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.
Nature Precedings : hdl:10101/npre.2008.1718.1 : Posted 25 Mar 2008
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.
2
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
Nature Precedings : hdl:10101/npre.2008.1718.1 : Posted 25 Mar 2008
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 coimmunolabeling 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.
3
Igor Buchwalow and Werner Boecker are at the Gerhard Domagk Institute of Pathology,
University of Muenster, 48149 Muenster, Germany.
e-mail: [email protected])
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).
Nature Precedings : hdl:10101/npre.2008.1718.1 : Posted 25 Mar 2008
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).
Nature Precedings : hdl:10101/npre.2008.1718.1 : Posted 25 Mar 2008