The main objectives of this proposal are: to increase the research skills of the applicant and to enhance the current understanding of how passive stretch may induce long term, structural cellular changes in muscle. The rehabilitation of patients with soft tissue contractures frequently involves the use of passive stretch. However a scientific rationale which might explain increased mobility after the application of passive stretch is lacking. The career development plan for the applicant includes a combination of coursework and hands-on laboratory experience focusing on increasing the applicant's expertise in molecular biology. Mentoring is achieved by a multidisciplinary research advisory committee which includes both clinicians (orthopaedic surgeons) and basic scientists with expertise in muscle biophysics, membrane biology and molecular biology. The research plan proposes to study the effects of passive stretch in a mammalian skeletal muscle cell culture. The central hypothesis is that stretch is perceived by the muscle fiber as a stimulatory signal and ultimately leads to myofibrillogenesis. Experiments are proposed to evaluate the role of the cytoskeleton and associated molecules, such as integrins, in the signaling mechanism and in organizing contractile structures. The following hypothesis will be tested: 1. Passive stretch enhances myoblast proliferation and subsequent myotube formation. 2. Passive stretch is a signal for cytoskeletal remodeling and ultimately for myofibrillogenesis. 3. Passive stretch regulates the mRNA and/or protein synthesis of cytoskeletal and cytoskeleton-related molecules 4. Passive stretch induces tyrosine phosphorylation of integrins.
The specific aims for this proposal are: 1. To determine the degree of proliferation, the rate of fusion, and the maturation of cultured mammalian skeletal myoblast/myofibers exposed to various regimens of passive stretch by using established cell biological and immunological methods. 2. To determine changes in the cellular location of cytoskeletal molecules (protein and mRNA) in cultured mammalian skeletal muscle fibers in response to stretch by using confocal immunofluorescent microscopy and in situ hybridization. 3. To determine whether passive stretch increases the expression of genes encoding cytoskeletal and cytoskeleton-related proteins by using western and northern blot analysis. 4. To elucidate whether tyrosine phosphorylation occurs as a result of passive stretch by using immunoprecipitation and western blot analysis. The results of this approach will be used to enhance the scientific basis of passive stretch as a rehabilitation technique. Ultimately this research should lead to improved treatments for patients with soft tissue contractures.
