Repair and replacement of damaged skeletal muscle in vertebrates requires the activity of a population of resident adult stem cells (satellite cells.) Following acute injury, these relatively rare and highly dispersed cells are required to respond by quickly becoming `activated', multiplying to produce a large pool of replacement myoblasts, relocating to the site of the injury, and differentiating to form new muscle fibers. The molecular and cellular processes required for adult myogenesis (regeneration) overlap with, but are largely distinct from, those active in the embryo when muscle is first formed. At the level of the cell's interactions with the extracellular environment, the requirements for satellite cell motility and migration within the tissue are a critical aspect of muscle regeneration that has not been well explored. In collaboration with Dr. George Davis, we have combined primary satellite cell culture on their host myofibers in a programmable 3D collagen matrix with timelapse videomicroscopy to develop a novel system for qualitatively and quantitatively examining satellite cell migration on their native substrate. Conditions can be altered by addition of exogenous stimuli such as potential mutagens, blocking of soluble or cell-surface proteins with antibodies or peptide mimetics, addition of pharmacological inhibitors of specific signaling pathways or cytoskeletal remodelers, specific infection of satellite cells with viral expression constructs, or use of fibers and satellite cells derived from targeted mutations;all of these can also be assayed both individually and in combination. We have used this system to assess the roles of soluble factors such as growth factors, chemokines and signaling lipids;extracellular matrix and adhesion factors;transmembrane signaling receptors and integrins;and intracellular modulators of the cytoskeleton. Our goal for this short-term exploratory grant is to extend and refine these results to build a working model of satellite cell migration in the context of these defined classes of signaling factors and effectors. Once we have identified critical interactions and points of control, we will continue on to ask how such activities are integrated within individual satellite cells to effect a coherent migration of the population of proliferating myoblasts towards an area of injury. Our broad, long-term goal is to understand how satellite cells detect, integrate, and respond appropriately in time and space to the transient and dynamic signaling environment that would constitute a muscle injury in vivo, with the biological robustness that is taken for granted in healthy muscle tissue and compromised in dystrophic muscle.
In addition to addressing an underexplored facet of basic research into the mechanisms of adult myogenesis, this project has high potential to contribute to the development of satellite cell- based therapies for diseases such as the muscular dystrophies. A critical area of concern in current adult myoblast and muscle-derived stem cell engraftment procedures is the unmet requirement for injected cells to, at least, disperse broadly from the injection site or, at best, actively home to either investigator-defined sites or sites of maximum damage. By providing insight into the motogenic stimuli, preferred migration substrate, and specific guidance cues used by satellite cells migrating in a system (single fiber culture) that could be expected to recapitulate many of the influences found in vivo, this work will ideally suggest potential avenues to modify current myoblast engraftment protocols to enhance their therapeutic effectiveness.
