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.

FAHFAs Regulate the Proliferation of C2C12 Myoblasts and Induce a Shift toward a More Oxidative Phenotype in Mouse Skeletal Muscle.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids reported to have antidiabetic and anti-inflammatory effects. Since skeletal muscle is a major target for insulin, the aim of this study is to explore for the first time the influence of several FAHFAs in C2C12 myoblasts and in skeletal muscle phenotype in mice. Here, we show that eleven FAHFAs belonging to different families inhibit C2C12 myoblast proliferation. In addition, all FAHFAs decreased mitochondrial cytochrome c oxidase activity without affecting reactive oxygen species production and the mitochondrial network. During C2C12 myoblasts differentiation, we found that two of the most active lipids, 9-PAHPA and 9-OAHPA, did not significantly affect the fusion index and the expression of myosin heavy chains. However, we found that three months' intake of 9-PAHPA or 9-OAHPA in mice increased the expression of more oxidative myosin in skeletal muscle without affecting skeletal muscle mass, number, and mean fiber area, mitochondrial activity, and oxidative stress parameters. In conclusion, our study indicated that the eleven FAHFAs tested decreased the proliferation rate of C2C12 myoblasts, probably through the inhibition of mitochondrial activity. In addition, we found that 9-PAHPA or 9-OAHPA supplementation in mice induced a switch toward a more oxidative contractile phenotype of skeletal muscle. These data suggest that the increase in insulin sensitivity previously described for these two FAHFAs is of muscular origin.

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.

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.

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.

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.

Endurance exercise decreases protein synthesis and ER-mitochondria contacts in mouse skeletal muscle.

Exercise is important to maintain skeletal muscle mass through stimulation of protein synthesis, which is a major ATP-consuming process for cells. However, muscle cells have to face high energy demand during contraction. The present study aimed to investigate protein synthesis regulation during aerobic exercise in mouse hindlimb muscles. Male C57Bl/6J mice ran at 12 m/min for 45 min or at 12 m/min for the first 25 min followed by a progressive increase in velocity up to 20 m/min for the last 20 min. Animals were injected intraperitoneally with 40 nmol/g of body weight of puromycin and euthanized by cervical dislocation immediately after exercise cessation. Analysis of gastrocnemius, plantaris, quadriceps, soleus, and tibialis anterior muscles revealed a decrease in protein translation assessed by puromycin incorporation, without significant differences among muscles or running intensities. The reduction of protein synthesis was associated with a marked inhibition of mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, a mechanism consistent with reduced translation initiation. A slight activation of AMP-activated protein kinase consecutive to the running session was measured but did not correlate with mTORC1 inhibition. More importantly, exercise resulted in a strong upregulation of regulated in development and DNA damage 1 (REDD1) protein and gene expressions, whereas transcriptional regulation of other recognized exercise-induced genes (IL-6, kruppel-like factor 15, and regulator of calcineurin 1) did not change. Consistently with the recently discovered role of REDD1 on mitochondria-associated membranes, we observed a decrease in mitochondria-endoplasmic reticulum interaction following exercise. Collectively, these data raise questions concerning the role of mitochondria-associated endoplasmic reticulum membrane disruption in the regulation of muscle proteostasis during exercise and, more generally, in cell adaptation to metabolic stress.NEW & NOTEWORTHY How muscles regulate protein synthesis to cope with the energy demand during contraction is poorly documented. Moreover, it is unknown whether protein translation is differentially affected among mouse hindlimb muscles under different physiological exercise modalities. We showed here that 45 min of running decreases puromycin incorporation similarly in 5 different mouse muscles. This decrease was associated with a strong increase in regulated in development and DNA damage 1 protein expression and a significant disruption of the mitochondria and sarcoplasmic reticulum interaction.

Indole, a Signaling Molecule Produced by the Gut Microbiota, Negatively Impacts Emotional Behaviors in Rats.

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota from tryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability.

Structure of protein emulsion in food impacts intestinal microbiota, caecal luminal content composition and distal intestine characteristics in rats.

SCOPE: Few studies have evaluated in vivo the impact of food structure on digestion, absorption of nutrients and on microbiota composition and metabolism. In this study we evaluated in rat the impact of two structures of protein emulsion in food on gut microbiota, luminal content composition, and intestinal characteristics. METHODS AND RESULTS: Rats received for 3 weeks two diets of identical composition but based on lipid-protein matrices of liquid fine (LFE) or gelled coarse (GCE) emulsion. LFE diet led to higher abundance, when compared to the GCE, of Lactobacillaceae (Lactobacillus reuteri) in the ileum, higher ß-diversity of the caecum mucus-associated bacteria. In contrast, the LFE diet led to a decrease in Akkermansia municiphila in the caecum. This coincided with heavier caecum content and higher amount of isovalerate in the LFE group. LFE diet induced an increased expression of (i) amino acid transporters in the ileum (ii) glucagon in the caecum, together with an elevated level of GLP-1 in portal plasma. However, these intestinal effects were not associated with modification of food intake or body weight gain. CONCLUSION: Overall, the structure of protein emulsion in food affects the expression of amino acid transporters and gut peptides concomitantly with modification of the gut microbiota composition and activity. Our data suggest that these effects of the emulsion structure are the result of a modification of protein digestion properties.