In Vivo Characterization of Mutant Myotilins
2012; Elsevier BV; Volume: 180; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.12.040
ISSN1525-2191
AutoresE. Keduka, Yukiko Hayashi, S. W. Shalaby, Hiroaki Mitsuhashi, S. Noguchi, Ikuya Nonaka, Ichizo Nishino,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoMyofibrillar 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 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.1Selcen D. Myofibrillar myopathies.Neuromuscul Disord. 2011; 21: 161-171Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar 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 BAG3.2Selcen D. Engel A.G. Myofibrillar myopathy.in: GeneReviews. 1997–2012http://www.ncbi.nlm.nih.gov/books/NBK1499Google Scholar, 3OlivÉ 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.Neuromuscul Disord. 2011; 21: 533-542Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar 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.4Salmikangas 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.Hum Mol Genet. 1999; 8: 1329-1336Crossref PubMed Scopus (171) Google Scholar, 5Parast M.M. Otey C.A. Characterization of palladin, a novel protein localized to stress fibers and cell adhesions.J Cell Biol. 2000; 150: 643-656Crossref PubMed Scopus (174) Google Scholar, 6Mykkänen O.M. Grönholm M. Rönty M. Lalowski M. Salmikangas P. Suila H. Carpén O. Characterization of human palladin, a microfilament-associated protein.Mol Biol Cell. 2001; 12: 3060-3073Crossref PubMed Scopus (119) Google Scholar, 7Bang 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.J Cell Biol. 2001; 153: 413-427Crossref PubMed Scopus (224) Google Scholar Myotilin is highly expressed in skeletal and cardiac muscle, and localizes to the Z-disk,4Salmikangas 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.Hum Mol Genet. 1999; 8: 1329-1336Crossref PubMed Scopus (171) Google Scholar which plays important roles in sarcomere assembly, actin filament stabilization, and muscle force transmission.8Faulkner G. Lanfranchi G. Valle G. Telethonin and other new proteins of the Z-disc of skeletal muscle.IUBMB Life. 2001; 51: 275-282Crossref PubMed Scopus (92) Google Scholar, 9Frank D. Kuhn C. Katus H.A. Frey N. The sarcomeric Z-disc: a nodal point in signalling and disease.J Mol Med (Berl). 2006; 84: 446-468Crossref PubMed Scopus (192) Google Scholar Myotilin interacts with several Z-disk–associated proteins, including α-actinin,4Salmikangas 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.Hum Mol Genet. 1999; 8: 1329-1336Crossref PubMed Scopus (171) Google Scholar filamin C,10van 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.J Cell Biol. 2000; 151: 235-248Crossref PubMed Scopus (168) Google Scholar, 11Gontier 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.J Cell Sci. 2005; 118: 3739-3749Crossref PubMed Scopus (104) Google Scholar FATZ,11Gontier 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.J Cell Sci. 2005; 118: 3739-3749Crossref PubMed Scopus (104) Google Scholar ZASP,12von 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.Mol Cell Biol. 2009; 29: 822-834Crossref PubMed Scopus (80) Google Scholar and MuRF ubiquitin ligase.13Witt 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.J Mol Biol. 2005; 350: 713-722Crossref PubMed Scopus (249) Google Scholar Myotilin also interacts with actin monomers and filaments through its Ig-like domains, which also mediate homodimerization.14Salmikangas 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.Hum Mol Genet. 2003; 12: 189-203Crossref PubMed Scopus (130) Google Scholar Previous studies have shown that myotilin can bundle actin filaments in vitro, acting alone or in collaboration with α-actinin and filamin C.4Salmikangas 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.Hum Mol Genet. 1999; 8: 1329-1336Crossref PubMed Scopus (171) Google Scholar, 14Salmikangas 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.Hum Mol Genet. 2003; 12: 189-203Crossref PubMed Scopus (130) Google Scholar, 15von Nandelstadh P. Grönholm M. Moza M. Lamberg A. Savilahti H. Carpén O. Actin-organising properties of the muscular dystrophy protein myotilin.Exp Cell Res. 2005; 310: 131-139Crossref PubMed Scopus (38) Google Scholar 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.12von 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.Mol Cell Biol. 2009; 29: 822-834Crossref PubMed Scopus (80) Google Scholar Missense mutations in MYOT have been associated with MFM,16Selcen D. Myofibrillar myopathies.Curr Opin Neurol. 2008; 21: 585-589Crossref PubMed Scopus (59) Google Scholar, 17Olivé M. Goldfarb L.G. Shatunov A. Fischer D. Ferrer I. Myotilinopathy: refining the clinical and myopathological phenotype.Brain. 2005; 128: 2315-2326Crossref PubMed Scopus (129) Google Scholar, 18Foroud 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.Neurology. 2005; 65: 1936-1940Crossref PubMed Scopus (72) Google Scholar limb girdle muscular dystrophy type 1A,17Olivé M. Goldfarb L.G. Shatunov A. Fischer D. Ferrer I. Myotilinopathy: refining the clinical and myopathological phenotype.Brain. 2005; 128: 2315-2326Crossref PubMed Scopus (129) Google Scholar, 19Hauser 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.Am J Hum Genet. 2002; 71: 1428-1432Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Hauser 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.Hum Mol Genet. 2000; 9: 2141-2147Crossref PubMed Scopus (247) Google Scholar and distal myopathy.21Penisson-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.Neuromuscul Disord. 2006; 16: 427-431Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 22Berciano 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.J Neurol Neurosurg Psychiatry. 2008; 79: 205-208Crossref PubMed Scopus (27) Google Scholar 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.23Shalaby 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.J Neuropathol Exp Neurol. 2009; 68: 701-707Crossref PubMed Scopus (21) Google Scholar All of the other previously reported MYOT mutations are located in exon 2 14Salmikangas 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.Hum Mol Genet. 2003; 12: 189-203Crossref PubMed Scopus (130) Google Scholar, 16Selcen D. Myofibrillar myopathies.Curr Opin Neurol. 2008; 21: 585-589Crossref PubMed Scopus (59) Google Scholar, 17Olivé M. Goldfarb L.G. Shatunov A. Fischer D. Ferrer I. Myotilinopathy: refining the clinical and myopathological phenotype.Brain. 2005; 128: 2315-2326Crossref PubMed Scopus (129) Google Scholar, 18Foroud 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.Neurology. 2005; 65: 1936-1940Crossref PubMed Scopus (72) Google Scholar, 24Reilich 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).J Neurol. 2011; 258: 1437-1444Crossref PubMed Scopus (24) Google Scholar, 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.25Mavroidis 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.FASEB J. 2008; 22: 3318-3327Crossref PubMed Scopus (36) Google Scholar, 26Wang 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.Circ Res. 2001; 89: 84-91Crossref PubMed Scopus (254) Google Scholar, 27Wang 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.Circulation. 2001; 103: 2402-2407Crossref PubMed Scopus (170) Google Scholar Some MFMs, including myotilinopathies, are late-onset and slowly progressive diseases.1Selcen D. Myofibrillar myopathies.Neuromuscul Disord. 2011; 21: 161-171Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 3OlivÉ 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.Neuromuscul Disord. 2011; 21: 533-542Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar 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,14Salmikangas 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.Hum Mol Genet. 2003; 12: 189-203Crossref PubMed Scopus (130) Google Scholar, 16Selcen D. Myofibrillar myopathies.Curr Opin Neurol. 2008; 21: 585-589Crossref PubMed Scopus (59) Google Scholar, 17Olivé M. Goldfarb L.G. Shatunov A. Fischer D. Ferrer I. Myotilinopathy: refining the clinical and myopathological phenotype.Brain. 2005; 128: 2315-2326Crossref PubMed Scopus (129) Google Scholar, 18Foroud 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.Neurology. 2005; 65: 1936-1940Crossref PubMed Scopus (72) Google Scholar, 23Shalaby 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.J Neuropathol Exp Neurol. 2009; 68: 701-707Crossref PubMed Scopus (21) Google Scholar, 24Reilich 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).J Neurol. 2011; 258: 1437-1444Crossref PubMed Scopus (24) Google Scholar only the Thr57Ile (T57I) mutation reproduces the pathological changes in transgenic mice after 12 months of age.28Garvey 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.Hum Mol Genet. 2006; 15: 2348-2362Crossref PubMed Scopus (41) Google Scholar 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. 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. 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.23Shalaby 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.J Neuropathol Exp Neurol. 2009; 68: 701-707Crossref PubMed Scopus (21) Google Scholar We cloned full-length human myotilin cDNA and generated mutant myotilin (mMYOT) by site-directed mutagenesis, as described previously.23Shalaby 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.J Neuropathol Exp Neurol. 2009; 68: 701-707Crossref PubMed Scopus (21) Google Scholar 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. 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.29Hayashi 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.J Clin Invest. 2009; 119: 2623-2633Crossref PubMed Scopus (311) Google Scholar 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. 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.29Hayashi 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.J Clin Invest. 2009; 119: 2623-2633Crossref PubMed Scopus (311) Google Scholar 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),30Thompson 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.J Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (241) Google Scholar 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). 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. 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). 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.23Shalaby 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.J Neuropathol Exp Neurol. 2009; 68: 701-707Crossref PubMed Scopus (21) Google Scholar 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). 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,23Shalaby 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.J Neuropathol Exp Neurol. 2009; 68: 701-707Crossref PubMed Scopus (21) Google Scholar with agarose-conjugated anti-Myc antibody (Santa Cruz Biotechnology). 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. 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.23Shalaby 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 pat
Referência(s)