Development of a High-Throughput Screen to Identify Small Molecule Enhancers of Sarcospan for the Treatment of Duchenne Muscular Dystrophy
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<Alpha>11<Beta>1 Integrin Is a Receptor for Interstitial Collagens
Developmental Biology 237, 116–129 (2001) doi:10.1006/dbio.2001.0363, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE ␣111 Integrin Is a Receptor for Interstitial provided by Elsevier - Publisher Connector Collagens Involved in Cell Migration and Collagen Reorganization on Mesenchymal Nonmuscle Cells Carl-Fredrik Tiger,* Francoise Fougerousse,† Gunilla Grundstro¨m,‡ Teet Velling,‡ and Donald Gullberg‡,1 *Department of Cell and Molecular Biology, Biomedical Center, Box 596, Uppsala University, S-75124 Uppsala, Sweden; †Laboratoire de Histoembryologie et de Cytoge´ne´tique, Faculte´ Cochin Port Royal, Paris 75014, France; and ‡Department of Medical Biochemistry and Microbiology, Biomedical Center, Box 582, Uppsala University, S-75123 Uppsala, Sweden ␣111 integrin constitutes a recent addition to the integrin family. Here, we present the first in vivo analysis of ␣11 protein and mRNA distribution during human embryonic development. ␣11 protein and mRNA were present in various mesenchymal cells around the cartilage anlage in the developing skeleton in a pattern similar to that described for the transcription factor scleraxis. ␣11 was also expressed by mesenchymal cells in intervertebral discs and in keratocytes in cornea, two sites with highly organized collagen networks. Neither ␣11 mRNA nor ␣11 protein could be detected in myogenic cells in human embryos. The described expression pattern is compatible with ␣111 functioning as a receptor for interstitial collagens in vivo. To test this hypothesis in vitro, full-length human ␣11 cDNA was stably transfected into the mouse satellite cell line C2C12, lacking endogenous collagen receptors. -
The Role of the Actin Cytoskeleton During Muscle Development In
THE ROLE OF THE ACTIN CYTOSKELETON DURING MUSCLE DEVELOPMENT IN DROSOPHILA AND MOUSE by Shannon Faye Yu A Dissertation Presented to the Faculty of the Louis V. Gerstner, Jr. Graduate School of the Biomedical Sciences in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy New York, NY Oct, 2013 Mary K. Baylies, PhD! Date Dissertation Mentor Copyright by Shannon F. Yu 2013 ABSTRACT The actin cytoskeleton is essential for many processes within a developing organism. Unsurprisingly, actin and its regulators underpin many of the critical steps in the formation and function of muscle tissue. These include cell division during the specification of muscle progenitors, myoblast fusion, muscle elongation and attachment, and muscle maturation, including sarcomere assembly. Analysis in Drosophila has focused on regulators of actin polymerization particularly during myoblast fusion, and the conservation of many of the actin regulators required for muscle development has not yet been tested. In addition, dynamic actin processes also require the depolymerization of existing actin fibers to replenish the pool of actin monomers available for polymerization. Despite this, the role of actin depolymerization has not been described in depth in Drosophila or mammalian muscle development. ! Here, we first examine the role of the actin depolymerization factor Twinstar (Tsr) in muscle development in Drosophila. We show that Twinstar, the sole Drosophila member of the ADF/cofilin family of actin depolymerization proteins, is expressed in muscle where it is essential for development. tsr mutant embryos displayed a number of muscle defects, including muscle loss and muscle misattachment. Further, regulators of Tsr, including a Tsr-inactivating kinase, Center divider, a Tsr-activating phosphatase, Slingshot and a synergistic partner in depolymerization, Flare, are also required for embryonic muscle development. -
Dystrophin Complex Functions As a Scaffold for Signalling Proteins☆
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1838 (2014) 635–642 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Review Dystrophin complex functions as a scaffold for signalling proteins☆ Bruno Constantin IPBC, CNRS/Université de Poitiers, FRE 3511, 1 rue Georges Bonnet, PBS, 86022 Poitiers, France article info abstract Article history: Dystrophin is a 427 kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and Received 27 May 2013 incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex Received in revised form 22 August 2013 (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins Accepted 28 August 2013 and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force trans- Available online 7 September 2013 duction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaf- fold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling Keywords: Dystrophin-associated protein complex (DAPC) processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. syntrophin The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle Sodium channel cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More TRPC channel generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. -
Dystrobrevin Alpha Gene Is a Direct Target of the Vitamin D Receptor in Muscle
64 3 Journal of Molecular M K Tsoumpra et al. Upregulation of dystrobrevin by 64:3 195–208 Endocrinology calcitriol RESEARCH Dystrobrevin alpha gene is a direct target of the vitamin D receptor in muscle Maria K Tsoumpra1, Shun Sawatsubashi2, Michihiro Imamura1, Seiji Fukumoto2, Shin’ichi Takeda1, Toshio Matsumoto2 and Yoshitsugu Aoki1 1Department of Molecular Therapy, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, Tokyo, Japan 2Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan Correspondence should be addressed to S Fukumoto: [email protected] Abstract The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its Key Words tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which f vitamin D functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response f muscle elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle f gene regulation cells occurs concomitantly with transcriptional regulation of key myogenic factors upon f receptor binding VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1–50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. -
Reviewreview Duchenne Muscular Dystrophy and Dystrophin: Pathogenesis and Opportunities for Treatment Third in Molecular Medicine Review Series Kristen J
reviewreview Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment Third in Molecular Medicine Review Series Kristen J. Nowak† & Kay E. Davies+ MRC Functional Genetics Unit, University of Oxford, UK Duchenne muscular dystrophy (DMD) is caused by mutations in protein. Becker muscular dystrophy (BMD; OMIM 300376)—a the gene that encodes the 427-kDa cytoskeletal protein dys- much milder form of the disease—is caused by a reduction in the trophin. Increased knowledge of the function of dystrophin and amount, or alteration in the size, of the dystrophin protein. The its role in muscle has led to a greater understanding of the high incidence of sporadic cases of DMD (1 in 10,000 sperm or pathogenesis of DMD. This, together with advances in the eggs) means that genetic screening will never eliminate this dis- genetic toolkit of the molecular biologist, are leading to many ease, so an effective therapy is highly desirable. This review sum- different approaches to treatment. Gene therapy can be marizes our understanding of the disease and the strategies that are achieved using plasmids or viruses, mutations can be corrected being developed for an effective treatment (Fig 1). using chimaeraplasts and short DNA fragments, exon skipping of mutations can be induced using oligonucleotides and Pathogenesis readthrough of nonsense mutations can be achieved using Dystrophin has a major structural role in muscle as it links the aminoglycoside antibiotics. Blocking the proteasome degrada- internal cytoskeleton to the extracellular matrix. The amino-terminus tion pathway can stabilize any truncated dystrophin protein, of dystrophin binds to F-actin and the carboxyl terminus to the and upregulation of other proteins can also prevent the dys- dystrophin-associated protein complex (DAPC) at the sarcolemma trophic process. -
Governs the Making of Photocopies Or Other Reproductions of Copyrighted Materials
Warning Concerning Copyright Restrictions The Copyright Law of the United States (Title 17, United States Code) governs the making of photocopies or other reproductions of copyrighted materials. Under certain conditions specified in the law, libraries and archives are authorized to furnish a photocopy or other reproduction. One of these specified conditions is that the photocopy or reproduction is not to be used for any purpose other than private study, scholarship, or research. If electronic transmission of reserve material is used for purposes in excess of what constitutes "fair use," that user may be liable for copyright infringement. University of Nevada, Reno The Role of Utrophin, Sarcospan, and Glycosyltransferase Activity in the Pathogenesis of Duchenne Muscular Dystrophy and a Representative Case Study A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Science in Biochemistry & Molecular Biology by Susan T. Alaei Josh Baker, Ph.D., Thesis Advisor May, 2013 UNIVERSITY OF NEVADA THE HONORS PROGRAM RENO We recommend that the thesis prepared under our supervision by Susan T. Alaei entitled The Role of Utrophin, Sarcospan, and Glycosyltransferase Activity in the Pathogenesis of Duchenne Muscular Dystrophy and a Representative Case Study be accepted in partial fulfillment of the requirements for the degree of Bachelor of Science in Biochemistry & Molecular Biology ______________________________________________ Josh Baker, Ph.D., Thesis Advisor ______________________________________________ Tamara Valentine, Ph.D., Director, Honors Program May 2013 i Abstract Duchenne Muscular Dystrophy is a degenerative muscle disease that is characterized by the breakdown of skeletal muscle as a result of membrane instability. A mutation in the dystrophin gene, one of the largest gene in the human genome, results in a complete lack of dystrophin in the membrane of skeletal muscle cells. -
Differentiation and Long-Term Survival of C2C12 Myoblast Grafts in Heart
Differentiation and long-term survival of C2C12 myoblast grafts in heart. G Y Koh, … , M H Soonpaa, L J Field J Clin Invest. 1993;92(3):1548-1554. https://doi.org/10.1172/JCI116734. Research Article Find the latest version: https://jci.me/116734/pdf Rapid Publication Differentiation and Long-Term Survival of C2C12 Myoblast Grafts in Heart Gou Young Koh, Michael G. Klug, Mark H. Soonpaa, and Loren J. Field Krannert Institute ofCardiology, Indiana University School ofMedicine, Indianapolis, Indiana 46202-4800 Abstract formation of chimeric myotubes expressing wild-type dystro- phin. We have assessed the ability of skeletal myoblasts to form In contrast to skeletal muscle, the myocardium lacks a re- long-term, differentiated grafts in ventricular myocardium. generative stem cell system. Furthermore, ventricular cardio- C2C12 myoblasts were grafted directly into the heart of synge- myocytes in the adult mammalian heart are permanently with- neic mice. Viable grafts were observed as long as 3 mo after drawn from the cell cycle (6, 7). Myofiber loss due to trauma implantation. Immunohistological analyses revealed the pres- or disease is consequently irreversible. These observations ence of differentiated myotubes that stably expressed the skele- prompted several groups to determine if targeted oncogene ex- tal myosin heavy chain isoform. Thymidine uptake studies indi- pression could induce cardiomyocyte proliferation. Expression cated that virtually all of the grafted skeletal myocytes were of the SV40 large T antigen oncogene in the hearts of trans- withdrawn from the cell cycle by 14 d after grafting. Graft genic mice (8-10) or in virally transfected rat cardiomyocytes myocytes exhibited ultrastructural characteristics typical of (11) resulted in a sustained proliferative response. -
Sarcospan-Dependent Akt Activation Is Required for Utrophin Expression and Muscle Regeneration
JCB: Article Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration Jamie L. Marshall,1 Johan Holmberg,1 Eric Chou,1 Amber C. Ocampo,1 Jennifer Oh,1 Joy Lee,1 Angela K. Peter,1 Paul T. Martin,3,4 and Rachelle H. Crosbie-Watson1,2 1Department of Integrative Biology and Physiology and 2Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095 3Center for Gene Therapy, The Research Institute, Nationwide Children’s Hospital, and 4Department of Pediatrics, Ohio State University College of Medicine and College of Public Health, Columbus, OH 43205 trophin is normally confined to the neuromus- sarcolemma. SSPN-null mice displayed delayed differen- cular junction (NMJ) in adult muscle and par- tiation after CTX injury caused by loss of utrophin and Akt U tially compensates for the loss of dystrophin in signaling. Treatment of SSPN-null mice with viral Akt mdx mice. We show that Akt signaling and utrophin increased utrophin and restored muscle repair after in- levels were diminished in sarcospan (SSPN)-deficient jury, revealing an important role for the SSPN-Akt-utro- muscle. By creating several transgenic and knockout phin signaling axis in regeneration. SSPN improved cell mice, we demonstrate that SSPN regulates Akt signal- surface expression of utrophin by increasing transportation ing to control utrophin expression. SSPN determined of utrophin and DG from endoplasmic reticulum/Golgi -dystroglycan (-DG) glycosylation by affecting levels membranes. Our experiments reveal functions of utro- of the NMJ-specific glycosyltransferase Galgt2. After phin in regeneration and new pathways that regulate cardiotoxin (CTX) injury, regenerating myofibers ex- utrophin expression at the cell surface. -
Large-Scale Spontaneous Self-Organization and Maturation Of
www.nature.com/scientificreports OPEN Large‑scale spontaneous self‑organization and maturation of skeletal muscle tissues on ultra‑compliant gelatin hydrogel substrates Joen H. Jensen1, Selgin D. Cakal1, Jingwen Li2, Christian J. Pless1, Carmen Radeke1, Morten Leth Jepsen1,3, Thomas E. Jensen2, Martin Dufva1,3* & Johan U. Lind1* Cellular self‑organization is the fundamental driving force behind the complex architectures of native tissue. Yet, attempts at replicating native tissue architectures in vitro often involve complex micro‑ fabrication methods and materials. While impressive progress has been made within engineered models of striated muscle, the wide adaptation of these models is held back by the need for specifc tools and knowhow. In this report, we show that C2C12 myoblasts spontaneously organize into highly aligned myotube tissues on the mm to cm scale, when cultured on sufciently soft yet fully isotropic gelatin hydrogel substrates. Interestingly, we only observed this phenomenon for hydrogels with Young’s modulus of 6 kPa and below. For slightly more rigid compositions, only local micrometer- scale myotube organization was observed, similar to that seen in conventional polystyrene dishes. The hydrogel‑supported myotubes could be cultured for multiple weeks and matured into highly contractile phenotypes with notable upregulation of myosin heavy chain, as compared to myotubes developed in conventional petri dishes. The procedure for casting the ultra‑soft gelatin hydrogels is straight forward and compatible with standardized laboratory tools. It may thus serve as a simple, yet versatile, approach to generating skeletal muscle tissue of improved physiological relevance for applied and basic research. Skeletal muscle comprises the largest tissue in the human body, and plays an essential role in locomotion as well as in metabolism and homeostasis1,2. -
Titin N2A Domain and Its Interactions at the Sarcomere
International Journal of Molecular Sciences Review Titin N2A Domain and Its Interactions at the Sarcomere Adeleye O. Adewale and Young-Hoon Ahn * Department of Chemistry, Wayne State University, Detroit, MI 48202, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-(313)-577-1384 Abstract: Titin is a giant protein in the sarcomere that plays an essential role in muscle contraction with actin and myosin filaments. However, its utility goes beyond mechanical functions, extending to versatile and complex roles in sarcomere organization and maintenance, passive force, mechanosens- ing, and signaling. Titin’s multiple functions are in part attributed to its large size and modular structures that interact with a myriad of protein partners. Among titin’s domains, the N2A element is one of titin’s unique segments that contributes to titin’s functions in compliance, contraction, structural stability, and signaling via protein–protein interactions with actin filament, chaperones, stress-sensing proteins, and proteases. Considering the significance of N2A, this review highlights structural conformations of N2A, its predisposition for protein–protein interactions, and its multiple interacting protein partners that allow the modulation of titin’s biological effects. Lastly, the nature of N2A for interactions with chaperones and proteases is included, presenting it as an important node that impacts titin’s structural and functional integrity. Keywords: titin; N2A domain; protein–protein interaction 1. Introduction Citation: Adewale, A.O.; Ahn, Y.-H. The complexity of striated muscle is defined by the intricate organization of its com- Titin N2A Domain and Its ponents [1]. The involuntary cardiac and voluntary skeletal muscles are the primary types Interactions at the Sarcomere. -
Biomechanical Forces Upregulate Myogenic Gene Induction in the Presence Or Absence of Inflammation - a Possible Role of Igfr1-Pi3k-Akt Pathway
BIOMECHANICAL FORCES UPREGULATE MYOGENIC GENE INDUCTION IN THE PRESENCE OR ABSENCE OF INFLAMMATION - A POSSIBLE ROLE OF IGFR1-PI3K-AKT PATHWAY DISSERTATION Presented in Partial Fulfillment for the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University by Ravi Chandran D.D.S., M.D.S. * * * * * The Ohio State University 2007 Approved by Dissertation Committee: Dr. Sudha Agarwal, Advisor _____________________________ Dr. Ning Quan Dr. Peter Reiser Advisor Oral Biology Graduate Dr. Denis Guttridge Program ABSTRACT C2C12 myoblasts proliferate in response to mitogens and, upon mitogen withdrawal, differentiate into multinucleated myotubes. Over the past decade, several studies have unraveled important mechanisms by which the four myogenic regulatory factors, MRF’s (Myod1, Myf5, myogenin, and MRF4/Myf6) control the specification and the differentiation of the muscle lineage. The members of bHLH transcription factors act synergistically with the myocyte enhancer binding factor-2 (MEF2) family of co-factors to induce synthesis of muscle restricted target genes. It is well established that Myod1 and Myf5 are required for commitment to the myogenic lineage, whereas myogenin plays a critical role in the expression of the terminal muscle phenotype previously established by Myod1 and Myf5, and MRF4 partly subserves both roles. In this study, we first induced C2C12 myoblasts to differentiate by transferring from growth medium (GM) to differentiation medium (DM) and characterizing the phenotype of C2C12 cells and examining the expression patterns of crucial myogenic factors and its targets genes. Later, by means of an in vitro model system, cyclic equibiaxial stretching and treatment with a proinflammatory cytokine, TNF-α, we have examined the effects of tensile forces imposed on C2C12 myoblasts. -
Pyropia Yezoensis Protein Protects Against TNF‑Α‑Induced Myotube Atrophy in C2C12 Myotubes Via the NF‑Κb Signaling Pathway
MOLECULAR MEDICINE REPORTS 24: 486, 2021 Pyropia yezoensis protein protects against TNF‑α‑induced myotube atrophy in C2C12 myotubes via the NF‑κB signaling pathway MIN‑KYEONG LEE1, YOUN HEE CHOI1,2 and TAEK‑JEONG NAM1 1Institute of Fisheries Sciences, Pukyong National University, Busan 46041; 2Department of Marine Bio‑Materials & Aquaculture, Pukyong National University, Busan 48513, Republic of Korea Received September 15, 2020; Accepted April 12, 2021 DOI: 10.3892/mmr.2021.12125 Abstract. The protein extracted from red algae genetic myopathies, such as muscular dystrophies, leading to Pyropia yezoensis has various biological activities, including a decrease in muscle strength and mass (2). Muscle atrophy anti‑inflammatory, anticancer, antioxidant, and antiobesity is caused by decreased protein synthesis and increased properties. However, the effects of P. yezoensis protein (PYCP) proteolysis, due primarily to hyperactivation of major cellular on tumor necrosis factor‑α (TNF‑α)‑induced muscle atrophy degradation pathways, including the ubiquitin‑proteasome are unknown. Therefore, the present study investigated the and autophagy‑lysosomal systems (3‑5). Proinflammatory protective effects and related mechanisms of PYCP against cytokines, such as tumor necrosis factor‑α (TNF‑α), interleukin TNF‑α‑induced myotube atrophy in C2C12 myotubes. (IL)‑1β, and IL‑6, have been shown to promote the breakdown Treatment with TNF‑α (20 ng/ml) for 48 h significantly reduced of myofibrillar proteins and reduce protein synthesis, leading myotube viability and diameter and increased intracellular directly to muscle loss (3‑5). Among the cytokines, TNF‑α reactive oxygen species levels; these effects were significantly induces ubiquitin‑dependent proteolysis by activating various reversed in a dose‑dependent manner following treatment intracellular factors, including reactive oxygen species (ROS), with 25‑100 µg/ml PYCP.