Lipid Accumulation in Dysferlin-Deficient Muscles
2014; Elsevier BV; Volume: 184; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2014.02.005
ISSN1525-2191
AutoresMiranda D. Grounds, Jessica R. Terrill, Hannah G. Radley‐Crabb, Terry Robertson, J. M. Papadimitriou, Simone Spuler, Tea Shavlakadze,
Tópico(s)Nuclear Structure and Function
ResumoDysferlin is a membrane associated protein involved in vesicle trafficking and fusion. Defects in dysferlin result in limb-girdle muscular dystrophy type 2B and Miyoshi myopathy in humans and myopathy in A/Jdys−/− and BLAJ mice, but the pathomechanism of the myopathy is not understood. Oil Red O staining showed many lipid droplets within the psoas and quadriceps muscles of dysferlin-deficient A/Jdys−/− mice aged 8 and 12 months, and lipid droplets were also conspicuous within human myofibers from patients with dysferlinopathy (but not other myopathies). Electron microscopy of 8-month-old A/Jdys−/− psoas muscles confirmed lipid droplets within myofibers and showed disturbed architecture of myofibers. In addition, the presence of many adipocytes was confirmed, and a possible role for dysferlin in adipocytes is suggested. Increased expression of mRNA for a gene involved in early lipogenesis, CCAAT/enhancer binding protein-δ, in 3-month-old A/Jdys−/− quadriceps (before marked histopathology is evident), indicates early induction of lipogenesis/adipogenesis within dysferlin-deficient muscles. Similar results were seen for dysferlin-deficient BLAJ mice. These novel observations of conspicuous intermyofibrillar lipid and progressive adipocyte replacement in dysferlin-deficient muscles present a new focus for investigating the mechanisms that result in the progressive decline of muscle function in dysferlinopathies. Dysferlin is a membrane associated protein involved in vesicle trafficking and fusion. Defects in dysferlin result in limb-girdle muscular dystrophy type 2B and Miyoshi myopathy in humans and myopathy in A/Jdys−/− and BLAJ mice, but the pathomechanism of the myopathy is not understood. Oil Red O staining showed many lipid droplets within the psoas and quadriceps muscles of dysferlin-deficient A/Jdys−/− mice aged 8 and 12 months, and lipid droplets were also conspicuous within human myofibers from patients with dysferlinopathy (but not other myopathies). Electron microscopy of 8-month-old A/Jdys−/− psoas muscles confirmed lipid droplets within myofibers and showed disturbed architecture of myofibers. In addition, the presence of many adipocytes was confirmed, and a possible role for dysferlin in adipocytes is suggested. Increased expression of mRNA for a gene involved in early lipogenesis, CCAAT/enhancer binding protein-δ, in 3-month-old A/Jdys−/− quadriceps (before marked histopathology is evident), indicates early induction of lipogenesis/adipogenesis within dysferlin-deficient muscles. Similar results were seen for dysferlin-deficient BLAJ mice. These novel observations of conspicuous intermyofibrillar lipid and progressive adipocyte replacement in dysferlin-deficient muscles present a new focus for investigating the mechanisms that result in the progressive decline of muscle function in dysferlinopathies. Muscular dystrophies represent a large group of inherited human muscle diseases that result from a diversity of gene defects that include many membrane-associated proteins such as dysferlin, caveolin-3, dystrophin, and members of the dystroglycan/sarcoglycan complex, as well as other proteins such as laminins (reviewed in Saini-Chohan et al1Saini-Chohan H.K. Mitchell R.W. Vaz F.M. Zelinski T. Hatch G.M. Delineating the role of alterations in lipid metabolism to the pathogenesis of inherited skeletal and cardiac muscle disorders: Thematic Review Series: Genetics of Human Lipid Diseases.J Lipid Res. 2012; 53: 4-27Crossref PubMed Scopus (42) Google Scholar and Bushby2Bushby K. Genetics and the muscular dystrophies.Dev Med Child Neurol. 2000; 42: 780-784Crossref PubMed Scopus (10) Google Scholar). This study is focused on dysferlinopathies that result from mutations in the dysferlin gene that was identified in 1998.3Anderson L.V. Davison K. Moss J.A. Young C. Cullen M.J. Walsh J. Johnson M.A. Bashir R. Britton S. Keers S. Argov Z. Mahjneh I. Fougerousse F. Beckmann J.S. Bushby K.M. Dysferlin is a plasma membrane protein and is expressed early in human development.Hum Mol Genet. 1999; 8: 855-861Crossref PubMed Scopus (240) Google Scholar, 4Urtizberea J.A. Bassez G. Leturcq F. Nguyen K. Krahn M. Levy N. Dysferlinopathies.Neurol India. 2008; 56: 289-297Crossref PubMed Scopus (57) Google Scholar These dysferlinopathies are a clinically heterogeneous group of disorders usually with onset in late teens and slow progression. They include limb-girdle muscular dystrophy type 2B and Miyoshi myopathy, with weakness mainly in proximal limb-girdle muscles or distal muscles, respectively.4Urtizberea J.A. Bassez G. Leturcq F. Nguyen K. Krahn M. Levy N. Dysferlinopathies.Neurol India. 2008; 56: 289-297Crossref PubMed Scopus (57) Google Scholar, 5Laval S.H. Bushby K.M. Limb-girdle muscular dystrophies–from genetics to molecular pathology.Neuropathol Appl Neurobiol. 2004; 30: 91-105Crossref PubMed Scopus (119) Google Scholar, 6Nguyen K. Bassez G. Krahn M. Bernard R. Laforet P. Labelle V. Urtizberea J.A. Figarella-Branger D. Romero N. Attarian S. Leturcq F. Pouget J. Levy N. Eymard B. Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes.Arch Neurol. 2007; 64: 1176-1182Crossref PubMed Scopus (201) Google Scholar, 7Lo H.P. Cooper S.T. Evesson F.J. Seto J.T. Chiotis M. Tay V. Compton A.G. Cairns A.G. Corbett A. MacArthur D.G. Yang N. Reardon K. North K.N. Limb-girdle muscular dystrophy: diagnostic evaluation, frequency and clues to pathogenesis.Neuromuscul Disord. 2008; 18: 34-44Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 8Amato A.A. Brown Jr., R.H. Dysferlinopathies.Handb Clin Neurol. 2011; 101: 111-118Crossref PubMed Scopus (35) Google ScholarDysferlin is a member of a large ferlin family of transmembrane proteins that are involved in protein vesicle trafficking and fusion,9Lek A. Evesson F.J. Sutton R.B. North K.N. Cooper S.T. Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair.Traffic. 2012; 13: 185-194Crossref PubMed Scopus (97) Google Scholar with dysferlin initially attracting attention because of its role in the resealing of experimentally damaged sarcolemma.10Bansal D. Miyake K. Vogel S.S. Groh S. Chen C.C. Williamson R. McNeil P.L. Campbell K.P. Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature. 2003; 423: 168-172Crossref PubMed Scopus (748) Google Scholar, 11Han R. Campbell K.P. Dysferlin and muscle membrane repair.Curr Opin Cell Biol. 2007; 19: 409-416Crossref PubMed Scopus (192) Google Scholar, 12Glover L. Brown Jr., R.H. Dysferlin in membrane trafficking and patch repair.Traffic. 2007; 8: 785-794Crossref PubMed Scopus (121) Google Scholar However, the extent to which such damage might normally occur in dysferlin-deficient muscles in vivo is unknown; although membrane resealing has proved a useful experimental tool, this may not be the primary cause of the dystropathology.13Lostal W. Bartoli M. Roudaut C. Bourg N. Krahn M. Pryadkina M. Borel P. Suel L. Roche J.A. Stockholm D. Bloch R.J. Levy N. Bashir R. Richard I. Lack of correlation between outcomes of membrane repair assay and correction of dystrophic changes in experimental therapeutic strategy in dysferlinopathy.PLoS One. 2012; 7: e38036Crossref PubMed Scopus (52) Google Scholar More recently, the localization of dysferlin in intracellular membranes such as T-tubules and sarcoplasmic reticulum14Al-Qusairi L. Laporte J. T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases.Skelet Muscle. 2011; 1: 26Crossref PubMed Scopus (107) Google Scholar with a potentially important role in calcium homeostasis related to excitation-contraction coupling has attracted attention.15Klinge L. Harris J. Sewry C. Charlton R. Anderson L. Laval S. Chiu Y.H. Hornsey M. Straub V. Barresi R. Lochmuller H. Bushby K. Dysferlin associates with the developing T-tubule system in rodent and human skeletal muscle.Muscle Nerve. 2010; 41: 166-173Crossref PubMed Scopus (76) Google Scholar, 16Roche J.A. Ru L.W. O'Neill A.M. Resneck W.G. Lovering R.M. Bloch R.J. Unmasking potential intracellular roles for dysferlin through improved immunolabeling methods.J Histochem Cytochem. 2011; 59: 964-975Crossref PubMed Scopus (18) Google Scholar, 17Waddell L.B. Lemckert F.A. Zheng X.F. Tran J. Evesson F.J. Hawkes J.M. Lek A. Street N.E. Lin P. Clarke N.F. Landstrom A.P. Ackerman M.J. Weisleder N. Ma J. North K.N. Cooper S.T. Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch.J Neuropathol Exp Neurol. 2011; 70: 302-313Crossref PubMed Scopus (72) Google Scholar Dysferlin has high expression in skeletal myofibers but is also present in many other tissues3Anderson L.V. Davison K. Moss J.A. Young C. Cullen M.J. Walsh J. Johnson M.A. Bashir R. Britton S. Keers S. Argov Z. Mahjneh I. Fougerousse F. Beckmann J.S. Bushby K.M. Dysferlin is a plasma membrane protein and is expressed early in human development.Hum Mol Genet. 1999; 8: 855-861Crossref PubMed Scopus (240) Google Scholar and cells, including macrophages18Nagaraju K. Rawat R. Veszelovszky E. Thapliyal R. Kesari A. Sparks S. Raben N. Plotz P. Hoffman E.P. Dysferlin deficiency enhances monocyte phagocytosis: a model for the inflammatory onset of limb-girdle muscular dystrophy 2B.Am J Pathol. 2008; 172: 774-785Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar and endothelium.19Sharma A. Yu C. Leung C. Trane A. Lau M. Utokaparch S. Shaheen F. Sheibani N. Bernatchez P. A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis.Arterioscler Thromb Vasc Biol. 2010; 30: 2196-2204Crossref PubMed Scopus (45) Google Scholar The precise role that dysferlin plays in mature myofibers in vivo remains unclear. A striking feature of the onset of dysferlinopathies in humans is that growing children and adolescents are often asymptomatic, although many are active in sports,20Angelini C. Peterle E. Gaiani A. Bortolussi L. Borsato C. Dysferlinopathy course and sportive activity: clues for possible treatment.Acta Myol. 2011; 30: 127-132PubMed Google Scholar with the disease manifesting within 1 to 2 years of cessation of growth,21Albrecht D.E. Garg N. Rufibach L.E. Williams B.A. Monnier N. Hwang E. Mittal P. 4th Annual Dysferlin Conference 11-14 September 2010, Washington, USA.Neuromuscul Disord. 2011; 21: 304-310Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar indicating a lesser role for dysferlin in such growing, elongating myofibers.22Grounds M.D. Shavlakadze T. Growing muscle has different sarcolemmal properties from adult muscle: a proposal with scientific and clinical implications: reasons to reassess skeletal muscle molecular dynamics, cellular responses and suitability of experimental models of muscle disorders.Bioessays. 2011; 33: 458-468Crossref PubMed Scopus (34) Google Scholar This is also supported by the late onset of dystropathology in a wide range of mouse models for dysferlinopathies, ranging from dysferlin-deficient or nulls to engineered-specific gene defects.23Ho M. Post C.M. Donahue L.R. Lidov H.G. Bronson R.T. Goolsby H. Watkins S.C. Cox G.A. Brown Jr., R.H. Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency.Hum Mol Genet. 2004; 13: 1999-2010Crossref PubMed Scopus (152) Google Scholar, 24Kobayashi K. Izawa T. Kuwamura M. Yamate J. The distribution and characterization of skeletal muscle lesions in dysferlin-deficient SJL and A/J mice.Exp Toxicol Pathol. 2010; 62: 509-517Crossref PubMed Scopus (25) Google Scholar, 25Kobayashi K. Izawa T. Kuwamura M. Yamate J. Dysferlin and animal models for dysferlinopathy.J Toxicol Pathol. 2012; 25: 135-147Crossref PubMed Scopus (35) Google Scholar, 26Hornsey M.A. Laval S.H. Barresi R. Lochmuller H. Bushby K. Muscular dystrophy in dysferlin-deficient mouse models.Neuromuscul Disord. 2013; 23: 377-387Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Many initial studies were performed in dysferlin-deficient SJL/J (Swiss) mice and then A/J (A/Jdys−/−) mice and more recently BLAJ mice (B6.A-Dysf[prmd]/GeneJ, whereby the A/Jdys−/− mutation was introduced into the C57Bl/6J background strain). The disease is generally mild in dysferlin-deficient mice, and all mice show a late onset, with minimal symptoms before 3 months of postnatal age, histological evidence of disease by 8 and 12 months in some muscles (especially psoas and also quadriceps), and more pronounced pathological changes by 19 months.27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google ScholarIn a recent analysis of the disease progression in A/Jdys−/− mice we observed abnormally high levels of fatty tissue that replaced up to 20% to 40% of myofibers in the severely affected psoas muscle (and to a lesser extent in quadriceps femoris), compared with normal A/J controls, at 8, 12, and 19 months of age.27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar We had previously observed this conspicuous fatty tissue in rectus femoris (quadriceps) muscles of 12-month-old dysferlin-deficient SJL/J mice in which fat can replace up to approximately 20% of myofibers (J. Torrisi and M. D. Grounds, unpublished data); the lipid replacement in old quadriceps of SJL/J mice has also been reported by others.25Kobayashi K. Izawa T. Kuwamura M. Yamate J. Dysferlin and animal models for dysferlinopathy.J Toxicol Pathol. 2012; 25: 135-147Crossref PubMed Scopus (35) Google Scholar It is unlikely that fat cell replacement of dysferlin-deficient myofibers is a consequence of the incidence of myonecrosis (and impaired regeneration), because the level of myonecrosis, classically identified by fragmentation of myofiber sarcoplasm with inflammatory cell infiltration, was relatively low (<1% of the total muscle area) in A/Jdys−/− mice aged up to 19 months.27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar It is noted that such fatty replacement of limb muscles is not seen in mdx mice (that lack dystrophin and are a model of Duchenne muscular dystrophy), even though they have much higher levels of myonecrosis throughout life (up to approximately 80% of myofibers damaged in young mice and approximately 8% in adult mice). Dysferlin-deficient muscles exhibit excellent myogenesis and readily form new muscle in response to experimental muscle damage in vivo28Roberts P. McGeachie J.K. Grounds M.D. The host environment determines strain-specific differences in the timing of skeletal muscle regeneration: cross-transplantation studies between SJL/J and BALB/c mice.J Anat. 1997; 191: 585-594Crossref PubMed Google Scholar, 29Mitchell C.A. McGeachie J.K. Grounds M.D. Cellular differences in the regeneration of murine skeletal muscle: a quantitative histological study in SJL/J and BALB/c mice.Cell Tissue Res. 1992; 269: 159-166Crossref PubMed Scopus (86) Google Scholar, 30Chiu Y.H. Hornsey M.A. Klinge L. Jorgensen L.H. Laval S.H. Charlton R. Barresi R. Straub V. Lochmuller H. Bushby K. Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy.Hum Mol Genet. 2009; 18: 1976-1989Crossref PubMed Scopus (85) Google Scholar; however, macrophages and other cells are also affected by dysferlin deficiency,18Nagaraju K. Rawat R. Veszelovszky E. Thapliyal R. Kesari A. Sparks S. Raben N. Plotz P. Hoffman E.P. Dysferlin deficiency enhances monocyte phagocytosis: a model for the inflammatory onset of limb-girdle muscular dystrophy 2B.Am J Pathol. 2008; 172: 774-785Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar and it appears that there may be an impaired resolution of the inflammatory response associated with regeneration in some models of injury.30Chiu Y.H. Hornsey M.A. Klinge L. Jorgensen L.H. Laval S.H. Charlton R. Barresi R. Straub V. Lochmuller H. Bushby K. Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy.Hum Mol Genet. 2009; 18: 1976-1989Crossref PubMed Scopus (85) Google Scholar, 31Roche J.A. Ru L.W. Bloch R.J. Distinct effects of contraction-induced injury in vivo on four different murine models of dysferlinopathy.J Biomed Biotechnol. 2012; 2012: 134031Crossref PubMed Scopus (17) Google Scholar The present study investigates the nature of this high fatty tissue replacement of myofibers and the steatosis within them, in severely affected dysferlin-deficient muscles27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar of A/Jdys−/− and BLAJ mice and in patients with dysferlinopathy, using a range of techniques, including Oil Red O and BODIPY staining and electron microscopy (EM).Materials and MethodsSources of Mouse and Human TissuesThe A/J dysferlin-deficient A/Jdys−/− mice were generously provided by Dr. Sandra Cooper (Children's Hospital at Westmead, Sydney, Australia) (as for Terrill et al27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar). Additional inbred strains of mice, including control normal A/J and C57Bl/6J (sometime referred to as C57), and dysferlin-deficient BLAJ mice (B6.A-Dysf[prmd]/GeneJ) were obtained from the Animal Resources Centre, Murdoch, Western Australia. Where required, mice were maintained at the Preclinical Animal Facility at the University of Western Australia on a 12-hour light/dark cycle, under standard conditions (at 23°C), with free access to meat-free rat and mouse diet (Specialty Feeds, Glen Forrest, Australia) and drinking water. All experiments were conducted in accordance with the guidelines of the National Health and Medical Research Council, Australia, and were approved by the Animal Ethics Committee of the University of Western Australia.The human biopsies and blood from patients with dysferlinopathy and other myopathies were obtained for diagnostic purposes in Berlin, and informed consent was given by all patients that stained sections and blood can be analyzed for scientific purposes: a summary of all patients is provided in Supplemental Table S1. For dysferlinopathies, muscles were examined from four patients: 1 (male, aged 40 years), 2 (male, aged 52 years), 3 (female, aged 44 years), and 4 (female, aged 32 years, with severe disease), and blood from two patients, 3 and 5 (female, aged 18 years), was used for standard lipid analysis. Information on four of five patients has been published (patients 1, 2, and 3 in Wenzel et al32Wenzel K. Carl M. Perrot A. Zabojszcza J. Assadi M. Ebeling M. Geier C. Robinson P.N. Kress W. Osterziel K.J. Spuler S. Novel sequence variants in dysferlin-deficient muscular dystrophy leading to mRNA decay and possible C2-domain misfolding.Hum Mutat. 2006; 27: 599-600Crossref PubMed Scopus (36) Google Scholar and patient 5 in Diers et al33Diers A. Carl M. Stoltenburg-Didinger G. Vorgerd M. Spuler S. Painful enlargement of the calf muscles in limb girdle muscular dystrophy type 2B (LGMD2B) with a novel compound heterozygous mutation in DYSF.Neuromuscul Disord. 2007; 17: 157-162Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar).Sampling and Processing of Mouse TissuesMost analyses were done with the affected psoas and quadriceps muscles from female dysferlin-deficient A/Jdys−/− and wild-type (control) A/J mice aged 3 to 12 months. Many of the tissues were from the same A/Jdys−/− and A/J mice as used in a previous study of histological quantification.27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar Some other muscles such as gastrocnemius were also examined. In addition, muscles from male A/Jdys−/− and A/J mice, from male and female dysferlin-deficient BLAJ mice, and normal control C57Bl/6J mice, plus from female dysferlin-deficient SJL/J mice, were used to confirm observations for sex and age and for different strains. The range of mice and analyses used are indicated in Supplemental Table S2.All mice were sacrificed by cervical dislocation while under terminal anesthesia (2% v/v Attane isoflurane; Bomac Animal Health Pty Limited, Hornsby, Australia). For RNA extraction muscles were snap-frozen in liquid nitrogen. For histological analyses muscles were either frozen in isopentane cooled in liquid nitrogen or fixed in 4% paraformaldehyde and embedded in paraffin. For transmission EM, muscles were placed into 2.5% glutaraldehyde in 0.05 mol/L cacodylate buffer (pH 7.4) at room temperature for 24 hours. Samples were postfixed in 1% osmium tetroxide, dehydrated in graded solutions of ethanol, infiltrated, and embedded in Araldite. Semithin sections were stained in 1% toluidine blue in 5% borax and were examined by light microscopy to select the samples for EM analysis. For these blocks, 50 nm ultra-thin sections were cut, mounted on 200 mesh thin-bar copper grids, double stained in uranyl acetate and lead citrate, and examined with a JEOL 1400 Transmission Electron Microscope at an accelerating voltage of 100 kV.Oil Red O, BODIPY, and NADH Histochemical StainingLipid was visualized on frozen muscle (8 μm sections) with the use of Oil Red O histochemical staining. In brief, transverse and longitudinal sections were air dried and fixed in 10% formalin. After 2 minutes of incubation in propylene glycol, sections were stained in 0.5% Oil Red O in propylene glycol for 10 minutes at 60°C. Sections were differentiated in 85% propylene glycol, rinsed in distilled water, and stained in hematoxylin for 30 seconds. A similar staining protocol was used for the frozen human muscle biopsies.BODIPY 493/503 (Invitrogen, Carlsbad, CA) is a nonpolar fluorophore that binds to neutral lipids and is used to identify and quantify lipids, including lipid droplets, in tissues with the use of fluorescent microscopy.34Singh R. Kaushik S. Wang Y. Xiang Y. Novak I. Komatsu M. Tanaka K. Cuervo A.M. Czaja M.J. Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135Crossref PubMed Scopus (2549) Google Scholar, 35Prats C. Donsmark M. Qvortrup K. Londos C. Sztalryd C. Holm C. Galbo H. Ploug T. Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine.J Lipid Res. 2006; 47: 2392-2399Crossref PubMed Scopus (80) Google Scholar After staining by using a standard protocol,35Prats C. Donsmark M. Qvortrup K. Londos C. Sztalryd C. Holm C. Galbo H. Ploug T. Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine.J Lipid Res. 2006; 47: 2392-2399Crossref PubMed Scopus (80) Google Scholar images were captured with a fluorescent Nikon Eclipse Ti microscope equipped with a Roper Industries CoolSNAP-HQ2 camera, a 450- to 490-nm excitation filter, a 515-nm emission barrier filter, and Nikon NIS-Elements software version 3.0. Quantification was performed with ImageJ software version 1.44 (NIH, Bethesda, MD), where image thresholds were adjusted, allowing the percentage of lipid in the whole muscle cross-sections to be measured.To identify fast and slow myofiber types, 8-μm frozen muscle sections were stained with a standard protocol for nicotinamide adenine dinucleotide–reduced (NADH). Sections were incubated with a solution that contained equal parts NADH solution (1.6 mg/mL 0.05 mol/L Tris buffer, pH 7.6), and nitro blue tetrazolium solution (2 mg/mL 0.05 mol/L Tris buffer, pH 7.6) for 30 minutes at 37°C. Slides were washed in H20, before multiple washes in acetone:H2O at increasing concentrations of acetone.RNA Extraction and RT-qPCRGene expression was quantified for CCAAT/enhancer binding protein (C/EBP)-δ and peroxisome proliferator activation receptor-γ, because these are associated with induction of lipogenesis and differentiation of adipocytes (reviewed in Shavlakadze et al36Shavlakadze T. Grounds M.D. Of bears, frogs, meat, mice and men: complexity of factors affecting skeletal muscle mass and fat.Bioessays. 2006; 28: 994-1009Crossref PubMed Scopus (67) Google Scholar). For RNA extraction, muscles were ground in liquid nitrogen, and RNA was extracted with Tri-Reagent (Sigma Chemical Co., St. Louis, MO) treated with RQ1 DNase 1 (Promega, Madison, WI) to remove DNA; reverse transcription was performed on 2 μg of RNA with the use of Moloney Murine Leukemia Virus Reverse Transcriptase (Promega). cDNA was purified with Ultra Clean PCR Clean-up kit (Mo Bio Laboratories, Carlsbad, CA), and quantitative RT-PCR (RT-qPCR) was performed on a Rotor Gene 6000 real-time rotary analyzer by using the QuantiFast SYBR Green RT-qPCR Kit (Qiagen, Valencia, CA). All primers were purchased from Qiagen and were mouse CCAAT/enhancer binding protein δ (Cebpd; QT00312809), peroxisome proliferator activation receptor gamma (Pparg; QT00100296), and hypoxanthine guanine phosphoribosyl transferase (Hprt; QT00166768) that was used as a reference gene to normalize mRNA levels of Cebpd and Pparg.ResultsLipid Accumulation in Dysferlin-Deficient MusclesA large area of lipid (approximately 20% to 40% of area) within muscles has previously been described for A/Jdys−/− mice,27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar and sections from the same A/Jdys−/− muscles stained with Oil Red O (Figure 1, A and C) showed extensive lipid compared with age- and sex-matched wild-type A/J muscles (Figure 1, B and D). The lipid was located in adipocytes between the myofibers and also appeared to be present as droplets within many myofibers in selected regions of the muscle. BODIPY fluorescent staining was used to quantify lipid in a range of female and male muscles, from different ages and strains (A/Jdys−/−, BLAJ, their respective normal controls, and SJL/J) (Supplemental Table S2), with data for BLAJ (and control) psoas muscles shown in Supplemental Figure S1. There did not appear to be any sex-dependent effect in the various strains, and BODIPY also stained the SJL/J muscles (not shown). There was also broad strain similarity, with approximately 20% lipid present in BLAJ psoas at 12 months which is similar to that previously reported for A/Jdys−/− psoas muscles at 8 and 12 months.27Terrill J.R. Radley-Crabb H.G. Iwasaki T. Lemckert F.A. Arthur P.G. Grounds M.D. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.FEBS J. 2013; 280: 4149-4164Crossref PubMed Scopus (123) Google Scholar Smearing of lipid can occur when the tissue is cryo-sectioned, making it difficult to determine the precise location of the lipid with the use of Oil Red O (or BODIPY). For this reason, the presence of lipid droplets within myofibers was confirmed by EM (Figure 2).Figure 2EM of dysferlin-deficient myofibers from mice. Psoas muscles were examined from two female A/Jdys−/− mice (A–E) and one BLAJ mouse (F) aged 8 months; the myofibers shown were distant from the presence of adipocytes. Many large lipid droplets (with varying extent of osmophilia) are present between myofibrils and closely associated with mitochondria (A–C). The lipid droplets were prominent in slow myofibers, as shown by adjacent fast (asterisk) myofiber (C). Occasional areas showed disorganized structure where intact myofilaments cannot be clearly discerned in the sarcoplasm (D) and large autophagosomes and lysosomes (white arrows) were present (E and F); some of the autophagosomes were filled with lipid vesicles (lipofuscin) (F, white arrows). Cell surface protrusions (black arrow) are also evident (F).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The proportion of fast and slow myofibers in quadriceps muscles was observed with NADH staining and appeared similar in control and dysferlin-deficient mice up to 12 months of age; shown for A/J mice in Supplemental Figure S2. Equivalent patterns of NADH staining were seen for quadriceps muscles of male BLAJ mice aged 3, 8, and 12 months (data not shown). It was noticed that the myofiber appearance in 12-month-old A/Jdys−/− and BLAJ muscles was disturbed compared with controls; this is more clearly visible in the high-power images for A/Jdys−/− muscles (Supplemental Figure S2, D and E).In all human muscles from patients with dysferlinopathy, strong staining by Oil Red O of lipid within many myofibers was evident (Figure 1, E and F), and lipid was pronounced also in many adipocytes in one severe case, patient 4 (Figure 1, G and H). Lipid droplets are rare in normal healthy human myo
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