Ph.D Maxence Jollet
email: maxence.jollet@ki.se.

Mahendra Mariadassou

Olivier Rué

Laurence Pessemesse

Vincent Ollendorff

Sofiane Ramdani

Barbara Vernus

Anne Bonnieu

Christelle Bertrand-Gaday

Bénédicte Goustard

Christelle Koechlin-Ramonatxo
email: christelle.ramonatxo@umontpellier.fr

Q4

Ph.D Telle Koechlin-Ramonatxo

Insight into the Role of Gut Microbiota in Duchenne Muscular Dystrophy

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Duchenne muscular dystrophy (DMD) is a progressive wasting disease of skeletal and cardiac muscles, and one of the most common recessive inherited genetic diseases (yearly incidence, 1:3500-1:5000 boys). In DMD, mutations in the DMD gene affect the proper production of the membrane-associated dystrophin protein, leading to weakening of the muscle cell membrane to mechanical stress during the contraction/relaxation cycles that promotes microlesions. These lesions can initially be associated with muscle pseudo-hypertrophy, especially in postural muscles (eg, triceps surae muscles), highlighting the muscle fiber regenerative capacities. The endless cycles of muscle necrosis and repair lead to fibrosis and progressive muscle weakness. Proximal skeletal muscles are the most affected: first, the locomotor muscles, then the trunk muscle, and finally the respiratory muscles and the heart, leading to quadriplegia and cardio-respiratory difficulties. Moreover, many secondary pathophysiological processes exacerbate muscle pathology in DMD: immunologic and inflammatory processes, [START_REF] Porter | A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice[END_REF][START_REF] Rosenberg | Immune-mediated pathology in Duchenne muscular dystrophy[END_REF] altered calcium homeostasis, [START_REF] Fong | AJPA-D-22-4. Gailly P: New aspects of calcium signaling in skeletal muscle cells: implications in Duchenne muscular dystrophy[END_REF]4 stress, [START_REF] Rando | Muscle cells from mdx mice have an increased susceptibility to oxidative stress[END_REF] and apoptosis and defective autophagy, [START_REF] Pauly | AMPK activation stimulates autophagy and ameliorates muscular dystrophy in the mdx mouse diaphragm[END_REF] as well as declines in mitochondrial number and function. [START_REF] Kuznetsov | Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse[END_REF][START_REF] Vila | Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy[END_REF] Actual therapies can be divided in two categories: primary therapy (to restore or partially restore functional dystrophin protein) and other therapeutic approaches (to improve muscle function and quality in patients with DMD). The current primary therapies are exon skipping, stop codon readthrough, gene addition, genome editing, and myoblast transplantation. [START_REF] Verhaart | Therapeutic developments for Duchenne muscular dystrophy[END_REF] In addition, many therapeutic avenues are being evaluated to improve muscle condition by targeting fibrosis, growth and regeneration processes, calcium homeostasis, or mitochondria biogenesis. [START_REF] Verhaart | Therapeutic developments for Duchenne muscular dystrophy[END_REF] In these therapeutic approaches, such as identification of small molecules [START_REF] Sheikh | Developing DMD therapeutics: a review of the effectiveness of small molecules, stop-codon readthrough, dystrophin gene replacement, and exon-skipping therapies[END_REF] or exercise, [START_REF] Hyzewicz | Comparison of experimental protocols of physical exercise for mdx mice and Duchenne muscular dystrophy patients[END_REF] an new strategy is emerging: nutraceuticals. [START_REF] Radley | Duchenne muscular dystrophy: focus on pharmaceutical and nutritional interventions[END_REF] Thanks to the current standards of care, many patients with DMD can now expect to live into their fourth decade of life. However, in the absence of a curative treatment, prevention of secondary processes that exacerbate DMD pathophysiology and offer improvement in the patients' quality of life are a priority. In this context, it is important to better understand the processes associated with DMD development and the links between the affected organs, including the possible crosstalk between gut microbiota and skeletal muscle.

The term "gut microbiota" describes the 10 14 bacteria housed in the digestive tract and classified in different species, families, and phyla. This bacterial community plays an important role in the host metabolism and health. Due to the functional crosstalk with other organs (eg, brain, heart, liver, adipose tissue), perturbations of the gut microbiota composition and function (ie, dysbiosis) have been associated with many diseases, such as brain disorders (eg, depression, autism, Alzheimer disease) and metabolic disorders (eg, obesity, type 2 diabetes, insulin resistance). Moreover, several studies have shown the implication of the intestinal microbiota in antitumor treatment effectiveness 13e15 and in cachexia (ie, the cancer-associated loss of skeletal muscle and adipose tissue). Indeed, Bindels et al [START_REF] Bindels | Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model[END_REF] found that the gut microbiota was depleted in a leukemic and cachexic mouse model. Interestingly, when the gut microbiota was normalized by oral supplementation of probiotics, the concentration in muscle of inflammation markers [IL-6, IL-4, monocyte chemoattractant protein-1 (MCP-1), and granulocyte colony-stimulating factor], autophagy markers (LC3 and cathepsin L), and proteolysis markers (atrogin1 and MAFbx) decreased. The probiotic treatment also prevented skeletal muscle inflammation and atrophy. Since this first study suggesting a gut microbiotaeskeletal muscle axis, accumulating evidence has highlighted the potential influence of gut microbiota on the skeletal muscle phenotype. Muscle mass is reduced in germ-free mice that lack microbiota compared with control mice. [START_REF] Hsu | Effect of intestinal microbiota on exercise performance in mice[END_REF][START_REF] Tung | Characterization of the serum and liver proteomes in gutmicrobiota-lacking mice[END_REF] Our laboratory showed that gut bacteria are necessary to optimize skeletal muscle function. Indeed, depletion of gut bacteria by treatment with broad-spectrum antibiotics led to a decrease in skeletal muscle endurance and to an alteration in glucose homeostasis, as indicated by the decreased expression of short-chain fatty acid chain (SCFA) and glucose transporters in the ileum, and reduced glycogen content in muscle. This phenotype was normalized after natural reseeding. [START_REF] Nay | Gut bacteria are critical for optimal muscle function: a potential link with glucose homeostasis[END_REF] Similarly, Yan et al [START_REF] Yan | Gut microbiota can transfer fiber characteristics and lipid metabolic profiles of skeletal muscle from pigs to germ-free mice[END_REF] observed that after transfer of gut microbiota from obese or lean pigs to germ-free mice, mice replicated the donor's skeletal muscle fiber profile.

Different pathways might be involved in the gut microbiotaeskeletal muscle axis. Several circulating mediators, such as pro-inflammatory cytokines, SCFA, and branched-chain amino acids, in relation with the gut microbiota composition, have recognized effects on skeletal muscle. For example, microbiota-derived SCFAs (eg, acetate, butyrate, propionate) are produced during a gut microbial fermentation process and could drive skeletal muscle toward an oxidative metabolism

Q9

. Similarly, in mice fed a high-fat diet, butyrate supplementation is associated with improved insulin sensitivity, increased peroxisome proliferatoreactivated receptor-g coactivator-1a and AMPactivated protein kinase activity (regulation of energy metabolism), and a higher proportion of type 1 fibers in skeletal muscle. [START_REF] Gao | Butyrate improves insulin sensitivity and increases energy expenditure in mice[END_REF] Moreover, the muscle-specific SCFA receptors FFAR3 and FFAR2 promote insulin sensitivity and modulate glucose uptake. These findings suggest that gut microbiota composition/function in DMD might be altered, with possible effects on the host's health and myopathy. However, very few exploratory data are available on intestinal smooth muscle in DMD, although dystrophin is expressed in smooth muscle, [START_REF] Vannucchi | Interstitial cells of Cajal, enteric neurons, and smooth muscle and myoid cells of the murine gastrointestinal tract express full-length dystrophin[END_REF][START_REF] Boland | Site-dependent pathological differences in smooth muscles and skeletal muscles of the adult mdx mouse[END_REF] and intestinal function is altered in patients with DMD. Moreover, gastrointestinal alterations, including aerophagia, gastroesophageal reflux, and constipation (affecting nearly 70% of patients for this last item), have been documented in these patients. [START_REF] Cascio | Gastrointestinal dysfunction in patients with Duchenne muscular dystrophy[END_REF] Gut bacterial community alteration in DMD could be potentiated by the fact that this chronic disease is associated with conditions that promote intestinal dysbiosis in other pathologic contexts. For instance, a sedentary lifestyle has deleterious effects on microbial composition, [START_REF] Cerdá | Gut microbiota modification: another piece in the puzzle of the benefits of physical exercise in health?[END_REF] and treatments prescribed to patients with DMD, particularly antibiotics to treat respiratory infections, [START_REF] Mangera | Practical approach to management of respiratory complications in neurological disorders[END_REF] deplete the intestinal microbiota. [START_REF] Viaud | The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide[END_REF] Therefore, to exclude these extrinsic factors, gut microbiota composition could be studied in mdx mice, an animal model of DMD. Some studies reported that, similar to patients with DMD, mdx mice exhibit increased intestinal peristalsis [START_REF] Baccari | Reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism[END_REF] and reduced fecal excretion, 28e30 particularly due to an alteration of the migrant motor complex responsible for mobility in the interdigestive period. [START_REF] Tameyasu | The effect of e-, i-, and n-nitric oxide synthase inhibition on colonic motility in normal and muscular dystrophy (mdx) mice[END_REF] The aims of the current study were: i) to characterize the gut microbiota composition in mdx mice and its changes during disease development by comparing the aand bdiversity of taxonomic profiles and gut microbiota abundance (16S rRNA gene metagenomic analysis) in mdx mice and control littermates at 8 weeks, 12 weeks, 6 months, and 1 year of age, ii) to measure plasma biomarkers that could mediate the skeletal muscleegut microbiota crosstalk, and iii) to investigate the intestine structure and contractile properties, as well as the expression in the ileum and muscle, of genes linked to bacterial-derived metabolites to identify microbiotaemuscle interactions in the mdx phenotype.

Materials and Methods

Q10

Animal Care

Stool Collection

Feces were collected directly from the anus of the mice at 8 weeks, 12 weeks, 6 months, and 1 year of age. Samples were then immediately frozen in liquid nitrogen before storage at e80 C.

Euthanasia and Sample Collection

Mice were fasted for 12 hours and then euthanized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). Blood was collected via inferior vena cava sampling and centrifuged at 2500 Â g for 15 minutes; the plasma supernatant was snap-frozen and stored at e80 C. Intestinal tissue samples (duodenum, jejunum, ileum, and colon), cecum content, and soleus and tibial anterior (TA) samples were collected, weighed, and immediately frozen in liquid nitrogen and stored at e80 C. Second TA samples were collected and placed directly in isopentane cooled in liquid nitrogen and stored at e80 C. Both extensor digitorum longus (EDL) muscles were carefully removed and tendons tied with braided surgical silk for future ex vivo contractility.

TA Staining by Immunohistochemistry

Serial Q11 transverse sections (10 mm thick) from liquid nitrogenecooled isopentane TA muscle samples embedded in optimal cutting temperature medium were obtained using a cryostat at e25 C and mounted on glass microscope slides. Sections were then washed in phosphate-buffered saline, blocked, and permeabilized with 0.1% Triton X-100 and 10% horse serum. Staining Q12 was performed with anti-laminin (L9393; 1/200; MilliporeSigma, Burlington, MA), anti-dystrophin (ab15277; 1/200; Abcam, Cambridge, United Kingdom), and Hoechst. This Q13 was followed by secondary antibody incubation with 546 donkey anti-rabbit (Fluoroprobes, Interchim; 1/500) for anti-laminin and Alexa 488 goat anti-rabbit (1/1000; Thermo Fisher Scientific, Waltham, MA) for anti-dystrophin. Whole

Ex Vivo Assessment of Contractility in EDL

Muscle contractile properties were assessed in ex vivo conditions as previously described. [START_REF] Ploquin | Lack of myostatin alters intermyofibrillar mitochondria activity, unbalances redox status, and impairs tolerance to chronic repetitive contractions in muscle[END_REF] This technique allows evaluation of the intrinsic muscle contractile properties. After 15 minutes' equilibration in the Krebs solution bath continually bubbled with 95% oxygen to 5% carbon dioxide (pH 7.4) and thermostatically maintained at 37 C, EDL samples were connected to a force transducer/length servomotor system (model 305B; Cambridge Instruments, Aurora Scientific Inc., Aurora, ON, Canada) and were stimulated along their entire length with platinum wire electrodes. The optimum muscle length (ie, the muscle length producing maximal twitch tension) was determined. All subsequent measurements were made at the optimum muscle length. The isometric tetanic tension was determined (701B Stimulator, Aurora Scientific Inc.) using stimulation trains of 500 milliseconds, with pulse duration of 0.5 millisecond at different frequencies, from 1 to 150 Hz. Stimulus trains were separated by a 1-minute interval. The maximum isometric tetanic tension was determined from the plateau of the frequency-tension curve. Three minutes after the tension-frequency determination, the resistance to fatigue was evaluated by using a low-frequency fatigue protocol of 50 Hz trains of 700 milliseconds delivered every 2 seconds for 5 minutes. The muscle fatigue index was defined as the time taken to produce a 50% reduction from the initial maximum power output. After all measurements, EDL samples were removed from the bath, trimmed of the connective tissue, blotted dry, and weighed. For Q15 each animal, the second EDL was placed in oxygenized Krebs solution throughout the procedure and was used in case of emergency.

Detection and Quantification of Plasma Proteins

One Q16 -quarter of the diluted plasma samples were analyzed by using the Meso Scale Discovery U-PLEX Metabolic Group biotinylated capture antibodies and corresponding detection antibodies for 11 proteins: IL-1b, IL-4, Il-6, tumor necrosis factor, MCP-1, ghrelin (active and total), glucagon-like peptide-1 (active and total), leptin, and peptide YY (total). Assays were performed following manufacturer's instructions using Meso Scale Discovery 96-well, 10-spot plates and recommended diluents, with all plasma samples being vortexed thoroughly before use. For experimental measurements with below detection limits, concentrations were considered as "0." Levels of circulating adiponectin were measured by using ELISA. Adiponectin doses were determined by using commercial ELISA kits (#DY1065; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Ex Vivo Jejunum Basal Contractile Function

Jejunum basal contractile properties were assessed ex vivo at 1 year of age. Specifically, 1 cm of the first part of the jejunum was connected to a force transducer/length servomotor system (model 305B; Cambridge Instruments, Aurora Scientific Inc.). The optimum smooth muscle length (ie, the muscle length producing maximal twitch tension) was determined. All subsequent measurements were made at optimum smooth muscle length. After 15 minutes' equilibration in Krebs solution continually bubbled with 95% oxygen to 5% carbon dioxide (pH 7.4) and thermostatically maintained at 37 C, the basal peristalsis was recorded at 200 Hz for 15 minutes. This method is adapted from Alves et al. [START_REF] Alves | Intestine of dystrophic mice presents enhanced contractile resistance to stretching despite morphological impairment[END_REF] To determine the period of peristalsis, the signal obtained with DMC version 3.500 software (Aurora Scientific Inc.) was extracted and imported to MATLAB 1.8.0_202 (MathWorks, Portola Valley, CA), in which a fast Fourier transform was used to convert the signal to a frequency domain. The Q17 peak frequency was then obtained as the frequency corresponding to the peak of the power spectrum. The period P was then estimated as the inverse of the peak frequency.

Histologic Analyses of the Small Intestine

The small intestine was removed, divided into two parts with the mucosal layer outward using a long wooden stick and immersed in a solution of 4% paraformaldehyde in phosphate buffer (0.1 mol/L, pH 7.4) for 4 hours at room temperature. [START_REF] Pereira | Comparison of two techniques for a comprehensive gut histopathological analysis: Swiss Roll versus intestine strips[END_REF] They were then rinsed in phosphate buffer and immersed in 20% sucrose in phosphate buffer (0.1 M, pH 7.4) for 24 hours at 4 C. Two parts of the intestine were opened longitudinally and coiled with the mucosal layer outward using a wooden stick, embedded in optimal cutting temperature medium, and frozen in isopentane cooled in liquid nitrogen. Swiss Rolls of intestine were cut in longitudinal sections into serial 10 mm thick slices and stained with hematoxylin and eosin and Alcian blue (stain goblets cells) for histologic analysis. Goblet cell density was determined by counting blue-stained cells per millimeter of intestine. Whole histologic sections were imaged with a digital slide scanner (MRI-INM platform; Nano-Zoomer 2.0-HT; Hamamatsu, Montpellier, France). The analysis was performed on NDP.view 2 software (Hamamatsu). Five fields of the same surface (7 mm 2 ) were captured, and all cells marked were counted and analyzed with ImageJ software version 1.53p

For structural analysis, Swiss Rolls of intestine were cut in longitudinal sections into serial 10 mm thick slices and stained with anti-laminin (L9393; 1/200; MilliporeSigma), anti-dystrophin (ab15277; 1/200; Abcam), and Hoechst. This was followed by secondary antibody incubation with 546 donkey anti-rabbit (Fluoroprobes; Interchim; 1/500) for anti-laminin and Alexa 488 goat anti-rabbit (1/1000; Thermo Fisher Scientific) for anti-dystrophin. The images were acquired with an automated imaging device (Axioscan; Zeiss, Oberkochen, Germany) provided by the facility imaging MRI-INM (Institute of Neuroscience, Montpellier, France).

mRNA Expression Analysis by Quantitative RT-PCR

Total RNA was isolated from ileum, TA, and soleus samples using TRIzol (15596-018, Invitrogen, Carlsbad, CA). RNA concentration was determined by spectrophotometric analysis (BioDrop DUO; BioDrop, Cambridge, UK), and purity was checked by calculating the OD 260nm /OD 280nm absorption ratio (>1.8). RNA quality was verified by using 1% agarose gel electrophoresis. Reverse Q18 transcription was performed with 2 mg of total RNA and the high-capacity cDNA Reverse Transcription Kit (catalog no. 4368813; Applied Biosystems) according to the manufacturer's instructions. One-tenth of the obtained cDNA was used in each PCR assay. real-time quantitative PCR analysis was performed using a Step One Plus detection system (AB Applied Biosystems) with 10 mL of Mastermix (PowerUp SYBR Green Master Mix, Scientific A25742; Thermo Fisher Scientific), 10 nmol/L of forward and reverse primers, 5 mL of diluted cDNA template, and water to a final volume of 15 mL. Forward and reverse primers are listed in Table 1 ½T1 . All PCR assays were performed in duplicate using the following cycling parameters: 50 C for 2 minutes, then 95 C for 2 minutes followed by 40 cycles of 95 C for 3 seconds and 60 C for 30 seconds. Relative mRNA levels were normalized to the levels of the housekeeping genes Arp, Tubulin-a for ileum samples, and Arp and Rps9 for TA and soleus samples. Results are expressed using the comparative cycle threshold method to generate DDC T values with template dilutions ranging from 10 1 to 10 6 copies. The PCR overall efficiency (E) was calculated from the standard curve slopes according to the equation E Z [10

DNA Extraction from Feces

Total cell DNA was extracted from 0.1 g of animal fecal material using the G'NOME Kit (BIO 10; MP Biomedicals, La Jolla, CA) with modifications. [START_REF] Mariat | The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age[END_REF] Fecal samples were homogenized in the supplied cell suspension solution. Cell lysis/denaturing solution was added, and samples were incubated at 55 C for 2 hours. To improve cell lysis, 0.1-mm diameter silica beads (750 mL) were added, and samples were mixed at the maximum speed in a Fast-Prep bench homogenizer (MP Biomedicals) for 4 minutes. Polyvinylpolypyrrolidone (15 mg) was added to ensure removal of polyphenol contamination that could inhibit the real-time quantitative PCR assays. Samples were vortexed and centrifuged at 20,000 Â g for 3 minutes, and supernatants were recovered. The remaining pellets were washed with 400 mL of TENP [50 mmol/L Tris (pH 8), 20 mmol/L EDTA (pH 8), 100 mmol/L NaCl, 1% Polyvinylpolypyrrolidone] and centrifuged at 20,000 Â g for 3 minutes. The washing step was repeated once, and the resulting supernatants pooled. Nucleic acids were precipitated by addition of one volume of isopropanol, incubation at À20 C for 20 minutes, and centrifugation at 20,000 Â g for 10 minutes. Pellets were resuspended in 400 mL of distilled water plus 100 mL of salt-out mixture and incubated at 4 C for 10 minutes. Samples were spun at maximum speed for 10 minutes, and DNA-containing supernatants were transferred to clean 1.5-mL microcentrifuge tubes. DNA was precipitated with two volumes of 100% ethanol at room temperature for 5 minutes, followed by centrifugation at 16,000 Â g for 5 minutes. DNA was resuspended in 150 mL of TE buffer and stored at À20 C.

Evaluation of Microbiota Composition by Sequencing

The V3-V4 region of the 16SrRNA genes was amplified using the bacterial primers 343F (5 0 -CTTTCCCTAC ACGACGCTCTTCCGATCTACGGRAGGCAGCAG-3 0 ) and 784R (5 0 -GGAGTTCAGACGTGTGCTCTTCCGAT CTTACCAGGGTATCTAATCCT-3 0 ) modified to add adaptors during the second PCR amplification. PCR assays were performed by using the MolTaq 16S DNA polymerase and the corresponding master mix (Molzym GmbH & Co. KG, Bremen, Germany). The PCR mix contained 10 ng of DNA, 1 mL of dNTPs (10 mmol/L), 1.25 mL each of forward and reverse primer (20 mM), and 0.5 mL of Taq polymerase in a total volume of 50 mL. The cycling program was as follows: 94 C for 3 minutes, followed by 40 cycles at 94 C for 15 seconds, 60 C for 30 seconds, 72 C

Ileum

Tubulin-a 5 0 -GTGGCCACGAGCATAGTTATT-3 0 5 0 -CTGGAACCCACGGTCATC-3 0 Soleus, TA Rps9 5 0 -ATCCGCCAACGTCACATTA-3 0 5 0 -TCTTCAGTCGCCTGGAC-3 0 Tight junction proteins and gut permeability Ileum Zo-1

5 0 -AAATCATCCGACTCCTCGTC-3 0 5 0 -CAGTTGGCTCCAACAAGGTAA-3 0 Ileum Zo-2 5 0 -CAGGCATGGAGGAGGTGA-3 0 5 0 -CACGGCAATTCCAAATCC-3 0 Ileum Cldn5 5 0 -ACGGGAGGAGCGCTTTAC-3 0 5 0 -GTTGGCGAACCAGCAGAG-3 0 Ileum Ocln 5 0 -TCCTCCTGGCTCAGTTGAA-3 0 5 0 -CCAGGCTCCCAAGATAAGC-3 0 Ileum Jam1 5 0 -AGAACAAAGAAAGGGACTGCAC-3 0 5 0 -ACCAGGAACGACGAGGTCT-3 0 SCFA receptors Ileum Ffar1 5 0 -AGGCGCTCTCCTCACACTC-3 0 5 0 -CTAGCCACATTGGAGGCATTA-3 0 Ileum, Soleus Ffar3 5 0 -CATCCTCCTGCCTGTACGAC-3 0 5 0 -ATACACAGGGGCACCATGA-3 0 Ileum, Soleus Ffar2 5 0 -TGCTCTGAAGAAGCCAATCA-3 0 5 0 -TTCTCCTCTGGTCCAGTGCT-3 0 LCFA receptor Ileum Ffar4 5 0 -TTGGTGTTGAGCGTCGTG-3 0 5 0 -CCAGCAGTGAGACGACAAAG-3 0 BCAA receptor Soleus Bcat2 5 0 -TGGAGACACTTTGAACACATGAGC-3 0 5 0 -TCTTTTGGACCCACATAGAAGC-3 0 Lipid metabolism Ileum, Soleus Angptl4 5 0 -GGGACCTTAACTGTGCCAAG-3 0 5 0 -GAATGGCTACAGGTACCAAACC-3 0 Glucose transporter Soleus Slc2a4 5 0 -GACGGACACTCCATCTGTTG-3 0 5 0 -GCCACGATGGAGACATAGC-3 0 Inflammation Ileum, Soleus Myd88 5 0 -GCCTTGTTAGACCGTGAGGAT-3 0 5 0 -CTAAGTATTTCTGGCAGTCCTCCT-3 0 Ileum, Soleus Tlr4 5 0 -GGACTCTGATCATGGCACTG-3 0 5 0 -CTGATCCATGCATTGGTAGGT-3 0 Ileum, Soleus Nfkb1 5 0 -TGAGGACGGGGTATGCAC-3 0 5 0 -TCACATGAAGTATTCCCAGGTTT-3 0 Soleus Trif 5 0 -CAGCTCAAGACCCCTACAGC-3 0 5 0 -CTCCCACACAGCCTCGTC-3 0 Adiponectin receptors Soleus, TA Adipor1 5 0 -GTTTGCCACTCCCAAGCA-3 0 5 0 -ACACCACTCAAGCCAAGTCC-3 0 Soleus, TA Adipor2 5 0 -TCTCAGTGGGACATGTTTGC-3 0 5 0 -AGGCCTAAGCCCACGAAC-3 0
Adipor1, adiponectin receptor 1; Adipor2, adiponectin receptor 2; Angptl4, fasting-induced adipose factor; Arp, acidic ribosomal phosphoprotein; BCAA, branched-chain amino acids; Bcat2, branched-chain-amino-acid aminotransferase; Cldn5, claudin-5; Ffar1, Free-fatty acid receptor 1; Ffar2, Free-fatty acid receptor 2; Ffar3, Free-fatty acid receptor 3; Ffar4, Free-fatty acid receptor 4; Jam1, junctional adhesion molecule A; LCFA, long-chain fatty acids; Myd88, myeloid differentiation primary response 88; Nfkb1, nuclear factor kappa B; Ocln, occludin; qPCR, real-time quantitative PCR; Rps9, 40S ribosomal protein S9; SCFA, short-chain fatty acids; Slc2a4, glucose transporter 4; TA, tibial anterior; Tlr4, Toll-like receptor 4; Trif, TIR-domain-containing adapter molecule 1; Zo-1, zonula occludens 1; Zo-2, zonula occludens 2.

Gut Microbiota and Mdx Physiopathology

The American Journal of Pathology -ajp.amjpathol.

Metagenomic Analysis

Sequencing data were demultiplexed at the Genopole Toulouse Midi-Pyrenees platform. Version 3.2.3 was used to produce abundance tables of operational taxonomic units (OTUs) and their taxonomic affiliation [START_REF] Escudié | FROGS: find, rapidly, OTUs with Galaxy Solution[END_REF] following author guidelines. [START_REF] Bernard | FROGS: a powerful tool to analyse the diversity of fungi with special management of internal transcribed spacers[END_REF] The most abundant sequences of each OTU were then matched with blastn to the Silva version 132 database. 38 Abundance tables and taxonomy files were imported into RStudio (version 1.2.1335), and phyloseq 1.28.0, 39 ggplot2 3.4.0, [START_REF] Wickham | ggplot2. Elegant Graphics For Data Analysis[END_REF] and custom scripts were used for data analysis. Samples were rarefied to even sampling depths before computing within-sample compositional diversities (Observed richness, Chao1, Shannon, and InvSimpson) and between-samples compositional diversity (UniFrac). Principal coordinates analysis was also performed on dissimilarity matrices to obtain a two-dimensional representation of the samples. Alpha diversity data were analyzed by using repeated measures analysis of variance. Permutational multivariate analysis of variance tests were performed on UniFrac matrices using 9999 random permutations and a significance level of 0.01. The relative abundances of phyla were compared by using repeated measures analysis of variance and GraphPad Prism version 10.0.0 for Windows (GraphPad Software, La Jolla, CA). As published by Segata et al, [START_REF] Segata | Metagenomic biomarker discovery and explanation[END_REF] the linear discriminant effect size (LEfSe) method was performed by combining the Kruskal-Wallis test or Wilcoxon rank-sum test with the linear discriminant analysis scores to estimate the effect size of differentially abundant features with biologic consistency and statistical significance (the a value was set at 0.05, and the linear discriminant analysis score threshold for discriminative features was >2.0) [START_REF] Segata | Metagenomic biomarker discovery and explanation[END_REF]

Statistical Analysis

All data are presented as means AE SEM. Statistical significance was checked by using two-way analysis of variance to compare the mdx and B10 groups at different time points; when not applicable, a t-test was used. For all statistical analyses, the significance level was set at 0.05. Data were analyzed by using GraphPad Prism version 10.0.0 for Windows.

Results

Reduced Gut Microbiota aand b-Diversities in mdx Genotype

Comparison of the Observed, Chao1, and InvSimpson indices obtained at different ages showed that a-diversity varied between genotypes. Specifically, the Observed (P < 0.01) and Chao1 (P < 0.05) indices were significantly reduced, whereas the InvSimpson index was increased, in mdx mice compared with wild-type B10 littermates (Figure 1 ½F1 A). The Shannon index was comparable between genotypes (Figure 1A). The graphical representation of bdiversity using principal coordinates analysis plots for the UniFrac distances showed a strong and significant effect of the genotype (P < 0.001) (Figure 1B). Separate analysis at each time point confirms that the separation of the genotype is significant at all time points (P < 0.001). The ordination plot shows that the first axis of the principal coordinates analysis corresponds to the genotype and accounts for almost 20% of the diversity (P < 0.0001) (Figure 1B).

Specific Taxonomic Modification of Main Phyla and Included Genera in mdx Genotype

Comparison of the abundance of the six main phyla during the first year of life revealed significant differences in gut microbiota composition in mdx and B10 mice. The abundance of Actinobacteria, Proteobacteria, and Tenericutes was significantly increased in mdx mice compared with B10 mice (Figure 2 ½F2 ). Moreover, the Deferribacteres phylum was only present in mdx mice (all ages tested) (Figure 2).

Analysis of the differential abundances using LEfSe highlighted 30 up-regulated OTUs (including eight unknown genera and species) in the gut microbiota of mdx mice compared with B10 mice. Conversely, nine OTUs (including eight unknown genera and species) were characteristic of B10 mice based on their linear discriminant analysis score compared with mdx mice (Figure 3 ½F3 A). Bacteroidaceae, Bacteroides, Alistipes, Rikenellaceae, Rhodospirillales, Rhodospirillaceae, Deferribacterales, Deferribacteraceae, and Deferribacteres were overexpressed by at least 3.6-fold in mdx mice. Conversely, the Lachnospiraceae NK4A136 group and Bacteroidales S24_7 group were decreased by almost 4.8-fold in B10 mice.

The cladogram representation of the LEfSe results according to the taxonomic rank (Figure 3B) allowed easy identification of the taxonomic branches (from phyla to OTUs) that were modified in mdx mice, as well as the extent of the modification. The abundance of the phylum Deferribacteres was increased in mdx mice, as was the class Deferribacteres, the order Deferribacterales, the family Deferribacteraceae, and the genus Mucispirillum. Similarly, two other main continuous taxonomic changes were identified in mdx mice: the phylum Proteobacteria, then the class (Alphaproteobacteria), order (Rhodospirillales), family (Rhodospirillaceae), and genus (unknown species); and the phylum Actinobacteria, then the class (Coriobacteriia), order (Coriobacteriales), family (Coriobacteriaceae), and genus (Enterorhabdus) (Figure 3B). For other phyla, some taxonomic ranks were altered; for instance, the prevalence of Bacteriodes and Bacteriodaceae belonging to the Bacteriodetes phylum was increased. Moreover, the Lachnospiraceae NK4A136 group from the Firmicutes phylum was reduced in B10 mice. No impact of dystrophin deficiency in the phylum Tenericutes was observed, however.

Low-Grade Circulating Inflammation and Reduced Adipokines Levels in mdx Genotype

Plasma biomarker analysis highlighted a low-grade inflammation in mdx mice characterized by higher levels

Gut Microbiota and Mdx Physiopathology

The American Journal of Pathology -ajp.amjpathol. of IL-6 (Figure 4 ½F4 A), tumor necrosis factor (Figure 4B), and MCP-1 (Figure 4C) compared with B10 mice. IL-1b involved in the inflammatory process was not modulated (Supplemental Table S1), and IL-4 was undetectable in both groups.

In addition, our data revealed a strong down-regulation of circulating adipokines such as adiponectin (Figure 4D) and leptin (Figure 4E) in mdx mice compared with their B10 littermates. Interestingly, as shown in Table 2 ½T2 , adiponectin and leptin explain 10% and 11.9%, respectively, of the gut microbiota signature of dystrophin-deficient mice.

The entero-endocrine hormone ghrelin differs between groups, with a normalization with age of circulating level in mdx mice compared with B10 mice (Supplemental Table S1). Nevertheless, glucagon-like peptide-1 and peptide YY measurements did not reveal differences between groups (Supplemental Table S1).

Slowed Down Gut Peristalsis in mdx Genotype, Not Likely Linked with the Dystrophin Protein Deficiency

In mdx mice, the limited amount of dystrophin protein synthesis is characterized by altered structure and function of skeletal muscle (Supplemental Figure S1A). Indeed Q20 , even with hypertrophy during the first 6 months of life (Supplemental Figure S1, BeD), hindlimb muscles revealed a force weakness from 8-weekeold worthening with aging (Supplemental Figure S1E).

Because gut microbiota is involved in intestinal smooth muscle motility, intestine morphology and function were investigated. Dystrophin staining by immunofluorescence revealed an absence of dystrophin in the intestine independently of the phenotype (Figure 5 ½F5 A). In addition, small intestine goblet cell density was assessed, as mucus production by the goblet cells is important for intestinal integrity. No difference was found in labeling and, consequently, in small intestine goblet cell density between the two phenotypes of mice (B10 mice, 52.1 AE 8.3 cells/mm 2 ; mdx mice, 54.1 AE 4.3 cells/mm 2 ) (Figure 5, B andC). The intestinal smooth muscle properties were assessed with the ex vivo contractility test, and the mean period of spontaneous basal contraction of jejunum was calculated. The period of peristalsis was significantly longer in 1-yeareold mdx mice than in B10 littermates, suggesting a slowing of peristalsis (P < 0.01) (Figure 5D).

Reduction of Ileum Gene Expressions Linked with Intestinal Functionality/Permeability in mdx Genotype

Analysis of the expression in ileum samples of various genes linked to intestinal functionality/permeability showed that Zo-1 and Zo-2 were significantly down-regulated in mdx mice compared with B10 mice (P < 0.05) (Figure 6, A and B

½F6

). Similarly, the gene encoding the SCFA receptor Ffar2 was down-regulated in mdx mice (P < 0.05) (Figure 6C). Conversely, Angptl4 was up-regulated in mdx mice (P < 0.005) (Figure 6D). Expression of genes encoding inflammatory markers, amino acid receptors, and lipid transporters were comparable between genotypes (data not shown).

Dysregulation of Muscle-Specific Receptors of Gut Bacterial Metabolites and Activation of the Muscle Tlr4/MyD88 Pathway in mdx Genotype

In TA samples, gene expression of Bcat2 (the skeletal muscleespecific isoform of transaminase 2) was strongly up-regulated in mdx mice compared with B10 mice at both ages (Figure 7 ½F7 A). In contrast, in oxidative soleus muscle, Bcat2 expression was down-regulated at both ages in mdx mice (12 weeks, 0.42 AE 0.2-fold change versus B10 mice; 1 year, 0.56 AE 0.07-fold change versus B10 mice; P < 0.01).

The adiponectin receptor 1 expression was also upregulated at both ages in mdx TA muscle (Figure 7B), whereas the nonmuscle-specific adiponectin receptor 2 was not significantly different (Figure 7C). Interestingly, in soleus muscle, adipor1 (12 weeks, 0.73 AE 0.16-fold change versus B10 mice; 1 year, 0.79 AE 0.08-fold change versus B10 mice; P < 0.01) and adipor2 (12 weeks, 0.64 AE 0.19fold change versus B10 mice; 1 year, 0.76 AE 0.06-fold change versus B10 mice; P < 0.001) were significantly down-regulated in mdx mice compared with B10 mice from 12 weeks old to 1 year old.

print & web 4C=FPO

Figure 3 Operational taxonomic unit abundances in mdx (C57BL10SnSc-DMD mdx /J) mice and B10 (wild-type C57BL10SnSc) mice. A: Linear discriminant analysis (LDA) effect size for the two genotype (all ages). The figure shows the microbial taxa, the abundance of which was significantly different between mdx (green) and B10 (red) mice. B: Cladogram representation of the microbiota composition in mdx and B10 mice. The cladogram plot shows the differences in the relative abundances of taxa at six levels between mdx and B10 mice. All plots were generated by using the online Galaxy Huttenhower Lab server. Each cycle represents a stratification from phylum (inner) up to genera (outer). n Z 10 per group per age.

Gut Microbiota and Mdx Physiopathology

The American Journal of Pathology -ajp.amjpathol.org 9 The mRNA level of Tlr4/Myd88 inflammation pathway showed a high activation in mdx mice muscles compared with those of B10 mice (Figure 8 ½F8

). The expression of the gene encoding the Toll-like receptor 4, which recognizes pathogen-associated molecular patterns and specifically bacterial lipopolysaccharides, was up-regulated in TA at both ages (Figure 8A) and soleus at 1 year old (B10 mice, 0.68 AE 0.09; mdx mice, 1.08 AE 0.08; interaction, P < 0.001) in mdx mice compared with B10 mice. In mdx mice, Myd88 also was significantly up-regulated in TA (Figure 8B) and soleus (12 weeks, 1.60 AE 0.9-fold change versus B10 mice; 1 year, 1.05 AE 0.2-fold change versus B10 mice; P Z 0.02) muscles. Angptl4, known to be up-regulated during inflammation, was highly up-regulated only in TA muscle (Figure 8C). The expression of other genes linked to gut microbiota metabolites (Table 1) was not different between genotypes (data not shown).

Discussion

This is the first study designed to explore gut microbiota in mdx mice to investigate the gut microbiotaeskeletal muscle crosstalk. Monitoring mdx mice and B10 wildtype littermates at 8 weeks, 12 weeks, 6 months, and 1 year of age highlighted a unique intestinal bacterial composition in mdx mice associated with an impairment of specific plasma and muscle inflammatory biomarker levels. We also confirmed a slowing of gut peristalsis independently of dystrophin deficiency in smooth muscle combined with the intestinal structure impairment in mdx mice. Taken together, these alterations might contribute to

Gut Microbiota and Mdx Physiopathology

The American Journal of Pathology -ajp.amjpathol. org Besides the gut microbiota diversity, the metagenomic analysis revealed strong taxonomic modifications. The abundances of four main phyla (Actinobacteria, Proteobacteria, Tenericutes, and Deferribacteres) were increased in mdx mice. For three of them, this concerned the phyla and also the included genera (LEfSe analysis): the Mucispirillum not in wild-type littermates, thus constituting a specific phylum and taxa related to dystrophin deficiency. This phylum includes six genera that are all Gram-negative bacteria. [START_REF] Alauzet | The phylum Deferribacteres and the genus Caldithrix[END_REF] To the best of our knowledge, these are original results, and no similar data have been published previously on this DMD model. Nevertheless, some hypotheses on the potential links between these microbiota abundance modifications and the dystrophic phenotype could be proposed based on findings of other pathologic models. For instance, in a mouse model of colorectal cancer, an increase in Mucispirillum schaedleri drives lipopolysaccharide production that is associated with an inflammatory response. [START_REF] Daniel | Functional changes in the gut microbiome contribute to transforming growth factor [beta]-deficient colon cancer[END_REF] In a model of ulcerative colitis (inflammatory bowel disease family), the active period of the disease is characterized by a larger abundance of Rhodospirillales. [START_REF] Yang | Gut microbiota drives the attenuation of dextran sulphate sodium-induced colitis by Huangqin decoction[END_REF] Clostridial are in close relationship with intestinal cells, to possibly modulating gut cells' immune processes. [START_REF] Lopetuso | Commensal Clostridia: leading players in the maintenance of gut homeostasis[END_REF] Some Enterorhabdusrelated species are known to degrade mucus and could expose the intestinal barrier to assault. [START_REF] Derrien | Mucin-bacterial interactions in the human oral cavity and digestive tract[END_REF] The combination of metagenomic and metatranscriptomic analyses might reveal the functional activity and implication in the dystrophic phenotype development of the specific commensal microbes identified in the current study.

The current study also found that in mdx mice, gut microbiota composition changes were associated with slowed intestinal motility, emphasized by the increased jejunum basal contraction wave period. This result is in accordance with a recent study by Singh et al [START_REF] Singh | Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice[END_REF] showing fewer full peristaltic waves in mdx mice associated with reduced contraction-stimulated force and mRNA expression of contractile proteins. Furthermore, dystrophin protein was not revealed by intestine staining, either in the control B10 group or in the mdx mice. Thus, the difference of intestinal microbiota between mdx and B10 is unlikely due to the absence of the dystrophin protein. In addition, the downregulation of genes encoding the tight junction proteins (Zo-1 and Zo-2), crucial for the epithelial barrier integrity maintenance [START_REF] Fasano | Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer[END_REF][START_REF] Ulluwishewa | Regulation of tight junction permeability by intestinal bacteria and dietary components[END_REF] observed in mdx ileum samples, indicates a potential impact on the epithelial barrier permeability. Furthermore, results on the gut microbiota suggest that intestine bacteria could play a role in the intestinal dysfunction observed in mdx mice. The Q22 unique gut microbiota signature in mdx mice with the over-representation of Gram-negative bacteria could promote inflammation in the lumen and disrupt the intestinal contractile properties, comforting the hypothesis of the low-grade circulating inflammation (discussed later). Moreover, Ffar2 (SCFA receptor) downregulation in mdx ileum highlights lower SCFA production that could alter the smooth muscle metabolism and contractile function. 50e52 Interestingly, Bcat2 profiles suggest a modulation of bioavailability of branched-chain amino acids. Because branched-chain amino acid production is partially regulated by the gut microbiota, the alteration of the gut microbiota in mdx mice could contribute to impairment in this production. Nevertheless, Bcat2 upregulation in TA and the opposite down-regulation in soleus indicate a more complicated and intricate response between muscle phenotypes, warranting further investigations.

The reduced Zo-1 and Zo-2 expression suggest an increased gut permeability that might lead to a leak of bacterial components in the circulatory system, thus contributing to low-grade inflammation, as confirmed by higher plasmatic levels of IL-6, tumor necrosis factor, and MCP-1 observed in mdx mice and/or bacterial infections in distant organs. [START_REF] Ghosh | Intestinal barrier dysfunction, LPS translocation, and disease development[END_REF] Furthermore, the LEfSe analysis showed an increase in lipopolysaccharide-producing Gram-negative bacteria (Deferribacteres phylum, Bacteroides genus) in mdx mice gut. Concomitantly, the gene expression of Tlr4 (receptor of lipopolysaccharide), myd88, and Angptl4 important proteins of the inflammation pathway were up-regulated in skeletal muscles of mdx mice, especially in the tibial anterior. Thus, alteration of gut microbiota associated with disruption of the intestinal barrier could worsen inflammation in dystrophindeficient skeletal muscle. Finally, circulating adipokine

Gut Microbiota and Mdx Physiopathology

The American Journal of Pathology -ajp.amjpathol. org dysregulation such as adiponectin, ghrelin, and leptin may indicate an adipose tissue disruption in mdx mice. Interestingly, the expression of adipor1, the suspected main driver of adiponectin cascade in skeletal muscle, [START_REF] Iwabu | Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2þ) and AMPK/SIRT1[END_REF][START_REF] Krause | Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function[END_REF] was altered in TA and soleus muscles from mdx mice (TA, up-regulation; soleus, down-regulation). The various patterns highlighted in mixed TA and oxidative soleus muscles might be explained by a different metabolism associated with the typology. Indeed, adiponectin electrophoresis in skeletal muscle has been shown to modulate myosin heavy chain genes toward oxidative phenotype. [START_REF] Abou-Samra | Potential therapeutic action of adiponectin in Duchenne muscular dystrophy[END_REF] Thus, adiponectin stimulation might be linked to muscle typology. Interestingly, plasma biomarker analysis also revealed a massive release of leptin in the blood flow at 1 year of age in B10 mice compared with mdx mice. Although this satiety hormone has not been studied intensively in the dystrophin-deficiency field, a lower level of leptin could be explained by the mdx fat metabolism impairment. [START_REF] Strakova | Integrative effects of dystrophin loss on metabolic function of the mdx mouse[END_REF] These findings raise questions on a third contributor as adipose tissue in the interorgan crosstalk.

To summarize, this original article observed for 1 year the gut microbiota signature in relation with the intestinal structure and function as well as blood biomarkers and skeletal muscle function in a context of dystrophin deficiency. These results showed a strong gut microbiota clustering between genotype, independently of age, with a modulation of four main phyla and genera related to inflammation with overall less diversity in mdx mice. Twenty percent of the b-diversity divergence was explained by the genotype, confirming the relationship between dystrophin deficiency and gut bacterial composition from birth. An over-representation of lipopolysaccharide-producing Gram-negative bacteria is shown in mdx mice, with reduced intestinal motility as well as gene expressions of ileum tight junction proteins, which suggest an increased intestinal porosity contributing to the low-grade inflammation. This is supported by the systemic inflammation, the upregulation of bacterial pro-inflammatory receptor Tlr4/ Myd88 in mdx muscles, and the adipose tissue secretion profile. Finally, the decrease of Ffar2 in the ileum might reveal a dysregulation of the gut microbiota SCFA production and bioavailability for skeletal muscles. This study highlights gut microbiota as a potential central metabolic organ in mdx physiopathology. [START_REF] Marullo | Microbes, metabolites and muscle: is the gut-muscle axis a plausible therapeutic target in Duchenne muscular dystrophy?[END_REF] Additional studies would be required to better understand gut microbiota involvement in dystrophy development/progression through the intestineeskeletal muscle crosstalk. This finding also encourages studies to develop novel approaches to address the gastrointestinal and muscle dysfunction in patients with DMD to improve the global therapeutic management of muscular dystrophies.

Q14

  histologic sections were imaged with an automated imaging device (MRI-INM; Axioscan; Zeiss, Oberkochen, Germany) and analyzed with ImageJ software version 1.53p (NIH, Bethesda, MD; http://imagej.nih.gov/ij).

print & web 4C=FPO Figure 1

 4C=FPO1 Figure 1 Dystrophin

Figure 2

 2 Figure 2 Four major gut microbiota phyla are increased in mdx (C57BL10SnSc-DMD mdx /J) mice. Relative

Figure 4

 4 Figure 4 mdx-Specific plasma profile associated with gut microbiota signature. AeE: Levels of IL-6 (A), tumor necrosis factor (TNF) (B), monocyte chemoattractant protein-1 (MCP-1) (C), adiponectin (D), and leptin (E) in mdx (C57BL10SnSc-DMD mdx /J) mice compared with B10 (wild-type C57BL10SnSc) mice at 12 weeks and 1 year of age. n Z 10 per group per age. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus B10 mice.

  worsen the physiopathology of the skeletal muscle in mdx mice. The Q21 impact of dystrophin deficiency on gut microbiota host. The lower Observed and Chao1 indices in mdx mice revealed reduced richness, and the increased InvSimpson index highlighted fewer dominant species and greater evenness. Thus, in mdx mice, the overall number of different OTUs and their abundance were significantly reduced. The nonsignificant difference of the Shannon index suggests that this overall lower diversity does not concern rare OTUs. Principal coordinates analysis plots for b-diversity clearly clustered the two genotypes, independently of age. Indeed, mdx genotype predicted 20% of b-diversity divergence, validating the theory that dystrophin deficiency affects bacterial composition from birth.

Figure 5

 5 Figure 5 Gut peristalsis and villi structure are impaired in 1-yeareold mice independently of dystrophin expression. A: Ileum staining with anti-laminin, anti-dystrophin, and Hoechst at 1 year old for both genotypes. B: General morphology of villi intestine in Swiss Roll. Sections were stained with hematoxylin and eosin (B) and Alcian blue (C) in 1-yeareold B10 (wild-type C57BL10SnSc) mice. D: Jejunum peristalsis at 1 year of age in mdx (C57BL10SnSc-DMD mdx /J) mice compared with B10 mice. n Z 8 per group (A); n Z 5 per group (B and C); n Z 10 per group. **P < 0.01 versus B10 mice. Scale bars: 50 mm (A); 500 mm (B and C). Magnified boxed area: Â20 (B and C).

  genus and Deferribacteraceae family in the Deferribacteres phylum; the Enterorhabdus genus and Coriobacteriaceae family in the Actinobacteria phylum; and the Rhdospirillaceae family in the Proteobacteria phylum were the most concerned. Interestingly, the Deferribacteres phylum and related taxa were only detected in stools from mdx mice and print & web 4C=FPO

Figure 6 Figure 7

 67 Figure 6 In mdx (C57BL10SnSc-DMD mdx /J) mice, alteration of gut microbiota composition is associated with gene expression dysregulation in ileum. A: Zo-1 (zonula occludens 1). B: Zo-2 (zonula occludens 2). C: Ffar2 (free-fatty acid receptor 2). D: Angptl4 (fasting-induced adipose factor). n Z 8 per group per age. *P < 0.05, **P < 0.01, ****P < 0.0001 versus B10 (wild-type C57BL10SnSc) mice.

Figure 8

 8 Figure 8 Up-regulation of the skeletal muscle inflammation Tlr4/Myd88 pathway in tibial anterior muscle of mdx (C57BL10SnSc-DMD mdx /J) mice. A: Tlr4 (Toll-like receptor 4). B: Myd88 (myeloid differentiation primary response 88). C: Angptl4 (fasting induce adipose factor). n Z 8 per group per age. ****P < 0.0001 versus B10 (wild-type C57BL10SnSc) mice .

  (À1/slope) ] e 1, and this value was >95% for all assays. The relative abundance of each sample was normalized according to the equation: Relative Quantity Z 2 ÀDDCT .

	Jollet et al
	4	374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 ajp.amjpathol.org 373 434

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Table 1

 1 Primers Used for qPCR in Ileum, Soleus, and TA

	Q31

  for 60 seconds, and a final extension at 72 C for 5 minutes. Sequencing was performed by using MiSeq technology (Illumina) at the Genopole Toulouse Midi-Pyrenees genomic facility (Toulouse, France).

		497
		498
		499
		500
		501
		502
		503
		504
		505
		506
		507
		508
		509
		510
		511
		512
		513
		514
		515
		516
		517
		518
		519
		520
		521
		522
		523
		524
		525
		526
		527
		528
		529
		530
		531
		532
		533
		534
		535
		536
		537
		538
		539
		540
		541
		542
		543
		544
		545
		546
		547
		548
		549
		550
		551
		552
		553
		554
		555
		556
		557
		558
	org	5

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Table 2

 2 Plasma Biomarkers and Their Effect on Gut Microbiota Signature

		Sum of
	Marker	squares R 2	Statistic P
	TNF (pg/mL)	0.2268 0.1328 2.144	0.006200
	Leptin (pg/mL)	0.2041 0.1195 1.901	0.01830
	Adiponectin (ng/mL)	0.1825 0.1069 1.675	0.04720
	MCP-1 (pg/mL)	0.1823 0.1068 1.673	0.03700
	Ghrelin active (pg/mL) 0.1253 0.1659 1.193	0.2032
	IL-4 (pg/mL)	0.07851 0.1219 0.6944 0.7855
	PYY (pg/mL)	0.1624 0.09510 1.471	0.08810
	GLP-1 active (pM)	0.1494 0.08752 1.343	0.1066
	IL-1b (pg/mL)	0.1155 0.08642 1.041	0.3317
	GLP-1 Total (pM)	0.1428 0.08362 1.278	0.1726
	IL-6 (pg/mL)	0.1345 0.07876 1.197	0.2147
	GLP-1, glucagon-like peptide-1; MCP-1, monocyte chemoattractant
	protein-1; PYY, peptide YY; TNF, tumor necrosis factor.
	Bold: P < 0.05.

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Acknowledgments

We thank the INRAE MIGALE bioinformatics facility (MIGALE, INRAE, 2020; Migale bioinformatics Facility, Jouy-en-Josas, France) for providing and storage resources. We thank Veronique Douard for her gift of phyla primers. We also thank the animal staff from the METAMUS DMEM platform facility, which belongs to the Montpellier Animal Facilities Network, as well as Pierre Delobel for his coding script regarding contractile properties files. We acknowledge the imaging facility MRI, member of the national infrastructure France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, "Investments for the Future"). The authors thank Elisabetta Andermarcher for expert manuscript editing.

Supported by a recurrent annual operating budget from the French National Research Institute for Agriculture, Food, and Environment and the University of Montpellier without specific grants Q32 .

Disclosure Statement

None declared. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Q24

Supplemental Data

Supplemental material for this article can be found at http://doi.org/10.1016/j.ajpath.2023.10.010.


Dany Jaffuel
email: dany.jaffuel@wanadoo.fr

Jean Pierre Mallet

François Roubille

Arnaud Bourdin

Muscle sympathetic nerve activity and adaptive servo-ventilation: questions remain

 for their efforts to clarify the effect of ASV on muscle sympathetic nerve activity (MSNA) in patients with central sleep apnoea (CSA) and heart failure with reduced left ventricular ejection fraction (HFrEF). Analysing data from an SERVE-HF ancillary study, they concluded that: 1) suppression of CSA with ASV did not seem to have a significant effect on chronic HFrEF-related sympathetic activation; and 2) simultaneous suppression of CSA and reduction in MSNA was associated with increased cardiovascular mortality. In our opinion, these conclusions might be different if some confounding factors were considered.

In addition to the limitation evoked by the authors (i.e. a type II error risk in the per-protocol analyses because of a lower number of patients in the ASV group (14 patients) than the calculated number of required subjects (16 patients)), we consider that the ASV efficiency on sleep disordered breathing (SDB) also needs to be questioned before acknowledging their conclusions. In the data reported in their table S3, 25% of patients adherent to ASV therapy had an apnoea-hypopnoea index (AHI) measured by polygraphy or polysomnography of at least 21.3 h-1 and a sleep time with peripheral oxygen saturation (SpO2) <90% of at least 43.5 min. It is a matter of fact that not only a residual AHI greater than 15 h-1 under continuous positive airway pressure treatment [START_REF] Arzt | Suppression of central sleep apnea by continuous positive airway pressure and transplant-free survival in heart failure: a post hoc analysis of the Canadian Continuous Positive Airway Pressure for Patients with Central Sleep Apnea and Heart Failure Trial (CANPAP)[END_REF] but also an SpO2 time greater than 22 min [START_REF] Oldenburg | Nocturnal hypoxaemia is associated with increased mortality in stable heart failure patients[END_REF] are associated with an excess in mortality of patients with CSA and chronic HFrEF.

Because TAMISIER et al. [START_REF] Tamisier | Effect of adaptive servo-ventilation for central sleep apnoea in systolic heart failure on muscle sympathetic nerve activity: a SERVE-HF randomised study[END_REF] initially hypothesised that ASV would reduce sympathetic tone by normalising breathing during sleep, the failure of ASV to address SDB for at least 25% of the population may have enhanced the risk of type II error. Reporting individual data and/or stratifying the data according to the efficacy of ASV on SDB before analysing variables such as MSNA, 24-h urinary catecholamine levels and cardiovascular mortality may help the journal readers to analyse this interesting SERVE-HF ancillary study. Stratified analyses of ongoing trials will need to take this ASV efficacy issue into account. Furthermore, these data reported by TAMISIER et al. [START_REF] Tamisier | Effect of adaptive servo-ventilation for central sleep apnoea in systolic heart failure on muscle sympathetic nerve activity: a SERVE-HF randomised study[END_REF] challenge the efficacy of ASV therapy in the SERVE-HF study [START_REF] Cowie | Adaptive servo-ventilation for central sleep apnea in systolic heart failure[END_REF]. It has been shown that the AHI reported by ASV devices is not as accurate as expected, hence the value of polysomnography monitoring under ASV treatment [START_REF] Silveira | Adaptive servoventilation device software in the assessment of residual respiratory events in patients with central or complex apnoeas[END_REF]. In the SERVE-HF study [START_REF] Cowie | Adaptive servo-ventilation for central sleep apnea in systolic heart failure[END_REF], ASV treated patients were considered effectively treated, but the measured AHI data were expressed as mean and minimum/maximum rather than median and quartile. This not only limits our ability to assess the true effectiveness of ASV therapy in the SERVE-HF study, but also to make a comparison with this ancillary study by TAMISIER et al. [START_REF] Tamisier | Effect of adaptive servo-ventilation for central sleep apnoea in systolic heart failure on muscle sympathetic nerve activity: a SERVE-HF randomised study[END_REF].

A limitation of this ancillary study pinpointed by the authors merits further detail. This ancillary study explored peripheral sympathetic vascular tone and not the cardiac sympathetic tone. In this regard, it is important to underline that peripheral sympathetic vascular tone measured using peroneal microneurography is not affected by medical treatments such as beta-blockers [START_REF] Azevedo | Nonselective versus selective beta-adrenergic receptor blockade in congestive heart failure: differential effects on sympathetic activity[END_REF]. This is both a strength and a limitation: a strength because it is possible to isolate the impact of ASV on sympathetic tone, but a limitation because in real life, the patient's sympathetic tone is the result of multiple factors, including drug treatment.

In conclusion, more than definitive answers, this ancillary study provides insights that should determine the design of future research to limit confounding factors. Pending the result of the ADVENT-HF study, new studies are needed in the modern era taking into account the evolution of medical treatments for these patients [START_REF] Mcdonagh | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure[END_REF] and in particular the potential impact of sacubritil/valsartan or gliflozin treatments on CSA [START_REF] Jaffuel | Sacubitril-valsartan initiation in chronic heart failure patients impacts sleep apnea: the ENTRESTO-SAS study[END_REF].

Conflict of interest:

A. Bourdin has nothing to disclose. D. Jaffuel reports personal fees for lectures and meeting attendance from Philips Healthcare and Lowenstein, personal fees for meeting attendance from Resmed, personal fees for lectures and meeting attendance, and non-financial support for board membership from SEFAM, grants and personal fees for lectures from Novartis, personal fees for lectures from Jazz, personal fees for advisory board work from Bioproject, personal fees for lectures and non-financial support for board membership from NOMICS, outside the submitted work. F. Roubille reports grants and personal fees from Novartis, personal fees from AstraZeneca, Servier, Boehringer, Vifor, Bayer, Pfizer, Novonordisk, Air Liquide, Abott and Lilly, outside the submitted work. J.P. Mallet has nothing to disclose.


Mohamed Amine Hebri

Abderrahmane Rebhaoui

Grégory Bauw

Jean-Philippe Lecointe

Stéphane Duchesne

Gianluca Zito

Abdenour Abdelli

Vincent Mallard

Adrien Maier

POWER DENSITY IMPROVEMENT OF AXIAL FLUX PERMANENT MAGNET SYNCHRONOUS MOTOR BY USING DIFFERENT MAGNETIC MATERIALS

This paper presents a study to improve the power density and efficiency of permanent magnet synchronous machines for electric traction applications with high operating speed. The studied axial flux permanent magnet motor is equipped of a yokeless dual-rotor. Several comparative studies have shown that this topology has the best power density and efficiency and that it allows a good cooling. This work is based on the use of different magnetic materials for an optimal design of the stator and rotor magnetic circuits to improve the motor performance. The candidate magnetic materials, perfectly adapted to the constraints of eMobility, are 3% Si-Fe Non-Oriented grain Electrical Steel (NOES), 3% Si-Fe Grain-Oriented Electrical Steel (GOES) and Iron-Cobalt Permendur electrical steel (Co-Fe). The main objective is to exploit, as far as possible, the optimal performances of each magnetic material in terms of high saturation flux density in order to reduce the geometrical dimensions of the stator teeth and rotor yoke and, consequently, to gain in overall motor mass. In addition, the benefit bring by the low specific losses is evaluated by calculating the overall efficiency of the machine.

I. INTRODUCTION

The road transport sector is a major source of greenhouse gas emissions, including CO2. For this reason, new restrictions and limitations of the CO2 emissions are implemented by the European Union in order to reach carbon neutrality in 2050 [START_REF] Auverlot | Les politiques publiques en faveur des véhicules à très faibles émissions, France stratégie[END_REF]. For example, in Europe and in 2020, CO2 emissions are limited to 95g/km on average for new cars [START_REF] Auverlot | Les politiques publiques en faveur des véhicules à très faibles émissions, France stratégie[END_REF]. This context leads car manufacturers to reduce their environmental impact in order to avoid penalties when the emissions exceed defined thresholds. As a result, and for several years, these manufacturers have been offering in their ranges hybrid cars (HEV), plug-in hybrids (PHEV), and fully battery electric cars (BEV). To make these cars economic and efficient and to improve the BEV autonomy, the manufacturers have the objective to improve the efficiency of the whole electric powertrain [START_REF] Auverlot | Les politiques publiques en faveur des véhicules à très faibles émissions, France stratégie[END_REF][START_REF] Cissé | Etat de l'art des topologies de machines électrques utilisées dans les véhicules électriques et hybrides[END_REF].

Improving efficiency requires to reduce losses in each element of the powertrain, as well as reducing its energy consumption due to the total weight of the components. This paper focuses on the electric motor by improving its efficiency and power to weight ratio. In automotive traction applications, the radial flux Permanent Magnet Synchronous Motors (PMSMs) are generally used due to its high-power density, high efficiency, compact size, and manufacturing simplicity [START_REF] Cissé | Etat de l'art des topologies de machines électrques utilisées dans les véhicules électriques et hybrides[END_REF][START_REF] Zhu | Electrical Machines and Drives for Electric, Hybrid, and Fuel Cell Vehicles[END_REF]. Axial flux PMSMs are good candidate as their power density may be even higher. To further improve the power density of axial-flux PMSM, there are several ways:

• the current density can be increased, but is limited by the thermal constraints and the cooling system [START_REF] Laidoudi | High-power density induction machines with increased windings temperature[END_REF]; • the maximum rotation speed can be increased, but is limited by mechanical constraints: the peripheral speed and the transmission system [START_REF] Pyrhonen | Design of Rotating Electrical Machines[END_REF]; • the use of permanent magnets with very high energy density are recommended (NdFeB, SmCo) [START_REF] Nagorny | Design aspects of a high speed permanent magnet synchronous motor / generator for flywheel applications[END_REF] but their environmental footprint are non-negligible; • the use of magnetic materials with high permeability, high saturation flux density and low specific losses is also a way to analyse, as Grain-Oriented Electrical Steel (GOES), or Iron-Cobalt Permendur electrical steel (CoFe) [START_REF] Rebhaoui | Analyse of Flux Density and Iron Loss Distributions in Segmented Magnetic Circuits Made with Mixed Electrical Steel Grades[END_REF][START_REF] Kowal | Comparison of Nonoriented and Grain-Oriented Material in an Axial Flux[END_REF][START_REF] Bloch | The use of iron-nickel and iron-cobalt alloys in electrical engineering, and especially for electrical motors[END_REF].

In this paper, the authors analyze the impact of using different electrical steels in the magnetic circuit of a yokeless dual-rotor axial flux PMSM. Conventionally, electric motors for automotive applications are designed with Non-Oriented grain Electrical Steel (NOES), which are well suited for rotating field applications [START_REF] Pyrhonen | Design of Rotating Electrical Machines[END_REF]. In this work, other magnetic materials is used as GOES. The latter is commonly applied in power transformer magnetic circuits [START_REF] Pyrhonen | Design of Rotating Electrical Machines[END_REF][START_REF] Fujisaki | Magnetic Material for Motor Drive Systems[END_REF]; it is characterized by a very high magnetic permeability, a high saturation flux density and low specific losses, compared to the conventional NOES. Similarly, Permendur CoFe, which is commonly used in aeronautical applications, is an attractive candidate due to its very high saturation flux density compared to NOES and GOES [START_REF] Bloch | The use of iron-nickel and iron-cobalt alloys in electrical engineering, and especially for electrical motors[END_REF]. The high level of saturation allows to reduce the size of the stator teeth and rotor yoke and, thus, to increase the power density at iso-torque. The low specific losses increase the efficiency of the machine [START_REF] Fujisaki | Magnetic Material for Motor Drive Systems[END_REF].

The paper is separated in four sections. First, a comparison between different candidate magnetic materials for e-mobility is presented, in terms of B-H curves (permeability and flux density level at saturation) and specific losses. Second, the studied motor is presented in its complete form and its equivalent 2-D Finite Element model, as well as the main parameters. In addition, the design approach and the integration of materials are explained. A brief presentation of the software and the simulation parameters is made. Third, the FEA numerical calculations results are presented in the form of mono-objective and multi objective optimization. The performance in the torque-speed plane is calculated for a selected structure. Four, the main results are presented and discussed in the conclusion.

II. MAGNETIC MATERIALS

Fig. 1-a shows the B-H curves for the three magnetic materials analyzed in this paper: NOES, GOES and the Permendur Hiperco50, which thicknesses are 0.20mm, 0.20mm and 0.15mm respectively. The measurements are obtained with a standardised Single Sheet Tester (SST) frame at 50 Hz.

The curves show that the GOES strips magnetized along the Rolling Direction (RD) have a high maximal permeability, about 31000, over 2 times more compared to NOES and Permendur (Fig. 1-b). On the other hand, GOES is highly anisotropic, which means that the performances in the Transverse Direction (TD) are much less interesting. The Permendur Hiperco50 sheet has a high saturation flux density value, over 2.25T, versus 1.83T for the conventionnel NOES and 2.03T for the GOES magnetized along the RD (Table I). Fig. 2 shows the specific losses for the different magnetic sheets when frequency is 500Hz. The GOES sheet magnetized along the RD presents the lowest iron losses, compared to the conventional NOES sheet, at iso-thickness.

In parallel, GOES strips of 0.20mm thickness have iron losses almost equal to the Co-Fe losses strips of 0.15mm thickness, at 500Hz and for flux density values lower than 1.8T. On the other hand, the performances of the GOES sheet following the TD are not interesting anymore.

The Permendur Hiperco50 steel becomes advantageous for the high frequency operating ranges because of its low thickness which limits the eddy current losses in the laminations. II. To improve power density and efficiency of the active parts of the studied axial flux, the authors propose to analyse the impact of using non-conventional materials for the magnetic stator and rotor circuits.

The strategy relies on the intrinsic properties of these materials but also on the topology of the magnetic circuit:

• It can be taken advantage of the Hiperco50 low thickness of 0.15mm to reduce the iron losses (eddy current losses) and consequently to improve the efficiency. Its high saturation flux density allows to reduce the size of the circuits. • GOES has good performance in the RD, which is usable in the stator teeth where the field lines are quasi-unidirectional along the axial direction as shown in fig. 4.

• In the rotor yoke, despite the rotating direction of the field (Fig. 4), possibility of using GOES can be studied.

The geometric parameters which are optimized are the rotor yoke width and the tooth width, respecting the necessary slot area for the wire arrangement, as well as the saturation levels of the sheets. The aim is to find the optimal geometrical dimensions for a high-power density, in the power range between 90kW and 105kW, at a given rotational speed of 10000rpm and at a given current density (15Arms/mm 2 ). The 3 magnetic materials described above NOES, GOES, and Hiperco50 (Fig. 1) are combined in the stator teeth and the rotor yoke, which lead to 9 configurations of magnetic circuits described in Table III. IV. FEA-2D MODELING

TABLE III COMBINATIONS FOR THE MAGNETIC CIRCUIT

Structures

The study is based on a Finite Element Analysis (FEA) using the commercial JMAG software. This software application allows to take into account both magnetic circuit saturation and magnetic anisotropy when GOES is modelled. The anisotropy is modelled with a diagonal permeability tensor based on measurements of the magnetic characteristics in RD and TD.

𝐵 = [ 𝜇 𝑥 0 0 𝜇 𝑦 ] × 𝐻 (1)
The calculation of iron losses is performed using the classical approximation of the Steinmetz model (taking into account the harmonics) which is based on standardized measurements. For GOES, the same model is used with bidirectional coefficients: (2

𝑃 𝑐𝑜𝑟𝑒_𝑙𝑜𝑠𝑠 = ∑ { (𝐾 ℎ
)
where i is the harmonic rank, N is the highest harmonic, f is the operating frequency, BRD, BTD are the maximum level of flux densities according to the RD and TD. The coefficients Kh, Ke, α, β, γ, and δ are determined with the measurement data.

The boundary conditions (i.e., periodicity and symmetry conditions) allow the machine geometry to be reduced to 3 slots/2 poles and to one rotor and a half stator (Fig. 5). This reduces the computational time. The mesh of 9304 elements is shown in Fig. 5; where the airgap is structured with 3 radial divisions of 0.43mm mesh size.

a) Parameter variations and reference results

In this part, the numerical results of the new structures presented in table III are shown. A comparison of the power densities and efficiencies between the different structures according to the different magnetic materials and the geometrical dimensions of the stator teeth and the rotor yoke is shown in Fig. 6 and7. The range of variation of the yoke thickness and the tooth width are respectively from 9 to 11mm and from 19 to 29mm. These limits varied depending on the structure, in order to satisfy the saturation flux density constraint in the magnetic circuits and the power range between 90 and 105kw.

The power density calculation only considers the active parts in the weight calculation. The calculation of the efficiency takes into account the iron losses, magnet losses and Joule losses considering the end coils and the skin effect/proximity effect.

For the full NOES conventional reference structure (Tn-Yn), the maximum power density is 9.39kW/kg obtained with a 24mm tooth width and a 12mm yoke width (Fig. 6-a). For the same structure, the maximum efficiency is 98.14%, obtained with a tooth width of 29mm and a rotor yoke width of 16mm (Fig. 7-a).

b) Power density optimization:

The Table IV and Fig. 6 allows to draws the following conclusions about the power density modifications :

-Keeping the NOES sheet in the rotor yoke, the stator teeth is replaced with non-conventional magnetic materials:

• The use of GOES and Hiperco50 sheets in the stator teeth allows to improve the power density. GOES (Tg-Yn) leads to obtain a maximum power density of 9.57kW/kg, 2% more compared to the reference structure and with a yoke width of 12mm and 22mm tooth width (Fig. 6-d);

• The use of the Hiperco50 sheet (Th-Yn) increases the power by 1.9% (9.56kW/kg) with a yoke thickness of 11mm and a tooth width of 19mm (fig. 6-g).

-Keeping the NOES in the stator teeth and replacing the rotor yoke with GOES and Hiperco50 also allow to improve the power density:

• By using the GOES (Tn-Yg), the maximum power density is 9.74kW/kg, i.e. an increase of 3.7%, with a yoke width of 11mm and a tooth width of 24mm (Fig. 6-b);

• Hiperco50 (Tn-Yh) leds to a maximum power density of 9.91kW/kg, with yoke width of 11mm and a tooth width of 19mm (fig. 6-g).

-When non-conventional materials is in both stator and rotor magnetic circuits:

• the Full GOES structure allows to have a maximum power density of 9.94kW/kg for a yoke width of 11mm and a tooth width of 22mm;

• A full Hiperco50 structure can give a max power density of 10.09kW/kg for a yoke width of 10mm and a tooth width of 20mm.

-For non-conventional mixed structures:

• GOES teeth and a Hiperco50 yoke lead to a power density of 10.11kW/kg for a yoke width of 10mm and a tooth width of 22mm

• Hiperco50 teeth and a GOES yoke provide 9.92kW/kg for a yoke width of 11mm and a tooth width of 20mm. The use of Hiperco50 sheets in the rotor yoke is more advantageous than NOES and GOES sheets. The best structures in terms of power density improvement are the mixed structure with GOES teeth and Hiperco50 yoke and the full Hiperco50 structure. The use of different non-conventional materials in the magnetic circuit (stator and rotor) can also improve the efficiency of the electric motor. To do that, a mono-objective optimization for different structures as a function of geometrical dimensions (tooth width, yoke width) is performed. Results are given in Fig. 7 and the maximum efficiencies obtained for each motor are given in Table V. The following conclusions can be written:

• The GOES in the rotor yoke is not advantageous, because the losses in the rotor increase; so this configuration is not adapted to this structure.

• On the other hand, using GOES in the stator leads to a strong improvement of the maximum efficiency with a benefit of more than 0.06%, compared to the NOES structure.

• Hiperco50 in the teeth allows to obtain a better improvement of the efficiency. The best structures in term of efficiency improvement are:

the mixt Hiperco50 tooth -NOES yoke structure (Th-Yn), with 25mm tooth width and 16mm yoke width, with an improvement of 0.10% compared to the NOES reference structure; the full Hiperco50 structure (Th-Yh), with 24mm tooth width and 15mm yoke width, with a 0.11% improvement. For a better design, a multi-objective optimization has been made in order to design the optimal structure in terms of efficiency and power density improvement, depending on the different magnetic materials and on the teeth and yoke geometric dimensions. Fig. 8 shows the Pareto fronts for the 9 structures described in Table III. The use of non-conventional magnetic materials, with full or mixed structures, always allows to improve the performances of the electric motor in terms of power density and efficiency, except for the structure with NOES conventional teeth and GOES yoke (Tn-Yg) for which only the power density is improved. The top three improved structures in power density and/or efficiency are:

• the full Hiperco50 structure (Th-Yh);

• the mixed GOES teeth -Hiperco50 yoke structure (Tg-Yh);

• the mixed Hiperco50 teeth -NOES yoke structure (Th-Yn). The mix GOES teeth -Hiperco50 yoke structure (Tg-Yh) is chosen for our motor, with a yoke width of 10mm and a tooth width of 22mm. It has been selected from the Pareto front (Fig. 8) and it provides the maximal power density of 10.11kW/kg and an efficiency of 98.08% at 10000rpm and a 130Arms current.

Chosen structure

The efficiency of the chosen motor at different operating points in the torque-speed plane is shown in Fig. 9. It is plotted for both operating modes: constant torque and flux weakening, using the MTPA control strategy. The map is calculated by FEA considering the iron losses, eddy current losses in the magnets and Joule losses considering coil-ends and the skin effect/proximity effect [START_REF] Rebhaoui | Using grain oriented electrical steel sheets for electric motor in automotive application: Tooth segmentation[END_REF]. The structure has a very high efficiency in the constant torque operating range (98.38%). It degrades with the flux weakening when the speed increases and at low load (89.57%). This is due to the increase in iron losses, losses in the permanent magnets and Joule effect losses at high frequency.

V. CONCLUSION

In this paper, the effects of using different magnetic materials (NOES, GOES and Hiperco50) in the magnetic circuit (stator-rotor) of a 12-tooth-pole dual-rotor and yokeless PMSM axial flux machine, are numerical analysed in order to improve the power density and the efficiency. The study is based on FEA-2D numerical modelling of the machine at the average radius and at the rated operating point. The principle of the study is to integrate different materials in the magnetic circuit, which gives full or mixed structures in the rotor and the stator. Mono-objective/multi-objective optimizations are performed for an optimal design with the goal of improving power density and/or efficiency by varying the geometrical dimensions of the stator teeth and the rotor yoke.

GOES in the stator teeth and Hiperco50 in the stator teeth and/or in the rotor yoke can improve power density and efficiency simultaneously, compared to a conventional NOES structure. The best structures in terms of power density and/or efficiency are:

• the GOES teeth -Hiperco50 yoke structure for the best power density; • the mixt of Hiperco50 teeth and NOES yoke for high efficiency; • the full Hiperco50 structure to get high power density and the best performance.

The GOES teeth -Hiperco50 yoke structure, which has the highest power density (10.11kW/kg), has been chosen to evaluate its efficiency in the torque-speed plane. The maximum efficiency of this structure can reach a high value of 98.38% at low average speed-load. This efficiency decreases with increasing speed. These conclusions result from numerical calculations based on implemented models. The latter may suffer from intrinsic errors, as the loss model or the anisotropic model for GOES.

For future work, a FEA-3D optimization including thermal and mechanical simulations will be carried out in order to quantify the gain. The experimental tests, on a prototype motor, will also be performed.

Fig. 1 .

 1 Fig.1. Performance of the different magnetic sheets used at 50Hz: a) B-H curves, b) relative permeabilityTABLE I MAGNETIC PROPERTIES Saturation flux density @ μr=100

Fig. 2 .

 2 Fig.2. Specific losses for the different magnetic sheets used at 500Hz III. DESIGN APPROACHThe studied axial flux dual-rotor and yokeless PMSM is shown in Fig.3-a. In order to simplify the study and the modelling, the authors has modelled a 2D structure at the average radius (Fig.3-b) of the axial flux PMSM. The main motor parameters are presented in TableII.

Fig. 3 .

 3 Fig.3. The PMSM structure studied: a) in 3D, b) in 2D at average radius

Fig. 4 .

 4 Fig.4. Flux lines in a pole of axial flux PMSM

Fig. 5

 5 Fig.5 Mesh of the studied structure V. FEA-2D RESULTS

Fig. 6 .

 6 Fig.6. Variation of power density as a function of the tooth width and the yoke width for: a) Tn-Yn, b) Tn-Yg, c) Tn-Yh, d) Tg-Yn, e) Tg-Yg, f) Tg-Yh, g) Th-Yn, h) Th-Yg, i) Th-Yh c) Efficiency optimization:

Fig. 7 .

 7 Fig.7. Variation of the efficiency as a function of the tooth-width and the yoke-width for: a) Tn-Yn, b) Tn-Yg, c) Tn-Yh, d) Tg-Yn, e) Tg-Yg, f) Tg-Yh, g) Th-Yn, h) Th-Yg, i) Th-Yh

Fig. 8 .

 8 Fig.8. Variation of the optimization criteria (Pareto front) e) Performance of the selected structure:

Fig. 8 .

 8 Fig.8. Efficiency maps of the selected motor.

TABLE II MOTOR PARAMETERS

 IIPARAMETERS

	Slot / Pole number	12 / 8
	Inner / Outer radius	60mm / 100mm
	Base rotational speed	10000rpm
	Number of series turns per phase	60
	Rated current	130Arms
	Airgap	1.3mm
	Magnet width	6.5mm

TABLE IV MAXIMAL

 IV POWER DENSITIES AND GAIN

	Structures	Max power density (kW/kg)	Gain (%)
	Tn-Yn	9.39	-
	Tn-Yg	9.74	3.72
	Tn-Yh	9.91	5.53
	Tg-Yn	9.57	1.91
	Tg-Yg	9.94	5.85
	Tg-Yh	10.11	7.66
	Th-Yn	9.56	1.81
	Th-Yg	9.92	5.64
	Th-Yh	10.09	7.45