In skeletal muscles, neuronal NO synthase (nNOS, designated also as NOS1) was reported to be restricted to the sarcolemma. With the advent of modern powerful immunocytochemical techniques this commonly accepted view appears to be a delusion and has to be re-evaluated

Igor Buchwalow

Werner Boecker

English

Nature Precedings

 1
SKELETAL MUSCLES 
Myth about neuronal nitric oxide synthase in the 
sarcolemma of skeletal muscles 
 
In skeletal muscles neuronal NO synthase (nNOS, designated also as NOS1) was 
reported to be restricted to the sarcolemma.  With the advent of modern powerful 
immunocytochemical techniques this commonly accepted view appears to be a delusion 
and has to be re-evaluated.   
 
In the earlier 90’s of the past century, NOS1 in skeletal muscle was originally reported to be 
localized around the border of some muscle fibers identified as type II (fast) fibers1.  In the 
following years, the circumferential NOS1 immunostaining pattern of muscle fibers, albeit 
without discriminating between fast and slow myofiber types, was reproduced by various 
groups2,3.  Positive NOS1 immunolabeling delineating the myofibers and the absence of 
sarcoplasmic immunostaining were interpreted by the above-quoted authors as proof for 
exclusive NOS1 localization in the sarcolemma.  Limited by the allowed space, we cited only 
a few publications supporting this commonly accepted point of view.  To the time of those 
studies, immunohistochemical techniques were not, however, what they are now.  Modern 
advances in immunohistochemistry, such as antigen retrieval4 and signal amplification5, 
permitted to localize all three NOS isoforms, including NOS1, also in sarcoplasmic 
compartments6,7.  Nevertheless the concept of exclusive sarcolemmal NOS1 localization is 
entertained in all current textbooks and reviews on myology and dystrophinopathies.   
 The main drawback of this concept is the fact that nobody has actually demonstrated 
the NOS immunolabeling of the sarcolemma.  Misleading statements of the above-quoted 
authors about the exclusive sarcolemmal NOS1 expression came up from misapprehension of 
the resolution limit of the light optic.  The sarcolemma, which measures only 5-8 nm wide, is 
much too small to be seen with the light or fluorescent microscope (whose limit of resolution 
is 0.2 μm).  This inability is a source of confusion to beginning students.  Moreover, the 
microscopical image of the layer of the fluorophore or chromogen deposits delineating muscle 
fibers after immunostaining varies from 0.5 to 2.0 μm, and this is a few orders of magnitude 
above the real thickness of the sarcolemma.  Therefore, the NOS1 immunostaining of this 
unidentified layer delineating muscle fibers can account for subsarcolemmally located 
mitochondria and caveolae as well as for the endomysium, if not for all of them together. 
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 Recently, our attention was attracted by an article of Suzuki et al8.  Double 
immunostaining of NOS1 and laminin performed by these authors showed striking parallels in 
the expression pattern of both proteins around myofibers.  This was regarded by the authors as 
proof for NOS1 targeting to the sarcolemma.  In fact, however, they provided evidence for 
NOS1 localization in the endomysium but, captured by the commonly accepted philosophy, 
they were unable to realize it.  The endomysium, unlike the sarcolemma, is a visibly (under 
light microscope) distinct structure, and the glycoprotein laminin is one of the constituents of 
the endomysium.  The endomysium, a layer of connective tissue that ensheaths a muscle fiber, 
is composed mostly from extracellular matrix produced by fibroblasts sparsely dispersed 
along the myofibers.  Along with laminin, the endomysium contains other extracellular matrix 
proteins like collagens.  This prompted us to reproduce the experiment of Suzuki et al8 with 
NOS1immunostaining of skeletal muscles using for co-immunolabeling another constituent of 
the endomysium - collagen IV.  As anticipated, the immunofluorescent labeling of NOS1 
revealed positive immunoreactivity in the endomysium visualized through co-
immunolabeling of collagen IV (Fig. 1).  In contrast to Suzuki et al8 and other groups who 
used standard indirect immunostaining technique, we applied in our experiment heat-induced 
antigen retrieval in conjunction with tyramide signal amplification as described elsewhere9.  
This permitted us to detect NOS1 expression also in the sarcoplasma of some myofibers 
apparently belonging to type II (fast) fibers10.  Conflicting conclusions reached by other 
groups about the absence of NOS1 in the sarcoplasma of myofibers are due to a low detection 
level in their experimental approaches, whereas misleading statements about the NOS1 
expression in the sarcolemma came up from misapprehension of the resolution limit of the 
light optic.   
 
Figure 1 ׀ Immunofluorescent co-labeling of NOS1 and collagen IV in skeletal muscles.  
(a) NOS1 immunolabeling reveals a pronounced circumferential immunostaining pattern of 
muscle fibers.  (b) As a marker of the endomysium, collagen IV immunostaining delineates 
myofibers in the same way as NOS1.  (c) Composite image resulting from merging three 
color components – blue for nuclear DAPI counterstaining, green for FITC-tyramide 
immunostaining of NOS1 and red for Cy3 immunostaining of collagen IV.  Antigen retrieval 
on paraffin sections, double immunolabeling and tyramide signal amplification for NOS1 
visualization were performed according to protocols described in Ref. 9.  50 µm scale bar for 
entire layout.    
 
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Igor Buchwalow and Werner Boecker are at the Gerhard Domagk Institute of Pathology, 
University of Muenster, 48149 Muenster, Germany.  
e-mail: buchwalo@uni-muenster.de)   
 
 1.  Kobzik, L., Reid, M. B., Bredt, D. S. & Stamler, J. S. Nature 372, 546-548 (1994). 
 2.  Brenman, J. E., Chao, D. S., Xia, H., Aldape, K. & Bredt, D. S. Cell 82, 743-752 
(1995). 
 3.  Crosbie, R. H. Nature Med. 7, 27-29 (2001). 
 4.  Shi, S. R., Cote, R. J. & Taylor, C. R. J. Histochem. Cytochem. 49, 931-938 (2001). 
 5.  Hasui, K. & Murata, F. Archives of Histology and Cytology 68, 1-17 (2005). 
 6.  Punkt, K. et al. Biochem. Biophys. Res. Comm. 348, 259-264 (2006). 
 7.  Rothe, F., Langnaese, K. & Wolf, G. Nitric Oxide 13, 21-35 (2005). 
 8.  Suzuki, N. et al. J. Clin. Invest. 117, 2468-2476 (2007). 
 9.  Buchwalow, I. B. et al. Biochem. Biophys. Res. Comm. 330, 615-621 (2005). 
 10.  Punkt, K. et al. Histochem. Cell Biol 125, 567-573 (2006). 
 
 
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Myth about neuronal nitric oxide synthase in the sarcolemma of skeletal muscles

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Myth about neuronal nitric oxide synthase in the sarcolemma of skeletal muscles

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