained by a decreased lipids transport potential (-50% FABP1 mRNA, O + F vs. O) inducing a down-regulation of regulatory elements of lipids metabolism / lipogenesis (-36% SREBP1c mRNA, O + F vs. O) but not to an increased oxidation (O + F not different from O and C for proteins and mRNA measured). Glucose metabolism was also differentially regulated by fibers supplementation, with an increased net hepatic release of glucose in the fasted state (diet × time effect, P<.05 at D56) that can be explained partially by a possible increased glycogen synthesis in the fed state (+82% GYS2 protein, O + F vs. O, P=.09). The direct role of short chain fatty acids on gluconeogenesis stimulation is questioned, with probably a short-term impact (D14) but no effect on a long-term (D56) basis.

Magnesium transport and homeostasis-related gene expression in skeletal muscle of young and old adults: analysis of the transcriptomic data from the PROOF cohort Study.

Magnesium (Mg2+) is critical for a number of biological processes and 25% body Mg2+ is located in the skeletal muscle. Mg2+ transport and homeostasis systems (MgTHs) regulate intracellular Mg2+ concentration and muscle MgTHs are thus related to whole body Mg2+ homeostasis. Nonetheless, few studies have investigated the regulation of muscle MgTHs under (patho)physiological conditions. Herein, we assessed the relationship between the expression of MgTHs genes (Trpm6, Trpm7, Magt1, Mrs2, Cnnm1-4, Slc41a1-3) and relevant pathways in human sarcopenia, which is one of the most dramatic physiologic changes affecting the human body. Transcriptomic data were compared between young adult (YO, 22 y, n = 11) and old (EL, 73 y, n = 13) men from the PROOF cohort. MgTH mRNA levels did not change with aging, with the exception of a slight decrease for Slc41a3. Nevertheless, interindividual variations of mRNA levels revealed strong correlations between MgTHs in the YO group, while few were maintained in the EL muscle. Moreover, in the YO muscle, different clusters of MgTH mRNAs strongly correlated with divers physiological (BMI, blood pressure) and muscle characteristics (intramyocellular droplets, capillarization); however, most correlations changed or disappeared in the EL muscle. Further investigations of the whole transcriptome identified several sets of mRNAs correlated with defined MgTHs. There again was a sharp difference between YO and EL muscles, as the number of mRNAs correlated with MgTHs strongly decreased with aging. Gene ontology analyses of these sets of correlated mRNAs revealed 6 biological processes common to YO and EL, 3 specific to the YO (RNA processing, translation, respiration), and 2 (regulation of catabolic process, Wnt signaling) to the EL muscle. Overall, these observations lead to questions about potential resilience to muscle Mg2+ homeostasis in the elderly.

Muscle Loss Associated Changes of Oxylipin Signatures During Biological Aging: An Exploratory Study From the PROOF Cohort.

Characterizations of the multiple mechanisms determining biological aging are required to better understand the etiology and identify early biomarkers of sarcopenia. Oxylipins refer to a large family of signaling lipids involved in the regulation of various biological processes that become dysregulated during aging. To investigate whether comprehensive oxylipin profiling could provide an integrated and fine characterization of the early phases of sarcopenia, we performed a quantitative targeted metabolomics of oxylipins in plasma of 81-year-old subjects from the PROOF cohort with decreased (n = 12), stable (n = 16), or increased appendicular muscle mass (n = 14). Multivariate and univariate analyses identified significant and concordant changes of oxylipin profiles according to the muscle status. Of note, 90% of the most discriminant oxylipins were derived from EPA and DHA and were increased in the sarcopenic subjects. The oxylipins signatures of sarcopenic subjects revealed subtle activation of inflammatory resolution pathways, coagulation processes, and oxidative stress as well as the inhibition of angiogenesis. Heat maps highlighted relationships between oxylipins and the cardiometabolic health parameters which were mainly lost in sarcopenic subjects. This exploratory study supports that targeted metabolomics of oxylipins could provide relevant and subtle characterization of early disturbances associated with muscle loss during aging.

Erratum: Polge, C., et al. UBE2E1 Is Preferentially Expressed in the Cytoplasm of Slow-Twitch Fibers and Protects Skeletal Muscles from Exacerbated Atrophy upon Dexamethasone Treatment. Cells 2018, 7, 214.

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Increased Serpina3n release into circulation during glucocorticoid-mediated muscle atrophy.

BACKGROUND: Glucocorticoids (GC) play a major role in muscle atrophy. As skeletal muscle is a secretory organ, characterization of the muscle secretome elicited by muscle atrophy should allow to better understand the cellular mechanisms and to identify circulating biomarkers of this condition. Our project aimed to identify the changes in the muscle secretome associated with GC-induced muscle atrophy and susceptible to translate into circulation. METHODS: We have identified the GC-induced changes in the secretome of C2 C12 muscle cells by proteomic analysis, and then, we have determined how these changes translate into the circulation of mice or human subjects exposed to high concentrations of GC. RESULTS: This approach led us to identify Serpina3n as one of the most markedly secreted protein in response to GC. Our original in vitro results were confirmed in vivo by an increased expression of Serpina3n in skeletal muscle (3.9-fold; P < 0.01) and in the serum (two-fold; P < 0.01) of mice treated with GC. We also observed increased levels of the human orthologue Serpina3 in the serum of Cushing's syndrome patients compared with healthy controls matched for age and sex (n = 9/group, 2.5-fold; P < 0.01). An increase of Serpina3n was also demonstrated in muscle atrophy models mediated by GC such as cancer cachexia (four-fold; P < 0.01), sepsis (12.5-fold; P < 0.001), or diabetes (two-fold; P < 0.01). In contrast, levels of Serpina3n both in skeletal muscle and in the circulation were reduced in several models of muscle hypertrophy induced by myostatin inhibition (P < 0.01). Furthermore, a cluster of data suggests that the regulation of muscle Serpina3n involves mTOR, an essential determinant of the muscle cell size. CONCLUSIONS: Taken together, these data suggest that Serpina3n may represent a circulating biomarker of muscle atrophy associated to GC and, broadly, a reflection of dynamic changes in muscle mass.

A muscle-specific MuRF1-E2 network requires stabilization of MuRF1-E2 complexes by telethonin, a newly identified substrate.

BACKGROUND: Muscle wasting is observed in the course of many diseases and also during physiological conditions (disuse, ageing). Skeletal muscle mass is largely controlled by the ubiquitin-proteasome system and thus by the ubiquitinating enzymes (E2s and E3s) that target substrates for subsequent degradation. MuRF1 is the only E3 ubiquitin ligase known to target contractile proteins (α-actin, myosins) during catabolic situations. However, MuRF1 depends on E2 ubiquitin-conjugating enzymes for ubiquitin chain formation on the substrates. MuRF1-E2 couples are therefore putative targets for preventing muscle wasting. METHODS: We focused on 14 E2 enzymes that are either expressed in skeletal muscle or up-regulated during atrophying conditions. In this work, we demonstrated that only highly sensitive and complementary interactomic approaches (surface plasmon resonance, yeast three-hybrid, and split green fluorescent protein) allowed the identification of MuRF1 E2 partners. RESULTS: Five E2 enzymes physically interacted with MuRF1, namely, E2E1, E2G1, E2J1, E2J2, and E2L3. Moreover, we demonstrated that MuRF1-E2E1 and MuRF1-E2J1 interactions are facilitated by telethonin, a newly identified MuRF1 substrate. We next showed that the five identified E2s functionally interacted with MuRF1 since, in contrast to the non-interacting E2D2, their co-expression in HEK293T cells with MuRF1 led to increased telethonin degradation. Finally, we showed that telethonin governed the affinity between MuRF1 and E2E1 or E2J1. CONCLUSIONS: We report here the first MuRF1-E2s network, which may prove valuable for deciphering the precise mechanisms involved in the atrophying muscle programme and for proposing new therapeutical approaches.