NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS
Naoki Suzuki, Norio Motohashi, Akiyoshi Uezumi, So-ichiro Fukada, Tetsuhiko Yoshimura, Yasuto Itoyama, Masashi Aoki, Yuko Miyagoe-Suzuki, Shin’ichi Takeda
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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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 4

Participation of nNOS in regulation of Foxo3a and upregulation of MuRF-1 and atrogin-1/MAFbx.

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Participation of nNOS in regulation of Foxo3a and upregulation of MuRF-1...
(A) The amounts of total Foxo3a, phosphorylated Foxo3a (p-Foxo3a), and intranuclear Foxo3a in wild-type and nNOS-null muscle during tail suspension were quantified (n = 5). Note that Foxo3a was dephosphorylated and accumulated in the myonuclei of wild-type mice but not of nNOS-null mice. *P < 0.05, Mann-Whitney. (B) Representative immunoblot analysis for Foxo3a, phosphorylated Foxo3a, Foxo1, and Foxo4 in total muscle extract, and Foxo3a in nuclear extracts in wild-type ground control (lane 1), wild-type tail-suspended (lane 2), nNOS-null ground control (lane 3), and nNOS-null tail-suspended (lane 4) muscles. α-Tubulin was used as a loading control. (C) mRNA levels of ubiquitin ligases (MuRF-1 and atrogin-1/MAFbx) (n = 5) were quantified by real-time RT-PCR. *P < 0.05, Mann-Whitney. (D) EMSA of NF-κB. Biotin-labeled double-stranded oligonucleotides containing NF-κB binding sites were incubated with nuclear extracts prepared from ground control and suspended muscles. An arrow indicates the DNA-protein complex. LPS was injected intraperitoneally into mice, and the muscle was used as a positive control for NF-κB binding activity. 5× cold, 5-fold excess of nonlabeled competitors.