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• W2034276422 abstract "Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy. Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy. The muscular dystrophies are a heterogeneous group of genetic disorders characterized by progressive muscle loss and weakness. The mechanisms that underlie muscular dystrophy are diverse, including defective regeneration, plasma membrane instability, and defective membrane repair. Dysferlin (DYSF) has been implicated in all of these processes.1Demonbreun A.R. Fahrenbach J.P. Deveaux K. Earley J.U. Pytel P. McNally E.M. Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy.Hum Mol Genet. 2011; 20: 779-789Crossref PubMed Scopus (57) Google Scholar, 2Bansal 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 (779) Google Scholar Autosomal recessive loss-of-function mutations in dysferlin cause three different forms of muscular dystrophy: limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy.3Liu J. Aoki M. Illa I. Wu C. Fardeau M. Angelini C. Serrano C. Urtizberea J.A. Hentati F. Hamida M.B. Bohlega S. Culper E.J. Amato A.A. Bossie K. Oeltjen J. Bejaoui K. McKenna-Yasek D. Hosler B.A. Schurr E. Arahata K. de Jong P.J. Brown Jr., R.H. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.Nat Genet. 1998; 20: 31-36Crossref PubMed Scopus (761) Google Scholar, 4Saito H. Suzuki N. Ishiguro H. Hirota K. Itoyama Y. Takahashi T. Aoki M. Distal anterior compartment myopathy with early ankle contractures.Muscle Nerve. 2007; 36: 525-527Crossref PubMed Scopus (18) Google Scholar, 5Bashir R. Britton S. Strachan T. Keers S. Vafiadaki E. Lako M. Richard I. Marchand S. Bourg N. Argov Z. Sadeh M. Mahjneh I. Marconi G. Passos-Bueno M.R. Moreira Ede S. Zatz M. Beckmann J.S. Bushby K. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42Crossref PubMed Scopus (559) Google Scholar Mutations in dysferlin become clinically evident in the second to third decade or later, with muscle weakness. An early characteristic feature of dysferlin mutations is massively elevated serum creatine kinase levels. A spectrum of myopathic changes can be seen in muscle biopsy specimens from humans with dysferlin mutations, including dystrophic features, such as fibrofatty replacement and inflammatory infiltrates. Dysferlin is a 230-kDa membrane-inserted protein that contains at least six cytoplasmic C2 domains. C2 domains mediate protein-protein interactions and, in some cases, directly bind phospholipids and calcium. The C2 domains of dysferlin are highly related to those found in the membrane trafficking and fusion protein synaptotagmins.6Chapman E.R. An S. Edwardson J.M. Jahn R. A novel function for the second C2 domain of synaptotagmin, Ca2+-triggered dimerization.J Biol Chem. 1996; 271: 5844-5849Crossref PubMed Scopus (185) Google Scholar Dysferlin is highly expressed in adult skeletal muscle, whereas it is expressed at lower levels in muscle precursor cells, myoblasts.1Demonbreun A.R. Fahrenbach J.P. Deveaux K. Earley J.U. Pytel P. McNally E.M. Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy.Hum Mol Genet. 2011; 20: 779-789Crossref PubMed Scopus (57) Google Scholar, 7Nagaraju 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 (114) Google Scholar, 8Vandré D.D. Ackerman 4th, W.E. Kniss D.A. Tewari A.K. Mori M. Takizawa T. Robinson J.M. Dysferlin is expressed in human placenta but does not associate with caveolin.Biol Reprod. 2007; 77: 533-542Crossref PubMed Scopus (40) Google Scholar On sarcolemma damage, dysferlin is found at the sites of membrane disruption and has been specifically implicated in resealing the sarcolemma.2Bansal 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 (779) Google Scholar Electron microscopy of skeletal muscle biopsy specimens from human dysferlin-mutant patients confirms discontinuity of the sarcolemma and reveals vesicles underneath the basal lamina, suggesting dysferlin plays an active role in vesicle fusion at the membrane lesion.9Gayathri N. Alefia R. Nalini A. Yasha T.C. Anita M. Santosh V. Shankar S.K. Dysferlinopathy: spectrum of pathological changes in skeletal muscle tissue.Indian J Pathol Microbiol. 2011; 54: 350-354Crossref PubMed Scopus (25) Google Scholar Dysferlin also has been shown to interact with a variety of cytosolic and membrane-associated binding partners, including MG53, caveolin-3, AHNAK, and annexins A1 and A2.10Lennon N.J. Kho A. Bacskai B.J. Perlmutter S.L. Hyman B.T. Brown Jr., R.H. Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.J Biol Chem. 2003; 278: 50466-50473Crossref PubMed Scopus (316) Google Scholar, 11Huang Y. Laval S.H. van Remoortere A. Baudier J. Benaud C. Anderson L.V. Straub V. Deelder A. Frants R.R. den Dunnen J.T. Bushby K. van der Maarel S.M. AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration.FASEB J. 2007; 21: 732-742Crossref PubMed Scopus (122) Google Scholar, 12Matsuda C. Hayashi Y.K. Ogawa M. Aoki M. Murayama K. Nishino I. Nonaka I. Arahata K. Brown Jr., R.H. The sarcolemmal proteins dysferlin and caveolin-3 interact in skeletal muscle.Hum Mol Genet. 2001; 10: 1761-1766Crossref PubMed Scopus (203) Google Scholar, 13Cai C. Weisleder N. Ko J.K. Komazaki S. Sunada Y. Nishi M. Takeshima H. Ma J. Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.J Biol Chem. 2009; 284: 15894-15902Crossref PubMed Scopus (209) Google Scholar Similar to dysferlin, MG53, caveolin-3, and the annexins have been implicated in membrane resealing, suggesting a large complex may act coordinately to seal the disrupted plasma membrane in a calcium-dependent manner.13Cai C. Weisleder N. Ko J.K. Komazaki S. Sunada Y. Nishi M. Takeshima H. Ma J. Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.J Biol Chem. 2009; 284: 15894-15902Crossref PubMed Scopus (209) Google Scholar, 14Jaiswal J.K. Marlow G. Summerill G. Mahjneh I. Mueller S. Hill M. Miyake K. Haase H. Anderson L.V. Richard I. Kiuru-Enari S. McNeil P.L. Simon S.M. Bashir R. Patients with a non-dysferlin Miyoshi myopathy have a novel membrane repair defect.Traffic. 2007; 8: 77-88Crossref PubMed Scopus (49) Google Scholar An increasing body of evidence suggests that dysferlin’s membrane-associated roles are not restricted to the sarcolemma. Dysferlin has been implicated in the development and maintenance of the transverse (T-) tubule, a muscle-specific membrane system essential for electromechanical coupling. The T-tubule is a membrane inversion of the sarcolemma that flanks the Z band of muscle, the anchor for sarcomeric proteins. Dysferlin associates with the T-tubule–like system in differentiated C2C12 myotubes,15Klinge L. Laval S. Keers S. Haldane F. Straub V. Barresi R. Bushby K. From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis.FASEB J. 2007; 21: 1768-1776Crossref PubMed Scopus (74) Google Scholar and dysferlin-null mouse muscle contains malformed T-tubules consistent with a role for dysferlin in the biogenesis and maintenance of the T-tubule system.16Klinge 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 (79) Google Scholar In mature muscle damaged by stretch, dysferlin localizes to T-tubules, suggesting a reparative function for dysferlin at the T-tubule.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 (73) Google Scholar Dysferlin belongs to a family of proteins, the ferlins, that contains six family members. Myoferlin is a dysferlin homologue, which is 76% identical at the amino acid level.18Davis D.B. Delmonte A.J. Ly C.T. McNally E.M. Myoferlin, a candidate gene and potential modifier of muscular dystrophy.Hum Mol Genet. 2000; 9: 217-226Crossref PubMed Scopus (142) Google Scholar Such as dysferlin, myoferlin also contains at least six calcium-sensitive C2 domains, a carboxy-terminal transmembrane domain, an Fer domain, and a DysF domain.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 (73) Google Scholar, 19Davis D.B. Doherty K.R. Delmonte A.J. McNally E.M. Calcium-sensitive phospholipid binding properties of normal and mutant ferlin C2 domains.J Biol Chem. 2002; 277: 22883-22888Crossref PubMed Scopus (159) Google Scholar Myoferlin is highly expressed in myoblasts and is markedly up-regulated in adult skeletal muscle on muscle damage.20Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Normal myoblast fusion requires myoferlin.Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar Myoferlin, such as dysferlin, is required for normal myoblast fusion and muscle growth through regulating steps of vesicle trafficking and endocytic recycling.1Demonbreun A.R. Fahrenbach J.P. Deveaux K. Earley J.U. Pytel P. McNally E.M. Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy.Hum Mol Genet. 2011; 20: 779-789Crossref PubMed Scopus (57) Google Scholar, 20Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Normal myoblast fusion requires myoferlin.Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar, 21Doherty K.R. Demonbreun A.R. Wallace G.Q. Cave A. Posey A.D. Heretis K. Pytel P. McNally E.M. The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion.J Biol Chem. 2008; 283: 20252-20260Crossref PubMed Scopus (86) Google Scholar Myoferlin, such as dysferlin, is required for the proper trafficking of and response to the insulin-like growth factor-1 receptor in muscle.22Demonbreun A.R. Posey A.D. Heretis K. Swaggart K.A. Earley J.U. Pytel P. McNally E.M. Myoferlin is required for insulin-like growth factor response and muscle growth.FASEB J. 2010; 24: 1284-1295Crossref PubMed Scopus (45) Google Scholar Myoferlin interacts with endocytic recycling proteins EHD1 and EHD2, as well as AHNAK.21Doherty K.R. Demonbreun A.R. Wallace G.Q. Cave A. Posey A.D. Heretis K. Pytel P. McNally E.M. The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion.J Biol Chem. 2008; 283: 20252-20260Crossref PubMed Scopus (86) Google Scholar, 23Posey Jr., A.D. Pytel P. Gardikiotes K. Demonbreun A.R. Rainey M. George M. Band H. McNally E.M. Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion.J Biol Chem. 2011; 286: 7379-7388Crossref PubMed Scopus (44) Google Scholar, 24Benaud C. Gentil B.J. Assard N. Court M. Garin J. Delphin C. Baudier J. AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.J Cell Biol. 2004; 164: 133-144Crossref PubMed Scopus (150) Google Scholar To date, no human forms of muscular dystrophy resulting from myoferlin mutations have been reported. However, mice lacking myoferlin show defects in muscle regeneration, establishing a role for myoferlin in muscle repair.20Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Normal myoblast fusion requires myoferlin.Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar We generated ferlin (FER) mice that carry both the dysferlin- and myoferlin-null loss of function mutations. We determined that FER mice have a more severe muscular dystrophy than dysferlin-null mice. In addition, FER muscle displays disorganization of the T-tubule system, dilated sarcoplasmic reticulum, and increased levels of serum glycerol. We revealed an enhanced sensitivity of Dysf, MKO, and especially FER myofibers to glycerol exposure, resulting in T-tubule vacuolation and disrupted membrane potential. Intramuscular glycerol injections into young FER muscle recapitulated the dystrophic phenotype characteristic of old FER muscle. Our data establish a role for both myoferlin and dysferlin in the biogenesis and remodeling of the sarcotubule system and suggest glycerol as a mediator of muscular dystrophy in dysferlin mutations. The naturally occurring dysferlin-null mice from the A/J strain were backcrossed for six generations to the 129/SV emst/J myoferlin mouse line, to generate FER mice with both the Dysf and MKO alleles.20Doherty K.R. Cave A. Davis D.B. Delmonte A.J. Posey A. Earley J.U. Hadhazy M. McNally E.M. Normal myoblast fusion requires myoferlin.Development. 2005; 132: 5565-5575Crossref PubMed Scopus (162) Google Scholar, 25Ho 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 (158) Google Scholar Mice were housed in a specific pathogen-free facility in accordance with the University of Chicago (Chicago, IL) Institutional Animal Care and Use Committee regulations. Muscles from 24-week-old mice were dissected and frozen in liquid nitrogen–cooled isopentane. Muscle sections were stained with H&E or anti-dystrophin (Ab15277; Abcam, Cambridge, MA), diluted 1:200. By using ImageJ (NIH, Bethesda, MD) particle analysis, the mean area was determined from >275 fibers from at least five fields from three different animals per genotype. The percentage of fibers with central nuclei was calculated from the number of fibers containing internalized nuclei in each image/the total number of fibers counted per image, standardized as a percentage. At least 2000 fibers per genotype were analyzed (n = 3 from each genotype). Statistical analysis was performed using Prism version 4 (Graphpad, La Jolla, CA). Images were captured using a Zeiss Axiophot microscope. Quadricep, tricep, abdominal, paraspinal, gluteus/hamstring, and gastrocnemius/soleus muscles from age-matched, wild-type (WT), Dysf, mice lacking myoferlin (MKO), and FER animals were dissected and frozen in liquid nitrogen–cooled isopentane (n ≥ 3 animals per genotype per age). Muscle sections were stained with H&E. Images were captured using a Zeiss Axiophot microscope. Serum was collected from age-matched, WT, Dysf, MKO, and FER animals from eye bleeds using heparinized capillary tubes (Fisher, Pittsburgh, PA) into serum separator tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged for 10 minutes at 8000 × g. The plasma fractions were frozen and stored at −80°C and then assayed later using the EnzyChrom CK Assay kit (ECPK-100; BioAssay Systems, Hayward, CA). Serum glycerol was determined with the Cayman Chemical Assay kits (number 10010755; Cayman Chemical, Ann Arbor, MI). Activity was measured in the FluoStar Optima plate reader (BMG Labtech, Cary, NC). Sixteen-month-old WT, Dysf, MKO, and FER mice were housed individually and were allowed to run on a free-running wheel over a period of 48 hours (ENV-004; Med Associates, St. Albans, VT). Wheel-running activity was continuously monitored through wireless transmitters and recorded using the Wireless Running Wheel Manager Data Acquisition Software version 1.5 (SOF-860; Med Associates). Wheel activity was analyzed from 5 PM to 5 AM in 1-minute bins. Mean wheel rotations were calculated from at least two nights. Kilometers per minute was calculated from the average kilometers per minute during activity. Quadricep muscles from 6-month-old, WT, Dysf, MKO, and FER mice were divided into sections and fixed with 4% paraformaldehyde blocked in 1× PBS containing 10% fetal bovine serum, and then immunostained. Anti-sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) 1 (clone CaF2-5D2; Developmental Studies Hybridoma Bank, Iowa City, IA) was used at a dilution of 1:100, and anti-dystrophin was used at a dilution of 1:200 (Ab15277; Abcam). Goat anti-mouse Alexa 594 (number A11005; Invitrogen, Grand Island, NY) was used at a dilution of 1:5000, and goat anti-rabbit Alexa 488 antibody (number A11008; Invitrogen) was used at a dilution of 1:5000. Slides were mounted with Vectashield (Burlingame, CA) with DAPI. Images were captured using a Zeiss Axiophot microscope. Proteins transferred to membranes were immunoblotted with anti-annexin A2 used at a dilution of 1:3000 (number 610068; BD Transduction, Franklin Lakes, NJ), rabbit polyclonal anti-Fer1L5 antibody23Posey Jr., A.D. Pytel P. Gardikiotes K. Demonbreun A.R. Rainey M. George M. Band H. McNally E.M. Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion.J Biol Chem. 2011; 286: 7379-7388Crossref PubMed Scopus (44) Google Scholar was used at a dilution of 1:3000, anti-dihydropuridine receptor (DHPR) (MA3-920; Pierce, Rockford, IL) was used at a dilution of 1:3000, and anti–caveolin-3 (number 610420; BD Transduction) was used at a dilution of 1:1000. Secondary antibodies, goat anti-rabbit and goat anti-mouse conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA), were used at a dilution of 1:5000. Blocking and antibody incubations were performed in StartingBlock T20 (Tris-buffered saline) Blocking Buffer (Pierce). ECL-Plus chemiluminescence (GE Healthcare, Piscataway, NJ) and Kodak Biomax MS film were used for detection. WT and FER flexor digitorum brevis (FDB) muscle was removed and incubated in collagenase II (number 17101-015; Invitrogen). After 2 to 3 hours, FDB bundles were moved to media containing 3% bovine serum albumin and 0.1% gentamicin for trituration. Free fibers were incubated at 37°C overnight and plated on Matrigel (number 356234; BD Bioscience) coated coverslips. Fibers were fixed in 4% paraformaldehyde, rinsed, and blocked in Super Block (number 37515; Pierce) with 0.1% Triton X-100 (Sigma, St. Louis). Anti-DHPR (number MA3-920; Pierce) was used at a dilution of 1:100. Goat anti-mouse Alexa 488 (number A11001; Invitrogen) was used at a dilution of 1:2000. Slides were mounted in Vectashield with DAPI. Images were acquired on the Marianas Yokogawa spinning disk confocal using a 100× oil objective. By using a standard aseptic surgery procedure, the tibialis anterior (TA) muscles from 8-week-old mice were injected with 50% or 1% glycerol mixed with HBSS (number 14025-092; Gibco, Grand Island, NY) with a sterile insulin syringe, as previously described.26Pisani D.F. Bottema C.D. Butori C. Dani C. Dechesne C.A. Mouse model of skeletal muscle adiposity: a glycerol treatment approach.Biochem Biophys Res Commun. 2010; 396: 767-773Crossref PubMed Scopus (48) Google Scholar Mice were sacrificed at 5 or 28 days after injection. Muscles were dissected and frozen in liquid nitrogen–cooled isopentane. FDB fibers were isolated from 8-week-old, WT, Dysf, MKO, and FER mice and plated on corning plates in Ringer’s solution in 2 mmol/L calcium. Fibers were stained with 10 μmol/L RH414 for 15 minutes before imaging at room temperature. Ringer’s solution with 100 mmol/L glycerol and 10 μmol/L RH414 (catolog no. T-1111; Molecular Probes, Grand Island, NY) was placed on the preloaded fibers for 30 minutes, whereas healthy fibers were located on the Marianas Yokogawa spinning disk confocal using a 100× oil objective (561 nm, 100 milliseconds, neutral density 20) and bright field (100 milliseconds, neutral density 20).27Krolenko S.A. Amos W.B. Brown S.C. Tarunina M.V. Lucy J.A. Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.J Muscle Res Cell Motil. 1998; 19: 603-611Crossref PubMed Scopus (20) Google Scholar After 30 minutes, the Ringer’s glycerol mixture was washed out using a gravity perfusion system and the solution was replaced with normal Ringer’s solution. Images were acquired immediately after washout. Images were taken every 1 minute for 15 minutes, 7 μm from the cell surface. Confocal images of WT, MKO, DYSF, and FER fibers, loaded with RH414 at time 0 and 15 minutes after glycerol withdrawal, were used to generate plot profiles, which were analyzed in Sigview (SignalLab) to calculate the fast Fourier transformation of the RH414 signal. The amplitude of the peak in the power spectrum correlates with the amount of regular T-tubule RH414 signal at the z-lines. The signal between peaks correlates with the amount of RH414 T-tubule RH414 staining found at the A-band. Muscles from WT, Dysf, MKO, and FER mice were dissected and placed in 4% paraformaldehyde. Muscles were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4 for 1hour at 4°C, rinsed, dehydrated in ethanol, and infiltrated overnight. Embedded samples were divided into sections and stained with 1% uranyl acetate, followed by lead citrate. For T-tubule staining, muscles were fixed in glutaraldehyde overnight, post-fixed in 2% osmium tetroxide containing 0.8% potassium ferrocyanide for 1 hour at room temperature, and processed as previously described.16Klinge 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 (79) Google Scholar Quadricep muscles from male and female 6-month-old WT, Dysf, MKO, and FER mice were prepared for electron microscopy analysis, as previously described. Tubular aggregates within myofibers were counted in at least 12 grid squares per animal, with each grid containing approximately 8 to 12 fibers. At least three animals were analyzed per genotype. Muscles from 6-month-old animals were stained with anti-dystrophin and anti-SERCA1, and imaged under immunofluorescence microscopy. SERCA1-positive aggregates were counted from >85 fibers and four samples per genotype. Quadricep muscles were divided into sections, rinsed with PBS, and then fixed with 10% formalin. Samples were rinsed, dehydrated with 60% isopropanol, and air dried. Lipids were stained with oil red O for 10 minutes, rinsed, and fixed. Images were captured using a Zeiss Axiophot microscope. Primary myoblasts, isolated similar to the methods described for neonatal WT, Dysf, MKO, and FER mice, were stained as previously described.22Demonbreun A.R. Posey A.D. Heretis K. Swaggart K.A. Earley J.U. Pytel P. McNally E.M. Myoferlin is required for insulin-like growth factor response and muscle growth.FASEB J. 2010; 24: 1284-1295Crossref PubMed Scopus (45) Google Scholar Lipid droplets were quantified from 75 myoblasts per genotype. Nine-month-old WT, Dysf, MKO, FER mice, two males and two females of each genotype, were imaged at the University of Florida (Gainesville). Magnetic resonance imaging (MRI) was performed in a 4.7-T horizontal bore magnet (Varian, Palo Alto, CA). The animals were anesthetized using 0.8 to 1 L/minute oxygen and isoflurane mixture (3% isoflurane) and maintained under 0.5% to 1% isoflurane for the duration of the MR procedure. Body temperature was maintained through an MR-compatible heating system that pumped heated air into the bore of the magnet, and respiratory rate was monitored for the duration of the scans (Small Animal Instruments, Inc., Stony Brook, NY). The hind limbs of each mouse were inserted into a custom-built solenoid 1H-coil (200 MHz) with a 2.0-cm internal diameter. Three-dimensional spin-echo images were acquired with the following parameters: repetition time, 1000 milliseconds; echo time, 20 milliseconds; echo train length, 8; matrix size, 192 × 192 × 128; field of view, 20 × 20 mm2. T1-weighted images were acquired using series of repetition times (6, 3, 2, 1, 0.5, and 0.25 seconds): echo time, 5.7 milliseconds; matrix size, 128 × 128; field of view, 20 × 20 mm2; number of slices, 4; slice thickness, 1.00 mm with gap of 1 mm. Mutliplanar MR images acquired from both hind limbs were converted to Digital Imaging and Communications on Medicine format using a custom-written IDL code for Varian data (ITT Visual Information Systems, Boulder, CO); subsequent regions of interest were measured with Osirix (Pixmeo, Geneva, Switzerland). Fatty tissue deposition behind the knee joint (popliteal fossa) was outlined on multiple images, and the volume of fatty tissue deposition was calculated based on these regions of interest. Statistical analyses were conducted with Prism using an unpaired t-test, unless otherwise noted. The naturally occurring dysferlin-null allele arose in the A/J mouse line housed in the Jackson Laboratory (Bar Harbor, ME) through a retrotransposon insertion into intron 4 of the dysferlin locus.25Ho 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 (158) Google Scholar We previously bred this allele into the 129/SV emst/J line through six successive generations to generate the dysferlin-null allele in the 129S/V emst/J background (referred to as Dysf).1Demonbreun A.R. Fahrenbach J.P. Deveaux K. Earley J.U. Pytel P. McNally E.M. Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy.Hum Mol Genet. 2011; 20: 779-789Crossref PubMed Scopus (57) Google Scholar This Dysf allele was crossed onto the 129/SV emst/J line carrying the myoferlin-null mutation (MKO) to generate a dysferlin-myoferlin double-null ferlin mouse line.20Doherty K.R. Cave A. Davis D.B. Delmonte A" @default.
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• W2034276422 title "Dysferlin and Myoferlin Regulate Transverse Tubule Formation and Glycerol Sensitivity" @default.
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