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.
Naoki Suzuki, Norio Motohashi, Akiyoshi Uezumi, So-ichiro Fukada, Tetsuhiko Yoshimura, Yasuto Itoyama, Masashi Aoki, Yuko Miyagoe-Suzuki, Shin’ichi Takeda
Submitter: Shin’ichi Takeda | takeda@ncnp.go.jp
National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
Published December 13, 2007
Thank you for your valuable comments.
We described in our article that appeared in J. Clin. Invest. (1) that nNOS is localized to the sarcolemma in normal conditions. It is widely accepted that in skeletal muscle, nNOS localizes immediately under the plasma membrane of the muscle fiber via association with the dystrophin-glycoprotein complex. As you pointed out, our interpretation of the immunohistochemstry (IHC) of nNOS in the paper is based on not only our IHC data but also on previous reports by ourselves and other researchers.
Most importantly, we have directly demonstrated that a considerable amount of nNOS protein is anchored to the sarcolemma through interaction with alpha1-syntrophin, a member of the dystrophin-glycoprotein complex, based on the experiments with alpha1-syntrophin–null mice. In the absence of alpha1-syntrophin, the sarcolemmal signal of nNOS is completely lost. Our cell fractionation and IHC data in J. Biol. Chem. (2) and J. Clin. Invest. papers are consistent with the model.
We, however, noticed that not all nNOS proteins are targeted to the sarcolemma. In our cell fractionation experiments, a considerable amount of nNOS is found in soluble fractions (S1–S3) even in ground controls. As you pointed out, nNOS would also associate with subsarcolemmally clustered mitochondria or caveolae.
One of our points in the J. Clin. Invest. paper is that the distribution of nNOS in the muscle fibers and its association partners quickly change in response to external or internal signals even in the presence of the intact dystrophin complex at the sarcolemma. Therefore, precise localization of nNOS remains to be determined in both physiological and atrophic conditions using several techniques, such as immunohistochemical approaches with high resolution.
Thank you for you consideration and understanding in advance.
References
1. Suzuki,N., Motohashi,N., Uezumi,A., Fukada,S.i., Yoshimura,T., Itoyama,Y., Aoki,M., Miyagoe-Suzuki,Y., and Takeda,S. 2007. NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS. J. Clin. Invest. 117:2468-2476.
2. Kameya S, Miyagoe Y, Nonaka I, Ikemoto T, Endo M, Hanaoka K, Nabeshima Y, Takeda S. (1999) a1-syntrophin gene disruption results in the absence of neuronal-type nitric-oxide synthase at the sarcolemma but does not induce muscle degeneration. J Biol Chem. 274(4): 2193-200.
Submitter: Igor B. Buchwalow | buchwalo@uni-muenster.de
Gerhard Domagk Institute of Pathology, University of Muenster, Domagkstr. 17, Muenster, Germany
Published December 13, 2007
To the Editor:
An interesting and highly informative article by Suzuki et al. (1) is unfortunately not free from serious drawbacks, such as a misapprehension of the resolution limit of fluorescent optics. Throughout their article, the authors write about nNOS targeting to the sarcolemma. However the sarcolemma, which measures only 80 Å wide, is much too small to be seen with the light or fluorescent microscope (whose limit of resolution is 2000 Å). This inability is a source of confusion to beginning students. Moreover, microscopical image of the layer of the immunofluorescent label delineating muscle fibers varies from 0.5 to 1.0 µm, and this is a few orders of magnitude above the real thickness of the sarcolemma. Therefore, the nNOS immunofluorescent staining of this unidentified layer delineating muscle fibers can also account for subsarcolemmally clustered mitochondria or caveolae, as well as for the endomysium, if not for all of them together. Curiously enough, Suzuki et al. (1) themselves provided evidence for nNOS localization in the endomysium but, captured by their philosophy, were unable to realize it. In Figures (1C and 7A) presented in their article, double immunostaining of nNOS and laminin-alpha2 shows striking parallels in the expression pattern of both proteins delineating myofibers, that is, in the endomysium. Actually, localization of nNOS in the endomysium constituted by the extracellular matrix can be well reconciled with reports that extracellular matrix metabolism is modulated through pathways dependent on NOS activity involved in the protection of matrix elements such as collagen from oxidative stress.
In the earlier 90’s of the past century, nNOS immunostaining delineating muscle fibers was indeed originally attributed exclusively to the sarcolemma (2), and the sarcoplasm was reported to be devoid of nNOS. But to the time of those studies, immunohistochemical techniques were not what they are now. Modern advances in immunohistochemistry, such as antigen retrieval and signal amplification, permit nowadays to localize all three NOS isoforms, including nNOS, in sarcoplasmic compartments (3- 5). But to their convenience, Suzuki et al. (1) opportunistically quote only the literature matching their technical level. The authentic scientific knowledge can only come through strict scientific method and not from speculative affirmation of theories. Knowledge of NOS compartmentalization in skeletal muscles is essential for understanding normal muscle functions and may have also implications in the interception of the NO signaling for designing new adjunctive therapies for muscular diseases.
References
1. Suzuki,N., Motohashi,N., Uezumi,A., Fukada,S.i., Yoshimura,T., Itoyama,Y., Aoki,M., Miyagoe-Suzuki,Y., and Takeda,S. 2007. NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS. J. Clin. Invest. 117:2468-2476.
2. Kobzik,L., Reid,M.B., Bredt,D.S., and Stamler,J.S. 1994. Nitric- Oxide in Skeletal-Muscle. Nature 372:546-548.
3. Buchwalow,I.B., Minin,E.A., Samoilova,V.E., Boecker,W., Wellner,M., Schmitz,W., Neumann,J., and Punkt,K. 2005. Compartmentalization of NO signaling cascade in skeletal muscles. Biochemical and Biophysical Research Communications 330:615-621.
4. Punkt,K., Schering,S., Loffler,S., Minin,E.A., Samoilova,V.E., Hasselblatt,M., Paulus,W., Muller-Werdan,U., Demus,U., Koehler,G. et al 2006. Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy. Biochemical and Biophysical Research Communications 348:259- 264.
5. Rothe,F., Langnaese,K., and Wolf,G. 2005. New aspects of the location of neuronal nitric oxide synthase in the skeletal muscle: A light and electron microscopic study. Nitric Oxide 13:21-35.