The Stress Protein/Chaperone Grp94 Counteracts Muscle Disuse Atrophy by Stabilizing Subsarcolemmal Neuronal Nitric Oxide Synthase

Aims: Redox and growth-factor imbalance fosters muscle disuse atrophy. Since the endoplasmic-reticulum chaperone Grp94 is required for folding insulin-like growth factors (IGFs) and for antioxidant cytoprotection, we investigated its involvement in muscle mass loss due to inactivity. Results: Rat soleus muscles were transfected in vivo and analyzed after 7 days of hindlimb unloading, an experimental model of muscle disuse atrophy, or standard caging. Increased muscle protein carbonylation and decreased Grp94 protein levels (p<0.05) characterized atrophic unloaded solei. Recombinant Grp94 expression significantly reduced atrophy of transfected myofibers, compared with untransfected and empty-vector transfected ones (p<0.01), and decreased the percentage of carbonylated myofibers (p=0.001). Conversely, expression of two different N-terminal deleted Grp94 species did not attenuate myofiber atrophy. No change in myofiber trophism was detected in transfected ambulatory solei. The absence of effects on atrophic untransfected myofibers excluded a major role for IGFs folded by recombinant Grp94. Immunoprecipitation and confocal microscopy assays to investigate chaperone interaction with muscle atrophy regulators identified 160kDa neuronal nitric oxide synthase (nNOS) as a new Grp94 partner. Unloading was demonstrated to untether nNOS from myofiber subsarcolemma; here, we show that such nNOS localization, revealed by means of NADPH-diaphorase histochemistry, appeared preserved in unloaded myofibers expressing recombinant Grp94, compared to those transfected with the empty vector or deleted Grp94 cDNA (p<0.02). Innovation: Grp94 interacts with nNOS and prevents its untethering from sarcolemma in unloaded myofibers. Conclusion: Maintenance of Grp94 expression is sufficient to counter unloading atrophy and oxidative stress by mechanistically stabilizing nNOS-multiprotein complex at the myofiber sarcolemma. Antioxid. Redox Signal. 20, 2479-2496.