Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin a Responsible for Congenital Muscular Dystrophy in a Patient

cells Article Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient Alice Barateau 1,*, Nathalie Vadrot 1, Onnik Agbulut 2, Patrick Vicart 1, Sabrina Batonnet-Pichon 1 and Brigitte Buendia 1 1 Unité de Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France; [email protected] (N.V.); [email protected] (P.V.); [email protected] (S.B.-P.); [email protected] (B.B.) 2 Biological Adaptation and Ageing, UMR CNRS 8256, Institut de Biologie Paris-Seine (IBPS), UPMC Univ Paris 06, Sorbonne Universités, 75005 Paris, France; [email protected] * Correspondence: [email protected]; Tel.: +33-157-277-958 Academic Editor: Thomas Dechat Received: 9 January 2017; Accepted: 20 April 2017; Published: 24 April 2017 Abstract: Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A. Keywords: lamin A; congenital muscular dystrophy; muscle fiber type transition; myosin heavy chain IIA 1. Introduction A-type lamins (A and C) are nuclear intermediate filament proteins. In mammalian cells, these proteins play multiple roles due to their assembly into polymers beneath the nuclear envelope as well as in the nucleoplasm and their capacity to bind multiple partners and chromatin [1,2]. Thus, A-type lamins regulate the shape and mechanical properties of nuclei and expression of genes related to cell proliferation and differentiation processes. Mutations in LMNA, which encodes lamins A and C, cause several diseases that eventually affect cardiac and/or skeletal muscle tissues [3]. Some mouse models have been developed to address the impact of mutant lamin proteins on different organs in vivo [4]. We previously showed that adeno-associated virus (AAV) expressing ectopic proteins can be directly injected into mouse skeletal muscles [5], providing a useful approach for assessing protein impact in vivo. Cells 2017, 6, 10; doi:10.3390/cells6020010 www.mdpi.com/journal/cells Cells 2017, 6, 10 2 of 14 The fast-twitch muscle tibialis anterior (TA) is poor in type I fibers expressing myosin heavy chain 1 (MHC I), which have oxidative metabolism, but rich in type II fibers expressing MHC II isoforms [6]. Among the latter, one can distinguish type IIA fibers (MHC IIA) with an oxidative metabolism, also named intermediate fibers, and type IIX and IIB fibers (MHC IIX and IIB) with a glycolytic metabolism. Of note, in humans, the Myh4 gene is present but the protein MHC IIB is not expressed, In addition, some fibers can express different myosin heavy chain isoforms resulting in hybrid fibers with various properties in terms of ATP usage and muscle contraction speeds [7]. Muscle fibers with an oxidative metabolism are more resistant to fatigue than those with a glycolytic metabolism. Of note, the proportion of muscle fiber types varies depending on muscle specificity and activity [7]. For instance, physical training to increase endurance favors a shift towards more fatigue-resistant MHC I and MHC IIA type fibers. In addition, physiopathological contexts can induce fiber type disproportion (FTD). In particular, congenital myopathies triggered by some LMNA mutations (p.Glu33del, p.Lys123del, p.Ser303Pro, p.Arg453Trp, p.Thr528Lys) can induce a particular form of muscle FTD characterized by type II fiber hypertrophy [8]. Similar FTD is also observed in other LMNA related myopathies such as limb-girdle muscle dystrophy (LMNA mutation p.T27I) [9]. Another type of FTD associated with congenital myopathies caused by mutations in other genes, including ACTA1, MYH7, or TMP2,3, is specified by an abnormally high amount of slow oxidative type I fibers that are also atrophic [8–10]. We recently identified one particular lamin A mutant, LMNA p.R388P, that is responsible for congenital muscular dystrophy (L-CMD) [11]. Here, we used AAV injections to rapidly assess the impact of R388P mutant lamin A on mouse skeletal muscle homeostasis. Our analysis included morphological features of TA fibers; relative oxidative potential of muscle fibers, using quantification of succinate dehydrogenase (SDH) enzyme; and fiber type identity based on content of different MHC isoforms. 2. Materials and Methods 2.1. Plasmid Production Plasmids encoding FLAG-tagged human WT and mutant lamin A under regulation of the CMV promoter [11] were used by the Therapeutic Research Institute—Institut de Recherche en Santé de l’Université de Nantes (IRS-UN), Centre de Production de Vecteurs viraux (CPV), Institut National de la santé et de la recherche médicale (INSERM), Unité Mixte de Recherche (UMR) 1089 (Nantes, France)—to generate and purify AAV2/2.CMV.FLAG.LA WT or R388P. Final viral preparations were stored in Dulbecco’s Phosphate-Buffered Saline (DPBS) (with Ca2+ and Mg2+), aliquoted in small volumes (50 µL), and stored at −80 ◦C. Particle titer (number of viral genomes) was determined by quantitative PCR poly A. 2.2. Intramuscular Delivery of AAV Vectors All animal protocols were approved by the institutional ethics committee and conducted according to French and European laws, directives, and regulations on animal care (European Commission Directive 86/609/European Economic Community (EEC); project number #5441 at the Comité d'éthique en expérimentation animale Charles Darwin N◦5). The Centre d’expérimentation Fonctionnelle (CEF) animal facility at Hopital Salpétrière in Paris is fully licensed by the competent French authorities and has animal welfare insurance. For intramuscular injections, 10-week-old female C57BL/6J mice (Janvier Labs, Le Genest-Saint-Isle, France) were randomized into two groups: AAV-FLAG-LA-WT and AAV-FLAG-LA-R388P. Animals were anesthetized by intraperitoneal injection of a mix of drugs with sedative and analgesic effects [10 mg/kg xylazine (Bayer, Puteaux, France) and 100 mg/kg ketamine (Merial, Gerland, France)]. Insulin syringes were used to inject each TA muscle with 70 µL of AAV (~1.75 × 1011 vg/70 µL). In figure captions, n corresponds to the number of muscles analyzed in two or more independent experiments. Cells 2017, 6, 10 3 of 14 2.3. Removal and Fixation of TA Muscles TA muscles were removed 4 weeks after injection, following euthanasia of mice by cervical dislocation. To perform immunohistochemical analyses, muscles were mounted in tragacanth gum (Fisher Scientific, Hampton, NH, USA) and frozen following different protocols. A one-step fixation protocol consisted of plunging the muscle for at least 1 min in isopentane precooled in liquid nitrogen. Alternatively, a three-step paraformaldehyde (PFA)-sucrose-isopentane protocol consisted of fixing the muscle with 4% paraformaldehyde in PBS for 1 h, followed by immersing it in three successive baths of gradually increasing concentrations of sucrose dissolved in PBS (5% sucrose for 2 h, 10% sucrose for 3 h, and 25% sucrose overnight) at 4 ◦C. Tissues were finally fixed in isopentane as described above. In parallel, to prepare protein or RNA extracts, muscles were transferred to microcentrifuge tubes and snap-frozen in liquid nitrogen. 2.4. Analysis of TA Muscle Protein Extracts Tissue from half of a snap-frozen TA muscle was resuspended in 145 µL of Laemmli sample buffer (without reducing agent) before heating at 95 ◦C for 10 min. Samples were homogenized with a Polytron homogenizer (Kinematica, Lucerne, Switzerland) for 15 s and finally centrifuged at 900× g (3100 rpm) for 3 min at 4 ◦C. Protein concentration in the resulting supernatant was measured using the Bradford assay. Finally, Dithiothreitol (DTT) (1 mM final) was added to the supernatant before loading 15 µg of total extracted proteins per well onto 10% polyacrylamide gels. Transfer of proteins from the gel to nitrocellulose membranes, incubation with antibodies, and visualization with enhanced chemiluminescence were done as previously described [11]. 2.5. Histology of TA Muscle Sections Sections were cut to 6-µm thickness using a Leica Biosystems CM1950 cryostat (Buffalo Grove, IL, USA), recovered on Superfrost plus glass microscope slides (Thermo Fisher Scientific, Waltham, MA, USA), and stored at −80 ◦C. Muscle sections were incubated at room temperature for 20 min before staining with hematoxylin and eosin, SDH, or Sirius Red. Images were taken with a Leica DM IL inverted

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