Adult skeletal muscles contain a heterogeneous group of fibers which can be characterized by their myosin isozymes, as well as by other properties. These fibers can change from one myosin to another, a transformation controlled both by the pattern of neural stimulation and the levels of several hormones. Changes in myosins also occur during development: from a seemingly homogeneous population of fibers and a single unique myosin, muscles diverge into a heterogeneous set of fibers with characteristic myosins. Yet, it is not known (a) if this original population of cells is truly homogeneous, (b) what factors are responsible for the initial diversity among fibers, or (c) what factors regulate the sequence of developmental myosins made prior to the synthesis of adult myosins. Complete answers to these questions are the long term goals of our research. The following are our more immediate aims. First, the initial diversity of fibers in the rat hindlimb will be studied to determine (a) how early diversity can be detected; (b) if the initiation of diversity is dependent on innervation; (c) if the sequence of myosins synthesized during development varies among fiber types; (d) if distinct fiber types respond differently to neural and hormonal cues. These studies will involve examination of myosin heavy chains and their mRNAs in individual fibers by immunohistochemistry with monoclonal antibodies and in situ hybridization with cDNAs. Muscles of innervated and aneural limbs and of animals with hormonal perturbations will be studied. Second, the synthesis, degradation and subcellular distribution of heavy chain mRNAs will be examined to determine whether changes in heavy chains during development are directly related to changes in the levels of their mRNAs or if post-transcriptional factors are involved in regulation. Isolation and in vitro translation of mRNA and analysis with specific cDNAs will be used. Third, the testosterone control of specific myosin and tropomyosin isoforms in the guinea pig temporalis will be examined to determine if testosterone (a) controls expression at transcription or translation; (b) affects the muscle or the motoneuron; (c) affects all fiber types similarly. Moreover, cDNAs will be used in Southern blots and will be sequenced to determine if the same myosin gene which is under androgen control in one muscle can be constitutively expressed in another. How these factors controlling diversity may go awry and lead to abnormal fiber types in human neuromuscular diseases will be the ultimate goal of this work.
