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In Vivo Characterization of Mutant Myotilins

  • Etsuko Keduka
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
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  • Yukiko K. Hayashi
    Correspondence
    Address reprint requests to Yukiko K. Hayashi, M.D, Ph.D., Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashicho, Kodaira, Tokyo, 187-8502, Japan
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
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  • Sherine Shalaby
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
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  • Hiroaki Mitsuhashi
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan

    Division of Genetics, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts
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  • Satoru Noguchi
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
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  • Ikuya Nonaka
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
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  • Ichizo Nishino
    Affiliations
    Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
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Published:February 20, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.040
      Myofibrillar myopathy (MFM) is a group of disorders that are pathologically defined by the disorganization of the myofibrillar alignment associated with the intracellular accumulation of Z-disk–associated proteins. MFM is caused by mutations in genes encoding Z-disk–associated proteins, including myotilin. Although a number of MFM mutations have been identified, it has been difficult to elucidate the precise roles of the mutant proteins. Here, we present a useful method for the characterization of mutant proteins associated with MFM. Expression of mutant myotilins in mouse tibialis anterior muscle by in vivo electroporation recapitulated both the pathological changes and the biochemical characteristics observed in patients with myotilinopathy. In mutant myotilin-expressing muscle fibers, myotilin aggregates and is costained with polyubiquitin, and Z-disk–associated proteins and myofibrillar disorganization were commonly seen. In addition, the expressed S60C mutant myotilin protein displayed marked detergent insolubility in electroporated mouse muscle, similar to that observed in human MFM muscle with the same mutation. Thus, in vivo electroporation can be a useful method for evaluating the pathogenicity of mutations identified in MFM.
      Myofibrillar myopathy (MFM) is a group of neuromuscular diseases with common morphological features such as disorganized myofibrillar alignment and accumulation of Z-disk–associated proteins.
      • Selcen D.
      Myofibrillar myopathies.
      Mutations in genes encoding Z-disk–associated proteins are known to cause MFM. Disease-associated mutations have been identified in six genes, including myotilin, desmin, αB-crystallin, ZASP, filamin C, and BAG3.
      • Selcen D.
      • Engel A.G.
      Myofibrillar myopathy.
      • OlivÉ M.
      • Odgerel Z.
      • Martínez A.
      • Poza J.J.
      • Bragado F.G.
      • Zabalza R.J.
      • Jericó I.
      • Gonzalez-Mera L.
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      • Lee H.S.
      • Armstrong J.
      • MaravÍ E.
      • Arroyo M.R.
      • Pascual-Calvet J.
      • Navarro C.
      • Paradas C.
      • Huerta M.
      • Marquez F.
      • Rivas E.G.
      • Pou A.
      • Ferrer I.
      • Goldfarb L.G.
      Clinical and myopathological evaluation of early- and late-onset subtypes of myofibrillar myopathy.
      Elucidation of their pathogenicity, however, is sometimes difficult.
      Myotilin (myofibrillar protein with titin-like immunoglobulin domains) is a 57-kDa protein with 10 exons encoded by the myotilin gene (MYOT) on chromosome 5q31. Myotilin consists of a unique serine-rich domain at the N-terminus and two Ig-like domains at the C-terminus.
      • Salmikangas P.
      • Mykkänen O.M.
      • Grönholm M.
      • Heiska L.
      • Kere J.
      • Carpén O.
      Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.
      • Parast M.M.
      • Otey C.A.
      Characterization of palladin, a novel protein localized to stress fibers and cell adhesions.
      • Mykkänen O.M.
      • Grönholm M.
      • Rönty M.
      • Lalowski M.
      • Salmikangas P.
      • Suila H.
      • Carpén O.
      Characterization of human palladin, a microfilament-associated protein.
      • Bang M.L.
      • Mudry R.E.
      • McElhinny A.S.
      • Trombitas K.
      • Geach A.J.
      • Yamasaki R.
      • Sorimachi H.
      • Granzier H.
      • Gregorio C.C.
      • Labeit S.
      Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.
      Myotilin is highly expressed in skeletal and cardiac muscle, and localizes to the Z-disk,
      • Salmikangas P.
      • Mykkänen O.M.
      • Grönholm M.
      • Heiska L.
      • Kere J.
      • Carpén O.
      Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.
      which plays important roles in sarcomere assembly, actin filament stabilization, and muscle force transmission.
      • Faulkner G.
      • Lanfranchi G.
      • Valle G.
      Telethonin and other new proteins of the Z-disc of skeletal muscle.
      • Frank D.
      • Kuhn C.
      • Katus H.A.
      • Frey N.
      The sarcomeric Z-disc: a nodal point in signalling and disease.
      Myotilin interacts with several Z-disk–associated proteins, including α-actinin,
      • Salmikangas P.
      • Mykkänen O.M.
      • Grönholm M.
      • Heiska L.
      • Kere J.
      • Carpén O.
      Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.
      filamin C,
      • van der Ven P.F.
      • Wiesner S.
      • Salmikangas P.
      • Auerbach D.
      • Himmel M.
      • Kempa S.
      • Hayess K.
      • Pacholsky D.
      • Taivainen A.
      • Schröder R.
      • Carpén O.
      • Fürst D.O.
      Indications for a novel muscular dystrophy pathway gamma-Filamin, the muscle-specific filamin isoform, interacts with myotilin.
      • Gontier Y.
      • Taivainen A.
      • Fontao L.
      • Sonnenberg A.
      • van der Flier A.
      • Carpen O.
      • Faulkner G.
      • Borradori L.
      The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins.
      FATZ,
      • Gontier Y.
      • Taivainen A.
      • Fontao L.
      • Sonnenberg A.
      • van der Flier A.
      • Carpen O.
      • Faulkner G.
      • Borradori L.
      The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins.
      ZASP,
      • von Nandelstadh P.
      • Ismail M.
      • Gardin C.
      • Suila H.
      • Zara I.
      • Belgrano A.
      • Valle G.
      • Carpen O.
      • Faulkner G.
      A class III PDZ binding motif in the myotilin and FATZ families binds enigma family proteins: a common link for Z-disc myopathies.
      and MuRF ubiquitin ligase.
      • Witt S.H.
      • Granzier H.
      • Witt C.C.
      • Labeit S.
      MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination.
      Myotilin also interacts with actin monomers and filaments through its Ig-like domains, which also mediate homodimerization.
      • Salmikangas P.
      • van der Ven P.F.
      • Lalowski M.
      • Taivainen A.
      • Zhao F.
      • Suila H.
      • Schröder R.
      • Lappalainen P.
      • Fürst D.O.
      • Carpén O.
      Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly.
      Previous studies have shown that myotilin can bundle actin filaments in vitro, acting alone or in collaboration with α-actinin and filamin C.
      • Salmikangas P.
      • Mykkänen O.M.
      • Grönholm M.
      • Heiska L.
      • Kere J.
      • Carpén O.
      Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.
      • Salmikangas P.
      • van der Ven P.F.
      • Lalowski M.
      • Taivainen A.
      • Zhao F.
      • Suila H.
      • Schröder R.
      • Lappalainen P.
      • Fürst D.O.
      • Carpén O.
      Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly.
      • von Nandelstadh P.
      • Grönholm M.
      • Moza M.
      • Lamberg A.
      • Savilahti H.
      • Carpén O.
      Actin-organising properties of the muscular dystrophy protein myotilin.
      Thus, myotilin is thought to play a role in anchoring and stabilizing actin filaments at the Z-disk, and is involved in the organization and maintenance of Z-disk integrity.
      • von Nandelstadh P.
      • Ismail M.
      • Gardin C.
      • Suila H.
      • Zara I.
      • Belgrano A.
      • Valle G.
      • Carpen O.
      • Faulkner G.
      A class III PDZ binding motif in the myotilin and FATZ families binds enigma family proteins: a common link for Z-disc myopathies.
      Missense mutations in MYOT have been associated with MFM,
      • Selcen D.
      Myofibrillar myopathies.
      • Olivé M.
      • Goldfarb L.G.
      • Shatunov A.
      • Fischer D.
      • Ferrer I.
      Myotilinopathy: refining the clinical and myopathological phenotype.
      • Foroud T.
      • Pankratz N.
      • Batchman A.P.
      • Pauciulo M.W.
      • Vidal R.
      • Miravalle L.
      • Goebel H.H.
      • Cushman L.J.
      • Azzarelli B.
      • Horak H.
      • Farlow M.
      • Nichols W.C.
      A mutation in myotilin causes spheroid body myopathy.
      limb girdle muscular dystrophy type 1A,
      • Olivé M.
      • Goldfarb L.G.
      • Shatunov A.
      • Fischer D.
      • Ferrer I.
      Myotilinopathy: refining the clinical and myopathological phenotype.
      • Hauser M.A.
      • Conde C.B.
      • Kowaljow V.
      • Zeppa G.
      • Taratuto A.L.
      • Torian U.M.
      • Vance J.
      • Pericak-Vance M.A.
      • Speer M.C.
      • Rosa A.L.
      Myotilin mutation found in second pedigree with LGMD1A.
      • Hauser M.A.
      • Horrigan S.K.
      • Salmikangas P.
      • Torian U.M.
      • Viles K.D.
      • Dancel R.
      • Tim R.W.
      • Taivainen A.
      • Bartoloni L.
      • Gilchrist J.M.
      • Stajich J.M.
      • Gaskell P.C.
      • Gilbert J.R.
      • Vance J.M.
      • Pericak-Vance M.A.
      • Carpen O.
      • Westbrook C.A.
      • Speer M.C.
      Myotilin is mutated in limb girdle muscular dystrophy 1A.
      and distal myopathy.
      • Penisson-Besnier I.
      • Talvinen K.
      • Dumez C.
      • Vihola A.
      • Dubas F.
      • Fardeau M.
      • Hackman P.
      • Carpen O.
      • Udd B.
      Myotilinopathy in a family with late onset myopathy.
      • Berciano J.
      • Gallardo E.
      • Dominguez-Perles R.
      • Garcia A.
      • Garcia-Barredo R.
      • Combarros O.
      • Infante J.
      • Illa I.
      Autosomal-dominant distal myopathy with a myotilin S55F mutation: sorting out the phenotype.
      We have previously identified a mutation p.Arg405Lys (R405K) in exon 9 in the second Ig-like domain of myotilin. The R405K mutant myotilin exhibited defective homodimerization and decreased interaction with α-actinin in a yeast 2-hybrid (Y2H) system.
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      All of the other previously reported MYOT mutations are located in exon 2
      • Salmikangas P.
      • van der Ven P.F.
      • Lalowski M.
      • Taivainen A.
      • Zhao F.
      • Suila H.
      • Schröder R.
      • Lappalainen P.
      • Fürst D.O.
      • Carpén O.
      Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly.
      • Selcen D.
      Myofibrillar myopathies.
      • Olivé M.
      • Goldfarb L.G.
      • Shatunov A.
      • Fischer D.
      • Ferrer I.
      Myotilinopathy: refining the clinical and myopathological phenotype.
      • Foroud T.
      • Pankratz N.
      • Batchman A.P.
      • Pauciulo M.W.
      • Vidal R.
      • Miravalle L.
      • Goebel H.H.
      • Cushman L.J.
      • Azzarelli B.
      • Horak H.
      • Farlow M.
      • Nichols W.C.
      A mutation in myotilin causes spheroid body myopathy.
      • Reilich P.
      • Krause S.
      • Schramm N.
      • Klutzny U.
      • Bulst S.
      • Zehetmayer B.
      • Schneiderat P.
      • Walter M.C.
      • Schoser B.
      • Lochmüller H.
      A novel mutation in the myotilin gene (MYOT) causes a severe form of limb girdle muscular dystrophy 1A (LGMD1A).
      , with p.Ser60Cys (S60C) being one of the most common mutations. The pathogenic effects of MYOT mutations and the disease mechanism involved remain poorly understood.
      Model animals, such as transgenic mice, have contributed to understanding of the critical pathogenic events in MFM.
      • Mavroidis M.
      • Panagopoulou P.
      • Kostavasili I.
      • Weisleder N.
      • Capetanaki Y.
      A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization.
      • Wang X.
      • Osinska H.
      • Klevitsky R.
      • Gerdes A.M.
      • Nieman M.
      • Lorenz J.
      • Hewett T.
      • Robbins J.
      Expression of R120G-alphaB-crystallin causes aberrant desmin and alphaB-crystallin aggregation and cardiomyopathy in mice.
      • Wang X.
      • Osinska H.
      • Dorn 2nd, G.W.
      • Nieman M.
      • Lorenz J.N.
      • Gerdes A.M.
      • Witt S.
      • Kimball T.
      • Gulick J.
      • Robbins J.
      Mouse model of desmin-related cardiomyopathy.
      Some MFMs, including myotilinopathies, are late-onset and slowly progressive diseases.
      • Selcen D.
      Myofibrillar myopathies.
      • OlivÉ M.
      • Odgerel Z.
      • Martínez A.
      • Poza J.J.
      • Bragado F.G.
      • Zabalza R.J.
      • Jericó I.
      • Gonzalez-Mera L.
      • Shatunov A.
      • Lee H.S.
      • Armstrong J.
      • MaravÍ E.
      • Arroyo M.R.
      • Pascual-Calvet J.
      • Navarro C.
      • Paradas C.
      • Huerta M.
      • Marquez F.
      • Rivas E.G.
      • Pou A.
      • Ferrer I.
      • Goldfarb L.G.
      Clinical and myopathological evaluation of early- and late-onset subtypes of myofibrillar myopathy.
      To reproduce clinical and pathological features in model animals for such late-onset mild myopathy is both labor intensive and time consuming. Among the 10 missense mutations identified to date in patients with myotilinopathy,
      • Salmikangas P.
      • van der Ven P.F.
      • Lalowski M.
      • Taivainen A.
      • Zhao F.
      • Suila H.
      • Schröder R.
      • Lappalainen P.
      • Fürst D.O.
      • Carpén O.
      Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly.
      • Selcen D.
      Myofibrillar myopathies.
      • Olivé M.
      • Goldfarb L.G.
      • Shatunov A.
      • Fischer D.
      • Ferrer I.
      Myotilinopathy: refining the clinical and myopathological phenotype.
      • Foroud T.
      • Pankratz N.
      • Batchman A.P.
      • Pauciulo M.W.
      • Vidal R.
      • Miravalle L.
      • Goebel H.H.
      • Cushman L.J.
      • Azzarelli B.
      • Horak H.
      • Farlow M.
      • Nichols W.C.
      A mutation in myotilin causes spheroid body myopathy.
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      • Reilich P.
      • Krause S.
      • Schramm N.
      • Klutzny U.
      • Bulst S.
      • Zehetmayer B.
      • Schneiderat P.
      • Walter M.C.
      • Schoser B.
      • Lochmüller H.
      A novel mutation in the myotilin gene (MYOT) causes a severe form of limb girdle muscular dystrophy 1A (LGMD1A).
      only the Thr57Ile (T57I) mutation reproduces the pathological changes in transgenic mice after 12 months of age.
      • Garvey S.M.
      • Miller S.E.
      • Claflin D.R.
      • Faulkner J.A.
      • Hauser M.A.
      Transgenic mice expressing the myotilin T57I mutation unite the pathology associated with LGMD1A and MFM.
      To screen for candidate mutations in MFM, a new method is required for demonstrating the pathogenicity of mutations. In the present study, we expressed mutant myotilin in mouse muscle by in vivo electroporation and were able to easily reproduce pathological changes similar to those observed in skeletal muscle from patients with MYOT mutations.

      Materials and Methods

      Clinical Materials

      All clinical materials used in this study were obtained for diagnostic purposes with written informed consent. The studies were approved by the Ethical Committee of the National Center of Neurology and Psychiatry.

      Genetic Analysis

      Genomic DNA was isolated from peripheral lymphocytes or muscle specimens of patients, using standard techniques. Sequencing and mutation analysis of MYOT were performed as described previously.
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.

      Plasmid Construction

      We cloned full-length human myotilin cDNA and generated mutant myotilin (mMYOT) by site-directed mutagenesis, as described previously.
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      A C→G substitution at nucleotide position 179 and a G→A substitution at nucleotide 1214 were introduced to obtain p.S60C and p.R405K, respectively. A schematic of the location of these mutations in the structure of the myotilin protein is given in Figure 1A. For expression in mammalian cells, cDNAs of wild-type myotilin (wtMYOT) or mMYOT (S60C or R405K) were subcloned into pCMV-Myc vector (Takara Bio, Shiga, Japan). All constructs were verified by sequencing. Primer sequences are available on request.
      Figure thumbnail gr1
      Figure 1Myotilin mutations and histopathological findings in myotilinopathy patients. A: Myotilin structure and disease-related mutations. p.Ser60Cys (S60C) is located in the serine-rich domain and p.Arg405Lys (R405K) is located in the second immunoglobulin (Ig)-like domain of myotilin. B–I: Pathological changes in muscles from patient 1 with MYOT S60C (B, D, F, and H) and from patient 2 with MYOT R405K (C, E, G, and I). B: Modified Gömöri trichrome (mGT) staining of biopsied skeletal muscle from patient 1 revealed markedly degenerated fibers with many spheroid protein inclusions (arrows). Some fibers had rimmed vacuoles (arrowhead). C: mGT staining of biopsied skeletal muscle from patient 2 revealed scattered fibers with rimmed vacuoles (arrowhead). D: NADH tetrazolium reductase (NADH-TR) staining of the serial section shown in B revealed markedly disorganized intermyofibrillar networks (arrows). E: NADH-TR staining of the serial section shown in C revealed disorganized intermyofibrillar networks (arrow). F–I: Coimmunostaining of muscles from patients using anti-myotilin (green) and anti-polyubiquitin (red) antibodies. F: Large accumulations of myotilin were observed in many fibers in patient 1. G: Small accumulations of myotilin were seen in some fibers in patient 2. Myotilin aggregates were positive for polyubiquitin in both patient 1 (H) and patient 2 (I). Scale bars: 50 μm (B–E); 20 μm (F–I).

      Cell Culture, Transfection, and Immunocytochemical Analysis

      C2C12 murine myoblast cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% carbon dioxide. The cells were transiently transfected using FuGENE HD transfection reagent (Roche Diagnostics, Indianapolis, IN), according to the manufacturer's instructions. Forty-eight hours after transfection, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100, and costained with anti-Myc antibody (Sigma-Aldrich) and rhodamine-labeled phalloidin (Wako Pure Chemical Industries, Osaka, Japan) to detect transfected myotilin and actin filaments, respectively, according to standard protocol.
      • Hayashi Y.K.
      • Matsuda C.
      • Ogawa M.
      • Goto K.
      • Tominaga K.
      • Mitsuhashi S.
      • Park Y.E.
      • Nonaka I.
      • Hino-Fukuyo N.
      • Haginoya K.
      • Sugano H.
      • Nishino I.
      Human PTRF mutations cause secondary deficiency of caveolins resulting in muscular dystrophy with generalized lipodystrophy.

      In Vivo Electroporation

      ICR mice were purchased from CLEA Japan (Fuji, Shizuoka, Japan). Animals were handled in accordance with the guidelines established by the Ethical Review Committee on the Care and Use of Rodents in the National Institute of Neuroscience, National Center of Neurology and Psychiatry. All mouse experiments were approved by the Committee. Five-week-old male ICR mice were anesthetized with diethyl ether, and the tibialis anterior (TA) muscles of mice were injected with 80 μg of purified Myc-tagged myotilin plasmid DNA. wtMYOT was injected to one side of TA muscle and mMYOT (S60C or R405K) was injected to the other side of TA muscle. In vivo transfection was performed using a square-wave electroporator (CUY-21SC; Nepa Gene, Ichikawa, Japan). A pair of electrode needles was inserted into the muscle to a depth of 3 mm to encompass the DNA injection sites. Each injected site was administered with three consecutive 50 ms-long pulses at the required voltage (50 to 90 V) to yield a current of 150 mA. After a 1-second interval, three consecutive pulses of the opposite polarity were administered. At 7 or 14 days after electroporation, mice were sacrificed by cervical dislocation, and TA muscles were isolated.

      Histochemical and Immunohistochemical Analyses

      Biopsied human muscles or electroporated mouse TA muscles were frozen in isopentane cooled in liquid nitrogen. Serial 10-μm cryosections were stained with modified Gömöri trichrome (mGT) and NADH-tetrazolium reductase (NADH-TR) and were subjected to a battery of histochemical methods. Immunohistochemistry was performed on serial 6-μm cryosections, as described previously.
      • Hayashi Y.K.
      • Matsuda C.
      • Ogawa M.
      • Goto K.
      • Tominaga K.
      • Mitsuhashi S.
      • Park Y.E.
      • Nonaka I.
      • Hino-Fukuyo N.
      • Haginoya K.
      • Sugano H.
      • Nishino I.
      Human PTRF mutations cause secondary deficiency of caveolins resulting in muscular dystrophy with generalized lipodystrophy.

      Antibodies

      The primary antibodies used in this study were as follows: actin (Kantoukagaku, Tokyo, Japan), α-actinin (Sigma-Aldrich), BAG3 (Abcam, Tokyo, Japan), αB-crystallin (StressGen Biotechnologies, Victoria, BC, Canada), desmin (PROGEN Biotechnik, Heidelberg, Germany), filamin C (kindly provided by A.H. Beggs),
      • Thompson T.G.
      • Chan Y.M.
      • Hack A.A.
      • Brosius M.
      • Rajala M.
      • Lidov H.G.
      • McNally E.M.
      • Watkins S.
      • Kunkel L.M.
      Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.
      c-Myc (Sigma-Aldrich), c-Myc (PROGEN Biotechnik), myotilin (Proteintech Group, Chicago, IL), polyubiquitinated protein (Biomol International-Enzo Life Sciences, Plymouth Meeting, PA), GAPDH (Advanced ImmunoChemical, Long Beach, CA), and horseradish peroxidase-labeled anti-c-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

      Evaluation of Aggregates

      Histochemical and immunohistochemical analyses were performed on cryosections of electroporated muscles sectioned at 500-μm intervals. The section containing the highest number of Myc-positive fibers (>100 fibers) was used. Myc-positive granules >1 μm in diameter were defined as aggregates. The Myc-positive fibers containing Myc-positive aggregates were counted among all Myc-positive fibers. Five mice each from the wtMYOT-, mMYOT S60C-, and mMYOT R405K-expressing groups were examined. To compare the number and size of Myc-positive aggregates per fiber, we measured the number and area of Myc-positive aggregates in 30 myofibers from each specimen using ImageJ software version 1.43 (NIH, Bethesda, MD). The results are presented as bar graphs (±SD) and histograms. Fifteen serial sections were immunoblotted to measure the amounts of electroporated Myc-tagged myotilin protein.

      Electron Microscopy

      For electron microscopy, cryosections (25 μm thick) of biopsied muscle with the S60C mutation (patient 1) were fixed with 2% glutaraldehyde in 100 mmol/L cacodylate buffer for 15 minutes on ice. After a shaking with a mixture of 4% osmium tetroxide, 1.5% lanthanum nitrate, and 200 mmol/L s-collidine for 1 to 2 hours, samples were embedded in epoxy resin. TA muscles of 5-week-old ICR mice were coelectroporated with pEGFP-C1 plasmid (Clontech, Tokyo, Japan), which encodes enhanced green fluorescent protein (EGFP), and with either Myc-wtMYOT or Myc-mMYOT (S60C or R405K) plasmid (40 μg each). As a control, pEGFP-C1 plasmid was electroporated alone. TA muscles were isolated 7 and 14 days after electroporation. EGFP-positive regions were trimmed under a fluorescence microscope and fixed with 2% glutaraldehyde in 100 mmol/L cacodylate buffer for 3 hours. After a shaking with a mixture of 4% osmium tetroxide, 1.5% lanthanum nitrate, and 200 mmol/L s-collidine for 2 to 3 hours, samples were embedded in epoxy resin. Semithin sections (1 μm thick) were stained with Toluidine Blue. Ultrathin sections (100 nm thick) were stained with uranyl acetate and lead citrate, and were analyzed at 120 kV using a Tecnai Spirit transmission electron microscope (FEI, Hillsboro, OR).

      Solubility and Immunoblot Assay

      To examine solubility of mutant myotilin, we used frozen biopsied muscles from human control subjects and from the two myotilinopathy patients, as well as TA muscles of six mice each from the wtMYOT-, mMYOT S60C-, and mMYOT R405K-expressing groups, at 14 days after electroporation. The 1.25-mm3 specimens of muscle were lysed and homogenized in 150 μL of radioimmunoprecipitation assay buffer containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and Roche complete protease inhibitor cocktail (Roche Diagnostics). The lysates were incubated at 4°C for 20 minutes with gentle rotation, and then centrifuged at 15,000 × g at 4°C for 20 minutes. The supernatants and precipitates were collected, and the protein concentrations of the supernatants were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Immunoblotting of the supernatant (detergent-soluble) and precipitate (detergent-insoluble) fractions was performed, as described previously.
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Immunoreactive complexes on the membranes were detected using enhanced chemiluminescence ECL Plus detection reagent (GE Healthcare, Chalfont St Giles, UK). Insolubility index was calculated as the ratio of the quantity of insoluble protein to the total quantity of proteins (the sum of soluble and insoluble proteins).

      Immunoprecipitation

      The 5-mm3 specimens of frozen electroporated mouse muscles isolated at 14 days after electroporation were lysed and homogenized in 0.6 mL of radioimmunoprecipitation assay buffer. The lysates were incubated at 4°C for 20 minutes with gentle rotation, and then centrifuged at 15,000 × g at 4°C for 20 minutes. The supernatants were collected, and their protein concentrations were adjusted using a protein assay kit (Bio-Rad Laboratories). Immunoprecipitation was performed as described previously,
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      with agarose-conjugated anti-Myc antibody (Santa Cruz Biotechnology).

      Statistical Analysis

      Differences between wtMYOT-, mMYOT S60C-, and mMYOT R405K-expressing mice were analyzed with GraphPad Prism version 5 (GraphPad Software, La Jolla, CA). Comparisons among groups were performed by one-way analysis of variance with post hoc Tukey's analysis. Data are expressed as means ± SD.

      Results

      Mutation Screening and Histochemical Analyses of Muscles from Patients

      We performed MYOT mutation screening in MFM patients and identified two patients with mutations. Patient 1, harboring a MYOT c.179C→G (p.S60C) mutation in exon 2, was a 63-year-old woman with a 6-year-long history of slowly progressive limb muscle weakness. Her mother (deceased) had had muscle weakness. The patient had difficulty in climbing stairs without support, and could not walk for long distances. Her serum creatine kinase level was elevated to 734 IU/L (reference, <200 IU/L). A biopsied specimen from the rectus femoris muscle showed marked variation in fiber size, with some necrotic fibers. Clusters of degenerated fibers with abnormal cytoplasmic inclusions were observed; some fibers with rimmed vacuoles were also seen (Figure 1B). Intermyofibrillar networks were markedly disorganized (Figure 1D). Under electron microscopy, electron-dense materials and cytoplasmic amorphous inclusions of various sizes were seen in some fibers (see Supplemental Figure S1 at http://ajp.amjpathol.org). Patient 2 was a 57-year-old woman harboring a MYOT c.1214G→A (p.R405K) mutation in exon 9. Detailed clinical symptoms have been described previously.
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      In brief, this patient had a 16-year-long history of slowly progressive proximal limb muscle weakness. Her serum creatine kinase level was mildly elevated (385 IU/I). A specimen from the vastus lateralis muscle showed marked variation in fiber size, scattered fibers with internal nuclei, and small angular fibers. Some fibers with rimmed vacuoles were seen (Figure 1C), and intermyofibrillar networks were disorganized (Figure 1E). Immunohistochemical analysis of muscle specimens from both patients revealed scattered fibers with strong immunoreactive accumulations of myotilin (Figure 1, F and G), which costained with polyubiquitin (Figure 1, H and I), α-B crystallin, BAG3, actin, desmin, and filamin C (see Supplemental Figure S2 at http://ajp.amjpathol.org).

      Mutant Myotilin Does Not Aggregate in Cultured Cells

      To examine the aggregation of mutant myotilins in cultured cells, C2C12 murine myoblasts were transfected with Myc-tagged wtMYOT (Myc-wtMYOT) or Myc-tagged mMYOT (Myc-mMYOT S60C or R405K). After 48 hours, immunostaining with anti-Myc antibody and rhodamine-labeled phalloidin revealed that the expressed Myc-wtMYOT, Myc-mMYOT S60C, and Myc-mMYOT R405K did not form abnormal protein aggregations, and they localized at actin stress fibers (Figure 2). Expression of mMYOT did not affect differentiation of C2C12 cells (data not shown).
      Figure thumbnail gr2
      Figure 2Expression of mutant myotilin in cultured cells. Immunofluorescence staining of transfected Myc-wtMYOT (A), Myc-mMYOT S60C (B), and Myc-mMYOT R405K (C) in C2C12 murine myoblasts. Merged images of Myc-tagged myotilin-expressing cells (green) costained for actin stress fibers (red), and nuclear staining with DAPI (blue). C2C12 myoblasts expressing mMYOT S60C (B) or R405K (C) did not exhibit protein aggregates, and the mutant myotilin colocalized with actin stress fibers similar to wtMYOT (A). Scale bar = 20 μm.

      Accumulation of Myotilin after Electroporation

      To investigate the roles of mutant myotilin, we performed in vivo electroporation to express Myc-wtMYOT or Myc-mMYOT (S60C or R405K) in mouse TA muscles. At 7 and 14 days after electroporation, Myc-positive granules with diameters >1 μm were observed in Myc-tagged myotilin-expressing myofibers (Figure 3A). Compared with wtMYOT-expressing myofibers, mMYOT-expressing myofibers contained more granular aggregates that were larger in size. At 7 days after electroporation, Myc-positive aggregates of wtMYOT, mMYOT S60C, and mMYOT R405K were observed in 14 ± 5%, 44 ± 7%, and 21 ± 4% of muscle fibers, respectively (Figure 3B). At 14 days after electroporation, the number of the fibers with aggregates increased to 22 ± 4% in wtMYOT, 50 ± 2% in mMYOT S60C, and 37 ± 3% in mMYOT R405K (Figure 3C). The number and size of Myc-positive aggregates in 30 randomly selected Myc-positive muscle fibers were much higher in mMYOT S60C and slightly higher in mMYOT R405K at 14 days after electroporation than at 7 days (see Supplemental Figure S3 at http://ajp.amjpathol.org). These data indicate that the expressed mutant myotilins, and mMYOT S60C in particular, are prone to aggregate in skeletal muscles. The amounts of expressed Myc-tagged myotilin proteins were approximately equal, as measured by immunoblotting (Figure 3D).
      Figure thumbnail gr3
      Figure 3Enhanced aggregation of mutant myotilins in mouse skeletal muscle. A: Immunohistochemical staining of Myc-wtMYOT (WT)-electroporated or Myc-mMYOT (S60C or R405K)-electroporated mouse TA muscles. At 7 and 14 days after electroporation, S60C and R405K formed many Myc-positive granular aggregates (arrows) in myofibers, compared with WT. More prominent protein aggregates were observed in the S60C-electroporated muscle. At 14 days after electroporation, S60C-expressing myofibers exhibited larger aggregates. Scale bars: 20 µm. B and C: The percentage of myofibers with Myc-positive aggregates in the electroporated fibers of the WT, S60C, and R405K expression groups (n = 5 mice per group). *P < 0.05; ***P < 0.001. D: Immunoblotting analysis of transfected Myc-tagged myotilin in 15 serial sections taken after the sections used for immunohistochemistry. GAPDH was used as a loading control.

      Myofibril Disorganization and Z-Disk Streaming in Muscles Expressing Mutant Myotilins

      To investigate the ultrastructural characteristics of mutant myotilin-electroporated muscles, we performed electron microscopy at 7 and 14 days after electroporation. In Toluidine Blue-stained longitudinal semithin sections, partial disorganization of the Z-disk was observed in both mMYOT S60C-expressing and mMYOT R405K-expressing TA muscles, but not in control or wtMYOT electroporated muscles (data not shown). Electron microscopy also revealed myofibril disorganization with disrupted Z-disk, such as Z-disk streaming and broadening, in mMYOT-expressing muscles (Figure 4, A and D). Variable-sized (1 to 8 μm in diameter) electron-dense material, with electron densities similar to that of the Z-disk, were also seen in mMYOT-expressing mouse muscles (Figure 4, B and E). The inclusions were occasionally associated with autophagic vacuoles (Figure 4, C and F). These ultrastructural findings were commonly observed in both mMYOT S60C- and mMYOT R405K-expressing mouse muscles.
      Figure thumbnail gr4
      Figure 4Electron microscopy of muscles expressing mutant myotilin. mMYOT S60C (A–C); mMYOT R405K (D–F). A and D: mMYOT-transfected muscle fibers exhibited myofibril disorganization with disrupted Z-disk; note broadening of Z-disks (A, brackets) and Z-disk streaming (D, asterisk). B and E: Variable-sized (1 to 8 μm in diameter) electron-dense inclusions (arrowheads) were seen in mMYOT-expressing muscles. C and F: Inclusions were occasionally associated with autophagic vacuoles (AV). B and C: Seven days after electroporation. A and D–F: Fourteen days after electroporation. Scale bars: 3.0 μm (B and E); 2.0 μm (C); 1.7 μm (A and D); 1.4 μm (F).

      Mutant Myotilin Aggregates Colocalize with Polyubiquitin and Other Z-Disk–Associated Proteins

      To compare the protein accumulations in human and mouse muscles, we performed immunohistochemical analysis. At 14 days after electroporation, some cytoplasmic inclusions were observed in mGT-stained sections of mMYOT-expressing muscles (Figure 5, A and B). Immunostaining of serial sections revealed that the inclusions were immunopositive for the Myc tag (Figure 5, A and B). The aggregates of Myc-mMYOT (S60C and R405K) strongly colocalized with polyubiquitin and αB-crystallin. Accumulations of other Z-disk–associated proteins were also observed, including BAG3, actin, desmin, and filamin C (Figure 5). These findings are similar to the observations made in the patients' muscles (Figure 1, F–I; see also Supplemental Figure S2 at http://ajp.amjpathol.org). In the electroporated muscles, Myc-wtMYOT aggregates also colocalized with Z-disk–associated proteins, including αB-crystallin, BAG3, actin, desmin, and filamin C (data not shown), whereas only few wtMYOT aggregates were immunopositive for polyubiquitin (Figure 6A).
      Figure thumbnail gr5
      Figure 5Mutant myotilin aggregates colocalize with polyubiquitin and other Z-disk–associated proteins in electroporated mouse muscle. mGT and immunohistochemical staining of mouse muscle expressing Myc-mMYOT S60C (A) or mMYOT R405K (B) at 14 days after electroporation. On mGT-stained sections of mMYOT-expressing muscles, cytoplasmic inclusions (arrows) were seen. The inclusions were immunopositive for the Myc tag in serial sections. The Myc-positive aggregates of S60C and R405K strongly colocalized with polyubiquitin (poly-Ub) and αB-crystallin (αBC). The aggregates were also immunopositive for BAG3, actin, desmin, and filamin C. Scale bars: 20 μm (A and B).
      Figure thumbnail gr6
      Figure 6Mutant myotilin displays marked detergent insolubility, along with polyubiquitinated proteins. A: At 14 days after electroporation of Myc-wtMYOT (WT) or Myc-mMYOT (S60C or R405K), Myc-mMYOT aggregates, particularly those of S60C, colocalized with polyubiquitin (polyUb) (arrows). The WT aggregates rarely costained with polyubiquitin. B–E Solubilities of myotilin, polyubiquitinated proteins, and other sarcomeric proteins in muscles from myotilinopathy patients (B and D) and from electroporated mice (C and E). GAPDH was used as a loading control. B: Immunoblotting of detergent-soluble and detergent-insoluble fractions of muscles from control subjects (C1 and C2) or myotilinopathy patients [P1 (patient 1) and P2 (patient 2)]. In the muscles from P1 with S60C, markedly increasing amounts of myotilin, polyubiquitinated proteins, and αB-crystallin were detected in the insoluble fraction, compared with muscles from control subjects. D: Quantification of myotilin insolubilities revealed highest insolubility in P1. C: Immunoblotting of detergent-soluble and detergent-insoluble fractions of WT, S60C, or R405K-expressing muscles at 14 days after electroporation. Increasing amounts of insoluble Myc-tagged myotilin proteins and polyubiquitinated proteins were observed in mMYOT-electroporated muscles, compared with WT. Particularly in S60C-electroporated muscles, the amounts of insoluble proteins were notably increased. E: Quantification of the insolubilities of electroporated Myc-tagged myotilin in the WT, S60C, and R405K expression groups (n = 6 mice per group). Insolubility of endogenous myotilin was measured using PBS-treated mouse muscles. Compared with WT, insolubilities of electroporated Myc-tagged myotilin were significantly increased in S60C and R405K. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar = 20 μm.

      Mutant Myotilin Proteins Display Marked Detergent Insolubility with Polyubiquitinated Proteins

      In the muscle specimens of the two myotilinopathy patients, myotilin aggregates exhibited positive staining for polyubiquitin (Figure 1; see also Supplemental Figure S3 at http://ajp.amjpathol.org). Similarly, in electroporated mouse muscles, mMYOT aggregates were positive for polyubiquitin, and polyubiquitin-positive aggregates were more prominently observed in mMYOT S60C-expressing muscles at 14 days after electroporation. On the other hand, only few aggregates of Myc-wtMYOT were positive for polyubiquitin (Figure 6A). This result suggests that mutant myotilin was ubiquitinated or that the expressed mutant myotilin induced the deposition of polyubiquitinated proteins in the muscles of patients and electroporated mice. To characterize these aggregates, we performed a solubility assay. The muscle specimen with the S60C mutation (patient 1) exhibited increased amounts of myotilin in the detergent-insoluble fraction, compared with the control specimens (Figure 6, B and D). Increasing amounts of polyubiquitinated proteins and αB-crystallin were also detected in the insoluble fraction. On the other hand, the solubilities of myotilin and other proteins, including polyubiquitin, in the muscle specimen with the R405K mutation (patient 2) were similar to those of controls (Figure 6B). Consistently, in the mouse muscles isolated at 14 days after electroporation, markedly increasing amounts of insoluble mMYOT S60C were observed (Figure 6C). In the PBS-injected control muscle, insolubility of endogenous myotilin was 31 ± 12%, whereas in the wtMYOT-, mMYOT S60C-, and mMYOT R405K-injected muscles, the Myc-tagged myotilin amounts in the insoluble fraction were 34 ± 10%, 69 ± 5%, and 48 ± 9%, respectively (Figure 6E). Insolubility of Myc-wtMYOT was similar to that of endogenous myotilin, but mMYOT, and S60C in particular, exhibited higher insolubility (Figure 6E).
      These results are consistent with the number of intracellular aggregates observed after electroporation. The amount of polyubiquitinated proteins was markedly increased in the insoluble fraction of mMYOT S60C-electroporated muscles, similar to that of the muscle with the S60C mutation (patient 1) (Figure 6, B and C). A slight increase in the amount of detergent-insoluble polyubiquitinated proteins was observed in mMYOT R405K-electroporated muscles (Figure 6C). The amounts of other Z-disk–associated proteins, including αB-crystallin, in the insoluble fraction did not exhibit an increase, even in mMYOT S60C-electroporated muscles (Figure 6C; see also Supplemental Figure S4, A and B, at http://ajp.amjpathol.org). We also performed an immunoprecipitation assay to examine whether myotilin was polyubiquitinated. Myc-tagged myotilin proteins were immunoprecipitated from the detergent-soluble fraction of the mouse muscles isolated at 14 days after electroporation. Polyubiquitin immunoreactivity was not detected in the immunoprecipitated proteins (see Supplemental Figure S4C at http://ajp.amjpathol.org), indicating that neither the wtMYOT nor the mMYOT proteins in the soluble fraction were polyubiquitinated.

      Discussion

      Patients with MFM, including myotilinopathy, exhibit variable clinical features. Some patients exhibit progressive weakness in proximal muscles, whereas others exhibit distal dominant muscle involvement. Cardiomyopathy, peripheral neuropathy, and respiratory insufficiency may be observed.
      • Selcen D.
      • Engel A.G.
      Myofibrillar myopathy.
      The diagnosis of MFM is generically based on characteristic pathological findings in biopsied muscles, namely, myofibrillar degradation and protein aggregation.
      • Selcen D.
      Myofibrillar myopathies.
      Histochemically, the most remarkable pathological changes were observed with mGT staining (Figure 1). Abnormal protein aggregates were observed, including amorphous, granular, or hyaline deposits of various sizes, shapes, and colors (dark blue, blue red, or dark green). The presence of rimmed and nonrimmed vacuoles was also a characteristic observation. Furthermore, NADH-TR staining revealed intermyofibrillar network disorganization. Attenuation or absence of NADH-TR activity in focal areas of myofibers is also observed in MFM.
      • Selcen D.
      Myofibrillar myopathies.
      • Schröder R.
      • Schoser B.
      Myofibrillar myopathies: a clinical and myopathological guide.
      Here, we have presented findings for myotilinopathy patients with similar clinical features but different pathological changes. Fibers with cytoplasmic inclusions and disorganized myofibrils were prominent in the patient with S60C mutation, and these inclusions were strongly immunoreactive for myotilin (Figure 1).
      Although transfected cultured cells did not show aggregations, our in vivo expression studies in mice were able to reproduce the pathological changes observed in myotilinopathy patients. Mutant myotilin caused enhanced protein aggregation in TA muscles within 1 to 2 weeks (Figure 3). The dark blue or dark green inclusions stained by mGT in mutant-expressing fibers (Figure 4) were similar to those observed in the myotilinopathy patients. Furthermore, mMYOT S60C-expressing myofibers exhibited a greater number of aggregates, which is consistent with the pathology of the patient with that mutation (patient 1). Of note, the size of mMYOT S60C aggregates markedly increased over time, suggesting that mutant myotilin may be resistant to protein degradation, as described previously for MFM-associated mutant desmin.
      • Liu J.
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      Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.
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      Aberrant protein aggregation is essential for a mutant desmin to impair the proteolytic function of the ubiquitin-proteasome system in cardiomyocytes.
      Focal disorganization of myofibrils, Z-disk streaming, and accumulation of electron-dense material near the Z-disk are characteristic electron microscopic findings in the muscles of MFM patients.
      • Olivé M.
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      Myotilinopathy: refining the clinical and myopathological phenotype.
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      Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.
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      Electron microscopy in myofibrillar myopathies reveals clues to the mutated gene.
      In the myotilinopathy patient, Z-disk streaming, numerous autophagic vacuoles
      • Olivé M.
      • Goldfarb L.G.
      • Shatunov A.
      • Fischer D.
      • Ferrer I.
      Myotilinopathy: refining the clinical and myopathological phenotype.
      and cytoplasmic amorphous inclusions were observed (see Supplemental Figure S2 at http://ajp.amjpathol.org). In the present study, expression of mMYOT by electroporation elicited myofibril disorganization and accumulation of electron-dense material, which are ultrastructural hallmarks of MFM (Figure 5). Autophagic vacuoles associated with inclusions were also observed in electroporated muscles. Disorganization of myofibrils starting from the Z-disk and material appearing to originate from the Z-disk are commonly observed in MFM patients,
      • Selcen D.
      • Ohno K.
      • Engel A.G.
      Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.
      • Claeys K.G.
      • Fardeau M.
      • Schröder R.
      • Suominen T.
      • Tolksdorf K.
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      • Stojkovic T.
      • Faulkner G.
      • Richard P.
      • Vicart P.
      • Udd B.
      • Voit T.
      • Stoltenburg G.
      Electron microscopy in myofibrillar myopathies reveals clues to the mutated gene.
      and these features were also observed in the mMYOT-electroporated muscles. These morphological findings imply that the presence of mutant myotilin can induce characteristic pathological features by affecting Z-disk structure.
      Ectopic accumulations of multiple proteins, including Z-disk–associated proteins, are typical pathological features of MFM.
      • Claeys K.G.
      • van der Ven P.F.
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      • Eymard B.
      • Dubourg O.
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      • Udd B.
      • Fardeau M.
      • Voit T.
      • Fürst D.O.
      Differential involvement of sarcomeric proteins in myofibrillar myopathies: a morphological and immunohistochemical study.
      • Olivé M.
      Extralysosomal protein degradation in myofibrillar myopathies.
      This study and previous reports
      • Shalaby S.
      • Mitsuhashi H.
      • Matsuda C.
      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      • Janué A.
      • Olivé M.
      • Ferrer I.
      Oxidative stress in desminopathies and myotilinopathies: a link between oxidative damage and abnormal protein aggregation.
      showed that myotilin-positive protein aggregates colocalize with ubiquitin and Z-disk–associated proteins (ie, αB-crystallin, BAG3, actin, desmin, and filamin C) in the muscles of myotilinopathy patients (Figure 1; see also Supplemental Figure S2 at http://ajp.amjpathol.org). It has been reported that the myotilin T57I transgenic mice develop progressive myofibrillar changes, including Z-disk streaming and accumulation of mutant myotilin with ubiquitin and Z-disk–associated proteins, similar to those observed in myotilinopathy patients.
      • Garvey S.M.
      • Miller S.E.
      • Claflin D.R.
      • Faulkner J.A.
      • Hauser M.A.
      Transgenic mice expressing the myotilin T57I mutation unite the pathology associated with LGMD1A and MFM.
      Expression of mMYOT elicited similar cytoplasmic aggregations in mouse skeletal muscle, and within 2 weeks the aggregates colocalized with polyubiquitin and other Z-disk–associated proteins. Our results indicate that mutant myotilin is able to nucleate aggregations of Z-disk–associated proteins in skeletal muscle.
      MFM is a proteinopathy (ie, a protein accumulation disease). In these diseases, protein aggregates are operationally defined by poor solubility in aqueous or detergent solvents.
      • Fink A.L.
      Protein aggregation: folding aggregates, inclusion bodies and amyloid.
      • Kopito R.R.
      Aggresomes, inclusion bodies and protein aggregation.
      Such insoluble protein aggregations are characteristic of many neurodegenerative diseases.
      • Ross C.A.
      • Poirier M.A.
      Protein aggregation and neurodegenerative disease.
      In the present study, we discovered that the mutant myotilin S60C protein, along with polyubiquitinated proteins, exhibited marked detergent insolubility in muscles from both the patient and electroporated mice. Mutant myotilin R405K protein showed increased, but lower, detergent insolubility in mice (Figure 6), which may be consistent with the observation that the muscle from the patient with the R405K mutation exhibited only mild protein aggregation (Figure 1). The different detergent insolubilities exhibited by the two MYOT mutations may closely correlate with the amounts of protein aggregation. Here, we confirmed the aggregation-prone property of mutant myotilin, which participates in the pathogenesis of myotilinopathy. Using an immunoprecipitation assay, we also showed that electroporated mMYOT was not ubiquitinated in the detergent-soluble fraction (see Supplemental Figure S4 at http://ajp.amjpathol.org). A previous study showed that transfected myotilin is degraded by the proteasome system in cultured cells.
      • von Nandelstadh P.
      • Soliymani R.
      • Baumann M.
      • Carpen O.
      Analysis of myotilin turnover provides mechanistic insight into the role of myotilinopathy-causing mutations.
      Our present findings show that ubiquitinated mutant myotilin can form insoluble aggregates. It is also possible that aggregation of insoluble ubiquitinated proteins is induced by the expression of mutant myotilin.
      Several causative genes have been identified for MFM; however, in previous studies no mutations were found in nearly half of the MFM patients.
      • Selcen D.
      • Engel A.G.
      Myofibrillar myopathy.
      To identify the unknown causative genes, easy methods are required for determining the pathogenicity of novel mutations. Some mutant proteins exhibit protein aggregation
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      • Vicart P.
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      Desmin myopathy.
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      A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy.
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      • Sewry C.
      • Bite A.V.
      • Engel A.G.
      Mutation in BAG3 causes severe dominant childhood muscular dystrophy.
      or biological dysfunction, including protein-protein interaction in vitro.
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      • Mitsuhashi H.
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      • Minami N.
      • Noguchi S.
      • Nonaka I.
      • Nishino I.
      • Hayashi Y.K.
      Defective myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
      • Sharma S.
      • Mücke N.
      • Katus H.A.
      • Herrmann H.
      • Bär H.
      Disease mutations in the “head” domain of the extra-sarcomeric protein desmin distinctly alter its assembly and network-forming properties.
      • Bär H.
      • Kostareva A.
      • Sjöberg G.
      • Sejersen T.
      • Katus H.A.
      • Herrmann H.
      Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation.
      • Bova M.P.
      • Yaron O.
      • Huang Q.
      • Ding L.
      • Haley D.A.
      • Stewart P.L.
      • Horwitz J.
      Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function.
      However, we could not detect any protein aggregation in mMYOT-expressing cultured cells (Figure 2). The difficulty of in vitro investigation may be responsible for the inability to identify Z-disk–associated proteins or mature Z-disk structures. Indeed, myotilin is expressed in later differentiated C2C12 myotubes with sarcomere-like structures.
      • Mologni L.
      • Moza M.
      • Lalowski M.M.
      • Carpén O.
      Characterization of mouse myotilin and its promoter.
      This suggests that mutant myotilin requires mature Z-disk and/or other sarcomeric proteins to cause aggregations. In such cases, in vivo examination is important for evaluating the pathogenicity of mutations. Because in vivo electroporation can reproduce the pathological changes observed in MFM patients within a short time, it is a useful and powerful tool for evaluating the pathogenicity of mutations in MFM.

      Acknowledgments

      We thank Dr. Alan H. Beggs (Children's Hospital Boston, Harvard Medical School) for the kind gift of anti-filamin C antibody and Dr. Satomi Mitsuhashi (Children's Hospital Boston, Harvard Medical School) for technical assistance in electron microscopy analysis.

      Supplementary data

      • Supplemental Figure S1

        Ultrastructural findings in muscle from myotilinopathy patient 1 with the MYOT S60C mutation. A: Cytoplasmic inclusions (asterisk) were observed in the muscle from patient 1. The boxed region is shown at higher magnification in the inset, showing details of the inclusion. B: Electron-dense material (arrows) was seen in muscle of patient 1. C: Higher-magnification view of the electron-dense material in B. Scale bars: 3.0 μm (A); 1.0 μm (B and C); 0.3 μm (A, inset).

      • Supplemental Figure S2

        Accumulated myotilin costains with polyubiquitin and other Z-disk–associated proteins in myotilinopathy patients. Strong immunoreactive accumulations of myotilin, polyubiquitin (poly-Ub), αB-crystallin (αBC), BAG3, actin, desmin, and filamin C were seen in the fibers of biopsied muscles from both patient 1 with the S60C mutation (A) and patient 2 with the R405K mutation (B). Scale bars: 20 μm (A and B).

      • Supplemental Figure S3

        Histograms of the size distribution of myotilin aggregates in electroporated mouse muscles, showing the number and area of Myc-positive aggregates per fiber in wtMYOT (WT) (A), mMYOT S60C-expressing (B), or mMYOT R405K-expressing (C) myofibers at 7 and 14 days after electroporation (30 myofibers per group). Compared with WT (A) and R405K (C), the Myc-positive aggregates were larger in S60C-expressing fibers (B) at 14 days. The number of Myc-positive mMYOT aggregates (B and C) was greater at 14 days after electroporation.

      • Supplemental Figure S4

        Solubilities of sarcomeric proteins and immunoprecipitation of electroporated myotilin. Shown is solubility of BAG3, actin, desmin, α-actinin, or filamin C in the muscles from myotilinopathy patients (A) and from electroporated mice (B). GAPDH was used as a loading control. A: Immunoblotting (IB) of detergent-soluble and detergent-insoluble fractions from the muscles of control subjects (C1 and C2) or myotilinopathy patients (P1 and P2). Solubilities of sarcomeric proteins showed no difference between the muscles from control subjects and patients. B: Immunoblotting of detergent-soluble and detergent-insoluble fractions of Myc-wtMYOT (WT) or Myc-mMYOT (S60C or R405K) from electroporated mouse muscles. In S60C- or R405K-electroporated muscles, solubility of each protein is similar to that of PBS-injected control or WT-electroporated muscles. C: Immunoprecipitation of Myc-tagged myotilin from detergent-soluble fraction of electroporated mouse muscle. At 14 days after electroporation, the detergent-soluble lysates were immunoprecipitated with an agarose-conjugated anti-Myc antibody. The lysates or immunoprecipitated fractions (IP) were detected with anti-Myc and anti-polyubiquitin (poly-Ub) antibodies.

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