Molecular Mechanism of Microgravity-Induced Skeletal Muscle Atrophy - Physiological Relevance of Cbl-b Ubiquitin Ligase (MyoLab) - 09.17.14
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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.
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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.
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
ISS Expedition Duration
March 2010 - September 2010
Previous ISS Missions
MyoLab is scheduled for its first operations on ISS Increment 23/24.
- 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.
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).
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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