NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS
Naoki Suzuki, … , Yuko Miyagoe-Suzuki, Shin’ichi Takeda
Naoki Suzuki, … , Yuko Miyagoe-Suzuki, Shin’ichi Takeda
Published September 4, 2007
Citation Information: J Clin Invest. 2007;117(9):2468-2476. https://doi.org/10.1172/JCI30654.
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Research Article

NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS

Abstract

Forkhead box O (Foxo) transcription factors induce muscle atrophy by upregulating the muscle-specific E3 ubiquitin ligases MuRF-1 and atrogin-1/MAFbx, but other than Akt, the upstream regulators of Foxos during muscle atrophy are largely unknown. To examine the involvement of the dystrophin glycoprotein complex (DGC) in regulation of Foxo activities and muscle atrophy, we analyzed the expression of DGC members during tail suspension, a model of unloading-induced muscle atrophy. Among several DGC members, only neuronal NOS (nNOS) quickly dislocated from the sarcolemma to the cytoplasm during tail suspension. Electron paramagnetic resonance spectrometry revealed production of NO in atrophying muscle. nNOS-null mice showed much milder muscle atrophy after tail suspension than did wild-type mice. Importantly, nuclear accumulation of dephosphorylated Foxo3a was not evident in nNOS-null muscle, and neither MuRF-1 nor atrogin-1/MAFbx were upregulated during tail suspension. Furthermore, an nNOS-specific inhibitor, 7-nitroindazole, significantly prevented suspension-induced muscle atrophy. The NF-κB pathway was activated in both wild-type and nNOS-null muscle during tail suspension. We also show that nNOS was involved in the mechanism of denervation-induced atrophy. We conclude that nNOS/NO mediates muscle atrophy via regulation of Foxo transcription factors and is a new therapeutic target for disuse-induced muscle atrophy.

Authors

Naoki Suzuki, Norio Motohashi, Akiyoshi Uezumi, So-ichiro Fukada, Tetsuhiko Yoshimura, Yasuto Itoyama, Masashi Aoki, Yuko Miyagoe-Suzuki, Shin’ichi Takeda

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Figure 1

nNOS disappears from the sarcolemma during tail suspension.

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nNOS disappears from the sarcolemma during tail suspension. (A) Weights ...
(A) Weights of soleus (Sol) and gastrocnemius (Gc) muscles from tail-suspended (sus) wild-type mice are normalized to body weight and are expressed as percentage of ground control (gro) (n = 15/group). *P < 0.05, Student’s t test. (B) Box and whiskers plot of myofiber diameter size of soleus. Boxes represent the middle 50% of the data, lines represent the median, and whiskers represent the range. More than 200 fibers were measured on laminin-α2 chain–stained cross sections. (C) Immunofluorescent staining for nNOS and laminin-α2 chain. Transverse muscle sections from ground control mice, tail-suspended mice, and mice after reloading for 7 days (reload 7d) were stained with anti-nNOS (green) and anti–laminin-α2 chain antibodies (red). Scale bar: 50 μm. (D) Western blot using anti-nNOS antibody on subcellular fractions of muscle extracts. P indicates insoluble pellet after sequential extraction of skeletal muscle homogenates with 100 mM NaCl (S1), 500 mM NaCl (S2), and 0.5% Triton X-100 (S3). GAPDH signals in S1 and Na/K-ATPase signals in P confirmed that our fractionation was correctly done. Fractionation and western blotting were repeated 5 times, and representative data are presented. Note the slight increase of nNOS levels in S1 fraction and loss of nNOS signal in insoluble P fraction during tail suspension. (E) Quantification of nNOS signals in S1 and P fractions of muscle extracts shown in D (n = 5 /group). The signals in S1 and P fractions were normalized to GAPDH or Na/K-ATPase, respectively. Mann-Whitney, *P < 0.05. (F) Levels of nNOS mRNA in muscles from ground control mice and tail-suspended mice for 2 weeks were evaluated by real-time PCR (n = 5/group). No significant difference was found by Mann-Whitney test. (G) Immunoprecipitation with caveolin-3 antibody and immunoblot with nNOS antibody, and vice versa, for ground control and tail suspension groups. (H) Immunoprecipitation with α1-syntrophin Ab and immunoblot with nNOS antibody, and vice versa. (I) Immunoprecipitation of dysferlin antibody and immunoblot with nNOS Ab, and vice versa. In G–I, flow-through fraction was also examined by western blotting with anti–α-tubulin antibody.