Program :    Biomedical Research and Countermeasures	Ground Research
Element :    Physiology
Activity Dependent Signal Transduction in Skeletal Muscle
Principal Investigator:
Susan L. Hamilton, Ph.D. Department of Molecular Physiology and Biophysics 400B Baylor College of Medicine One Baylor Plaza Houston, TX 77030	Phone: (713) 798-3894 Email: susanh@bcm.tmc.edu Fax: (713) 798-5441 Congressional District: TX-25
Co-Investigator(s):
Armstrong, Robert B. Reid, Michael B. Texas A&M University Baylor College of Medicine
Monitoring Center: NSBRI	Solicitation: NSBRI
Initial Funding Date: 1997	Expiration: 2000
Students Funded Under Research: 0	Post-Doctoral Associates: 1
Task Description:
Significant remodeling of skeletal muscle, especially slow twitch muscle fiber occurs in astronauts upon extended exposure to microgravity. This leads to decreased ability of these muscles to function upon re-exposure to a larger gravitational load. To develop effective countermeasures, the molecular mechanisms producing these changes need to be identified. Our long-term goal is to define the early signal transduction events whereby removal of load triggers muscle remodeling/atrophy, and to identify appropriate countermeasures. Our working hypothesis is that decreased levels of the calcineurin (phosphatase), play an important role in the remodeling and atrophy that occurs with unloading of weight bearing muscles. Hind limb unloading (HLU) leads an increase in resting Ca2+ levels in the soleus possibly due to increase in reactive oxygen species (ROS) that alters the Ca2+ leak properties of the sarcoplasmic reticulum Ca2+ release channel. In the current work, we will evaluate the changes in signal transduction pathways activated by this increase in Ca2+, and evaluate the ability of eccentric exercise protocols to prevent HLU resulting mass and signal transduction changes. We postulate that increased resting Ca2+ activates calpains, leading to proteolysis of calcineurin and NFAT; reduced nuclear translocation of NFAT; coactivation of MEF2 and finally fiber type switching / muscle mass decreases. Earlier, we demonstrated that hind limb unloading leads to early decrease in calcineurin and NFAT protein levels. We will analyze whether the decreases in calcineurin and NFAT arise from increased turnover or decreased synthesis by determining the mRNA levels, protein amounts, and subcellular localization of calcineurin isoforms and NFAT as a function of time after hind limb unloading. Ca2+ signaling is likely to involve other Ca2+ dependent pathways apart from calcineurin/NFAT pathway, and crosstalk with other pathways not directly regulated by Ca2+. Proteomics (2D electrophoresis/ mass spectrometry) and Gene array technology will be utilized to assess the effects of hind limb unloading and increased resting Ca2+ on other Ca2+ dependent proteins (PKC, rac, CaMK, RYR1, DHPR, SERCA, etc). We will assess the effects of the eccentric exercise protocols during conditioning, prior to, and during hindlimb unloading, on muscle strength, mass and levels of proteins shown to change with HLU (calcineurin, NFAT, MEF-2, etc). The proposed objectives should contribute to our understanding of molecular mechanisms of disuse atrophy and provide effective countermeasures to ease muscle atrophy during weightlessness in long-term space flight.
1, We demonstrated an increase in resting Ca2+ as supposed to altered E-C coupling that leads to changes in muscle function during muscle unloading. 2. We investigated the role of various modulators like calmodulin and FKBP-12 in excitation-contraction coupling. 3. We addressed the question of how the increase in [Ca2+]i alters muscle function. A number of signaling pathways known to be altered by changes in intracellular Ca2+ were investigated. These include pathways known to be involved in regulation of muscle mass and fiber type. Our work indicates muscle unloading leads to decreases in the levels of calcineurin, NFAT (both soleus and gastrocnemius), and MEF-2. 4. We demonstrated that muscle unloading leads to changes in calpain activity and its sub-cellular distribution, a pathway that could explain the loss of proteins during muscle unloading.
These studies should contribute to our understanding of molecular mechanisms of disuse atrophy.
FY00 Publications, Presentations, and Other Accomplishments:
Rodney G.G., Moore C.P., Williams B.Y., Zhang J.Z., Krol J., Pedersen S.E., Hamilton S.L. ''Calcium Binding to Calmodulin Leads to an N- Terminal Shift in its Binding Site on the Ryanodine Receptor.'' J Biol Chem, (October 16, 2000). Pate P., Mochca-Morales J., Wu Y., Zhang J.Z., Rodney G.G., Serysheva I.I., Williams B.Y., Anderson M.E. Hamilton S.L. ''Determinants for calmodulin binding on voltage-dependent Ca2+ channels.'' J Biol Chem, 275(50):39786-92, (December 15, 2000). Rodney G.G., Williams B.Y., Strasburg G.M., Beckingham K., Hamilton S.L. ''Regulation of RYR1 activity by Ca(2+) and calmodulin.'' Biochemistry, 39(26):7807-12, (July 4, 2000). Hamilton, S.L., Serycheva, I., Strasberg, G.M. ''Calmodulin and excitation- contraction coupling.'' News in Physiological Sciences, Volume 15, (December, 2000). Rao, V.L., Papineni, C.P., Ingalls, J., Hamilton, S.L. ''Hindlimb suspension increases resting Ca2+ but decreases calcineurin levels.'' 44th Annual Meeting, Biophysical Society, USA (February 16, 2000).

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