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The control and effects of Foxo1 in skeletal muscle.

机译：Foxo1在骨骼肌中的控制和作用。

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摘要

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.
机译：在骨骼肌中，转录因子Foxo1和Foxo3A控制介导肌肉萎缩的蛋白质的表达，在肌肉消瘦的情况下，对Foxo1和Foxo3A的核浓度和核/胞质运动具有治疗意义。在这里，我们使用在培养的成年小鼠骨骼肌纤维中腺病毒表达的Foxo-GFP融合蛋白来表征Foxo1-GFP响应于IGF-1 / PI3K / Akt途径激活的核外排的时间过程，以确定时间过程抑制Foxo1-GFP在此途径中的核内流，并探讨Foxo1对肌纤维收缩的影响。通过免疫细胞化学测定，内源性Foxo1在肌纤维中的定位与Foxo1-GFP的定位一致。 Leptomycin B（LMB）对核输出载体CRM1的抑制将Foxo1捕获在细胞核中，并导致Foxo1的核积累相对较快，这与在应用LMB之前在控制条件下Foxo1的核/细胞质穿梭率较高有关，单向流入和流出的平衡接近。表达的Foxo3A-GFP的穿梭速度比Foxo1-GFP慢20倍。与仅表达GFP的纤维相比，表达Foxo1-GFP的纤维表现出收缩能力，动作电位的异常传播以及对电刺激的钙瞬变消融。使用膜荧光染料对T管系统进行评估，该膜系统参与了动作电位的径向和纵向传播，揭示了在过表达Foxo1-GFP的纤维中完整的T管网络。有趣的是，在Foxo1-GFP纤维中进行长期的IGF-1处理可恢复正常钙瞬变，表明Foxo1易位会影响涉及动作电位产生和/或传播的蛋白质的表达。在不存在IGF-1的情况下，过表达Foxo1的纤维中Nav1.4的表达也下降。我们得出的结论是，Foxo1的过度活跃通过降低Nav1.4的表达和其他可能的方式阻止了正常的肌肉对电刺激的反应。我们的方法允许在各种实验条件下对活肌纤维中的Foxo1和Foxo3A核质运动进行定量动力学表征，以及Foxo1对肌肉电生理的影响。

著录项

  • 作者

    Schachter, Tova Neustadt.;

  • 作者单位

    University of Maryland, Baltimore.;

  • 授予单位 University of Maryland, Baltimore.;
  • 学科 Chemistry Biochemistry.;Biology Physiology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 103 p.
  • 总页数 103
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 地球物理学;
  • 关键词

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