Skeletal muscle development is a finely orchestrated process requiring integration of multiple cell times in time and space to form each of the 200+ muscles found in adult mammals, each of which is unique in its size, shape, and location as well as in the functional connections it forms with other organ systems such as nerves and tendons, innervation, and contractile properties. When the integrity of a muscle is disrupted by acute damage or disease, skeletal muscle is remarkable not only in its capacity to rapidly and efficiently regenerate lost cell number and mass, but in the extent to which the pre-existing patterns intrinsic to each muscle are recapitulated. One aspect of patterning that is critical for the specific function of any given muscle is the unique proportion and arrangement of fast and slow myofibers to provide the necessary balance of force and endurance. Distinct muscle fiber types are present in limb muscles, which can be defined on the basis of their glycolytic vs. oxidative capacity, twitch force generation, motor neuron connectivity, and myosin heavy chain gene expression and are broadly defined as 'slow' (expressing Type I MyHC) and 'fast' (expressing Type II MyHC). Preferential cell-cell associations based on origin or associations with fast vs. slow muscle have been observed both among myogenic cells and between myogenic cells and other cell types for more than fifty years, but no molecular explanation for any of the cases where it occurs have been found. Recent data showing that classical repulsive guidance signaling by Eph/ephrin family members modifies stem cell behavior in skeletal muscle led to the hypothesis that it could be responsible for cell sorting during myogenesis, innervation, and regeneration as a mechanism to preserve fiber type identity. A single repulsive guidance ligand, ephrin-A3, is expressed on all and every slow myofiber in adult limb muscle, and is not expressed by any fast myofiber. Expression of two ephrin-A3 receptors, EphA3 and EphA8, are found in a complementary pattern, associated with all and only fast myofibers and no slow myofibers and appear to identify myoblasts and neuronal cells, respectively. Because the usual effect of Eph-ephrin interactions is cellular de-adhesion and repulsion, expression data would support a model in which 'fast' cells are prevented from interacting with slow myofibers via specific Eph/ephrin interactions. This model will be tested both during initial muscle development and patterning and following injury to adult muscle or nerve, primarily by comparing fiber type profiles in wild type, ephrin-A3 loss-of-function and ephrin-A3 gain-of-function muscles in vivo. Because fiber type-specific loss of muscle groups is associated with both frailty due to aging and muscle dysfunction in metabolic or neuromuscular disease, this novel approach to understanding how fiber type patterns are initially specified then recapitulated will provide novel targets that may be modified muscle fiber types in situ to preserve muscle mass and function.
This proposal addresses the potential role of a single repulsive guidance ligand, ephrin-A3, in specifying slow muscle fibers by preventing either fusion by 'fast' myoblasts or innervation by fast motor neurons during development or repair. Our goal is to identify a simple and consistent mechanism that defines and maintains slow myofiber identity, which will not only answer long-standing questions in basic muscle biology but may suggest novel approaches to maintaining or rescuing muscle mass during aging, metabolic disorders, or neuromuscular disease.
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