Molecular Mechanism of Microgravity-Induced Skeletal Muscle Atrophy - Physiological Relevance of Cbl-b Ubiquitin Ligase (MyoLab) - 07.15.14

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Science Objectives for Everyone Molecular Mechanism of Microgravity-Induced Skeletal Muscle Atrophy - Physiological Relevance of Cbl-b Ubiquitin Ligase (MyoLab) studies a rat muscle gene modified cell line to determine the effects of microgravity.

Science Results for Everyone

Astronauts and your grandparents have something in common: muscle loss. MyoLab studies the effects of space on rat muscle genes to find out how microgravity affects biochemical signaling in skeletal muscle. The results match other findings that microgravity conditions increased stress-dependent muscle signaling. Recent studies identified several of these signaling chemicals, but more research is needed narrow down the specific ones. Traditional space workouts have not proven very effective with reducing muscle loss.  This research also examines novel countermeasures, including enzymes and growth hormones.



The following content was provided by Takeshi Nikawa, Ph.D., M.D., and is maintained in a database by the ISS Program Science Office.
Information provided courtesy of the Japan Aerospace and Exploration Agency (JAXA).

Experiment Details

OpNom

Principal Investigator(s)

  • Takeshi Nikawa, Ph.D., M.D., University of Tokushima, Tokushima, Japan

  • Co-Investigator(s)/Collaborator(s)
  • Yuji Okumura, Sagami Women's University, Japan
  • Katsuya Hirasaka, Ph. D., University of Tokushima, Tokushima, Japan
  • Shinichi Takeda, National Center of Neurology and Phychiatry, Japan
  • Kazumi Ishido, Tokushima Bunri University, Japan
  • Atsushi Higashibata, JAXA, Japan

  • Developer(s)
    Information Pending
    Sponsoring Space Agency
    Japan Aerospace Exploration Agency (JAXA)

    Sponsoring Organization
    Information Pending

    Research Benefits
    Information Pending

    ISS Expedition Duration
    March 2010 - September 2010

    Expeditions Assigned
    23/24

    Previous ISS Missions
    MyoLab is scheduled for its first operations on ISS Increment 23/24.

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    Experiment Description

    Research Overview

    • A gene modified cell line from rat muscle cell (L-6) which can be grown as an attached culture in the MeasExp culture chamber, will be launched at ambient temperature using a “MeasExp CO2 bag,” which maintains an adequate concentration of CO2.


    • After medium exchange (5 +/- 1 day) using “MeasExp Solution Exchanger” by a crewmember, the cells are cultivated using the Cell Biology Experiment Facility (CBEF) at 37 degrees Celsius for 12 days.


    • After 10 days (+/-2 days) of incubation, another medium exchange, including IGF-1 for half of the chambers, incubation should be carried out for one day.


    • After a total of 11 days (+/-2 days) of incubation, all chambers are washed by a PBS (Phosphate Buffered Saline) solution, exchanged by RNAlaterTM for preserving RNA, and frozen to recover (in MELFI and STS freezer).


    • After recovery, cells are analyzed for microgravity effects using RT-PCR (Reverse Transcription-Polymerase Chain Reaction - the most sensitive technique for mRNA detection and quantitation currently available) assay, DNA microaray and western blotting techniques.

    Description
    The number of bedridden elderly people in Japan is remarkably increasing, which can be considered as a serious social problem. However, there is no effective countermeasure for muscle atrophy (decrease in muscle mass), which is a main cause for bedridden conditions. The few countermeasures for unloading mediated muscle atrophy include: rehabilitation, diet and drugs. The MyoLab payload will focus on the inhibition of Cbl-b-mediated ubiquitination (enzyme found in humans) to improve IGF-1 (insulin-like growth hormone) resistance of skeletal muscle cells. Ubiquitin ligase Cbl-b is inhibited by focusing on the competitively inhibitory function of oligopeptides (molecules containing a small number of peptides).

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    Applications

    Space Applications
    Information Pending

    Earth Applications
    Information Pending

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    Operations

    Operational Requirements
    Information Pending

    Operational Protocols
    Information Pending

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    Results/More Information

    Researchers investigated the transcription factors that regulate Cbl-b expression using rat L6 myoblasts and differentiated myotubes. The biological relevance of Cbl-b expression as a sensor of unloading is strengthened by the findings that both oxidative stress and 3-D-clinorotation induced Cbl-b expression in L6 myoblasts and myotubes. These findings suggest that increased levels of ROS link mechanical stress to downstream signaling pathways. In the present study, we observed that H2O2 treatment promoted the binding of Egr to the 5'-franking region of Cbl-b gene. Moreover, 3-D-clinorotation and H2O2 each induced the expression of Cbl-b in a manner accompanied by the early expression of Egrs 1-3. This is consistent with the findings of another laboratory using Egr-2 or Egr-3 knockout mice. The results obtained in Egr knockdown studies (siRNA) confirm that Egr transcription factors play a major role in 3-D-clinorotation-mediated Cbl-b induction. Together, these data uncover the molecular mechanism through which mechanical unloading is transduced into biochemical signaling in skeletal muscle.  Several lines of evidence in diverse cell types point to the involvement of Egr transcription factors in the response to mechanical stress. Egr expression induced by 3-D-clinorotation occurs within 90 minutes of stimulation, indicating that the Egr genes are in close temporal proximity to the mechanical stress “receptor.” Consistent with the role of oxidants as the second messengers of Egr activation and downstream unloading responses, the ERK1/2 pathway, a common target of oxidative signaling, was activated by 3-D-clinorotation and H2O2. Together, these results are consistent with the findings of other laboratories; they showed that immobilization or tail suspension increased oxidative stress-dependent signaling in rat skeletal muscles. Recent studies have identified several signaling molecules, such as ASK1, that mediate oxidative stress-dependent activation of MAPK signaling. An important area for further investigation will be to identify the molecules that regulate ROS production in distinct cellular compartments (plasma membrane, mitochondria) in response to unloading. It is anticipated that these molecules may be the direct receptors/sensors for unloading stress. This hypothesis is supported by previous finding that the disrupted expression of cytoskeletal genes, especially mitochondria-anchoring protein genes, is associated with large imbalances in the expression of genes encoding diverse members of the electron transport system in the mitochondria of space-flown skeletal muscle. 

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    Results Publications

      Mukai R, Horikawa H, Fujikura Y, Kawamura T, Nemoto H, Nikawa T, Terao J.  Prevention of Disuse Muscle Atrophy by Dietary Ingestion of 8-Prenylnaringenin in Denervated Mice. PLOS ONE. 2012 September 19; 7(9): e45048. DOI: 10.1371/journal.pone.0045048.

      Tanaka H, Shimazawa M, Kimura M, Takata M, Tsuruma K, Yamada M, Takahashi H, Hozumi I, Niwa J, Iguchi Y, Nikawa T, Sobue G, Inuzuka T, Hara H.  The potential of GPNMB as novel neuroprotective factor in amyotrophic lateral sclerosis. Scientific Reports. 2012 August 13; 2(573): 11 pp. DOI: 10.1038/srep00573.

      Abe T, Hirasaka K, Kagawa S, Kohno S, Ochi A, Utsunomiya K, Sakai A, Ohno A, Teshima-Kondo S, Okumura Y, Oarada M, Maekawa Y, Terao J, Mills EM, Nikawa T.  Cbl-b Is a Critical Regulator of Macrophage Activation Associated With Obesity-Induced Insulin Resistance in Mice. Diabetes. 2013 January 24; 62(6): 1957-1969. DOI: 10.2337/db12-0677. PMID: 23349502.

      Kohno S, Yamashita Y, Abe T, Hirasaka K, Oarada M, Ohno A, Teshima-Kondo S, Higashibata A, Choi I, Mills EM, Okumura Y, Terao J, Nikawa T.  Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages. Journal of Applied Physiology. 2012 March 1; 112(10): 1773-1782. DOI: 10.1152/japplphysiol.00103.2012.

      Nishisho T, Yukata K, Matsui Y, Matsuura T, Higashino K, Suganuma K, Nikawa T, Yasui N.  Angiogenesis and myogenesis in mouse tibialis anterior muscles during distraction osteogenesis: VEGF, its receptors, and myogenin genes expression. Journal of Orthopaedic Research. 2012 November; 30(11): 1767-1773. DOI: 10.1002/jor.22136.

      Utsunomiya K, Owaki K, Okumura Y, Yano M, Oto T, Suzuki E, Tamura S, Abe T, Kohno S, Ohno A, Hirasaka K, Teshima-Kondo S, Nikawa T.  An Intracellular Fragment of Osteoactivin Formed by Ectodomain Shedding Translocated to the Nucleoplasm and Bound to RNA Binding Proteins. Bioscience, Biotechnology, and Biochemistry. 2012; 76(12): 2225-2229. DOI: 10.1271/bbb.120515.

      Teshima-Kondo S, Ochi A, Kohno S, Abe T, Utsunomiya K, Nagano H, Suto T, Tomida C, Yamagishi N, Hirasaka K, Maita A, Okumura Y, Nikawa T.  Space flight/bedrest immobilization and bone. Development of inhibitors for atrophy caused by unloading stress. Clinical Calcium. 2012 December; 22(12): 1879-1885. DOI: CliCa121218791885. PMID: 23187081. [Japanese]

      Abe T, Kohno S, Yama T, Ochi A, Suto T, Hirasaka K, Ohno A, Teshima-Kondo S, Okumura Y, Oarada M, Choi I, Mukai R, Terao J, Nikawa T.  Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b    . International Journal of Endocrinology    . 2013; 2013(907565): 1-11. DOI: 10.1155/2013/907565.

      Yano S, Masuda D, Kasahara H, Omori K, Higashibata A, Asashima M, Ohnishi T, Yatagai F, Kamisaka S, Furusawa T, Higashitani A, Higashitani A, Majima HJ, Nikawa T, Wakabayashi K, Takahashi H, Suzuki H, Shimazu T, Fukui K, Hattori A, Tanigaki F, Shirakawa M, Nakamura T, Yoshimura Y, Suzuki N, Ishioka N.  Excellent Thermal Control Ability of Cell Biology Experiment Facility (CBEF) for Ground-Based Experiments and Experiments Onboard the Kibo Japanese Experiment Module of International Space Station. Biological Sciences in Space. 2012; 26: 12-20. DOI: 10.2187/bss.26.12.

      Oarada M, Tsuzuki T, Nikawa T, Kohno S, Hirasaka K, Gonoi T.  Refeeding with a high-protein diet after a 48 h fast causes acute hepatocellular injury in mice. British Journal of Nutrition    . 2012 May; 107(10): 1435-1444. DOI: 10.1017/S0007114511004521. PMID: 21902856.

      Lago CU, Nowinski SM, Rundhaug JE, Pfeiffer M, Kiguchi K, Hirasaka K, Yang X, Abramson EM, Bratton SB, Rho O, Colavitti R, Kenaston MA, Nikawa T, Trempus C, DiGiovanni J, Fischer SM, Mills EM.  Mitochondrial respiratory uncoupling promotes keratinocyte differentiation and blocks skin carcinogenesis. Oncogene. 2012 January 23; 31(44): 4725-4731. DOI: 10.1038/onc.2011.630. PMID: 22266853.

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