Skeletal muscle plasticity is characterized by dynamic functional and structural remodeling but very little is known about the regulatory mechanisms involved. The skeletal muscle sarco [or endo] plasmic reticulum calcium ATPase (SERCA1) gene is a major determinant of intracellular calcium fluxes and it is strongly transactivated by reduced contractile activity in the presence of intact innervation. The long-term goal of this project is to elucidate the molecular mechanisms of SERCA1 transactivation in muscle. The cis-regulatory and corresponding trans-acting factors will be identified which are responsible for the transactivation of SERCA1 during unloading-disuse atrophy. In vivo transcriptional activity of the SERCA1 promoter will be studied using reporter gene assays following direct muscle injection of chimeric plasmid DNA where different portions of the promoter are linked to a reporter gene. The approach will involve deletion analysis of these constructs as well as constructs containing mutations at specific regulatory sites, and rearrangements of proposed enhancer sequences thought to be regulating promoter activity. Gel shift assays to detect protein-DNA binding activity will be performed as well as strategies to either over express or reduce expression of putative trans-acting factors in vivo. A strength of this approach is that it will uncover molecular mechanisms involved in the transactivation of SERCA1 in the in vivo setting.
A second aim will be to identify transcriptional mechanisms of SERCA1 regulation by calcium in cultured myocytes and compare this to the in vivo mechanism. The same SERCA1 promoter-reporter constructs used in vivo will transiently transfected in cultured myocytes to determine the cis-elements sensitive to calcium. Trans-acting factors will be identified using conventional strategies: protein-DNA binding assays and over expression of wild type, mutant and chimeric GAL4 transcription factors. Candidate upstream pathways will be identified via treatment of cells with specific kinase/phosphatase inhibitors in the presence of the identified calcium sensitive SERCA1 reporter. Candidate signaling proteins will be tested for involvement by over expression in the presence of the calcium sensitive SERCA1 reporter. An understanding of the regulation of SERCA1 is of fundamental importance to understanding a variety of cellular processes and disease states (e.g. congestive heart failure), and in developing pharmaceuticals for effecting improvements in muscle function due to atrophy.
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