Abstract submissions to News Directions in Biology and Disease of Skeletal Muscle, April 23-26, Dallas, Texas

Abstract Title: Gene Expression Profiling to Monitor Therapeutic and Adverse Effects of Antisense Therapies for Duchenne Muscular Dystrophy
Presenting/First Author: 't Hoen, Peter A (Postdoc - Degree Year: 4)
Department, Institution: Center for Human and Clinical Genetics, Leiden University Medical Center
Address: PO Box 9600 LEIDEN, 2300 RC Netherlands
Phone/Fax: +31 71 5269421 / +31 71 5268285
Email: [email protected]
Abstract Theme: Therapies
Author(s): 't Hoen, Peter A (Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC LEIDEN)
van der Wees, Caroline G (Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC LEIDEN)
Aartsma-Rus, Annemieke (Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC LEIDEN)
Goyenvalle, Aurélie (Genethon & CNRS UMR 8115, 1, rue de l'Internationale, Evry, France)
Garcia, Luis (Genethon & CNRS UMR 8115, 1, rue de l'Internationale, Evry, France)
van Ommen, Gertjan (Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC LEIDEN)
den Dunnen, Johan T (Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC LEIDEN)
van Deutekom, Judith C (Center for Human and Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC LEIDEN)
Body of Abstract: We used expression profiling to monitor the efficacy and possible adverse effects of antisense therapies for Duchenne muscular dystrophy (DMD). Muscles from mdx mice were injected with antisense constructs that restore the open reading frame in the Dmd gene. Polyethylenimine (PEI)-complexed antisense oligonucleotides (AONs) were more effective than uncomplexed, F127- or Optison-complexed AONs. In contrast to F127 and Optison, PEI induced the expression of many immune genes, reflecting an aggravation of the inflammation present in untreated mdx mice. The level of transcript correction was too low to cause a significant shift towards wild-type expression patterns. Injection with adenoassociated virus-expressed antisense sequences resulted in much higher production of dystrophin. Depending on the efficacy, the expression of genes known to be elevated in muscular dystrophies, partly or completely returned to wild-type expression levels. Reductions in inflammation and fibrosis were among the most prominent changes observed. We conclude that expression profiling is a powerful tool to monitor desired and adverse effects of new therapeutic approaches in preclinical research and clinical trials.
Funded by Prinses Beatrix Fonds.


 
Abstract Title: Towards Clinical Studies on Antisense-mediated Exon Skipping in Duchenne Muscular Dystrophy.
Presenting/First Author: 't Hoen, Peter-Bram A (Postdoc - Degree Year: )
Department, Institution: Center for Human and Clinical Genetics, Leiden University Medical Center
Address: Einthovenweg 20- Postbus 9600 Leiden, 2300 RC Netherlands
Phone/Fax: +31-71-526 9421 / +31-71-526 8285
Email: [email protected]
Abstract Theme: Therapies
Author(s): 't Hoen, Peter-Bram A (Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands)
Heemskerk, Hans (Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands)
de Winter, Christa (Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands)
Verschuuren, Jan J (Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands)
van Kuik, Petra (Prosensa BV, Leiden, The Netherlands)
de Kimpe, Sjef (Prosensa BV, Leiden, The Netherlands)
Platenburg, Gerard J (Prosensa BV, Leiden, The Netherlands)
van Deutekom, Judith C (Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands)
Body of Abstract: For Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs)have demonstrated to be an efficient and relatively simple and safe alternative to gene replacement approaches. By inducing specific exon skipping, AONs allow restoration of the open reading frame of mutated DMD transcripts and turn on the synthesis of internally truncated, but largely functional dystrophins. In the first half of 2006, a clinical study, based on intramuscular injections of an AON, will be undertaken in a selected group of DMD patients, of which the set-up will be discussed. To facilitate full-body treatment of DMD patients, we are now focusing on the development of a safe and efficient systemic AON-delivery method. Using the mdx mouse model, we compare efficacy, persistence and biodistribution of an AON targeting the mutated exon 23. We have obtained high exon 23 skipping and dystrophin levels (up to 45%) in different muscles, including diaphragm and heart. We determined the p hysiological stability of the AON in the different organs and tissues, and noted a half life of 10 days in skeletal muscle. The overall therapeutic effect was further demonstrated by decreased creatin kinase levels and improved muscle performances in RotaRod studies.


 
Abstract Title: The role of alpha-syntrophin in assembly of the neuromuscular junction signaling scaffold
Presenting/First Author: Adams, Marvin E (Faculty)
Department, Institution: Physiology and Biophysics, University of Washington
Address: 1959 Pacific ST NE Seattle, WA 98195 United States
Phone/Fax: 206 543-9094 / 206 68500619
Email: [email protected]
Abstract Theme: Sarcolemmal Excitability
Author(s): Adams, Marvin E (University of Washington, Seattle, WA 98195)
Froehner, Stanley C (University of Washington, Seattle, WA 98195)
 
Body of Abstract: Dystrophin and its homologues utrophin and dystrobrevin are concentrated at the postsynaptic neuromuscular junction (NMJ) where they form scaffolding complexes with the adapter protein, alpha-syntrophin. The NMJs of alpha-syntrophin null mice have reduced levels and abnormal distribution of acetylcholine receptors(AChRs). These mice lack both utrophin and neuronal nitric oxide synthase (nNOS) at the NMJ. We generated transgenic mice that express alpha-syntrophin lacking the PDZ domain or the first pleckstrin homology (PH) domain and tested if the mutated alpha-syntrophin restores the NMJ scaffold in alpha-syntrophin null mice. We find that the PDZ domain of syntrophin is required for nNOS localization to the NMJ and for proper distribution of AChRs. Likewise the AChRs remain abnormally distributed when only the PH domain of alpha-syntrophin is missing. Surprisingly, nNOS localization at the synapse (and sarcolemma) could not be restored by alpha-syntrophin mi ssing the first PH domain despite the presence of a functional PDZ domain. Utrophin localization to the NMJ did not require the PDZ or PH domain. The domains of alpha-syntrophin play distinct roles in synapse formation/maintenance at the NMJ. Supported by NIH and MDA.


 
Abstract Title: Identification of the cofilin-2 gene, CFL2, as a probable sixth gene for nemaline myopathy
Presenting/First Author: Agrawal, Pankaj B (Faculty)
Department, Institution: Medicine, Children's Hospital
Address: 300 Longwood Ave Boston, MA 02115 United States
Phone/Fax: 6179192153 / 6177300253
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Agrawal, Pankaj B (Children's Hospital and Harvard Medical School, Boston)
Schweikert, Rebecca (Children's Hospital and Harvard Medical School, Boston)
Darras, Basil T (Children's Hospital and Harvard Medical School, Boston)
Beggs, Alan H (Children's Hospital and Harvard Medical School, Boston)
 
Body of Abstract: Nemaline Myopathy (NM) is characterized by skeletal muscle weakness and presence of nemaline bodies. Causative mutations have been described in NEB, ACTA1, TPM2, TPM3, and TNNT1 genes. Cofilin-2 (CFL2) is a candidate gene for NM as it encodes a thin filament protein involved in actin regulation. We screened 132 unrelated NM cases and found a homozygous missense CFL2 mutation in two siblings who had typical NM. Microscopically, the proband’s muscle showed moderate numbers of nemaline bodies. The proband and her affected sibling were homozygous for c.103G>A mutation resulting in the Ala35Thr change. An unaffected sibling and both parents, who are first cousins, were heterozygous. The mutation was not seen in 282 controls including 91 from the same geographical region. On comparing the expression of cofilin-2 in the patient with that in age-matched normal skeletal muscle, the patient’s muscle had dramatically lower amounts of cofilin-2 on immunofluorescence, and 2-D SDS-PAGE with Western blot. Expression of both normal and mutant cofilin-2 in E. coli demonstrated that the mutant protein was considerably less soluble than the wild type, supporting that CFL2 likely represents a sixth gene responsible for NM.


 
Abstract Title: Regulation of satellite cell growth and differentiation by extracellular heparan sulfate 6-O endosulfatases
Presenting/First Author: Ai, Xingbin (Faculty)
Department, Institution: cell & Dev. Biol, Boston Biomedical Research Institute
Address: 64 Grove Street Watertown, MA 02476 United States
Phone/Fax: 6176587869 / 6179721759
Email: [email protected]
Abstract Theme: Repair Mechanisms
Author(s): Ai, Xingbin (Boston Biomedical Research Institute, Watertown, MA 02476)
Langsdorf, Ally (Boston Biomedical Research Institute, Watertown, MA 02476)
Emerson, Charles P (Boston Biomedical Research Institute, Watertown, MA 02476)
 
Body of Abstract: Satellite cells are quiescent “stem” cells in adult skeletal muscles that can be activated upon injury to repair damaged muscle under physiological and disease conditions. Although signals involved in the growth and differentiation of satellite cells are known, the regulatory mechanisms that coordinate the activities of these signals in intact and regenerating muscles have not been characterized. Both intact and injured muscles express a number of growth factors that require 6-O sulfated heparan sulfate (HS) to form functional signaling complexes to induce the proliferation and differentiation of satellite cells. We recently have identified a novel family of HS endosulfatases (Sulfs) that catalytically remove HS 6-O sulfates to repress growth factor signaling. Mouse endosulfatase 1 (MSulf1) and MSulf2 are differentially expressed by quiescent and activated satellite cells, but not by muscle fibers. Loss of MSulfs leads to delayed myofiber differentiation and prolo nged satellite cell proliferation in response to FGF2. We are investigating roles of MSulf1 and MSulf2 in satellite cell growth and differentiation using tissue culture model and cardiotoxin-induced acute injury model. This work is supported by MDA.


 
Abstract Title: Rigid spine muscular dystrophy caused by a homozygous mutation in the 3’ UTR Selenocystein Insertion Sequence Element of Selenoprotein N
Presenting/First Author: Allamand, Valérie (Faculty)
Department, Institution: Institut de Myologie- GH Pitié-Salpétriè, INSERM U582
Address: 47 Boulevard de l'Hôpital Paris Cedex 13, 75651 France
Phone/Fax: +33 1 42 16 57 43 / +33 1 42 16 57 00
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Allamand, Valérie (INSERM U582, Institut de Myologie, IFR 14, Groupe Hospitalier Pitié-Salpêtrière, Université Pierre et Marie Curie, Paris, France)
Richard, Pascale (Assistance Publique-Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, UF Cardiogénétique et Myogénétique, Service de Biochimie B, Paris, France)
Lescure, Alain (CNRS-Université Louis Pasteur, UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France)
Ferreiro, Ana (INSERM U582, Institut de Myologie, IFR 14, Groupe Hospitalier Pitié-Salpêtrière, Université Pierre et Marie Curie, Paris, France)
Krol, Alain (CNRS-Université Louis Pasteur, UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France)
Pellegrini, Nadine (Hôpital Raymond Poincaré, Garches, France)
Urtizberea, Andoni J (Hôpital Raymond Poincaré, Garches, France)
Guicheney, Pascale (INSERM U582, Institut de Myologie, IFR 14, Groupe Hospitalier Pitié-Salpêtrière, Université Pierre et Marie Curie, Paris, France)
Body of Abstract: Selenoprotein N (SelN) was the first selenoprotein shown to be involved in a genetic disorder. The SEPN1-related myopathy regroups four autosomal recessive disorders: Rigid Spine Muscular Dystrophy (RSMD1), multiminicore disease, desmin-related myopathy with Mallory Body-like inclusions and congenital fiber type disproportion.
Here we report the first mutation in the Selenocysteine Insertion Sequence (SECIS) of SelN, in a patient presenting a classical although rather mild form of RSMD1. In cultured fibroblasts mRNA and protein levels were greatly decreased. We also demonstrated that this mutation abolishes SECIS-binding protein 2 (SBP2) binding to SECIS in vitro, thereby preventing cotranslational incorporation of selenocysteine and SelN synthesis.
This is the first mutation identified in the “non-Watson-Crick” quartet of the SECIS element, a hairpin structure located in the 3’ UTR allowing, through interactions with trans-acting partners, the insertion of a selenocysteine residue at the UGA codon. The identification of this mutation reveals the structural basis for a novel mechanism leading to SEPN1-related myopathy.

Supports: INSERM, AFM, GIS-Institut des Maladies Rares, Assistance Publique-Hôpitaux de Paris.


 
Abstract Title: Studies of sarcoglycan trafficking and localization in Drosophila
Presenting/First Author: Allikian, Michael J (Faculty)
Department, Institution: Medicine, University of Chicago
Address: 5841 S. Maryland, MC6088 Chicago, IL 60637 United States
Phone/Fax: 773 702-2684 / 773 702-2681
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Allikian, Michael J (University of Chicago, Chicago, IL 60637)
Bhabha, Gira (University of Chicago, Chicago, IL 60637)
McNally, Elizabeth M (University of Chicago, Chicago, IL 60637)
 
Body of Abstract: The dystrophin glycoprotein complex (DGC) is essential for plasma membrane stability in skeletal muscle myofibers and cardiomyocytes. Mutations in the genes encoding dystrophin and its associated proteins, the sarcoglycans, lead to muscular dystrophy in humans and in mouse models. Trafficking of the sarcoglycan complex to the sarcolemma is a critical step for skeletal myofiber and cardiomyocyte maintenance. However, little is known about the molecular mediators of this process. To identify proteins necessary for sarcoglycan localization and interaction, we created transgenic Drosophila that express a properly localized and labeled delta-sarcoglycan subunit. We subsequently carried out a genetic screen using this transgenic fly line and identified two different deficiencies that harbor genes necessary for proper sarcoglycan processing and localization. These deficiencies, both on chromosome 2, encompass many loci. One is comprised of 21 genes, the other is su bstantially larger and contains 130 genes. For the smaller region, lines were obtained that are mutant for each of the known genes in the interval. For the larger deletion, we opted to examine lines that had smaller deletions to narrow down the interval of interest.


 
Abstract Title: Viral expression of IGF-IB drives proliferation at the expense of hypertrophy in mdx skeletal muscle
Presenting/First Author: Barton, Elisabeth R (Faculty)
Department, Institution: Anatomy and Cell Biology, University of Pennsylvania School of Dental Medicine
Address: 240 S. 40th Street Philadelphia, PA 19104 United States
Phone/Fax: 215-573-0887 / 215-573-2324
Email: [email protected]
Abstract Theme: Regulation of Muscle Mass
Author(s): Barton, Elisabeth R (University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104)
Feng, Jessie (University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104)
Ma, Rong-Ine (University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104)
Tian, Zuozhen (University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104)
 
Body of Abstract: Alternative splicing of the insulin-like growth factor I gene gives rise to at least 4 peptide classes, leading to the production of an identical IGF-I protein but different E-peptide extensions. We have recently shown that viral delivery of murine IGF-IA and IGF-IB produce hypertrophy in young growing animals, yet only IGF-IA can increase muscle mass in mature animals. This raises the possibility that the IGF-IB target is only available during the growth phase, and could be activated satellite cells. We tested this hypothesis using viral delivery of either IGF-IA or IGF-IB in mature mdx mice (which have activated satellite cells due to ongoing degeneration/regeneration) and in mature wildtype mice. Hypertrophy was observed in mdx and wildtype muscles treated with IGF-IA. In contrast, muscles treated with IGF-IB produced no hypertrophy, and IGF-IB treated mdx muscles had large regions of unresolved proliferation. These results are consistent with IGF-IB mediated p roliferation via satellite cell targets. It suggests that transient expression of IGF-IB in concert with damage might boost proliferation of these cells to enhance repair. However, constant expression of this isoform may be detrimental to completion of repair in muscle.


 
Abstract Title: Designing oligonucleotides containing CpG modifications to recruit specific repair mechanisms leads to improved gene correction efficiency in skeletal muscles of mdx mice.
Presenting/First Author: Bertoni, Carmen (Faculty)
Department, Institution: Neurology, Stanford University
Address: 300 Pasteur drive Room A-343 Stanford, CA 94304 United States
Phone/Fax: (650) 493 5000 Ext.62073 / (50)858 3935
Email: [email protected]
Abstract Theme: Therapies
Author(s): Bertoni, Carmen (Stanford University, Stanford, CA 94304)
Rustagi, Arjun (Stanford University, Stanford, CA 94304)
Rando, Thomas A (Stanford University, Stanford, CA 94304)
 
Body of Abstract: Gene editing mediated by single stranded oligonucleotides (ssODNs) represents an appealing option to DMD since it has the potential to treat both single point mutations as well as deletions that cause a frame shift of the dystrophin mRNA. We have focused on the development of new vectors capable of activating specific repair mechanisms to direct the correction of mutations in genomic DNA. The methyl binding protein 4 (MBD4) takes an active role in DNA repair and is highly expressed in muscles. MBD4 contains a specific glycosilase capable of recognizing a T to G transversion at CpG sites and direct the conversion of the thymine into methylcytosine. CpG modifications were introduced on the mutating base of the targeting oligonucleotide in the attempt to mimic a deamination of methylcytosine which results in the activation of MBD4. The ability of these modified ssODNs to increase gene repair was studied in mdx muscle cells in vitro and in vivo. The level of dystrophi n protein expression was significantly increased by the use of these modified ssODNs. Our studies demonstrated the specific recruitment of the MBD4-mediated repair pathway. Correction of the dystrophin gene was shown at the genomic level and was stable over time.


 
Abstract Title: Improved Quality of Life and Reduced Health Care Needs for Boys with Duchenne Muscular Dystrophy treated with Long-term Deflazacort
Presenting/First Author: Biggar, Doug W (Faculty)
Department, Institution: Pediatrics, Bloorview Kids Rehab
Address: 150 Kilgour Road Toronto, ON M4G 1R8 Canada
Phone/Fax: 416-424-3813 / 416-424-3840
Email: [email protected]
Abstract Theme: Therapies
Author(s): Biggar, Doug W (Bloorview Kids Rehab, Toronto, Ontario, Canada. M4G IR8)
Harris, Vivien A (Bloorview Kids Rehab, Toronto, Ontario, Canada. M4G IR8)
Eliasoph, Laila R (Bloorview Kids Rehab, Toronto, Ontario, Canada. M4G IR8)
Gonzales, Angela N (Bloorview Kids Rehab, Toronto, Ontario, Canada. M4G IR8)
MacLeod, Kathleen A (Bloorview Kids Rehab, Toronto, Ontario, Canada. M4G IR8)
 
Body of Abstract: ABSTRACT: We compare the clinical outcomes of 74 boys, 10 to 18 years of age with DMD: 40 were treated (T) and 34 were not treated (NT) with daily deflazacort. Boys-T were able to rise from supine to standing, climb 4 stairs and walk 10 metres without aids, 3-5 years longer than boys-NT. At 10 years, boys-T had significantly better pulmonary function than boys-NT and at 15 years, 8 of 34 boys-NT required nocturnal ventilation (T=0). Thirty of 34 boys-NT had a spinal curve greater than 20° compared to 4 of 40 boys-T. Most boys-NT required assistance with feeding after spine surgery compared to none of the boys-T. At 18 years, 7 of 34 boys-NT had lost 25% or more of their body weight (T=0) and 4 of those 7 boys required a gastric feeding tube. At 18 years, 20 of 34 boys-NT had cardiac left ventricular ejection fractions less than 45% compared to 4 of 40 boys-T. Twelve of 34 boys-NT died in their second decade (17.6 ± 1.7 years) primarily of cardio respiratory comp lications. Two of 40 boys-T died at 13 and 18 years of age from cardiac failure. The boys-T were significantly shorter, did not have excessive weight gain and 22 of 40 had asymptomatic cataracts. Long bone fractures occurred in 25% of boys in both the T and NT groups. We conclude that long-term deflazacort treatment has a very significant impact on health, quality of life and health care needs for boys with DMD and their families, and is usually associated with few side effects.


 
Abstract Title: Differential expression of iron metabolism proteins in normal human skeletal and cardiac muscle
Presenting/First Author: Boyer, Philip J (Faculty)
Department, Institution: Pathology/Neuropath., Univ. of Texas Southwestern
Address: 5323 Harry Hines Blvd., Room H2.132 Dallas, TX 75390-9073 United States
Phone/Fax: 214-648-7170 / 214-648-2077
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Boyer, Philip J (Univ. of Texas Southwestern, Dallas, TX 75390)
Dellinger, Bonnie B (Penn State Univ., Hersehy, PA 17033)
Simmons, Zachary (Penn State Univ., Hersehy, PA 17033)
Carey, Evan P (Univ. of Texas Southwestern, Dallas, TX 75390)
Piner0, Diego J (Penn State Univ., Hersehy, PA 17033)
Connor, James R (Penn State Univ., Hersehy, PA 17033)
 
Body of Abstract: This study characterizes the distribution of the various iron metabolism proteins in striated muscle. Frozen sections from histologically normal skeletal muscle biopsies (N=10) and hearts obtained at autopsy (N=5) were evaluated by Perls' iron stain and by immunohistochemistry using antibodies against divalent metal transporter 1 (DMT1), ferroportin 1 (FP1), transferrin, transferrin receptor, and the hemochromatosis gene product (HFE). Type I and type II fibers were distinguished using ATPase histochemistry and myosin heavy chain immunohistochemistry. Iron was diffusely present in all myofibers. Skeletal myofibers were labeled by all markers with differentially strong expression of HFE and FP1 noted in type I and II fibers, respectively. In cardiac muscle, strong expression of both HFE and FP1 was noted; distinct localization of DMT1 and HFE was present at intercalated disks. The differential expression of HFE and FP1 in type I and II myofibers likely reflects di fferent iron requirements. Significant expression of HFE implies an important role for this protein in iron metabolism in striated muscle and is likely relevant to the accumulation of iron in cardiac muscle in hemochromatosis. (Support: Kimmel Family Philanthropic Fund)


 
Abstract Title: Severe muscular dystrophy in mice that lack the alpha7 integrin and dystrophin: a new dystrophic mouse model
Presenting/First Author: Burkin, Dean J (Faculty)
Department, Institution: Pharmacology, University of Nevada
Address: 1664 N. Virginia Reno, NV 89557 United States
Phone/Fax: 775-784-6288 / 775-784-1620
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Burkin, Dean J (Univ. of Nevada, Reno, NV 89557)
Rooney, Jachinta E (Univ. of Nevada, Reno, NV 89557)
Welser, Jennifer V (Univ. of Nevada, Reno, NV 89557)
Body of Abstract: The dystrophin glycoprotein complex and alpha7beta1 integrin are transmembrane receptors in skeletal muscle that provide molecular continuity between laminin in the extracellular matrix and the cell cytoskeleton. Loss of dystrophin results in Duchenne Muscular Dystrophy (DMD). Mutations in the alpha7 integrin gene cause congenital myopathy. The alpha7beta1 integrin is increased in the skeletal muscle of DMD patients and mdx mice. This observation led to the suggestion that dystrophin and the alpha7beta1 integrin might have complementary functional and structural roles. To test this hypothesis, we generated mice lacking both dystrophin and alpha7 integrin (mdx/alpha7-/-). mdx/alpha7-/- mice developed early onset muscular dystrophy highly akin to DMD patients and died between 2-4 weeks of age. Skeletal muscle fibers from mdx/alpha7-/- mice exhibited loss of membrane integrity, increased centrally located nuclei and mononuclear cell infiltrate compared to mdx or alph a7 integrin null animals. Loss of dystrophin and/or alpha7 integrin resulted in altered expression of laminin-alpha2 chain. These results point to complementary roles for dystrophin and the alpha7beta1 integrin in maintaining the functional integrity of skeletal muscle.


 
Abstract Title: Paired-like homeodomain transcription factor 1 (Pitx1) regulates genes involved in muscle atrophy
Presenting/First Author: Chen, Yi-Wen (Faculty)
Department, Institution: Center for Genetic Medicine Research, Children’s National Medical Center
Address: 111 Michigan Avenue, N.W. Washington, DC 20010 United States
Phone/Fax: 202-884-6025 / 202-884-6014
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Chen, Yi-Wen (Children’s National Medical Center, Washington, DC 20010)
Kostek, Matthew (Children’s National Medical Center, Washington, DC 20010)
Li, Ling (Children’s National Medical Center, Washington, DC 20010)
Shi, Rongye (Children’s National Medical Center, Washington, DC 20010)
Body of Abstract: Paired-like homeodomain transcription factor 1 (Pitx1) plays a critical role in specification of hindlimb identity, however its role in postnatal skeletal muscle is not known. By comparing expression profiles of facioscapulohumeral muscular dystrophy (FSHD) to 125 muscle profiles of 11 neuromuscular disorders, we showed PITX1 was specifically up-regulated 11 fold (p<0.01) and 13.2 (p<0.001) in affected and unaffected FSHD muscles, respectively. Further in vivo functional studies showed Pitx1 induced the ubiquitin-proteasome pathways, including up-regulation of Foxo1a and atrogin 1. Interestingly, although not up-regulated in other neuromuscular diseases, we found PITX1, FOXO1A and atrogin 1 was up-regulated in muscles undergoing atrophy after two days of inactivity. Using luciferase assay, we further showed that cotransfecting Pitx1 expression vector and a Foxo1a promoter fragment containing consensus Pitx1 binding sites leaded to 8 fold up-regulation (p<0.001 ) of luciferase activities. Our results suggest that PITX1 is likely to be involved in the early skeletal muscle atrophy process through regulating FOXO1A, an upstream regulator of atrogin 1 and the ubiquitin-ligase proteasome pathway.


 
Abstract Title: Muscle LIM Protein cooperates with titin to maintain the structural integrity of Drosophila muscle
Presenting/First Author: Clark, Kathleen A (Faculty)
Department, Institution: Biology, University of Utah
Address: 2000 Circle of Hope Salt Lake City, UT 84112 United States
Phone/Fax: 801 581 4793 / 801 581 2175
Email: [email protected]
Abstract Theme: Sarcomere Cytoskeleton
Author(s): Clark, Kathleen A (Univ. of Utah, Salt Lake City, UT 84112)
Bland, Jennifer M (Univ. of Utah, Salt Lake City, UT 84112)
Beckerle, Mary C (Univ. of Utah, Salt Lake City, UT 84112)
 
Body of Abstract: Muscle LIM Protein (MLP) is found at Z-lines and muscle termini in mature muscle, but is nuclear during muscle differentiation. Thus, MLP may have important roles as both a structural protein and regulator of gene expression. Mutations in MLP lead to cardiomyopathy, and MLP is potently upregulated in skeletal muscle during eccentric exercise; however, its cellular functions remain unknown.
We have undertaken the study of an MLP homolog in Drosophila melanogater. We generated null mutations in mlp84B and found that the protein is essential for post-embryonic muscle function. The mlp84B mutants cannot fully contract their body wall muscles during pupariation, and make a long, thin pupal case. Most animals arrest development at this point, and exhibit only limited muscle contractions. A few mutants eclose as adults, but have impaired flight. We looked for mutations in other genes that would modify the mlp84B phenotype. Flies null for mlp84B and heterozygous for a mutation in D-titin show marked enhancement of the mlp84B muscle phenotypes. Surprisingly, the mlp84B mutant muscles on their own do not have any observable structural defects, but co-reduction in D-titin activity produces a severe disruption of muscle organization.


 
Abstract Title: A Novel Fluorescent Nanocircuit for Measuring Conformational Heterogeneity of Myosin Bound to Thin Filaments
Presenting/First Author: Coffee, Pilar G (Grad)
Department, Institution: Biology, University of North Texas
Address: PO Box 305220 Denton, TX 76203 United States
Phone/Fax: 940-565-2683 / 940-565-4136
Email: [email protected]
Abstract Theme: Sarcomere Cytoskeleton
Author(s): Coffee, Pilar G (University of North Texas, Denton, TX 76203)
Root, Douglas D (University of North Texas, Denton, TX 76203)
 
Body of Abstract: A novel fluorescent nanocircuit assay is developed to study the dynamics of the interactions of actomyosin using LRET. Excitation of the CS-124 antenna with a pulsed laser excites a terbium chelate on the lever arm which transfers energy to its acceptor, Cy5-ADP on myosin, which in turn relays the energy to Cy7-Troponin T on the thin filament. The nanocircuit is broken if any of the three components is farther apart than their Ro distance for energy transfer to occur, so the decay of signal from the terminal acceptor reports on the conformation of myosin S1 bound near troponin while the decays of previous fluors of the circuit report on the average conformations of all S1 regardless of their position. The nanocircuit has allowed us to distinguish pre- versus post-powerstroke conformations of skeletal myosin S1 when it is bound near troponin on the thin filament. Additionally, it has allowed us to detect changes in the relationship of troponin T relative to myos in in the presence and absence of muscle contraction triggering molecule calcium. A speculative model is suggested that myosins binding adjacent to the troponin complex are unable to complete their powerstroke but might contribute to the activation of the thin filament.


 
Abstract Title: Identification of putative in vivo substrates of calpain 3 by using comparative proteomics of overexpressing transgenic and non-transgenic mice
Presenting/First Author: Cohen, Niaz (Postdoc - Degree Year: 2)
Department, Institution: Neurology and Pediatrics, University of California, Los Angeles
Address: 635 Young Dr. South Los Angeles, CA 90095-7334 United States
Phone/Fax: 310-794-5225 / 310-206-1998
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Cohen, Niaz (University of California, Los Angeles, USA)
Kudryashova, Elena (University of California, Los Angeles, USA)
Kramerova, Irina (University of California, Los Angeles, USA)
Anderson (deceased), Louise (University of Newcastle, UK)
Beckmann, Jacques (University of Lausanne, Lausanne, Switzerland)
Bushby, Katherine (Institute of Human Genetics, International Center for Life, Newcastle upon Tyne, UK)
Spencer, Melissa (University of California, Los Angeles, USA)
 
Body of Abstract: Calpain 3 (CAPN3) is a calcium dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To understand the physiological function of CAPN3 we compared the proteomes of transgenic mice that over express CAPN3 (CAPN3 Tg) and their non-transgenic (non-Tg) counterparts. We first examined known dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of 10 possible substrates for CAPN3, classified into two categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyse MLC1 in vit ro. These studies are the first to identify possible substrates for CAPN3 in vivo, and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.


 
Abstract Title: Evaluation of the olopatadine effect in the dystrophinopathy progression in mdx mice submitted to physical activity
Presenting/First Author: Costa, Maria Cristina R (Faculty)
Department, Institution: Medicine, University of Ribeirao Preto
Address: Av Costabile Romano 2201 Ribeirao Preto, 14096-900 Brazil
Phone/Fax: 55-16-3603-6795 / 55-16-3919-3028
Email: [email protected]
Abstract Theme: Therapies
Author(s): Costa, Maria Cristina R (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Araujo, Daniela D (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Pires, Francisco Souza M (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Fuzisaki, Tatiana T (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Matioli, Fabiana (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Cerri, Daniel G (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Couto, Lucelio B (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Bizario, Joao Carlos S (Univ. of Ribeirao Preto, Muscular Dystrophy Research Center - AADM, Ribeirao Preto, 14096-900, Brazil)
Body of Abstract: Duchenne Muscular Dystrophy (DMD) is a progressive neuromuscular disease caused by dystrophin absence. The mdx mouse, a DMD animal model, shows milder phenotype, intensified by physical activity. Inflammatory response and immune cells contribute to muscle degeneration and/or regeneration. Mast cells are recruited to injury sites and liberate mediators that increase collagen production, attract other inflammatory cells and intensify tissue damage. Olopatadine, an H1-histamine antagonist and a mast cell degranulation inhibitor, decreases Ca2+ influx and the liberation of histamine, tryptase, leukotrienes and prostaglandins. In this study, we have evaluated the effect of olopatadine in mdx mice. Four-week old mice were characterized in the beginning and in the end of a physical activity program during five weeks in relation to histopathological analyses of gastrocnemius and diaphragm and serum creatine kinase dosage. Nine week old mice treated with olopatadine showe d amelioration of muscular condition, with less inflammatory infiltrates and necrosis foci, decreased degeneration/regeneration ratio and centrally nucleated myofibers. Quantitative analyses are in progress in order to reinforce the benefic effect of olopatine in DMD.


 
Abstract Title: Ageing, Skeletal Muscle and Sprint-Force Training
Presenting/First Author: Cristea, Alexander D (Undergrad)
Department, Institution: Neurovetenskap, Inst f neurofysiologi
Address: Akademiska Sjukhuset Uppsala Uppsala, 751 85 Sweden
Phone/Fax: (46)18 6119338 / (46)18500952
Email: [email protected]
Abstract Theme:  
Author(s): Cristea, Alexander (University of Uppsala, Uppsala; Clinincal Neurofysiology, University Hospital, Uppsala)
Larsson, Lars (Clinincal Neurofysiology, University Hospital, Uppsala)
 
Body of Abstract: The potential of sprint-force training to ameliorate sarcopenia was explored. The relationship between age, training and skinned single fiber characteristics and force production were determined in 18- to 77-yr-old male sprinters. The maximum force generated by the single muscle fibers was higher (P<0.01) in the younger than in the older subjects. Type I and IIa MyHC fibers had a larger cross sectional area in younger than in elderly (P<0.05). The specific tension in single fibers expressing the type I and IIa MyHC isoforms did not significantly differ between younger and older subjects. The V0 of type I MyHC fibers was lower (P<0.05) in older than in younger subjects, but there was no ageing-related difference in the shortening velocity of muscle fibers expressing the IIa MyHC isoform. The sprint-trained athletes experienced the earlier reported ageing-related reduction in the size of fast fibers and a lower area and V0 of type I MyHC fibers. At the same, the mu scle fiber and strength characteristics were preserved at a high level in the oldest runners, underlining the favorable impact of sprint exercise on ageing skeletal muscle.
Supported by NIH, Swedish Research Council, Swedish National Centre for Research in Sports


 
Abstract Title: Association between AKT signaling and hypertrophic response of Duchenne and limb-girdle muscular dystrophies
Presenting/First Author: Crosbie, Rachelle H (Faculty)
Department, Institution: Physiological Science, UCLA
Address: 621 Charles E. Young Drive South Los Angeles, CA 90095-1606 United States
Phone/Fax: 310-794-2103 / 310-206-9184
Email: [email protected]
Abstract Theme: Therapies
Author(s): Crosbie, Rachelle H (University of California, Los Angeles)
Peter, Angela K (University of California, Los Angeles)
 
Body of Abstract: Dystrophic muscle undergoes repeated cycles of degeneration/regeneration, characterized by the presence of hypertrophic fibers. In order to elucidate the signaling pathways that govern these events, we investigated Akt activation in normal and dystrophic muscle. Akt is activated in neonatal muscle and in actively dividing myoblasts, supporting a developmental role for Akt signaling. Akt activation was detected at very early, prenecrotic stages of disease pathogenesis and maximal activation was observed during peak stages of muscle hypertrophy. Duchenne muscular dystrophy patients exhibit a similar pattern of Akt activation. Mice with sarcoglycan-deficient muscular dystrophy possess more severe muscle pathology and display elevated Akt signaling. However, the highest levels of Akt activation were found in dystrophin-utrophin deficient muscle with very advanced dystrophy. We propose that Akt may serve as an early biomarker of disease and that Akt activation mediates hypertrophy in muscular dystrophy. Current investigations are focused on introducing constitutively-active and dominant-negative Akt into prenecrotic mdx mice to determine how early modification of Akt activity influences disease pathogenesis.


 
Abstract Title: Overexpression of Microspan, a Novel Component of the Sarcoplasmic Reticulum, Causes Severe Muscle Pathology with Triad Abnormalities
Presenting/First Author: Crosbie, Rachelle H (Faculty)
Department, Institution: Physiological Science, UCLA
Address: 621 Charles E Young Drive South Los Angeles, CA 90024 United States
Phone/Fax: 310-794-2103 / 310-206-3987
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Crosbie, Rachelle H (Univ. of California, Los Angeles, CA 90095)
Peter, Angela K (Univ. of California, Los Angeles, CA 90095)
Heighway, Jim (Univ. of Liverpool, Cancer Research Centre, Liverpool, UK)
Espinoza, Erica (Univ. of California, Los Angeles, CA 90095)
Miller, Gaynor (Univ. of California, Los Angeles, CA 90095)
 
Body of Abstract: Sarcospan is a core component of the dystrophin-glycoprotein complex. We describe the isolation of an alternatively-spliced isoform of sarcospan, which we designate ‘microspan’ based on its small size relative to sarcospan. Microspan has two transmembrane domains, is not an integral component of the dystrophin-glycoprotein complex and is not perturbed by the loss of dystrophin. Microspan protein is detected at the sarcoplasmic reticulum using indirect immunofluorescence and immunoelectron microscopy. Furthermore, microspan purifies with skeletal muscle sarcoplasmic reticulum and not transverse tubules. Mice engineered to overexpress microspan display severe kyphosis and die at approximately eight weeks of age. Levels of ryanodine receptor, dihydropyridine receptor, and SERCA-1 are greatly reduced in microspan transgenic muscle. Furthermore, electron microscopy reveals that microspan overexpression causes a dramatic perturbation in triad structure. Our findings suggest that microspan is an important component of the sarcoplasmic reticulum and may contribute to excitation-contraction coupling.


 
Abstract Title: Sarcospan Overexpression in Skeletal Muscle Perturbs the Dystrophin-Glycoprotein Complex and Causes Severe Muscle Pathology
Presenting/First Author: Crosbie, Rachelle H (Faculty)
Department, Institution: Physiological Science, UCLA
Address: 621 Charles E Young Drive South Los Angeles, CA 90095 United States
Phone/Fax: 310-794-2103 / 310-206-3987
Email: [email protected]
Abstract Theme: Plasma Membrane Stability/Repair
Author(s): Crosbie, Rachelle H (Univ. of California, Los Angeles, CA 90095)
Espinoza, Erica (Univ. of California, Los Angeles, CA 90095)
Mehta, Swapan (Univ. of California, Los Angeles, CA 90095)
Miller, Gaynor (Univ. of California, Los Angeles, CA 90095)
 
Body of Abstract: Sarcospan is a tetraspanin-like component of the dystrophin-glycoprotein complex (DGC). In order to elucidate the role of sarcospan in muscle, we generated transgenic mice that overexpress sarcospan (~10-fold) in skeletal muscle. Sarcospan transgenic mice exhibit kyphosis and die prematurely between 8 and 10 weeks of age. At 4 weeks of age, sarcospan transgenic mice are smaller, less motile, and display an abnormal waddling gait. Histological analysis reveals that sarcospan overexpression causes severe muscle pathology, including variation in fiber size, fibrosis, and mild central nucleation. Using serum creatine kinase and Evans Blue dye assays, we show that sarcospan transgenic muscle fibers display normal sarcolemma integrity. Increases in sarcospan expression cause upregulation of the entire DGC and caveolin-3 at the sarcolemma. Surprisingly, we were unable to purify the DGC from sarcospan transgenic muscle. Biochemical fractionation demonstrates that sarcospa n clusters the DGC and into insoluble protein aggregates within the membrane, which also affects structure of the extracellular matrix. These perturbations lead to severe muscle pathology reminiscent of congenital muscular dystrophy.


 
Abstract Title: Effect of Sarcospan Expression on Dystroglycan Function
Presenting/First Author: Crosbie, Rachelle H (Faculty)
Department, Institution: Physiological Science, UCLA
Address: 621 Charles E Young Drive South Los Angeles, CA 90095 United States
Phone/Fax: 310-794-2103 / 310-206-3987
Email: [email protected]
Abstract Theme: Sarcomere Cytoskeleton
Author(s): Crosbie, Rachelle H (Univ. of California, Los Angeles, CA 90095)
Miller, Gaynor (Univ. of California, Los Angeles, CA 90095)
Felix, Ricardo (Cinvestav-IPN, Mexico)
Mehta, Swapan (Univ. of California, Los Angeles, CA 90095)
 
Body of Abstract: Several observations suggest that dystroglycan and sarcospan may interact. We set out to probe whether sarcospan affects dystroglycan function by creating stable myoblast cell lines with either elevated or reduced levels of sarcospan. Myoblasts are an excellent model system for such studies since they express dystroglycan and sarcospan, but not the sarcoglycans. Myoblasts with reduced sarcospan are small and round with few filapodia. In contrast, myoblasts with elevated sarcospan are very large and exhibit many filapodia. Cell surface area was quantitated by whole cell patch clamp, demonstrating that sarcospan overexpressing cells are 30% larger than controls while sarcospan-deficient cells are 30% smaller. Sarcospan-deficiency doubled cell division rates and sarcospan overexpression reduced cell growth rates. Sarcospan stable cell lines never differentiated into myotubes. Dystroglycan levels were coordinately regulated with sarcospan. Upregulation of dystroglycan led to elevated numbers of focal adhesion contacts and dramatic increases in laminin binding. Reduction of dystroglycan was associated with fewer focal adhesions and suppressed laminin binding. These data suggest that sarcospan functions to regulate dystroglycan.


 
Abstract Title: Therapeutical benefit of AAV-mediated injection of a mutated propeptide of myostatin in calpain 3 deficient mice
Presenting/First Author: Daniele, Nathalie (Faculty)
Department, Institution: UMR8115, Genethon
Address: 1, rue de l'internationale Evry, 91000 France
Phone/Fax: 33 1 69 47 29 87 / 33 1 60 77 86 98
Email: [email protected]
Abstract Theme: Regulation of Muscle Mass
Author(s): Daniele, Nathalie (Genethon, CNRS UMR 8115, Evry 91000, France)
Bartoli, Marc (Genethon, CNRS UMR 8115, Evry 91000, France)
Poupiot, Jerome (Genethon, CNRS UMR 8115, Evry 91000, France)
Fougerousse, Francoise (Genethon, CNRS UMR 8115, Evry 91000, France)
Vulin, Adeline (Genethon, CNRS UMR 8115, Evry 91000, France)
Garcia, Luis (Genethon, CNRS UMR 8115, Evry 91000, France)
Danos, Olivier (Genethon, CNRS UMR 8115, Evry 91000, France)
Richard, Isabelle (Genethon, CNRS UMR 8115, Evry 91000, France)
Body of Abstract: Myostatin (GDF8) is a negative regulator of muscle mass which inhibition has been proposed as a therapeutical strategy for muscle wasting conditions. Indeed, blocking myostatin action through different strategies has been proved beneficial for the pathophysiology of the dystrophin–deficient mdx mouse. We tested the inhibition of myostatin by AAV-mediated expression of a mutated myostatin propeptide fused to the murine secreted alkaline phophatase in wild-type and two animal models of muscular dystrophies. The results in wild-type mice indicated that the fusion is permissive for the secretion of mSeAP and induce a hypertrophy. Unexpectedly, in the highly regenerative Sgca-null mice, expression is not maintained, suggesting that inhibition of myostatin does not improve survival of the muscle fibers. However, in calpain 3 deficient mice, a boost in muscle mass and a muscle strengthening were obtained, suggesting that myostatin inhibition could constitute of a therape utical strategy for this pathology.


 
Abstract Title: CNS abnormalities in myotonic dystrophy types 1 and 2
Presenting/First Author: Day, John W (Faculty)
Department, Institution: Neurology and Institute Human Genetics, University of Minnesota
Address: 420 Delaware St. SE Minneapolis, MN 55455 United States
Phone/Fax: 612-625-6180 / 612-625-8488
Email: [email protected]
Abstract Theme:  
Author(s): Day, John W (Department of Neurology, University of Minnesota, Minneapolis, MN 55455)
Dalton, Joline C (Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455)
Franc, Daniel (Department of Psychiatry, University of Minnesota, Minneapolis, MN 55455)
Rodriguez, Craig P (Department of Psychiatry, University of Minnesota, Minneapolis, MN 55455)
Wozniak, Jeff R (Department of Psychiatry, University of Minnesota, Minneapolis, MN 55455)
Ranum, Laura P (Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455)
Lim, Kelvin O (Department of Psychiatry, University of Minnesota, Minneapolis, MN 55455)
 
Body of Abstract: CNS dysfunction can be severe in muscular dystrophy, but the molecular pathophysiology is often unknown. To define CNS changes in myotonic dystrophy, we studied subjects with both DM1 and DM2. Demonstrations of molecular and clinical parallels of DM1 and DM2, each of which is caused by a transcribed but untranslated repeat expansion, indicate that features common to both forms result from toxic effects of the RNA expansions. Whether this mechanism also causes the CNS aspects of DM, which differ in DM1 and DM2, remains unclear.
Neuropsychological tests and MRI scans were performed on 5 strictly defined adult-onset DM1, 3 DM2, and 8 healthy control subjects. Quantitative volumetric measures of grey matter, white matter, and CSF showed significantly less grey matter volume in both DM groups compared to controls. Diffusion tensor imaging showed significantly reduced inferior frontal fractional anisotropy (FA, a measure of white matter integrity) in both DM groups compared to controls. Consistent with the frontal white matter changes, there was a trend toward reduced working memory in both DM groups compared to controls. Determining CNS changes common to DM1 and DM2 will help define features caused by the toxic RNA mechanism.


 
Abstract Title: Muscle-derived nitric oxide modulates adult neurogenesis in the brains of mdx mice.
Presenting/First Author: Deng, Bo (Grad)
Department, Institution: Physiological Science, UCLA
Address: 621 Charles E. Young Drive South Los Angeles, CA 90095 United States
Phone/Fax: 310-206-8389 / 310-825-8489
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Deng, Bo (Univ. of California, Los Angeles, CA 90095)
Tidball, James G (Univ. of California, Los Angeles, CA 90095)
 
Body of Abstract: Duchenne muscular dystrophy (DMD) patients display significant cognitive defects with IQs about 1 standard deviation below normal. However, the mechanisms underlying these defects are obscure. Learning deficits are well-correlated with perturbations in adult neurogenesis in the dentate gyrus (DG), which can be modulated by nitric oxide (NO). NO is not only a local signaling molecule, it can also have systemic, regulatory roles, which suggests that systemic NO may modulate adult neurogenesis. In DMD patients, there is an approximately 80% reduction in NOS activity in muscle as well as in plasma NO concentrations. Mdx mice, the animal model of DMD, display similar reduction in muscle NOS activity and learning deficits. Therefore, we propose that loss of systemic, muscle-derived NO causes defects in adult neurogenesis in the DG of mdx mice. We have found that mdx mice display enhanced cell proliferation and decreased neuronal differentiation in the DG, which can be rescued to wild-type levels by expression of a muscle-specific nNOS transgene. Our data strongly support our hypothesis, providing new insights into the mechanisms underlying the learning deficits in DMD patients. (Supported by the Muscular Dystrophy Association)


 
Abstract Title: Aberrant expression of lymphatic vessel proteins in laminin alpha2-deficient mouse muscle suggests that laminin alpha2 is required for normal lymphangiogenesis in muscle.
Presenting/First Author: Dominov, Janice A (Faculty)
Department, Institution: Neuromuscular Diseases, Boston Biomedical Research Institute
Address: 64 Grove Street Watertown, MA 02472 United States
Phone/Fax: 617-658-7739 / 617-972-1761
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Dominov, Janice A (Boston Biomedical Research Institute, Harvard Medical School)
Wardrop, Katherine E (Boston Biomedical Research Institute)
 
Body of Abstract: An early feature of laminin alpha2-deficient congenital muscular dystrophy is transient inflammation of muscle tissue. We are studying early pathogenesis of laminin alpha2-deficient (dy) mouse muscles using tenascin-C and lymphatic vessel expression as indicators of inflammation. We have found that expression of the lymphatic vessel marker LYVE-1 is dramatically reduced in dy muscles as early as 7 days after birth. Other lymphatic markers, podoplanin and Prox1, are also aberrantly expressed in dy muscles compared with controls. Podoplanin is up-regulated in the space surrounding dy fibers while Prox1 is up-regulated within muscle fibers themselves. Expression of the blood vessel marker CD31 is not altered in dy muscles. Results suggest that laminin alpha2 is required for normal lymphangiogenesis in skeletal muscle. Lymphatic vessels transport proteins, fluids and cells from tissues to lymph nodes and help recruit immune cells into damaged tissue to promote repair. An abnormal lymphatic system in dy muscles could significantly impair both the inflammatory process and muscle regeneration. These observations provide evidence for additional complexity in the underlying cause of laminin alpha2-deficient muscle pathology. Support: NIAMS


 
Abstract Title: Application of amphiphillic polymers for gene delivery
Presenting/First Author: Doran, Timothy J (Faculty)
Department, Institution: Muscular Dystrophy, Carolinas Medical Center
Address: 1000 Blythe Blvd Charlotte, NC 28203 United States
Phone/Fax: 704-446-6237 / 704-355-1679
Email: [email protected]
Abstract Theme: Therapies
Author(s): Doran, Timothy J (MDA/ALS Center, Cannon Research Center, Carolinas Medical Center, Charlotte, NC)
Gonsalves, Ken (Department of Chemistry, UNC Charlotte, Charlotte, NC)
Yengo, Chris M (Department of Biology, UNC Charlotte, Charlotte, NC)
Lu, Qi L (MDA/ALS Center, Cannon Research Center, Carolinas Medical Center, Charlotte, NC)
 
Body of Abstract: Non ionic copolymers consisting of hydrophobic and hydrophilic blocks in a triblock configuration hold a great potential for gene and nucleic acid delivery. They are easily synthesized and can be manipulated to produce a wide array of polymers with highly diverse physical properties and potential interactions with target transgenes or oligonucleotides. The efficiency can be improved by incorporating functional groups critical for gene delivery which will induce DNA encapsulation and protection. Preliminary studies found that polymers improve efficiency of gene delivery in vivo and in vitro differentially. The polymers also bind to nucleic acid differentially. Both peak excitation and stopped-flow kinetic studies revealed that the interaction is different from those seen in cationic polymers which condense nucleic acid. Systematic investigation into the interaction between polymer and nucleic acid in connection with the effect of polymers on gene delivery could re veal the relationship between structure and function and lead to new designs of polymers that should increase delivery efficiency, provide specific targeting of cells, and reduce toxicity.


 
Abstract Title: Loss of cyclophilin D improves cardiac pathology in delta-sarcoglycan deficient mice, but has no effect on skeletal muscle pathology
Presenting/First Author: Douglas, Millay P (Grad)
Department, Institution: Molecular Cardiovascular Biology, Cincinnati Children's Hopital Medical Center
Address: 3333 Burnet Ave. CHRF Rm. 3029 Cincinnati, OH 45229 United States
Phone/Fax: 513-636-2467 / 513-636-5958
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Millay, Douglas P (Univ. of Cincinnati, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229-3039)
Parsons, Stephanie A (Univ. of Cincinnati, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229-3039)
McNally, Elizabeth M (The Univ. of Chicago, Chicago, IL 60637)
Molkentin, Jeffery D (Univ. of Cincinnati, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229-3039)
 
Body of Abstract: Increased Ca2+ influx is hypothesized to initiate dystrophic myofiber loss through activation of degradative enzymes and/or cell death signaling pathways. Cyclophilin D directly regulates Ca2+-dependent necrosis through its interaction with the mitochondrial permeability transition pore. We generated mice lacking delta-sarcoglycan (scgd-/-) and cyclophilin D (Ppif-/-) in order to block mitochondrial-driven necrosis. Cell death indicators, central nucleation, and histological analysis revealed no differences in the skeletal muscles of scgd-/-Ppif-/- mice compared to scgd-/- mice. However, scgd-/-Ppif-/- mice had improved cardiac function and preliminary data suggests less cardiac fibrosis. Our results indicate that loss of cyclophilin D is not effective in altering skeletal muscle pathology in scgd-/- mice, but that cardiac muscle is benefited. However, other types of muscular dystrophy could utilize a mitochondrial-dependent necrotic pathway. Hence, we crossed the Ppif-/- mouse with a mouse model of congenital muscular dystrophy due to laminin-2A deficiency. Preliminary data from this cross will also be presented to better suggest the overall importance of mitochondrial-dependent necrosis in muscular dystrophy.


 
Abstract Title: AAV-mediated Microdystrophin Expression Reduces Muscular Dystrophy in Symptomatic Utrophin/dystrophin Double Knock-out Mice
Presenting/First Author: Duan, Dongsheng (Faculty)
Department, Institution: Molecular Microbiology and Immunology, University of Missouri
Address: One Hospital Dr. Columbia, MO 65212 United States
Phone/Fax: 573-884-9584 / 573-882-4287
Email: [email protected]
Abstract Theme: Therapies
Author(s): Yue, Yongping (University of Missouri, Columbia, MO)
Liu, Mingju (University of Missouri, Columbia, MO)
Duan, Dongsheng (University of Missouri, Columbia, MO)
 
Body of Abstract: AAV-mediated microdystrophin expression has shown encouraging results in ameliorating in mdx muscle pathology. Unfortunately, mdx mice do not display the same phenotype as human patients. Utrophin/dystrophin double knock-out (u-dko) mice were developed to mold severe dystrophic changes in human patients. In this study we evaluated therapeutic effect of the ?R4-R23/?C microdystrophin gene (?R4/?C) after AAV-6 gene transfer in neonatal u-dko muscle. At two months after gene transfer, percentage of centrally nucleated myofiber was reduced from 89.2% to 3.4% and muscle weight was normalized. We have also demonstrated for the first time that microdystrophin can eliminate interstitial fibrosis, macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch and tetanic specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the ?R4/?C microgene holds great promise in preventing muscular dystrophy.


 
Abstract Title: Increased cytokines, chemokines, and extracellular matrix proteins in 21-day mdx and mdx:utrophin-/- serum and diaphragm muscles
Presenting/First Author: Evans, Nicholas P (Grad)
Department, Institution: Human Nutrition Foods and Exercise, Virginia Tech
Address: 338 Wallace Hall Blacksburg, VA 24060 United States
Phone/Fax: 540-231-9048 / 540-231-3916
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Evans, Nicholas P (Dept. Human Nutrition Foods and Exercise, Virginia Tech, Blacksburg VA 24060)
Call, Jarrod A (Dept. Human Nutrition Foods and Exercise, Virginia Tech, Blacksburg VA 24060)
Niday, Ashley K (Dept. of Engineering Sci. and Mechanics, Virginia Tech, Blacksburg VA 24060)
Voelker, Kevin A (Dept. Human Nutrition Foods and Exercise, Virginia Tech, Blacksburg VA 24060)
Wolff, Andrew V (Dept. Mechanical Engineering, Virginia Tech, Blacksburg VA 24060)
Grange, Robert W (Dept. Human Nutrition Foods and Exercise, Virginia Tech, Blacksburg VA 24060)
 
Body of Abstract: Dystrophin-deficient muscles (i.e., Duchenne muscular dystrophy) are characterized by immune/inflammatory responses and fiber necrosis. Cytokines and chemokines are important signaling molecules that regulate the immune/inflammatory response. We hypothesized the concentration of inflammatory cytokines, chemokines and extracellular matrix proteins in serum and diaphragm muscles of mdx (n=2) and mdx:utrophin-/- (n=3) mice would be elevated vs. control (n=3) during early maturation (e.g., age 21-d). The cytokines IL1α and IL-10 were increased ≥ 3-fold in mdx serum, while IL1α, and IL10 were increased 3 to 4-fold in both mdx:utrn-/- serum and diaphragm (P≥0.05). The chemokines MCP1 and MCP5 were increased 2 to 10-fold in mdx and mdx:utrn-/- serum (P≥0.05), but only MCP5 was increased in mdx:utrn-/- diaphragm (P≥0.05). Rantes was increased 8-fold in mdx:utrn-/- diaphragm (P≥0.05). The extracellular matrix protein MMP3 was increas ed 3-fold in mdx and mdx:utrn-/- diaphragm (P≥0.05), but OPN and MMP9 were unchanged. These data suggest that immune/inflammatory signaling mechanisms may be important mediators in early disease progression to target for therapeutic intervention. Research supported by NIH grant AR049881.


 
Abstract Title: Systemic rAAV6-microdystrophin administration enhances skeletal muscle function and extends lifespan in the dystrophin-/utrophin- mouse model of severe muscular dystrophy
Presenting/First Author: Gregorevic, Paul (Postdoc - Degree Year: 2001)
Department, Institution: Neurology, The University of Washington
Address: 1959 NE Pacific Street Seattle, WA 98195 United States
Phone/Fax: (206) 221-5412 / (206) 616-8272
Email: [email protected]
Abstract Theme: Therapies
Author(s): Gregorevic, Paul (Dept of Neurology, Univ of Washington, Seattle, WA 98195)
Blankinship, Michael J (Dept of Neurology, Univ of Washington, Seattle, WA 98195)
Minami, Elina (Dept of Pathology, Univ of Washington, Seattle, WA 98195)
Allen, James M (Dept of Neurology, Univ of Washington, Seattle, WA 98195)
Adams, Marvin E (Dept of Physiology and Biophysics, Univ of Washington, Seattle, WA 98195)
Froehner, Stanley C (Dept of Physiology and Biophysics, Univ of Washington, Seattle, WA 98195)
Murry, Charles E (Dept of Pathology, Univ of Washington, Seattle, WA 98195)
Chamberlain, Jeffrey S (Dept of Neurology, Univ of Washington, Seattle, WA 98195)
Body of Abstract: Gene replacement for the treatment of Duchenne muscular dystrophy (DMD) has not been rigorously assessed for therapeutic potential because traditional gene transfer systems do not efficiently transduce key skeletal muscles in mammals. Also, previous studies have typically assessed local interventions in the dystrophin-deficient mdx mouse, which poorly models the severe dystrophy and shortened lifespan of DMD patients. We have established that IV delivery of recombinant adeno-associated viral (rAAV) vectors facilitates systemic gene transfer in mice. This advance has enabled us to rigorously evaluate administration of a microdystrophin construct as a treatment for DMD in the dystrophin-/utrophin- mouse, which exhibits severe muscular dystrophy and ~75% shortened lifespan compared with wildtype mice. Systemic rAAV6-microdystrophin administration restored dystrophin expression in limb and respiratory muscles, reducing indices of muscle disease, and increasing mus cle function. Treated mice also exhibited considerably extended lifespan. These data demonstrate that systemic rAAV6-microdystrophin administration can alleviate pathology associated with muscular dystrophy body-wide in mice, and may hold potential for treatment of DMD.


 
Abstract Title: Anti-inflammatory drugs, or increased IGF-1 expression, reduce necrosis of dystrophic muscle.
Presenting/First Author: Grounds, Miranda D (Faculty)
Department, Institution: Anatomy & Human Biology, University of Western Australia
Address: Stirling Highway, Crawley Perth, WA 6009 Australia
Phone/Fax: (618) 6488 3486 / (618) 6488 1051
Email: [email protected]
Abstract Theme: Plasma Membrane Stability/Repair
Author(s): Grounds, Miranda D (University of Western Australia, Australia.)
Radley, Hannah (University of Western Australia, Australia.)
Shavlakadze, Thea (University of Western Australia, Australia.)
Gebski, Bjanka (University of Western Australia, Australia.)
Torrisi, Jo (University of Western Australia, Australia.)
Davies, Marilyn (University of Western Australia, Australia.)
Bogoyevitch, Marie (University of Western Australia, Australia.)
 
Body of Abstract: Dystrophic myofibres with defective dystroglycan complexes are susceptible to sarcolemma damage. Little is known about the balance between repair of minor damage and myofibre necrosis. Inflammatory cytokines and cells can exacerbate initial damage and we have shown reduced necrosis of dystrophic mdx muscle following a range of anti-inflammatory interventions including silencing TNF-alpha using antibodies (Remicade and cV1q) or the soluble receptor (Enbrel), the depletion of neutrophils, the blocking of mast cell degranulation (Cromolyn), the blocking of complement C5a, and exposure to the corticosteroid prednisolone. These data support an important role for inflammation in the exacerbation of muscular dystrophy and provide new drug interventions to reduce the clinical severity of Duchenne Muscular Dystrophy. Conversely, we have shown that over-expression of IGF-1 specifically within dystrophic myofibres of MLC.IGF-1:Ea/mdx also reduces necrosis (see http://school .anhb.uwa.edu.au/personalpages/grounds/). As TNF-alpha can block IGF-1 signaling, the mechanisms of action of both TNF-alpha and IGF-1 combined with interactions between these signaling pathways is a central focus of our ongoing research.


 
Abstract Title: Mechanisms for dystrophin expression in BMD patients with premature stop codons and frameshift mutations in DMD exon 1
Presenting/First Author: Gurvich, Olga L (Postdoc - Degree Year: 2005)
Department, Institution: Human Genetics, Universtiy of Utah
Address: 15 North 2030 East Salt Lake City, UT 84112 United States
Phone/Fax: 801-587-7849 / 801-581-6463
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Gurvich, Olga L (Department of Human Genetics, University of Utah, Salt Lake City, UT, USA)
Aggarwal, Gaurav (Department of Human Genetics, University of Utah, Salt Lake City, UT, USA)
Anderson, Christine B (Department of Human Genetics, University of Utah, Salt Lake City, UT, USA)
Howard, Michael T (Department of Human Genetics, University of Utah, Salt Lake City, UT, USA)
Hart, Kim (Department of Human Genetics, University of Utah, Salt Lake City, UT, USA)
Weiss, Robert B (Department of Human Genetics, University of Utah, Salt Lake City, UT, USA)
Flanigan, Kevin M (Departments of Neurology and Pathology, University of Utah, Salt Lake City, UT, USA)
 
Body of Abstract: A Becker Muscular Dystrophy (BMD) patient who can ambulate at age 62 carries a premature stop codon mutation (c.9G>A; Trp3X) in exon 1 of the DMD gene. Three potential mechanisms may explain the phenotype: readthrough of the premature UGA codon; upregulation of alternate full-length DMD isoforms; or translational initiation at a downstream methionine codon.
Muscle biopsy was analyzed by immunofluorescence (IF) and immunoblot (IB), and by quantitative RT-PCR of Dp427m, Dp427p, and Dp427c mRNAs. Readthrough was determined using a dual luciferase reporter system.
Only a low level (<1%) translational read-through of the Trp3X stop codon was detected. Upregulation (2.5 fold) of Dp427c alone was noted. IF and IB showed significant dystrophin expression with dys1, dys2, and dys3 antibodies, but not with ManEx1a (recognizing an exon 1-encoded epitope specific to the Dp427m isoform); a decrease in size was suggested by IB. Approaches to verify the downstream initiation hypothesis are currently being developed.
We have identified only four more patients with exon 1 mutations (two Trp3X, Trp4X, and c.15delA). All have BMD, which leads us to postulate that a mechanism(s) exists which ameliorates the detrimental effect of exon 1 mutations.


 
Abstract Title: Genetic Modifiers of Muscular Dystrophy
Presenting/First Author: Heydemann, Ahlke (Faculty)
Department, Institution: Medicine, University of Chicago
Address: 4218 N Bell #2 Chicago, IL 60618 United States
Phone/Fax: 773 702 2684 / 773 702 2681
Email: [email protected]
Abstract Theme:  
Author(s): Heydemann, Ahlke (The University of Chicago, Chicago, IL 60637)
Huber, Jill M (The University of Chicago, Chicago, IL 60637)
Demonbreun, Alexis (The University of Chicago, Chicago, IL 60637)
Hadhazy, Michele (The University of Chicago, Chicago, IL 60637)
McNally, Elizabeth M (The University of Chicago, Chicago, IL 60637)
 
Body of Abstract: Limb Girdle Muscular Dystrophy 2C is variable in its presentation and severity in both humans and mice (Sgcg). To assess the genetic contribution to phenotypic variation in muscular dystrophy, we initiated a study to identify genetic modifiers. Sgcg mutant mice were backcrossed heterozygously for ten generations into four different genetic background strains: 129/SVEMS+/J (g129), C57BL/6J (gC57), DBA 2J (gDBA) and CD1 VAF+ (gCD1). After the tenth generation, we assayed homozygous mouse skeletal, abdominal, diaphragm and heart muscles for Evan’s Blue Dye (EBD) uptake and hydroxyproline (HOP) content. EBD is a measure of increased membrane permeability. HOP content measures the amount of collagen and fibrosis. Both characteristics are hallmarks of muscular dystrophy. All muscles including heart were affected by dye uptake and enhanced fibrosis. g129 mice displayed less EBD uptake and less HOP deposition, and gDBA were the most severely affected by these two a ssays. We have now analyzed over 100 F2 Sgcg null mice generated from g129 x gDBA crosses, followed by F1 intercrosses. From these mice, cohorts that display divergent values for three characteristics will be analyzed using genome wide scans. Funding: NIH RO1.


 
Abstract Title: A Genetic Study in a C. elegans Model of Muscular Dystrophy
Presenting/First Author: Hongkyun, Kim (Faculty)
Department, Institution: Neurology, Gallo Research Center
Address: 5858 Horton Street, suite 200 Emeryville, CA 94608 United States
Phone/Fax: 510-985-3162 / 510-985-3101
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Kim, Hongkyun (Gallo Research Center, Department of Neurology, University of California, San Francisco, CA 94608)
Wu, Julia (Gallo Research Center, Department of Neurology, University of California, San Francisco, CA 94608)
Rogers, Matthew J (Gallo Research Center, Department of Neurology, University of California, San Francisco, CA 94608)
Pierce-Shimomura, Jonathan T (Gallo Research Center, Department of Neurology, University of California, San Francisco, CA 94608)
McIntire, Steven L (Gallo Research Center, Department of Neurology, University of California, San Francisco, CA 94608)
 
Body of Abstract: C. elegans is an established model organism that possesses most of the components of the DGC. DGC mutations can result in progressive muscle degeneration and a unique defect in the movement of C. elegans. Using this model system, we have pursued genetic screens to identify new components of the DGC and to understand the mechanisms underlying the muscle degeneration. First, we performed a forward genetic screen to identify mutants that exhibit the same locomotory phenotype as the known DGC mutants. This screen led to the identification of novel genes, including a plasma membrane acetylcholine/choline transporter that is expressed in body muscle and interacts with the DGC. Second, we have screened directly for mutations resulting in age-dependent muscle degeneration. C. elegans is semi-transparent, allowing the direct visualization of muscle degeneration in aging animals. Thus far, we have identified several mutations that cause muscle degeneration without d efects in muscle formation, as is observed in the DGC mutants. These studies may lead to a better understanding of muscle disease and the mechanisms that lead to muscle degeneration in patients with muscular dystrophies.


 
Abstract Title: Expression of clathrin heavy chain isoform CHC22 in the inflammatory myopathies
Presenting/First Author: HOSHINO, SACHIKO (Postdoc - Degree Year: 2002)
Department, Institution: GW Hooper Foundation, UCSF
Address: 513 Parnassus Avenue San Francisco, CA 94143-0552 United States
Phone/Fax: 415 476 6405 / 415 476 6185
Email: [email protected]
Abstract Theme:  
Author(s): Hoshino, Sachiko (University of California San Francisco, CA, 94143-0552)
Ishii, Akiko (University of Tsukuba, Tsukuba City, Japan, 305-8577)
Ohkoshi, Norio (Tsukuba University of Technology, Tsukuba City, Japan, 305-8520)
Esk, Christopher (University of California San Francisco, CA, 94143-0552)
Brodsky, Frances M (University of California San Francisco, CA, 94143-0552)
 
Body of Abstract: The CHC22 isoform of clathrin heavy chain is expressed primarily in skeletal muscle and has a distinct distribution from that of the ubiquitous form of clathrin heavy chain (CHC17) that mediates vesicular membrane traffic. Relative to CHC17, CHC22 protein levels are elevated during myoblast differentiation and in regenerating rat muscle fibers, suggesting a role for CHC22 in muscle development and/or repair. To determine whether CHC22 protein was elevated in regenerating human muscle fibers, its expression was analyzed in two inflammatory myopathies, polymyositis (PM) and dermatomyositis (DM). In these diseases, regenerating fibers express increased levels of class I histocompatibility molecules, as well as developmental myosin heavy chain. The distribution of these markers relative to CHC22, the H & E phenotype of the fibers and the fiber type (determined by ATPase reaction) were analyzed. Compared to controls (N=4), the staining for both CHC22 and class I h istocompatibility molecules was increased in muscle samples from PM (N=8) and DM (N=4) patients. The localization of the increased expression of both markers was coincident and was mainly confined to regenerating fibers. All of the fibers in which CHC22 expression was detected were type II fibers. These observations support the implication of CHC22 in a membrane traffic pathway involved in myogenesis or muscle repair and suggest relevance of its function to human muscle disease.


 
Abstract Title: Selenoprotein N: Evidence for a novel cis-acting stimulator of Selenocysteine insertion
Presenting/First Author: Howard, Michael T (Faculty)
Department, Institution: Human Genetics, University of Utah
Address: 15 N 2030 E, Rm 2100 Salt Lake City, UT 84105 United States
Phone/Fax: 801-585-1927 / 801-585-3910
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Howard, Michael (University of Utah, Salt Lake City, UT 85105)
Aggarwal, Gaurav (University of Utah, Salt Lake City, UT 85105)
Anderson, Christine (University of Utah, Salt Lake City, UT 85105)
Flanigan, Kevin (University of Utah, Salt Lake City, UT 85105)
Atkins, John (University of Utah, Salt Lake City, UT 85105)
 
Body of Abstract: Mutations in SEPN1 are associated with several early onset myopathies including, rigid spine muscular dystrophy, multiminicore disease and desmin-related myopathy with mallory body-like inclusions. Expression of SEPN1 requires redefinition of an in-frame UGA codon to encode for incorporation of the 21st amino acid, selenocysteine. Selenocysteine insertion is known to require a specialized selenocysteine insertion sequence (SECIS) located in the 3’ UTR of the mRNA. Using a reconstituted in vitro translation system, and a cell based dual luciferase reporter assay, we have identified a second cis-acting modulator of selenocysteine insertion efficiency, termed Selenocysteine Redefinition Element (SRE), comprised of a stem loop located 6 nts 3’of the SEPN1 UGA selenocysteine codon. Directed mutagenesis and phylogenetic analysis supports the importance of this stem loop and sequences located 5’ to it. The SRE is sufficient to induce high level readthrough of the UGA cod on in the absence of the SECIS element, and acts to facilitate selenocysteine insertion mediated by the 3’UTR SECIS element and SECIS binding protein 2. Potential regulatory roles for this new stimulator of selenocysteine insertion will be presented.


 
Abstract Title: mdm Muscular Dystrophy: Interactions with Calpain 3 and a Novel Functional Role for Titin’s N2A Domain
Presenting/First Author: Huebsch, Kimberly A (Postdoc - Degree Year: 2003)
Department, Institution: NA, The Jackson Laboratory
Address: 600 Main Street Bar Harbor, ME 04609 United States
Phone/Fax: (207) 288-6503 / (207) 288-6073
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Huebsch, Kimberly A (The Jackson Laboratory, Bar Harbor, ME 04609)
Kudryashova, Elena (Department of Neurology and Pediatrics, and Duchenne Muscular Dystrophy Research Center, Los Angeles, CA 90095)
Wooley, Christine M (The Jackson Laboratory, Bar Harbor, ME 04609)
Sher, Roger B (The Jackson Laboratory, Bar Harbor, ME 04609)
Seburn, Kevin L (The Jackson Laboratory, Bar Harbor, ME 04609)
Spencer, Melissa J (Department of Neurology and Pediatrics, and Duchenne Muscular Dystrophy Research Center, Los Angeles, CA 90095)
Cox, Gregory A (The Jackson Laboratory, Bar Harbor, ME 04609)
 
Body of Abstract: Human tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2A?83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter lifespan and more severe muscular dystrophy. However, in a direct genetic test of CAPN3’s role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.


 
Abstract Title: Identification of novel utrophin-interacting proteins
Presenting/First Author: Humston, Jill L (Grad)
Department, Institution: Physiology, University of Wisconsin-Madison
Address: Rm 127 470 N. Charter St. Madison, WI 53706 United States
Phone/Fax: 608-265-3440 / (608) 265-5512
Email: [email protected]
Abstract Theme:  
Author(s): Humston, Jill L (Graduate program in Molecular and Cellular Pharmacology, University of Wisconsin, Madison, 53706)
Rybakova, Inna N (Department of Physiology, University of Wisconsin, Madison, 53706)
Vestling, Martha M (Department of Chemistry, University of Wisconsin, Madison, 53706)
Ervasti, James M (Department of Physiology, University of Wisconsin, Madison, 53706)
 
Body of Abstract: Dystrophin and utrophin have a number of well-established molecular partners. However, the large size and multi-domain structure of dystrophin and utrophin lead us to hypothesize that novel interacting proteins remain to be identified. We utilized full-length recombinant Flag-utrophin as an affinity probe to isolate interacting proteins from lysates of normal skeletal muscle. This pull-down approach was validated by co-elution of syntrophin, a known dystrophin and utrophin binding partner. Proteins specifically bound to Flag-utrophin were identified based on the array of tryptic peptides detected by MALDI-TOF mass spectrometry. Interestingly, we identified two proteins previously localized to costameres, but not known to directly bind utrophin. We also identified several proteins previously implicated in acetylcholine receptor clustering at the neuromuscular junction, the primary location of utrophin in normal muscle. Our results support the use of whole protein a ffinity chromatography in combination with mass spectrometry to identify new molecular partners of utrophin. This approach is also directly applicable for identifying novel molecular partners of full-length dystrophin.


 
Abstract Title: Elevated gamma-actin in skeletal muscle alters expression of genes associated with cellular metabolism
Presenting/First Author: Jaeger, Michele A (Grad)
Department, Institution: Physiology, University of Wisconsin-Madison
Address: 1300 University Ave Madison, WI 53706 United States
Phone/Fax: 608-265-3440 / 608-265-5512
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Jaeger, Michele A (Graduate Program in Cellular and Molecular Biology, Univ. of Wisconsin-Madison)
Fitzsimons, Daniel P (Department of Physiology, Univ. of Wisconsin-Madison)
Sonnemann, Kevin J (Department of Physiology, Univ. of Wisconsin-Madison)
Mitchell, Stephanie (Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC)
Hoffman, Eric P (Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC)
Ervasti, James M (Department of Physiology, Univ. of Wisconsin-Madison)
 
Body of Abstract: Our lab has previously shown that cytoplasmic gamma-actin protein levels are elevated 10-fold in dystrophin-deficient mdx skeletal muscle. To determine the role of increased gamma-actin in the pathophysiology of dystrophic muscle, we generated transgenic mice over-expressing gamma-actin specifically in skeletal muscle. Force generation, levels of necrosis, regeneration, and membrane damage in transgenic mice were not different from wild-type, indicating that elevated gamma-actin does not contribute to classic mdx pathology. However, microarray analysis of wild-type, transgenic and mdx skeletal muscle indicated that elevated levels of gamma-actin contribute to the altered transcriptional profiles reported in mdx mice. The majority of genes differentially expressed in both transgenic and mdx mice were associated with cellular metabolism. Most interestingly, many nuclear-encoded mitochondrial transcripts were down-regulated, suggesting that elevated gamma-actin ma y contribute to the altered energy metabolism observed in mdx muscle.

This work was supported by the NIH (AR049899) and NRSA T32 HL07936 from the UW Cardiovascular Research Center.


 
Abstract Title: DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression
Presenting/First Author: Jamie, Margolis M (Grad)
Department, Institution: Genetics, Cell Biology and Genetics, University of Minnesota
Address: MMC 206 Mayo 8206 420 Delaware Minneapolis, MN 55455 United States
Phone/Fax: 612-626-3521 / 612-625-8488
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Margolis, Jamie M (University of Minnesota, Minneapolis 55455)
Schoser, Benedikt G (Ludwig Maximilians University, Munich 80336)
Moseley, Melinda L (University of Minnesota, Minneapolis 55455)
Day, John W (University of Minnesota, Minneapolis 55455)
Ranum, Laura P (University of Minnesota, Minneapolis 55455)
 
Body of Abstract: Myotonic dystrophy type 2 (DM2) is caused by a large CCTG expansion mutation (mean = 5,000 CCTGs; range=75-11,000) in intron 1 of the zinc finger protein 9 (ZNF9) gene. Although an RNA gain of function mechanism involving DM1 CUG or DM2 CCUG expansion transcripts is now well established, still debated are the potential role that flanking sequences within the DMPK 3’UTR may have on disease pathogenesis and whether or not decreased expression of DMPK, ZNF9 or neighboring genes at these loci contribute to disease. To address these questions in DM2, we have examined the nucleic acid content of the ribonuclear inclusions and the effects of these expansions on ZNF9 expression. Using cell lines haploid and homozygous for the expansion and skeletal muscle biopsy tissue, we demonstrate that pre-mRNAs containing large CCUG expansions are normally spliced and exported from the nucleus, that the expansions do not decrease ZNF9 expression at the mRNA or protein level, and that the ribonuclear inclusions contain the CCUG expansion, but not flanking intronic sequences. These data support a gain of function RNA model in which the accumulation of the CCUG expansion itself is sufficient to cause the multisystemic features of DM2.


 
Abstract Title: Protective role of proteolytic clearance of troponin T fragments in muscle cells
Presenting/First Author: Jin, J.-P. (Faculty)
Department, Institution: Medicine, Northwestern University
Address: 2650 Ridge Ave., SB515 Evanston, IL 60201 United States
Phone/Fax: (847)570-1960 / (847)570-1865
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Jin, J.-P. (Northwestern University)
Wang, Xin (Northwestern University)
 
Body of Abstract: A nonsense mutation at codon Glu180 in the exon 11 of slow skeletal muscle TnT gene causes an autosomal-recessive nemaline myopathy. The truncated slow TnT has substantially lower affinity for binding to tropomyosin due to the loss of one of two tropomyosin-binding sites. Inefficient incorporation into myofilament resulted in the instability of mutant slow TnT in the patient muscle. Rapid degradation of the truncated slow TnT provides a protective mechanism to avoid potential dominant-negative effect in careers.
Free TnT shows toxicity to cells. To investigate the role of the proteolytic clearance in preventing cytotoxicity of non-incorporated TnT in muscle cells, we studied expression and cellular toxicity of TnT. The results demonstrated toxic effects of TnT fragments in both bacterial and eukaryotic cells. Therefore, a peak release of TnT fragments during acute myocardial infarction or skeletal muscle fatigue and injury may impose toxic effects if it exceeds the proteolytic clearance capacity. The potentially apoptotic effect of TnT fragments may be a pathogenic factor in muscle diseases. To identify TnT peptide sequences responsible for the toxic effect and to understand the underlying mechanism has a clinical significance.


 
Abstract Title: Selective deletion of the N-terminal variable region of cardiac troponin T in ischemia-reperfusion by myofibril-associated ?-calpain cleavage
Presenting/First Author: Jin, J.-P. (Faculty)
Department, Institution: Medicine, Northwestern University
Address: 2650 Ridge Ave., SB515 Evanston, IL 60201 United States
Phone/Fax: 847 570-1960 / 847 570-1865
Email: [email protected]
Abstract Theme: Cardiac Pathology
Author(s): Jin, J.-P. (Northwestern University)
Zhang, Zhiling (Northwestern University)
Biesiadecki, Brandon (Northwestern University)
 
Body of Abstract: An N-terminal truncated cardiac TnT(cTnT-ND) is found in myocardial ischemia reperfusion. It is generated by selectively removing the N-terminal variable region and preserving the conserved core structure of TnT. Triton X-100 extraction of cardiac muscle strips promoted production of cTnT-ND, indicating a myofibril-associated proteolytic activity. Supporting a role of ?-calpain in producing cTnT-ND in myocardial ischemia reperfusion, calpain inhibitors decreased the level of cTnT-ND in Triton-extracted myofibrils. ?-calpain treatment of cardiac myofibril effectively reproduced cTnT-ND. In contrast, ?-calpain treatment of isolated cardiac TnT resulted in non-specific degradation, suggesting that this restricted proteolysis of TnT is relevant to the physiological structures of the myofilament. Triton X-100 treatment of transgenic mouse cardiac myofibrils over-expressing fast skeletal muscle TnT produced similar N-terminal truncations of the exogenous and endogenous TnT. With the established functional effect of removing the N-terminal variable region of TnT, this ?-calpain-mediated proteolytic modification of TnT represents an acute mechanism to adjust muscle contractility under stress conditions.


 
Abstract Title: Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex
Presenting/First Author: Judge, Luke M (Grad)
Department, Institution: Neurology, University of Washington
Address: 1959 NE Pacific St. Room K233 HSC Seattle, WA 98195-7720 United States
Phone/Fax: (206) 221-5412 / (206) 616-8272
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Judge, Luke M (Univ. of Washington, Seattle, WA 98195)
Haraguchi, Miki (Univ. of Washington, Seattle, WA 98195)
Chamberlain, Jeffrey S (Univ. of Washington, Seattle, WA 98195)
 
Body of Abstract: DMD is caused by mutations in dystrophin, which is required for assembly of the dystrophin-glycoprotein complex (DGC) and which provides a mechanical link between the cytoskeleton and the extracellular matrix. Several proteins in the DGC also participate in signaling cascades, but the relative contribution of signaling and mechanical dysfunction to myofiber necrosis is unclear. To examine this question we generated transgenic mice that express the dystrophin isoform Dp116 in skeletal muscle. Dp116 cannot link to the cytoskeleton but did assemble the DGC. Expression of Dp116 mildly increased the severity of dystrophy in mdx4cv muscle, likely due to displacement of utrophin from the sarcolemma. Therefore, we crossed Dp116 transgenic mice with mice deficient for both dystrophin and utrophin (mdx:utrn-/-). Dp116:mdx:utrn-/- muscles were similarly dystrophic although muscle mass was increased compared to control mdx:utrn-/-. Unexpectedly, expression of Dp116 pre vented growth retardation, joint contractures, kyphosis, and premature death. We propose that expression of Dp116 and restoration of the DGC does not prevent myofiber necrosis but compensates for muscle damage by maintaining muscle mass and regenerative capacity.


 
Abstract Title: Regulation of Membrane-Associated Beta-Catenin by Calpain 3 During Terminal Stages of Myogenic Differentiation
Presenting/First Author: Kramerova, irina (Faculty)
Department, Institution: Neurology, UCLA
Address: 635 Charles Young Dr. South Los Angeles, CA 90095 United States
Phone/Fax: (310)267-4582 / (310)206-1998
Email: [email protected]
Abstract Theme:  
Author(s): Kramerova, Irina (Univ. of California, Los Angeles, CA 90095)
Kudryashova, Elena (Univ. of California, Los Angeles, CA 90095)
Wu, Benjamin (Univ. of California, Los Angeles, CA 90095)
Spencer, Melissa J (Univ. of California, Los Angeles, CA 90095)
 
Body of Abstract: Mutations in the cysteine protease calpain 3 (C3) cause LGMD2A. We have showed that myoblasts isolates from C3 knockout (C3KO) mice were able to fuse; however, sarcomere formation was disrupted. Here we show that C3KO myotubes have an increased myonuclear index and that C3 acts during myogenesis to specifically control levels of membrane-associated but not cytoplasmic beta-catenin. In the absence of C3, beta-catenin abnormally accumulates at the membrane coincident with increased levels of M-cadherin. Given the role of M-cadherin in myoblast fusion, this finding might explain increased myonuclear index in C3KO cultures. Consistent with these observations, we found an increased myonuclei in adult C3KO muscles even though the fibers are smaller than wild type. In addition, replacement of beta1 integrin isoforms, that normally accompanies myotube maturation, did not occur properly in C3KO myotubes. However, this is not a primary defect since neither of these isoforms are substrates for C3. These data suggest that the persistence of fusion that we observed in C3KO cells, possibly due to accumulation of beta-catenin and M-cadherin, inhibits subsequent steps of differentiation such as integrin isoform switching and sarcomere assembly.


 
Abstract Title: Measuring calpain 3 proteolytic activity using an in vitro substrate
Presenting/First Author: Laure, Lydie (Grad)
Department, Institution: UMR8115, Genethon
Address: 1, rue de l'internationale Evry, GA 91000 France
Phone/Fax: 33 1 69 47 28 85 / 33 1 60 77 86 98
Email: [email protected]
Abstract Theme:  
Author(s): Laure, Lydie (Généthon, CNRS-UMR 8115, Evry 91000, France)
Milic, Astrid (Généthon, CNRS-UMR 8115, Evry 91000, France)
Daniele, Nathalie (Généthon, CNRS-UMR 8115, Evry 91000, France)
Lochmueller, Hanns (Friedrich-Baur-Institute, München, Germany)
Mora, Marina (Istituto Nazionale Neurologico “C. Besta”,, Milano, Italy.)
Comi, Giacomo (Fondazione Ospedale Maggiore, Milano, Italy)
Moggio, Maurizio (Fondazione Ospedale Maggiore, Milano, Italy)
Richard, Isabelle (Généthon, CNRS-UMR 8115, Evry 91000, France)
Body of Abstract: Limb-girdle muscular dystrophy type 2A (LGMD2A; OMIM 253600) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependant protease. In order to improve presently available LGMD2A diagnosis, we developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 that can still be a substrate for active calpain 3 molecules that may be present in the sample to be tested. A total of 80 human biopsies consisting of 43 LGMD2A muscles and 37 controls have been analyzed with this assay. In all controls, normal calpain 3 level and activity were observed. A reduced or absent proteolytic activity was found in 32 (74%) of the LGMD2A biopsies. In the remaining 12 biopsies, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties, possibly recognition of substrates , should be impaired to give rise to the LGMD2A phenotype. Although the false positive results are possible, we believe that this assay is a quick and simple approach that improves LGMD2A diagnosis as it is able to test at once several calpain 3 properties.


 
Abstract Title: Myosin function in patients with a IIa myosin missense mutation (Glu-706 to Lys).
Presenting/First Author: Li, Mingxin (Grad)
Department, Institution: Department of clinical neurophysiology, Academic Hospital, Uppsala University
Address: Akademiska Sjukhuset Ing 85, 3tr Uppsala, 751 85 Sweden
Phone/Fax: +46(0)18 611 3432 / +01146(0)18 556 106
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Li, M. (Depart. of Clinical Neurophysiology, Uppsala Univ., Uppsala, SE-75185, Sweden)
Lionikas, A. (Center for Developmental and Health Genetics, The Pennsylvania State Univ., Univ. Park, Pennsylvania 16802, USA)
Yu, F. (Center for Developmental and Health Genetics, The Pennsylvania State Univ., Univ. Park, Pennsylvania 16802, USA)
Tajshargi, H. (Depart. of Pathology, Sahlgrenska Univ. Hospital, Goteborg, Sweden)
Oldfors, A. (Depart. of Pathology, Sahlgrenska Univ. Hospital, Goteborg, Sweden)
Larsson, L. (Depart. of Clinical Neurophysiology, Uppsala Univ., Uppsala, SE-75185, Sweden)
 
Body of Abstract: Objective: Analyze the pathogenic events leading to the progressive muscle weakness in the adults with a E706K mutation in the head of the myosin heavy chain (MyHC) Methods: Maximum velocity of unloaded shortening (V0) and maximum force normalized to fiber cross-sectional area (specific tension) were measured in single cell segments. Myosin function was also studied without the confounding influence of structural and regulatory proteins in a single muscle fiber in vitro motility assay. Sensitive electrophoretic methods were used to determine MyHC isoform expression in the fiber segment. Results: A dramatic impairment in the function of the IIa MyHC isoform was observed in the single muscle fiber in vitro motility assay, while the other MyHC isoforms were spared. Investigation of contractility at the single fiber level revealed a dramatic decrease in the number of preparations fulfilling the criteria for acceptance irrespective of MyHC isoform expression. Conclu sions: The pathogenesis of the MyHC IIa E706K myopathy involves defective function of the mutated myosin. The myopathy encompasses alterations in the structural integrity of all muscle cells irrespective of MyHC isoform expression.


 
Abstract Title: Transfection of Adult Desmin-Null Muscles with GFP-Desmin Plasmid Restores Sarcomere Alignment and Stress Generation
Presenting/First Author: Lieber, Richard L (Faculty)
Department, Institution: Orthopaedics and Bioengineering, University of California, San Diego
Address: 9500 Gilman Drive La Jolla, CA 92093-9151 United States
Phone/Fax: 858-534-0162 / 858-553-4381
Email: [email protected]
Abstract Theme: Sarcomere Cytoskeleton
Author(s): Lieber, Richard L (University of California, San Diego)
Palmisano, Michelle G (University of California, San Diego)
Bremner, Shannon (University of California, San Diego)
Shah, Sameer B (University of California, San Diego)
Ryan, Allen F (University of California, San Diego)
 
Body of Abstract: In the absence of desmin, muscles demonstrate pathology characterized by a disorganized Z-disk and a 25% decrease in stress produced. This could be due directly to the desmin loss or to secondary changes due to development in the absence of desmin. We transfected desmin-null muscles with a GFP-desmin plasmid to test the hypothesis that reintroduction of desmin directly reverses these effects.

GFP-desmin plasmids were introduced into the desmin-null mice tibialis anterior (TA) by electroporation. Muscle physiological testing was then performed and passive mechanics were performed on single cells isolated from these muscles while imaging by confocal microscopy.

Z-disk phase shift variance (i.e. “waviness" of Z-disk) was significantly greater for desmin-null fibers (KO) (0.017±0.001 sq µm/µm) and transfected desmin-null fibers not expressing GFP-desmin (KO-D) (0.019±0.001), compared to wild-type (WT) (0.005±0.001) and transfected desmin-null fibers expressing GFP-desmin (KO+D) (0.006±0.001; p<0.001). Similarly, stress generated was significantly less for KO muscles (160±41 kPa) and KO-D (146±85), compared to WT (205±32) and KO+D (189±44; p<0.05). Stress generated was positively correlated with the degree of transfection (r2=0.82,p<.0001).

These results show that the GFP-desmin chimeric protein integrated into the myofibrillar lattice and reversed the structural and functional alterations induced by the absence of desmin.


 
Abstract Title: Alpha7 beta1 integrin promotes muscle cell adhesion, proliferation and decreases apoptosis without disrupting normal transcription
Presenting/First Author: Liu, Jianming (Grad)
Department, Institution: Department of Cell & Developmental Biolo, University of illinois at urbana-champaign
Address: B107 Chemical and Life Sciences Laboratory (CLSL), 601 S. Goodwin Avenue urbana, IL 61801 United States
Phone/Fax: (217) 333-6058 / (217) 244-1648
Email: [email protected]
Abstract Theme: Therapies
Author(s): Liu, Jianming (Department of Cell and Development Biology,University of Illinois at Urbana- Champaign, Urbana, IL 61801)
Berry, Suzanne E (Department of Cell and Development Biology,University of Illinois at Urbana- Champaign, Urbana, IL 61801)
Kaufman, Stephen J (Department of Cell and Development Biology,University of Illinois at Urbana- Champaign, Urbana, IL 61801)
 
Body of Abstract: Alpha7 beta1 integrin is one of the two major complexes in skeletal muscle that link laminin in the extracellular matrix and the actin cytoskeleton. The integrin can functionally compensate for the loss of the dystrophin-associated glycoprotein complex (DGC) in mice with severe muscular dystrophy. Alleviation of dystrophy in mdx/utr(-/-) mice by additional integrin alpha7 demonstrates the potential of enhanced integrin as a therapy for muscular dystrophy. We report here that inducible expression of 3-fold more integrin alpha7 in C2C12 myoblasts promotes adhesion to laminin and proliferation. Apoptosis, resulting from staurosoprin treatment, also decreases in the presence of more integrin alpha7. Affymetrix array analysis shows that neither a 3-fold increase of alpha7 integrin in C2C12 myoblasts nor a 8-fold increase in skeletal muscle tissue alters their transcriptomes. We conclude that enhanced integrin alpha7 expression in skeletal muscle does not disrupt normal skeletal muscle transcription at levels that achieve functional improvements in dystrophic mice. This suggests that increasing integrin levels in muscular dystrophy may be therapeutic and without additional side effects. Supported by the NIH and MDA.


 
Abstract Title: NKT cells and Osteopontin Correspond with the Inflammatory Response in Mdx Muscular Dystrophy
Presenting/First Author: Lopez, Sylvia A (Grad)
Department, Institution: Neurology and Pediatrics, University of California, Los Angeles
Address: 635 Charles E. Young Dr. South, NRB 404 Los Angeles, CA 90059 United States
Phone/Fax: (310) 267-4582 / (310) 206-1998
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Lopez, Sylvia A (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 and Duchenne Muscular Dystrophy Research Center, UCLA)
Montecino-Rodriguez, Encarnacion (Department of Pathology and Laboratory Medicine and Duchenne Muscular Dystrophy Research Center, UCLA)
Wu, Ben G (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 and Duchenne Muscular Dystrophy Research Center, UCLA)
Kramerova, Irina (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 and Duchenne Muscular Dystrophy Research Center, UCLA)
Kudryashova, Elena (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 and Duchenne Muscular Dystrophy Research Center, UCLA)
Hoffman, Eric P (Childrens National Medical Center, Washington D.C.)
Micelli, Carrie (Department of Microbiology and Molecular Genetics, UCLA)
Spencer, Melissa (David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 and Duchenne Muscular Dystrophy Research Center, UCLA)
Body of Abstract: Muscle inflammation is a secondary feature that exacerbates Duchenne Muscular Dystrophy (DMD). The goal of this investigation was to characterize immune cells infiltrating dystrophic muscle with the purpose of identifying specific T cell populations that might be targeted for immunotherapeutics. Flow cytometry of purified lymphocytes from mdx mice showed that the Vbeta8.1/8.2 T cell receptor (TCR) rearrangement represented approximately 30% of all T cells and that these Vbeta8.1/8.2 cells expressed NK cell surface markers, suggesting that they are NKT cells. Cell sorting confirmed that Vbeta8.1/8.2+ cells expressed the invariant Valpha14 TCR chain and MIP-2, a chemokine not expressed by conventional T cells. Sorted NKT cells also expressed osteopontin (OPN), a pro-inflammatory cytokine. Examination of DMD biopsies confirmed the presence of both NKT cells and OPN. Interestingly, OPN was highly expressed by invading immune cells and muscle fibers. Mdx mice were trea ted with asialo GM1, an antibody that inhibits NK and NKT cell activity. This treatment caused reductions in serum OPN and splenic IL-4. These studies suggest that NKT cells infiltrate dystrophic muscle and express cytokines that modulate the immune response.


 
Abstract Title: Differences in myogenesis and sarcolemmal repair after single and repetitive muscle strains
Presenting/First Author: Lovering, Richard M (Faculty)
Department, Institution: Physiology, Univ of Maryland, School of Medicine, Dept. of Physiology
Address: 685 W. Baltimore St., HSF-1, Rm 580 Baltimore, MD 21201 United States
Phone/Fax: 410-409-1077 / 410-706-8341
Email: [email protected]
Abstract Theme: Plasma Membrane Stability/Repair
Author(s): Lovering, Richard M (University of Maryland School of Medicine, Baltimore, MD)
Roche, Joseph A (University of Maryland School of Medicine, Baltimore, MD)
Bloch, Robert J (University of Maryland School of Medicine, Baltimore, MD)
De Deyne, Patrick G (DePuy Biologics/Soft Tissue Technologies, Raynham, MA)
 
Body of Abstract: We determined if myogenic cell proliferation in skeletal muscle was equally important to recovery of contractile function after different contraction-induced injuries. Tibialis anterior (TA) muscles of anesthetized male rats were injured by a single lengthening contraction under large strain (1R), or multiple lengthening contractions under small strain (MR). Gamma irradiation was used to prevent proliferation. Maximal tetanic tension was assessed after injury and on days 3, 7, 14, and 21 during recovery. Both 1R and MR protocols caused comparable injuries, as measured by a loss of force, but irradiation inhibited recovery of force only after the MR protocol. Centrally nucleated fibers were only present after MR, and only in unirradiated muscle. Evans blue dye injected before injury was retained by myofibers recovering from 1R, but was lost after MR. Dye-labeled fibers after 1R initially lost sarcolemmal dystrophin, but eventually regained it, although they retained dye. Our results suggest that myogenesis is necessary for recovery after multiple lengthening contractions, but not after a single maximal lengthening contraction. Instead, recovery after 1R involves membrane resealing and replacement of damaged proteins.


 
Abstract Title: Body-wide restoration of functional levels of dystrophin by intravenous delivery of antisense oligonucleotides alleviates pathology in dystrophic mice
Presenting/First Author: Lu, Qi L (Faculty)
Department, Institution: Muscular Dystrophy Laboratory, Carolinas Medical Center
Address: 1542 Garden Terrace Charlotte, NC 28203 United States
Phone/Fax: 7043551701 / 7043551679
Email: [email protected]
Abstract Theme: Therapies
Author(s): Lu, Qi L (Qi Long Lu, Carolinas Medical Center, Charlotte, NC, USA)
Alter, Julia (Julia Alter, Imperial College, London Univ. London, UK)
Lou, Fang (Fang Lou, Univ. Hertfordshire, UK)
Partridge, Terence A (Terence A. Partridge, Children's National Medical Center, Washington DC)
 
Body of Abstract: Duchenne muscular dystrophy (DMD) results from non-sense or frame-shifting mutations in the dystrophin gene. The majority of these mutations can be corrected by antisense oligonucleotide (AON)-mediated exon skipping, which creates shortened, but in-frame transcripts and produces at least partially functional proteins. 2’-O-methyl phosphorothioate AONs (2OMeAONs) induced functional levels of dystrophin within individual muscles by local injection, but therapeutically inadequate levels of dystrophin in body-wide skeletal muscles when delivered systemically. With several different chemistries for AON, it is now possible to induce dystrophin expression up to levels in individual muscles with functional significance. Importantly, methods of systemic delivery applicable to clinic is able to restore dystrophin expression up to 50% of normal level in all skeletal muscles with clear indications of functional improvements including reduced rates in central nucleation of mus cle fibers, improved force generation and decreased serum CK levels. Thus exon skipping provides a realistic hope for the treatment of a majority of DMD patients.


 
Abstract Title: Congenital Myopathy with Internal Myonuclei and Perinuclear Aggregates: Reducing Body Myopathy without cytoplasmic bodies?
Presenting/First Author: Maiti, Baijayanta (Grad)
Department, Institution: Human Genetics, University of Utah
Address: 533 EIHG, 15 N 2030 E Salt Lake City, UT 84112 United States
Phone/Fax: 801-585-0246 / 801-581-6463
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Maiti, Baijayanta (Dept. of Human Genetics, University of Utah)
Chin, Steven S (Dept. of Pathology, University of Utah School of Medicine)
Flanigan, Kevin M (Depts. of Human Genetics, Pathology, and Neurology, University of Utah School of Medicine)
 
Body of Abstract: Reducing body myopathy (RBM) is a rare congenital disorder marked by cytoplasmic reducing bodies (RBs) and dense perinuclear osmiophilic aggregates. We report the clinical and pathological findings from two cases of congenital myopathy. One shows the classic features of RBM; the other shows RBM-like perinuclear aggregates, but no cytoplasmic RBs. Case 1 presented with weakness by age 2. Biopsy at age 4 revealed non-specific myopathic changes but no reducing bodies. Biopsy 16 months later revealed widespread cytoplasmic reducing bodies, seen by both light and electron microscopy, and typical perinuclear aggregates. Case 2 is the more severely affected of two siblings with weakness. Muscle biopsy at age 2 and again at age 8 showed the presence of many internal nuclei, and no cytoplasmic bodies. Electron microscopy revealed many nuclei surrounded by perinuclear aggregates typical of RBM. This may be a variant of RBM, or it may represent RBM in evolution, with the presence of perinuclear aggregation as the earliest feature preceding development of cytoplasmic RBs. However, given the absence of RBs in serial biopsies (six years apart) in a profoundly weak patient, this may represent a novel congenital myopathy.


 
Abstract Title: Myotonic dystrophy type 2: a reversible multisystemic murine model of CCUG RNA toxicity
Presenting/First Author: Margolis, Jamie M (Grad)
Department, Institution: Genetics, Cell Biology and Genetics, University of Minnesota
Address: MMC 206 Mayo 8206 420 Delaware Minneapolis, MN 55455 United States
Phone/Fax: 612-626-3521 / 612-625-8488
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Margolis, Jamie M (University of Minnesota, Minneapolis 55455)
Kang, Yuan-Lin (University of Minnesota, Minneapolis 55455)
Day, John W (University of Minnesota, Minneapolis 55455)
Ranum, Laura P (University of Minnesota, Minneapolis 55455)
 
Body of Abstract: Myotonic dystrophy type 2 (DM2) is caused by a large CCTG repeat in intron 1 of the zinc finger protein 9 gene. There is growing evidence that DM1 and DM2 are caused by an RNA gain of function mechanism in which the repeats themselves cause the multisystemic features of these diseases. To investigate the role that expression of the CCUG transcripts has on the DM phenotype, we developed a tetracycline inducible murine model of DM2. The expression of a CCTG-300 is driven by a tet-responsive element minimal CMV promoter as part of a non-coding transcript. These TRE-CCTG expansion lines were crossed to CMV driven tet activator mice, creating doubly transgenic mice that ubiquitously express the CCUG expansion. H&E staining of muscle from these doubly transgenic animals shows variation in fiber size and centrally located nuclei. Additionally, skeletal muscle shows ribonuclear inclusions, and aberrant splicing of the insulin receptor. In a separate series of experiments the TRE-CCUG expansion lines will be crossed to two different BAC transgenics engineered to drive expression of the tTA using the endogenous human DMPK or ZNF9 promoters to determine how differences in the expression pattern of the repeat affect the phenotype.


 
Abstract Title: Titin-cap knockdown by RNA interference inhibits myogenic regulatory factors in C2C12 cells
Presenting/First Author: Markert, Chad D (Postdoc - Degree Year: 2004)
Department, Institution: Veterinary Biosciences, University of Missouri--Columbia
Address: 1600 E Rollins Rd, Rm E102 Columbia, MO 65211 United States
Phone/Fax: 573-884-6731 / 573-884-6890
Email: [email protected]
Abstract Theme: Regulation of Muscle Mass
Author(s): Markert, Chad (Department of Biomedical Sciences, College of Veterinary Medicine, Univ. of Missouri--Columbia)
Ning, Jie (Department of Biomedical Sciences, College of Veterinary Medicine, Univ. of Missouri--Columbia)
Zhu, Zhifang (Department of Biomedical Sciences, College of Veterinary Medicine, Univ. of Missouri--Columbia)
Staley, JT (Department of Physical Medicine and Rehabilitation, Univ. of Missouri--Columbia)
Stoker, Aaron (Department of Veterinary Medicine and Surgery, Univ. of Missouri--Columbia)
Omolewu, Rachel Gee, James M (The Dalton Cardiovascular Research Center, Univ. of Missouri--Columbia)
Childers, Martin K (Department of Physical Medicine and Rehabilitation, Univ. of Missouri--Columbia)
Body of Abstract: Null mutation of titin-cap (T-cap) causes limb-girdle muscular dystrophy type 2G (LGMD2G). Patients with LGMD2G develop muscle wasting and weakness, and lose the ability to walk by the third decade. Previous findings suggest that T-cap regulates myostatin, a key regulator of muscle growth. PURPOSE: We tested the hypothesis that T-cap knockdown with RNA interference increases myostatin level and leads to impaired muscle cell differentiation. METHODS: C2C12 myoblasts were grown to confluence in GM (0 D) and then shifted to DM for 5 Days. Cells were treated with T-cap siRNA duplex constructs and analyzed for RNA and protein expression using Northern blots, real-time RT-PCR and Western immunoblots. RESULTS: siRNA treatment decreased T-cap RNA (p<0.05) and protein expression in differentiating muscle cells. Subsequent expression of myostatin increased, while decreases were observed in the myogenic regulatory factors MyoD and myogenin. CONCLUSIONS: Our findings suppo rt the hypothesis that T-cap knockdown with RNA interference increases myostatin levels and impairs normal muscle cell differentiation. This process likely occurs by repression of myogenic regulatory factors. Funded, in part, by the MU Research Board.


 
Abstract Title: Clinico-pathological Spectrum of FKRP Related Diseases
Presenting/First Author: Muntoni, Francesco (Faculty)
Department, Institution: Pediatrics, Imperial College, Hammersmith Hospital
Address: Du Cane Road London, W12ONN England
Phone/Fax: 00442083833295 / 00442087462187
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Muntoni, Francesco (Department of Pediatrics, Imperial College, Hammersmith Hospital, London, England)
Brockington, Martin (Department of Pediatrics, Imperial College, Hammersmith Hospital, London, England)
Torelli, Silvia (Department of Pediatrics, Imperial College, Hammersmith Hospital, London, England)
Kaluarachchi, Manuja R (Department of Pediatrics, Imperial College, Hammersmith Hospital, London, England)
Brown, Susan C (Department of Pediatrics, Imperial College, Hammersmith Hospital, London, England)
 
Body of Abstract: Defective glycosylation of alpha dystroglycan is a recently identified mechanism responsible for muscular dystrophy (MD). Mutations in 6 glycosyltransferases result in different conditions, ranging in severity from congenital onset MD and severe brain and eye structural involvement, to onset of weakness in adolescence or adult life. Originally severity was thought to be dependent on the mutated gene i.e. POMT1; POMT2; POMGnT1; fukutin; FKRP or LARGE. However, recent data suggests that phenotype is due to the effect of the mutation on the enzymatic function of the protein rather than the gene involved. Mutations in FKRP are the best example of this spectrum of phenotypes which range from Walker-Warburg severity to adult onset LGMD2I. At the protein level the expression of the glycosylated alpha-dystroglycan broadly correlates with disease severity suggesting a direct or indirect role for FKRP in dystroglycan processing. We have studied the sub-cellular localization of FKRP using different antibodies; and performed in-situ hybridization studies on its pattern of expression in the brain. These studies will improve our understanding of the functional role of FKRP and provide us with insight into the pathogenesis of FKRP-related MDs.


 
Abstract Title: Early increases in 20s Proteasome activity in fast and slow twitch muscle of rats exposed to moderate psychological stress
Presenting/First Author: Myburgh, Kathryn H (Faculty)
Department, Institution: Physiological Sciences, Stellenbosch University
Address: Merriman Avenue Stellenbosch, 7602 South Africa
Phone/Fax: 27 21 8083149 / 27 21 8083145
Email: [email protected]
Abstract Theme: Regulation of Muscle Mass
Author(s): Myburgh, Kathryn H (Univ. Stellenbosch, Stellenbosch 7602, South Africa)
Neethling, Ian (Univ. Stellenbosch, Stellenbosch 7602, South Africa)
Smith, Carine (Univ. Stellenbosch, Stellenbosch 7602, South Africa)
 
Body of Abstract: We exposed adult male Wistar rats to incremental, intermittent stress to determine the effect on skeletal muscle 20s proteasome activity and atrophy of the soleus and extensor digitorum longus (EDL).
Six control rats had no intervention (C). Six rats were exposed to restraint stress for 7 days (S7). Group S14, exposed to the same stress in wk 1, had restraint stress or randomly applied novel stressors (cage tilt; soiled bedding; tail exposure to heat) in wk 2. Stress exposure was 30 min for days 1-2, increasing to 3x 30 min for days 5-14. A 4th group, stressed similarly to S14, also exercised carrying weights in wk 2 (S14E7-14).
After 1 wk, S7 lost 47 ±15 g (8 %) of body mass with no change in C (P<0.01). But, soleus to body mass ratio (C: 0.45 ±0.06 vs. S7: 0.49 ±0.03 mg/g) and EDL to body mass ratio (C: 0.42 ±0.09 vs. S7: 0.42 ±0.03 mg/g) did not differ. Stress exposure increased 20s proteasome activity by 24.8% in soleus and 39.2% in EDL (both P<0.05). S14 and S14E7-14 results will be available shortly.
Preliminary evidence suggests that moderate psychological stress upregulates the 20s proteasome before atrophy.
Approval: Univ Research Ethics Committee; Funding: National Research Foundation


 
Abstract Title: Aberrant splicing of dystrophin and alpha-dystrobrevin in myotonic dystrophy
Presenting/First Author: Nakamori, Masayuki (Grad)
Department, Institution: neurology, Osaka university graduate school of medicine
Address: 2-2, D-4 Suita, Osaka, 565-0871 Japan
Phone/Fax: 81-6-6879-3571 / 81-6-6879-3579
Email: [email protected]
Abstract Theme: Sarcolemmal Excitability
Author(s): Nakamori, Masayuki (Osaka Univ., Suita 565-0871, Japan)
Kimura, Takashi (Osaka Univ., Suita 565-0871, Japan)
Fujimura, Harutoshi (Toneyama National Hospital, Toyonaka 560-8552, Japan)
Aoike, Futoshi (Osaka Univ., Suita 565-0871, Japan)
Takahashi, Masanori P (Osaka Univ., Suita 565-0871, Japan)
Sakoda, Saburou (Osaka Univ., Suita 565-0871, Japan)
 
Body of Abstract: In myotonic dystrophy type 1 (DM1), misregulation of alternative mRNA splicing has been attributed to some of its clinical features including myotonia. However, the cause of muscle wasting, a core symptom of DM, has still been unknown. The abnormalities of cytoskeletal proteins are responsible for some forms of progressive muscular dystrophies. Alpha-dystrobrevin (DB), a component of dystrophin-associated protein complex, and dystrophin have been shown to have alternative splicing isoforms. Therefore, we examined the splicing abnormalities of these cytoskeletal proteins in muscles from DM1 using RT-PCR. We also quantified the mRNA and protein for DB by real time PCR and Western blot. We found significant existence of alternatively spliced isoforms of dystrophin and DB in DM1. The total amount of mRNA for DB2 was significantly increased in DM1 than in control. Moreover the expression of aberrantly spliced DB isoform was confirmed using a specific antibody. These re sults suggest the possibility of functional defect in structural proteins in DM1 muscle, which might results in structural vulnerability of the sarcolemma or disturbance of signaling pathway.

Supported by Ministry of Health, Labour and Welfare, Japan (17A-10) and JSPS.


 
Abstract Title: Role of the MEF2-regulated gene myospryn in striated muscle
Presenting/First Author: Naya, Frank J (Faculty)
Department, Institution: Biology, Boston University
Address: 24 Cummington Street Boston, MA 02215 United States
Phone/Fax: 617-353-2469 / 617-353-6340
Email: [email protected]
Abstract Theme: Sarcomere Cytoskeleton
Author(s): Naya, Frank J (Boston University, Boston, MA 02215)
Reynolds, Joseph G (Boston University, Boston, MA 02215)
McCalmon, Sarah (Boston University, Boston, MA 02215)
Tomczyk, Thomas (Boston University, Boston, MA 02215)
 
Body of Abstract: The MEF2 transcription factor plays an important role in muscle gene expression and differentiation. A novel MEF2-regulated gene, myospryn, was identified by our lab by expression profiling using mef2a knockout mice which exhibit severe cytoarchitectural defects in cardiac muscle. The myospryn gene encodes a muscle-specific protein that interacts with alpha-actinin and is localized to the costamere, an elaborate protein network at the periphery of the Z-disc. Myospryn interacts with dysbindin which itself associates with alpha-dystrobrevin, a protein within the dystrophin-glycoprotein complex (DGC). Furthermore, myospryn transcripts are down-regulated in dystrophic muscle. A recent bioinformatics analysis by our lab has revealed that Myospryn is related to the protein kinase A (PKA) anchoring protein (AKAP) family. Indeed, co-immunoprecipitation assays have shown a direct interaction between Myospryn and PKA. Moreover, immunostaining of adult mouse skeletal muscle shows co-localization of endogenous Myospryn and PKA. These studies set the foundation for further investigating the potential role of Myospryn directing PKA signaling events to modulate the function of the DGC. Supported by NIH/NHLBI R01 HL73304.


 
Abstract Title: Morphology, and EDL stress production and passive mechanical properties of maturing mdx and mdx:utrophin-/- mice
Presenting/First Author: Niday, Ashley K (Faculty)
Department, Institution: HNFE, Virginia Tech
Address: 338 Wallace Hall Blacksburg, VA 24060 United States
Phone/Fax: 540-231-9048 / 540-231-3916
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Niday, Ashley K (Dept. of Engineering Science and Mechanices, Blacksburg, VA 24060)
Wolff, Andrew V (Dept. of Mechanical Engineering, Blacksburg, VA 24060)
Voelker, Kevin A (Dept. of Human Nutrition, Foods and Exercise, Blacksburg, VA 24060)
Call, Jarrod A (Dept. of Human Nutrition, Foods and Exercise, Blacksburg, VA 24060)
Evans, Nick P (Dept. of Human Nutrition, Foods and Exercise, Blacksburg, VA 24060)
Granata, Kevin P (Dept. of Engineering Science and Mechanices, Blacksburg, VA 24060)
Grange, Robert W (Dept. of Human Nutrition, Foods and Exercise, Blacksburg, VA 24060)
 
Body of Abstract: The purpose of this study was to (1) profile body mass, and extensor digitorum longus (EDL) muscle mass and length and, (2) determine stress production and passive mechanical properties (i.e., stiffness and damping) of maturing Control (C), mdx (M) and mdx: utrophin-/- (M:U-/-) EDL at ages 14-, 21-,28- and 35-d. Our aim was to identify specific ages for more detailed analysis of disease mechanisms. EDL muscles were subjected to passive stretch protocols in vitro. Force profiles were fit to a 3 parameter viscoelastic muscle model. At 14-d, all physical characteristics across the 3 genotypes were similar; at ages 21-35-d C and M:U-/- were similar, but at 21-d, M body and muscle masses were larger than C and M:U-/- values (P≤0.05). Stress production at 14 and 28-d was similar for all genotypes, but was depressed for M:U-/- vs C at 21-d, and for M and M:U-/- vs C at age 35-d (P≤0.05). Series elastic stiffness of the 3 genotypes were not different at ages 14-28-d, but M series stiffness was greater at 35-d vs C. M parallel elastic stiffness was similar to C at each age, except at 28-d (>C; P≤0.05). These time course data will be useful to target specific ages to explore dystrophic mechanisms. Research supported by NIH grant AR049881.


 
Abstract Title: Transcription factors in atrophy caused by post-synaptic block of neuromuscular transmission and muscle unloading in rats muscle
Presenting/First Author: Nordquist, Jenny (Postdoc - Degree Year: 2002)
Department, Institution: Neuroscience, Uppsala University
Address: Akademiska sjukhuset, ing. 85, 3 tr Uppsala, 751 85 Sweden
Phone/Fax: +46 18 611 34 32 / +46 18 55 61 06
Email: [email protected]
Abstract Theme:  
Author(s): Nordquist, Jenny (Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden.)
Höglund, Anna-Stina (Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden.)
Norman, Holly (Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden.)
Dworkin, Barry (Hershey Medical Center, Hershey, Pennsylvania, USA)
Larsson, Lars (Department of Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden.)
 
Body of Abstract: The muscle wasting associated with modern intensive care has a negative effect on rehabilitation and quality of life in patients recovering from critical illness. To identify the mechanisms behind this form of muscle wasting, we analysed the nuclear concentrations of a number of transcription factors in extensor digitorum longus (EDL) and soleus muscles in a rat intensive care unit (ICU) model. The rats were sedated, treated with a post-synaptic neuromuscular blocker and mechanically ventilated for 7-14 days. A significant muscle wasting and an up-regulation of the ubiquitin ligases MAFbx and MuRF1 followed the treatment. Bcl-3 displayed a concomitant decrease in concentration, suggesting kB controlled gene expression not involving NFkB p65. The nuclear levels of GR and TRalpha1 were altered and are also suggested as potential mediators of the MAFbx- and MuRF1-induction in the absence of induced Foxo1. We believe that this model, and the strategy of quantifying nu clear proteins, will provide a valuable tool for further, more detailed, analyses of the muscle wasting occurring in patients kept on a mechanical ventilator.
The study was supported by NIH (AR45627, AR47318, AG14731), AFM and the Swedish Research Council (8651).


 
Abstract Title: Longitudinal measurements of myofibrillar protein and mRNA expression in patients with Acute Quadriplegic Myopathy
Presenting/First Author: Norman, Holly (Grad)
Department, Institution: Neurophysiology, Uppsala University
Address: Akademiska Sjukhuset, Ing 85, 3 tr Uppsala, 751 85 Sweden
Phone/Fax: 46-18-6113432 / 46-18-556106
Email: [email protected]
Abstract Theme:  
Author(s): Norman, H (Uppsala University, Uppsala, Sweden)
Andersson, P (Uppsala University, Uppsala, Sweden)
Nordquist, J (Uppsala University, Uppsala, Sweden)
Zackrisson, H (Karolinska Institute, Stockholm, Sweden)
Larsson, L (Uppsala University, Uppsala, Sweden)
 
Body of Abstract: Acute Quadriplegic Myopathy (AQM) is an acquired myopathy caused by pharmacological treatment used in anaesthesiology and the intensive care and is characterized by severe muscle wasting and paralysis, and a loss of myosin (MyHC) and myosin binding proteins (MyBP). Degradation of thick filament proteins has been suggested to cause the muscle wasting in AQM. Based on previous observations in our group we hypothesize that downregulation of proteins synthesis at the transcriptional level is equally important in causing the loss of muscle mass and myofibrillar proteins. Therefore myofibrillar protein and mRNA expression, i.e., MyHC, actin, and MyBP, were measured during the acute and recovery stages of AQM. At the protein level, a loss of MyHC and MyBP was observed during muscle paralysis according to immunoblots and gel electrophoreses. Protein levels returned to normal after 1-2 months recovery. At the transcriptional level, decreased levels of mRNA for the domi nating MyHC isoforms and actin, but not for the MyBP, were observed during the acute stage of AQM according to real-time PCR. It is suggested that the decreased MyHC synthesis at the transcriptional level is a strong contributing factor in the development of AQM.


 
Abstract Title: Impact of chronic post-synaptic block of neuromuscular transmission, muscle unloading and mechanical ventilation on skeletal muscle protein and mRNA expression
Presenting/First Author: Norman, Holly S (Grad)
Department, Institution: Clinical Neurophysiology, Uppsala University
Address: Inst f Neurovetenskap, klinisk neurofysiologi, Akademiska Sjukhuset, Ing 85, 3tr Uppsala, 751 85 Sweden
Phone/Fax: 011-46-18-6113432 / 011-46-18-556106
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Norman, Holly (Uppsala University, Uppsala, Sweden)
Nordquist, J (Uppsala University, Uppsala, Sweden)
Andersson, P (Uppsala University, Uppsala, Sweden)
Ansved, T (Karolinska Institute, Stockholm, Sweden)
Tang, X (Hershey Medical Center at the Pennsylvania State University College of Medicine, Hershey, Pennsylvania)
Dworkin, B (Hershey Medical Center at the Pennsylvania State University College of Medicine, Hershey, Pennsylvania)
Larsson, L (Uppsala University, Uppsala, Sweden)
 
Body of Abstract: We developed a rodent model to study muscle atrophy found in the intensive care unit (ICU). Rats were paralyzed by block of neuromuscular transmission (NMB) and mechanically ventilated for 1-2 weeks. The effects on protein and mRNA expression of sarcomeric proteins, i.e., myosin heavy chain (MyHC), actin, myosin binding proteins -C (MyBP-C) and - H (MyBP-H) in fast- and slow-twitch limb, respiratory and masticatory muscles, were studied. At the protein level, a decreased MyHC:actin ratio was observed in both fast- and slow-twitch hindlimb, a masticatory and in one respiratory (intercostal) muscles, but not in the other (diaphragm). At the mRNA level, decreased expression of MyHC isoform(s) was observed in the hindlimb and intercostal muscles, but not in the diaphragm and masseter. The MyBP-C mRNA expression was decreased in the limb muscles, but otherwise remained unaffected, whereas MyBP-H increased for all muscles. Differing effects on protein and mRNA expre ssion between the ICU model and a model with denervated hindlimb muscles are also reported. The NMB rat is a useful model for studying muscle wasting and impaired muscle function in immobilized ICU patients.


 
Abstract Title: A gene for speed: The ACTN3 R577X polymorphism influences muscle performance
Presenting/First Author: North, Kathryn N (Faculty)
Department, Institution: Neurogenetics, University of Sydney
Address: Hawkesbury Road, Westmead Sydney, 2145 Australia
Phone/Fax: +61-438-601654 / +61-2-98453389
Email: [email protected]
Abstract Theme: Sarcomere Cytoskeleton
Author(s): North, Kathryn N (University of Sydney, NSW, Australia)
Macarthur, Daniel G (University of Sydney, NSW, Australia)
Yang, Nan (Children's Hospital at Westmead, Sydney, NSW, Australia)
Seto, Jane T (Children's Hospital at Westmead, Sydney, NSW, Australia)
Raftery, Joanna (Children's Hospital at Westmead, Sydney, NSW, Australia)
Kee, Anthony J (Children's Medical Research Institute, Sydney, NSW, Australia)
Hardeman, Edna C (Children's Medical Research Institute, Sydney, NSW, Australia)
Gunning, Peter W (Children's Hospital at Westmead, Sydney, NSW, Australia)
Body of Abstract: Alpha-actinin-3 is a component of the Z-disc in fast skeletal muscle fibres. Intriguingly, a common nonsense variant in the human ACTN3 gene (R577X) results in complete deficiency of alpha-actinin-3 in ~18% of the general population.We have recently demonstrated a significantly lower frequency of XX homozygotes amongst elite sprint athletes, suggesting that the presence of alpha-actinin-3 is required for optimal fast fibre function. This finding has been supported by a number of recent studies demonstrating associations between R577X genotype and muscle strength and response to training. The ACTN3 R577X polymorphism thus represents a genetic factor influencing muscle performance in humans.
We have developed an ACTN3 KO mouse model that replicates human alpha-actinin-3 deficiency. The closely related protein, α-actinin-2, is able to compensate for the absence of α-actinin-3 in sarcomere assembly and function at baseline physiological conditions. However, there is a marked increase in sarcomeric damage following eccentric exercise in KO mice compared to WT, suggesting that α-actinin-3 protects muscle from exercise-induced damage, and explaining the association between R577X and muscle function in humans.


 
Abstract Title: Unravelling the thin filament: mechanisms of weakness in intranuclear rod myopathy
Presenting/First Author: North, Kathryn N (Faculty)
Department, Institution: Neurogenetics, University of Sydney
Address: Children's Hospital, Hawkesbury Road, Westmead Sydney, 2145 Australia
Phone/Fax: +61-438-601654 / +61-2-98453389
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): North, Kathryn N (University of Sydney, NSW, Australia)
Domazetovska, Ana (University of Sydney, NSW, Australia)
Ilkovski, Biljana (Chidren's Hospital at Westmead, Sydney, Australia)
Valova, Val A (Children's Medical Research Institute, Sydney, Australia)
Hardeman, Edna C (Children's Medical Research Institute, Sydney, Australia)
Gunning, Peter W (Chidren's Hospital at Westmead, Sydney, Australia)
Yang, Nan (Chidren's Hospital at Westmead, Sydney, Australia)
Cooper, Sandra T (Chidren's Hospital at Westmead, Sydney, Australia)
Body of Abstract: Mutations at Val163 in ACTA1 result in congenital myopathy with rods containing actin and α-actinin confined to the nucleus and minimal disruption of sarcomeric structure. We are investigating mechanisms underlying intranuclear rod formation and their effects on cell function. By MALDI-TOF MS the mutant V163M ACTA1 allele is expressed at lower relative levels to the wild type allele. The expression levels of the mutant allele varied between affected patients within the same family and correlated with severity of symptoms. Transfection studies in C2C12 myoblasts show significant increase in the ability of the V163M actin mutant to incorporate into large actin filaments. By live-cell imaging, intranuclear aggregates form inside the nucleus and are dynamic structures. We also demonstrate that the ‘trapping’ of alpha-actinin into intranuclear aggregates has an adverse effect on cell viability and division. We have now generated a knock-in mouse model of the V163M mutation in ACTA1. These mice are viable, but a proportion die in the first week of life, likely due to poor feeding associated with a myopathy. We are now using the animal model to further investigate the pathogenesis of weakness in our patients.


 
Abstract Title: Regulation of muscle contraction in a novel myopathy associated with a mutation in the beta-tropomyosin (TPM2) gene
Presenting/First Author: Ochala, Julien (Postdoc - Degree Year: 2005)
Department, Institution: Neuroscience, Clinical Neurophysiology, Uppsala University
Address: University Hospital, Entrance 85, 3rd floor Uppsala, SE-751 85 Sweden
Phone/Fax: +46 18 611 9338 / +46 18 55 6106
Email: [email protected]
Abstract Theme:  
Author(s): Ochala, Julien (Uppsala University, Uppsala 751 85, Sweden)
Kimber, Eva (Sahlgrenska University Hospital, Goteborg 413 45, Sweden)
Tajshargi, Homa (Sahlgrenska University Hospital, Goteborg 413 45, Sweden)
Tulinius, Mar (Sahlgrenska University Hospital, Goteborg 413 45, Sweden)
Oldfors, Anders (Sahlgrenska University Hospital, Goteborg 413 45, Sweden)
Larsson, Lars (Uppsala University, Uppsala 751 85, Sweden)
 
Body of Abstract: A novel beta-tropomyosin (beta-Tm) TPM2 mutation, R133W, in exon 4 associated with muscle weakness and distal arthrogryposis was recently identified in a woman and her daughter. In spite of muscle weakness, vastus lateralis biopsies revealed only minor morphological changes and type I fiber predominance. No nemaline rods were observed. The aim of the study was to explore the mechanisms underlying the motor handicap in patients with the beta-Tm mutation. Maximum force normalized to fiber cross-sectional area (SF), maximum unloaded shortening velocity (Vo), and tension-pCa relationship were measured in chemically skinned vastus lateralis muscle fibers (n = 105) from the 2 patients and from 4 healthy control subjects. A 30% decrease in SF and a 40% increase in Vo were observed (p<0.05) in patients type I muscle cells where the beta-Tm is dominating. The force-pCa relationship did not differ between patient and control fibers. Further, unregulated actin filament speed propelled by myosin was similar between patient and control fibers. Thus, the beta-Tm mutation appears to have a significant effect on the kinetics of the cross-bridge cycling causing muscle weakness, without affecting the Ca2+-activation of contractile proteins.


 
Abstract Title: Transcriptional profile of transgenic mice expressing an expanded CUG repeat
Presenting/First Author: Osborne, Robert J (Postdoc - Degree Year: 2)
Department, Institution: Neurology, University of Rochester
Address: 575 Elmwood Ave Rochester, NY 14642 United States
Phone/Fax: 585 461 4791 / 585 273 1255
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Osborne, Robert J (University of Rochester, NY, 14642)
Welle, Stephen (University of Rochester, NY, 14642)
Thornton, Charles A (University of Rochester, NY, 14642)
 
Body of Abstract: HSALR transgenic mice expressing CUG expansion RNA in skeletal muscle exhibit myotonia, myopathy and misregulated alternative splicing characteristic of myotonic dystrophy type 1 (DM1). In an attempt to identify additional pathways affected by repeat expansion RNA, we used expression microarrays to compare skeletal muscle in two independent founder lines of HSALR mice and in wild-type (WT) mice. We identified 105 probe sets that were differentially expressed in both transgenic lines compared to WT (P≤0.01, fold-change ≥2). We postulated that some expression changes resulted from responses to myotonic discharges or nonspecific muscle injury. To eliminate these genes from consideration, we also studied adr (ClC-1 null) mice and their WT controls. adr mice have severe myotonia and modest up-regulation of regeneration dependent genes. After filtering out probe sets showing similar changes in adr and HSALR mice, we were left with 53 genes that were up- regulated and 16 genes that were down-regulated. Of note, none of these genes have been previously implicated in DM pathogenesis. The function of these genes suggests signaling pathways that might be involved in the DM disease process.


 
Abstract Title: Muscle specific delivery of the alpha sarcoglycan gene provides functional and morphological correction of limb girdle muscular dystrophy type 2D
Presenting/First Author: Pacak, Christina A (Grad)
Department, Institution: Molecular Genetics and Microbiology, University of Florida
Address: 1600 SW Archer Rd, ARB RG_150 Gainesville, FL 32610 United States
Phone/Fax: 352-377-6257 / 352-392-8885
Email: [email protected]
Abstract Theme: Therapies
Author(s): Pacak, Christina A (Dept. of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida, Gainesville, FL, 32610)
Gaidosh, Gabriel (Dept. of Physiology, Powell Gene Therapy Center, University of Florida, Gainesville, FL, 32610)
Germain, Sean (Dept. of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL, 32610)
Lewis, Melissa A (Dept. of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL, 32610)
Campbell, Kevin P (HHMI, University of Iowa)
Walter, Glenn A (Dept. of Physiology, Powell Gene Therapy Center, University of Florida, Gainesville, FL, 32610)
Byrne, Barry J (Dept. of Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL, 32610)
 
Body of Abstract: Limb Girdle Muscular Dystrophy (LGMD) describes a group of inherited diseases that are the physical consequence of mutations in genes encoding proteins involved in maintaining skeletal muscle membrane stability. LGMD-2D is the result of mutations in the alpha sarcoglycan (sgca) gene. Here we describe a gene delivery approach employing a muscle specific promoter expressing the human sgca gene in combination with the adeno-associated virus 1 capsid to provide functional and morphological correction of LGMD-2D. Delivery of our construct to adult sgca-/- mice has demonstrated successful establishment of the sarcoglycan complex in muscle cell membranes and reduced fiber damage. Using a T2 weighted MRI technique, we observed the prevention of lesion development in lower hind-limbs following delivery of our construct to sgca-/- mouse neonates. Force mechanics measurements performed on extensor digitorum longus muscles from mice treated as adults or neonates showed c orrection of the typical resistance to stretch LGMD-2D phenotype. Both our gene delivery scheme and T2 weighted MRI method for non-invasively assessing muscle integrity are clinically applicable techniques warranting further progression towards implementation in humans.


 
Abstract Title: Characterization of the cardiac phenotype in a mouse model of Pompe Disease and correction using AAV-mediated gene delivery
Presenting/First Author: Pacak, Christina A (Grad)
Department, Institution: Molecular Genetics and Microbiology, University of Florida
Address: 1600 SW Archer Rd, ARB RG_150 Gainesville, FL 32610 United States
Phone/Fax: 352-846-2887 / 352-392-8885
Email: [email protected]
Abstract Theme: Cardiac Pathology
Author(s): Pacak, Christina A (Molecular Genetics and Microbiology, Powell Gene Therpay Center, Gainesville, FL 32610)
Mah, Cathryn S (Cellular and Molecular Therapy, Powell Gene Therapy Center, Gainesville, FL, 32610)
Cresawn, Kerry O (Pediatric Cardiology, Powell Gene Therapy Center, Gainesville, FL, 32610)
Lewis, Melissa A (Pediatric Cardiology, Powell Gene Therapy Center, Gainesville, FL, 32610)
Germain, Sean (Pediatric Cardiology, Powell Gene Therapy Center, Gainesville, FL, 32610)
Byrne, Barry J (Pediatric Cardiology, Powell Gene Therapy Center, Gainesville, FL, 32610)
 
Body of Abstract: Pompe Disease is a form of muscular dystrophy and metabolic myopathy caused by mutations in the acid alpha glucosidase (GAA) gene and results in dilated cardiomyopathy and skeletal muscle weakness. Insufficient amounts of GAA leads to the accumulation of glycogen in lysosomes and consequent cellular dysfunction. We have observed development of the cardiac pathology in Pompe mice (GAA-/-) through analysis of ECG traces, MRI data and histological analysis. Similar to the patient population an age-related progression of disease occurs in the mouse model. By 2 weeks of age periodic acid Schiff (PAS) staining shows abnormal amounts of glycogen in lysosomes of cardiac cells. Mitochondrial aggregation and lysosome enlargement are evident in electron microscopy images. Through ECG analysis we observe a shortened PR interval at 3 months mimicking the conduction phenotype in human patients. MRI data shows an increased myocardial mass by 12 months. A treatment approa ch using intra-venous (IV) delivery of either recombinant adeno-associated virus (rAAV) serotypes 1 or 9 carrying the CMV-hGAA construct to GAA-/- neonates results in restoration of GAA activity and ameliorate the cardiac phenotypes observed by PAS, ECG, and MRI analysis.


 
Abstract Title: Overcoming Immune Rejection in Myoblast Transplant Therapy through Hematopoietic Cell Transplantation
Presenting/First Author: Parker, Maura H (Postdoc - Degree Year: 2)
Department, Institution: Transplantation Biology, Fred Hutchinson Cancer Research Center
Address: 1100 Fairview Ave. N. Seattle, WA 98109 United States
Phone/Fax: 206-667-1623 / 206-667-6124
Email: [email protected]
Abstract Theme: Therapies
Author(s): Parker, Maura H (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109)
Storb, Rainer (Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109)
Kuhr, Christian (Department of Surgery, University of Washington, Seattle, WA 98195)
Tapscott, Stephen J (Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109)
 
Body of Abstract: Duchenne Muscular Dystrophy (DMD) is the most common and severe form of muscular dystrophy in humans. The goal of satellite cell-derived myoblast transplant therapy (MTT) for DMD is to increase dytrophin expression in existing fibers, and provide a stem cell source for future regeneration. The major limitation to the success of MTT has been immune rejection of transplanted myoblasts. We asked if generating a chimeric DMD-affected dog, in which the immune system is reconstituted with DLA-identical donor-derived hematopoietic cells, could provide a more effective platform for myoblast transplant therapy. DMD affected canines were subjected to myeloblative conditioning prior to hematopoietic cell transplant, generating a fully chimeric dog. The hematopoietic cells did not detectably contribute to either skeletal muscle or the satellite cell population. However, intramuscular injection of donor-derived satellite cell-derived myoblasts results in robust and stable expr ession of dystrophin in DMD affected skeletal muscle. This establishes chimeric recipients as a viable model for addressing myoblast transplantation in an immune tolerant, random-bred, large animal model of Duchenne muscular dystrophy.


 
Abstract Title: Genetic Disruption of Calcineurin Improves Skeletal Muscle Pathology and Cardiac Function in a Mouse Model of Limb-Girdle Muscular Dystrophy
Presenting/First Author: Parsons, Stephanie A (Postdoc - Degree Year: 2003)
Department, Institution: Physiology, University of Pennsylvania
Address: 1115 Abramson, 3615 Civic Center Blvd. Philadelphia, PA 19446 United States
Phone/Fax: 215-898-0046 / 215-746-3684
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Parsons, Stephanie A (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039)
Millay, Douglas P (University of Cincinnati, Cincinnati, OH, 45267)
Sargent, Michelle A (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039)
Naya, Francisco J (Boston University, Boston, MA, 02215)
McNally, Elizabeth M (The University of Chicago, Chicago, IL 60637)
Molkentin, Jeffery D (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039)
 
Body of Abstract: Calcineurin (Cn) is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase that regulates differentiation-specific gene expression in diverse tissues, including control of fiber-type in skeletal muscle. Cn is also thought to regulate dystrophic muscle pathogenesis. For instance, the Cn inhibitor cyclosporine A can delay muscle regeneration in the dystrophin-deficient mdx mouse, while expression of activated Cn can reduce disease severity and increase utrophin expression. Here we examined the role of Cn in the more severe delta-sarcoglycan-/- (scgd-/-) mouse model of limb-girdle muscular dystrophy. In contrast to mdx mice, genetic deletion of a loxP-targeted CnB1 gene using a skeletal muscle-specific cre allele in the scgd-/- background substantially reduced skeletal muscle degeneration and histopathology compared with scgd-/- mice. A similar decrease in disease manifestation was also observed in scgd-/- CnAbeta gene-targeted mice in both skeletal muscle and heart. Conversely, greater Cn expression augmented cardiac fibrosis, decreased cardiac function, and increased fat replacement in various skeletal muscles. Collectively, our results suggest that Cn inhibition may benefit limb-girdle type of muscular dystrophies.


 
Abstract Title: Aberrant Golgi Complex Organization in Dystrophin-Deficient Skeletal Muscle Fibers
Presenting/First Author: Percival, Justin M (Postdoc - Degree Year: 2002)
Department, Institution: Physiology and Biophysics, University of Washington
Address: 1959 NE Pacific St Seattle, WA 98195 United States
Phone/Fax: 206 543 9094 / 206 685 0619
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Percival, Justin M (University of Washington, WA 98195)
Froehner, Stanley C (University of Washington, WA 98195)
 
Body of Abstract: Interest in the Golgi complex (GC) in skeletal muscle has emerged from studies showing that defective glycosylation causes congenital muscular dystrophy. Glycosylation is a well-established function of the GC. We investigated whether GC abnormalities also occur in the mdx mouse model for Duchenne Muscular Dystrophy. In wild type muscle fibers the GC has an unconventional punctate morphology in contrast to the ribbon-like GC organization of adjacent non-muscle cells. The GC shows a fiber type-specific distribution and is predominantly localized just below the sarcolemma. The GC is heavily concentrated beneath post-synaptic membranes in a fiber type-specific manner, but is reduced and mislocalized in mdx muscles. In both dystrophic and regenerating muscle fibers surface GC puncta are reduced. Increased numbers of enlarged GC puncta are found in the core of regenerating fibers. Aberrant GC organization occurs prior to necrosis and may represent an unrecognized early event in the development of dystrophy. Together these findings demonstrate a secondary disruption of GC organization in dystrophin-deficient muscles. Alterations in Golgi organization may disrupt normal trafficking pathways and contribute to the dystrophic phenotype.


 
Abstract Title: Molecular basis for skeletal muscle group diversity: How many ‘colors’ and what kind?
Presenting/First Author: Porter, John D (Faculty)
Department, Institution: Channels, Synapses, & Circuits Cluster, National Institute of Neurological Disorders and Stroke
Address: 6001 Executive Blvd, NINDS/NSC 2142 Bethesda, MD 20878 United States
Phone/Fax: 301-496-1917 / 301-402-1501
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Porter, John D (Case Western Reserve University and University Hospitals of Cleveland)
Merriam, Anita P (Case Western Reserve University and University Hospitals of Cleveland)
Feuerman, Jason (Case Western Reserve University and University Hospitals of Cleveland)
Gong, Bendi (Case Western Reserve University and University Hospitals of Cleveland)
Kaminski, Henry J (Case Western Reserve University and University Hospitals of Cleveland)
 
Body of Abstract: Skeletal muscles are not created equal. The muscle allotype concept states that muscle groups may differ by more than just their content of highly conserved muscle fiber types. It is important to understand the breadth and consequences of allotype-specific traits, as current neuromuscular disease models do not account for the not-uncommon heterogeneity of muscle group responses. Here, we used genome-wide expression profiling to characterize a diverse set of adult murine skeletal muscles. We asked the question—how many muscle colors/allotypes and what kind? Our approach used a genome-wide perspective that facilitates definitive allotype identification. We identified (a) a consensus skeletal muscle transcriptome but also (b) clear-cut differentiation of four putative muscle allotypes based upon the aggregate genetic distance between muscle groups. Data strongly support the notion that skeletal muscle group traits are not a simple result of the aggregate properties o f highly conserved fiber types. These data represent an important step toward defining a novel classification scheme that can provide a conceptual basis for future studies of the differential development, function, and pathogenesis of muscle groups. Support: NEI.


 
Abstract Title: NIH translational research program in muscular dystrophy
Presenting/First Author: Porter, John D (Faculty)
Department, Institution: Channels, Synapses, & Circuits Cluster, National Institute of Neurological Disorders and Stroke
Address: 6001 Executive Blvd, NINDS/NSC 2142 Bethesda, MD 20878 United States
Phone/Fax: 301-496-1917 / 301-402-1501
Email: [email protected]
Abstract Theme:  
Author(s): Porter, John D (National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, MD)
Nuckolls, Glen (National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), Bethesda, MD)
Miller, Thomas (National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, MD)
 
Body of Abstract: NINDS, NIAMS, and NICHD have released a program that facilitates the translation of mechanistic knowledge into new therapies for muscular dystrophy (MD). Although pathogenic mechanisms are known for many MDs, there is no consensus as to which strategy, or combination of strategies, may prove successful in reducing patient morbidity and mortality. This program solicits applications for exploratory/developmental grants (R21; PAR-06-203) and milestone-driven cooperative agreements (U01; PAR-06-044) to develop efficacious treatments for MD and supports the development of drugs, biologics, and devices in cells and animals, but excludes basic or mechanistic work and non-exempt human studies. Awards can support the full spectrum of preclinical development activities from preliminary studies through IND or IDE application to the FDA. Applications for support are reviewed in a special peer review environment in a context of other preclinical development projects. Experimen tal approaches should address one or more of the MDs, and be based upon established disease mechanisms or in strategies previously shown to have potential efficacy in MD. Potential applicants are encouraged to make early contact with an NIH program director listed here.


 
Abstract Title: Spectrin mutations cause destabilitzation of membrane proteins and Purkinje cell degeneration in spinocerebellar ataxia type 5
Presenting/First Author: Ranum, Laura (Faculty)
Department, Institution: Genetics, Cell Biology and Development, University of Minnesota
Address: MMC 206, 420 Delaware St. S.E. Minneapolis, MN 55455 United States
Phone/Fax: 612 624-0901 / 612 625-8488
Email: [email protected]
Abstract Theme:  
Author(s): Ranum, Laura (University of Minnesota, Minneapolis)
Dick, Katherine (University of Minnesota, Minneapolis)
Gincel, Dan (Johns Hopkins University, Baltimore MD)
Zuelke, Christine (University of Lübeck, Germany)
Brice, Alexis (INSERM and UniversitySalpetriere Hospital, Paris France)
Rothstein, Jeffrey (Johns Hopkins University, Baltimore MD)
Day, John (University of Minnesota, Minneapolis)
Yoshio, Ikeda (University of Minnesota, Minneapolis)
Body of Abstract: One unexplained feature of Duchenne muscular dystrophy is its effect on the central nervous system, which involves variably severe cognitive impairment. The mechanisms by which the absence of dystrophin cause neuronal loss or dysfunction remain unknown. We report that mutations in beta-III spectrin, a similar plasma-membrane associated cytoskeletal protein, cause cerebellar Purkinje cell degeneration in spinocerebellar ataxia type 5. Two families have separate in-frame deletions in the 3rd spectrin repeat and a third family has a missense mutation in the actin/ARP1 binding region. Beta-III spectrin, which is highly expressed in Purkinje cells, stabilizes the glutamate transporter EAAT4 at the surface of the plasma membrane. Dramatic differences in EAAT4 were found by Western and cell fractionation in SCA5 autopsy tissue and cell culture studies demonstrate that wildtype but not mutant β-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutat ions are a novel cause of neurodegenerative disease that affect the stabilization of membrane proteins involved in glutamate signaling. These results may help determine how Duchenne muscular dystrophy, and other disorders involving cytoskeletal proteins, cause CNS effects.


 
Abstract Title: Chronic treatment with the beta-adrenoceptor agonist formoterol deleteriously affects cardiac function in rats
Presenting/First Author: Ryall, James G (Postdoc - Degree Year: 2006)
Department, Institution: Physiology, The University of Melbourne
Address: Grattan Street Melbourne, 3010 Australia
Phone/Fax: 613-8344-8942 / 613-8344-5818
Email: [email protected]
Abstract Theme: Cardiac Pathology
Author(s): Ryall, James G (The University of Melbourne, Victoria, 3010, Australia)
Schertzer, Jonathan D (The University of Melbourne, Victoria, 3010, Australia)
Allen, Andrew (The University of Melbourne, Victoria, 3010, Australia)
Lynch, Gordon S (The University of Melbourne, Victoria, 3010, Australia)
 
Body of Abstract: The beta-agonist formoterol has been shown to elicit skeletal muscle hypertrophy and may have therapeutic potential for muscle wasting conditions. However, their use in clinical practice requires a better understanding of the possible effects of activation of beta-adrenoceptors in the heart. We tested the hypothesis that low-dose administration of formoterol would not cause cardiac hypertrophy and associated cardiovascular complications in rats. Male rats were anesthetized and implanted with a radio telemeter to monitor cardiovascular function during 4 weeks of formoterol administration (25 micro g/kg/day, i.p.). Treatment reduced systolic and diastolic blood pressure (9% and 14%, respectively), increased heart rate (19%) and decreased diastolic relaxation time (22%), in the first few days of administration. However, after treatment, heart rate was reduced(12%) and diastolic relaxation increased (14%). These findings indicate that even low-dose formoterol admini stration affects cardiovascular parameters, and warrants closer scrutiny before clinical application can be advocated.
Supported by the Muscular Dystrophy Association (USA), the Australian Research Council and the Australian Association of Gerontology.


 
Abstract Title: Dystrophin and utrophin: sequence homologues with distinct functional mechanisms
Presenting/First Author: Rybakova, Inna (Faculty)
Department, Institution: Physiology, UW
Address: 1300 University Av. Madison, WI 53706 United States
Phone/Fax: 608-265-3440 / 608-265-5512
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Rybakova, Inna N (Dept. of Physiology, University of Wisconsin Medical School, Madison, WI 53706)
Humston, Jill L (Dept. of Physiology, University of Wisconsin Medical School, Madison, WI 53706)
Sonnemann, Kevin J (Dept. of Physiology, University of Wisconsin Medical School, Madison, WI 53706)
Ervasti, James M (Dept. of Physiology, University of Wisconsin Medical School, Madison, WI 53706)
 
Body of Abstract: Dystrophin and utrophin are homologous proteins that link the actin cytoskeleton to the extracellular matrix. We previously demonstrated that the actin binding region of full-length utrophin spans from its N-terminus through the first 10 spectrin-like repeats. Here, we defined the molecular epitopes of dystrophin-actin interaction and directly compared the actin binding properties of dystrophin and utrophin. According to our data, dystrophin and utrophin both bound alongside actin filaments with sub-micromolar affinities. Both proteins stabilized F-actin from depolymerization with similar efficacies, but did not appear to compete for binding sites on actin. We found that dystrophin binding to actin was sensitive to increasing ionic strength while utrophin binding was unaffected. Dystrophin construct spanning from the N-terminus through repeat 10 bound actin with low affinity and near 1:1 stoichiometry as previously measured for the isolated N-terminus. In contra st, a construct spanning from the N-terminus through repeat 17 bound actin with properties most similar to full-length dystrophin. We conclude that dystrophin and utrophin are functionally homologous actin binding proteins but act through distinct modes of contact.


 
Abstract Title: Eosinophilia, a mediator of fibrosis in dystrophin-deficient muscle
Presenting/First Author: Sokolow, Sophi (Postdoc - Degree Year: 2)
Department, Institution: Physiological Science, University of California at Los Angeles
Address: 621 Charles Young Drive S Los Angeles, CA 90095 United States
Phone/Fax: +13102068390 / +13108258489
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Sokolow, Sophie (University of California, Los Angeles, CA 90095)
Wehling-Henricks, Michelle (University of California, Los Angeles, CA 90095)
Lee, James J (Mayo Clinic Arizona, Scottsdale, AZ 85259)
Tidball, James G (University of California, Los Angeles, CA 90095)
 
Body of Abstract: Eosinophils (eos) are inflammatory cells in dystrophin-deficient muscle that have unknown significance in the pathology of muscular dystrophy. Major basic protein (MBP) is a major cytolytic protein that is released by eos that can lyse target cells, suggesting that MBP may mediate eos lysis of dystrophin-deficient, mdx mouse muscles. We confirmed eos’ cytotoxicity by showing that these cells lyse myotubes in culture. We tested whether eos contribute to mdx pathology by generating a mutant mouse model that is deficient in dystrophin and major basic protein (mMBP-1) genes. We observed that MBP+/+ eos invade mdx fibers and are localized at sites of necrotic fibers. However, the MPB-/-/mdx muscles show no reduction in muscle fiber injury in vivo, although they invade mdx muscle. MBP deficiency has no effect on the concentrations of macrophages, neutrophils or CD4+ T-cells in mdx muscle, but causes a large increase in cytotoxic T-cells (CTLs) in mdx diaphragm and qua driceps. Finally, we showed that the absence of MBP causes a significant reduction in diaphragm and hamstring fibrosis at 14 months of age. These findings show that eosinophilia promotes fibrosis of dystrophic muscle by a mechanism that may involve CTLs.


 
Abstract Title: Gamma-actin is not required for muscle development but its absence leads to a novel form of centronuclear myopathy
Presenting/First Author: Sonnemann, Kevin J (Postdoc - Degree Year: 2005)
Department, Institution: Physiology, University of Wisconsin-Madison
Address: 1300 University Ave Madison, WI 53704 United States
Phone/Fax: 608.265.3440 / 608.265.5512
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Sonnemann, Kevin J (Department of Physiology, University of Wisconsin, Madison, WI 53706)
Fitzsimons, Daniel P (Department of Physiology, University of Wisconsin, Madison, WI 53706)
Patel, Jitandrakumar R (Department of Physiology, University of Wisconsin, Madison, WI 53706)
Liu, Yewei (Department of Biochemistry, University of Maryland, Baltimore, MD 21201)
Schneider, Martin F (Department of Biochemistry, University of Maryland, Baltimore, MD 21201)
Moss, Richard L (Department of Physiology, University of Wisconsin, Madison, WI 53706)
Ervasti, James M (Department of Physiology, University of Wisconsin, Madison, WI 53706)
 
Body of Abstract: Cytoplasmic gamma-actin is hypothesized to play important roles in myogenesis and sarcomere assembly. In mature skeletal muscle gamma-actin localizes to costameres. Here, we show that muscle development proceeded normally in skeletal muscle-specific gamma-actin knockout (Actg1-msKO) mice. In 1-month-old mice, Actg1-msKO muscle fibers were regular in shape and appearance and displayed properly assembled sarcomeres. However, Actg1-msKO mice displayed an overt myopathy characterized by hind limb joint contractures. Focal areas of necrosis and regeneration progressed from low levels at 3 months of age to widespread incidence by 12 months of age. Actg1-msKO mice exhibited deficits in whole-body tension and isometric twitch force in isolated hind limb muscles. Surprisingly, these pathologies were observed without the fibrosis, inflammation, and membrane damage typical of muscular dystrophies but are consistent with a novel centronuclear myopathy. Our data demonstrate a n important role for gamma-actin in adult skeletal muscle and describe a new mouse model to explore a structural basis for centronuclear myopathies. Supported by grants from the MDA, AHA, NIH 049899, and NRSA T32 HL07936 from the UW Cardiovascular Research Center.


 
Abstract Title: Impaired Inward Rectifier K+ Conductance in Ba+2-Poisoned Muscle: Insights into the Pathophysiology of Hypokalemic Paralysis
Presenting/First Author: Struyk, Arie F (Faculty)
Department, Institution: Neurology, University of Texas-Southwestern Medical Center
Address: 6000 Harry Hines Blvd Dallas, TX 75390 United States
Phone/Fax: 214 648-7453 / 214 648-7451
Email: [email protected]
Abstract Theme: Sarcolemmal Excitability
Author(s): Struyk, Arie F (University of Texas-Southwestern Medical Center, Dallas, TX 75390)
Cannon, Stephen C (University of Texas-Southwestern Medical Center, Dallas, TX 75390)
 
Body of Abstract: Ba+2 poisoning induces sarcolemmal depolarization accompanied by hypokalemia, similar to paralytic attacks that occur in familial Hypokalemic Periodic Paralysis (HypoPP), Andersen-Tawil syndrome (ATS), and thyrotoxicosis. We measured resting K+ currents from Ba+2-poisoned mouse skeletal muscle under voltage-clamp. Ba+2 depolarized fibers and selectively blocked a strong inward rectifying K+ conductance (gIRK), consistent with the notion that gIRK has a major influence on VREST. The pathological implications of this dependence were explored. As expected, VREST became hyperpolarized in low external K+ (2 mM), but was depolarized in 1 mM K+. We propose this paradoxical depolarization response in very low K+ is related to the leftward shift in gIRK. In support of this idea, partial block of gIRK with Ba+2 altered the threshold, such that sarcolemmal depolarization could be elicited by exposure to 2 mM K+. Model simulations derived from these data illustrate the relationship between gIRK and VREST stability. These findings suggest that altered gIRK influence over VREST may contribute to the depolarization-induced paralysis found in HypoPP, ATS, and thyrotoxicosis.


 
Abstract Title: Decreased stiffness and power generation from muscle lacking muscle LIM protein
Presenting/First Author: Swank, Douglas (Faculty)
Department, Institution: Center for Biotechnology, Rensselaer Polytechnic Institute
Address: 110 8th Street Troy, NY 12180 United States
Phone/Fax: 518-276-4174 / 518-276-2851
Email: [email protected]
Abstract Theme: Cardiac Pathology
Author(s): Swank, Douglas (Center for Biotechnology, Rensselaer Polytechnic Institute, Troy, NY)
Lesage, Heather (Dept of Mol. Physiology, University of Vermont, Burlington, VT)
Clark, Kathleen (Huntsman Cancer Institute, University of Utah, Salt Lake City, UT)
 
Body of Abstract: Muscle LIM protein (MLP) is located in the Z-line of sarcomeres where it is hypothesized to be involved in sensing muscle stretch. Mutations in human MLP lead to cardiac hypertrophy or dilated cardiomyopathies. We generated a Drosophila line null for MLP, Def/Def, through the use of deficiency chromosomes. MLP null flies that survive to adulthood are unable to fly. Transgenic expression of one copy of the mlp84B gene in the deficiency background, P[mlp+],Def/Def, rescues flight ability (flight index (FI) of 2.8, wing beat frequency (WBF) of 150 Hz), although not to wild type levels (FI of 5.4, WBF of 184 Hz). Transgenic expression of 2 MLP transgenes, P[mlp+, mlp+],Def/Def, increased WBF (162 Hz) compared to P[mlp+],Def/Def, but no improvement in flight ability was observed. Mechanical analysis of skinned flight muscle fibers showed a 30% decrease in oscillatory power production for fibers lacking MLP compared to P[mlp+, mlp+],Def/Def fibers. MLP null fibers disp layed decreased passive, active, and rigor stiffness compared to P[mlp+, mlp+], Def/Def fibers. We conclude that MLP is either a parallel compliant element in the z-disk, or it interacts with D-titin to help maintain muscle stiffness. Supported by NIH AR51473 to D.M.S.


 
Abstract Title: Transplantation of SM/C-2.6+ satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice
Presenting/First Author: Takeda, Shin'ichi (Faculty)
Department, Institution: Department of Molecular Therapy, National Institute of Neuroscience, NCNP
Address: 4-1-1 Ogawa-higashi Kodaira, 187-8502 Japan
Phone/Fax: +81-42-346-1720 / +81-42-346-1750
Email: [email protected]
Abstract Theme: Therapies
Author(s): Takeda, Shin'ichi (Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan)
Ikemoto, Madoka (Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan)
Fukada, So-ichiro (Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan)
Uezumi, Akiyoshi (Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan)
Masuda, Satoru (Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan)
Miyoshi, Hiroyuki (Subteam for Manipulation of Cell Fate, BRC, RIKEN Tsukuba Institute, Ibaraki, Japan)
Yamamoto, Hiroshi (Department of Immunology, Osaka University, Osaka, Japan)
Miyagoe-Suzuki, Yuko (Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan)
Body of Abstract: Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by?mutations in the dystrophin gene and satellite cells are expected to be an ideal cell source for cell therapy of DMD. We developed a cell sorting system to purify quiescent satellite cells from intact mouse skeletal muscle using a recently developed novel monoclonal antibody SM/C-2.6 (Exp. Cell Res. 296: 245-55, 2004). First, to determine the regenerative capacity of SM/C-2.6+ cells, we injected three kinds of cells, fresh isolated quiescent satellite cells, passaged satellite cells and primary myoblasts prepared by preplating technique from GFP-Tg mice into skeletal muscles of dystrophin-deficient mdx mice. Grafting of fresh isolated SM/C-2.6+ cells resulted in a large number of GFP-positive fibers at 4 weeks after the injection, whereas grafting of the same number of SM/C-2.6+ cells passaged in vitro and primary myoblasts gave rise to significantly fewer GFP-positive fibers. Surpr isingly, SM/C-2.6+ cells passaged once in culture after isolation showed almost the same regenerative capacity with those of fresh SM/C-2.6. Next, to test whether autologous, genetically corrected satellite cells may represent a possible tool for the therapy of DMD, we isolated SM/C-2.6+ cells from skeletal muscles of mdx mice and transduced them with a lentiviral vector expressing micro-dystrophin CS1 (Mol Ther 10: 821-8, 2004). We, then, transplanted them into mdx muscles and found that many fibers expressed micro-dystrophin on the sarcolemma four weeks after the injection. These data indicate that mdx SM/C-2.6+ cells transduced with a micro-dystrophin gene by a lentiviral vector represents a promising approach to cell therapy of DMD.


 
Abstract Title: Regulation of emerin interactions by tyrosine phosphorylation
Presenting/First Author: Tifft, Kathryn E (Grad)
Department, Institution: Cell Biology, Johns Hopkins School of Medicine
Address: 725 North Wolfe Street, WBSB G-11 Baltimore, MD 21205 United States
Phone/Fax: 410-614-2654 / 410-955-4129
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Tifft, Kathryn E (Johns Hopkins School of Medicine, Baltimore, MD)
Wilson, Katherine L (Johns Hopkins School of Medicine, Baltimore, MD)
 
Body of Abstract: Emery-Dreifuss muscular dystrophy (EDMD) is characterized by weakening of selected skeletal muscles, contractures of major tendons, and cardiac conduction defects. Loss of the inner nuclear membrane protein emerin causes X-linked EDMD. Emerin regulates gene expression during muscle development, potentially via direct binding to transcriptional regulators like BAF, GCL, Lmo7, Btf, and mRNA splicing factor YT521B. Emerin also binds structural proteins (lamin A, actin, and Nesprins) suggesting additional roles in nuclear structure. The binding sites in emerin for many partners overlap suggesting regulation of emerin binding partners and emerin functions. Five labs identified eleven tyrosine residues in emerin that are phosphorylated in vivo. We confirmed that emerin is tyrosine phosphorylated in HeLa cells and discovered that tyrosine phosphorylation decreases in the presence of Src inhibitors. We are testing the hypothesis that phosphorylation of specific tyrosine r esidues in emerin regulates specific partners using single missense mutations at known sites of tyrosine phosphorylation. Understanding how emerin is regulated by tyrosine phosphorylation may provide novel insight into emerin function and the mechanism of EDMD disease.


 
Abstract Title: Myotubularin knockdown results in down-regulation of genes that may be important for cell size control and regulation of oxidative state
Presenting/First Author: Tomczak, Kinga K (Postdoc - Degree Year: 2000)
Department, Institution: Genetics and Genomics, Children's Hospital Boston and Harvard Medical Shool
Address: 300 Longwood Avenue Boston, MA 02130 United States
Phone/Fax: 617-919-2155 / 617-730-0253
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Tomczak, Kinga K (Genomics Program and Division of Genetics, Children’s Hospital Boston and Harvard Medical School, Boston, USA)
Pierson, Christopher R (Genomics Program and Division of Genetics, Children’s Hospital Boston and Harvard Medical School, Boston, USA)
Zerra, Patricia E (Genomics Program and Division of Genetics, Children’s Hospital Boston and Harvard Medical School, Boston, USA)
Buj-Bello, Anna (Molecular Pathology, IGBMC, Illkirch, France)
Sanoudou, Despina (IIBEAA, Athens, Greece)
Laporte, Jocelyn (Molecular Pathology, IGBMC, Illkirch, France)
Mandel, Jean-Louis (Molecular Pathology, IGBMC, Illkirch, France)
Beggs, Alan H (Genomics Program and Division of Genetics, Children’s Hospital Boston and Harvard Medical School, Boston, USA)
Body of Abstract: MTM1 encodes myotubularin, a phosphoinositide phosphatase that is thought to play a role in vesicular trafficking and is mutated in X-linked myotubular myopathy (XLMTM). Mtm1 expression was knocked down (KD) in C2C12 cells using siRNA and expression profiling was performed at 3 time points of muscle differentiation: day -2 (myoblasts), day +2 (myogenic fusion) and day +6 (mature myotubes). The cells fused as expected on day 2 and showed well-differentiated myotubes on day 6 confirming no gross effects of KD. T-tests and geometric fold change analyses revealed 27 genes that were consistently up- or down-regulated in Mtm1 KD. The microtubule associated gene, doublecortin (Dcx) was up-regulated in Mtm1 KD cells and absent in controls at all time points. Among down-regulated transcripts, Car3 (carbonic anhydrase 3) and mitochondrial Txn2 (thioredoxin 2) are thought to be involved in protection of the cell from oxidative damage while two other genes Gdf5 (growth differ entiation factor 5) and Igfbp4 (insulin growth factor binding protein 4) are involved in regulation of growth. Decreased expression of Car3, Txn2, Igfbp4 and Gdf5 was confirmed by quantitative real-time PCR in KD cells and in Mtm1 knock-out mouse muscles. Alterations in these genes suggest a role of myotubularin in the control of cell or fiber size and regulation of the oxidative state. The pathogenic mechanism linking alterations in the expression of these genes with Mtm1 KD and XLMTM is on going.


 
Abstract Title: Systemic gene delivery of of micro-dystrophin prevents acute heart failure in mdx mice
Presenting/First Author: Townsend, DeWayne (Postdoc - Degree Year: 2003)
Department, Institution: Molecular and Integrative Physiology, University of Michigan
Address: 7712 Med Sci II, 1301 E Catherine St Ann Arbor, MI 48109 United States
Phone/Fax: 734-764-9920 / 734-647-6461
Email: [email protected]
Abstract Theme: Plasma Membrane Stability/Repair
Author(s): Townsend, DeWayne (Univ. of Michigan, Ann Arbor, MI 48109)
Blankinship, Mike J (Univ. of Washington, Seattle, WA, 98195)
Allen, James M (Univ. of Washington, Seattle, WA, 98195)
Chamberlain, Jeffery S (Univ. of Washington, Seattle, WA, 98195)
Metzger, Joseph M (Univ. of Michigan, Ann Arbor, MI 48109)
 
Body of Abstract: Duchenne muscular dystrophy (DMD) is a debilitating and fatal disease, that is characterized by both skeletal muscle pathology and cardiomyopathy, resulting from deletions of dystrophin (DYS). None of the truncated forms of dystrophin have been functionally assessed in cardiac tissue. A single intravenous injection of high titer (~10^12 vector genomes/mouse) adeno-associated virus-micro-DYS (3.7 kb) resulted in strong and uniform expression of micro-DYS at 10 weeks post injection. These mice show supra-normal levels of several dystrophin-associated proteins. The cardiac function of virus injected mice was assessed by pressure-volume catheterization at baseline and during a 30 minute dobutamine challenge. At baseline conditions the end-diastolic volume of the mdx mouse is significantly smaller compared to controls (20±2 ul (n=10) vs. 31±3 ul (n=8); P<0.01); this geometry is corrected by the expression of micro-DYS (31±3 ul (n=8); P>0.05 vs. Control). Furthermore, w ithout therapeutic intervention, 70% of mdx mice develop acute heart failure during a dobutamine challenge; this decompensation is alleviated by expression of micro-DYS (n=8). Thus micro-DYS is able to redress both acute and chronic aspects of dystrophic cardiomyopathy.


 
Abstract Title: FRG1 interacts with spliceosomal proteins
Presenting/First Author: Van der Maarel, Silvère M (Faculty)
Department, Institution: Human Genetics, Leiden University Medical Center
Address: Albinusdreef 2 Leiden, AZ 2333 ZA Netherlands
Phone/Fax: +31 71 526 9480 / +31 71 526 8285
Email: [email protected]
Abstract Theme:  
Author(s): van der Maarel, Silvère M (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
van Koningsbruggen, Silvana (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
Straasheijm, Kirsten R (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
Sterrenburg, Ellen (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
Klooster, Rinse (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
de Graaf, Natascha (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
Dauwerse, Hans G (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
Frants, Rune R (Dept. of Human Genetics, Leiden University Medical Center, Leiden, Netherlands)
Body of Abstract: Facioscapulohumeral muscular dystrophy (FSHD) is caused by an epigenetic mechanism involving contraction of the D4Z4 repeat and loss of transcriptional control over one or more genes. FRG1 is considered a candidate gene for FSHD and the FRG1 protein localizes to nucleoli, Cajal bodies and speckles. It has been suggested to be a component of the spliceosome but its exact function is unknown. Transgenic mice muscle-specifically overexpressing FRG1 have muscular dystrophy and upregulation of FRG1 correlates with mis-splicing of specific pre-mRNAs. Missplicing is also observed in our FRG1 stable cell lines, but not in skeletal muscle of FSHD patients.
By computational analysis, yeast-two-hybrid screens, and co-immunoprecipitation and co-localization studies, we have identified known and novel proteins that associate with FRG1. All of these have a direct or indirect role in pre-mRNA splicing. In addition, artificially induced nucleolar aggregates of VSV-FRG1 specifically sequester proteins involved in pre-mRNA splicing, but not in other processes of RNA biogenesis. Our data confirm the presence of FRG1 in the human spliceosome and strongly support a role for FRG1 in splicing.

Supported by NIAMS, MDA, FSHD Foundation and IOP.


 
Abstract Title: Which is the calcium channel partner of triadin TRISK 32 ?
Presenting/First Author: Vassilopoulos, Stéphane - (Undergrad)
Department, Institution: DRDC- CEA Grenoble, INSERM U607 - CCFP
Address: 17 rue des Martyrs Grenoble, 38054 France
Phone/Fax: 33 4 38 78 58 39 / 33 4 38 78 50 41
Email: [email protected]
Abstract Theme: Sarcolemmal Excitability
Author(s): Vassilopoulos, Stéphane - (Inserm U607 - CCFP- CEA Grenoble, 38054 Grenoble, France)
Oddoux, Sarah - (Inserm U607 - CCFP- CEA Grenoble, 38054 Grenoble, France)
Brocard, Julie - (Inserm U607 - CCFP- CEA Grenoble, 38054 Grenoble, France)
Fauré, Julien - (Inserm U607 - CCFP- CEA Grenoble, 38054 Grenoble, France)
Lunardi, Joël - (Inserm U607 - CCFP- CEA Grenoble, 38054 Grenoble, France)
Marty, Isabelle - (Inserm U607 - CCFP- CEA Grenoble, 38054 Grenoble, France)
 
Body of Abstract: Internal calcium release is performed via two major calcium release channels: Ryanodine receptor (RyR) or IP3-receptor (IP3R). In skeletal muscle, release of calcium leading to contraction is performed by the calcium release complex, which involves RyR1 as the intracellular calcium channel. Other proteins are associated to RyR1 in this complex, among which triadin. We have cloned four triadin isoforms: Trisk 95, Trisk 51, Trisk 49 and Trisk 32. Trisk 32 is not only expressed in skeletal muscle, it is also the major cardiac triadin isoform. We have shown that in skeletal muscle, Trisk 32 is only partially associated with RyR1, but could be associated with IP3R. In cardiac muscle on the contrary, Trisk 32 has been shown to be associated with RyR2. In the present work, we studied the respective localization of RyR1, Trisk 32 and IP3R using immunofluorescent labeling, and their association by immunoprecipitation in rat skeletal muscle. We performed the same study on rat cardiac muscle, to analyze the respective localization of RyR2, Trisk 32 and IP3R and their possible association. We wish to identify through this study the calcium release channel partner of Trisk 32, both in heart and in skeletal muscle.


 
Abstract Title: Proteomic analysis of CAPN3 deficient mice implicates CAPN3 in myofibrillar protein turnover
Presenting/First Author: Venkatraman, Gayathri (Postdoc - Degree Year: 2004)
Department, Institution: Neurology, University of California at Los Angeles
Address: NRB 1, Room 404, 635 Charles Young Drive South Los Angeles, CA 90095 United States
Phone/Fax: 310-267-4582 / 310-206-1998
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Venkatraman, Gayathri (Department of Neurology, Univ. of California, Los Angeles)
Gomes, Aldrin V (Department of Physiolgy and Medicine, University of California, Los Angeles)
Spencer, Melissa J (Department of Neurology, Univ. of California, Los Angeles)
 
Body of Abstract: Calpain 3 (CAPN3) is a calcium-dependent, cysteine protease predominantly found in skeletal muscle. Mutations in CAPN3 cause Limb Girdle muscular dystrophy type 2A. Previously, we showed that CAPN3 knockout (KO) mice have reduced accumulation of ubiquitinated proteins during muscle remodeling, and that this reduction is not due to enhanced proteasome activity. These studies suggest that CAPN3 acts upstream of the ubiquitin-proteasome pathway during muscle protein turnover. To further investigate proteins targeted for ubiquitination by CAPN3, we compared ubiquitinated proteins from wildtype (WT) and CAPN3 deficient mice. Ubiquitinated proteins were isolated by affinity purification, and analyzed by LC-MS/MS. Over 200 proteins were identified in the ubiquitinated extracts from WT and KO muscles. Immunoblotting confirmed the MS data which suggested that myosin heavy chain, myomesin and actin are all significantly reduced in the ubiquitinated fraction from CAPN3 KO m ice. The down regulation of the major muscle sarcomeric proteins in the ubiquitinated fraction of the CAPN3 KO muscle suggests a role for CAPN3 during myofibrillar protein turnover. Supported by NIH AR48177 to MJS.


 
Abstract Title: Prevention of oculopharyngeal muscular dystrophy-associated aggregation of nuclear poly(A)-binding protein with an intracellularly expressed camelid-derived antibody domain
Presenting/First Author: Verheesen, Peter (Faculty)
Department, Institution: Molecular Biology, Ablynx nv
Address: Technologiepark 4 Ghent, 9000 Belgium
Phone/Fax: +3292610622 / +3292610628
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Verheesen, Peter (Univ. of Utrecht, Utrecht, The Netherlands)
de Kluijver, Anna (Univ. of Utrecht, Utrecht, The Netherlands)
van Koningsbruggen, Silvana (Leiden University Medical Center, Leiden, The Netherlands)
de Haard, Hans (Ablynx nv, Ghent, Belgium)
van Ommen, Gert-Jan (Leiden University Medical Center, Leiden, The Netherlands)
Verrips, Theo (Univ. of Utrecht, Utrecht, The Netherlands)
van der Maarel, Silvère (Leiden University Medical Center, Leiden, The Netherlands)
 
Body of Abstract: Oculopharyngeal muscular dystrophy (OPMD) is regarded a paradigm for protein aggregation disorders. It is caused by extensions of the N-terminal polyalanine stretch of the nuclear polyA-binding protein 1 (PABPN1) causing the presence of intranuclear aggregates in skeletal muscle. Intranuclear aggregation of mutant PABPN1 is also observed in transgenic mouse and cell models for OPMD supporting a direct role for protein aggregation in pathogenesis.
We have isolated camelid-derived antibody reagents (VHH) against different epitopes in PABPN1, which specifically detect and label endogenous PABPN1 in cell lysates, cultured cells and muscle sections. When expressed intracellularly as intrabodies in a model for OPMD, aggregation was prevented in a dose-dependent manner. These intrabodies also have curative properties as they could reduce already existing aggregates. Given the domain specificity of VHH-mediated aggregation interference, this approach facilitates definition of the nucleation kernel in aggregation-prone proteins, thus providing etiological insight into this and other protein aggregation disorders. It may also provide useful therapeutic agents.
Supported by IOP Genomics/Senter Novem, the NIH, and E.C.


 
Abstract Title: Sarcoglycan expression is restored in delta-sarcoglycan null mice by muscle-derived stem cell transplantation
Presenting/First Author: Wallace, Gregory Q (Postdoc - Degree Year: 2005)
Department, Institution: Medicine, University Of Chicago
Address: 5841 S Maryland Chicago, IL 60637 United States
Phone/Fax: 7737022864 / 7737022681
Email: [email protected]
Abstract Theme:  
Author(s): Wallace, Gregory Q (Department of Medicine, University of Chicago, Chicago, Illinois, USA)
Lapidos-Cielo, Karen A (Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois, USA)
Chen, Yiyin E (Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois, USA)
McNally, Elizabeth M (Department of Medicine, University of Chicago, Chicago, Illinois, USA)
 
Body of Abstract: Muscular dystrophies are characterized by progressive muscle degeneration that overcomes muscle’s ability to regenerate. Stem cell transplantation is a promising therapeutic strategy for functional correction of dystrophic muscle. Use of stem cells has the dual advantage of delivering normal copies of the disease-causing gene while adding cells to the regenerative cell pool. Ongoing challenges in optimizing a stem cell-based therapy approach include identifying cells with potent myogenic activity, maintaining long-term survival of transplanted cells, and avoiding immune responses. We have isolated mononuclear cells from muscle of adult, male wildtype mice and injected them directly into muscles of delta-sarcoglycan null female mice. Donor cells were fractionated by Hoechst dye staining, centrifugation through a Ficoll gradient, or by cell size immediately prior to injection. Four weeks or six months after transplantation, mice were sacrificed and donor-derived mus cle fibers were scored. We have identified a subset of adult, muscle-derived stem cells that can yield up to 13% donor-derived muscle fibers and survive at least six months in vivo.
Research supported by Muscular Dystrophy Association and National Institutes of Health.


 
Abstract Title: Local calcium release events in wildtype and dystrophic adult myofibers
Presenting/First Author: Ward, Christopher W (Faculty)
Department, Institution: OSAHN, Univ. Maryland
Address: 755 W Lombard St Baltimore, MD 21201 United States
Phone/Fax: 410-706-3618 / 410-706-2222
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Ward, Christopher W (University of Maryland Baltimore)
Voelker, Kevin (Virginia Tech, Blacksburg, VA)
Grange, Robert W (Virginia Tech, Blacksburg, VA)
Shtifman, Alexander (St Elizabeth's Hospital, Tufts Univ., MA)
 
Body of Abstract: Local calcium (Ca2+) release events (LCRE) are spatially restricted elevations in Ca2+ arising from a small cluster of ryanodine receptor(RyR) Ca2+ channels in the sarcoplasmic reticulum (SR). In intact adult mammalian myofibers solely expressing RyR1, spontaneous LCRE are suppressed and rarely visualized. Recently, spontaneous LCRE in intact FDB myofibers was revealed secondary to osmotic stress (Wang et al., Nat. Cell. Bio, 2005) with an enhanced occurrence in MDX myofibers; a disease in which an alteration in calcium homeostasis is a hallmark of the pathogenesis. Having verified these results qualitatively, we determined that external [Ca2+] modulates the occurrence of LCRE as well as global SR Ca2+ release during osmotic and ionic stress in WT and MDX myofibers. In addition we have verified that in MDX myofibers, sarcolemma permeability to Ca2+ is ~ 2 fold higher than WT controls. We hypothesize that under stress, MDX myofibers have an increased Ca2+ influx wh ich contributes to the occurrence of LCRE. Independent of the sarcolemma pathway, permeablized MDX myofibers demonstrate an increase in LCRE frequency and altered properties which may suggest an alteration in RyR dependent Ca2+ release as a contributing factor.


 
Abstract Title: Ribonuclear inclusions in junctional nuclei and anterior horn cells in myotonic dystrophy type I
Presenting/First Author: Wheeler, Thurman M (Postdoc - Degree Year: )
Department, Institution: Neurology, University of Rochester
Address: 601 Elmwood Ave., Box 673 Rochester, NY 14642 United States
Phone/Fax: 5852731456 / 5852731255
Email: [email protected]
Abstract Theme: Molecular Basis of Muscle Dysfunction
Author(s): Wheeler, Thurman M (University of Rochester, Rochester, NY 14642)
Krym, Matt C (University of Rochester, Rochester, NY 14642)
Thornton, Charles A (University of Rochester, Rochester, NY 14642)
 
Body of Abstract: A characteristic feature of muscle pathology in myotonic dystrophy (DM) is the finding of severely atrophic fibers that are nearly devoid of cytoplasm, similar to those seen in denervated muscle. This observation has prompted speculation that DM1 may lead to instability of the NMJ. However, whether DMPK is expressed at the motor endplate is controversial. To address this question, we used fluorescent in situ hybridization (FISH) to examine the expression pattern of expanded repeat RNA. First, in HSALR transgenic mice that express skeletal actin mRNA with an expanded CUG in the 3’ UTR, nuclear RNA foci are abundant in extrajunctional myonuclei but minimal to absent in junctional nuclei. Of note, HSALR mice display some histologic features similar to DM but they do not develop nuclear clumps. Next, FISH in human DM1 muscle clearly demonstrates nuclear foci in junctional nuclei and motor neurons, indicating that DMPK RNA is expressed on both sides of the endpla te. These results indicate an important difference between the HSALR transgenic model and human DM1, and suggest that specialization of junctional nuclei involves downregulation of genes encoding myofibrillar proteins. Analysis of muscle sections confirms that DMPK protein accumulates at the neuromuscular junction. Finally, muscleblind-1 protein forms foci that colocalize with expanded repeat RNA in junctional nuclei and motor neurons, suggesting susceptibility to RNA-mediated toxicity. These observations raise the possibility that expression of CUG expansion RNA may compromise the stability or function of the NMJ in DM1.


 
Abstract Title: Prevention of Lethal Muscular Dystrophy in a Severe DMD Mouse Model via Human Retinal Dystrophin Transgene: Implications for DMD Gene Therapy
Presenting/First Author: White, Robert A (Faculty)
Department, Institution: Medical Research, Children's Mercy Hospital, Pediatric Research Center, 4th Fl
Address: 2401 Gillham Kansas City, MO 64108 United States
Phone/Fax: 816-983-6502 / 816-983-6501
Email: [email protected]
Abstract Theme: Therapies
Author(s): White, Robert A (Children's Mercy Hospitals & Clinics, Kansas City, MO)
McNulty, Steven G (Children's Mercy Hospitals & Clinics, Kansas City, MO)
Modrcin, Ann C (Children's Mercy Hospitals & Clinics, Kansas City, MO)
Rinaldi, Robert J (Children's Mercy Hospitals & Clinics, Kansas City, MO)
Gaedigk, Roger (Children's Mercy Hospitals & Clinics, Kansas City, MO)
Law, Douglas J (University of Missouri-Kansas City)
Fitzgerald-Gustafson, Kathleen M (University of Kansas Medical Center, Kansas City, KS)
Hauschka, Stephen D (University of Washington, Seattle, WA)
Body of Abstract: Duchenne muscular dystrophy (DMD) is a progressive muscle disease characterized by lack of dystrophin expression, progressive muscle weakness, and death by the third decade. The most commonly used mouse model of DMD is the muscular dystrophy mdx mouse. This mutant has underlying muscle disease but appears physically normal with an ordinary lifespan possibly due to compensatory expression of utrophin in these mice. In contrast to mdx mice, double mutant mice (DM), deficient for both dystrophin and utrophin (mdx/Y, utrn-/-), present a model that most resembles DMD. These mice die prematurely at 3 months of age and suffer from severe muscle weakness, pronounced growth retardation, and severe spinal curvature. The capacity of human retinal dystrophin (Dp260) to compensate for the absence of muscle dystrophin was tested in DM mice. Functional outcomes were assessed by muscle histology, EMG, MRI, mobility studies and measurement of weight and longevity. MCK promoter-dri ven transgenic expression of Dp260 in DM mice converts their disease course from a severe, lethal muscular dystrophy to a viable, mild myopathic phenotype. These studies have important clinical implications for the possible treatment of DMD, via gene therapy approaches.


 
Abstract Title: Nanopolymer-oligonucleotides for the induction of dystrophin expression in mdx mice
Presenting/First Author: Williams, Jason H (Grad)
Department, Institution: Pharmacology & Physiology, Drexel University College of Medicine
Address: 245 N 15th St Philadelphia, PA 19118 United States
Phone/Fax: 215-762-4979 / 215-762-2299
Email: [email protected]
Abstract Theme: Therapies
Author(s): Williams, Jason H (Drexel University College of Medicine, Philadelphia, PA 19102)
Sirsi, Shashank R (Drexel University College of Medicine, Philadelphia, PA 19102)
Nicolai, Jackie (Drexel University College of Medicine, Philadelphia, PA 19102)
Latta, Daniel R (Drexel University College of Medicine, Philadelphia, PA 19102)
Lutz, Gordon J (Drexel University College of Medicine, Philadelphia, PA 19102)
 
Body of Abstract: Antisense oligonucleotides (AOs) have previously been shown to modulate dystrophin pre-mRNA splicing, causing “skipping” of mutated dystrophin exons, and production of partially to fully functional protein. Thus, AOs are hopeful drug candidates for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. In our recent report we showed that a PEG-PEI copolymer comprised of low Mw PEI2K functioned as an effective AO carrier, producing wide-spread appearance of dystrophin-positive fibers after intramuscular injection into mdx mice, with no apparent cytotoxicity. We now show evidence of dystrophin expression at extended time points. These data suggest that PEG-PEI copolymers are able to sequester and protect AOs from degradation, and may facilitate sustained delivery of AOs to myonuclei over long durations. Overall, PEG-PEI copolymers represent a flexible nucleotide delivery system with controllable size and adjustable unpackaging properties. Additional optimization of nanopolymer-oligonucleotide properties will likely improve transfection efficiency and promote wider usage of this beneficial carrier compound.


 
Abstract Title: Passive mechanical properties of maturing EDL are not affected by lack of dystrophin
Presenting/First Author: Wolff, Andrew V (Grad)
Department, Institution: Mechanical Engineering, Virginia Tech
Address: 338 Wallace Hall Blacksburg, VA 24061 United States
Phone/Fax: 540-525-0178 / 540-231-3916
Email: [email protected]
Abstract Theme: Congenital Muscular Dystrophy and Nuclear Membrane Diseases
Author(s): Wolff, Andrew V (Department of Mechanical Engineering, Virginia Tech, Blacksburg, VA)
Niday, Ashley K (Department of Engineering Science and Mechanics, Virginia Tech, Blacksburg, VA)
Voelker, Kevin A (Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA)
Call, Jarrod A (Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA)
Evans, Nick P (Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA)
Granata, Kevin P (Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA)
Grange, Robert W (Department of Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA)
 
Body of Abstract: To better understand the role that mechanical weakness might play in the onset of Duchenne muscular dystrophy, the mechanical properties of maturing dystrophic and control skeletal muscles should be systematically assessed. The purpose of this study was to determine (1) if the passive mechanical properties of maturing dystrophic muscles were different from control; and, (2) if different, when during maturation did these properties change? At ages prior to and following the overt onset of the dystrophic process (14-35 days), control and dystrophic extensor digitorum longus (EDL) muscles were subjected to two passive stretch protocols at 5% strain with two strain rates in vitro. Force profiles at the instantaneous strain rate were fit to a three parameter viscoelastic muscle model to determine two parameters of stiffness and a damping parameter. The dystrophic and control EDL muscles exhibited similar passive mechanical properties at each age. These results sug gest a functional threshold for dystrophic muscle below which damage may be minimized. Determining this threshold could have important clinical implications for treatments of muscular dystrophy involving physical activity. Research supported by NIH grant AR049881.


 
Abstract Title: A C-terminal skeletal muscle sodium channel mutation associated with myotonia disrupts fast inactivation
Presenting/First Author: Wu, Fen-Fen (Postdoc - Degree Year: 2001)
Department, Institution: Neurology, UT Southwestern Medical Center
Address: 6000 Harry Hines Blvd Dallas, TX 75390 United States
Phone/Fax: 214-648-7450 / 214-648-7451
Email: [email protected]
Abstract Theme: Sarcolemmal Excitability
Author(s): Wu, Fen-Fen (Univ of Texas Southwestern Medical Center, Dallas, TX 75390)
Gordon, Erynn (Children’s National Medical Center, Washington, DC 20010)
Hoffman, Eric P (Children’s National Medical Center, Washington, DC 20010)
Cannon, Stephen C (Univ of Texas Southwestern Medical Center, Dallas, TX 75390)
 
Body of Abstract: Missense mutations in the skeletal muscle sodium channel alpha-subunit gene (SCN4A) are associated with clinically overlapping diseases caused by alterations in sarcolemmal excitability, which may result in myotonia or periodic paralysis. A cold-aggravated myotonia patient was screened 24 exons of SCN4A by DHPLC and sequencing. Two novel changes were found: T323M in the DIS5-S6 loop and F1705I in the C-terminus. The functional impact was assessed by recording whole-cell Na+ currents from HEK cells. T323M currents were indistinguishable from WT. Fast inactivation (FI) was impaired for F1705I by an 8.6 mV rightwards shift in voltage dependence and a 2-fold slowing in the inactivation rate. Recovery from FI was not altered, nor was an increase in the persistent current. Activation and slow inactivation were not affected. These data suggest that T323M is a benign polymorphism, whereas F1705I results in FI defects, which are often observed for myotonia. This is the first example of a SCN4A C-terminal mutation associated with human disease. Like the cardiac disorders (LQT3 or Brugada syndrome) and epilepsy (GEFS+) associated with C-terminal mutations in other NaV channels, the defect of F1705I was a partial disruption of FI.