• The control and effects of Foxo1 in skeletal muscle

      Schachter, Tova Neustadt; Schneider, Martin F. (2012)
      In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins which mediate muscle atrophy, making the nuclear concentration and nuclear/cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the IGF-1/PI3K/Akt pathway, to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway, and to explore the effects of Foxo1 on contraction of muscle fibers. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier CRM1 by Leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear/cytoplasmic shuttling of Foxo1 under control conditions prior to LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles about 20 fold more slowly than Foxo1-GFP. Fibers expressing Foxo1-GFP exhibit an inability to contract, abnormal propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared to fibers expressing GFP alone. Evaluation of the T-tubule system, the membranous system involved in the radial and longitudinal propagation of the action potential, using a membrane fluorescent dye, revealed an intact T-tubule network in fibers over-expressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment in Foxo1-GFP fibers induced recovery of normal calcium transients, indicating that Foxo1 translocation affects the expression of proteins involved in the generation and/or propagation of action potentials. A decrease in Nav1.4 expression in fibers overexpressing Foxo1 was also observed in the absence of IGF-1. We conclude that overactivity of Foxo1 prevents the normal muscle responses to electrical stimulation by decreasing expression of Nav1.4 and possibly other means. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions, as well as the effects of Foxo1 on the electrophysiology of muscle.