Supplementary figure, tables and results Supplementary Figure S1. Flowchart of the 6 studies Supplementary Table S1. Transitions and collision energies used for the quantification (Bold) and confirmation (Italic) of AAH on the triple quadrupole instrument. Supplementary Table S2. Active compounds (extract, molecule and family), used quantity and radical scavenger activity results of marinade used in the study 1. Supplementary Table S3. Effect of grape-olive marinade and of cooking on lipid peroxidation (TBARs) and on iron levels in fresh red meats. Data are presented as mean {plus minus} SD. a two-way ANOVA assessing marinade and cooking factors. Supplementary Table S4. Effect of meat, marinade and cooking on heme content and lipoperoxidation biomarkers in feces from rats fed a no-meat control diet or experimental red meats for 14 days. Data are presented as mean {plus minus} SD. a The effect of meat intake was evaluated by a one-way ANOVA and the effect of marinated and cooked meat intake by two-way ANOVA.

The ubiquitin-proteasome system (UPS) degrades most cell proteins and is involved in the control of many major biological functions. The ubiquitination/deubiquitination machinery is responsible for the specific tagging and proofreading of substrates degraded by the 26S proteasome, but ubiquitination itself also serves other functions. Poly-ubiquitinated substrates are recognized by the 26S proteasome and degraded into peptides, but mammalian cells contain multiple ubiquitin- and/or proteasome-dependent pathways. The precise role of these systems in respiratory diseases is still unclear. The proteasome is activated in mechanical-ventilation associated diaphragmatic atrophy and in patients with chronic obstructive pulmonary diseases who exhibit muscle wasting.

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.

AbstractBackgroundLoss 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.MethodsMuscle 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.ResultsWe 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.ConclusionsWe 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.

Abstract Red meat is probably carcinogenic to humans (WHO/IARC class 2A), in part through heme iron-induced lipoperoxidation. Here, we investigated whether red meat promotes carcinogenesis in rodents and modulates associated biomarkers in volunteers, speculating that an antioxidant marinade could suppress these effects via limitation of the heme induced lipid peroxidation. We gave marinated or non-marinated beef with various degrees of cooking to azoxymethane-initiated rats, Min mice, and human volunteers (crossover study). Mucin-depleted foci were scored in rats, adenoma in Min mice. Biomarkers of lipoperoxidation were measured in the feces and urine of rats, mice, and volunteers. The organoleptic properties of marinated meat were tested. Fresh beef increased colon carcinogenesis and lipoperoxidation in rats and mice and lipoperoxidation in humans. Without an adverse organoleptic effect on meat, marinade normalized peroxidation biomarkers in rat and mouse feces, reduced peroxidation in human feces and reduced the number of Mucin-depleted foci in rats and adenoma in female Min mice. This could lead to protective strategies to decrease the colorectal cancer burden associated with red meat consumption. Cancer Prev Res; 11(9); 569–80. ©2018 AACR.

Change in author names (order). [...]

Introduction: Muscle atrophy is frequently encountered in diseased patients. It contributes to patient’s frailty and is associated with an increased risk of death. Studies using animal models suggest the involvement of the Ubiquitin Proteasome System (UPS) in renal failure-induced muscle atrophy. However, this remains to be established in humans. Another important goal is to detect markers that may help fighting against muscle atrophy through nutritional or pharmacological strategies. Indeed, it is very difficult to counteract the increased proteolysis when it is established. Our objectives were (i) to identify the proteolytic systems activated in chronic hemodialysis (HD) or lung cancer (LC) patients, i.e. pathologies having a different etiology and (ii) to identify markers specific to the activation of muscle atrophy processes independently of the pathology per se. Methods: Muscle biopsies (n = 7 per group) were obtained upon programmed surgery. mRNA and protein levels were determined using qRT-PCR, immunoblotting and proteomic approaches. Results: We found that the UPS and autophagy were activated in both HD and LC patients. Mass spectrometry analysis identified > 1700 proteins. Main component analysis revealed 3 distinct protein expression profiles corresponding to the 3 groups studied. We identified 106 proteins that were significantly modified (decreased or increased) in both HD and LC patients compared to controls (CT). Hierarchical cluster analysis showed that expression levels of these proteins distinguished diseased (HD or LC) vs. CT patients. Orthogonal partial least square discriminant analysis confirmed these results. Conclusion: We demonstrated that the UPS and autophagy were activated during long-term disease in humans. We also found a set of proteins whose expression levels may be specific of the atrophying process. These proteins constitute potential biomarkers witnessing the activation of muscle atrophy and/or potential therapeutic targets.

Skeletal muscle mass is reduced during many diseases or physiological situations (disuse, aging), which results in decreased strength and increased mortality. Muscle mass is mainly controlled by the ubiquitin-proteasome system (UPS), involving hundreds of ubiquitinating enzymes (E2s and E3s) that target their dedicated substrates for subsequent degradation. We recently demonstrated that MuRF1, an E3 ubiquitin ligase known to bind to sarcomeric proteins (telethonin, α-actin, myosins) during catabolic situations, interacts with 5 different E2 enzymes and that these E2-MuRF1 couples are able to target telethonin, a small sarcomeric protein, for degradation. Amongst the E2s interacting with MuRF1, E2E1 was peculiar as the presence of the substrate was necessary for optimal MuRF1-E2E1 interaction. In this work, we focused on the putative role of E2E1 during skeletal muscle atrophy. We found that E2E1 expression was restricted to type I and type IIA muscle fibers and was not detectable in type IIB fibers. This strongly suggests that E2E1 targets are fiber-specific and may be strongly linked to the contractile and metabolic properties of the skeletal muscle. However, E2E1 knockdown was not sufficient for preserving the protein content in C2C12 myotubes subjected to a catabolic state (dexamethasone treatment), suggesting that E2E1 is not involved in the development of muscle atrophy. By contrast, E2E1 knockdown aggravated the atrophying process in both catabolic C2C12 myotubes and the Tibialis anterior muscle of mice, suggesting that E2E1 has a protective effect on muscle mass.

Une perte de masse musculaire complique de nombreuses situations pathologiques telles le cancer ou l'insuffisance rénale. Elle contribue au syndrome de fragilité et est associée à un risque accru de décès. Les études conduites sur des modèles animaux ont révélées le rôle prépondérant de l'activation des systèmes de protéolyse musculaire et notamment du système ubiquitine protéasome (UPS). Cependant, les études conduites chez l'homme restent rares. L'objectif de ce travail était donc d'identifier les mécanismes communs qui caractérisent les muscles en situation d'atrophie chez l'homme. Des biopsies musculaires chirurgicales ont été réalisées au cours d'interventions programmées chez des patient ayant un cancer bronchique (LC, n = 7), hémodialysés chroniques (HD, n = 7) ou indemnes de pathologies (CT, n = 7). Une quantification de l'expression de gènes des systèmes protéolytiques ubiquitine protéasome, de l'autophagie et de l'apoptose a été réalisée par qRT-PCR. Une analyse globale du protéome musculaire soluble a été conduite par Shot-Gun en spectrométrie de masse. Nous avons retrouvé une augmentation de l'expression de plusieurs acteurs de l'UPS et de l'autophagie chez les patients LC et HD, et de marqueurs d'apoptose chez les patients HD. La spectrométrie de masse nous a permis d'identifier plus de 1700 protéines. Une ACP a révélé 3 profils protéomiques différents correspondant aux trois groupes de patients. Une O-PLS-DA a été utilisée pour créer un modèle permettant de distinguer les muscles des patients malades de ceux des patients sains. Les protéines qui contribuaient le plus au modèle ont été retenues. Une analyse fonctionnelle a permis de déterminer que ces protéines étaient impliquées dans la protéolyse, le métabolisme des acides aminés, la glycolyse, la phosphorylation oxydative, la défense contre le stress oxydant, la contraction musculaire, la matrice extracellulaire et l'organisation du cytosquelette musculaire. Une analyse de réseau de co-expression a retrouvé des résultats concordant. Enfin, une étude de réseau d'interaction est en faveur d'une activation de la voie Wnt-bêtacaténine. Cette étude démontre chez l'homme, l'exécution d'un programme d'atrophie musculaire commun indépendamment de la pathologie causale. Il existe une activation de l'UPS mais aussi de l'autophagie. Elle s'accompagne de modification métaboliques caractérisée par une augmentation de la glycolyse et une dysfonction mitochondriale. Une augmentation du stress oxydant participerait à l'induction de ces changements. L'activation continue de la voie Wnt serait à l'origine de fibrose musculaire. Les mécanismes en jeu sont autant de pistes pour la mise au point de traitements de l'atrophie musculaire.

Periods of immobilization or acute inactivity are associated with weakness and/or frailty, and further contribute to muscle atrophy and elevated healthcare costs. Muscle wasting is often associated with mitochondria (mt) alterations and autophagy is required for muscle mass maintenance through the elimination of defective mitochondria. Tibialis anterior (TA) and gastrocnemius (GA) rat muscles exhibit different mitochondria-associated apoptotic responses to immobilization and recovery (1). We therefore investigated the regulation of mitochondria quality control in the TA and the GA following 8 days of immobilization (I8) and 6 days of remobilization (R6) in rats. The mtDNA / genomic DNA ratio decreased in both muscles at I8 and R6, suggesting a decrease in mitochondria abundance. The levels for the fission protein Fis1 were elevated in the TA at I8 and in both muscles at R6, while levels for the fusion proteins Opa1 and Mfn2 were elevated at R6 only in the TA. Thus, fusion and fission processes were imbalanced during immobilization and remobilization, underwent muscle-specific alterations, and followed a different temporal regulation in the TA and the GA. The Pink-Parkin dependent selective pathway for addressing altered mitochondria to autophagy also showed a muscle-specific regulation. Indeed, only the immobilized and/or remobilized GA exhibited elevated mRNA levels for Pink and Parkin and protein levels for VDAC1. However, P62 protein levels increased during immobilization in both muscles at I8 and remained elevated only in the GA during remobilization. However, although this suggest a selective addressing altered mitochondria to autophagy in both muscles, Rab32 mRNA levels and LC3 lipidation increased only in the TA during immobilization and remobilization. Altogether, we suggest that the addressing altered mitochondria for elimination following immobilization involved different muscle-specific mechanisms that should be further investigated. They may include either a different temporal regulation of the Pink-Parkin dependent pathway in the GA and the TA and/or a muscle specific regulation of the LC3 dependent pathway in the TA and the GA. The latter may imply the formation of mitochondria-derived vesicles in the GA that would be directly driven to late endosomes independently of LC3. Reference (1) Slimani L, Micol D, Amat J, Delcros G, Meunier B, Taillandier D, Polge C, Bechet D, Dardevet D, Picard B, Attaix D, Listrat A, Combaret L. The worsening of tibialis anterior muscle atrophy during recovery post-immobilization correlates with enhanced connective tissue area, proteolysis, and apoptosis. Am J Physiol Endocrinol Metab. 2012, 303, E1335-47.

Abstract 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.

BackgroundSkeletal muscle protein loss is an adaptive response to various patho-physiological situations, and the ubiquitin proteasome system (UPS) is responsible for the degradation of the bulk of muscle proteins. The role of E2 ubiquitin-conjugating enzymes is still poorly understood in skeletal muscle.MethodsWe screened for E2s expression levels in C2C12 myotubes submitted to the catabolic glucocorticoid dexamethasone (Dex).ResultsOne micromolar Dex induced an accumulation of proteasome substrates (polyUb conjugates) and an overexpression of the muscle-specific E3 ligase MuRF1 and of six E2 enzymes, UBE2A, UBE2B, UBE2D1, UBE2D2, UBE2G1, and UBE2J1. However, only MuRF1 and UBE2B were sensitive to mild catabolic conditions (0.16M Dex). UBE2B knockdown induced a sharp decrease of total (-18%) and K48 (-28%) Ub conjugates, that is, proteasome substrates, indicating an important role of UBE2B in the overall protein breakdown in catabolic myotubes.ConclusionsInterestingly, these results indicate an important role of UBE2B on muscle protein homeostasis during catabolic conditions.

TGF-β1 (transforming growth factor β1) was considered to play a critical role in the forming of hypertrophic scars. Smad, as a kind of signal downstream mediators, can modulate the functions of TGF-β1. Smad7 can regulate TGF-β1/Smad pathway and present negative feedbacks, which prevents fibrosis mediated by TGF-β1. Nonetheless, the mechanisms related to Smad7 activity in regulating hypertrophic scarring are hardly known. The studies have shown that Smad7 decrease induced by the increase of Smurf2 (Smad ubiquitination regulatory factor 2, an E3 ubiquitin ligase of Smad7) ubiquitination degradation plays a part in fibrosis. We thus made a hypothesis that Smad7 could not inhibit TGF-β1 because Smurf2 ubiquitin degradation was increased in hypertrophic scar fibroblasts. In our research, it was discovered that there was an increase in Smad7 mRNA levels but no increase in Smad7 protein levels in the fibroblasts of hypertrophic scars after TGF-β1 treatment. The ubiquitination activity and degradation of Smad7 protein were increased in the fibroblasts of hypertrophic scars compared with the fibroblasts of normal skin. Enhanced degradation of Smad7 protein in the fibroblasts of hypertrophic scars was prevented by proteasome inhibitors MG132 / MG115. Furthermore, it was found that TGF-β1 stimulation increased Smad7 protein expression after silencing Smurf2 gene in hypertrophic scar fibroblasts, and enhanced Smad7 degradation was prevented in hypertrophic scar fibroblasts after Smurf2 was silenced. It was implied that ubiquitin degradation mediated by Smurf2 might contribute to decreased Smad7 protein levels following TGF-β1 stimulation in the fibroblasts of hypertrophic scars.

s of the 9th International Conference on Cachexia, Sarcopenia and Muscle Wasting, Berlin, Germany, 10–11 December 2016 (part 1) 1-01 New formulation based on anti-atrophic peptides and dendrimers for skeletal muscle atrophy treatment Johanna Ábrigo, Valeria Márquez-Miranda, Juan C. Rivera, Ingrid Araya-Durán, Javier Aravena, Nicolas Pacheco, Fernando D. González-Nilo & Claudio Cabello-Verrugio Laboratory of Biology and Molecular Physiopathology, Universidad Andres Bello, CBIB, Universidad Andrés Bello, IMII, Santiago, Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile, Fundación Fraunhofer Chile Research, SantiagoChile Background: Loss of muscle strength and myofibrillar proteins are key features in skeletal muscle atrophy by disuse. One of the main mechanisms involved is the over-activation of ubiquitin–proteasome pathway (UPP). Angiotensin-(1-7) [Ang-(1-7)], a vasoactive peptide with anti-atrophic activity in skeletal muscle, is rapidly degraded in vivo and inefficient as treatment therapy. Many peptide-delivery strategies have been studied, including direct injection or administration using osmotic pumps. Dendrimers are promising vehicles for the protection and transport of numerous bioactive molecules, being hydroxyl poly(amidoamine) (PAMAM-OH) suggested as an safe drug carriers without toxic effects during in vitro and in vivo applications. Aim: The aim of the study is to evaluate the effect of Ang(1-7)/PAMAM-OH dendrimer complex intraperitoneally (IP) administered in skeletal muscle atrophy induced by disuse. Methods: C57/BL10J mice were IP injected with vehicle (PBS), Ang-(1-7), PAMAM-OH, and Ang-(1-7)/PAMAM-OH, and 24 h after, immobilized in the lower hindlimb for 24 h or 14 days. Gastrocnemius muscle was extracted, and maximal isometric strength was measured by electrophysiological analyses. Histological analyses were made by hematoxylin eosin stain. Muscle fiber cross-sectional area (CSA) was determined estimating the minimal Feret’s diameter of cryosections stained with Wheat Germ Agglutinin (WGA). Myosin Heavy Chain (MHC) levels were determined by western blot. Atrogin-1 and MuRF-1 were evaluated by RT-qPCR. Results: IP administration of Ang-(1-7)/PAMAM-OH complex, but not Ang-(1-7) alone, avoided the decrease of muscle strength, diminution of fiber diameter and decrease of MHC protein levels in the skeletal muscle, induced by disuse. Ang-(1-7)/PAMAM-OH also prevented the increment of atrogin-1 and MuRF-1 expression. Conclusion: Ang-(1-7) IP administrated as complex with PAMAM-OH dendrimer avoided the atrophic effects in skeletal muscle induced by disuse. Ang-(1-7)/PAMAM-OH complex can be an efficient method in therapy for treatment of skeletal muscle atrophy. Funding: AFM #16670; FONDECYT #1120380, 1161646; IMII #P09-016-F; UNAB DI-741-15/N; PhD Scholarship CONICYT; F.G.N.; FCR-CSB 09CEII-6991, ACT1107, and RED CYTED 214RT0482; CINV. 1-02 Endotoxin-induced autophagy dependent on Beclin1/Bcl2 complex is decreased by Angiotensin(1-7) in skeletal muscle Juan Carlos Rivera, Johanna Abrigo, Mario Chiong, Michael Bader, Robson A. Santos, Enrique Brandan, María José Acuña & Claudio Cabello-Verrugio Laboratorio de Biología y Fisiopatología Molecular, Universidad Andrés Bello, IMII, ACCDiS, Universidad de Chile, Santiago, Chile, Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany, National Institute in Science and Technology in Nanobiopharmaceutics, UFMG, Belo HorizonteBrazil, CARE, Department of Cell and Molecular Biology, Universidad Católica, Santiago, Chile Background and Aim: Autophagy is a key mechanism in endotoxin-induced cachexia by lipopolysaccharide (LPS). The vasoactive peptide of non-classical axis of renin angiotensin system, Angiotensin-(1-7) [Ang-(1-7)] and Mas receptor have anti-atrophic effects in cachexia, however, is still unknown the effect on autophagy and mechanism in cachectic skeletal muscle. Methods: C57BL/6 J (WT) or KO Mas receptor (KO) mice were treated with LPS in absence or presence of Ang-(1-7), and we evaluated autophagic proteins. C2C12 culture cells exposed to LPS and Ang-(1-7), and we analyze autophagic and MAPK proteins, and autophagosome formation. Results: Our results showed that Ang-(1-7) decreased the increment of LPS-induced LC3II/LC3I ratio protein levels in ABSTRACTS © 2016 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society of Sarcopenia, Cachexia and Wasting Disorders Journal of Cachexia, Sarcopenia and Muscle 2016; 7: 626–662 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12164 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. diaphragm muscle compared to control of WT mice. In KO Mas mice, Ang-(1-7) lost this effect. In culture cells exposed to LPS, Ang-(1-7) produced a reduction on the autophagy flux, LC3II/LC3I ratio and the amount of autophagosome. Interestingly, LPS increased autophagy by the disruption of the Beclin1/Bcl2 complex, while Ang-(1-7) restored its formation. Finally, Ang-(1-7) reduced LPS-activated p38MAPK and JNK, two signalling pathways involved in the separation of the Beclin1/Bcl2 complex in presence of LPS. Conclusion: We suggest that Ang-(1-7) is a new regulator of autophagy in endotoxin-induced cachexia by a mechanism dependent on MAPK and Beclin1/Bcl2 complex. Funding: Association-Francaise Contre Les Myopathies AFM #16670; FONDECYT #1161646, 1120380; IMII #P09-016-F; UNAB DI-741-15/N. Conicyt Scholarship #21141242 and #21161353. 1-03 Identification of E2 enzymes involved in MuRF1dependent skeletal muscle atrophy Cécile Polge, Agnès Claustre, Christiane Deval, Daniel Béchet, Lydie Combaret, Didier Attaix & Daniel Taillandier Unité de Nutrition Humaine (UMR 1019 INRA/Clermont Université), CRNH Auvergne 63000 Clermont-FerrandFrance Background and aims: The Ubiquitin Proteasome System (UPS) is the major actor of muscle wasting during various physio-pathological situations. This system involves an enzymatic cascade E1, E2, and E3. The targeting specificity of the UPS relies on the capacity of the system to first recognize (E3s) and then label (E2s) the proteins to be degraded generally with a polyubiquitin chain. E2–E3 interactions are therefore crucial as they determine the fate of the substrates. In the past 15 years, numerous experiments have contributed to depict an incomplete picture of the mechanisms responsible for myofibrillar proteolysis. This includes the discovery of muscle-specific E3 ligases (e.g. MuRF1) and the identification of the signaling pathways involved. Our main objective was to identify the E2-MuRF1 couples involved in the targeting of myofibrillar proteins in atrophying muscles. Methods: We focused on 14 E2 enzymes that are either abundant in skeletal muscle and/or up-regulated in atrophying conditions. We used control fa-C2C12 myotubes (expressing flag-actin) treated or not with dexamethasone (Dex, 1μM) and muscles from hindlimb suspensed rats to determine the expression levels of these enzymes. The MuRF1 cognate E2 enzymes were identified by knockdown and interactomic approaches. Results: Dex treatment increased mRNA levels of UBE2A, UBE2B, UBE2D1, UBE2D2, UBE2G1, and UBE2J1. UBE2A did not interact with MuRF1 and was not involved in contractile protein degradation. We next demonstrated that UBE2B was involved in α-actin and myosin heavy-chain destabilization in fa-C2C12 myotubes. However, this process was restricted to the cytoplasmic fraction and was MuRF1-independent. By contrast with previous assumptions, we found that UBE2D2 is not the MuRF1 partner for α-actin degradation in cellulo and is probably not involved in muscle wasting. Conclusions: UBE2B is the first E2 involved in contractile protein targeting, this action being presumably downstream of MuRF1 action. 1-04 Repetitive pulmonary inflammation in emphysematous mice induces sustained muscle wasting due to impaired muscle mass recovery Judith JM Ceelen, Annemie MWJ Schols, Stefan J van Hoof, Chiel C de Theije, Frank Verhaegen & Ramon CJ Langen Maastricht University Medical Center, Department of Respiratory Medicine, MaastrichtThe Netherlands, Department of Radiation Oncology (MaastRO) Background and aims: Exacerbations in COPD are often accompanied by pulmonary inflammation, and associated with increased prevalence of weight loss and muscle wasting. Emphysema-associated muscle atrophy may result from the cumulative effects of acute muscle mass loss during disease exacerbations, and subsequent impaired muscle regrowth. The aim of this study was to test whether muscle mass recovery following muscle atrophy induced by pulmonary inflammation is impaired in emphysematous mice and culminates in sustained muscle wasting. Methods: Emphysema was induced by 3 weekly intra-tracheal (IT) elastase instillations. Subsequently, 3 weekly boluses of ITLPS were administered to mimic a repetitive pulmonary inflammation-driven disease exacerbation. Using micro conebeam CT-scans, emphysema was verified, and muscle mass changes were monitored and correlated to muscle strength. At 2 and 3 days following the first IT-LPS administration and 7 days after the third IT-LPS bolus, skeletal muscle was collected for analyses. Results: Irrespective of emphysema, muscle weight and strength was reduced 48 h after the first bolus IT-LPS and recovered thereafter. mRNA and protein levels of genes of the ubiquitin-proteasome pathway (UPS) and the autophagy-lysosomal pathway (ALP) were upregulated 48 h following IT-LPS. In contrast, mTOR signaling was reduced 48 h post-induction of pulmonary inflammation in control and emphysematous mice. Importantly, muscle strength recovery following subsequent IT-LPS challenges was impaired in emphysematous mice, resulting in a sustained decre

Muscle wasting prevails in numerous diseases (e.g. diabetes, cardiovascular and kidney diseases, COPD,…) and increases healthcare costs. A major clinical issue is to devise new strategies preventing muscle wasting. We hypothesized that a long-term docosahexaenoic acid (DHA) supplementation prior to fasting may preserve muscle mass in vivo. Six-wk-old C57BL/6 mice were fed a DHA-enriched or a control diet for 8 weeks and then fasted for 48 h. The effect of DHA on i) muscle energy stores (glycogen, triglycerides (TG)), ii) muscle mass, and iii) Akt and AMPK signaling pathways involved in the control of protein and energy metabolism has been addressed. The regulation of the formation and the fate of lipid stores has been also evaluated. Feeding mice a DHA-enriched diet prior to fasting elevated muscle glycogen contents without any change in TG levels, reduced muscle wasting, blocked the 55 % decrease in Akt phosphorylation, and reduced by 30-40% the activation of AMPK, ubiquitination or autophagy. The DHA-enriched diet fully abolished the fasting induced-mRNA over-expression of the endocannabinoid receptor-1. Finally, DHA prevented or modulated the fasting-dependent increase in muscle mRNA levels for Rab18, PLD1 and perilipins, which determine the formation and fate of lipid droplets, in parallel with muscle sparing. These data suggest that long-term DHA supplementation increased energy stores that can be efficiently mobilized, and thus preserved muscle mass in response to fasting through the regulation of Akt- and AMPK-dependent signaling pathways for reducing proteolysis activation. Whether a nutritional strategy aiming at increasing energy status may shorten recovery periods in clinical settings remains to be tested