Muscle cells express a number of small stress responsive proteins called small heat shock proteins that are known to be critical to cell survival, growth, and repair. However, how these proteins accomplish their tasks is not understood. Proposed studies will examine the regulation and function of these proteins in muscle cells responding to low oxygen conditions that occur during injury, exercise and hibernation. The work will be conducted using zebrafish larvae as a model system because they can be used to study the organization and function of fluorescently labeled proteins in living tissues. Data resulting from these studies are expected to provide novel insight into the mechanisms underlying how muscle cells respond to stress. Such insights have implications for understanding or modifying the limits of muscle performance, engineering, adaptation and repair in a wide variety of organisms. In addition to the new information generated, the project will also benefit society by promoting teaching, training and learning, and by broadening the participation of underrepresented groups in science. Outreach educational workshops with area high schools will be developed and strengthened through conducting hands-on research projects with zebrafish larvae. Student conceptual understanding will be assessed through the use of pre-and post-workshop evaluations. Opportunities will also be created for high school students to visit and work in laboratories of active investigators at Washington State University and the neighboring University of Idaho. A summer internship will be provided annually to area high school science teachers who will learn methods for zebrafish husbandry and analysis of zebrafish embryonic development applicable to a K-12 classroom environment.
The overall goal of the studies is to test the hypothesis that the heat shock inducible transcription factor, HSF1, regulates expression of small heat shock proteins, which dynamically associate with cytoskeletal filaments in a phosphorylation-dependent manner in hypoxic muscle cells to preserve cytoskeletal integrity and muscle function. The proposal includes three specific aims. First the role of HSF1 in regulating muscle specific small heat shock protein expression in response to hypoxia will be assessed. To accomplish this, the function of transcription factor HSF1 will be manipulated and effects on hypoxia inducible small heat shock protein expression analyzed. Mutagenesis of small heat shock protein promoters will also be conducted to identify motifs required for hypoxia-induced expression. Second, phosphorylation-dependent interaction kinetics of small heat shock proteins with the cytoskeleton will be analyzed in hypoxic muscle cells. Mutagenesis of serine/threonine residues will be conducted and effects on hypoxia inducible phosphorylation established. Dynamic interactions of wild-type and phosphorylation site mutant heat shock proteins with the cytoskeleton and each other will be examined in control and hypoxic muscles. Finally, small heat shock protein expression will be altered in muscles, and cytoarchitecture and function of muscle cells responding to hypoxia will be assessed. To accomplish this aim, zebrafish lines that over-, and under-express small heat shock proteins in skeletal muscle will be created. Muscle cell architecture and function will be assayed in larvae and adults under control conditions and after exposure to low oxygen levels.
