Cell Mechanosensing-2 (Cell Mechanosensing-2) - 10.08.14
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Cell Mechanosensing-2 is a continuing investigation to identify gravity sensors in skeletal muscle cells and help develop countermeasures to muscle atrophy, a key space health issue. Scientists believe that the lack of mechanical stress from gravity causes tension fluctuations in the plasma membrane of skeletal muscle cells, in turn changing the expression of key proteins and genes and allowing muscles to atrophy. Muscle cells from rats and kidney cells from African clawed frogs are given fluorescent gene markers and then attached to an extracellular matrix under different tensions.
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JAXA TKSC Space Environment Utilization Center, Tsukuba, , Japan
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
ISS Expedition Duration
March 2014 - March 2015
Previous ISS Missions
Myo Lab experiment in Increment 23/24, Cell Mechanosensing-1 experiment in Increment 35/36.
Skeletal muscle is sensitive to mechanical stress. For example, it easily atrophies under microgravity conditions, while exercise stimulates its growth. Several lines of investigation suggest that skeletal muscle cells possess unique sensors for mechanical stress. However, this sensing mechanism of mechanical stress remains unknown. Based on previous studies, it is hypothesized that tension fluctuation in salcolemma (the plasma membrane of skeletal muscle cells) plays an important role in sensing mechanical stress, such as microgravity. Mechanical stress causes changes in tension fluctuation in the salcolemma, resulting in the activation of channels and/or proteases on membrane surface. They transduce intracellular signaling in skeletal muscle cells to express muscle atrophy-associated ubiquitin ligases (atrogenes), including Cbl-b, MAFbx/atrogin-1, and MuRF-1. We have already reported two candidate transducers, MSCL and MSPL. The former is a mechanosensitive channel with a large pore (MSCL) which induces a Calcium Ion (Ca2+) influx. The latter is Mosaic Serine Protease, large form (MSPL) which mediates the shedding of bioactive membrane molecules. In this study, we will clarify the mechanism on how the tension fluctuation in the salcolemma regulates activities of such transducers during microgravity conditions. We will cultureL6 myotubes/myoblastic cells and A6 amphibian cells on extracellular matrices with different rigidity in the ‘Kibo’ module of the International Space Station. Additionally, the activities of MSCL and MSPL will be measured, as well as the myotube formation of L6 cells. Furthermore, it is hoped that development of inhibitors of these molecules to combat microgravity-induced muscle atrophy will be possible. Finally, the fluorescence signal from recombinant cellular organelles will be observed.
Mechanical stress changes in tension fluctuation in cell membrane, resulting in activation of channels and/or proteases on membrane surface. The experiment will clarify the mechanism how the tension fluctuation in cell membrane regulate activities of such transducers during microgravity conditions. The experiment will culture L6 myotubes/myoblastic cells on extracellular matrix with different rigidity in 'Kibo' module of the International Space Station, so that microgravity conditions induce distinct tension fluctuation in cell membrane. Finally, PIs will measure the activities of channels and/or proteases on membrane, and myotube formation of L6 cells. Furthermore, we will develop inhibitors of these molecules, to combat microgravity-induced muscle atrophy.
Cell Mecahonsensing-1 will continue to Cell Mechanosensing-2 which utilizes GFP recombinant cells with fluorescence microscope observation.
JAXA CBEF, Measurement Experiment Culture Chambers and Measurement Experiment Cages are used for incubation. Measurement Experiment Solution Exchangers are used for medium/fixative exchange.
Loss of muscle strength and tone, and impaired recovery after return to Earth, are long-standing space medicine challenges that resist exercise and other counter-measures in microgravity. Investigations like Cell Mechanosensing-2 will help reveal the mechanical and chemical links that alter normal cell functions and cause muscle atrophy so researchers can develop inhibitors to combat microgravity-induced muscle atrophy.
People subjected to long periods of bed rest and inactivity, such as the elderly or patients after surgery, experience severe muscle atrophy that is difficult to reverse. Some forms of muscular dystrophy also appear to parallel aspects of muscle wasting in space. Understanding the biochemical mechanisms at work will lead to improved therapeutic approaches for these disabilities.
The samples (L6 cells and A-6 cells) are incubated in JAXA CBEF with micro-gravity and artificial 1g environment. L6 cells and A-6 cells are cultured in Measurement Experiment/ Cell Experiment Culture Chambers in CBEF. L6 and A-6 cells are observed using fluorescence microscopy and downlinked images. Lastly, the cells are fixed with Paraformaldehyde for recovery (at +2oC).
Launch active L6 cells and A-6 cells at ambient temperature, and then starts incubation in CBEF soon after docking. After 3 days of incubation, 2 chambers are observed and cells fixed using Cell Experiment Fixation Cylinder, then kept at 2oC in MELFI. Other chambers are continuously observed over the next 4 days