Insight into the Role of Gut Microbiota in Duchenne Muscular Dystrophy: An Age-Related Study in Mdx Mice.

Dystrophin deficiency alters the sarcolemma structure, leading to muscle dystrophy, muscle disuse, and ultimately death. Beyond limb muscle deficits, patients with Duchenne muscular dystrophy have numerous transit disorders. Many studies have highlighted the strong relationship between gut microbiota and skeletal muscle. The aims of this study were: i) to characterize the gut microbiota composition over time up to 1 year in dystrophin-deficient mdx mice, and ii) to analyze the intestine structure and function and expression of genes linked to bacterial-derived metabolites in ileum, blood, and skeletal muscles to study interorgan interactions. Mdx mice displayed a significant reduction in the overall number of different operational taxonomic units and their abundance (α-diversity). Mdx genotype predicted 20% of ß-diversity divergence, with a large taxonomic modification of Actinobacteria, Proteobacteria, Tenericutes, and Deferribacteres phyla and the included genera. Interestingly, mdx intestinal motility and gene expressions of tight junction and Ffar2 receptor were down-regulated in the ileum. Concomitantly, circulating inflammatory markers related to gut microbiota (tumor necrosis factor, IL-6, monocyte chemoattractant protein-1) and muscle inflammation Tlr4/Myd88 pathway (Toll-like receptor 4, which recognizes pathogen-associated molecular patterns) were up-regulated. Finally, in mdx mice, adiponectin was reduced in blood and its receptor modulated in muscles. This study highlights a specific gut microbiota composition and highlights interorgan interactions in mdx physiopathology with gut microbiota as the potential central metabolic organ.

Transient Changes of Metabolism at the Pronuclear Stage in Mice Influences Skeletal Muscle Phenotype in Adulthood.

Skeletal muscle has a remarkable plasticity, and its phenotype is strongly influenced by hormones, transcription factors, and physical activity. However, whether skeletal phenotype can be oriented or not during early embryonic stages has never been investigated. Here, we report that pyruvate as the only source of carbohydrate in the culture medium of mouse one cell stage embryo influenced the establishment of the muscular phenotype in adulthood. We found that pyruvate alone induced changes in the contractile phenotype of the skeletal muscle in a sexually dependent manner. For male mice, a switch to a more glycolytic phenotype was recorded, whereas, in females, the pyruvate induced a switch to a more oxidative phenotype. In addition, the influence of pyruvate on the contractile phenotypes was confirmed in two mouse models of muscle hypertrophy: the well-known myostatin deficient mouse (Mstn-/-) and a mouse carrying a specific deletion of p43, a mitochondrial triiodothyronine receptor. Finally, to understand the link between these adult phenotypes and the early embryonic period, we assessed the levels of two histone H3 post-translational modifications in presence of pyruvate alone just after the wave of chromatin reprogramming specific of the first cell cycle. We showed that H3K4 acetylation level was decreased in Mstn-/- 2-cell embryos, whereas no difference was found for H3K27 trimethylation level, whatever the genotype. These findings demonstrate for the first time that changes in the access of energy substrate during the very first embryonic stage can induce a precocious orientation of skeletal muscle phenotype in adulthood.

Gut bacteria are critical for optimal muscle function: a potential link with glucose homeostasis.

Gut microbiota is involved in the development of several chronic diseases, including diabetes, obesity, and cancer, through its interactions with the host organs. It has been suggested that the cross talk between gut microbiota and skeletal muscle plays a role in different pathological conditions, such as intestinal chronic inflammation and cachexia. However, it remains unclear whether gut microbiota directly influences skeletal muscle function. In this work, we studied the impact of gut microbiota modulation on mice skeletal muscle function and investigated the underlying mechanisms. We determined the consequences of gut microbiota depletion after treatment with a mixture of a broad spectrum of antibiotics for 21 days and after 10 days of natural reseeding. We found that, in gut microbiota-depleted mice, running endurance was decreased, as well as the extensor digitorum longus muscle fatigue index in an ex vivo contractile test. Importantly, the muscle endurance capacity was efficiently normalized by natural reseeding. These endurance changes were not related to variation in muscle mass, fiber typology, or mitochondrial function. However, several pertinent glucose metabolism markers, such as ileum gene expression of short fatty acid chain and glucose transporters G protein-coupled receptor 41 and sodium-glucose cotransporter 1 and muscle glycogen level, paralleled the muscle endurance changes observed after treatment with antibiotics for 21 days and reseeding. Because glycogen is a key energetic substrate for prolonged exercise, modulating its muscle availability via gut microbiota represents one potent mechanism that can contribute to the gut microbiota-skeletal muscle axis. Taken together, our results strongly support the hypothesis that gut bacteria are required for host optimal skeletal muscle function.

New evidence of exercise training benefits in myostatin-deficient mice: Effect on lipidomic abnormalities.

Myostatin (Mstn) inactivation or inhibition is considered as a promising treatment for various muscle-wasting disorders because it promotes muscle growth. However, myostatin-deficient hypertrophic muscles show strong fatigability associated with abnormal mitochondria and lipid metabolism. Here, we investigated whether endurance training could improve lipid metabolism and mitochondrial membrane lipid composition in mice where the Mstn gene was genetically ablated (Mstn-/- mice). In Mstn-/- mice, 4 weeks of daily running exercise sessions (65-70% of the maximal aerobic speed for 1 h) improved significantly aerobic performance, particularly the endurance capacity (up to +280% compared with untrained Mstn-/- mice), to levels comparable to those of trained wild type (WT) littermates. The expression of oxidative and lipid metabolism markers also was increased, as indicated by the upregulation of the Cpt1, Ppar-δ and Fasn genes. Moreover, endurance training also increased, but far less than WT, citrate synthase level and mitochondrial protein content. Interestingly endurance training normalized the cardiolipin fraction in the mitochondrial membrane of Mstn-/- muscle compared with WT. These results suggest that the combination of myostatin inhibition and endurance training could increase the muscle mass while preserving the physical performance with specific effects on cardiolipin and lipid-related pathways.

Retinoic acid maintains human skeletal muscle progenitor cells in an immature state.

Muscle satellite cells are resistant to cytotoxic agents, and they express several genes that confer resistance to stress, thus allowing efficient dystrophic muscle regeneration after transplantation. However, once they are activated, this capacity to resist to aggressive agents is diminished resulting in massive death of transplanted cells. Although cell immaturity represents a survival advantage, the signalling pathways involved in the control of the immature state remain to be explored. Here, we show that incubation of human myoblasts with retinoic acid impairs skeletal muscle differentiation through activation of the retinoic-acid receptor family of nuclear receptor. Conversely, pharmacologic or genetic inactivation of endogenous retinoic-acid receptors improved myoblast differentiation. Retinoic acid inhibits the expression of early and late muscle differentiation markers and enhances the expression of myogenic specification genes, such as PAX7 and PAX3. These results suggest that the retinoic-acid-signalling pathway might maintain myoblasts in an undifferentiated/immature stage. To determine the relevance of these observations, we characterised the retinoic-acid-signalling pathways in freshly isolated satellite cells in mice and in siMYOD immature human myoblasts. Our analysis reveals that the immature state of muscle progenitors is correlated with high expression of several genes of the retinoic-acid-signalling pathway both in mice and in human. Taken together, our data provide evidences for an important role of the retinoic-acid-signalling pathway in the regulation of the immature state of muscle progenitors.

Myostatin deficiency is associated with lipidomic abnormalities in skeletal muscles.

Myostatin (Mstn) deficiency leads to skeletal muscle overgrowth and Mstn inhibition is considered as a promising treatment for muscle-wasting disorders. Mstn gene deletion in mice also causes metabolic changes with decreased mitochondria content, disturbance in mitochondrial respiratory function and increased muscle fatigability. However the impact of MSTN deficiency on these metabolic changes is not fully elucidated. Here, we hypothesized that lack of MSTN will alter skeletal muscle membrane lipid composition in relation with pronounced alterations in muscle function and metabolism. Indeed, phospholipids and in particular cardiolipin mostly present in the inner mitochondrial membrane, play a crucial role in mitochondria function and oxidative phosphorylation process. We observed that Mstn KO muscle had reduced fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and ß-HAD activities) and impaired lipogenesis (decreased triglyceride and free fatty acid content), indicating a role of mstn in muscle lipid metabolism. We further analyzed phospholipid classes and fatty acid composition by chromatographic methods in muscle and mitochondrial membranes. Mstn KO mice showed increased levels of saturated and polyunsaturated fatty acids at the expense of monounsaturated fatty acids. We also demonstrated, in this phenotype, a reduction in cardiolipin proportion in mitochondrial membrane versus the proportion of others phospholipids, in relation with a decrease in the expression of phosphatidylglycerolphosphate synthase and cardiolipin synthase, enzymes involved in cardiolipin synthesis. These data illustrate the importance of lipids as a link by which MSTN deficiency can impact mitochondrial bioenergetics in skeletal muscle.

Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models.

INTRODUCTION: The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. METHODS: We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KOLee ) and (ii) the Grobet model (KOGrobet ), and measured the contraction of tibialis anterior muscle in situ. RESULTS: Absolute maximal isometric force was increased in 6-month-old KOLee and KOGrobet mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KOLee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KOGrobet mice, whereas specific maximal power was reduced only in male KOLee mice. CONCLUSIONS: Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017.

REDD1 deletion prevents dexamethasone-induced skeletal muscle atrophy.

REDD1 (regulated in development and DNA damage response 1) has been proposed to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) during in vitro hypoxia. REDD1 expression is low under basal conditions but is highly increased in response to several catabolic stresses, like hypoxia and glucocorticoids. However, REDD1 function seems to be tissue and stress dependent, and its role in skeletal muscle in vivo has been poorly characterized. Here, we investigated the effect of REDD1 deletion on skeletal muscle mass, protein synthesis, proteolysis, and mTORC1 signaling pathway under basal conditions and after glucocorticoid administration. Whereas skeletal muscle mass and typology were unchanged between wild-type (WT) and REDD1-null mice, oral gavage with dexamethasone (DEX) for 7 days reduced tibialis anterior and gastrocnemius muscle weights as well as tibialis anterior fiber size only in WT. Similarly, REDD1 deletion prevented the inhibition of protein synthesis and mTORC1 activity (assessed by S6, 4E-BP1, and ULK1 phosphorylation) observed in gastrocnemius muscle of WT mice following single DEX administration for 5 h. However, our results suggest that REDD1-mediated inhibition of mTORC1 in skeletal muscle is not related to the modulation of the binding between TSC2 and 14-3-3. In contrast, our data highlight a new mechanism involved in mTORC1 inhibition linking REDD1, Akt, and PRAS40. Altogether, these results demonstrated in vivo that REDD1 is required for glucocorticoid-induced inhibition of protein synthesis via mTORC1 downregulation. Inhibition of REDD1 may thus be a strategy to limit muscle loss in glucocorticoid-mediated atrophy.

Glutathione peroxidase 3, a new retinoid target gene, is crucial for human skeletal muscle precursor cell survival.

Protection of satellite cells from cytotoxic damages is crucial to ensure efficient adult skeletal muscle regeneration and to improve therapeutic efficacy of cell transplantation in degenerative skeletal muscle diseases. It is therefore important to identify and characterize molecules and their target genes that control the viability of muscle stem cells. Recently, we demonstrated that high aldehyde dehydrogenase activity is associated with increased viability of human myoblasts. In addition to its detoxifying activity, aldehyde dehydrogenase can also catalyze the irreversible oxidation of vitamin A to retinoic acid; therefore, we examined whether retinoic acid is important for myoblast viability. We showed that when exposed to oxidative stress induced by hydrogen peroxide, adherent human myoblasts entered apoptosis and lost their capacity for adhesion. Pre-treatment with retinoic acid reduced the cytotoxic damage ex vivo and enhanced myoblast survival in transplantation assays. The effects of retinoic acid were maintained in dystrophic myoblasts derived from facioscapulohumeral patients. RT-qPCR analysis of antioxidant gene expression revealed glutathione peroxidase 3 (Gpx3), a gene encoding an antioxidant enzyme, as a potential retinoic acid target gene in human myoblasts. Knockdown of Gpx3 using short interfering RNA induced elevation in reactive oxygen species and cell death. The anti-cytotoxic effects of retinoic acid were impaired in GPx3-inactivated myoblasts, which indicates that GPx3 regulates the antioxidative effects of retinoic acid. Therefore, retinoid status and GPx3 levels may have important implications for the viability of human muscle stem cells.

Absence of hyperplasia in Gasp-1 overexpressing mice is dependent on myostatin up-regulation.

BACKGROUND/AIMS: Overexpression of Gasp-1, an inhibitor of myostatin, leads to a hypermuscular phenotype due to hypertrophy rather than hyperplasia in mice. However to date, the cellular and molecular mechanisms underlying this phenotype are not investigated. METHODS: Skeletal muscles of overexpressing Gasp-1 mice, called Tg(Gasp-1) mice, were analyzed by histological methods. Satellite cell-derived myoblasts from these mice were used to investigate the molecular mechanisms. RESULTS: We demonstrated that hypertrophy in Tg(Gasp-1) mice was related to a myonuclear accretion during the first 3 postnatal weeks and an activation of the pro-hypertrophic Akt/mTORC/p70S6K signaling. In accordance with these results, we showed that overexpressing Gasp-1 primary myoblasts proliferated faster and myonuclei average per myotube was increased during differentiation. Molecular analysis revealed that Gasp-1 overexpression resulted in increased myostatin expression related to its auto-regulation. Despite its inhibition, myostatin led to Pax7 deregulation through its non-canonical Erk1/2 signaling pathway. Consistent with this, inhibition of Erk1/2 signaling pathway as well as neutralization of secreted myostatin rescue the Pax7 expression in overexpressing Gasp-1 myoblasts. CONCLUSION: Our study shows that myostatin is able to act independently of its canonical pathway to regulate the Pax7 expression. Altogether, our results indicate that myostatin could regulate muscle development despite its protein inhibition.

Lack of myostatin alters intermyofibrillar mitochondria activity, unbalances redox status, and impairs tolerance to chronic repetitive contractions in muscle.

Loss of myostatin (mstn) function leads to a decrease in mitochondrial content, a reduced expression of cytochrome c oxidase, and a lower citrate synthase activity in skeletal muscle. These data suggest functional or ultrastructural mitochondrial abnormalities that can impact on muscle endurance characteristics in such phenotype. To address this issue, we investigated subsarcolemmal and intermyofibrillar (IMF) mitochondrial activities, skeletal muscle redox homeostasis, and muscle fiber endurance quality in mstn-deficient mice [mstn knockout (KO)]. We report that lack of mstn induced a decrease in the coupling of IMF mitochondria respiration, with significantly higher basal oxygen consumption. No lysis of mitochondrial cristae or excessive swelling were observed in mstn KO mice compared with wild-type (WT) mice. Concerning redox status, mstn KO gastrocnemius exhibited a significant decrease in lipid peroxidation levels (-56%; P < 0.01 vs. WT) together with a significant upregulation of the antioxidant glutathione system. In contrast, superoxide dismutase and catalase activities were altered in mstn KO, gastrocnemius and soleus with a reduction of up to 80% compared with WT animals. The force production observed after contractile endurance test was significantly lower in extensor digitorum longus and soleus muscles of mstn KO mice compared with the controls (17 ± 3 and 36 ± 5% vs. 28 ± 4 and 56 ± 5%, respectively, P < 0.05). Together, these findings indicate that, besides an increased skeletal muscle mass, genetic mstn inhibition has differential effects on redox homeostasis and mitochondrial function that would have functional consequences on muscle response to endurance exercise.

Myostatin deficiency is associated with an increase in number of total axons and motor axons innervating mouse tibialis anterior muscle.

INTRODUCTION: Myostatin (Mstn) is a secreted protein that acts as a negative regulator of skeletal muscle mass. However, a critical evaluation of neuromuscular aspects of hypertrophied muscles induced by Mstn deficiency has not been done. METHODS: We compared the tibialis anterior muscle-nerve interrelationships in wild-type and Mstn-null mice of both genders by immunohistochemical analyses, which allowed us to count the number of total axons and motor axons and estimate the size of motor units and the innervation ratio of the tibialis anterior muscle (TAm). RESULTS: There was an increase in the number of total axons and motor axons, and higher values in both the motor unit size and the innervation ratio of Mstn-null TAm compared with those of wild-type TAm. CONCLUSIONS: We found that myostatin is involved either directly in the control of neuromuscular interrelationships or indirectly through its effect on muscle size.

Calpastatin overexpression in the skeletal muscle of mice prevents clenbuterol-induced muscle hypertrophy and phenotypic shift.

Accumulating evidence suggests that the calpain/calpastatin system is involved in skeletal muscle remodelling induced by ß(2) -adrenoceptor agonist treatment. In addition to other pathways, the Akt/mammalian target of rapamycin (mTOR) pathway, controlling protein synthesis, and the calcium/calmodulin-dependent protein kinase 2 (CamK2) and AMP-activated protein kinase (AMPK) pathways, recently identified as calpain substrates, could be relevant in ß(2) -adrenoceptor agonist-induced skeletal muscle remodelling. In the present study we investigated muscle hypertrophy and phenotypic shifts, as well as the molecular response of components of the Akt/mTOR pathway (i.e. Akt, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 (rpS6), CamK2 and AMPK), in response to calpastatin overexpression in the skeletal muscle of mice treated with 1 mg/kg per day clenbuterol for 21 days. Using gene electrotransfer of a calpastatin expression vector into the tibialis anterior of adult mice, we found that calpastatin overexpression attenuates muscle hypertrophy and phenotypic shifts induced by clenbuterol treatment. At the molecular level, calpastatin overexpression markedly decreased calpain activity, but was ineffective in altering the phosphorylation of Akt, 4E-BP1 and rpS6. In contrast, calpastatin overexpression increased the protein expression of both total AMPK and total CamK2. In conclusion, the results support the contention that the calpain/calpastatin system plays a crucial role in skeletal muscle hypertrophy and phenotypic shifts under chronic clenbuterol treatment, with AMPK and CamK2 probably playing a minor role. Moreover, the calpastatin-induced inhibition of hypertrophy under clenbuterol treatment was not related to a decreased mTOR-dependent initiation of protein translation.

Myostatin gene inactivation increases post-mortem calpain-dependent muscle proteolysis in mice.

Myostatin deficiency leads to extensive skeletal muscle hypertrophy, but its consequence on post-mortem muscle proteolysis is unknown. Here, we compared muscle myofibrillar protein degradation, and autophagy, ubiquitin-proteasome and Ca2+-dependent proteolysis relative to the energetic and redox status in wild-type (WT) and myostatin knock-out mice (KO) during early post-mortem storage. KO muscles showed higher degradation of myofibrillar proteins in the first 24 h after death, associated with preserved antioxidant status, compared with WT muscles. Analysis of key autophagy and ubiquitin-proteasome system markers indicated that these two pathways were not upregulated in post-mortem muscle (both genotypes), but basal autophagic flux and ATP content were lower in KO muscles. Proteasome and caspase activities were not different between WT and KO mice. Conversely, calpain activity was higher in KO muscles, concomitantly with higher troponin T and desmin degradation. Altogether, these results suggest that calpains but not the autophagy, proteasome and caspase systems, explain the difference in post-mortem muscle protein proteolysis between both genotypes.

Wnt4 activates the canonical ß-catenin pathway and regulates negatively myostatin: functional implication in myogenesis.

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical ß-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells.

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56(+) fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H(2) O(2) )-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.

Myostatin inactivation increases myotube size through regulation of translational initiation machinery.

Myostatin deficiency leads in skeletal muscle overgrowth but the precise molecular mechanisms underlying this hypertrophy are not well understood. In this study, to gain insight into the role of endogenous myostatin in the translational regulation, we used an in vitro model of cultured satellite cells derived from myostatin knock-out mice. Our results show that myostatin knock-out myotubes are larger than control myotubes and that this phenotype is associated with an increased activation of the Akt/mTOR signaling pathway, a known regulator of muscle hypertrophy. These results demonstrate that hypertrophy due to myostatin deficiency is preserved in vitro and suggest that myostatin deletion results in an increased protein synthesis. Accordingly, the rates of global RNA content, polysome formation and protein synthesis are all increased in myostatin-deficient myotubes while they are counteracted by the addition of recombinant myostatin. We furthermore demonstrated that genetic deletion of myostatin stimulates cap-dependent translation by positively regulating assembly of the translation preinitiation complex. Together the data indicate that myostatin controls muscle hypertrophy in part by regulating protein synthesis initiation rates, that is, translational efficiency.

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment.

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that ß2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

Does Physical Inactivity Induce Significant Changes in Human Gut Microbiota? New Answers Using the Dry Immersion Hypoactivity Model.

Gut microbiota, a major contributor to human health, is influenced by physical activity and diet, and displays a functional cross-talk with skeletal muscle. Conversely, few data are available on the impact of hypoactivity, although sedentary lifestyles are widespread and associated with negative health and socio-economic impacts. The study aim was to determine the effect of Dry Immersion (DI), a severe hypoactivity model, on the human gut microbiota composition. Stool samples were collected from 14 healthy men before and after 5 days of DI to determine the gut microbiota taxonomic profiles by 16S metagenomic sequencing in strictly controlled dietary conditions. The α and ß diversities indices were unchanged. However, the operational taxonomic units associated with the Clostridiales order and the Lachnospiraceae family, belonging to the Firmicutes phylum, were significantly increased after DI. Propionate, a short-chain fatty acid metabolized by skeletal muscle, was significantly reduced in post-DI stool samples. The finding that intestine bacteria are sensitive to hypoactivity raises questions about their impact and role in chronic sedentary lifestyles.

Autophagy in farm animals: current knowledge and future challenges.

Autophagy (a process of cellular self-eating) is a conserved cellular degradative process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. Surprisingly, little attention has been paid to the role of this cellular function in species of agronomical interest, and the details of how autophagy functions in the development of phenotypes of agricultural interest remain largely unexplored. Here, we first provide a brief description of the main mechanisms involved in autophagy, then review our current knowledge regarding autophagy in species of agronomical interest, with particular attention to physiological functions supporting livestock animal production, and finally assess the potential of translating the acquired knowledge to improve animal development, growth and health in the context of growing social, economic and environmental challenges for agriculture.Abbreviations: AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ASC: adipose-derived stem cells; ATG: autophagy-related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BVDV: bovine viral diarrhea virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DAP: Death-Associated Protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; Gln: Glutamine; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IF: immunofluorescence; IVP: in vitro produced; LAMP2A: lysosomal associated membrane protein 2A; LMS: lysosomal membrane stability; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MDBK: Madin-Darby bovine kidney; MSC: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NDV: Newcastle disease virus; NECTIN4: nectin cell adhesion molecule 4; NOD1: nucleotide-binding oligomerization domain 1; OCD: osteochondritis dissecans; OEC: oviduct epithelial cells; OPTN: optineurin; PI3K: phosphoinositide-3-kinase; PPRV: peste des petits ruminants virus; RHDV: rabbit hemorrhagic disease virus; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy.