Diffraction patterns and analysis

Conclusion and perspectives

Summary GIFAD is a powerful surface structure analytical tool providing online characterization of crystalline surface. It's one of most sensitive technique to the topmost layer. The interactions responsible for the scattering of the neutral projectile is similar to the ones repelling the tip of an atomic force microscope (AFM), the Pauli repulsion[3] and the long range attraction [3].

In this respect GIFAD is similar to an AFM with a perfect tip but operating in the reciprocal space because deflection angles are measured. Using helium as projectile and quantum scattering theory, GIFAD has demonstrated spatial accuracy close to a pm on the topology of the electronic density.

Perspective and future work

One important application of GIFAD is monitoring of the growth in MBE environment especially working under high temperature. We are looking for situations where a charge transfer at the surface could be triggered by a temperature variation or a E or B field. GIFAD would then be able to track online the associated change of the valence electron density profile. Reference

Results and discussion

The Quantum binary collision model(QBCM):

Simplified Model

Laue Ab initio calculation:

2).Detector with high quantum efficiency ~50%, all scattered particles recorded on a ~80mm MCP 3). Nondestructive and no charging effect 4). High spatial accuracy close to pm on the topology of the electronic density 1).High temperature tolerable up to 1000K (LiF)

Advantages:

Spatial resolution: ~pm

Peng Pan and Philippe Roncin

E⊥=100meV

Quantum Theory in Coll. with Andrey Borisov (ISMO)

  FIG 3. Quasi polar transform of the raw diffraction image in panel d). Panel b) polar scattering profile. Experimental data are fitted by using Gaussian and log-normal distributions. Panel c) corresponds to the intensity in a narrow horizontal band centered on the specular reflection [3].

  [1]. Atkinson, Paola, et al. "Dynamic grazing incidence fast atom diffraction during molecular beam epitaxial growth of GaAs." Applied Physics Letters 105.2 (2014): 021602. [2]. Debiossac, M., et al. "Combined experimental and theoretical study of fast atom diffraction on the β 2 (2× 4) reconstructed GaAs (001) surface." Physical Review B 90.15 (2014): 155308. [3]. Pan P. et.al. Polar inelastic profiles in fast atom diffraction at surfaces. Phys. Rev. B(accepted 2021) [4]. Brivio, G. P., and M. I. Trioni. "The adiabatic molecule-metal surface interaction: Theoretical approaches." Reviews of Modern Physics 71.1 (1999): 231. [5]. Debiossac M. et.al. Refraction of fast Ne atoms in the attractive well of a LiF (001) surface. JPCL. 2020

  FIG 5. Rocking curves for Ne atoms scattered along the ⟨110⟩ direction [5]. The intensity of the j th diffracted beam is shown as a function of E ⊥ , the energy of the motion in the (y, z) plane. For the degenerate ± j diffraction orders, the summed intensity is shown. Diffracted intensities were measured during a scan of the projectile energy (blue triangles) or of the incidence angle (red circles). The black line is for theoretical results. The very narrow peaks calculated at low energies reveal the bound state resonances.


Sabrina Ben Larbi

Marielle Saclier

Aurélie Fessard

Gaëtan Juban

Bénédicte Chazaud
email: benedicte.chazaud@inserm.fr

Histological analysis of Tibialis Anterior muscle of DMD mdx4Cv mice from 1 to 24 months

Keywords:

BACKGROUND:

The mdx-C57/B6 mouse model does not show the clinical signs of Duchenne muscular dystrophy (DMD), although muscles exhibit hallmarks of permanent regeneration and alterations in muscle function. The DMD mdx4Cv strain exhibits very few revertant dystrophin positive myofibers, making that model suitable for studies on gene and cell therapies.

OBJECTIVE:

The study appraises the histological evolution of the Tibialis Anterior muscle of WT and DMD mdx4Cv mutant from 1 to 24 months. METHODS: Histological analysis included a series of immunostainings of muscle sections for assessing tissue features (fibrosis, lipid deposition, necrosis) and cellular characteristics (size of myofibers, number and distribution of myonuclei, number of satellite cells, vessels, macrophages).

RESULTS: None of the investigated cell types (satellite cells, endothelial cells, macrophages)

showed variations in their density within the tissue in both WT and DMD mdx4Cv muscle.

However, analyzing their number per myofiber showed that in DMD mdx4Cv , myofiber capillarization was increased from 1 to 6 months as compared with WT muscle, then dropped from 12 months. Macrophage number did not vary in WT muscle and peaked at 6 months in DMD mdx4Cv muscle. The number of satellite cells per myofiber did not vary in WT muscle while it remained high in DMD mdx4Cv muscle, starting to decrease from 12 months and being significantly lower at 24 months of age. Myofiber size was not different in DMD mdx4Cv from WT except at 24 months, when it strongly decreased in DMD mdx4Cv muscle. Necrosis and lipid deposition were rare in DMD mdx4Cv muscle. Fibrosis did not increase with age in DMD mdx4Cv muscle and was higher than in WT at 6 and 12 months of age.

CONCLUSION: As a whole, the results show a strong decrease of the myofiber size at 24 months, and an increased capillarization until 6 months of age in DMD mdx4Cv as compared with the WT. Thus, DMD mdx4Cv mice poorly recapitulates histological DMD features, and its use should take into account the age of the animals according to the purpose of the investigation.

INTRODUCTION

Duchenne muscular dystrophy (DMD) is a progressive X-linked neuromuscular disorder due to mutations in the DMD gene, that encodes for dystrophin. Dystrophin belongs to a large transmembrane complex (dystrophin associated glycoprotein complex) that links the intracellular cytoskeleton of the myofiber to the extracellular matrix. The absence of dystrophin leads to recurrent myofiber damages [START_REF] Petrof | Dystrophin protects the sarcolemma from stresses developed during muscle contraction[END_REF], that trigger the activation of satellite cells, or muscle stem cell (MuSC)s and their entry into the myogenesis program, in attempt to repair the damaged myofibers [START_REF] Yin | Satellite cells and the muscle stem cell niche[END_REF]. Although highly efficient in normal skeletal muscle, the process of regeneration eventually fails in DMD due to chronic myofiber damage [START_REF] Petrof | Dystrophin protects the sarcolemma from stresses developed during muscle contraction[END_REF], leading to the replacement of the muscle parenchyma by fibrosis and lipid deposition. Moreover, a pro-fibrotic environment is associated with a deficit in myogenesis [START_REF] Mazala | TGFbeta-driven muscle degeneration and failed regeneration underlie disease onset in a DMD mouse model[END_REF]. The most used model for DMD is the mdx mouse, which bears a nonsense mutation in the Dmd gene [START_REF] Sicinski | The molecular basis of muscular dystrophy in the mdx mouse: a point mutation[END_REF]. On the contrary to human, mdx mice do not show clinical signs of the disease, move and live normally. However, mdx mice are easy to breed and the mdx hindlimb muscle shows signs of permanent regeneration associated with alterations in muscle function when specifically stimulated [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF][START_REF] Bobet | Force and stiffness of old dystrophic (mdx) mouse skeletal muscles[END_REF][START_REF] Anderson | Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse[END_REF].

Therefore, despite its limitations, the mdx mouse is widely used as a model for understanding the physiopathological mechanisms sustaining DMD and as a preclinical model to test therapeutic strategies.

Most of the longitudinal studies describing mdx skeletal muscle tissue all life long were mainly done with C57/BL10 mice, the original background of the mdx mouse (here after referred as B10-mdx) [START_REF] Coulton | The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation[END_REF][START_REF] Dimario | Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle[END_REF][START_REF] Chamberlain | Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma[END_REF][START_REF] Massopust | Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss[END_REF]. More recent studies have been using a C57/B6 background, and the DMD mdx4Cv and DMD mdx5Cv mutants because they show a low frequency of reversion mutations [START_REF] Im | Differential expression of dystrophin isoforms in strains of mdx mice with different mutations[END_REF].

A crucial parameter to consider when evaluating mdx muscle phenotype or function is the age of the animal. Of most importance, B10-mdx mice show an episode of acute myofiber degeneration at 3-4 weeks of age (at time of weaning) that leads to an important process of regeneration [START_REF] Cullen | Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic (mdx) mouse. Comparison with Duchenne muscular dystrophy[END_REF][START_REF] Dangain | Muscle development in mdx mutant mice[END_REF][START_REF] Tanabe | Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse[END_REF]. Thereafter, continuous cycles of damage and regeneration lead to more than 80-90% of myofibers exhibiting hallmarks of regeneration at 3 months of age [START_REF] Tanabe | Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse[END_REF][START_REF] Torres | The mutant mdx: inherited myopathy in the mouse. Morphological studies of nerves, muscles and end-plates[END_REF]. Second, from several pioneers studies that investigated B10-mdx muscle at various ages (from 10 to 24 months), it was admitted that time accelerates the dystrophic process, although high variations were observed between the studies [START_REF] Coulton | The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation[END_REF][START_REF] Dimario | Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle[END_REF][START_REF] Pastoret | Further aspects of muscular dystrophy in mdx mice[END_REF][START_REF] Lefaucheur | Phenotype of dystrophinopathy in old mdx mice[END_REF][START_REF] Pastoret | mdx mice show progressive weakness and muscle deterioration with age[END_REF]. As a result, the literature in the field encompasses studies using a variety of time ranges using the mdx model, animals being considered as "old" from 10 to 22 months.

One of the most commonly used readouts of muscle homeostasis are histological parameters, such as the number and size of myofibers, the number of satellite cells, of vessels, etc. Having established a series of samples that were used for previous studies, we took the opportunity to perform a histological comparative analysis of these samples, which were Tibialis Anterior (TA) muscle of the C57/B6 DMD mdx4Cv mouse strain, from 1 to 24 months of age. TA muscle is one of the mostly used for investigation of MuSC biology. The B6 background is also very popular because of the use of many transgenic strains for the study of muscle biology. In the DMD mdx4Cv model, a C to T transition in exon 53 at position 7916 creates a premature ochre stop codon (CAA to TAA) [START_REF] Chapman | Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice[END_REF]. DMD mdx4Cv mouse strain exhibits low frequency of reversion mutations, rendering that model suitable for gene and cell therapy in preclinical investigations [START_REF] Im | Differential expression of dystrophin isoforms in strains of mdx mice with different mutations[END_REF].

MATERIALS AND METHODS

Mice experiments and histology. WT and DMD mdx4Cv (20) on C57BL/6J background males from 1 to 24 months of age were used according to the French legislation (Approval from local Animal Care and Use Committee was obtained under ref CEE A34.BC-RM.053.12). 5 mice were used at each time point. The TA muscle was recovered and was frozen in liquid nitrogenprecooled isopentane, and stored at -80°C until use. Ten micrometer-thick cryosections were made and used for stainings and immunostainings.

Histological stainings. Hematoxylin-eosin (HE) was used for morphological observation of the muscle tissue. To label lipids, sections were stained with Sudan Black solution (199664, Sigma) for 2 h and were counterstained with Hemalun (MHS80, Sigma) for 1 min. Whole muscle sections were reconstituted with ImageJ software after recording at x10 objective using a Zeiss axioskop microscope and an Axiocam ICC5 zeiss camera. The area of black staining (Sudan black staining) was measured using ImageJ software using the AutoThreshold Yen and Analyze/histogram function.

Immunolabeling for satellite cells. Muscle sections were fixed with paraformaldehyde (4%) for 10 min at room temperature and permeabilized with Triton X-100 (0.5%) before acidic antigen retrieval was performed (Citrate buffer 10 mM at 90°C for 5 min). Slides were incubated with primary antibodies against Pax7 (1/50, Developmental Studies Hybridoma Bank, DSHB) overnight at 4°C, then washed with PBS and further incubated with FITC-conjugated secondary antibodies (1/200, Jackson ImmunoResearch Laboratories). A biotin-conjugated secondary antibody (1/200 Vector laboratory, BA-2000) revealed by a DTAF-conjugated streptavidin (1/1000, Beckman Coulter, PN IM0307) was used to amplify the signal as previously described [START_REF] Theret | AMPKalpha1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis[END_REF]. Muscle sections were then incubated with anti-laminin antibodies (1/100, L9393 sigma) that stains all basal membranes, for 2 h at 37°C, washed and further incubated with Cy3-conjugated secondary antibodies (1/200, Jackson ImmunoResearch Laboratories) for 45 min at 37°C. Sections were washed with PBS, incubated in Hoechst solution for 10 sec, and then mounted with Fluoromount (FP483331, Interchim). About 12 pictures covering all areas of the muscle section were taken at x20 objective using Zeiss Z1 imager microscope and a Photometrics CoolSnap camera. The number of muscle stem cells (Pax7 pos ) was manually counted using ImageJ software as well as the number of myofibers (thanks to laminin immunostaining). Results are given in number of cells/myofiber or in number of cells/mm 2 of muscle section.

Immunolabeling for macrophages. Muscle sections were directly incubated with primary antibodies against F4/80 (1/200 ab6640 Abcam) overnight at 4°C, then washed with PBS and further incubated with Cy3-conjugated secondary antibodies (1/200 Jackson ImmunoResearch Laboratories). Muscle sections were then fixed with paraformaldehyde (4%) for 10 min at room temperature and permeabilized with Triton X-100 (0.5%) before immunolabeling for laminin as described above for satellite cells. About 12 pictures covering all areas of the muscle section were taken at x20 objective using Zeiss Z1 imager microscope and a Photometrics CoolSnap camera. The number of macrophages (F4/80 pos ) was manually counted using ImageJ software as well as the number of myofibers (thanks to laminin immunostaining). Results are given in number of cells/myofiber or in number of cells/mm 2 of muscle section.

Immunolabeling for endothelial cells. Muscle sections were fixed with paraformaldehyde (4%) for 10 min at room temperature and permeabilized with Triton X-100 (0.5%) before incubation with primary antibodies against CD31 (1/200, ab7388, Abcam) overnight at 4°C, then washed with PBS and further incubated with Cy3-conjugated secondary antibodies (1/200, Jackson ImmunoResearch Laboratories). Muscle sections were then treated for the detection of laminin as described above for satellite cells. About 12 pictures covering all areas of the muscle section were taken at x20 objective using Zeiss Z1 imager microscope and a Photometrics CoolSnap camera. The number of capillaries (CD31 pos ) was manually counted using ImageJ software as well as the number of myofibers (thanks to laminin immunostaining).

Results are given in number of cells/myofiber or in number of cells/mm 2 of muscle section.

Immunolabeling for collagen 1. Muscle sections were fixed with paraformaldehyde (4%) for 10 min at room temperature and permeabilized with Triton X-100 (0.5%) before incubation with primary antibodies against collagen 1 (1310-01, Southern Biotech) overnight at 4°C, then washed with PBS and further incubated with Cy3-conjugated secondary antibodies (1/200, Jackson ImmunoResearch Laboratories) and mounted with Fluoromount (FP483331, Interchim). About 12 pictures covering all areas of the muscle section were taken at x20 objective using Zeiss Z1 imager microscope and a Photometrics CoolSnap camera. Fibrosis was quantified after collagen I immunolabelling as in [START_REF] Juban | AMPK Activation Regulates LTBP4-Dependent TGF-beta1 Secretion by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy[END_REF]. Whole muscle sections were automatically scanned at × 10 of magnification using an Axio Observer.Z1 (Zeiss) connected to a CoolSNAP HQ2 CCD Camera (photometrics).

Immunolabeling for damaged myofibers. Muscle sections were fixed with paraformaldehyde (4%) for 10 min at room temperature and permeabilized with Triton X-100 (0.5%) before incubation with donkey anti-mouse FITC-conjugated IgGs (Jackson ImmunoResearch Laboratories) overnight at 4°C, then washed with PBS and mounted with Fluoromount (FP483331, Interchim). Whole muscle sections were automatically scanned at × 10 of magnification using an Axio Observer.Z1 (Zeiss) connected to a CoolSNAP HQ2 CCD Camera (photometrics). The area of IgG positive myofibers was manually delineated. Results are given in % of the total muscle section area.

Analysis of myofiber CSA. Whole muscle sections were automatically scanned at × 10 of magnification using an Axio Observer.Z1 (Zeiss) connected to a CoolSNAP HQ2 CCD Camera (photometrics). Myofiber cross-section area (CSA) was calculated on whole muscle sections using Open-CSAM software based on laminin staining as previously described [START_REF] Desgeorges | Open-CSAM, a new tool for semi-automated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle[END_REF].

Analysis of myonuclei.

12 pictures covering all areas of the muscle section immunolabelled for laminin were taken at x20 objective using Zeiss Z1 imager microscope and a Photometrics CoolSnap camera. The number of myonuclei per myofiber (distinguishing the myofibers with peripheral versus central nuclei) was manually counted using ImageJ software.

Statistics.

For each time point, 5 mice were analyzed in a non-blinded way by two independent experimenters. Statistical analyses included One-way Anova after checking normality or Man-Whitney test for non-parametric data. P<0.05 was considered significant.

RESULTS AND DISCUSSION

TA muscles from WT and DMD mdx4Cv from 1 to 24 months were proceeded for histological stainings and immunostainings. While DMD mdx4Cv muscle showed several signs of necrosis, inflammation, heterogeneity in fiber size and signs of regeneration as compared with WT, there was no obvious macroscopic changes in the muscles with age (HE staining examples are given in Figure Suppl1).

Myofibers.

Laminin immunolabeling (Figure 1A) was used to analyze various parameters of myofibers.

After the huge regeneration process observed at 3 weeks, the number of regenerating myofibers increases in mdx muscles, reaching about 80% after a few weeks and remaining high until 2 years of age [START_REF] Pastoret | mdx mice show progressive weakness and muscle deterioration with age[END_REF][START_REF] Pastoret | Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse[END_REF]. We evaluated the number of myonuclei/myofiber in both nonregenerating myofibers (that present a peripheral location of their nuclei) and regenerating/regenerated myofibers (exhibiting myonuclei in a central location) in about 12 pictures taken randomly in the whole section. In WT muscle, myonuclei were mainly present at the periphery of myofibers (Figure 1B). Rare myonuclei were present in a central position, reflecting isolated fusion events all life long, with no increase with age (Figure 1B). In DMD mdx4Cv muscles, the number of myonuclei dramatically increased in myofibers with central myonuclei from 3 months of age (+272% vs 1 month), likely reflecting the transition from an acute to a chronic regenerating state of the muscle (Figure 1B). Concomitantly, the number of nuclei in myofibers exhibiting only peripheral nuclei declined (Figure 1B). It was previously reported that myofibers isolated from Soleus, Extensor Digitorum Longus and Flexor Digitorum Brevis (FDB) muscles show abnormalities from 4 months of age, which increase after 6 months to reach 90% of myofibers (30% in FDB) [START_REF] Head | Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice[END_REF]. Adding a level of complexity in the analysis of mdx muscle using transversal sections, both peripheral and central nuclei were observed along the same myofiber in B10-mdx muscle [START_REF] Massopust | Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss[END_REF][START_REF] Head | Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice[END_REF].

Next, the number and size of myofibers were evaluated on entire muscle sections. In both WT and DMD mdx4Cv muscles, the number of myofibers per unit area decreased from 1 to 3 months. Then, it did not vary in WT while in DMD mdx4Cv muscle, the number of myofibers increased at 24 months (Figure 1C). Similarly, a previous study indicated no high variation between 3 and 12 months of age in B10-mdx hindlimb muscles [START_REF] Pastoret | Further aspects of muscular dystrophy in mdx mice[END_REF].

Myofiber size is a popular feature to assess skeletal muscle regeneration. We used a semiautomated tool to measure myofiber CSA in regenerating conditions on entire muscle sections [START_REF] Desgeorges | Open-CSAM, a new tool for semi-automated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle[END_REF]. Results of the mean CSA (Figure 1D) and CSA distribution (Figure 1E,F) showed that in both WT and DMD mdx4Cv muscles, CSA increased at 3 months (+157 and 182% vs 1 month, respectively). Thereafter, the mean myofiber CSA did not vary until a very late time point, i.e. at 24 months (-59% vs 12 months), where the smallest CSA was observed in DMD mdx4Cv muscles, in accordance with the increased number of myofibers at this last time-point (Figure 1C). This decrease was not observed in WT muscles, although the distribution of myofiber CSA showed an increased number of smaller myofibers at 24 months (Figure 1E). In DMD mdx4Cv muscles, the distribution of myofiber CSA varied according to the CSA mean, with the smallest myofibers being observed at 1 and 24 months, while bigger myofibers were observed in 6-month-old animals (Figure 1F). Previous studies reported myofiber hypertrophy during the first months of life of B10-mdx, likely in response to the huge degenerating process occurring at 3-5 weeks of age [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF][START_REF] Pastoret | Further aspects of muscular dystrophy in mdx mice[END_REF][START_REF] Mouisel | Muscle weakness and atrophy are associated with decreased regenerative capacity and changes in mTOR signaling in skeletal muscles of venerable (18-24-month-old) dystrophic mdx mice[END_REF]. Another study showed an increase of the myofiber CSA at 12 months [START_REF] Pastoret | Further aspects of muscular dystrophy in mdx mice[END_REF]. However, other analyses indicated that at 10 months of age, B10-mdx muscles exhibit an increase of both small and large myofibers [START_REF] Massopust | Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss[END_REF][START_REF] Louboutin | Morphometric analysis of mdx diaphragm muscle fibres. Comparison with hindlimb muscles[END_REF]. It is likely that this great heterogeneity is due to myofiber branching, which has been repeatedly reported [START_REF] Anderson | Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse[END_REF][START_REF] Head | Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice[END_REF] to increase with age [START_REF] Tamaki | Three-dimensional cytoarchitecture of complex branched fibers in soleus muscle from mdx mutant mice[END_REF] and to be very important after 2 years [START_REF] Massopust | Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss[END_REF][START_REF] Chan | Branched fibers in dystrophic mdx muscle are associated with a loss of force following lengthening contractions[END_REF]. Myofiber branching is associated with an alteration of calcium signaling and of excitation/contraction coupling, leading to defects in myofiber function [START_REF] Head | Branched fibres in old dystrophic mdx muscle are associated with mechanical weakening of the sarcolemma, abnormal Ca2+ transients and a breakdown of Ca2+ homeostasis during fatigue[END_REF][START_REF] Lovering | Malformed mdx myofibers have normal cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+ signaling[END_REF]. Myofiber branching is observed in aged normal skeletal muscle, with about 15% of myofibers exhibiting 2 branches [START_REF] Pichavant | Incidence and severity of myofiber branching with regeneration and aging[END_REF]. In contrast, myofiber branching is a frequent event in B10-mdx muscle, since 100% of myofibers of EDL muscle are branched at 17 months of age [START_REF] Boldrin | Satellite cells from dystrophic muscle retain regenerative capacity[END_REF], and this may explain the small myofiber CSA that we observed at 24 months.

Muscle Stem Cells.

MuSCs have been particularly investigated in the dystrophic context. We performed Pax7 immunolabeling to count total (both quiescent and activated) MuSCs in WT and DMD mdx4Cv muscles (Figure 2A). When counting the number of MuSCs per area unit, no variation was observed from 1 to 24 months of age in both strains, although the number of MuSCs was always much higher in DMD mdx4Cv than in WT muscle (Figure 2B, Figure Suppl2A,B). When counting the number of MuSCs per myofiber, the only significant difference was observed at 24-month-old in DMD mdx4Cv muscles (-41%) (Figure 2C, Figure Suppl2A,B), when muscles exhibited the most myofibers. These results suggest that the number of MuSCs did not vary with age in TA DMD mdx4Cv muscle. Studies using the same Myf5 nlacZ lineage tracing model and similar isolated EDL single fiber technique reported opposite results with either an increase [START_REF] Boldrin | Satellite cells from dystrophic muscle retain regenerative capacity[END_REF] or a decrease of the number of satellite cells with age [START_REF] Jiang | Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy[END_REF]. Thus, the evolution of the number of MuSCs in mdx strains should be carefully monitored depending on the technique used, genetic background and sex of the animal.

Endothelial cells.

Skeletal muscle is highly vascularized. Moreover, endothelial cells exert specific effects on

MuSC differentiation and myogenesis [START_REF] Latroche | Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages[END_REF]. Immunolabeling for CD31 (PECAM1) allowed to evaluate the number of blood vessels and capillaries (Figure 3A). The number of vessels per surface unit was not significantly altered from 2 to 24 months (Figure 3B). However, when reporting the number of capillaries per myofiber, there was an increase until 6 months in myofiber capillarization in DMD mdx4Cv muscles, as compared with WT, followed by an important drop between 6 and 12 months of age in DMD mdx4Cv animals (-47%) while no variation was observed in WT muscle (Figure 3C and Figure Suppl2C,D). These results are consistent with previous studies showing a reduced number of vessels irrigating each myofiber, anatomical alterations with anastomosis, that is associated with a defect in perfusion in one-year-old DMD mdx4Cv [START_REF] Latroche | Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice[END_REF] and with a low number of capillaries per myofiber at 24 months [START_REF] Mouisel | Muscle weakness and atrophy are associated with decreased regenerative capacity and changes in mTOR signaling in skeletal muscles of venerable (18-24-month-old) dystrophic mdx mice[END_REF]. No specific explanation is available to explain the capillarization drop from 12 months.

Macrophages.

Macrophages have been shown to be present in dystrophic B10-mdx muscle from early stages (5 weeks) to advanced age (12 months) [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF][START_REF] Ikeda | Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy[END_REF]. A chronic inflammatory response signature was detected in 8-week-old B10-mdx muscle using microarrays [START_REF] Porter | A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice[END_REF]. Macrophages are important cells during muscle regeneration but were shown to play both beneficial and adverse roles in mdx muscle of various backgrounds [START_REF] Juban | AMPK Activation Regulates LTBP4-Dependent TGF-beta1 Secretion by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy[END_REF][START_REF] Mojumdar | Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2[END_REF][START_REF] Zhao | CCR2 deficiency does not provide sustained improvement of muscular dystrophy in mdx5cv mice[END_REF]. Immunolabeling for the panmacrophage marker F4/80 (Figure 3D) showed that the number of macrophages per unit area was not altered from 2 to 24 months of age in both strains (Figure 3E). However, the number of macrophages was much higher in DMD mdx4Cv muscles than in WT muscles, all life long (from 41 to 91 fold) (Figure E,F). When counting the number of macrophages per myofiber, no change was observed in WT muscle (Figure 3F). In DMD mdx4Cv muscles, an increase was observed at 6 months where macrophages were at least x1.35 fold more numerous than at any other time point (Figure 3F, Figure Suppl2E,F). Apart this time point, the number of macrophages remained constant, notably at advanced age, indicating that the number of macrophages did not increase with age.

Necrosis.

In DMD, myofibers undergo chronic cycles of damage/degeneration and regeneration. Muscle sections were labelled with anti-mouse IgGs allows to detect leaky myofibers that uptake serum proteins [START_REF] Arecco | Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation[END_REF]. It was shown that myofibers that appear necrotic in HE staining are positive for plasma protein labelling or for Evans blue dye, although some positively labelled myofibers may appear intact in HE staining [START_REF] Straub | Animal Models for Muscular Dystrophy Show Different Patterns of Sarcolemmal Disruption[END_REF][START_REF] Grounds | Biomarkers for Duchenne muscular dystrophy: myonecrosis, inflammation and oxidative stress[END_REF]. Only a few reports quantified myonecrosis in mdx muscle. While important necrosis is observed at 3 weeks, its extent is dramatically reduced few days later [START_REF] Hodgetts | Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with Etanercept in mdx mice[END_REF]. Figure 4 shows that the number of myofibers labeled with antimouse IgGs was very low, never reaching more that 0.25% of the total WT muscle area (Figure 4 A,B). In DMD mdx4Cv muscles, the number of positive myofibers was higher but still did not represent more than 1% of the area of the whole muscle section (Figure 4A,B). Similarly, Pastoret et al. previously showed that only very few degenerating myofibers are present in B10-mdx muscles from 1 to 24 months [START_REF] Pastoret | mdx mice show progressive weakness and muscle deterioration with age[END_REF].

Lipid deposition.

Lipid deposition due to adipocyte infiltration is, with fibrosis, a hallmark of muscle degeneration in DMD muscle, where the parenchyma, i.e., the myofibers, is replaced by fatty-fibrotic tissue, leading to muscle weakness. Sudan Black stains lipid deposits (Figure 4C). Overall, the extent of lipid deposition in WT and DMD mdx4Cv muscles was extremely low, accounting for no more than 0.35% of the whole muscle section area (Figure 4D). An increase of lipid deposits was observed in DMD mdx4Cv muscles at 24 months as compared with the other time points and WT (Figure 4D and Figure Suppl3A). As previously shown in the B10 background, no lipid deposition is present in the mdx muscle at any age, making an important difference with human DMD muscle [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF][START_REF] Lefaucheur | Phenotype of dystrophinopathy in old mdx mice[END_REF][START_REF] Coulton | The mdx mouse skeletal muscle myopathy: II. Contractile properties[END_REF].

Fibrosis.

Fibrosis is a major adverse process in DMD since excess of collagen deposit hampers muscle function. It was shown that at the time of diagnosis, endomysial fibrosis is a bad prognosis of motor outcome in DMD patients years later [START_REF] Desguerre | Endomysial Fibrosis in Duchenne Muscular Dystrophy: A Marker of Poor Outcome Associated With Macrophage Alternative Activation[END_REF]. However, B10-mdx hindlimb muscles present no or little fibrosis, contrary to the diaphragm [START_REF] Stedman | The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy[END_REF][START_REF] Goldspink | Age-related changes in collagen gene expression in the muscles of mdx dystrophic and normal mice[END_REF]. While some studies reported no fibrosis in hindlimb muscle until 9-11 months of age [START_REF] Cullen | Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic (mdx) mouse. Comparison with Duchenne muscular dystrophy[END_REF][START_REF] Coulton | The mdx mouse skeletal muscle myopathy: II. Contractile properties[END_REF], others mentioned an increase of fibrosis at 23 months but it was not quantified [START_REF] Lefaucheur | Phenotype of dystrophinopathy in old mdx mice[END_REF]. Evaluation of the area of collagen deposition in the TA muscle (Figure 4E and Figure Suppl3B) showed that in DMD mdx4Cv muscles, the amount of "fibrosis" accounted for about 14 to 18% of the muscle field area, and remained stable from 1 month to 24 months of age (Figure 4F), with no evidence of increased fibrosis with age in the whole muscle section (Figure Suppl3). The amount of fibrosis was higher in DMD mdx4Cv than in WT muscles at 6 and 12 months of age, but was not significantly different in 24-month-old animals, since collagen area also increased in WT muscle at that time point (+184% vs 12 months) (Figure 4F).

CONCLUSION

Altogether, these results show that the histology of the TA muscle of DMD mdx4Cv mice on C57/BL6 background showed modest variations in absolute numbers of the various muscle cell types with age. Myofiber CSA increased at 3 months to remain stable until 24 months when it was strongly reduced. None of the cell types evaluated in this study showed a variation in their density within the muscle tissue. However, analyzing their number relative to the number of myofibers indicated that the number of MuSCs decreased at 24 months, that myofiber capillarization was high until 6 months before dropping at later ages. Necrosis and lipid deposition were very rare in the DMD mdx4Cv muscle tissue, even at late time points. Finally, collagen I deposition did not increase with age. Overall, the main variations were observed at 24 months of age, when the number of myofibers was strongly increased, probably due to increased branching, inducing a mathematical decrease of all cellular parameters. These results are in accordance with the DMD mdx4Cv mice behavior that move, behave and breed as well as normal mice when maintained in normal conditions. These results also indicate that the use of the mdx model has limitations in mimicking DMD since there is no obvious worsening of the muscle histology with time. This should be taken into account depending on the type of biological investigations and the purpose of the study. Studies aiming at investigating degenerative myopathies in general may prefer other models, such as sarcoglycan deficient mice, that show a natural evolution of the pathology (49), or, for DMD pathology the more recently described DBA/2-mdx, where stronger features of degenerative myopathies are observed [START_REF] Fukada | Genetic background affects properties of satellite cells and mdx phenotypes[END_REF][START_REF] Coley | Effect of genetic background on the dystrophic phenotype in mdx mice[END_REF]. In this model, a link between TGF-b pathway, fibrotic areas and myogenesis deficit was evidenced in muscles from young adults (3) while some pathological features appear less severe in older animals [START_REF] Fukada | Genetic background affects properties of satellite cells and mdx phenotypes[END_REF][START_REF] Van Putten | Natural disease history of the D2-mdx mouse model for Duchenne muscular dystrophy[END_REF], suggesting that in the DBA/2 model also, the age of the animals should be considered depending on the purpose of the study.

Figure 1 .

 1 Figure 1. Analysis of myofibers in WT and DMD mdx4Cv muscle. TA muscle sections from 1to 24-month-old WT and DMD mdx4Cv were immunolabelled for laminin (green) and stained with Hoechst (nuclei) (A). From this immunolabeling, the number and location of nuclei within myofibers (B), the number of myofibers per mm 2 (C), mean CSA (D), and CSA distribution (E-

Figure 2 .

 2 Figure 2. Analysis of satellite cells in WT and DMD mdx4Cv muscle. TA muscle sections from 1-to 24-month-old WT and DMD mdx4Cv were immunolabelled for Pax7 (green) and for laminin (red) and stained with Hoechst (nuclei) (A). From this immunolabeling, the number of Pax7 pos cells/mm 2 (B) and the number of Pax7 pos cells/myofiber (C) were evaluated. Values are given

Figure 3 .

 3 Figure 3. Analysis of endothelial cells and macrophages in WT and DMD mdx4Cv muscle.TA muscle sections from 1 to 24-month-old WT and DMD mdx4Cv were immunolabelled for CD31

Figure Suppl1 . 25 Figure Suppl2 .

 Suppl125Suppl2 Figure Suppl1. Histology of WT and DMD mdx4Cv muscle. TA muscle sections from 1-to 24month-old WT and DMD mdx4Cv mice were stained for HE (A,C) or immunolabelled for laminin (green) (B, D). Examples of large areas of muscle sections are given for various ages. Bars =

Figure Suppl3 .

 Suppl3 Figure Suppl3. Lipid deposition and fibrosis in WT and DMD mdx4Cv muscle. (A) Example of Sudan Black staining of 24-mo old DMD mdx4Cv muscle at higher magnification showing rare black lipid droplets. Bar = 50 µm. (B) TA muscle sections from 3-to 24-month-old were

ACKNOWLEDGMENTS

This work used samples that were established for previous studies that were funded by grants from the Framework Programme FP7 Endostem (under grant agreement 241440), AFM-Telethon (grant 16029 and MyoNeurAlp Alliance), Fondation pour la Recherche Médicale (Equipe FRM DEQ20140329495).

Conflict of Interest

The authors have no conflict of interest to report. Necrosis was evaluated after staining with anti-mouse IgGs (A), as the percentage of total muscle section area (B). Lipid deposition was evaluated after staining with Sudan Black (C), as the percentage of total muscle section area (D). Fibrosis was evaluated after immunolabeling for Collagen I (E), as the percentage of total field area (F). Values are given in means ± SEM of 5 experiments (one black circle represents one mouse). P value of Anova analysis is provided on the upper left corner of the graph. Post-hoc comparisons show significant differences for * vs WT 1 mo, $ vs mdx 1 mo, £ vs mdx 24 mo, j vs WT 12 mo and § mdx vs WT at same age. Blue=Hoechst. Bars in A, C= 100 µm, in E= 50 µm.


Marielle Saclier

Sabrina Ben Larbi

Ha My Ly

Eugénie Moulin

Rémi Mounier

Bénédicte Chazaud

Gaëtan Juban
email: gaetan.juban@univ-lyon1.fr

Interplay between myofibers and pro-inflammatory macrophages controls muscle damage in mdx mice

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Adult skeletal muscle completely regenerates after an injury, thanks to the muscle stem cells (MuSCs) or satellite cells, that sustain adult myogenesis. This includes activation, proliferation, commitment into terminal myogenesis, differentiation and fusion to form new myofibers. The regeneration power of adult skeletal muscle is very high and damaged muscle fully recovers its functions within a few weeks. While MuSCs are absolutely indispensable for skeletal muscle regeneration, surrounding cells also play important roles in this process, including endothelial cells, fibro/adipogenic progenitors (FAPs) and immune cells [START_REF] Bentzinger | Cellular dynamics in the muscle satellite cell niche[END_REF].

Particularly, macrophages are crucial for efficient muscle regeneration. As in other damaged tissues, the first macrophages to invade the injured area are pro-inflammatory damage-associated macrophages that mainly arise from circulating monocytes. Inflammatory damage-associated macrophages express pro-inflammatory effectors that stimulate MuSC proliferation [START_REF] Saclier | Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration[END_REF], while limiting FAP expansion [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF][START_REF] Lemos | Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors[END_REF]. Phagocytosis of debris participates to the resolution of inflammation, which is crucial to start tissue repair and regeneration [START_REF] Arnold | Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis[END_REF]. Resolution of inflammation is characterized by a progressive switch of the inflammatory profile exhibited by macrophages, that become anti-inflammatory or recovery macrophages. This process is controlled by several molecular effectors, including the metabolic sensor AMPK [START_REF] Mounier | AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration[END_REF], the IGF1 pathway [START_REF] Tonkin | Monocyte/Macrophage-derived IGF-1 Orchestrates Murine Skeletal Muscle Regeneration and Modulates Autocrine Polarization[END_REF], the p38 regulator MKP-1 (Perdiguero et al., 2014), and the transcription factors C/EBPβ [START_REF] Ruffell | A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair[END_REF] and NFIX [START_REF] Saclier | The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration[END_REF]. These antiinflammatory macrophages exert a variety of effects on surrounding cells: they stimulate the last steps of myogenesis, including differentiation and fusion [START_REF] Saclier | Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration[END_REF], they activate FAPs to remodel the extracellular matrix (ECM) [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF][START_REF] Lemos | Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors[END_REF], and favor angiogenesis, which occurs concomitantly to myogenesis [START_REF] Latroche | Coupling between myogenesis and mngiogenesis during skeletal muscle regeneration is stimulated by restorative macrophages[END_REF].

Post-acute injury skeletal muscle regeneration is a highly regulated process, during which the various stages described above must be well coordinated in space and time. The situation is drastically different in degenerating myopathies, such as Duchenne Muscular Dystrophy (DMD), during which multiple injuries occur asynchronously throughout the muscle. Skeletal muscle in degenerative myopathies fails to regenerate because of these permanent and asynchronous cycles of degeneration-regeneration due to mutations in the dystrophinglycoprotein complex, which ensures myofiber integrity [START_REF] Lapidos | The dystrophin glycoprotein Complex: signaling strength and integrity for the sarcolemma[END_REF]. In these disorders, recurrent myofiber damage leads to chronic inflammation and the eventual establishment of fibrosis. Indeed, it was shown that asynchronous injuries in normal muscle trigger a chronic inflammatory status in the muscle associated with a fibrotic signature, reminiscent of what is observed in degenerative myopathies [START_REF] Dadgar | Asynchronous remodeling is a driver of failed regeneration in Duchenne muscular dystrophy[END_REF].

Macrophages are associated with fibrosis in mdx muscle, the mouse model for DMD [START_REF] Vidal | Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta /alternative macrophage activation pathway[END_REF]. Inhibition of the pro-inflammatory NF-κB pathway is associated with a lower macrophage number and a better phenotype of the muscle [START_REF] Acharyya | Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy[END_REF]. Similarly, preventing the entry of circulating monocytes into the muscle temporarily improves muscle histology and function [START_REF] Liang | The dual CCR2/CCR5 chemokine receptor antagonist Cenicriviroc reduces macrophage infiltration and disease severity in Duchenne muscular dystrophy (Dmdmdx-4Cv) mice[END_REF][START_REF] Mojumdar | Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2[END_REF][START_REF] Wehling | A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice[END_REF].

However this is detrimental on a longer term [START_REF] Zhao | CCR2 deficiency does not provide sustained improvement of muscular dystrophy in mdx5c mice[END_REF], showing the pivotal role of macrophages during muscle regeneration, and the necessity to target the right subset to prevent disease progression. Recently, we showed in an experimentally fibrotic mdx mouse model, that fibrosis is associated with pro-inflammatory macrophages [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF].

Functionally, these macrophages stimulate matrix production by fibroblastic cells through the abnormal secretion of latent TGFβ, due to an increased expression of the gene encoding a Latent TGF-beta binding protein (LTBP), that is involved in latent TGFβ export into the ECM.

Interestingly, pharmacological skewing of these macrophages towards an anti-inflammatory phenotype using an AMPK activator improves muscle phenotype and function [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF].

The standard treatment for DMD patients consists of Glucocorticoids (GCs), which have been shown to delay disease progression and ambulation loss [START_REF] Mcdonald | Longterm effects of glucocorticoids on function, quality of life, and survival in patients with Duchenne muscular dystrophy: a prospective cohort study[END_REF].

However, chronic daily exposure to GCs is associated with adverse effects including obesity, metabolic changes and muscle atrophy [START_REF] Schakman | Glucocorticoid-induced skeletal muscle atrophy[END_REF][START_REF] Wood | Short stature and pubertal delay in Duchenne muscular dystrophy[END_REF]. Therefore, alternative therapeutic strategies are required. Hydrogen Sulfide (H2S) is an endogenously synthetized small gaseous signaling molecule that is freely permeable to cell membrane [START_REF] Wang | Gasotransmitters: growing pains and joys[END_REF]. H2S is involved in various biological processes, including inflammation.

Alteration of H2S levels have been associated with several diseases in human or animal models, including sepsis, diabetes or rheumatoid arthritis [START_REF] Fagone | Gasotransmitters and the immune system: Mode of action and novel therapeutic targets[END_REF]. H2S has emerged as a promoter of the resolution of inflammation through the regulation of macrophage inflammatory state [START_REF] Sun | The role of hydrogen sulphide signalling in macrophage activation[END_REF][START_REF] Wallace | Hydrogen Sulfide: An Endogenous Mediator of Resolution of Inflammation and Injury[END_REF]. Interestingly, H2S-releasing molecules, like NaHS, have been shown to reduce skeletal muscle atrophy in a mouse model of diabetes [START_REF] Lu | Hydrogen sulphide ameliorating skeletal muscle atrophy in db/db mice via Muscle RING finger 1 S-sulfhydration[END_REF] and to decrease muscle fibrosis after contusion-induced injury [START_REF] Zhao | Hydrogen sulfide alleviates skeletal muscle fibrosis via attenuating inflammation and oxidative stress[END_REF]. Moreover, H2S-releasing molecules have shown promising results in cardiovascular disorders and arthritis with reduced adverse effects [START_REF] Wallace | A proof-of-concept, Phase 2 clinical trial of the gastrointestinal safety of a hydrogen sulfide-releasing anti-inflammatory drug[END_REF][START_REF] Wallace | Hydrogen Sulfide-Releasing Therapeutics: Translation to the Clinic[END_REF].

In the present study, we investigated the interaction between myofibers and macrophages in the context of degenerative myopathies. As the macrophage phenotype is modulated by their close environment, we tested the hypothesis that dystrophic muscle fibers may themselves alter macrophage status, using ex vivo coculture experiments. Then, we analyzed the possibility to modulate the macrophage phenotype in vivo using NaHS, an H2S donor, to ameliorate dystrophic muscle phenotype. Finally, we identified the metabolic sensor AMPK as an essential NaHS mediator in macrophages.

Results

DMD-derived myofibers trigger pro-inflammatory profile of macrophages

The role of myofibers on macrophage polarization was investigated by coculturing wild-type bone marrow derived macrophages (BMDMs) with single fibers isolated from wild-type and dystrophic muscle (Fig. 1A). Wild-type myofibers increased the expression of 2 out of 4 proinflammatory markers analyzed by resting macrophages (Fig. 1B-C, +23% for TNFα and +37% for COX2 as compared with control). However, in the presence of mdx myofibers, the number of BMDMs expressing pro-inflammatory markers was strongly increased: it increased the expression of 3 out of 4 pro-inflammatory markers: TNFα, CCR2 and COX2 (Fig. 1B-C, 45, 30 and 58% increase as compared with control, respectively). Importantly, this was not associated with a difference in mortality between wild-type and mdx myofibers after 3 days of culture (Fig. S1). No modification of the expression of the anti-inflammatory markers CD301 and ARG1 by BMDMs was observed when cocultured with wild-type or mdx myofibers (Fig. 1B-C). These results show that mdx myofibers skew macrophages towards a pro-inflammatory phenotype.

Thus, not only the cycles of injury/regeneration, but also the dystrophic myofibers themselves sustain the inflammation in mdx muscles, highlighting the relevance of modulating inflammation as a therapeutic strategy.

NaHS treatment reduces pro-inflammatory macrophages in dystrophic muscle

In an attempt to modulate the macrophage inflammatory status, we treated 3-month-old mdx mice with NaHS, an H2S donor. Because of the short half-life of H2S in vivo [START_REF] Calvert | Novel insights into hydrogen sulfide-mediated cytoprotection[END_REF], NaHS was delivered intraperitoneally daily for 3 weeks (Fig. 2A). NaHS treatment induced an important decrease in the number of macrophages per myofiber in the Tibialis Anterior (TA) muscle (Fig. 2B, -42%) and in the diaphragm (Fig. 2C, -56%). More precisely, in TA from NaHS treated mice, a strong decrease in the number of macrophages expressing proinflammatory markers was observed (Fig. 2D-E, -35 and -31% for TNFα and iNOS, respectively) together with a decrease of the fibrosis-associated ARG1 marker (Fig. 2D-E, -35%), and an increase of the anti-inflammatory marker CD206 (Fig. 2E,+61%). The expression of CCR2 and CD301 was not affected (Fig. S2). Similarly, macrophages expressing proinflammatory markers were reduced in the diaphragm (Fig. 2F, -23 and -31% for TNFα and iNOS, respectively), although the expression of ARG1 and CD206 was not affected in this muscle (Fig. 2F). These results show that NaHS treatment specifically reduces the number of pro-inflammatory macrophages in mdx mice muscles.

Pharmacological dampening of macrophage pro-inflammatory phenotype by NaHS reduces myofiber branching in mdx mice

To study the impact of pro-inflammatory macrophage reduction in dystrophic muscles, several myogenic parameters were analyzed after NaHS treatment. Immunostainings performed on TA transversal sections from treated mdx muscles did not show a modification of the number of satellite cells per fiber as determined by Pax7 immunostaining (Fig. S3A), or of the proportion of newly formed myofibers that expressed eMHC+ (Fig. S3B). However, NaHS treatment led to a decreased number of nuclei per myofiber in the TA (Fig. 3A-B and Fig. S3C, -20 to -33% depending on the section level), as well as in the diaphragm (Fig. 3C, -32%), suggesting a reduction in cell fusion. Importantly, this was confirmed by longitudinal analyses on mono-myofibers isolated from TA that showed a decrease in the number of nuclei/µm along the length of myofibers from NaHS-treated animals (Fig. 3D-E, -8%). Muscle regeneration after an injury is associated with myofiber branching [START_REF] Pichavant | Incidence and severity of myofiber branching with regeneration and aging[END_REF], which is characterized by myofibers containing two or more cytoplasmically continuous strands. Mdx mice harbor an increased proportion of branched myofibers as compared with wild-type [START_REF] Bockhold | Aging normal and dystrophic mouse muscle: analysis of myogenicity in cultures of living single fibers[END_REF], likely due to the permanent cycles of injury/regeneration, which is correlated with a decrease in muscle force production [START_REF] Chan | Branched fibers in dystrophic mdx muscle are associated with a loss of force following lengthening contractions[END_REF]. Interestingly, isolated myofibers from NaHS-treated mdx mice were characterized by a decrease in myofiber branching (Fig. 3F-G, 16% decrease). Altogether, these results show that dampening the proinflammatory status of macrophages is associated with a reduction of myofiber branching and of the number of myonuclei per myofiber, indicative of a reduction of muscle damage.

NaHS treatment improves dystrophic muscle

Next, we evaluated the consequence of dampening the macrophage pro-inflammatory status by NaHS treatment on dystrophic muscle features. Hematoxylin-eosin (HE) staining of muscle transversal sections showed a strong decrease in the damaged areas in NaHS-treated mice in the TA (Fig. 4A-B and Fig. S4A, -50 to -55% depending on the section level) and the diaphragm (Fig. 4C, -58%). This was associated with a decrease in muscle fibrosis, as shown by the reduction in the areas expressing Collagen1 (Coll1) in the TA (Fig. 4D-E and Fig. S4B, -9 to -10% depending on the section level), but not in the diaphragm (Fig. 4F). Finally, the mean cross-sectional area (CSA) of myofibers, which is correlated with muscle force production, was increased in the TA (Fig. 4G-H Taken together, these results show that the reduction of pro-inflammatory macrophages by NaHS treatment is associated with an amelioration of the dystrophic muscle phenotype.

AMPKa1 is required for NaHS-induced dampening of pro-inflammatory macrophages

The AMPK metabolic sensor plays a critical role in macrophage phenotypic shift during skeletal muscle regeneration [START_REF] Mounier | AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration[END_REF]. As AMPK was previously shown to be activated by NaHS in a microglia cell line [START_REF] Zhou | CaMKKβ-dependent activation of AMP-activated protein kinase is critical to suppressive effects of hydrogen sulfide on neuroinflammation[END_REF], we asked whether NaHSmediated pro-inflammatory macrophage dampening was mediated through AMPK activation.

Treatment of wild-type BMDMs by NaHS increased the phosphorylation at Thr172 of the AMPK catalytic a subunit after 1 and 2 h (Fig. 5A-B, +61 and +89% as compared with untreated control, respectively), as did the AMPK allosteric activator 991 (Fig. 5A-B, +80 and +110% as compared with untreated control, respectively), indicating AMPK activation. Next, we investigated the functional role of AMPK activation in NaHS-mediated macrophage skewing towards an anti-inflammatory phenotype. To this aim, we used AMPKa1 -/-BMDMs, which harbor a knockout in the gene coding for the AMPK a1 subunit, the only catalytic subunit expressed in macrophages (Sag et al., 2008) (Fig. 5C). As expected, NaHS-induced AMPK activation was associated with a decreased expression of the pro-inflammatory markers TNFa and CCL3 (Fig. 5D, -14 and -11%, respectively), and an increased expression of the antiinflammatory markers CD206 and CD163 (Fig. 5E, +19 and +11%, respectively). These NaHS-mediated effects on macrophage inflammatory status were completely abrogated in AMPKa1 - /-BMDMs (Fig. 5D). Finally, we used an in vitro model of adult myogenesis where conditioned medium generated from BMDMs was transferred onto MuSCs to stimulate their fusion into multinucleated myotubes (Fig. 5F). Conditioned medium from NaHS-treated wild-type BMDMs increased MuSC fusion into myotubes (Fig. 5G-H, +22%), whereas this was not observed with conditioned medium derived from NaHS-treated AMPKa1 -/-BMDMs (Fig. 5G-H).

These results identify AMPK activation through the phosphorylation of its a1 catalytic subunit as a critical downstream target of NaHS in macrophages.

Discussion

In the present study, we analyzed the interaction between myofibers and macrophages in the context of DMD. We showed that the dystrophic myofibers themselves skew resting macrophages towards a pro-inflammatory phenotype. As alterations in the secretome of myotubes generated by myoblasts isolated from mdx mice [START_REF] Duguez | Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion[END_REF] or DMD patients [START_REF] Lecompte | Skeletal muscle secretome in Duchenne muscular dystrophy: a pivotal anti-inflammatory role of adiponectin[END_REF] have already been described, this suggests that in the context of degenerative myopathies, not only the degenerating/regenerating events lead to chronic inflammation, but the myofibers themselves contribute to the maintenance of the proinflammatory environment that alters macrophage properties and favors the generation of a pro-fibrotic population. Although the molecular effectors secreted by mdx myofibers contributing to the sustained inflammation remain to be precisely characterized, Duguez et al. showed an increased secretion of various proteins, including secreted protein acidic and rich in cysteine (SPARC), by myotubes derived from mdx-isolated myoblasts [START_REF] Duguez | Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion[END_REF].

As in vitro treatment of peritoneal macrophages with recombinant SPARC was shown to stimulate the expression of pro-inflammatory genes such as Tnfa (Toba et al., 2015), its increased secretion by mdx myofibers could contribute to the skewing of resting macrophages towards a pro-inflammatory phenotype and, therefore, to fibrosis establishment in DMD muscle. Indeed, we previously showed that pro-inflammatory macrophages promote fibrosis in DMD muscle [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF]. Moreover, modulating the inflammatory status of this population towards an anti-inflammatory phenotype improves DMD muscle function [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF]. Therefore, identification of specific anti-inflammatory strategies appears as a relevant therapeutic approach to alleviate muscle damage.

H2S is a gasotransmitter that exhibit anti-inflammatory properties, in part through the regulation of macrophage inflammatory status [START_REF] Sun | The role of hydrogen sulphide signalling in macrophage activation[END_REF][START_REF] Wallace | Hydrogen Sulfide: An Endogenous Mediator of Resolution of Inflammation and Injury[END_REF]. H2S donors have been widely used in vivo using animal models to treat various pathological conditions [START_REF] Fagone | Gasotransmitters and the immune system: Mode of action and novel therapeutic targets[END_REF]. This includes reduction by the H2S donor NaHS of skeletal muscle atrophy in a mouse model of diabetes [START_REF] Lu | Hydrogen sulphide ameliorating skeletal muscle atrophy in db/db mice via Muscle RING finger 1 S-sulfhydration[END_REF], or the decrease of muscle fibrosis after contusion-induced injury [START_REF] Zhao | Hydrogen sulfide alleviates skeletal muscle fibrosis via attenuating inflammation and oxidative stress[END_REF]. Interestingly, various therapeutics like Zofenopril have been shown to exert their effect through the indirect release of H2S [START_REF] Fagone | Gasotransmitters and the immune system: Mode of action and novel therapeutic targets[END_REF][START_REF] Wallace | Hydrogen Sulfide-Releasing Therapeutics: Translation to the Clinic[END_REF]. Recently, clinical studies have shown the safety and efficiency of H2S-releasing molecules in human. Patients with congestive heart failure exhibit a deficit of H2S in the blood. Treatment with the H2S donor SG1002 attenuated the level of brain natriuretic peptide, a stress marker of cardiac function, thus reducing the severity of and potentially preventing heart failure [START_REF] Polhemus | A Novel Hydrogen Sulfide Prodrug, SG1002, Promotes Hydrogen Sulfide and Nitric Oxide Bioavailability in Heart Failure Patients[END_REF]. Similarly, ATB-346, an H2Sreleasing derivative of the nonsteroidal anti-inflammatory drug Naproxen, reduced the symptoms severity in osteoarthritis patients with reduced adverse effects as compared to Naproxen alone [START_REF] Wallace | Hydrogen Sulfide-Releasing Therapeutics: Translation to the Clinic[END_REF][START_REF] Wallace | A proof-of-concept, Phase 2 clinical trial of the gastrointestinal safety of a hydrogen sulfide-releasing anti-inflammatory drug[END_REF].

At the molecular level, the H2S donor NaHS was shown to favor microglia inflammatory shift towards an anti-inflammatory phenotype [START_REF] Du | Downregulation of cystathionine β-synthase/hydrogen sulfide contributes to rotenone-induced microglia polarization toward M1 type[END_REF][START_REF] Zhou | CaMKKβ-dependent activation of AMP-activated protein kinase is critical to suppressive effects of hydrogen sulfide on neuroinflammation[END_REF] through the activation of AMPK [START_REF] Zhou | CaMKKβ-dependent activation of AMP-activated protein kinase is critical to suppressive effects of hydrogen sulfide on neuroinflammation[END_REF]. Interestingly, our previous work identified AMPK as a crucial activator of macrophage inflammatory shift during normal skeletal muscle regeneration [START_REF] Mounier | AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration[END_REF]. In the present study, we identified AMPK as a critical mediator of NaHSinduced macrophage skewing that allow the acquisition of a phenotype associated with muscle repair. Although the molecular cascade leading to AMPK activation by NaHS in macrophages remains to be established, the resolvin Annexin A1 (ANXA1) may be involved in this process. Indeed, NaHS-induced down-regulation of pro-inflammatory markers expression in BMDMs following LPS stimulation requires the resolvin Annexin A1 [START_REF] Brancaleone | Annexin A1 mediates hydrogen sulfide properties in the control of inflammation[END_REF], and we recently showed ANXA1 to trigger AMPK activation in macrophages through the binding of its G protein-coupled formyl peptide receptor (FPR) 2 [START_REF] Mcarthur | Annexin A1 drives macrophage skewing to accelerate muscle regeneration through AMPK activation[END_REF].

Thus, in an attempt to counteract the exacerbated macrophage pro-inflammatory phenotype induced by dystrophic myofibers, we treated mdx mice with NaHS. We showed that NaHStreated mdx mice exhibited a decrease in the total number of macrophages in the TA and the diaphragm. More specifically, in the TA, we observed a reduction in the number of macrophages expressing pro-inflammatory markers as well as the ARG1 fibrotic marker, together with an increase in the anti-inflammatory CD206 marker, suggesting a skewing towards an anti-inflammatory phenotype and a reduction of the pro-fibrotic macrophage population [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF]. This was associated with a global muscle phenotype amelioration characterized by a reduced myofiber necrosis and reduced fibrosis, together with an increased myofiber size. In the diaphragm, NaHS treatment also induced a reduction of macrophages expressing pro-inflammatory markers but no effect was observed on ARG1 and CD206 markers. This was associated with a decreased myofiber necrosis and an increased myofiber size, but no fibrosis reduction. These discrepancies observed between the TA and the diaphragm may be explained by the more severe phenotype exhibited by the diaphragm, notably the higher level of fibrosis that may require extended treatment duration to be reduced.

Globally, these results are in accordance with the beneficial effect observed on dystrophic muscle function by preventing the entry of circulating monocytes at early time points [START_REF] Liang | The dual CCR2/CCR5 chemokine receptor antagonist Cenicriviroc reduces macrophage infiltration and disease severity in Duchenne muscular dystrophy (Dmdmdx-4Cv) mice[END_REF][START_REF] Mojumdar | Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2[END_REF][START_REF] Wehling | A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice[END_REF], or by skewing macrophage towards an anti-inflammatory phenotype [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF]. These results confirm that the modulation of macrophage inflammatory status is a relevant therapeutic approach to relieve inflammation and muscle damage in degenerative myopathies.

The amelioration of the muscle phenotype by NaHS treatment was characterized by a decrease in the number of nuclei per myofiber together with a reduction of myofiber branching.

Branched myofibers are barely observed in normal skeletal muscle but they appear after regeneration following acute injury [START_REF] Pichavant | Incidence and severity of myofiber branching with regeneration and aging[END_REF]. They are observed in DMD patients [START_REF] Bell | Histopathological changes in Duchenne muscular dystrophy[END_REF]) and in DMD fibers generated in vitro [START_REF] Tanoury | Prednisolone rescues Duchenne Muscular Dystrophy phenotypes in human pluripotent stem cells-derived skeletal muscle in vitro[END_REF], as well as in the mdx mouse model [START_REF] Head | Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice[END_REF][START_REF] Lefaucheur | Phenotype of dystrophinopathy in old mdx mice[END_REF]. Branched myofibers are characterized by an impairment of calcium signaling and Excitation-Contraction coupling, causing myofiber function defects and higher fragility [START_REF] Head | Branched fibres in old dystrophic mdxmuscle are associated with mechanical weakening of the sarcolemma, abnormal Ca2+ transients and a breakdown of Ca2+ homeostasis during fatigue[END_REF][START_REF] Lovering | Malformed mdx myofibers have normal cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+ signaling[END_REF]. While the mechanisms resulting in myofiber branching are still unclear (defect in final fusion between forming myofibers, or normal process that is exacerbated in dystrophic muscle due to continuous regeneration processes), a recent study showed in the mdx EDL muscle that the increased number of myonuclei per myofiber is concomittant to myofiber branching [START_REF] Massopust | Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss[END_REF]. Accordingly, the concomitant reduction of the number of nuclei per myofiber and of branching observed in NaSH-treated mice suggests an improvement in muscle homeostasis.

To conclude, the present study identified a novel property of dystrophic myofibers in the DMD mouse model mdx, associated with a regulatory loop between myofibers and macrophages. Dystrophic myofibers skew macrophages towards a pro-inflammatory phenotype that in turn contributes to myofiber damage, consequently increasing myofiber branching and fragility, thus favoring muscle damage and fibrosis. Finally, this work identified H2S-releasing molecules as potential therapeutic strategies to dampen inflammation in DMD and improve dystrophic muscle phenotype through AMPK activation.

Materials and methods

Mice experiments. DMD mdx4Cv [START_REF] Chapman | Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice[END_REF] mice on C57BL/6J background and 129;B6-Prkaa1 tm (AMPKa1 -/-) were bred and used according to French legislations. The protocols were approved by the Ethical committee from Université Claude Bernard Lyon 1 (CEEA55). Experiments were conducted on males at 10-12 weeks of age. NaHS (1 mg/kg, Sigma-Aldrich) or PBS was injected daily intraperitoneally in mdx mice for 3 weeks and muscles were harvested the day after the last injection.

Histology and immunofluorescence analyses in mouse. Fascia of TA muscles was removed, then TA and diaphragm were frozen in nitrogen-chilled isopentane and kept at -80°C until use. 8 µm-thick cryosections were prepared for HE staining and immunolabelings. For immunolabelings, cryosections were permeabilized 10 min in 0.5 % Triton X-100 and saturated in 2% bovine serum albumin (BSA) for 1 h at room temperature. For identification of regenerating myofibers, saturated cryosections were co-labeled with primary antibodies directed against embryonic myosin (eMHC, sc-53091, Santa Cruz, 1/100) and Laminin (L9393, Sigma Aldrich, 1/200). For macrophage double immunolabelings, cryosections were labeled with antibodies (from Abcam unless indicated) against F4/80 (ab6640 or ab74383, 1/400) overnight at 4°C and labeling using the second antibody was performed for 2 h at 37°C. Antibodies were directed against Arginase 1 (sc-18355, Santa Cruz, 1/100), CCR2 (ab32144, 1/200), CD206 (sc-58987, Santa Cruz, 1/100), CD301 (ab59167, 1/200), Collagen I (ab292, ab34710 or Biotech 131001, 1/200), Cox2 (ab2372, 1/50), iNOS (ab15323, 1/25), Laminin (L9393, Sigma Aldrich, or ab78287, 1/200) and TNFα (ab34839, 1/50). Secondary antibodies were coupled to FITC, Cy3 or Cy5 (Jackson Immunoresearch Inc, 1/200). Muscle stem cells were labeled as previously described [START_REF] Théret | AMPKα1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis[END_REF] using an antibody directed against Pax7 (AB_528428, DSHB, 1/10). HE-stained muscle sections were recorded with a Nikon E800 microscope at 20X magnification connected to a QIMAGING camera. Damaged areas were measured as previously described [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF]. Fluorescent immunolabelings were recorded with a DMI 6000 Leica microscope connected to a Coolsnap camera at 20X magnification. For each condition of each experiment, at least 8-10 fields chosen randomly were counted. The number of labeled macrophages was calculated using the Image J software and was expressed as a percentage of total macrophages. Areas of Collagen1 were calculated with ImageJ software as previously described [START_REF] Juban | AMPK activation regulates LTBP4-dependent TGF-beta;1 secretion by pro-inflammatory macrophages and controls fibrosis in duchenne muscular dystrophy[END_REF]. Cross Section Area (CSA) was determined on whole muscle sections labeled by laminin antibody using Open-CSAM program as previously described [START_REF] Desgeorges | Open-CSAM, a new tool for semiautomated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle[END_REF]. For quantification in the TA of the number of nuclei per myofiber, damaged areas, fibrotic areas and CSA, 2 different cross sections were analyzed in TA muscles. The first and second cross sections were harvested 5 and 10 mm from the tendon base, respectively.

Murine BMDM isolation and culture. Macrophages were derived from murine bone marrow precursors as previously described [START_REF] Mounier | AMPKα1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration[END_REF]. Briefly, total bone marrow was obtained by flushing femur and tibiae from wild-type C57BL/6J mice with Dubecco's Modified Eagle's Medium (DMEM). Cells were cultured in DMEM containing 20% heat inactivated Fetal Bovine Serum (FBS), 30% of L929 cell line-derived conditioned medium (enriched in CSF-1), 2.5 μg/mL of fungizone and 100 U/mL of penicillin/streptomycin for 6-7 days. To generate conditioned medium, 110,000 cells/cm 2 were treated with 500 µM NaHS for 2 days. Cells were then washed twice with PBS and incubated for 24 h in DMEM F-12 medium (Gibco) without serum. Medium was collected, centrifuged for 10 min at 500 g and supplemented with 2% Horse serum (HS, Gibco) before transfer onto MuSC culture (see below).

Murine BMDM immunophenotypic characterization. 54,000 cells/cm 2 were seeded on glass coverslips ant treated by 500 µM of NaHS for 2 days. Cells were fixed for 10 min in 4% formaldehyde, permeabilized 10 min in 0.5 % Triton X-100 and saturated in 2% BSA for 1 h at room temperature. They were then labeled as described above with anti-TNFα (ab34839, 1/50), anti-CCL3 (sc-1383, Santa Cruz, 1/50), anti-CD206 (ab64693, 1/50) or anti-CD163 (bs-2527R, Bioss, 1/100) primary antibodies revealed by secondary antibodies coupled to FITC or Cy3 (Jackson Immunoresearch Inc, 1/200). After nuclei staining with Hoechst, cells were mounted in Fluoromount (Interchim) and 10 to 12 random fields were imaged on a Nikon E800 microscope at 10X magnification connected to a QIMAGING camera.

Cocultures of macrophages with isolated single myofibers. Single myofibers of extensor digitorum longus (EDL) muscle were isolated from wild-type and mdx mice as previously described (Le [START_REF] Grand | Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration[END_REF] and were cocultured as 15 myofibers/well in 96 wells containing 14000 cells/cm 2 of wild-type bone-marrow derived macrophages (prepared as described above) for 3 days. Macrophages were immunolabelled as described above for the immunofluorescence on muscle sections. For each condition of each experiment, about 10-12 fields chosen randomly were recorded with a DMI 6000 Leica microscope connected to a Coolsnap camera (Photometrics) at 20X magnification.

Isolation of single myofibers from TA muscle. Fascia of TA muscles was removed and muscles were fixed for 45 min in 4% formaldehyde. After 2 quick washes and a 30 min wash in PBS, muscles were transferred into PBS containing 0.5% Triton X-100. 30 to 40 single myofibers were gently isolated by manual dissociation using tweezers, incubated with 1µg/mL of Hoechst to label their nuclei, and mounted on slides. Slides were observed on an Axio Observer.Z1 (Zeiss) connected to a CoolSNAP HQ2 CCD Camera (Photometrics) and the branched myofibers were numerated. For the quantification of the number of nuclei, between 30 and 40 images corresponding to 7,000-9,000 nuclei from 6 to 10 randomly chosen single myofibers were acquired and the number of nuclei was normalized by the myofiber length.

Isolation and culture of MuSCs. Muscle hindlimbs from adult males were dissociated and digested first for 45 min at 37°C under agitation in Ham's F-10 medium (Gibco) containing 10% HS (Gibco) and 300 U/mL collagenase II (Gibco), followed by a second digestion for 30 min at 37°C under agitation in Ham's F-10 medium (Gibco) containing 10% HS (Gibco), 150 U/mL collagenase II (Gibco) and 1,7 U/mL dispase (Gibco). MuSCs were isolated with Satellite Cell Isolation Kit (Miltenyi Biotec) using LS Columns (Miltenyi Biotec) on a MidiMACS separator (Miltenyi Biotec), and seeded on gelatin-coated flasks at 2,000 cells/cm 2 in DMEM F-12 (Gibco) supplemented with 20% FBS, 2% Ultroser G (Pall Gelman Sciences) and 100 U/mL of penicillin/streptomycin.

MuSC fusion assay.

MuSCs were seeded on Growth Factor Reduced Matrigel (Corning) at 10,000 cells/cm 2 in DMEM F-12 (Gibco) supplemented with 20% FBS, 2% Ultroser G (Pall Gelman Sciences) and 100 U/mL of penicillin/streptomycin overnight and culture medium was replaced by macrophage-derived conditioned medium (see above). After 48 h, cells were fixed for 10 min in 4% formaldehyde, permeabilized 10 min in 0.5 % Triton X-100 and saturated in 2% BSA for 1 h at room temperature. They were then labeled and mounted as described above with an anti-desmin antibody (ab32362, 1/200). Ten to 12 random fields were imaged on a Nikon E800 microscope at 10X magnification connected to a QIMAGING camera.

Western blot. Two million macrophages were seeded in 6-well plates. One h before treatment, culture medium was replaced by DMEM without red phenol (Gibco) supplemented with 10% charcoal stripped FBS (Gibco) and cells were treated with 10 µM 991 (SpiroChem) or 500 µM NaHS (Sigma Aldrich) for 1 or 2 h. Proteins were isolated in lysis buffer containing 50 mM Tris-HCL (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose, 1% triton, 20 mM glycerol-2-phosphate disodium, 50 mM NaF, 0.5 mM PMSF, 1 mM benzamidine, 1 mM Na3VO4 and 1% cocktail phosphatase inhibitor 3 (Sigma-Aldrich, P0044) for 30 min on ice and centrifuged for 10 min at 16,100 g to remove debris. Twenty five µg of proteins were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane which was probed with antibodies against p-AMPKa (#2531, Cell Signaling, 1/1000), AMPKa (#2532, Cell Signaling, 1/1000), or b-actin (A5316, Sigma-Aldrich, 1/2000). Blots were revealed on a Chemidoc imager (Bio-Rad) using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and signal intensity was quantified using Image Lab Software (Bio-Rad).

Statistical Analyses.

All experiments were performed using at least three different cultures or animals in independent experiments. Statistical tests were performed using GraphPad Prism. Depending on the experimental setup, Student's t test, one-way or two-way ANOVA with multiple comparisons were used for statistical analyses. C

  and Fig.S4C, +18 to +27% depending on the section level) and the diaphragm (Fig.4I, +25%). Precisely, a decreased proportion of the smallest fibers (Fig.4G-H and Fig.S4C, -28% of fibers with CSA <500 μm 2 on both TA section levels; Fig.4I, -23% of fibers with CSA <250 μm 2 in the diaphragm) and a concomitant increased proportion of the bigger fibers (Fig.4G-H and Fig.S4C, +40 to +47% of fibers with CSA >5000 μm 2 depending on the TA section level; Fig.4I, +155% of fibers with CSA between 2000 and 2250 μm 2 in the diaphragm) were observed in NaHS-treated mdx mice.

Figure 1 .

 1 Figure 1. Dystrophic myofibers trigger pro-inflammatory macrophages. (A) Diagram depicting the cocultures of BMDMs with isolated single myofibers. (B-C) BMDMs that were

Figure 2 .

 2 Figure 2. NaHS treatment reduces pro-inflammatory macrophages in mdx muscle. (A)

Figure 3 .

 3 Figure 3. NaHS treatment reduces muscle damage in mdx mice. Mdx mice were treated daily with PBS or NaHS for 3 weeks (as described in Fig.2A), TA and diaphragm muscles were

Figure 4 .

 4 Figure 4. NaHS treatment ameliorates dystrophic muscle phenotype. Mdx mice were

Figure 5 .

 5 Figure 5. AMPKa1 mediates NaHS-induced acquisition of the macrophage anti-

  Figure S1

Acknowledgements

This work was funded by the Framework Programme FP7 Endostem (under grant agreement 241440), Association Française contre les myopathies (MyoNeurAlp Alliance) and Fondation pour la Recherche Médicale (Equipe FRM DEQ20140329495).

Conflict of interest

The authors declare that they have no conflict of interest

Author contribution

Conceptualization: GJ, BC, RM; Methodology: MS, SBL, GJ, HML, EM, BC; Analysis: SBL, GJ, MS, BC; Writing original draft: GJ, MS; Writing review & editing: GJ, MS, BC, RM; Supervision: GJ, BC; Funding Acquisition: BC, GJ.

Supplementary figure legends

Figure S1. Viability of isolated myofibers in culture. BMDMs were cultured with wild-type (WT) or mdx myofibers for 3 days (as described in Fig. 1A) and live myofibers (i.e., myofibers that were not hypercontracted) were numerated. are means ± sem of 5 cultures.

Statistical analysis was performed by Student's t test.


Bruno Giraudeau

Corinne Alberti

Is the randomized trial design adapted to population health intervention research?

Randomized trials are frequently used in clinical research and considered the gold standard, but they are less common in population health intervention research (PHIR). We discuss issues that are sometimes shared and sometimes distinct between PHIR and clinical research, notably the randomization unit, design, standardization of the intervention, outcome(s) and ethical issues. In the end, both PHIR and clinical research share the common aim of assessing interventions, and randomized trials should be more widely used in PHIR, provided that how they are planned and conducted is adapted to the PHIR context.

Population health interventions are defined as "policies, programs and resource distribution approaches that impact a number of people by changing the underlying conditions of risk and reducing health inequities" (1). Evaluating such interventions is one of the aims of population health intervention research (PHIR) whose final goal is, as stated by the Canadian Institutes of Health Research, "to produce knowledge about policy and program interventions that operate within or outside of the health sector and have the potential to impact health at the population level" [START_REF] Hawe | What is population health intervention research? Can J Public Health[END_REF]. In the field of clinical research, clinical interventions are often assessed with randomized trials, which are considered the gold standard because they are well suited to limit bias [START_REF] Grimes | An overview of clinical research: the lay of the land[END_REF]. How a randomized clinical trial should be planned, conducted and analyzed is well known, as are the associated legal and ethical issues. Obviously, these general and theoretical well-known rules must be adapted for each specific randomized trial: conducting a trial is indeed far from being as simple as is described in books, and it requires flexibility.

In PHIR, most interventions are complex: they correspond to a series of interrelated events occurring within a broader system that they are in constant interaction with [START_REF] Hawe | Complex interventions: how 'out of control' can a randomised controlled trial be?[END_REF]. A recent literature review illustrated that randomized trials can be used to assess these interventions, although several other approaches are also common [START_REF] Minary | Which design to evaluate complex interventions? Toward a methodological framework through a systematic review[END_REF]. However, conducting a randomized trial in the population health field requires adapting the clinical randomized trial rules to a non-clinical context as follows. First, in most clinical trials, the unit of randomization is the patient whereas individual randomization is rarely well-adapted to PHIR.

Because interventions of interest are generally delivered at a group level as opposed to an individual level, randomizing groups such as schools, health centers, geographical areas, etc. is mandatory. Doing so avoids group contamination, which would occur due to interactions between members of a common cluster being allocated to different groups. The trial then becomes a cluster randomized trial. Second, most randomized trials are planned as parallel trials, but other designs may be of interest in PHIR, notably the stepped-wedge cluster randomized trial [START_REF] Hemming | The stepped wedge cluster randomised trial: rationale, design, analysis, and reporting[END_REF]. In a stepped-wedge cluster design, clusters are randomly allocated to different sequences, each sequence defining the timing of cluster switch from the control to the experimental condition. Thus, all clusters have the opportunity to receive the intervention at some point, which may help reluctant stakeholders participate and may help the logistics of the study because the intervention's roll-out is staggered [START_REF] Hemming | Reflection on modern methods: when is a stepped-wedge cluster randomized trial a good study design choice?[END_REF]. Third, in clinical trials, standardizing the intervention is generally the rule (besides, we may have weight-or bodysurface-area-adapted drug doses), but tailoring the intervention to individual participant needs or the local context is common in complex interventions [START_REF] Hawe | Complex interventions: how 'out of control' can a randomised controlled trial be?[END_REF]. As an example, rather than using a common information kit, information may be provided differently among centres. This refers to the concept of "standardization by function" as compared with "standardization by form" (4). The core components of the interventions nevertheless need to be well specified and clearly described using for instance the TIDieR checklist [START_REF] Hoffmann | Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide[END_REF]. They need to be common to all clusters and participants allocated to the intervention group; otherwise, it would hamper the interpretation of the trial results. A consequence of such a tailoring is that in PHIR, beyond assessing the intervention effect in terms of a priori-specified outcomes, process evaluations are welcome to better understand the results obtained and to link implementation variations to effect variations [START_REF] Craig | Developing and evaluating complex interventions: the new Medical Research Council guidance[END_REF]. They focus on how the intervention has been implemented to better understand the involved mechanisms (as a pathophysiology study would do in a clinical context) and thus provide information valuable to scale-up the intervention [START_REF] Moore | Process evaluation of complex interventions: Medical Research Council guidance[END_REF].

Fourth, in clinical research, it is widely advised to have a unique a priori-specified primary outcome, although some exceptions are accepted (11). Doing so allows for specifying sample size on the basis of a quantitative hypothesis and offers the opportunity for a clear-cut conclusion based on the sole primary outcome result. In PHIR, such an approach is probably reductive. Because interventions are complex and stakeholders numerous, intervention effectiveness may be appraised differently depending on the stakeholder's goal. Results may also be obtained from both quantitative and qualitative analyses (notably regarding its interaction with the context) for a complete overview of the intervention effect. Obviously, strict rules generally applied in clinical research need to be used, and some guidance on this topic would be welcome. Fifth, while individual randomization ensures group comparability things are more complex with cluster randomization. The number of clusters is often small which favours chance imbalance. Otherwise, in most cluster trials, participants are recruited after cluster randomization without any blinding, due to the real nature of the interventions.

This favours group imbalance and bias [START_REF] Giraudeau | Preventing bias in cluster randomised trials[END_REF]. Statistical analysis then requires some form adjustment and cluster randomized trials are often analysed more as an observational study than as a randomized trial. Sixth, any study involving human beings must be conducted in accordance with international ethical principles and local regulatory guidelines. A lot has been done on this topic since World War II, and the very aim is to ensure participants' protection and also protect their autonomy, which is achieved by informed consent. However, existing regulatory guidelines do not apply well to PHIR, which raise "distinct ethical challenges relative to clinical medical interventions, primarily because of their focus on prevention (rather than treatment) and their intended impact on the health of a population as a whole (rather than the individual)" [START_REF] Buchanan | Public Health Interventions: Ethical Implications[END_REF]. Guidelines have been published on the specific case of the cluster randomized trial, notably defining who is a study participant and under which condition a waiver of individual consent may be accepted [START_REF] Weijer | The Ottawa Statement on the Ethical Design and Conduct of Cluster Randomized Trials[END_REF]. However, they have been developed through the prism of the methodological feature of cluster randomization, which surely does not cover the whole specificities of PHIR. More work is undoubtedly needed, as is an accustoming of ethics committees to PHIR, which differs from clinical research. Finally, although admittedly, randomized trials provide the highest level of evidence in clinical research, it is generally agreed that this is not the only way to assess an intervention's impact.

The parachute trial is a tremendous parody that illustrates the limitations of randomized trials [START_REF] Yeh | Parachute use to prevent death and major trauma when jumping from aircraft: randomized controlled trial[END_REF]. Impossibility to randomize is not a prerogative of PHIR: it also exists in clinical research. In 1965, Hill listed 9 criteria allowing to conclude causation rather than association in an observational rather than randomized context [START_REF] Hill | The environment and disease: association or causation?[END_REF]. Although debatable and debated, these criteria are still used, and surely, there is room to revisit these criteria in the specific context of PHIR.

In the end, both PHIR and clinical research share the same objective of assessing intervention effects. Fundamentals are common: studies need to be comparative and conducted to prevent bias as much as possible. Although "the most common type of population health research remains the non-interventional observational study" [START_REF] Martin | Population health intervention research: myths and misconceptions[END_REF], the randomized design is clearly an option and should be encouraged.

Contributors

BG wrote the first draft. CA critically revised the draft for important intellectual content, and gave final approval of the version to be published.

Conflict of interests:

We declare that we have no conflicts of interest.

Funding: no funding.