Small heat shock proteins (sHsps) are a large, but poorly understood, family of proteins. In vertebrates and many invertebrates, sHsps are constitutively found in muscle cells where they may play roles in homeostasis and injury repair. Preliminary results obtained in the Shelden laboratory have revealed striking, stress-dependent recruitment of the ubiquitous small heat shock protein, Hsp27, to muscle myofibrils. This research focuses on defining the mechanism and functional significance of this recruitment and specifically addresses the following questions: 1) How is Hsp27 regulated when it associates with myofibrils, and what is the effect of regulation on its function?, 2) What are the kinetics of association between Hsp27 and myofibrils during injury? and, 3) What is the target for Hsp27 within myofibrils? The project will employ immunolocalization as well as ulltrastructural and biochemical approaches to examine stress-induced redistribution of Hsp27 and phosphoserine mutants of Hsp27 in zebrafish embryos. Completion of this work will substantially advance the understanding of Hsp27 regulation and function in muscle cells. These studies are also expected to advance general understanding of sHsp functions and response of muscle cells to stress and injury, especially in vivo. Such knowledge may ultimately be used to enhance muscle growth, or ameliorate injury and age-dependent muscle dysfunction in commercial or clinical settings, and permit a better understanding of adaptations to environmental stress in poikilotherm (cold blooded) animals. In addition, the project will support outreach and educational activities to students in area elementary schools and junior and senior high school students at a local summer science camp program.
The overall goal of our three year project was to understand the regulation and function of heat shock proteins in the zebrafish, a model for the study of warm-water ray-finned species and other vertebrates including mammals. Our particular focus was a small heat shock protein called Hsp27 which was known from other species but poorly investigated in fish. In addition, funding was provided in part to provide educational and research opportunities to K-12 students and undergraduates. Results of our studies demonstrated that Hsp27 has a significant role in supporting the development of craniofacial muscles during vertebrate embryogenesis. The involvement of Hsp27, a stress responsive gene product, in the development of these structures leads us to hypothesize that unnatural stimulation of Hsp27 expression or function could result in developmental defects with serious consequences for organismal survival. Our studies also revealed that Hsp27 expression is enhanced in zebrafish embryos responding to hypoxic injury, and that its expression in tissues responding to hypoxia is regulated by a stress activated transcription factor known as HSF1. Because HSF1 is not generally considered to be a significant player in the response of tissues to hypoxic injury, these results may lead to new insights into the manner in which tissues respond to hypoxia, how these responses can be augmented, and why they fail to fully address hypoxic injury under some conditions. Other studies demonstrated that Hsp27 interacts directly with the giant protein, titin, during the response of zebrafish cardiac muscle tissues to stress. This had not been previously demonstrated for Hsp27 in other studies. Because titin serves to anchor the muscle specific motor protein, myosin, in its proper location in muscle fibers, our data lead us to hypothesize that Hsp27 protects the function of injured muscle tissues by maintaining the integrity of titin and therefore the position of myosin within cells. Results of these studies have implications for understanding how the functional integrity of muscle cells is maintained after injury. Our project supported the production of two graduate theses, as well as the laboratory research training of eight other graduate students, six undergraduate students and two high-school students. Funding of this project also supported three annual research workshops on developmental toxicology held with classes of approximately 20 advanced high school students/year in the neighboring Moscow Idaho community.
