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Total Absence of Dystrophin Expression Exacerbates Ectopic Myofiber Calcification and Fibrosis and Alters Macrophage Infiltration Patterns

Open ArchivePublished:November 11, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.09.021
      Duchenne muscular dystrophy (DMD) causes severe disability and death of young men because of progressive muscle degeneration aggravated by sterile inflammation. DMD is also associated with cognitive and bone-function impairments. This complex phenotype results from the cumulative loss of a spectrum of dystrophin isoforms expressed from the largest human gene. Although there is evidence for the loss of shorter isoforms having impact in the central nervous system, their role in muscle is unclear. We found that at 8 weeks, the active phase of pathology in dystrophic mice, dystrophin-null mice (mdxβgeo) presented with a mildly exacerbated phenotype but without an earlier onset, increased serum creatine kinase levels, or decreased muscle strength. However, at 12 months, mdxβgeo diaphragm strength was lower, whereas fibrosis increased, compared with mdx. The most striking features of the dystrophin-null phenotype were increased ectopic myofiber calcification and altered macrophage infiltration patterns, particularly the close association of macrophages with calcified fibers. Ectopic calcification had the same temporal pattern of presentation and resolution in mdxβgeo and mdx muscles, despite significant intensity differences across muscle groups. Comparison of the rare dystrophin-null patients against those with mutations affecting full-length dystrophins may provide mechanistic insights for developing more effective treatments for DMD.
      Duchenne muscular dystrophy (DMD) is a severely debilitating and invariably fatal X-linked neuromuscular disorder, which results from mutations in the DMD gene.
      • Hoffman E.P.
      • Fischbeck K.H.
      • Brown R.H.
      • Johnson M.
      • Medori R.
      • Loire J.D.
      • Harris J.B.
      • Waterston R.
      • Brooke M.
      • Specht L.
      • Kupsky W.
      • Chamberlain J.
      • Caskey C.T.
      • Shapiro F.
      • Kunkel L.M.
      Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy.
      DMD is the largest human gene known, encoding multiple structurally diverse isoforms of dystrophin.
      • Hoffman E.P.
      • Brown R.H.
      • Kunkel L.M.
      Dystrophin: the protein product of the duchenne muscular dystrophy locus.
      Three full-length transcripts, comprising 79 exons, encode 427-kDa proteins while further intragenic promoters
      • Ahn A.H.
      • Kunkel L.M.
      The structural and functional diversity of dystrophin.
      drive expression of progressively truncated variants (Figure 1A).
      Figure thumbnail gr1
      Figure 1Dystrophin isoform expression in mdx versus mdxβgeo muscle. A: DMD mutation location and their effects on predicted isoform expression in mdx and mdxβgeo. The mdx mouse carries a point mutation in exon 23, whereas mdxβgeo harbors an insertion disrupting the reading frame downstream from exon 63. B: Western blot analysis of dystrophin protein expression in 8-week muscles showing loss of the Dp427 isoform in mdx samples and loss of all isoforms from mdxβgeo, which confirm it to be a complete dystrophin knockout. Triplicate bands shown represent lysates from three different animals, and actin is shown as a protein loading control.
      The current central hypothesis states that Duchenne muscular dystrophy pathology is caused by the loss of the full-length dystrophin (Dp427) in myofibers, where it anchors the dystrophin-associated protein complex, linking the extracellular matrix, the sarcolemma, and the intracellular cytoskeleton. This assembly is considered critical for muscle function and survival. Therefore, all the current preclinical and clinical therapeutic approaches are aimed at dystrophin restoration in differentiated muscle cells.
      However, there is growing evidence that DMD mutations produce a range of significant cell-autonomous abnormalities in both human and mouse myogenic cells, suggesting a much earlier onset of pathology and explaining impaired muscle regeneration.
      • Dumont N.A.
      • Wang Y.X.
      • Von Maltzahn J.
      • Pasut A.
      • Bentzinger C.F.
      • Brun C.E.
      • Rudnicki M.A.
      Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division.
      • Dumont N.A.
      • Wang Y.X.
      • Rudnicki M.A.
      Intrinsic and extrinsic mechanisms regulating satellite cell function.
      • Yeung D.
      • Zablocki K.
      • Lien C.-F.
      • Jiang T.
      • Arkle S.
      • Brutkowski W.
      • Brown J.
      • Lochmuller H.
      • Simon J.
      • Barnard E.A.
      • Górecki D.C.
      Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.
      • Young C.N.J.
      • Brutkowski W.
      • Lien C.F.
      • Arkle S.
      • Lochmüller H.
      • Zabłocki K.
      • Górecki D.C.
      P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment.
      • Young C.N.J.
      • Sinadinos A.
      • Lefebvre A.
      • Chan P.
      • Arkle S.
      • Vaudry D.
      • Gorecki D.C.
      A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.
      • Yablonka-Reuveni Z.
      • Anderson J.E.
      Satellite cells from dystrophic (Mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers.
      • Blau H.M.
      • Webster C.
      • Pavlath G.K.
      Defective myoblasts identified in Duchenne muscular dystrophy.
      The severity of DMD-associated cognitive impairment correlates with the cumulative loss of dystrophin isoforms expressed in the central nervous system, thus suggesting a prominent functional role for these shorter isoforms in brain cells.
      • Taylor P.J.
      • Betts G.A.
      • Maroulis S.
      • Gilissen C.
      • Pedersen R.L.
      • Mowat D.R.
      • Johnston H.M.
      • Buckley M.F.
      Dystrophin gene mutation location and the risk of cognitive impairment in duchenne muscular dystrophy.
      ,
      • Masubuchi N.
      • Shidoh Y.
      • Kondo S.
      • Takatoh J.
      • Hanaoka K.
      Subcellular localization of dystrophin isoforms in cardiomyocytes and phenotypic analysis of dystrophin-deficient mice reveal cardiac myopathy is predominantly caused by a deficiency in full-length dystrophin.
      However, little attention has been given to the potential role of shorter dystrophins controlled by the intergenic promoters, and few in-depth comparisons between the full-length and the dystrophin-null muscle phenotypes have been undertaken. Interestingly, the proportion of patients with a severe motor and cognitive phenotype has been shown to correlate with mutations affecting all dystrophins.
      • Desguerre I.
      • Christov C.
      • Mayer M.
      • Zeller R.
      • Becane H.M.
      • Bastuji-Garin S.
      • Leturcq F.
      • Chiron C.
      • Chelly J.
      • Gherardi R.K.
      Clinical heterogeneity of Duchenne muscular dystrophy (DMD): definition of sub-phenotypes and predictive criteria by long-term follow-up.
      Gene mutations causing DMD disrupt the reading frame and include large deletions (68%), duplications (11%), and smaller rearrangements and point mutations (20%).
      • Aartsma-Rus A.
      • Ginjaar I.B.
      • Bushby K.
      The importance of genetic diagnosis for Duchenne muscular dystrophy.
      Initial analyses indicated that the DMD gene mutation hot spots are located in the regions encoding the full-length isoforms. However, although large deletions and duplications have a nonrandom distribution with the two identifiable hot spots, small insertions/deletions and point mutations are distributed along the entire gene,
      • Juan-Mateu J.
      • Gonzalez-Quereda L.
      • Rodriguez M.J.
      • Baena M.
      • Verdura E.
      • Nascimento A.
      • Ortez C.
      • Baiget M.
      • Gallano P.
      DMD mutations in 576 dystrophinopathy families: a step forward in genotype-phenotype correlations.
      thus affecting multiple isoforms.
      Interestingly, there are little data documenting the expression of the so-called nonmuscle dystrophin isoforms in muscle. Given that myogenic cells are affected by DMD mutations and are known to express some of these truncated isoforms (eg, Dp71), we hypothesized that null DMD mutations may alter functions of myogenic cells and thus affect the phenotype. Therefore, the consequences of total loss of DMD expression were investigated. The muscle pathology was compared in the most widely used animal model of DMD—the mdx mouse, lacking full-length isoforms because of a point mutation in exon 23,
      • Bulfield G.
      • Siller W.G.
      • Wight P.A.
      • Moore K.J.
      X chromosome-linked muscular dystrophy (mdx) in the mouse.
      against the mdxβgeo dystrophin-null mouse with the reading-frame disruption downstream of exon 63, which is present in all dystrophins and therefore with all isoforms being ablated. This mouse, unlike models generated by chemical mutagenesis, is a true pan-dystrophin knockout with no revertant fibers present.
      • Wertz K.
      • Füchtbauer E.M.
      Dmd(mdx-βgeo): a new allele for the mouse dystrophin gene.

      Materials and Methods

      Animals

      The male mdx and mdxβgeo wild-type control mice (C57Bl10 and C57Bl6, respectively) were used in accordance with institutional Ethical Review Board and the Home Office (United Kingdom) approvals. The C57Bl10 and C57Bl6 strains derived from the common origin,
      • Petkov P.M.
      • Ding Y.
      • Cassell M.A.
      • Zhang W.
      • Wagner G.
      • Sargent E.E.
      • Asquith S.
      • Crew V.
      • Johnson K.A.
      • Robinson P.
      • Scott V.E.
      • Wiles M.V.
      An efficient SNP system for mouse genome scanning and elucidating strain relationships.
      and it has been demonstrated that the mdx mutation on the C57Bl6 background shows the same pathology as the original Bl10 strain.
      • McGreevy J.W.
      • Hakim C.H.
      • McIntosh M.A.
      • Duan D.
      Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy.
      All mice were maintained under pathogen-free conditions and in a controlled environment (12-hour light/dark cycle, 19°C to 23°C ambient temperature, and 45% to 65% humidity). Mice were euthanized by carbon dioxide inhalation, and cells and muscles were dissected and used for protein extraction or frozen in isopentane prechilled in liquid nitrogen for cryosectioning.

      Antibodies and Reagents

      The following antibodies were used at 1:1000: antidystrophin (ab15277; Abcam, Cambridge, UK), antiactin (A2066; Sigma-Aldrich, Gillingham, UK), anti-F4/80 (ab6640; Abcam), and anti-CD68 (ab125212; Abcam). All other chemicals were purchased from Sigma-Aldrich or Fisher Scientific (Loughborough, UK).

      Serum Creatine Kinase Level Measurement

      Blood samples were collected, allowed to coagulate, and centrifuged for 10 minutes at 2500 × g. Sera isolated immediately after centrifugation were analyzed for the creatine kinase levels using the Creatine Kinase Activity Assay Kit (CK-NAC; Randox Laboratories Ltd., Crumlin, UK), according to manufacturer's instructions.

      Force Measurements in Diaphragms ex Vivo

      Whole diaphragms from 4-month–old wild-type and dystrophic mice were excised, and contractile force strength was measured following the Treat Neuromuscular Disorders Neuromuscular Network (TREAT-NMD) standard operating procedures (https://treat-nmd.org/research-overview/preclinical-research/experimental-protocols-for-dmd-animal-models, last accessed September 26, 2019) and as previously described.
      • Young C.N.J.
      • Sinadinos A.
      • Lefebvre A.
      • Chan P.
      • Arkle S.
      • Vaudry D.
      • Gorecki D.C.
      A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.
      Essentially, diaphragms were placed into Krebs-Ringer solution. Sutures were tied and muscle then attached to an immobile plastic clamp with the central triangular section of the diaphragm being used for testing. Contractile force was measured using a mechanical force transducer (ADInstruments, Oxford, UK), amplifier, and data acquisition setup. Excitation was achieved via local field potentials through platinum electrodes in oxygenated (95% O2, 5% CO2) Krebs-Ringer solution, at a constant temperature (37°C). After incremental stretching to establish the optimal excitation-to-force generation length and confirmation of the appropriate voltage twitch stimulus, diaphragm sections were subjected to a 140-V (2-millisecond) stimulus train at 100-Hz frequency for 0.5 to 1 second. The test regimen involved collecting six twitch responses, followed by six tetanic trains, with a 2-minute rest period between each. All forces were normalized to muscle wet weight and expressed as Newtons per gram of tissue (N/g).

      Grip Strength Test

      In this and all other in vivo tests, investigators (N.C. and Scott Rodaway) were blinded with respect to the sample group allocation. The grip strength test was performed, as previously described
      • Al-Khalidi R.
      • Panicucci C.
      • Cox P.
      • Chira N.
      • Róg J.
      • Young C.N.J.
      • McGeehan R.E.
      • Ambati K.
      • Ambati J.
      • Zabłocki K.
      • Gazzerro E.
      • Arkle S.
      • Bruno C.
      • Górecki D.C.
      Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism.
      and according to the TREAT-NMD protocol (http://www.treat-nmd.eu/downloads/file/sops/sma/SMA_M.2.1.002.pdf, last accessed September 26, 2019). Essentially, mice were held by the tail and slowly approached to a metallic grid (6 × 6-cm) connected to a force sensor gauge (FG-5000A; Lutron Electronic, London, UK). Once the animal gripped the grid by its forelimbs, a gentle horizontal traction was applied to the tail until the animal let the grid go. The maximal force was recorded over two trials with a 1-minute intertrial interval. Strength was estimated by the mean of both trials.

      RNA-Sequencing Analysis

      Total RNA was extracted from tibialis anterior (TA) of 7-week–old C57BL/10 and mdx male mice (n = 4), quality controlled, and sequenced, as previously described.
      • Young C.N.J.
      • Sinadinos A.
      • Lefebvre A.
      • Chan P.
      • Arkle S.
      • Vaudry D.
      • Gorecki D.C.
      A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.
      Quality control of raw reads was performed using fastQC version 0.11.7 (Babraham Institute, Cambridge, UK; http://www.bioinformatics.babraham.ac.uk/projects/fastqc, last accessed September 26, 2019). Reads were trimmed using trim-galore version 0.4.4 (Babraham Institute; https://www.bioinformatics.babraham.ac.uk/projects/trim_galore, last accessed September 26, 2019) with parameters to remove adapter sequence and low-quality sequence tails. Trimmed reads were mapped against the GRCm38 Mus musculus genome from Ensembl using the STAR version 2.5.3a modified universal RNA-seq aligner
      • Dobin A.
      • Davis C.A.
      • Schlesinger F.
      • Drenkow J.
      • Zaleski C.
      • Jha S.
      • Batut P.
      • Chaisson M.
      • Gingeras T.R.
      STAR: ultrafast universal RNA-seq aligner.
      with the following parameters: --outSAMmultNmax 300 --outSAMstrandField intronMotif. Properly paired reads that mapped uniquely to the genome, with a mapping quality >20, were retained for further analyses.
      Differential expression analysis was conducted using the DESeq2 package
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      in R version 3.5.1 (http://www.R-project.org, last accessed September 26, 2019).
      R Core Team: R: A language and environment for statistical computing.
      Gene models were taken from Ensembl version 91, and read counts over unique genes were quantified using the summarizeOverlaps() function in the GenomicAlignments package
      • Lawrence M.
      • Huber W.
      • Pagès H.
      • Aboyoun P.
      • Carlson M.
      • Gentleman R.
      • Morgan M.T.
      • Carey V.J.
      Software for computing and annotating genomic ranges.
      using parameters mode = Union, singleEnd = FALSE, ignore.strand = FALSE, fragments = FALSE, preprocess.reads = invertStrand. P values were adjusted for multiple testing by using the Benjamini and Hochberg false discovery rate correction.
      • Hochberg B.
      Controlling the false discovery rate: a practical and powerful approach to multiple testing.
      The whole muscle RNA-seq data can be accessed from Array Express (https://www.ebi.ac.uk/arrayexpress/experiments; accession code E-MTAB-7698).

      Histologic Stains

      Hematoxylin and eosin, Alizarin red (AR), periodic acid–Schiff, Von Kossa, oil red O, and picrosirius red staining methods followed standard operating procedures from TREAT-NMD–recommended protocols available online (http://treat-nmd.eu/research/preclinical/dmd-sops, last accessed September 26, 2019). All staining was performed using cryosections (10 μm thick), air dried onto poly-l-lysine–coated glass slides (Fisher Scientific). Slides were mounted in DPX or aqueous media, coverslipped, and imaged (Axiozoom V.16; Zeiss, Cambridge, UK). Representative images per genotype are shown, whereas montages, where n = 30 to 40, were constructed and assessed using ImageJ version 2.0.0-rc-69/1.52p (Fiji, https://fiji.sc, last accessed September 26, 2019) counting tools.
      • Abràmoff M.D.
      • Magalhães P.J.
      • Ram S.J.
      Image processing with imageJ.

      Whole-Body Tissue Clearing, Imaging, and Analysis

      Clearing procedure was performed as described previously.
      • Bozycki L.
      • Łukasiewicz K.
      • Matryba P.
      • Pikula S.
      Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy.
      Briefly, animals were deeply anesthetized with i.p. injection of lethal dose of sodium pentobarbital (100 mg/kg), subjected to cardiac perfusion, and subjected to fixation followed by 2 to 3 days of clearing with CUBIC reagent-1
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Kishino F.
      • Tawara T.
      • Watanabe T.M.
      • Yokoyama C.
      • Onoe H.
      • Eguchi M.
      • Yamaguchi S.
      • Abe T.
      • Kiyonari H.
      • Shimizu Y.
      • Miyawaki A.
      • Yokota H.
      • Ueda H.R.
      Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.
      or reagent-1A (Riken, http://cubic.riken.jp/data/CUBIC_clearing_protocol_with_Reagent-1A.pdf, last accessed September 26, 2019) clearing solutions and 1 day of 0.03% (w/v) AR staining dissolved in fresh clearing solution. Finally, specimens were placed for 2 to 3 days of gentle shaking with fresh clearing solution at 37°C in an incubator to remove the excess of unbound AR. Images were collected with customized light-sheet apparatus and analyzed according to a previously described protocol.
      • Bozycki L.
      • Łukasiewicz K.
      • Matryba P.
      • Pikula S.
      Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy.

      Immunolocalization and Morphologic Analysis

      Frozen muscle was transferred to a cryostat chamber and allowed to equilibrate to −20°C. Cryosections (10 μm thick) were then cut from the middle third of the sample and collected on poly-l-lysine (0.5 mg/mL)–coated glass slides. Sections were allowed to air dry for several hours. Samples were fixed in a 2% to 4% w/v paraformaldehyde solution in Tris-buffered saline/Tween 20 for 15 minutes at 4°C, followed by two washes in phosphate-buffered saline/Tween 20. The primary antibody incubation in phosphate-buffered saline/Tween 20 containing 10% v/v serum was applied for 2 hours at room temperature or overnight at 4°C. Three 5-minute Tris-buffered saline/Tween 20 washes were applied before secondary antibody incubation in phosphate-buffered saline/Tween 20 and 10% v/v serum containing Hoechst (1:1000) fluorescent nuclear counterstain for 1 hour at room temperature. Sections were finally washed three times for 30 minutes before mounting in FluorSave (Merk Millipore, Watford, UK) fluorescence mounting medium. Either entire cross-sections through the midportion of TA muscles were captured in their entirety using Axiozoom V.16 (Zeiss) or whole cross-sections were made of montaged ×20 magnification fields of view. For quantification of immunofluorescence cells, a semiautomated (unbiased) method using a thresholding macro designed in ImageJ software (Fiji) was used. Numbers were then expressed per unit of area. For diaphragms, counts per unit area for each animal were derived by averaging the counts from five fields of view encompassing a significant portion of each diaphragm cross-section. Counts were also made using the threshold and analyze particles functions of ImageJ software.

      Western Blot Analysis

      Proteins were extracted, resolved, and blotted, as described previously.
      • Al-Khalidi R.
      • Panicucci C.
      • Cox P.
      • Chira N.
      • Róg J.
      • Young C.N.J.
      • McGeehan R.E.
      • Ambati K.
      • Ambati J.
      • Zabłocki K.
      • Gazzerro E.
      • Arkle S.
      • Bruno C.
      • Górecki D.C.
      Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism.
      ,
      • Sinadinos A.
      • Young C.N.J.
      • Al-Khalidi R.
      • Teti A.
      • Kalinski P.
      • Mohamad S.
      • Floriot L.
      • Henry T.
      • Tozzi G.
      • Jiang T.
      • Wurtz O.
      • Lefebvre A.
      • Shugay M.
      • Tong J.
      • Vaudry D.
      • Arkle S.
      • DoRego J.C.
      • Górecki D.C.
      P2RX7 purinoceptor: a therapeutic target for ameliorating the symptoms of duchenne muscular dystrophy.
      Blots were blocked in 5% w/v nonfat milk powder in 1× Tris-buffered saline (50 mmol/L Tris, 150 mmol/L NaCl, and 0.01% v/v Tween-20; Sigma-Aldrich), for 1 hour before probing with primary antibody diluted in the same blocking buffer (overnight at 4°C or 2 hours at room temperature), then washed (three times) with 1× Tris-buffered saline/Tween 20 for 10 minutes and incubated with the appropriate horseradish peroxidase–conjugated secondary antibody: anti-mouse 1:10:000 (A4416; Sigma-Aldrich) or anti-rabbit 1:5000 (A6154; Sigma-Aldrich) overnight at 4°C or 1 hour at room temperature. Specific protein bands were visualized using Luminata Classico or Forte chemiluminescent substrates (WBLUC0500 or WBLUF0500, respectively; Merck Millipore), and images were obtained using a ChemiDoc MP system (Bio-Rad, Hertfordshire, UK). Densitometric analyses were performed using the integrated density measurement function of ImageJ software. All experiments were repeated at least three times in triplicate, throughout.

      X-Ray Micro–Computed Tomography

      Quadriceps were placed within a 1.5-mL tube (Eppendorf, Stevenage, UK) and supported by a polyurethane foam saturated in 70% ethanol. Muscles were imaged using a Zeiss Xradia 520 Versa X-ray microscope (Zeiss) operating at an energy of 50 kV, a power of 4 W, and a tube current of 80 μA; and a Zeiss LE1 filter was positioned directly after the X-ray source. A 0.4× objective lens was used with an X-ray source (sample distance of 20 mm) and a detector (sample distance of 105 mm). A total of 1601 X-ray projection images were collected over 360 degrees at equal intervals with an isotropic voxel size of 11 μm. The exposure time for each projection was 2 seconds. The projections were reconstructed using the manufacturer's integrated software, which uses a filtered back projection reconstruction algorithm. The individual tomography scans were quantified using the threshold function in ImageJ
      • Abràmoff M.D.
      • Magalhães P.J.
      • Ram S.J.
      Image processing with imageJ.
      and visualized in three dimensions using TXM3DViewer (Zeiss).

      Statistical Analysis

      Results are reported as means ± SD, where n refers to number of independent experiments (3 to 6). Significance scores were based on Kruskal-Wallis with post-hoc Dunn's test for nonparametric multiple comparisons; one-way analysis of variance with post-hoc Tukey test for normal multiple comparisons; and unpaired t-tests for individual comparisons, with Mann-Whitney post-hoc test for nonparametric t-tests (GraphPad Prism8; GraphPad Software, San Diego, CA). For cumulative frequency distribution, Kolmogorov-Smirnov test was used. Differences were considered statistically significant at P < 0.05.

      Results

      Dystrophic Pathology in mdxβgeo Dystrophin-Null Mice

      Muscle pathology in the mdx muscles begins to present at 2 to 3 weeks, reaching maximum intensity in leg muscles at approximately 8 weeks, before plateauing at approximately 12 to 16 weeks.
      • Suelves M.
      • Vidal B.
      • Serrano A.L.
      • Tjwa M.
      • Roma J.
      • López-Alemany R.
      • Luttun A.
      • De Lagrán M.M.
      • Díaz M.À.
      • Jardí M.
      • Roig M.
      • Dierssen M.
      • Dewerchin M.
      • Carmeliet P.
      • Muñoz-Cánoves P.
      uPA deficiency exacerbates muscular dystrophy in MDX mice.
      ,
      • Yucel N.
      • Chang A.C.
      • Day J.W.
      • Rosenthal N.
      • Blau H.M.
      Humanizing the mdx mouse model of DMD: the long and the short of it.
      However, the mdx mouse diaphragm shows progressive pathology
      • Stedman H.H.
      • Sweeney H.L.
      • Shrager J.B.
      • Maguire H.C.
      • Panettieri R.A.
      • Petrof B.
      • Narusawa M.
      • Leferovich J.M.
      • Sladky J.T.
      • Kelly A.M.
      The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.
      and, therefore, this muscle closely represents the human condition.
      To identify the potential phenotypic differences resulting from the absence of all versus full-length isoforms, mdx and mdxβgeo (dystrophin-null) mice (Figure 1) were compared following the TREAT-NMD standard operating procedures.
      • Aartsma-Rus A.
      • van Putten M.
      Assessing functional performance in the mdx mouse model.
      ,
      • Briguet A.
      • Courdier-Fruh I.
      • Foster M.
      • Meier T.
      • Magyar J.P.
      Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse.
      At 8 weeks (the peak of pathology), morphologic analysis of leg muscles revealed a significant shift in myofiber cross-sectional area toward smaller fibers in dystrophic muscle in the order of wild-type (BL10) > mdx > mdxβgeo (Figure 2, A and B ). A significant reduction in the average ferret diameter followed the same trend (Kruskal-Wallis with Dunn's test) (Figure 2C). Central nucleation was significantly elevated in mdxβgeo compared with mdx (Mann-Whitney test, P = 0.0159) (Figure 2D). At 8 weeks, serum creatine kinase levels (Figure 2E) were not significantly different (Mann-Whitney test, P = 0.4127) between the two dystrophic strains, indicating that loss of short dystrophins did not exacerbate sarcolemma damage. Grip strength in vivo (Figure 2F) and diaphragm strength ex vivo (Figure 2G) were equally reduced in both dystrophic strains at 8 weeks. Yet, there was an age-dependent difference between mdxβgeo and mdx: In 12-month–old animals, diaphragm contractile force strength showed small but significant increase in mdx preparations (unpaired t-test, t = 6.572, df = 4, P = 0.0028), but no increase was found in mdxβgeo (unpaired t-test, t = 0.6558, df = 4, P = 0.5478) (Figure 2G). Furthermore, fibrosis (Figure 2H) and fat accumulations (Figure 2I) were both found elevated in 12-month–old mdxβgeo diaphragms compared with age-matched mdx.
      Figure thumbnail gr2
      Figure 2Morphologic and functional alteration in mdx versus mdxβgeo muscle. AC: Morphometric analysis of 8-week tibialis anterior muscles reveals a shift in fiber size (A) in the order of: wild-type (Wt) > mdx > mdxβgeo, which was found to be consistent for the average fiber area (B) and feret diameter (C). Insets: Example of fiber threshold and the analysis using ImageJ version 2.0.0-rc-69/1.52p (Fiji). D: Numbers of centrally nucleated fibers are significantly elevated: mdxβgeo > mdx > Wt. E: Serum creatine kinase (CK) levels indicative of sarcolemma stability are not significantly altered in mdxβgeo compared with mdx. F: Grip strength at 8 weeks is reduced by approximately 50% in both mdx and mdxβgeo compared with Wt, and diaphragm contractile force is also low in both mdx and mdxβgeo. G: In contrast, at 12 months, increases in maximum force were recorded in both Wt and mdx, but not in mdxβgeo, diaphragms, which remain at a basal level. H and I: Picrosirius red staining for collagen (H) and oil red O staining for fat (I) in 8-week versus 12-month diaphragms reveal significant increases in older animals: mdxβgeo > mdx > Wt. Data are expressed as means ± SD (BI). n = 3 to 5 (AI). *P < 0.05, ***P < 0.001, and ****P < 0.0001. Scale bars = 250 μm (H and I). FOV, field of view.

      Total Dystrophin Loss Exacerbates Ectopic Calcification of Dystrophic Muscle Fibers

      Muscles from mdxβgeo mice do not express any dystrophin isoforms (Figure 1B) or truncated variants in revertant fibers (Figure 3A). In contrast, at 8 weeks, striking opaque fibers, particularly prominent in diaphragms but detectable in all major skeletal muscle groups, were found to be notably more abundant in mdxβgeo than in mdx mice (Figure 3, B–D). The appearance of these fibers closely resembled ectopic calcification reported previously in the mdx, mdx/Utrophin double knockout and the humanized-mdx mouse models,
      • Kikkawa N.
      • Ohno T.
      • Nagata Y.
      • Shiozuka M.
      • Kogure T.
      • Matsuda R.
      Ectopic calcification is caused by elevated levels of serum inorganic phosphate in mdx mice.
      • Sohn J.
      • Lu A.
      • Tang Y.
      • Wang B.
      • Huard J.
      Activation of non-myogenic mesenchymal stem cells during the disease progression in dystrophic dystrophin/utrophin knockout mice.
      • Isaac C.
      • Wright A.
      • Usas A.
      • Li H.
      • Tang Y.
      • Mu X.
      • Greco N.
      • Dong Q.
      • Vo N.
      • Kang J.
      • Wang B.
      • Huard J.
      Dystrophin and utrophin “double knockout” dystrophic mice exhibit a spectrum of degenerative musculoskeletal abnormalities.
      • Wada E.
      • Yoshida M.
      • Kojima Y.
      • Nonaka I.
      • Ohashi K.
      • Nagata Y.
      • Shiozuka M.
      • Date M.
      • Higashi T.
      • Nishino I.
      • Matsuda R.
      Dietary phosphorus overload aggravates the phenotype of the dystrophin-deficient mdx mouse.
      in the golden retriever muscular dystrophy dog,
      • Barthélémy I.
      • Uriarte A.
      • Drougard C.
      • Unterfinger Y.
      • Thibaud J.L.
      • Blot S.
      Effects of an immunosuppressive treatment in the GRMD dog model of Duchenne muscular dystrophy.
      ,
      • Liu J.M.K.
      • Okamura C.S.
      • Bogan D.J.
      • Bogan J.R.
      • Childers M.K.
      • Kornegay J.N.
      Effects of prednisone in canine muscular dystrophy.
      and, more important, in DMD patients.
      • Larcher T.
      • Lafoux A.
      • Tesson L.
      • Remy S.
      • Thepenier V.
      • François V.
      • Le Guiner C.
      • Goubin H.
      • Dutilleul M.
      • Guigand L.
      • Toumaniantz G.
      • De Cian A.
      • Boix C.
      • Renaud J.-B.
      • Cherel Y.
      • Giovannangeli C.
      • Concordet J.-P.
      • Anegon I.
      • Huchet C.
      Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy.
      To confirm, it was first verified if calcium- and phosphorus-containing deposits were present in these opaque diaphragm fibers using AR (Figure 4A) and Von Kossa (Figure 4B) stains, respectively. Scanning electron microscopy energy-dispersive X-ray spectroscopy electron backscatter analysis (Figure 4C) confirmed the presence of mineral deposits containing both calcium (Figure 4D) and phosphate (Figure 4E) with a molar ratio of 3:2 (Figure 4F), consistent with tricalcium phosphate [Ca3(PO4)2].
      • Vallet-Regí M.
      Ceramics for medical applications.
      Figure thumbnail gr3
      Figure 3Muscle fiber mineralization is linked to the loss of dystrophin expression in mdx and mdxβgeo. A: Immunohistochemistry staining for dystrophin in 8-week tibialis anterior (TA) muscle sections confirms the mdx to express dystrophin in a small number of revertant fibers. In contrast, mdxβgeo animals display no revertant fibers, in keeping with the molecular alteration in these animals. B: On dissection, significant white striations (arrows) are observed in the diaphragms of mdxβgeo, which are also found in mdx, albeit at much lower levels, but not in controls. Heightened diaphragm hypercontraction is also consistently observed in the order of: mdxβgeo > mdx > wild-type (Wt), represented by the enlarged region of translucent connective tissue in the center. C: Striations are found in all skeletal muscle groups of dystrophic mice but at different levels, with proximal muscles (quadriceps and gluteus) being affected more than distal muscles, such as TA. D: Heart muscles are affected, albeit showing slightly different striation patterns than skeletal muscles (). Arrows highlight regions of ectopic calcification (white streaks). Scale bars = 100 μm (A).
      Figure thumbnail gr4
      Figure 4Histochemical and mineral analyses in 8-week–old mdx and mdxβgeo muscles. A and B: Alizarin red (A) and Von Kossa (B) staining demonstrates that the white striations in diaphragm sections contain calcium and phosphate, respectively. CF: Electron backscatter diffraction analysis of diaphragm sections from 8-week mdxβgeo (C) identifies colocalization of calcium (D) and phosphate (E) in electron-dense fibers, with the calcium/phosphate ratio of 1.50 (F), consistent with the presence of tricalcium phosphate [Ca3(PO4)2] or hydroxyapatite, which has a ratio of 1.6729. n = 3 mice (AF). Scale bars = 250 μm (AE).
      The striated appearance of calcified fibers showed regions of calcification with distinct patterning, sometimes along the length of almost entire fiber, sometimes in short regions of otherwise unaltered fiber (Figure 3C and Supplemental Video S1). Muscle groups most severely affected with ectopic calcification were diaphragm (Figure 3B) and the proximal limb (quadriceps and gluteus) with a consistently milder phenotype in the distal groups (TA and gastrocnemius) (Figure 3C). More important, this ectopic calcification was also found in cardiac muscles of mdxβgeo (Figure 3D and Supplemental Figure S1), which, to our knowledge, is the first demonstration of this abnormality in a DMD model.

      Whole-Body Musculature Analysis of Ectopic Calcification in mdx and mdxβgeo

      The initial study revealed significant differences in ectopic calcifications between various muscle groups, indicating the need for systematic comparisons. To screen for and quantify ectopic mineralization in various muscle groups of the entire animal, previously optimized whole-body tissue optical clearing method
      • Bozycki L.
      • Łukasiewicz K.
      • Matryba P.
      • Pikula S.
      Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy.
      was applied. Such an approach, when combined with AR staining, allowed us to demonstrate excessive accumulation of ectopic calcifications in mdxβgeo versus mdx and confirm complete absence of these in control animals (Figure 5, A–D). Thereby, calcified deposits were observed particularly abundant within mdxβgeo diaphragms (Figure 5B) but also in skeletal muscles of the laryngopharynx, forelimb, lumbar region, pelvic region, and hind limbs. Next, a customized light-sheet setup was used to perform detailed three-dimensional imaging of isolated muscles from three distinct body regions (ie, spinalis pars lumborum, biceps femoris, and triceps brachii) (Figure 5, E and F). When compared with mdx, every mdxβgeo muscle was characterized by a higher percentage of tissue mineralization, with differences being particularly striking in triceps brachii, where ectopic calcification reached 11.59% in mdxβgeo and 0.36% in mdx [percentage of mineralization: unpaired t-test t(4) = 5.32; P < 0.01] (Figure 5F). In contrast, the difference was not found to be statistically significant in spinalis pars lumborum [unpaired t-test t(4) = 2.62; P = 0.058] (Figure 5F) and biceps femoris [unpaired t-test t(4) = 0.97; P = 0.386] (Figure 5F). Cumulative frequency distribution analysis showed different distribution of calcified deposits in triceps brachii and spinalis pars lumborum muscles from mdxβgeo mice in comparison to mdx mice (Figure 5F).
      Figure thumbnail gr5
      Figure 5Whole-body and three-dimensional muscle analysis of ectopic calcifications in mdx and mdxβgeo. Whole-body tissue clearing and Alizarin red staining show distribution of ectopic calcification across the entire musculature. A: Representative bright-field (top panels) and epifluorescent images (bottom panels) reveal sites of myofiber calcification and allow detailed comparative imaging of the affected body regions. B: Epifluorescent images of the selected planes from A demonstrate higher prevalence of calcifications in mdxβgeo versus mdx with a complete absence of deposits in the control mouse. Arrowheads indicate clusters of calcium deposits in laryngopharynx (1 to 3), forelimb (4 to 6), diaphragm (7 to 9), lumbar region (10 to 12), and hind limb (13 to 15). C and D: Spinalis pars lumborum from macroscopically prescreened mice was isolated and imaged in crossed polarized light (C) and light-sheet fluorescence microscopy (D). E and F: Three-dimensional light-sheet data allow us to reconstruct distribution of sites of ectopic calcification (E) and quantify its pattern in muscles of mdxβgeo and mdx—presented herein as percentage mineralization and cumulative frequency distributions in triceps brachii, spinalis pars lumborum, and biceps femoris (F). Unpaired t-test and two-sample Kolmogorov-Smirnov test were performed. Data are expressed as means ± SD (F). n = 3 mice per group (F). *P < 0.05. Scale bar = 5 mm (D). Original magnification, ×4 (AE).
      Further confirmation of muscle fiber calcification was undertaken using AR staining of TA (Figure 6, C and D ) and diaphragm (Figure 6, A and B) sections and particle analysis–based quantification of threshold images using ImageJ software (Figure 6E). Significantly elevated numbers and percentages of calcified fibers were confirmed in diaphragms (Figure 6F), whereas TA was confirmed to be less affected by the ectopic calcification. Finally, ectopic calcification in isolated 8-week–old quadriceps mdx and mdxβgeo muscles were visualized in three dimensions under the X-ray microscope (Xradia; Zeiss) (Figure 6, G–J, and Supplemental Video S1).
      Figure thumbnail gr6
      Figure 6Quantification of muscle fiber mineralization in mdx versus mdxβgeo. A and B: Alizarin red (AR) staining was quantified in mdx (A) and mdxβgeo (B) diaphragms (Diap.) at 8 weeks of age. C and D: Representative images of tibialis anterior sections from mdx (C) and mdxβgeo (D) are shown to illustrate the difference in severity between different muscle groups. E: Alizarin red images were thresholded, a mask was generated in ImageJ software, and fibers displaying an arbitrarily assigned positive value at or above the threshold level were counted using the ImageJ version 2.0.0-rc-69/1.52p (Fiji) particle analysis function. F: A significant increase in absolute numbers and percentage of mineralized muscle fibers is found in mdxβgeo compared with age-matched mdx diaphragms. GJ: Striations along the entire length of fibers were analyzed in whole muscle mineralization analysis using three-dimensional (3D) X-ray imaging. G and H: Quadriceps (QUAD.) from 8-week–old mdx (G) and mdxβgeo (H) in 3D rendering reveals two different patterns of mineralization: one diffuse and globular and the other striated (left and right sides of tissue shown in H, respectively). I and J: Representative Z-sections for mdx and mdxβgeo are shown, respectively. Data are expressed as means ± SD (F). n = 3 (F). ****P < 0.0001. Scale bars = 5 mm (GJ). Original magnification, ×4 (AE).

      Age of Onset and Evolution of Ectopic Muscle Mineralization in mdx and mdxβgeo

      The onset and progression of muscle pathology in the mdx muscle are well documented with cycles of degeneration and regeneration and significant sterile inflammation between 3 and 12 weeks of age, followed by a significant reduction of symptoms from 12 weeks onward. The exception is diaphragm, where the pathology is progressive and thus resembles human disease.
      • Suelves M.
      • Vidal B.
      • Serrano A.L.
      • Tjwa M.
      • Roma J.
      • López-Alemany R.
      • Luttun A.
      • De Lagrán M.M.
      • Díaz M.À.
      • Jardí M.
      • Roig M.
      • Dierssen M.
      • Dewerchin M.
      • Carmeliet P.
      • Muñoz-Cánoves P.
      uPA deficiency exacerbates muscular dystrophy in MDX mice.
      ,
      • Yucel N.
      • Chang A.C.
      • Day J.W.
      • Rosenthal N.
      • Blau H.M.
      Humanizing the mdx mouse model of DMD: the long and the short of it.
      Aforementioned exacerbation of ectopic mineralization in mdxβgeo led us to assess whether total dystrophin ablation triggers an earlier onset of dystrophic damage with ectopic calcification. To test this hypothesis, AR staining intensities were analyzed in 2- and 4-week–old mdx and mdxβgeo diaphragm muscle sections. Two-week–old muscles were found to be visually devoid of detectable calcifications (Figure 7, A–C), but at 4 weeks, white striations were clearly beginning to form in limb and diaphragm muscles (Figure 7, D–F). Quantification of AR staining in diaphragm sections confirmed first calcified fibers to appear somewhere between 2 and 4 weeks of age but equally in both mdx and mdxβgeo animals (Figure 7J). Given the nature of the ectopic calcification, it could be expected to worsen with age, particularly in diaphragms. However, analyses in 3- and 6-month–old mice showed the calcified fibers could no longer be found (Figure 7K). The diaphragm appearance, with thickening and opacity (Figure 7, G–I), may be due to ongoing inflammation and emerging fibrosis, which are pathologic hallmarks of 12-month–old diaphragms. Indeed, picrosirius red staining for collagen (Figure 2H) revealed the presence of fibrosis.
      Figure thumbnail gr7
      Figure 7Timing and evolution of muscle fiber mineralization in mdxβgeo muscles. AC: At 2 weeks, muscles appear normal with no visible striations. DF: By 4 weeks, light striations begin to appear (arrows). GI: After a peak at approximately 2 months (G), calcium-containing fibers (arrows) disappear at approximately 10 to 12 weeks (H), and are replaced by connective tissue (I). Note the increased opacity of the diaphragm with increasing age (G to I progression). J: Quantification of Alizarin red (AR)–positive fibers across ages. K: Quantification of AR staining in 2-, 3-, and 6-month–old diaphragm sections, confirming the absence of mineralization. Data are expressed as means ± SD (J and K). n = 3 (J and K). ****P < 0.0001.

      Differences in Macrophage Distribution and Association with Mineralized Fibers in mdx versus mdxβgeo Diaphragms

      Inflammation is the well-known pathologic hallmark of DMD. It affects muscle regeneration but also degeneration and fibrosis,
      • Villalta S.A.
      • Rosenberg A.S.
      • Bluestone J.A.
      The immune system in Duchenne muscular dystrophy: friend or foe.
      ,
      • Villalta S.A.
      • Nguyen H.X.
      • Deng B.
      • Gotoh T.
      • Tidbal J.G.
      Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.
      Of the inflammatory cells found in mdx muscles, macrophages play a significant yet complex role: Their depletion results in the reduction or exacerbation of pathology, depending on the stage of disease.
      • Kikkawa N.
      • Ohno T.
      • Nagata Y.
      • Shiozuka M.
      • Kogure T.
      • Matsuda R.
      Ectopic calcification is caused by elevated levels of serum inorganic phosphate in mdx mice.
      ,
      • Villalta S.A.
      • Nguyen H.X.
      • Deng B.
      • Gotoh T.
      • Tidbal J.G.
      Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.
      ,
      • Farini A.
      • Meregalli M.
      • Belicchi M.
      • Battistelli M.
      • Parolini D.
      • D'Antona G.
      • Gavina M.
      • Ottoboni L.
      • Constantin G.
      • Bottinelli R.
      • Torrente Y.
      T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse.
      The RNA-seq data (Array Express code E-MTAB-7698) identified significant contribution of macrophage genes to the altered inflammatory gene expression profile in mdx muscles (Supplemental Figure S2). Furthermore, a recent study has demonstrated that inorganic phosphate (Pi) can specifically activate macrophages to prevent ectopic calcification.
      • Villa-Bellosta R.
      • Hamczyk M.R.
      • Andrés V.
      Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate.
      Given that the evolution of calcified muscle fibers mirrored the onset and cessation of inflammation in mdx muscle, the immune cells in the muscle sections were analyzed. F4/80 staining for macrophages was markedly different in the two dystrophic strains: mdx muscle showed scattered staining with numerous macrophage puncta spread throughout the tissue and only some larger puncta of intense staining (Figure 8, C and G ). In contrast, mdxβgeo muscles displayed large F4/80-positive puncta, which colocalized perfectly with mineralized fibers and appeared almost uniquely and intricately associated with them (Figure 8, A, B, D–F, and H). Often, macrophages were tightly associated with what appeared to be partially degraded fibers (Figure 8B). The CD68 and osteopontin staining colocalization in these macrophages indicated their predominantly M1 phenotype (Figure 8, I–K). CD68 macrophage targeting of ectopic calcium deposits was also increased in mdxBgeo, with significantly increased clustering of CD68 (Figure 8, L and M).
      Figure thumbnail gr8
      Figure 8Differential macrophage distribution and association with mineralized fibers in mdx and mdxβgeo muscles. A: Confocal image showing F4/80 (red) macrophage marker and cell nuclei (blue) staining combined with mineral deposits visualized in bright field in mdxβgeo diaphragms. B: Calcified fibers can be seen saturated with macrophages (arrow). C and D: Macrophage distribution differs between mdx and mdxβgeo muscles: In mdx diaphragm (C), macrophages can be seen distributed throughout the tissue with some areas of increased infiltration, whereas in mdxβgeo, macrophages appear to be predominantly associated with calcified fibers (D). EH: Confocal images of F4/80 staining without bright field, corresponding with A through D, respectively. IK: Higher-magnification images showing CD68 (red) marker colocalization with osteopontin (green), indicating the predominantly M1 phenotype of macrophages associated with calcified fibers. L: ImageJ-based (Fiji) quantification of images showing the CD68 staining to localize to fewer (left panel) but larger (right panel) puncta in mdxβgeo, confirming macrophage clustering at sites of mineralization. M: Significant differences in CD68 puncta number and size are found between mdx and mdxβgeo (left and middle panels), but total CD68 intensity in mdxβgeo is not found to be significantly different to that of mdx (right panel). Data are expressed as means ± SD (M). n = 3 (M). ****P < 0.0001. Scale bars: 250 μm (A, C, D, E, G, and H); 50 μm (B and F); 100 μm (IK). FOV, field of view.
      In conclusion, total loss of dystrophin expression in the mouse model of DMD specifically exacerbated ectopic myofiber calcification, altered macrophage infiltration, and aggravated subsequent fibrosis.

      Discussion

      There is evidence that absence of the full-length (427-kDa) dystrophin in the fully differentiated myofibers may not necessarily cause the dystrophic phenotype.
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      RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology.
      ,
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      • Campbell K.P.
      Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle.
      In contrast, Dp427 has been shown to play a role in satellite cells
      • Dumont N.A.
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      • Pasut A.
      • Bentzinger C.F.
      • Brun C.E.
      • Rudnicki M.A.
      Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division.
      ,
      • Dumont N.A.
      • Wang Y.X.
      • Rudnicki M.A.
      Intrinsic and extrinsic mechanisms regulating satellite cell function.
      ,
      • Yablonka-Reuveni Z.
      • Anderson J.E.
      Satellite cells from dystrophic (Mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers.
      ,
      • Xie X.
      • Tsai S.Y.
      • Tsai M.J.
      COUP-TFII regulates satellite cell function and muscular dystrophy.
      ,
      • Alexakis C.
      • Partridge T.
      • Bou-Gharios G.
      Implication of the satellite cell in dystrophic muscle fibrosis: a self-perpetuating mechanism of collagen overproduction.
      and there are clear data that a lack of DMD gene expression affects various important functions of myoblasts, including cell proliferation, differentiation, energy metabolism, and signaling.
      • Yeung D.
      • Zablocki K.
      • Lien C.-F.
      • Jiang T.
      • Arkle S.
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      • Brown J.
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      • Simon J.
      • Barnard E.A.
      • Górecki D.C.
      Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.
      ,
      • Young C.N.J.
      • Sinadinos A.
      • Lefebvre A.
      • Chan P.
      • Arkle S.
      • Vaudry D.
      • Gorecki D.C.
      A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.
      ,
      • Young C.N.J.
      • Chira N.
      • Róg J.
      • Al-Khalidi R.
      • Benard M.
      • Galas L.
      • Chan P.
      • Vaudry D.
      • Zabłocki K.
      • Górecki D.C.
      Sustained activation of P2X7 induces MMP-2-evoked cleavage and functional purinoceptor inhibition.
      These and other findings indicate that dystrophic pathology starts much earlier than has been suggested
      • Merrick D.
      • Stadler L.K.J.
      • Larner D.
      • Smith J.
      Muscular dystrophy begins early in embryonic development deriving from stem cell loss and disrupted skeletal muscle formation.
      and point at the importance of the loss of dystrophin expression in myogenic cells, dysfunction of which determines abnormalities of muscle regeneration and therefore disease progression. Because Dp71 dystrophin has been found in undifferentiated myogenic cells,
      • Howard P.L.
      • Dally G.Y.
      • Ditta S.D.
      • Austin R.C.
      • Worton R.G.
      • Klamut H.J.
      • Ray P.N.
      Dystrophin isoforms Dp71 and Dp427 have distinct roles in myogenic cells.
      we hypothesized that Dmd gene mutations eliminating expression of this isoform may further alter functions of myogenic cells and thus affect the dystrophic phenotype. Therefore, the muscle pathology was compared in the most widely used animal model of DMD—the mdx mouse, lacking full-length isoforms because of a stop mutation in exon 23,
      • Bulfield G.
      • Siller W.G.
      • Wight P.A.
      • Moore K.J.
      X chromosome-linked muscular dystrophy (mdx) in the mouse.
      against the mdxβgeo dystrophin-null mouse.
      • Wertz K.
      • Füchtbauer E.M.
      Dmd(mdx-βgeo): a new allele for the mouse dystrophin gene.
      The latter DMD model is interesting as it has no observed dystrophin-positive revertant fiber clusters
      • Farini A.
      • Meregalli M.
      • Belicchi M.
      • Battistelli M.
      • Parolini D.
      • D'Antona G.
      • Gavina M.
      • Ottoboni L.
      • Constantin G.
      • Bottinelli R.
      • Torrente Y.
      T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse.
      and allows complex phenotypes to be investigated. Notably, mutation hot spots of large deletions and duplications are located in the regions encoding the full-length isoforms. However, small insertions/deletions and point mutations are distributed along the entire gene
      • Juan-Mateu J.
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      • Baena M.
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      • Baiget M.
      • Gallano P.
      DMD mutations in 576 dystrophinopathy families: a step forward in genotype-phenotype correlations.
      ; and these would affect the full spectrum of dystrophins. However, there are little data evaluating the role of the shorter dystrophin isoforms in muscle.
      Our analyses revealed a slightly exacerbated phenotype in mdxβgeo, especially in older mice. However, these dystrophin-null mice did not show an earlier onset of the dystrophic pathology, which might have been expected given that Dp71 was found expressed in muscle development.
      • Wertz K.
      • Füchtbauer E.M.
      Dmd(mdx-βgeo): a new allele for the mouse dystrophin gene.
      ,
      • Sarig R.
      • Mezger-Lallemand V.
      • Gitelman I.
      • Davis C.
      • Fuchs O.
      • Yaffe D.
      • Nudel U.
      Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development.
      The muscle pathology being similar to that in mdx mice was in agreement with the previous study in Cre-loxP mouse, in which the DMD gene was deleted.
      • Kudoh H.
      • Ikeda H.
      • Kakitani M.
      • Ueda A.
      • Hayasaka M.
      • Tomizuka K.
      • Hanaoka K.
      A new model mouse for Duchenne muscular dystrophy produced by 2.4 Mb deletion of dystrophin gene using Cre-loxP recombination system.
      Moreover, no increase in serum creatine kinase levels, indicative of sarcolemma permeability, suggested a different role for this short isoform. Interestingly, the most striking alteration in mdxβgeo was the ectopic calcification. Ectopic calcifications have been reported previously in mdx
      • Kikkawa N.
      • Ohno T.
      • Nagata Y.
      • Shiozuka M.
      • Kogure T.
      • Matsuda R.
      Ectopic calcification is caused by elevated levels of serum inorganic phosphate in mdx mice.
      ,
      • Geissinger H.D.
      • Prasada Rao P.V.V.
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      Calcified muscles have been linked to increased Pi levels, and serum Pi was found elevated in mdx mice.
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      In turn, calcium precipitate inhibition with pyrophosphate and bisphosphonate has already shown therapeutic promise in DMD.
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      However, it is not clear whether ectopic calcification is linked to the intracellular calcium accumulation, which resulted in the calcium hypothesis of DMD damage. Assuming that these events are connected, the exacerbated calcification in the dystrophin-null muscle suggests that the calcium influx via permeable sarcolemma solely due to the absence of Dp427 is an insufficient explanation.
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      Indeed, although elevated calcium levels in muscle fibers are sufficient to induce dystrophic-like changes,
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      Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy.
      this can occur independently of membrane instability.
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      Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism.
      Ectopic calcification was also found in mdxβgeo hearts but without obvious histologic deterioration compared with mdx. These data also agree with observations of cardiac histopathology not being significantly different between mdx and the Cre-loxP DMD-null mice.
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      Subcellular localization of dystrophin isoforms in cardiomyocytes and phenotypic analysis of dystrophin-deficient mice reveal cardiac myopathy is predominantly caused by a deficiency in full-length dystrophin.
      Interestingly, Dp71 in cardiomyocytes is located exclusively in the T-tubules.
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      Subcellular localization of dystrophin isoforms in cardiomyocytes and phenotypic analysis of dystrophin-deficient mice reveal cardiac myopathy is predominantly caused by a deficiency in full-length dystrophin.
      Given that most of the calcium enters the cell via T-tubules, absence of Dp71 could affect this function and contribute to ectopic calcification.
      Ectopic calcifications in dystrophic muscle appeared at 3 to 4 weeks in both mdx and mdxβgeo mice, increasing in number up until 8 to 12 weeks, beyond which calcified myofibers were replaced by fibrosis, which is one of the hallmarks of this disease. Thus, calcification follows the course of mdx pathology in limb muscles and in the diaphragm, one mdx muscle that most closely reproduces disease progression in humans. The timing of calcified fibers being replaced by fibrosis was also approximately week 12. Therefore, calcification seems to have the same temporal pattern of presentation and resolution in all dystrophic muscle, despite significant differences in intensity across different muscle groups (Figure 5).
      The cycles of degeneration and regeneration in mdx muscle are concomitant with immune cell infiltration. These immune cells are attracted by the danger-associated molecular patterns released from damaged muscle, and they play important roles in the pathology: they can contribute to damage but are also involved in clearing the cellular debris and releasing factors facilitating satellite cell activation and therefore promoting muscle regeneration. Moreover, in the chronic disease, inflammation is also linked to fibrosis.
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      and total ablation exacerbated the disease.
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      Given that the evolution of calcified muscle fibers mirrored the onset and cessation of inflammatory cell infiltrates in mdx muscle and the important role of macrophages, these cells were analyzed in relation to calcification. The distribution of macrophages was markedly different in mdx versus mdxβgeo muscles, with a close colocalization of F4/80 staining puncta with mineralized fibers in the latter. Moreover, the staining often appeared crescent shaped, around what looked like partially digested fibers (Figure 8B). In view that Pi-induced macrophages can evoke anticalcification actions, which are mediated by increased availability of extracellular ATP and pyrophosphate,
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      Dystrophin and utrophin “double knockout” dystrophic mice exhibit a spectrum of degenerative musculoskeletal abnormalities.
      the dystrophic muscle would offer ideal conditions for their activation. However, markers expressed on cells in mdxβgeo muscles suggested that these had predominantly the M1 phenotype, whereas the Pi-induced macrophages were shown to adopt a phenotype resembling the M2 subtype.
      • Isaac C.
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      Dystrophin and utrophin “double knockout” dystrophic mice exhibit a spectrum of degenerative musculoskeletal abnormalities.
      Of course, macrophages are known for their ability to change phenotype in response to environmental signals, so functional interplay between populations preventing calcification and eliminating calcified deposits is possible. Manipulating macrophage functions should provide further insight into their role in this process.
      Understanding these phenomena may also aid in identifying new therapeutic approaches. Furthermore, ectopic calcification is associated with pathologic outcomes in many human disorders apart from DMD, including osteoarthritis,
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      Articular cartilage calcification of the humeral head is highly prevalent and associated with osteoarthritis in the general population.
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      Macrophage subsets in atherosclerosis.
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      • van de Rijn M.
      • Debiec-Rychter M.
      Macrophage infiltration and genetic landscape of undifferentiated uterine sarcomas.
      renal disease,
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      • Chonchol M.
      Vascular calcification in end-stage renal disease.
      fibrodysplasia ossificans progressiva,
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      Activation of hedgehog signaling by loss of GNAS causes heterotopic ossification.
      and soft tissue impact trauma,
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      Post-traumatic ectopic calcification in the muscles of athletes: a review.
      where macrophage-specific roles are already established.
      Given these mouse model data and the correlation of severity of patients' cognitive impairment with the loss of shorter dystrophins both suggest a prominent functional role for these isoforms, comparison of muscle pathology in dystrophin-null patients against those with mutations affecting full-length dystrophins only is clearly warranted. Mouse with selective ablation of Dp71 is not dystrophic
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      • Davis C.
      • Fuchs O.
      • Yaffe D.
      • Nudel U.
      Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development.
      but presents with retinal channel abnormality,
      • Dalloz C.
      • Sarig R.
      • Fort P.
      • Yaffe D.
      • Bordais A.
      • Pannicke T.
      • Grosche J.
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      • Sahel J.
      • Nudel U.
      • Rendon A.
      Targeted inactivation of dystrophin gene product Dp71: phenotypic impact in mouse retina.
      early cataract formation,
      • Fort P.E.
      • Darche M.
      • Sahel J.A.
      • Rendon A.
      • Tadayoni R.
      Lack of dystrophin protein Dp71 results in progressive cataract formation due to loss of fiber cell organization.
      and vomeronasal nerve defasciculation.
      • Takatoh J.
      • Kudoh H.
      • Kondo S.
      • Hanaoka K.
      Loss of short dystrophin isoform Dp71 in olfactory ensheathing cells causes vomeronasal nerve defasciculation in mouse olfactory system.
      In contrast, transgenic overexpression of Dp71 resulted in more severe muscle disease.
      • Cox G.A.
      • Sunada Y.
      • Campbell K.P.
      • Chamberlain J.S.
      Dp71 can restore the dystrophin-associated glycoprotein complex in muscle but fails to prevent dystrophy.
      ,
      • Greenberg D.S.
      • Sunada Y.
      • Campbell K.P.
      • Yaffe D.
      • Nudel U.
      Exogenous Dp71 restores the levels of dystrophin associated proteins but does not alleviate muscle damage in mdx mice.
      Therefore, it may not be the absence of Dp71 but altered expression of dystrophin isoforms at a critical time point or/and at a specific location that causes the pathology.
      Understanding the mechanism of this abnormality may contribute to the development of more effective treatments not only for DMD but a range of diseases.

      Acknowledgments

      We thank Dr. Slawomir Pikula for advice on mineral deposit analysis, Gianluca Tozzi (Zeiss Global Centre, University of Portsmouth) for help with X-ray micro-computed tomography, and Scott Rodaway for help with in vivo experiments.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Mineralization of cardiomyocytes in mdxβgeo. Alizarin red staining confirms deposits seen during dissection of 8-week–old mdxβgeo hearts to stain positively for calcium (arrows). Original magnification, ×4.
      • Supplemental Figure S2

        Heat map of immune response and inflammatory genes most significantly differentially expressed between mdx and control muscles. Heat map showing genes involved in immune response and inflammation functions that show significant differential expression between mdx and wild-type (Wt) samples. Read counts were normalized to both the gene length and the library depth to generate fragment per kilobase mapped values. Normalized read counts were scaled such that each row has mean 0 and SD 1 to highlight changes in expression between Wt and mdx samples. Genes showing high expression are shown in red, whereas those showing low expression are shown in blue.

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