The level of expression of a wide range of muscle fiber type-specific genes in any given skeletal muscle fiber is modulated by the frequency and pattern of activity imposed on that fiber. Fiber protein phenotype is thus molded by the cumulative activity the fiber has undergone over the previous weeks or months. The underlying mRNA levels for the corresponding genes are altered on the shorter time scale of days or even hours. The overall goal of this project is to gain further insight into the molecular signaling mechanisms underlying activity dependent activation of slow fiber type-specific gene expression at the transcriptional level in adult fast skeletal muscle fibers using our novel, recently developed system in which calcium, NFATc and other downstream signaling pathways involved in regulating fiber type-specific gene expression may be monitored and manipulated in living, fully differentiated adult skeletal muscle fibers maintained and electrically stimulated in culture.
Our aims will be: (1) To determine the mechanism(s) underlying the marked stimulus pattern dependence of the translocation of the transcription factor NFATc into the nucleus after cytosolic dephosphorylation by the Ca 2+ dependent phosphatase calcineurin, and into nuclear foci in isolated adult skeletal muscle fibers. (2) To formulate and use a mathematical computer simulation model for the activity pattern dependent intracellular movement and activity of NFATc, as well as for other possible related signaling pathways, including MEF2, for regulation of slow fiber type gene transcription. (3) To use experiments and computer simulation to characterize the effects of various molecular steps in the NFATc signaling pathway on stimulus pattern dependent NFATc translocation. (4) To characterize the activity pattern dependence and signaling pathways regulating the nuclear to cytoplasmic translocation of class 2 histone deacetylases (HDACs), which bind to and repress the activity of intranuclear MEF2, a transcription factor which activates slow fiber type-specific genes and which may be de-repressed by HDAC movement out of the nucleus. (5) To use a custom """"""""mini"""""""" microarray to determine the roles of stimulation pattern, calcineurin, NFATc, MEF2 and other signals that may be necessary or sufficient for the activation of slow fiber type-specific gene expression. The studies proposed here should have important bearing on the effects of use and disuse on muscle fiber gene expression. ? ?
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