DOCSLIB.ORG

Differentiation and Long-Term Survival of C2C12 Myoblast Grafts in Heart

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
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that virtually all of the grafted skeletal myocytes were of the SV40 large T antigen oncogene in the hearts of trans- withdrawn from the cell cycle by 14 d after grafting.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. Elevated differentiated myotubes. Graft formation and the associated expression of c-myc during embryogenesis increased develop- myocardial remodeling did not induce overt cardiac arrhyth- mental cardiomyocyte hyperplasia in transgenic mice (12). mia. This study indicates that the myocardium can serve as a Cardiomyocyte proliferation was also observed following retro- stable platform for skeletal myoblast grafts. The long-term sur- viral transduction of ras or myc in embryonic rat ( 13) or avian vival, differentiated phenotype, and absence of sustained prolif- (14) cardiomyocytes. Thus targeted oncogene expression, at erative activity observed in myoblast grafts raise the possibility least in some instances, can bypass the cues that usher cell cycle that similar grafting approaches may be used to replace dis- withdrawal during normal cardiomyocyte development. Pres- eased myocardium. Furthermore, the genetic tractability of ently it is not clear if targeted oncogene expression can induce myoblasts could provide a useful means for the local delivery of proliferation in adult cardiomyocytes. recombinant molecules to the heart. (J. Clin. Invest. 1993. Given the absence of significant regenerative potential in 92:1548-1554.) Key words: skeletal myoblasts * intracardiac mammalian myocardium, we have previously conducted a se- grafts * cell transplantation * gene therapy * myocardial repair ries of experiments to determine if cardiomyocytes manipu- lated in vitro could form intracardiac grafts ( 15). These experi- Introduction ments utilized AT- 1 cardiomyocytes, which were derived from It is now well established that the regenerative capacity of skele- transgenic mice expressing the SV40 large T antigen oncogene tal muscle resides in the satellite cells first described by Mauro in the atrium (8, 16). AT-l cardiomyocytes retain the capacity in 1961 (1). Trauma induces satellite cell proliferation and for proliferation in culture while displaying a differentiated phe- subsequent differentiation into new (or regenerated) muscle notype ( 17, 18). Our results showed that AT- I cardiomyocytes fibers. Satellite cell (myoblast) lines that retain the capacity to formed long-term, differentiated intracardiac grafts without differentiate into myotubes in culture have been isolated from having a deleterious effect on cardiac function. However, the a number of species, including mouse and human. Several unchecked proliferation of grafted AT- 1 cells limits the poten- groups have shown that myoblasts have the capacity to fuse to tial value of these grafts for both myocardial repair and long- preexisting fibers when introduced directly into the skeletal term delivery of recombinant molecules. muscle of a syngeneic host (2, 3). Thus, skeletal muscle regen- The successful use of skeletal myoblasts in the grafting ex- eration can be engendered via heterokaryon formation be- periments mentioned above prompted us to test their capacity tween host myotubes and histocompatible donor myoblasts. to generate intracardiac grafts. In this study we addressed three This observation has been exploited in studies with mdx mice issues. First, we determined if C2C12 myoblasts could form (4) and Duchenne's patients (5), where the introduction of stable grafts in a syngeneic myocardium. Second, we ascer- myoblasts carrying a normal dystrophin gene resulted in the tained the proliferative and differentiated status ofgrafted cells. Third, we determined if the presence of C2C12 grafts had a deleterious effect on host cardiac function. Our results indicate Address reprint requests to Loren J. Field, Krannert Institute of Cardi- that C2C 12 differentiate into myotubes when ology, 1111 West 10th Street, Indianapolis, IN 46202-4800. myoblasts Receivedfor publication 5 March 1993 and in revisedform 26 April grafted into the heart. The absence of tritiated thymidine up- 1993. take indicates that the grafted cells are withdrawn from the cell cycle by 14 days after implantation. There was no overt effect J. Clin. Invest. on cardiac function in animals bearing differentiated myotube ©D The American Society for Clinical Investigation, Inc. grafts. The implication of these results with respect to the po- 0021-9738/93/09/1548/07 $2.00 tential replacement of myocardial tissue and to the long-term Volume 92, September 1993, 1548-1554 delivery of recombinant proteins to the heart is discussed. 1548 G. Y. Koh, M. G. Klug, M. H. Soonpaa, and L. J. Field Methods Results C2C12 cell culture and myocardial grafting protocol. C2Cl2 myoblasts Several myoblast cell lines have been described that, as exem- were obtained from American Type Culture Collection (Rockville, plified by C2C 12 cells, have the capacity to differentiate into MD). Cells were maintained in the undifferentiated state by culturing myotubes in culture (21 ). C2C1 2 myoblasts were derived from at low density in high glucose DME supplemented with 20% FBS, 1% cultured explants of injured thigh muscle of C3H mice. When chicken embryo extract, 100 U/ml penicillin, and 100 ,g/ml strepto- maintained in serum-rich the mycin. For some studies, myogenic differentiation was induced by cul- media, myoblasts proliferate rap- an turing in DME supplemented with 2% horse serum and antibiotics. idly and retain undifferentiated phenotype. However, when Immediately before injection, myoblasts were harvested with trypsin, cultured in serum-poor media, myogenic differentiation is in- washed three times with serum-free DME, and directly injected into duced. The C2C1 2 cells withdraw from the cell cycle and fuse, the ventricular myocardium of adult syngeneic C3Heb/FeJ mice thereby forming multinucleated myotubes. Myogenic differen- (Jackson Laboratories, Bar Harbor, ME) under open heart surgery as tiation is also induced, as evidenced by the appearance of nu- described ( 15, 19). 4-10 X 104 cells were injected in a volume of 2-3 ,1 merous muscle-specific gene products. Thus, in this model, using a plastic syringe fitted with a 30-gauge needle. proliferation and myogenic differentiation are mutually exclu- Histology. Hearts were removed following cervical dislocation and sive (22). Myoblast differentiation in vitro is thought to mimic cryoprotected in 30% sucrose, embedded and sectioned at 10 Am with a satellite cell mediated myofiber regeneration in vivo. cryomicrotome as described (20). For hematoxylin and eosin (H and We have used C2C1 2 myoblasts to generate E)' staining, sections were postfixed in acetone/methanol ( 1:1) and long-term stained according to manufacturer's specifications (Sigma Diagnostics, grafts in the adult heart. Myoblasts were injected directly into St. Louis, MO). For immunohistology, methanol-fixed sections the myocardium of syngeneic C3Heb/FeJ mice and the viabil- (-20'C, 10 min) were reacted with the monoclonal anti-skeletal myo- ity of the grafted material was assessed. 100% ( 13/ 13) of the sin heavy chain antibody (MY-32; Sigma Chemical Corp., St. Louis, mice receiving intracardiac implants ofC2C 12 myoblastsdevel- MO) followed by rhodamine-conjugated sheep anti-mouse IgG oped grafts in the heart. Viable grafts were observed as long as 3 F(ab')2 fragment (Boehringer Mannheim, Indianapolis, IN), and visu- mo after implantation, the latest time point assayed to date. In alized by epifluorescence. For [3H]thymidine incorporation, mice all instances, the grafted material was not encapsulated (Fig. 1, were given a single bolus injection of isotope (400 MCi at 28 Ci/mM, A, D, F, and H). The differentiated status of the grafted C2C 12 Amersham, Arlington Heights, IL) and 18 h later killed by cervical cells was determined dislocation. by immunohistological assay with an The heart was removed, cryoprotected in 30% sucrose, chain This embedded,
Load more

Recommended publications
  • Development of a High-Throughput Screen to Identify Small Molecule Enhancers of Sarcospan for the Treatment of Duchenne Muscular Dystrophy
    UCLA UCLA Previously Published Works Title Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy. Permalink https://escholarship.org/uc/item/85z6k8t7 Journal Skeletal muscle, 9(1) ISSN 2044-5040 Authors Shu, Cynthia Kaxon-Rupp, Ariana N Collado, Judd R et al. Publication Date 2019-12-12 DOI 10.1186/s13395-019-0218-x Peer reviewed eScholarship.org Powered by the California Digital Library University of California Shu et al. Skeletal Muscle (2019) 9:32 https://doi.org/10.1186/s13395-019-0218-x RESEARCH Open Access Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy Cynthia Shu1,2,3, Ariana N. Kaxon-Rupp2, Judd R. Collado2, Robert Damoiseaux4,5 and Rachelle H. Crosbie1,2,3,6* Abstract Background: Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Methods: Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting.
    [Show full text]
  • <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 ␣11␤1 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 ␣11␤1 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 ␣11␤1 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • Culturing C2C12 Myotubes on Micromolded Gelatin Hydrogels Accelerates Myotube Maturation Lance T
    Denes et al. Skeletal Muscle (2019) 9:17 https://doi.org/10.1186/s13395-019-0203-4 RESEARCH Open Access Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation Lance T. Denes1†, Lance A. Riley2†, Joseph R. Mijares2, Juan D. Arboleda1, Kendra McKee1, Karyn A. Esser2*† and Eric T. Wang1*† Abstract Background: Skeletal muscle contributes to roughly 40% of lean body mass, and its loss contributes to morbidity and mortality in a variety of pathogenic conditions. Significant insights into muscle function have been made using cultured cells, in particular, the C2C12 myoblast line. However, differentiation of these cells in vitro typically yields immature myotubes relative to skeletal muscles in vivo. While many efforts have attempted to improve the maturity of cultured myotubes, including the use of bioengineered substrates, lack of molecular characterization has precluded their widespread implementation. This study characterizes morphological, molecular, and transcriptional features of C2C12 myotubes cultured on crosslinked, micropatterned gelatin substrates fabricated using previously established methods and compares them to myotubes grown on unpatterned gelatin or traditional plasticware. Methods: We used immunocytochemistry, SDS-PAGE, and RNAseq to characterize C2C12 myotubes grown on micropatterned gelatin hydrogels, unpatterned gelatin hydrogels, and typical cell culture substrates (i.e., plastic or collagen-coated glass) across a differentiation time course. The ability to form aligned sarcomeres and myofilament protein concentration was assessed. Additionally, the transcriptome was analyzed across the differentiation time course. Results: C2C12 myotubes grown on micropatterned gelatin hydrogels display an increased ability to form aligned sarcomeres as well as increased contractile protein content relative to myotubes cultured on unpatterned gelatin and plastic.
    [Show full text]
  • Tumor Necrosis Factor-K Exerts Interleukin-6-Dependent and -Independent E¡Ects on Cultured Skeletal Muscle Cells
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1542 (2002) 66^72 www.bba-direct.com Tumor necrosis factor-K exerts interleukin-6-dependent and -independent e¡ects on cultured skeletal muscle cells Bele¨n Alvarez a, LeBris S. Quinn b,S|¨lvia Busquets a, Maria T. Quiles c, Francisco J. Lo¨pez-Soriano a, Josep M. Argile¨s a;* a Cancer Research Group, Departament de Bioqu|¨mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain b Geriatric Research, Education and Clinical Center, VA Puget Sound Health Care System, American Lake Division, Tacoma, WA 98493, USA c Hospital Vall d'Hebron, Barcelona, Spain Received 5 March 2001; received in revised form 2 October 2001; accepted 4 October 2001 Abstract In vivo studies have shown that cancer-associated skeletal muscle wasting (cachexia) is mediated by two cytokines, tumor necrosis factor-K (TNF) and interleukin-6 (IL-6). It has been unclear from these studies whether TNF exerts direct effects on skeletal muscle and/or whether these effects are mediated via IL-6. Previous studies from our laboratory have shown that TNF induces IL-6 mRNA expression in cultured skeletal muscle cells. To further investigate the relationship between TNF and IL-6, the effects of TNF and IL-6 on protein and DNA dynamics in murine C2C12 skeletal myotube cultures were determined. At 1000 U/ml, TNF induced 30% increases in protein and DNA content. The effects of TNF on protein accumulation were inhibited by aphidicolin, an inhibitor of DNA synthesis.
    [Show full text]
  • Engineering Contractile 2D and 3D Human Skeletal and Cardiac Muscle Microtissues
    Engineering Contractile 2D and 3D Human Skeletal and Cardiac Muscle Microtissues Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical engineering Rebecca Marie Duffy B.S., Biomedical Engineering, Virginia Commonwealth University Carnegie Mellon University Pittsburgh, PA August, 2016 Dedication In memory of Sue, Phil, and Bill. You are always in my heart Thank you to my parents, Jerry and Lisa, for always encouraging me to do my best. I wouldn't be the person I am today without all of the valuable lessons you've taught me - following through on things you start, understanding how to juggle priorities, the importance of family, how to stay true to yourself, and how to stand up for yourself. You've always told me you'd be proud of me as long as I did my best...and it looks like you've served me pretty well with that attitude. I'm so grateful for all of the sacrifices the two of you have made (that I'm probably unaware of) to make my life better and give me all of the best opportunities you could over the years. Thank you to my grandparents, Shelby and Jimmy - you two are such an important part of my life, and I want to thank you for busting your butts to raise two amazing kids and for reminding me to have perspective on how lucky I have been to be blessed with the opportunities life has given me. My grandparents Big Ed, Margaret, and BK. You all were also testaments to working hard to achieve your goals and provide for your families, and I just hope I continue to make you proud.
    [Show full text]
  • Myosin Heavy Chain Is Stabilized by BCL-2 Interacting Cell Death Suppressor (BIS) in Skeletal Muscle
    OPEN Experimental & Molecular Medicine (2016) 48, e225; doi:10.1038/emm.2016.2 & 2016 KSBMB. All rights reserved 2092-6413/16 www.nature.com/emm ORIGINAL ARTICLE Myosin heavy chain is stabilized by BCL-2 interacting cell death suppressor (BIS) in skeletal muscle Jin Hong1,2, Jun-Sub Park1,2, Hyun Lee1,2, Jaemin Jeong3, Hye Hyeon Yun4,5, Hye Yun Kim4,5, Young-Gyu Ko1,2 and Jeong-Hwa Lee4,5 BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle. Experimental & Molecular Medicine (2016) 48, e225; doi:10.1038/emm.2016.2; published online 1 April 2016 INTRODUCTION processes, including apoptosis, stress responses, macroauto- Skeletal and cardiac muscles are dynamic organs that are phagy, chaperone-assisted selective autophagy, cell adhesion constantly adapting in response to mechanical loading and and migration and senescence.9–17 BIS is ubiquitously metabolic changes.
    [Show full text]
  • Assessing Differentiation Potential of C2C12 Myoblastic Cells On
    Assessing differentiation potential of C2C12 myoblastic cells on hydrogels, and development of stimulation device to induce contraction on regular and micropatterned C2C12 cells Submitted By: Adriana Martinez-Betancourt ____________________________________________ Jeffrey Lessard ____________________________________________ Kien Dao ____________________________________________ Yow-Chyuan Yeh ____________________________________________ Submitted to: Professor Sakthikumar Ambady __________________________________________ Profressor Raymond Page __________________________________________ April 26, 201 Page | 1 Abstract We examined the differentiation characteristics of C2C12 mouse myoblasts on polyacrylamide hydrogels of varying stiffness’ in comparison to widely used polystyrene tissue culture plates. The benchmarks we used included formation of myotube fibers, BrdU incorporation, myosin heavy chain expression, and fiber contraction. As a method of observing fiber contraction, we designed, built, and used an electrical stimulation device which allowed us to run a current through the cells after they had differentiated while viewing and recording the cells in real time. The data suggest that cells grown on hydrogels proliferate less but differentiate more similar to those in vivo. Page | 2 Table of Contents Abstract ......................................................................................................................................................... 2 Acknowledgements ......................................................................................................................................
    [Show full text]