One major outstanding question in muscle biology involves determining the molecular mechanisms by which alterations in mechanical load lead to changes in muscle fiber phenotype. A quintessential marker of myofiber phenotype and function is the myosin heavy chain (MyHC) since it is a major determinate of maximum unloaded velocity of shortening (Vmax) of skeletal muscle fibers. By developing a mouse model of decreased mechanical loading [hindlimb nonweight bearing (NWB)] to investigate transcriptional regulation of the betaMyHC gene we have previously identified in vivo the first NWB responsive promoter region (-450 to -294). We also showed that this NWB region contains a negative element (-332 to -311; termed dbetaNRE-S) that binds two different proteins identified ONLY in NWB-soleus nuclear extracts. Thus, the major focus of this proposal is to identify the nuclear factor(s) that transcriptional repress betaMyHC gene expression under NWB conditions, and to test their possible role in fiber-specific expression (FSE). Proposed experiments at the fine level involve: 1) isolating cDNAs encoding nuclear transcription factor(s) that bind the dbetaNRE-S using expression cloning. Northern analysis will determine developmental, tissue and FSE pattern of the nuclear factor(s). The gross level involves the generation and analyses of transgenic mice harboring: 1) a transgene carrying PCR site- directed mutation of the dbetaNRE-S element to determine its authenticity as an NWB-E. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase (CAT) specific activity in NWB-soleus muscle extract, and 2) transgenes overexpressing cDNA(s) encoding nuclear factor(s) specifically targeted to striated muscle. RNA and protein analyses will assess the impact of cDNA overexpression on whole striated muscle phenotype, which will be correlated to whole muscle and single fiber functional analysis. The proposed experiments will identify and test the in vivo function of nuclear factor(s) and betaMyHC cis-element(s) involved in NWB induced transcriptional repression of betaMyHC gene expression and possibly its FSE. In vivo overexpression of nuclear factor(s) will identify their potential roles in regulation of other muscle gene(s), muscle atrophy and development. These experiments are expected to identify potential DNA/protein targets for therapies aimed at providing counter-measures against the slow-to-fast muscle phenotype and debilitating loss of postural function induced by altered mechanical loads resulting from disease, space flight or extended bed rest.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR047197-04
Application #
6649868
Study Section
Respiratory and Applied Physiology Study Section (RAP)
Program Officer
Nuckolls, Glen H
Project Start
2000-08-01
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
4
Fiscal Year
2003
Total Cost
$340,750
Indirect Cost
Name
University of Missouri-Columbia
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
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Tsika, Richard W; Schramm, Christine; Simmer, Gretchen et al. (2008) Overexpression of TEAD-1 in transgenic mouse striated muscles produces a slower skeletal muscle contractile phenotype. J Biol Chem 283:36154-67
Ji, Juan; Tsika, Gretchen L; Rindt, Hansjorg et al. (2007) Puralpha and Purbeta collaborate with Sp3 to negatively regulate beta-myosin heavy chain gene expression during skeletal muscle inactivity. Mol Cell Biol 27:1531-43
Tsika, Gretchen; Ji, Juan; Tsika, Richard (2004) Sp3 proteins negatively regulate beta myosin heavy chain gene expression during skeletal muscle inactivity. Mol Cell Biol 24:10777-91
Karasseva, Natalia; Tsika, Gretchen; Ji, Juan et al. (2003) Transcription enhancer factor 1 binds multiple muscle MEF2 and A/T-rich elements during fast-to-slow skeletal muscle fiber type transitions. Mol Cell Biol 23:5143-64
Tsika, Richard W; McCarthy, John; Karasseva, Natalia et al. (2002) Divergence in species and regulatory role of beta -myosin heavy chain proximal promoter muscle-CAT elements. Am J Physiol Cell Physiol 283:C1761-75
Vyas, D R; McCarthy, J J; Tsika, G L et al. (2001) Multiprotein complex formation at the beta myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness. J Biol Chem 276:1173-84