157 results on '"Bloch RJ"'
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2. Dystrophin in a membrane skeletal network: localization and comparison to other proteins
- Author
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Dmytrenko, GM, primary, Pumplin, DW, additional, and Bloch, RJ, additional
- Published
- 1993
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3. Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations
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Luther, PW, primary, Yip, RK, additional, Bloch, RJ, additional, Ambesi, A, additional, Lindenmayer, GE, additional, and Blaustein, MP, additional
- Published
- 1992
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4. Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle
- Author
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Porter, GA, primary, Dmytrenko, GM, additional, Winkelmann, JC, additional, and Bloch, RJ, additional
- Published
- 1992
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5. Sarcolemmal reorganization in facioscapulohumeral muscular dystrophy.
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Reed P, Porter NC, Strong J, Pumplin DW, Corse AM, Luther PW, Flanigan KM, and Bloch RJ
- Published
- 2006
6. Acetylcholine receptor clustering in rat myotubes: requirement for CA2+ and effects of drugs which depolymerize microtubules
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Bloch, RJ, primary
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- 1983
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7. Loss of acetylcholine receptor clusters induced by treatment of cultured rat myotubes with carbachol
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Bloch, RJ, primary
- Published
- 1986
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8. Skeletal muscle adaptations following eccentric contractions are not mediated by keratin 18.
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Ganjayi MS, Frank SW, Krauss TA, York ML, Bloch RJ, and Baumann CW
- Abstract
The molecular mechanisms that drive muscle adaptations after eccentric exercise training are multifaceted and likely impacted by age. Previous studies have reported that many genes and proteins respond differently in young and older muscles following training. Keratin 18 (Krt18), a cytoskeletal protein involved in force transduction and organization, was found to be upregulated after muscles performed repeated bouts of eccentric contractions, with higher levels observed in young muscle compared to older muscle. Therefore, the purpose of this study was to determine if Krt18 mediates skeletal muscle adaptations following eccentric exercise training. The anterior crural muscles of Krt18 knockout (KO) and wild-type (WT) mice were subjected to either a single bout or repeated bouts of eccentric contractions, with isometric torque assessed across the initial and final bouts. Functionally, Krt18 KO and WT mice did not differ prior to performing any eccentric contractions (p≥0.100). Muscle strength (tetanic isometric torques) and the ability to adapt to eccentric exercise training were also consistent across strains at all time points (p≥0.169). Stated differently, immediate strength deficits and the recovery of strength following a single or multiple bouts of eccentric contractions were similar between Krt18 KO and WT mice. In summary, the absence of Krt18 does not impede the muscle's ability to adapt to repeated eccentric contractions, suggesting it is not essential for exercise-induced remodeling.
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- 2024
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9. Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca 2+ signaling.
- Author
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Muriel J, Lukyanenko V, Kwiatkowski TA, Li Y, Bhattacharya S, Banford KK, Garman D, Bulgart HR, Sutton RB, Weisleder N, and Bloch RJ
- Abstract
Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca
2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)- Published
- 2024
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10. Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice.
- Author
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O'Neill A, Martinez AL, Mueller AL, Huang W, Accorsi A, Kane MA, Eyerman D, and Bloch RJ
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- Adult, Humans, Male, Mice, Female, Animals, Heterografts, Transplantation, Heterologous, Muscle, Skeletal pathology, Cardiotoxins, Muscular Dystrophy, Facioscapulohumeral pathology
- Abstract
Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl
2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.- Published
- 2024
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11. Elevated Ca 2+ at the triad junction underlies dysregulation of Ca 2+ signaling in dysferlin-null skeletal muscle.
- Author
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Lukyanenko V, Muriel J, Garman D, Breydo L, and Bloch RJ
- Abstract
Dysferlin-null A/J myofibers generate abnormal Ca
2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J. Physiol., 2017). Ca2+ waves are typically associated with Ca2+ -induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+ . EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft., Competing Interests: LB is employed by Regeneron Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lukyanenko, Muriel, Garman, Breydo and Bloch.)- Published
- 2022
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12. The C2 domains of dysferlin: roles in membrane localization, Ca 2+ signalling and sarcolemmal repair.
- Author
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Muriel J, Lukyanenko V, Kwiatkowski T, Bhattacharya S, Garman D, Weisleder N, and Bloch RJ
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- Dysferlin genetics, Muscle, Skeletal metabolism, Sarcolemma metabolism, C2 Domains, Membrane Proteins genetics, Membrane Proteins metabolism
- Abstract
Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca
2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)- Published
- 2022
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13. Desmin interacts with STIM1 and coordinates Ca2+ signaling in skeletal muscle.
- Author
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Zhang H, Bryson VG, Wang C, Li T, Kerr JP, Wilson R, Muoio DM, Bloch RJ, Ward C, and Rosenberg PB
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- Animals, Calcium Signaling, Cells, Cultured, Desmin biosynthesis, Membrane Proteins biosynthesis, Membrane Proteins genetics, Mice, Microscopy, Electron, Transmission, Models, Animal, Muscle, Skeletal ultrastructure, Sarcoplasmic Reticulum metabolism, Stromal Interaction Molecule 1 biosynthesis, Desmin genetics, Gene Expression Regulation, Muscle, Skeletal metabolism, RNA genetics, Stromal Interaction Molecule 1 genetics
- Abstract
Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca2+ entry (SOCE) in skeletal muscle and, thereby, coordinates Ca2+ homeostasis, Ca2+-dependent gene expression, and contractility. STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca2+ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca2+ signaling in skeletal muscle.
- Published
- 2021
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14. Biomechanical Properties of the Sarcolemma and Costameres of Skeletal Muscle Lacking Desmin.
- Author
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Garcia-Pelagio KP and Bloch RJ
- Abstract
Intermediate filaments (IFs), composed primarily by desmin and keratins, link the myofibrils to each other, to intracellular organelles, and to the sarcolemma. There they may play an important role in transfer of contractile force from the Z-disks and M-lines of neighboring myofibrils to costameres at the membrane, across the membrane to the extracellular matrix, and ultimately to the tendon ("lateral force transmission"). We measured the elasticity of the sarcolemma and the connections it makes at costameres with the underlying contractile apparatus of individual fast twitch muscle fibers of desmin-null mice. By positioning a suction pipet to the surface of the sarcolemma and applying increasing pressure, we determined the pressure at which the sarcolemma separated from nearby sarcomeres, P
separation , and the pressure at which the isolated sarcolemma burst, Pbursting . We also examined the time required for the intact sarcolemma-costamere-sarcomere complex to reach equilibrium at lower pressures. All measurements showed the desmin-null fibers to have slower equilibrium times and lower Pseparation and Pbursting than controls, suggesting that the sarcolemma and its costameric links to nearby contractile structures were weaker in the absence of desmin. Comparisons to earlier values determined for muscles lacking dystrophin or synemin suggest that the desmin-null phenotype is more stable than the former and less stable than the latter. Our results are consistent with the moderate myopathy seen in desmin-null muscles and support the idea that desmin contributes significantly to sarcolemmal stability and lateral force transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Garcia-Pelagio and Bloch.)- Published
- 2021
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15. µ-Crystallin: A thyroid hormone binding protein.
- Author
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Kinney CJ and Bloch RJ
- Subjects
- Animals, Carrier Proteins genetics, Carrier Proteins metabolism, Crystallins genetics, Crystallins metabolism, Humans, Membrane Proteins genetics, Membrane Proteins metabolism, Thyroid Hormones genetics, Thyroid Hormones metabolism, mu-Crystallins, Thyroid Hormone-Binding Proteins, Carrier Proteins physiology, Crystallins physiology, Membrane Proteins physiology, Mental Disorders genetics, Mental Disorders metabolism, Nervous System Diseases genetics, Nervous System Diseases metabolism, Thyroid Hormones physiology
- Abstract
µ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the CRYM gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in CRYM can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more CRYM mRNA than the median level. Ablation of the Crym gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of Crym in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via β-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with CRYM , an important modulator of metabolism, are reviewed., (© 2021 Christian J. Kinney et al., published by Sciendo.)
- Published
- 2021
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16. μ-Crystallin in Mouse Skeletal Muscle Promotes a Shift from Glycolytic toward Oxidative Metabolism.
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Kinney CJ, O'Neill A, Noland K, Huang W, Muriel J, Lukyanenko V, Kane MA, Ward CW, Collier AF, Roche JA, McLenithan JC, Reed PW, and Bloch RJ
- Abstract
μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T
3 and T4 . Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym . We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6-147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)- Published
- 2021
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17. Meeting report: the 2020 FSHD International Research Congress.
- Author
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Kyba M, Bloch RJ, Dumonceaux J, Harper SQ, van der Maarel SM, Sverdrup FM, Wagner KR, van Engelen B, and Chen YW
- Subjects
- Animals, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Humans, Muscular Dystrophy, Facioscapulohumeral genetics, Muscular Dystrophy, Facioscapulohumeral physiopathology, Muscular Dystrophy, Facioscapulohumeral metabolism
- Published
- 2020
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18. Skeletal muscle cell transplantation: models and methods.
- Author
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Mueller AL and Bloch RJ
- Subjects
- Animals, Humans, Mice, Mice, Inbred NOD, Mice, SCID, Cell Transplantation methods, Muscle, Skeletal metabolism
- Abstract
Xenografts of skeletal muscle are used to study muscle repair and regeneration, mechanisms of muscular dystrophies, and potential cell therapies for musculoskeletal disorders. Typically, xenografting involves using an immunodeficient host that is pre-injured to create a niche for human cell engraftment. Cell type and method of delivery to muscle depend on the specific application, but can include myoblasts, satellite cells, induced pluripotent stem cells, mesangioblasts, immortalized muscle precursor cells, and other multipotent cell lines delivered locally or systemically. Some studies follow cell engraftment with interventions to enhance cell proliferation, migration, and differentiation into mature muscle fibers. Recently, several advances in xenografting human-derived muscle cells have been applied to study and treat Duchenne muscular dystrophy and Facioscapulohumeral muscular dystrophy. Here, we review the vast array of techniques available to aid researchers in designing future experiments aimed at creating robust muscle xenografts in rodent hosts.
- Published
- 2020
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19. Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle.
- Author
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Muriel JM, O'Neill A, Kerr JP, Kleinhans-Welte E, Lovering RM, and Bloch RJ
- Subjects
- Animals, Female, Intermediate Filaments ultrastructure, Keratin-18 deficiency, Keratin-18 genetics, Keratin-19 genetics, Keratin-19 metabolism, Male, Mice, Knockout, Muscle, Skeletal ultrastructure, Signal Transduction, Intermediate Filaments metabolism, Isometric Contraction, Keratin-18 metabolism, Muscle Strength, Muscle, Skeletal metabolism
- Abstract
Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18 -knockout ( Krt18 -KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18 -null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.
- Published
- 2020
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20. Muscle xenografts reproduce key molecular features of facioscapulohumeral muscular dystrophy.
- Author
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Mueller AL, O'Neill A, Jones TI, Llach A, Rojas LA, Sakellariou P, Stadler G, Wright WE, Eyerman D, Jones PL, and Bloch RJ
- Subjects
- Animals, Homeodomain Proteins genetics, Humans, Mice, Muscle, Skeletal pathology, Disease Models, Animal, Heterografts, Muscular Dystrophy, Facioscapulohumeral genetics, Myoblasts transplantation
- Abstract
Aberrant expression of DUX4, a gene unique to humans and primates, causes Facioscapulohumeral Muscular Dystrophy-1 (FSHD), yet the pathogenic mechanism is unknown. As transgenic overexpression models have largely failed to replicate the genetic changes seen in FSHD, many studies of endogenously expressed DUX4 have been limited to patient biopsies and myogenic cell cultures, which never fully differentiate into mature muscle fibers. We have developed a method to xenograft immortalized human muscle precursor cells from patients with FSHD and first-degree relative controls into the tibialis anterior muscle compartment of immunodeficient mice, generating human muscle xenografts. We report that FSHD cells mature into organized and innervated human muscle fibers with minimal contamination of murine myonuclei. They also reconstitute the satellite cell niche within the xenografts. FSHD xenografts express DUX4 and DUX4 downstream targets, retain the 4q35 epigenetic signature of their original donors, and express a novel protein biomarker of FSHD, SLC34A2. Ours is the first scalable, mature in vivo human model of FSHD. It should be useful for studies of the pathogenic mechanism of the disease as well as for testing therapeutic strategies targeting DUX4 expression., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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21. Effect of Ibuprofen on Skeletal Muscle of Dysferlin-Null Mice.
- Author
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Collier AF, Gumerson J, Lehtimäki K, Puoliväli J, Jones JW, Kane MA, Manne S, O'Neill A, Windish HP, Ahtoniemi T, Williams BA, Albrecht DE, and Bloch RJ
- Subjects
- Animals, Dysferlin genetics, Mice, Mice, Knockout, Time Factors, Dysferlin deficiency, Ibuprofen pharmacology, Muscle, Skeletal drug effects
- Abstract
Ibuprofen, a nonsteroidal anti-inflammatory drug, and nitric oxide (NO) donors have been reported to reduce the severity of muscular dystrophies in mice associated with the absence of dystrophin or α -sarcoglycan, but their effects on mice that are dystrophic due to the absence of dysferlin have not been examined. We have tested ibuprofen, as well as isosorbide dinitrate (ISDN), a NO donor, to learn whether used alone or together they protect dysferlin-null muscle in A/J mice from large strain injury (LSI) induced by a series of high strain lengthening contractions. Mice were maintained on chow containing ibuprofen and ISDN for 4 weeks. They were then subjected to LSI and maintained on the drugs for 3 additional days. We measured loss of torque immediately following injury and at day 3 postinjury, fiber necrosis, and macrophage infiltration at day 3 postinjury, and serum levels of the drugs at the time of euthanasia. Loss of torque immediately after injury was not altered by the drugs. However, the torque on day 3 postinjury significantly decreased as a function of ibuprofen concentration in the serum (range, 0.67-8.2 µ g/ml), independent of ISDN. The effects of ISDN on torque loss at day 3 postinjury were not significant. In long-term studies of dysferlinopathic BlAJ mice, lower doses of ibuprofen had no effects on muscle morphology, but reduced treadmill running by 40%. Our results indicate that ibuprofen can have deleterious effects on dysferlin-null muscle and suggest that its use at pharmacological doses should be avoided by individuals with dysferlinopathies., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2018
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22. Absence of synemin in mice causes structural and functional abnormalities in heart.
- Author
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García-Pelagio KP, Chen L, Joca HC, Ward C, Jonathan Lederer W, and Bloch RJ
- Subjects
- Aging pathology, Animals, Calcium Signaling, Cytoskeletal Proteins metabolism, Electrocardiography, Heart Ventricles pathology, Intermediate Filament Proteins metabolism, Mice, Myocardial Contraction, Phosphorylation, Pressure, Sarcolemma metabolism, Intermediate Filament Proteins deficiency, Myocardium metabolism, Myocardium pathology
- Abstract
Cardiomyopathies have been linked to changes in structural proteins, including intermediate filament (IF) proteins located in the cytoskeleton. IFs associate with the contractile machinery and costameres of striated muscle and with intercalated disks in the heart. Synemin is a large IF protein that mediates the association of desmin with Z-disks and stabilizes intercalated disks. It also acts as an A-kinase anchoring protein (AKAP). In murine skeletal muscle, the absence of synemin causes a mild myopathy. Here, we report that the genetic silencing of synemin in mice (synm -/-) causes left ventricular systolic dysfunction at 3months and 12-16months of age, and left ventricular hypertrophy and dilatation at 12-16months of age. Isolated cardiomyocytes showed alterations in calcium handling that indicate defects intrinsic to the heart. Although contractile and costameric proteins remained unchanged in the old synm -/- hearts, we identified alterations in several signaling proteins (PKA-RII, ERK and p70S6K) critical to cardiomyocyte function. Our data suggest that synemin plays an important regulatory role in the heart and that the consequences of its absence are profound., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
- Full Text
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23. Coupling of excitation to Ca 2+ release is modulated by dysferlin.
- Author
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Lukyanenko V, Muriel JM, and Bloch RJ
- Subjects
- Animals, Calcium Channels, L-Type physiology, Dysferlin genetics, Mice, Knockout, Osmotic Pressure physiology, Ryanodine Receptor Calcium Release Channel physiology, Sarcoplasmic Reticulum physiology, Thiazepines pharmacology, Calcium physiology, Dysferlin physiology, Muscle Fibers, Skeletal physiology
- Abstract
Key Points: Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca
2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca2+ channels protect voltage-induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+ -free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca2+ leak., Abstract: Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca2+ leak from the SR into the cytoplasm., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)- Published
- 2017
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24. Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1).
- Author
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Desmond PF, Labuza A, Muriel J, Markwardt ML, Mancini AE, Rizzo MA, and Bloch RJ
- Subjects
- Animals, Ankyrins genetics, COS Cells, Chlorocebus aethiops, HEK293 Cells, Humans, Muscle Proteins genetics, Proteolipids genetics, Sarcoplasmic Reticulum genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Ankyrins metabolism, Muscle Proteins metabolism, Proteolipids metabolism, Sarcoplasmic Reticulum metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
SERCA1, the sarco(endo)plasmic reticulum Ca
2+ -ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca2+ levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca2+ -ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca2+ -ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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25. Deficiency of the intermediate filament synemin reduces bone mass in vivo.
- Author
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Moorer MC, Buo AM, Garcia-Pelagio KP, Stains JP, and Bloch RJ
- Subjects
- Animals, Biomarkers blood, Bone Diseases, Metabolic metabolism, Bone Diseases, Metabolic physiopathology, Cancellous Bone physiology, Cell Differentiation physiology, Cyclin D1 metabolism, Femur physiology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Muscle, Skeletal metabolism, Muscle, Skeletal physiology, Osteoblasts metabolism, Osteoblasts physiology, RNA, Messenger metabolism, Bone Density physiology, Cancellous Bone metabolism, Femur metabolism, Intermediate Filament Proteins metabolism, Intermediate Filaments metabolism, Osteogenesis physiology
- Abstract
While the type IV intermediate filament protein, synemin, has been shown to play a role in striated muscle and neuronal tissue, its presence and function have not been described in skeletal tissue. Here, we report that genetic ablation of synemin in 14-wk-old male mice results in osteopenia that includes a more than 2-fold reduction in the trabecular bone fraction in the distal femur and a reduction in the cross-sectional area at the femoral middiaphysis due to an attendant reduction in both the periosteal and endosteal perimeter. Analysis of serum markers of bone formation and static histomorphometry revealed a statistically significant defect in osteoblast activity and osteoblast number in vivo. Interestingly, primary osteoblasts isolated from synemin-null mice demonstrate markedly enhanced osteogenic capacity with a concomitant reduction in cyclin D1 mRNA expression, which may explain the loss of osteoblast number observed in vivo. In total, these data suggest an important, previously unknown role for synemin in bone physiology., (Copyright © 2016 the American Physiological Society.)
- Published
- 2016
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26. Neuromuscular electrical stimulation promotes development in mice of mature human muscle from immortalized human myoblasts.
- Author
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Sakellariou P, O'Neill A, Mueller AL, Stadler G, Wright WE, Roche JA, and Bloch RJ
- Subjects
- Adult, Animals, Biomarkers metabolism, Cell Line, Cell Proliferation, Cell Survival, Graft Survival, Heterografts, Humans, Male, Mice, Inbred NOD, Mice, Transgenic, Muscular Dystrophy, Facioscapulohumeral metabolism, Myoblasts, Skeletal metabolism, Myoblasts, Skeletal pathology, Time Factors, Cell Differentiation, Electric Stimulation methods, Muscle Development, Muscular Dystrophy, Facioscapulohumeral pathology, Myoblasts, Skeletal transplantation, Neuromuscular Junction, Peroneal Nerve
- Abstract
Background: Studies of the pathogenic mechanisms underlying human myopathies and muscular dystrophies often require animal models, but models of some human diseases are not yet available. Methods to promote the engraftment and development of myogenic cells from individuals with such diseases in mice would accelerate such studies and also provide a useful tool for testing therapeutics. Here, we investigate the ability of immortalized human myogenic precursor cells (hMPCs) to form mature human myofibers following implantation into the hindlimbs of non-obese diabetic-Rag1 (null) IL2rγ (null) (NOD-Rag)-immunodeficient mice., Results: We report that hindlimbs of NOD-Rag mice that are X-irradiated, treated with cardiotoxin, and then injected with immortalized control hMPCs or hMPCs from an individual with facioscapulohumeral muscular dystrophy (FSHD) develop mature human myofibers. Furthermore, intermittent neuromuscular electrical stimulation (iNMES) of the peroneal nerve of the engrafted limb enhances the development of mature fibers in the grafts formed by both immortal cell lines. With control cells, iNMES increases the number and size of the human myofibers that form and promotes closer fiber-to-fiber packing. The human myofibers in the graft are innervated, fully differentiated, and minimally contaminated with murine myonuclei., Conclusions: Our results indicate that control and FSHD human myofibers can form in mice engrafted with hMPCs and that iNMES enhances engraftment and subsequent development of mature human muscle.
- Published
- 2016
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27. Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle.
- Author
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Desmond PF, Muriel J, Markwardt ML, Rizzo MA, and Bloch RJ
- Subjects
- Amino Acid Sequence, Animals, Ankyrins genetics, COS Cells, Chlorocebus aethiops, Immunoprecipitation, Models, Molecular, Molecular Sequence Data, Muscle Proteins chemistry, Protein Structure, Tertiary, Proteolipids chemistry, Rabbits, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Ankyrins chemistry, Muscle, Skeletal metabolism, Sarcoplasmic Reticulum metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ~2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca(2+) affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca(2+) in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca(2+) misregulation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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28. Myofiber damage precedes macrophage infiltration after in vivo injury in dysferlin-deficient A/J mouse skeletal muscle.
- Author
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Roche JA, Tulapurkar ME, Mueller AL, van Rooijen N, Hasday JD, Lovering RM, and Bloch RJ
- Subjects
- Animals, Disease Models, Animal, Dysferlin, Inflammation metabolism, Macrophages metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Knockout, Muscle, Skeletal injuries, Muscle, Skeletal metabolism, Muscular Dystrophies, Limb-Girdle metabolism, Inflammation pathology, Macrophages pathology, Muscle, Skeletal pathology, Muscular Dystrophies, Limb-Girdle pathology
- Abstract
Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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29. Myopathic changes in murine skeletal muscle lacking synemin.
- Author
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García-Pelagio KP, Muriel J, O'Neill A, Desmond PF, Lovering RM, Lund L, Bond M, and Bloch RJ
- Subjects
- Animals, Biomechanical Phenomena, Costameres metabolism, Costameres pathology, Genotype, Intermediate Filament Proteins genetics, Male, Mice, Inbred C57BL, Mice, Knockout, Muscle Fatigue, Muscle Fibers, Fast-Twitch metabolism, Muscle Fibers, Fast-Twitch pathology, Muscle, Skeletal pathology, Muscle, Skeletal physiopathology, Muscular Diseases etiology, Muscular Diseases genetics, Muscular Diseases pathology, Muscular Diseases physiopathology, Phenotype, Running, Sarcolemma metabolism, Sarcolemma pathology, Intermediate Filament Proteins deficiency, Isometric Contraction, Muscle Strength, Muscle, Skeletal metabolism, Muscular Diseases metabolism
- Abstract
Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle., (Copyright © 2015 the American Physiological Society.)
- Published
- 2015
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30. Muscle structure influences utrophin expression in mdx mice.
- Author
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Banks GB, Combs AC, Odom GL, Bloch RJ, and Chamberlain JS
- Subjects
- Animals, Calcium-Binding Proteins biosynthesis, Dystrophin-Associated Proteins biosynthesis, Elapid Venoms, Inflammation immunology, Macrophages immunology, Male, Membrane Proteins biosynthesis, Mice, Mice, Inbred mdx, Mice, Knockout, Muscle Proteins biosynthesis, Muscle, Skeletal metabolism, Muscular Dystrophy, Duchenne mortality, Muscular Dystrophy, Duchenne physiopathology, Sarcolemma metabolism, Sarcomeres physiology, Desmin genetics, Dystrophin genetics, Muscle, Skeletal pathology, Muscular Dystrophy, Duchenne genetics, Utrophin biosynthesis
- Abstract
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin-/- mice. An ∼ 2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼ 91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin-/-, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.
- Published
- 2014
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31. Dysferlin at transverse tubules regulates Ca(2+) homeostasis in skeletal muscle.
- Author
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Kerr JP, Ward CW, and Bloch RJ
- Abstract
The class of muscular dystrophies linked to the genetic ablation or mutation of dysferlin, including Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MM), are late-onset degenerative diseases. In lieu of a genetic cure, treatments to prevent or slow the progression of dysferlinopathy are of the utmost importance. Recent advances in the study of dysferlinopathy have highlighted the necessity for the maintenance of calcium handling in altering or slowing the progression of muscular degeneration resulting from the loss of dysferlin. This review highlights new evidence for a role for dysferlin at the transverse (t-) tubule of striated muscle, where it is involved in maintaining t-tubule structure and function.
- Published
- 2014
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32. Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane.
- Author
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Kerr JP, Ziman AP, Mueller AL, Muriel JM, Kleinhans-Welte E, Gumerson JD, Vogel SS, Ward CW, Roche JA, and Bloch RJ
- Subjects
- Animals, Antihypertensive Agents pharmacology, Calcium Channels, L-Type genetics, Calcium Channels, L-Type metabolism, Cell Membrane pathology, Diltiazem pharmacology, Dysferlin, Membrane Proteins genetics, Mice, Mice, Mutant Strains, Muscle Contraction drug effects, Muscle Contraction genetics, Muscle Fibers, Skeletal pathology, Muscular Dystrophies, Limb-Girdle genetics, Muscular Dystrophies, Limb-Girdle pathology, Necrosis genetics, Necrosis metabolism, Necrosis pathology, Calcium Signaling, Cell Membrane metabolism, Membrane Proteins metabolism, Muscle Fibers, Skeletal metabolism, Muscular Dystrophies, Limb-Girdle metabolism, Stress, Physiological
- Abstract
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 (Ca(2+)) 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 [Ca(2+)] or blocking L-type Ca(2+) 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 Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.
- Published
- 2013
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33. Structural and functional evaluation of branched myofibers lacking intermediate filaments.
- Author
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Goodall MH, Ward CW, Pratt SJP, Bloch RJ, and Lovering RM
- Subjects
- Action Potentials genetics, Animals, Desmin genetics, Intermediate Filaments chemistry, Intermediate Filaments pathology, Keratin-19 genetics, Mice, Mice, Inbred C57BL, Mice, Inbred mdx, Mice, Knockout, Muscle Fibers, Fast-Twitch chemistry, Muscle Fibers, Fast-Twitch pathology, Mutation, Neuromuscular Junction genetics, Structure-Activity Relationship, Desmin deficiency, Intermediate Filaments physiology, Keratin-19 deficiency, Muscle Fibers, Fast-Twitch physiology
- Abstract
Intermediate filaments (IFs), composed of desmin and keratins, link myofibrils to each other and to the sarcolemma in skeletal muscle. Fast-twitch muscle of mice lacking the IF proteins, desmin and keratin 19 (K19), showed reduced specific force and increased susceptibility to injury in earlier studies. Here we tested the hypothesis that the number of malformed myofibers in mice lacking desmin (Des(-/-)), keratin 19 (K19(-/-)), or both IF proteins (double knockout, DKO) is increased and is coincident with altered excitation-contraction (EC) coupling Ca(2+) kinetics, as reported for mdx mice. We quantified the number of branched myofibers, characterized their organization with confocal and electron microscopy (EM), and compared the Ca(2+) kinetics of EC coupling in flexor digitorum brevis myofibers from adult Des(-/-), K19(-/-), or DKO mice and compared them to age-matched wild type (WT) and mdx myofibers. Consistent with our previous findings, 9.9% of mdx myofibers had visible malformations. Des(-/-) myofibers had more malformations (4.7%) than K19(-/-) (0.9%) or DKO (1.3%) myofibers. Confocal and EM imaging revealed no obvious changes in sarcomere misalignment at the branch points, and the neuromuscular junctions in the mutant mice, while more variably located, were limited to one per myofiber. Global, electrically evoked Ca(2+) signals showed a decrease in the rate of Ca(2+) uptake (decay rate) into the sarcoplasmic reticulum after Ca(2+) release, with the most profound effect in branched DKO myofibers (44% increase in uptake relative to WT). Although branched DKO myofibers showed significantly faster rates of Ca(2+) clearance, the milder branching phenotype observed in DKO muscle suggests that the absence of K19 corrects the defect created by the absence of desmin alone. Thus, there are complex roles for desmin-based and K19-based IFs in skeletal muscle, with the null and DKO mutations having different effects on Ca(2+) reuptake and myofiber branching.
- Published
- 2012
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34. Optimization of large gel 2D electrophoresis for proteomic studies of skeletal muscle.
- Author
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Reed PW, Densmore A, and Bloch RJ
- Subjects
- Adult, Animals, Cells, Cultured, Humans, Molecular Weight, Muscle Fibers, Skeletal chemistry, Muscle Proteins chemistry, Muscle, Skeletal cytology, Myoblasts chemistry, Proteolysis, Rats, Rats, Sprague-Dawley, Reproducibility of Results, Sensitivity and Specificity, Solubility, Thiourea chemistry, Urea chemistry, Electrophoresis, Gel, Two-Dimensional methods, Muscle Proteins analysis, Muscle, Skeletal chemistry, Proteomics methods
- Abstract
We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
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35. Hydrophobic residues in small ankyrin 1 participate in binding to obscurin.
- Author
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Willis CD, Oashi T, Busby B, Mackerell AD Jr, and Bloch RJ
- Subjects
- Alanine genetics, Alanine metabolism, Amino Acid Sequence, Animals, Ankyrins genetics, Binding Sites, Hydrophobic and Hydrophilic Interactions, Leucine genetics, Leucine metabolism, Molecular Dynamics Simulation, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Rats, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Surface Plasmon Resonance, Ankyrins chemistry, Ankyrins metabolism, Guanine Nucleotide Exchange Factors chemistry, Guanine Nucleotide Exchange Factors metabolism, Muscle Proteins chemistry, Muscle Proteins metabolism
- Abstract
Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc₆₃₁₆₋₆₃₄₅ and Obsc₆₂₃₁₋₆₂₆₀. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc₆₃₁₆₋₆₃₄₅. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc₆₂₃₁₋₆₂₆₀. Alanine mutagenesis of the five hydrophobic residues present in Obsc₆₃₁₆₋₆₃₄₅ showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc₆₂₃₁₋₆₂₆₀ had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc₆₃₁₆₋₆₃₄₅-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc₆₃₁₆₋₆₃₄₅. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc₆₃₁₆₋₆₃₄₅ for sAnk1 and to the dominant role exhibited by this sequence in binding.
- Published
- 2012
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36. Influences of desmin and keratin 19 on passive biomechanical properties of mouse skeletal muscle.
- Author
-
Shah SB, Love JM, O'Neill A, Lovering RM, and Bloch RJ
- Subjects
- Aging, Animals, Cell Membrane chemistry, Cell Membrane physiology, Cytoskeleton chemistry, Cytoskeleton physiology, Desmin physiology, Female, Intermediate Filaments chemistry, Intermediate Filaments physiology, Keratin-19 physiology, Mice, Mice, Transgenic, Muscle, Skeletal physiology, Muscular Diseases genetics, Muscular Diseases pathology, Weight-Bearing, Desmin genetics, Keratin-19 genetics, Muscle, Skeletal chemistry, Structure-Activity Relationship
- Abstract
In skeletal muscle fibers, forces must be transmitted between the plasma membrane and the intracellular contractile lattice, and within this lattice between adjacent myofibrils. Based on their prevalence, biomechanical properties and localization, desmin and keratin intermediate filaments (IFs) are likely to participate in structural connectivity and force transmission. We examined the passive load-bearing response of single fibers from the extensor digitorum longus (EDL) muscles of young (3 months) and aged (10 months) wild-type, desmin-null, K19-null, and desmin/K19 double-null mice. Though fibers are more compliant in all mutant genotypes compared to wild-type, the structural response of each genotype is distinct, suggesting multiple mechanisms by which desmin and keratin influence the biomechanical properties of myofibers. This work provides additional insight into the influences of IFs on structure-function relationships in skeletal muscle. It may also have implications for understanding the progression of desminopathies and other IF-related myopathies.
- Published
- 2012
- Full Text
- View/download PDF
37. Distinct effects of contraction-induced injury in vivo on four different murine models of dysferlinopathy.
- Author
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Roche JA, Ru LW, and Bloch RJ
- Subjects
- Animals, Dysferlin, Histocytochemistry, Inflammation, Macrophages pathology, Membrane Proteins genetics, Membrane Proteins physiology, Mice, Mice, Inbred Strains, Mice, Transgenic, Muscle Contraction, Muscle Fibers, Skeletal pathology, Muscle, Skeletal cytology, Muscle, Skeletal pathology, Necrosis, Torque, Disease Models, Animal, Muscle, Skeletal injuries, Muscular Dystrophies, Limb-Girdle pathology
- Abstract
Mutations in the DYSF gene, encoding dysferlin, cause muscular dystrophies in man. We compared 4 dysferlinopathic mouse strains: SJL/J and B10.SJL-Dysf(im)/AwaJ (B10.SJL), and A/J and B6.A-Dysf(prmd)/GeneJ (B6.A/J). The former but not the latter two are overtly myopathic and weaker at 3 months of age. Following repetitive large-strain injury (LSI) caused by lengthening contractions, all except B6.A/J showed ~40% loss in contractile torque. Three days later, torque in SJL/J, B10.SJL and controls, but not A/J, recovered nearly completely. B6.A/J showed ~30% torque loss post-LSI and more variable recovery. Pre-injury, all dysferlinopathic strains had more centrally nucleated fibers (CNFs) and all but A/J showed more inflammation than controls. At D3, all dysferlinopathic strains showed increased necrosis and inflammation, but not more CNFs; controls were unchanged. Dystrophin-null DMD(mdx) mice showed more necrosis and inflammation than all dysferlin-nulls. Torque loss and inflammation on D3 across all strains were linearly related to necrosis. Our results suggest that (1) dysferlin is not required for functional recovery 3 days after LSI; (2) B6.A/J mice recover from LSI erratically; (3) SJL/J and B10.SJL muscles recover rapidly, perhaps due to ongoing myopathy; (4) although they recover function to different levels, all 4 dysferlinopathic strains show increased inflammation and necrosis 3 days after LSI.
- Published
- 2012
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38. Muscle contractility and cell motility.
- Author
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Jin JP, Bloch RJ, Huang X, and Larsson L
- Subjects
- Animals, Humans, Mice, Molecular Motor Proteins physiology, Myocardial Contraction physiology, Cell Movement physiology, Muscle Contraction physiology
- Published
- 2012
- Full Text
- View/download PDF
39. Lack of correlation between outcomes of membrane repair assay and correction of dystrophic changes in experimental therapeutic strategy in dysferlinopathy.
- Author
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Lostal W, Bartoli M, Roudaut C, Bourg N, Krahn M, Pryadkina M, Borel P, Suel L, Roche JA, Stockholm D, Bloch RJ, Levy N, Bashir R, and Richard I
- Subjects
- Animals, Bystander Effect genetics, Dependovirus genetics, Dysferlin, Female, Gene Deletion, Gene Expression Regulation genetics, Humans, Male, Membrane Fusion genetics, Membrane Proteins deficiency, Membrane Proteins genetics, Mice, Mice, Transgenic, Muscle Proteins genetics, Muscles metabolism, Muscles pathology, Muscles physiopathology, Muscular Dystrophies, Limb-Girdle therapy, Phenotype, Sarcolemma metabolism, Sarcolemma pathology, Treatment Outcome, Cell Membrane metabolism, Cell Membrane pathology, Genetic Therapy methods, Muscular Dystrophies, Limb-Girdle genetics, Muscular Dystrophies, Limb-Girdle pathology
- Abstract
Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fulfilling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.
- Published
- 2012
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40. Synemin isoforms differentially organize cell junctions and desmin filaments in neonatal cardiomyocytes.
- Author
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Lund LM, Kerr JP, Lupinetti J, Zhang Y, Russell MA, Bloch RJ, and Bond M
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- Actinin metabolism, Animals, Animals, Newborn, Cell Adhesion physiology, Intermediate Filament Proteins chemistry, Intermediate Filament Proteins genetics, Intermediate Filaments physiology, Isomerism, Primary Cell Culture, RNA, Messenger metabolism, RNA, Small Interfering pharmacology, Rats, Sarcomeres physiology, Solubility, Vinculin metabolism, Desmin physiology, Intercellular Junctions physiology, Intermediate Filament Proteins physiology, Myocytes, Cardiac cytology, Myocytes, Cardiac physiology
- Abstract
Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and β forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell-cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas β-synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell-cell junctions, with no effect on sarcomeric organization. Solubility assays showed that β-synemin was soluble and interacted with sarcomeric α-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that β-synemin mediates the association of desmin IFs with Z disks, whereas α-synemin stabilizes junctional complexes between cardiomyocytes.
- Published
- 2012
- Full Text
- View/download PDF
41. Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation.
- Author
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Roche JA, Ford-Speelman DL, Ru LW, Densmore AL, Roche R, Reed PW, and Bloch RJ
- Subjects
- Animals, Antigens, CD analysis, Antigens, Differentiation, Myelomonocytic analysis, Green Fluorescent Proteins genetics, Macrophages, Male, Mice, Mice, Inbred C57BL, Muscle Development genetics, Muscle Development physiology, Muscle Strength genetics, Satellite Cells, Skeletal Muscle cytology, Satellite Cells, Skeletal Muscle physiology, Transgenes, Electroporation, Gene Transfer Techniques, Muscle, Skeletal cytology, Muscle, Skeletal physiology
- Abstract
Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.
- Published
- 2011
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- View/download PDF
42. Unmasking potential intracellular roles for dysferlin through improved immunolabeling methods.
- Author
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Roche JA, Ru LW, O'Neill AM, Resneck WG, Lovering RM, and Bloch RJ
- Subjects
- Animals, Dysferlin, Humans, Male, Membrane Proteins analysis, Mice, Muscle Proteins analysis, Muscle, Skeletal ultrastructure, Muscular Dystrophies diagnosis, Rats, Rats, Sprague-Dawley, Fluorescent Antibody Technique methods, Membrane Proteins metabolism, Muscle Proteins metabolism, Muscle, Skeletal chemistry
- Abstract
Mutations in the DYSF gene that severely reduce the levels of the protein dysferlin are implicated in muscle-wasting syndromes known as dysferlinopathies. Although studies of its function in skeletal muscle have focused on its potential role in repairing the plasma membrane, dysferlin has also been found, albeit inconsistently, in the sarcoplasm of muscle fibers. The aim of this article is to study the localization of dysferlin in skeletal muscle through optimized immunolabeling methods. We studied the localization of dysferlin in control rat skeletal muscle using several different methods of tissue collection and subsequent immunolabeling. We then applied our optimized immunolabeling methods on human cadaveric muscle, control and dystrophic human muscle biopsies, and control and dysferlin-deficient mouse muscle. Our data suggest that dysferlin is present in a reticulum of the sarcoplasm, similar but not identical to those containing the dihydropyridine receptors and distinct from the distribution of the sarcolemmal protein dystrophin. Our data illustrate the importance of tissue fixation and antigen unmasking for proper immunolocalization of dysferlin. They suggest that dysferlin has an important function in the internal membrane systems of skeletal muscle, involved in calcium homeostasis and excitation-contraction coupling.
- Published
- 2011
- Full Text
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43. Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1.
- Author
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Ackermann MA, Ziman AP, Strong J, Zhang Y, Hartford AK, Ward CW, Randall WR, Kontrogianni-Konstantopoulos A, and Bloch RJ
- Subjects
- Animals, Ankyrins genetics, Muscle Proteins genetics, Muscle Proteins metabolism, Proteolipids genetics, Proteolipids metabolism, Rats, Sarcoplasmic Reticulum genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Ankyrins metabolism, Muscle, Skeletal metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca(2+) channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca(2+) were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.
- Published
- 2011
- Full Text
- View/download PDF
44. Determinants of the repeated-bout effect after lengthening contractions.
- Author
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Dipasquale DM, Bloch RJ, and Lovering RM
- Subjects
- Adaptation, Physiological physiology, Animals, Cumulative Trauma Disorders etiology, Male, Muscle, Skeletal pathology, Range of Motion, Articular, Rats, Rats, Sprague-Dawley, Tarsus, Animal, Cumulative Trauma Disorders physiopathology, Cumulative Trauma Disorders prevention & control, Muscle Contraction physiology, Muscle, Skeletal physiopathology
- Abstract
Objective: Stresses to skeletal muscle often result in injury. A subsequent bout of the same activity performed days or even weeks after an initial bout results in significantly less damage. The underlying causes of this phenomenon, termed the "repeated-bout effect" (RBE), are unclear. This study compared the protective effect of two different injury protocols on the ankle dorsiflexors in the rat. We hypothesized that the RBE would occur soon after the initial injury and persist for several weeks and that the RBE would occur even if the second injury was performed under different biomechanical conditions than the first., Design: In this controlled laboratory study, the dorsiflexor muscles in the left hind limbs of adult male Sprague-Dawley rats (N = 75) were subjected to ten repetitions of large-strain lengthening contractions or 150 repetitions of small-strain lengthening contractions., Results: Both protocols induced a significant (P < 0.001) and similar loss of isometric torque (approximately 50%) after the first bout of contractions. The RBE occurred as early as 2 days after the injury and remained high for 14 days (P < 0.001) but diminished by 28 days and was lost by 42 days. The small-strain contractions offered a protective effect against a subsequent large-strain contraction, but not vice versa. Although the RBE did not occur sooner than day 2, the early recovery after a second large-strain injury performed 8 hrs after the first was 2-fold greater than after a single injury., Conclusions: The RBE is both rapid in onset and prolonged, and some, but not all, injuries can protect against different types of subsequent injury.
- Published
- 2011
- Full Text
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45. Crystallin-gazing: unveiling enzymatic activity.
- Author
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Reed PW and Bloch RJ
- Subjects
- Animals, Crystallins metabolism, Humans, Ketamine metabolism, Oxidoreductases Acting on CH-NH Group Donors metabolism, Thyroid Hormones metabolism, mu-Crystallins, Crystallins physiology, Thyroid Hormones physiology
- Published
- 2011
- Full Text
- View/download PDF
46. Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies.
- Author
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Busby B, Oashi T, Willis CD, Ackermann MA, Kontrogianni-Konstantopoulos A, Mackerell AD Jr, and Bloch RJ
- Subjects
- Ankyrins chemistry, Ankyrins genetics, Guanine Nucleotide Exchange Factors chemistry, Guanine Nucleotide Exchange Factors genetics, Humans, Maltose-Binding Proteins genetics, Maltose-Binding Proteins metabolism, Molecular Dynamics Simulation, Muscle Proteins chemistry, Muscle Proteins genetics, Mutagenesis, Site-Directed, Mutation genetics, Protein Conformation, Protein Serine-Threonine Kinases, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Rho Guanine Nucleotide Exchange Factors, Surface Plasmon Resonance, Ankyrins metabolism, Guanine Nucleotide Exchange Factors metabolism, Models, Molecular, Muscle Proteins metabolism, Muscle, Skeletal metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
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47. Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes.
- Author
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Cappello RE, Estrada-Gutierrez G, Irles C, Giono-Cerezo S, Bloch RJ, and Nataro JP
- Subjects
- Actinin metabolism, Actins metabolism, Bacterial Toxins genetics, Bacterial Toxins metabolism, Cell Line, Enterotoxins genetics, Enterotoxins metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Flow Cytometry, Focal Adhesion Protein-Tyrosine Kinases metabolism, Humans, Immunoblotting, Immunoprecipitation, Paxillin metabolism, Plasmids, Serine Endopeptidases genetics, Serine Endopeptidases metabolism, Spectrin metabolism, Vinculin metabolism, Bacterial Toxins toxicity, Enterotoxins toxicity, Escherichia coli physiology, Escherichia coli Proteins toxicity, Focal Adhesions drug effects, Serine Endopeptidases toxicity
- Abstract
Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
48. Physiology, structure, and susceptibility to injury of skeletal muscle in mice lacking keratin 19-based and desmin-based intermediate filaments.
- Author
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Lovering RM, O'Neill A, Muriel JM, Prosser BL, Strong J, and Bloch RJ
- Subjects
- Animals, Desmin genetics, Female, Intermediate Filament Proteins genetics, Intermediate Filament Proteins metabolism, Keratin-19 genetics, Male, Mice, Mice, Knockout, Motor Activity physiology, Muscle Contraction physiology, Muscle Fibers, Fast-Twitch metabolism, Muscle Fibers, Fast-Twitch ultrastructure, Muscle, Skeletal injuries, Sarcolemma metabolism, Sarcolemma ultrastructure, Desmin metabolism, Intermediate Filaments metabolism, Keratin-19 metabolism, Muscle, Skeletal physiology, Muscle, Skeletal ultrastructure
- Abstract
Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.
- Published
- 2011
- Full Text
- View/download PDF
49. Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice.
- Author
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García-Pelagio KP, Bloch RJ, Ortega A, and González-Serratos H
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Mice, Knockout, Costameres metabolism, Dystrophin deficiency, Dystrophin metabolism, Muscle Fibers, Skeletal metabolism, Sarcolemma metabolism
- Abstract
We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8-3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.
- Published
- 2011
- Full Text
- View/download PDF
50. The actin binding domain of βI-spectrin regulates the morphological and functional dynamics of dendritic spines.
- Author
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Nestor MW, Cai X, Stone MR, Bloch RJ, and Thompson SM
- Subjects
- Animals, Animals, Newborn, Binding Sites, CA1 Region, Hippocampal metabolism, Cell Shape, Dendritic Spines ultrastructure, Microfilament Proteins, Nerve Tissue Proteins, Protein Isoforms, Rats, Spectrin metabolism, rac GTP-Binding Proteins metabolism, Actins metabolism, Dendritic Spines physiology, Spectrin physiology
- Abstract
Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.
- Published
- 2011
- Full Text
- View/download PDF
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