The long-term objective of the investigation proposed here is the characterization of the mechanisms regulating the structure of myotendinous junctions (MTJs). MTJs are structurally and functionally specialized sites for transmission of force between muscle and tendon and for addition and deletion of sarcomeres during muscle growth and remodeling. MTJs are also primary sites of injury during eccentric contractions and exhibit early defects in Duchenne muscular dystrophy. Notwithstanding the fundamental physiological importance of MTJs in muscle growth, force transmission, muscle injury and disease, there are no published findings concerning mechanisms regulating the structure of MTJs. Substantial preliminary findings implicate the following hypothetical mechanism for regulating MTJ structure growth, repair and remodeling. Hypothesis: Perturbations of muscle (including modified use, injury or growth) stimulate macrophages at MTJs to secrete interleukin-1Beta (IL-1Beta). IL-1Beta then stimulates macrophages and fibroblasts at the MTJ to secrete platelet derived growth factor (PDGF). PDGF binds to PDGF-receptors concentrated at MTJs. PDGF-receptors autophosphorylate on tyrosine and also phosphorylate tyrosine on some or all of the following proteins involved in linking thin filaments to the muscle cell membrane at MTJs: talin, vinculin, paxillin and integrin Beta1. Phosphorylation of PDGF-receptor causes internalization and degradation of the receptor-ligand complex, thereby providing a negative control for the PDGF-mediated process. Phosphorylation of MTJ structural proteins(s) results in dissociation of myofibrils from the MTJ membrane. PDGF binding also results in activation of proteases, called calpains, that are concentrated at the MTJ. Calpains then act on their substrates at the MTJ, talin and vinculin, thereby contributing to dissociation of myofibrils from the membrane. PDGF-receptors are also substrates for calpain, providing another negative feedback mechanism for the PDGF- stimulated process. Finally, PDGF binding by muscle stimulates type IV collagenase release from the muscle, thereby degrading extracellular components of the MTJ to enable growth or remodeling. The hypothetical mechanism will be tested using hindlimb-suspended rat muscle that undergoes MTJ remodeling during reloading following suspension. The mechanism also will be examined in L6 rat myoblast using in vitro manipulations. The remodeling process will be examined using biochemical, immunological, morphological and biomechanical assays.
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