Several chronic diseases and conditions associated with prolonged muscle inactivity can induce muscle atrophy. Regardless the cause, skeletal muscle atrophy results in muscular weakness and a decreased quality of life. Mechanical unloading that occurs with limb casting, splinting, bed rest, and spaceflight result in a substantial loss of force generating capacity, linked to a decrease in muscle fiber cross-sectional area. Recent experiments demonstrated that disassembly of skeletal muscle in response to mechanical unloading is accelerated by translocation of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the sarcoplasm. Elevated oxidative stress from mitochondria and possibly Nox2 may result in a loss or translocation of nNOS from the sarcolemma. However, the "escalator system" that detaches and transports nNOSµ is unknown. Therefore, the aim of this study is to identify the influence of reactive oxygen species on regulation of membrane proteins (dysferlin, annexin-2, annexin-6, and MG53) and the nNOSµ translocation in unloaded skeletal muscle. Fischer-344 rats will be used to model disuse via hindlimb unloading. Briefly, the hindlimbs of the hindlimb unloading (HU) group will be elevated to a spinal orientation of 55° above horizontal. Peptide Nox2 inhibition will be accomplished via gp91ds-tat (3 ¼g/ml) with a scrambled sequence used as a control. The following groups of 12 week old will be tested: (n=10/group): (1) ambulatory controls with saline injections (CON), (2) ambulatory rats + gp91ds-tat (CG), (3) hindlimb unloading + saline injections (HUS), (4) hindlimb unloading + gp91ds-tat (HUN). Following 7 days of unloading, soleus muscle will be snap frozen. Markers of oxidative stress, Nox2, nNOS translocation, FoxO3a phosphorylation [Thr-32], membrane repair proteins, and morphology (fiber cross sectional area, fiber-type) will be assessed using immunofluorescence, coimmunoprecipitation, hematoxylin and eosin stains, and Western blot analysis. One way ANOVA with Fisher's LSD used post-hoc will be used to distinguish among group means. The threshold for statistical significance will be set at ± = 0.05.
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