The Experts below are selected from a list of 2163 Experts worldwide ranked by ideXlab platform
Eric P. Hoffman - One of the best experts on this subject based on the ideXlab platform.
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membrane stabilization by modified steroid offers a potential therapy for muscular dystrophy due to Dysferlin deficit
Molecular Therapy, 2018Co-Authors: Sen Chandra Sreetama, Kanneboyina Nagaraju, Eric P. Hoffman, Goutam Chandra, Jack H Van Der Meulen, Mohammad Mahad Ahmad, Peter Suzuki, Shivaprasad BhuvanendranAbstract:Mutations of the DYSF gene leading to reduced Dysferlin protein level causes limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin facilitates sarcolemmal membrane repair in healthy myofibers, thus its deficit compromises myofiber repair and leads to chronic muscle inflammation. An experimental therapeutic approach for LGMD2B is to protect damage or improve repair of myofiber sarcolemma. Here, we compared the effects of prednisolone and vamorolone (a dissociative steroid; VBP15) on Dysferlin-deficient myofiber repair. Vamorolone, but not prednisolone, stabilized Dysferlin-deficient muscle cell membrane and improved repair of Dysferlin-deficient mouse (B6A/J) myofibers injured by focal sarcolemmal damage, eccentric contraction-induced injury or injury due to spontaneous in vivo activity. Vamorolone decreased sarcolemmal lipid mobility, increased muscle strength, and decreased late-stage myofiber loss due to adipogenic infiltration. In contrast, the conventional glucocorticoid prednisolone failed to stabilize Dysferlin deficient muscle cell membrane or improve repair of Dysferlinopathic patient myoblasts and mouse myofibers. Instead, prednisolone treatment increased muscle weakness and myofiber atrophy in B6A/J mice—findings that correlate with reports of prednisolone worsening symptoms of LGMD2B patients. Our findings showing improved cellular and pre-clinical efficacy of vamorolone compared to prednisolone and better safety profile of vamorolone indicates the suitability of vamorolone for clinical trials in LGMD2B.
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Molecular Pathogenesis of Genetic and Inherited Diseases Dysferlin Deficiency Enhances Monocyte Phagocytosis A Model for the Inflammatory Onset of Limb-Girdle Muscular Dystrophy 2B
2015Co-Authors: Kanneboyina Nagaraju, Rashmi Rawat, Edina Veszelovszky, Rachana Thapliyal, Akanchha Kesari, Susan Sparks, Nina Raben, Paul Plotz, Eric P. HoffmanAbstract:Dysferlin deficiency causes limb-girdle muscular dys-trophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflam-mation is often present in Dysferlin deficiency, and patients are frequently misdiagnosed as having poly-myositis. Because monocytes normally express dys-ferlin, we hypothesized that monocyte/macrophage dysfunction in Dysferlin-deficient patients might con-tribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL Dysferlin-deficient mouse model. Dysferlin-defi-cient monocytes showed increased phagocytic activ-ity compared with control cells. siRNA-mediated inhi
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Dysferlin deficiency enhances monocyte phagocytosis a model for the inflammatory onset of limb girdle muscular dystrophy 2b
American Journal of Pathology, 2008Co-Authors: Kanneboyina Nagaraju, Rashmi Rawat, Edina Veszelovszky, Rachana Thapliyal, Akanchha Kesari, Nina Raben, Susan E Sparks, Paul H Plotz, Eric P. HoffmanAbstract:Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in Dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express Dysferlin, we hypothesized that monocyte/macrophage dysfunction in Dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL Dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of Dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-α. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-α, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not Dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in Dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in Dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.
Robert H. Brown - One of the best experts on this subject based on the ideXlab platform.
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Membrane blebbing as an assessment of functional rescue of Dysferlin-deficient human myotubes via nonsense suppression.
Journal of applied physiology (Bethesda Md. : 1985), 2010Co-Authors: Bingjing Wang, Zhaohui Yang, Becky K. Brisson, Huisheng Feng, Zhi-qian Zhang, Ellen Welch, Stuart W. Peltz, Elisabeth R. Barton, Robert H. Brown, H. Lee SweeneyAbstract:Mutations that result in the loss of the protein Dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using Dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires Dysferlin. Based on this, we developed an in vitro assay to assess rescue of Dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for Dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in Dysferlin and produce sufficient functional Dysferlin (∼15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for Dysferlin-deficient patients harboring nonsense mutations.
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Dysferlin overexpression in skeletal muscle produces a progressive myopathy
Annals of Neurology, 2009Co-Authors: Louise E Glover, Robert H. Brown, Roderick T. Bronson, Kimberly Newton, Gomathi Krishnan, Alexandra Boyle, Lisa S KrivickasAbstract:Objective The dose-response effects of Dysferlin transgenesis were analyzed to determine if the Dysferlin-deficient myopathies are good candidates for gene replacement therapy.
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Disruption of muscle membrane and phenotype divergence in two novel mouse models of Dysferlin deficiency
Human Molecular Genetics, 2004Co-Authors: Cristina M Post, Gregory A. Cox, Leah Rae Donahue, Hart G.w. Lidov, Roderick T. Bronson, Holly Goolsby, Simon C. Watkins, Robert H. BrownAbstract:Limb girdle muscular dystrophy type 2B and Miyoshi myopathy are clinically distinct forms of muscular dystrophy that arise from defects in the Dysferlin gene. Here, we report two novel lines of Dysferlin-deficient mice obtained by (a) gene targeting and (b) identification of an inbred strain, A/J, bearing a retrotransposon insertion in the Dysferlin gene. The mutations in these mice were located at the 3' and 5' ends of the Dysferlin gene. Both lines of mice lacked Dysferlin and developed a progressive muscular dystrophy with histopathological and ultrastructural features that closely resemble the human disease. Vital staining with Evans blue dye revealed loss of sarcolemmal integrity in both lines of mice, similar to that seen in mdx and caveolin-3 deficient mice. However, in contrast to the latter group of animals, the Dysferlin-deficient mice have an intact dystrophin glycoprotein complex and normal levels of caveolin-3. Our findings indicate that muscle membrane disruption and myofiber degeneration in Dysferlinopathy were directly mediated by the loss of Dysferlin via a new pathogenic mechanism in muscular dystrophies. We also show that the mutation in the A/J mice arose between the late 1970s and the early 1980s, and had become fixed in the production breeding stocks. Therefore, all studies involving the A/J mice or mice derived from A/J, including recombinant inbred, recombinant congenic and chromosome substitution strains, should take into account the Dysferlin defect in these strains. These new Dysferlin-deficient mice should be useful for elucidating the pathogenic pathway in Dysferlinopathy and for developing therapeutic strategies.
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Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.
The Journal of biological chemistry, 2003Co-Authors: Niall J. Lennon, Alvin T. Kho, Brian J. Bacskai, Sarah L. Perlmutter, Bradley T. Hyman, Robert H. BrownAbstract:Mutations in the Dysferlin gene cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. We report here the results of expression profile analyses and in vitro investigations that point to an interaction between Dysferlin and the Ca2+ and lipid-binding proteins, annexins A1 and A2, and define a role for Dysferlin in Ca2+-dependent repair of sarcolemmal injury through a process of vesicle fusion. Expression profiling identified a network of genes that are co-regulated in Dysferlinopathic mice. Co-immunofluorescence, co-immunoprecipitation, and fluorescence lifetime imaging microscopy revealed that Dysferlin normally associates with both annexins A1 and A2 in a Ca2+ and membrane injury-dependent manner. The distribution of the annexins and the efficiency of sarcolemmal wound-healing are significantly disrupted in Dysferlin-deficient muscle. We propose a model of muscle membrane healing mediated by Dysferlin that is relevant to both normal and dystrophic muscle and defines the annexins as potential muscular dystrophy genes.
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The sarcolemmal proteins Dysferlin and caveolin-3 interact in skeletal muscle
Human molecular genetics, 2001Co-Authors: Chie Matsuda, Megumu Ogawa, Yukiko K. Hayashi, Kiichi Arahata, Ichizo Nishino, Masashi Aoki, Ikuya Nonaka, Kumiko Murayama, Robert H. BrownAbstract:Dysferlin is a surface membrane protein in skeletal muscle whose deficiency causes distal and proximal, recessively inherited, forms of muscular dystrophy designated Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B), respectively. The function of Dysferlin is not defined. Caveolin-3 is another skeletal muscle membrane protein which is important in the formation of caveolae and whose mutations cause dominantly inherited limb girdle muscular dystrophy type 1C (LGMD1C). We report that Dysferlin co-immunoprecipitates with caveolin-3 from biopsied normal human skeletal muscles. We also describe abnormal localization of Dysferlin in muscles from patients with LGMD1C including novel missense mutation (T64P) in the human caveolin-3 gene (CAV3). The immunoprecipitation data are consistent with the parallel observation that Dysferlin immunostaining is not normal in LGMD1C muscles. Amino acid sequence analysis of the Dysferlin protein reveals seven sites that correspond to caveolin-3 scaffold-binding motifs, and one site that is a potential target to bind the WW domain of the caveolin-3 protein. This is the first description of a possible Dysferlin interacting protein; it suggests the hypothesis that one function of Dysferlin may be to interact with caveolin-3 to subserve signaling functions of caveolae.
Kanneboyina Nagaraju - One of the best experts on this subject based on the ideXlab platform.
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membrane stabilization by modified steroid offers a potential therapy for muscular dystrophy due to Dysferlin deficit
Molecular Therapy, 2018Co-Authors: Sen Chandra Sreetama, Kanneboyina Nagaraju, Eric P. Hoffman, Goutam Chandra, Jack H Van Der Meulen, Mohammad Mahad Ahmad, Peter Suzuki, Shivaprasad BhuvanendranAbstract:Mutations of the DYSF gene leading to reduced Dysferlin protein level causes limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin facilitates sarcolemmal membrane repair in healthy myofibers, thus its deficit compromises myofiber repair and leads to chronic muscle inflammation. An experimental therapeutic approach for LGMD2B is to protect damage or improve repair of myofiber sarcolemma. Here, we compared the effects of prednisolone and vamorolone (a dissociative steroid; VBP15) on Dysferlin-deficient myofiber repair. Vamorolone, but not prednisolone, stabilized Dysferlin-deficient muscle cell membrane and improved repair of Dysferlin-deficient mouse (B6A/J) myofibers injured by focal sarcolemmal damage, eccentric contraction-induced injury or injury due to spontaneous in vivo activity. Vamorolone decreased sarcolemmal lipid mobility, increased muscle strength, and decreased late-stage myofiber loss due to adipogenic infiltration. In contrast, the conventional glucocorticoid prednisolone failed to stabilize Dysferlin deficient muscle cell membrane or improve repair of Dysferlinopathic patient myoblasts and mouse myofibers. Instead, prednisolone treatment increased muscle weakness and myofiber atrophy in B6A/J mice—findings that correlate with reports of prednisolone worsening symptoms of LGMD2B patients. Our findings showing improved cellular and pre-clinical efficacy of vamorolone compared to prednisolone and better safety profile of vamorolone indicates the suitability of vamorolone for clinical trials in LGMD2B.
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annexin a2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle
Human Molecular Genetics, 2017Co-Authors: Aurelia Defour, Sushma Medikayala, Jack H Van Der Meulen, Marshall W Hogarth, Nicholas Holdreith, Apostolos Malatras, William Duddy, Jessica F Boehler, Kanneboyina NagarajuAbstract:Repair of skeletal muscle after sarcolemmal damage involves Dysferlin and Dysferlin-interacting proteins such as annexins. Mice and patient lacking Dysferlin exhibit chronic muscle inflammation and adipogenic replacement of the myofibers. Here, we show that similar to Dysferlin, lack of annexin A2 (AnxA2) also results in poor myofiber repair and progressive muscle weakening with age. By longitudinal analysis of AnxA2-deficient muscle we find that poor myofiber repair due to the lack of AnxA2 does not result in chronic inflammation or adipogenic replacement of the myofibers. Further, deletion of AnxA2 in Dysferlin deficient mice reduced muscle inflammation, adipogenic replacement of myofibers, and improved muscle function. These results identify multiple roles of AnxA2 in muscle repair, which includes facilitating myofiber repair, chronic muscle inflammation and adipogenic replacement of Dysferlinopathic muscle. It also identifies inhibition of AnxA2-mediated inflammation as a novel therapeutic avenue for treating muscle loss in Dysferlinopathy.
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Molecular Pathogenesis of Genetic and Inherited Diseases Dysferlin Deficiency Enhances Monocyte Phagocytosis A Model for the Inflammatory Onset of Limb-Girdle Muscular Dystrophy 2B
2015Co-Authors: Kanneboyina Nagaraju, Rashmi Rawat, Edina Veszelovszky, Rachana Thapliyal, Akanchha Kesari, Susan Sparks, Nina Raben, Paul Plotz, Eric P. HoffmanAbstract:Dysferlin deficiency causes limb-girdle muscular dys-trophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflam-mation is often present in Dysferlin deficiency, and patients are frequently misdiagnosed as having poly-myositis. Because monocytes normally express dys-ferlin, we hypothesized that monocyte/macrophage dysfunction in Dysferlin-deficient patients might con-tribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL Dysferlin-deficient mouse model. Dysferlin-defi-cient monocytes showed increased phagocytic activ-ity compared with control cells. siRNA-mediated inhi
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greg cells a Dysferlin deficient myogenic mouse cell line
Experimental Cell Research, 2012Co-Authors: Glen Humphrey, Kanneboyina Nagaraju, Antoine De Morree, Elena Mekhedov, Paul S Blank, Gulcin Pekkurnaz, Joshua ZimmerbergAbstract:The Dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for Dysferlin. Dysferlin is a large (~200kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of Dysferlin function in muscle cells, we have established a Dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for Dysferlinopathy. GREG cells have no detectable Dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in Dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for Dysferlin deficiency.
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Dysferlin deficiency enhances monocyte phagocytosis a model for the inflammatory onset of limb girdle muscular dystrophy 2b
American Journal of Pathology, 2008Co-Authors: Kanneboyina Nagaraju, Rashmi Rawat, Edina Veszelovszky, Rachana Thapliyal, Akanchha Kesari, Nina Raben, Susan E Sparks, Paul H Plotz, Eric P. HoffmanAbstract:Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in Dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express Dysferlin, we hypothesized that monocyte/macrophage dysfunction in Dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL Dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of Dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-α. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-α, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not Dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in Dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in Dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.
Kevin P. Campbell - One of the best experts on this subject based on the ideXlab platform.
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dystrophin deficiency exacerbates skeletal muscle pathology in Dysferlin null mice
Skeletal Muscle, 2011Co-Authors: Renzhi Han, Dimple Bansal, Jennifer R Levy, Erik P Rader, Kevin P. CampbellAbstract:Mutations in the genes coding for either dystrophin or Dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with β-dystroglycan. This link extends to the extracellular matrix by β-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because Dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in Dysferlin-null mice and allow further characterization of the role of Dysferlin in skeletal muscle. To test our hypothesis, we generated dystrophin/Dysferlin double-knockout (DKO) mice by breeding mdx mice with Dysferlin-null mice and analyzed the effects of a combined deficiency of Dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. The DKO mice exhibited more severe muscle pathology than either mdx mice or Dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in Dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of Dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or Dysferlin-null muscles, suggesting that Dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles. The results of our study suggest that Dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of Dysferlin in membrane repair during lengthening contractions. Dystrophin/Dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat Dysferlin deficiency.
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genetic ablation of complement c3 attenuates muscle pathology in Dysferlin deficient mice
Journal of Clinical Investigation, 2010Co-Authors: Renzhi Han, Dimple Bansal, Steven A. Moore, Ellie M Frett, Jennifer R Levy, Erik P Rader, John D Lueck, Daniel Beltranvalero De Bernabe, John A Faulkner, Kevin P. CampbellAbstract:Mutations in the Dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as Dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood. Although muscle inflammation is widely recognized in Dysferlinopathy and Dysferlin is expressed in immune cells, the contribution of the immune system to the pathology of Dysferlinopathy remains to be fully explored. Here, we show that the complement system plays an important role in muscle pathology in Dysferlinopathy. Dysferlin deficiency led to increased expression of complement factors in muscle, while muscle-specific transgenic expression of Dysferlin normalized the expression of complement factors and eliminated the dystrophic phenotype present in Dysferlin-null mice. Furthermore, genetic disruption of the central component (C3) of the complement system ameliorated muscle pathology in Dysferlin-deficient mice but had no significant beneficial effect in a genetically distinct model of muscular dystrophy, mdx mice. These results demonstrate that complement-mediated muscle injury is central to the pathogenesis of Dysferlinopathy and suggest that targeting the complement system might serve as a therapeutic approach for this disease.
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Dysferlin mediated membrane repair protects the heart from stress induced left ventricular injury
Journal of Clinical Investigation, 2007Co-Authors: Renzhi Han, Dimple Bansal, Katsuya Miyake, Paul L Mcneil, Viviane P Muniz, Robert M Weiss, Kevin P. CampbellAbstract:Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that Dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of Dysferlin in the heart remains to be determined. Here, we demonstrate that Dysferlin is also involved in cardiomyocyte membrane repair and that Dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in Dysferlin-null mice and increases Evans blue dye uptake in Dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and Dysferlin leads to early onset cardiomyopathy. Our results suggest that Dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of Dysferlin.
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defective membrane repair in Dysferlin deficient muscular dystrophy
Nature, 2003Co-Authors: Dimple Bansal, Katsuya Miyake, Steven S Vogel, Severine Groh, Chienchang Chen, Roger A Williamson, Paul L Mcneil, Kevin P. CampbellAbstract:Muscular dystrophy includes a diverse group of inherited muscle diseases characterized by wasting and weakness of skeletal muscle1. Mutations in Dysferlin are linked to two clinically distinct muscle diseases, limb-girdle muscular dystrophy type 2B and Miyoshi myopathy, but the mechanism that leads to muscle degeneration is unknown2,3. Dysferlin is a homologue of the Caenorhabditis elegans fer-1 gene, which mediates vesicle fusion to the plasma membrane in spermatids4. Here we show that Dysferlin-null mice maintain a functional dystrophin–glycoprotein complex but nevertheless develop a progressive muscular dystrophy. In normal muscle, membrane patches enriched in Dysferlin can be detected in response to sarcolemma injuries. In contrast, there are sub-sarcolemmal accumulations of vesicles in Dysferlin-null muscle. Membrane repair assays with a two-photon laser-scanning microscope demonstrated that wild-type muscle fibres efficiently reseal their sarcolemma in the presence of Ca2+. Interestingly, Dysferlin-deficient muscle fibres are defective in Ca2+-dependent sarcolemma resealing. Membrane repair is therefore an active process in skeletal muscle fibres, and Dysferlin has an essential role in this process. Our findings show that disruption of the muscle membrane repair machinery is responsible for Dysferlin-deficient muscle degeneration, and highlight the importance of this basic cellular mechanism of membrane resealing in human disease.
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Intracellular accumulation and reduced sarcolemmal expression of Dysferlin in limb--girdle muscular dystrophies.
Annals of neurology, 2000Co-Authors: F. Piccolo, Steven A. Moore, Gwendolyn C. Ford, Kevin P. CampbellAbstract:Dysferlin has recently been identified as a novel gene involved in limb-girdle muscular dystrophy type 2B (LGMD2B) and its allelic disease, Miyoshi myopathy. The predicted structure of Dysferlin suggests that it is a transmembrane protein possibly involved in membrane fusion. Thus, unlike previously identified structural proteins in muscular dystrophy, Dysferlin is likely involved in a novel pathogenic mechanism for this disease. In this study, we have analyzed the expression of Dysferlin in skeletal muscle of patients with disruptions in the dystrophin-glycoprotein complex and patients with a clinical diagnosis of LGMD2B or Miyoshi myopathy. We show expression of Dysferlin at the sarcolemma in normal muscle and reduced sarcolemmal expression along with accumulation of intracellular staining in dystrophic muscle. Electron microscopy in Miyoshi myopathy biopsies suggests that the cytoplasmic staining could be a result of the abundance of intracellular vesicles. Our results indicate that Dysferlin expression is perturbed in LGMD and that both mutations in the Dysferlin gene and disruption of the dystrophin-glycoprotein complex can lead to the accumulation of Dysferlin within the cytoplasm.
Robert J. Bloch - One of the best experts on this subject based on the ideXlab platform.
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Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Jaclyn P. Kerr, Steven S Vogel, Andrew P. Ziman, Amber L. Mueller, Joaquin Muriel, Emily Kleinhans-welte, Jessica D. Gumerson, Christopher W. Ward, Joseph A. Roche, Robert J. BlochAbstract:Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein Dysferlin. Studies of Dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but Dysferlin’s association with calcium (Ca2+) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that Dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, Dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca2+] or blocking L-type Ca2+ channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of Dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of Dysferlin as a t-tubule protein that stabilizes stress-induced Ca2+ signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.
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unmasking potential intracellular roles for Dysferlin through improved immunolabeling methods
Journal of Histochemistry and Cytochemistry, 2011Co-Authors: Joseph A. Roche, Richard M Lovering, Andrea Oneill, Wendy G Resneck, Robert J. BlochAbstract:The DYSF gene encodes Dysferlin, a 230-kDa protein that is absent or severely reduced in patients with limb girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal myopathy with anterior tibial onset, skeletal muscle-wasting syndromes collectively referred to as Dysferlinopathies (Urtizberea et al. 2008). Inheritance is autosomal recessive, and disease-causing mutations have been identified across the DYSF gene (Nguyen et al. 2005). Since its discovery, Dysferlin has been referred to as a plasma membrane protein that is also found in cytoplasmic vesicles (Anderson et al. 1999; Bansal et al. 2003), largely because it appears enriched at the sarcolemma in cross sections of snap-frozen, unfixed muscle. The accumulation of subsarcolemmal vesicles in Dysferlinopathic muscle, and studies of muscle fibers cultured from mice lacking Dysferlin that involve laser wounding or other damaging treatments, have suggested that Dysferlin’s function is to repair disrupted plasma membranes (Bansal and Campbell, 2004; Glover and Brown, 2007; Han and Campbell, 2007). Recent reports suggest that cytoplasmic Dysferlin may be present, at least in part, in the transverse tubules (t-tubules) of skeletal muscle (Ampong et al. 2005; Lostal et al. 2010; Waddell et al. 2011). This location suggests that Dysferlin is required for maintaining the integrity of the t-tubules or perhaps for their coupling with the junctional sarcoplasmic reticulum (SR). Here, we report an improved method for immunolocalizing Dysferlin in the internal membranes of rat, mouse, and human skeletal muscles. Our methods rely on the “unmasking” of epitopes on Dysferlin by heating fixed cryosections of muscle in mildly acidic citrate buffer. Heat-induced antigen retrieval (AR) in citrate buffers improves the labeling of several muscle proteins (Mundegar et al. 2008), but its effectiveness for Dysferlin have not been documented. Using our modification of this method, we show that Dysferlin is much more abundant in the intracellular membranes of skeletal muscle fibers than it is at the sarcolemma and that the small amount of Dysferlin at the sarcolemma is present where the t-tubules insert, rather than at other sarcolemmal domains that are enriched in dystrophin (Williams and Bloch 1999). We show further that intracellular Dysferlin concentrates in a reticulum that flanks the Z-disks of each sarcomere, consistent with its presence in t-tubules, the junctional SR, or both. We conclude that the absence of Dysferlin from intracellular membranes, rather than or in addition to its absence from the sarcolemma, contributes to the mechanisms underlying Dysferlinopathies.
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extensive mononuclear infiltration and myogenesis characterize recovery of Dysferlin null skeletal muscle from contraction induced injuries
American Journal of Physiology-cell Physiology, 2010Co-Authors: Joseph A. Roche, Richard M Lovering, Renuka Roche, Patrick W Reed, Robert J. BlochAbstract:We studied the response of Dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that Dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of Dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and Dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the Dysferlin-null muscles, which recovered more slowly. Both control and Dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that Dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in Dysferlin-null mice.
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genetic manipulation of Dysferlin expression in skeletal muscle novel insights into muscular dystrophy
American Journal of Pathology, 2009Co-Authors: Douglas P Millay, Joseph A. Roche, Robert J. Bloch, Elizabeth M Mcnally, Marjorie Maillet, Michelle A Sargent, Jeffery D MolkentinAbstract:Mutations in the gene DYSF, which codes for the protein Dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle degenerative disease in mice. Dysferlin is a Ca(2+)-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of Dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle-specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The Dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf(-/-) A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of Dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex-lacking Scgd (delta-sarcoglycan) null mouse, indicating that Dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of Dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.
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impaired recovery of Dysferlin null skeletal muscle after contraction induced injury in vivo
Neuroreport, 2008Co-Authors: Joseph A. Roche, Richard M Lovering, Robert J. BlochAbstract:The protein, Dysferlin, mediates sarcolemmal repair in vitro, implicating defective membrane repair in Dysferlinopathies. To study the role of Dysferlin in vivo, we assessed contractile function, sarcolemmal integrity, and myogenesis before and after injury from large-strain lengthening contractions in Dysferlin-null and control mice. We report that Dysferlin-null muscles produce higher contractile torque, and are equally susceptible to initial injury but recover from injury more slowly. Two weeks after injury, control muscles retain fluorescein dextran and do not show myogenesis. Dysferlin-null muscles do not retain fluorescein dextran, and show necrosis followed by myogenesis. Our data indicate that recovery of control muscles from injury primarily involves sarcolemmal repair whereas recovery of Dysferlin-null muscles primarily involves myogenesis without repair and long-term survival of myofibers.