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Nitric oxide synthase I (NOS-I) is deficient in the sarcolemma of striated muscle fibers in patients with Duchenne muscular dystrophy, suggesting an association with dystrophin
Acta histochem. (lena) 98, 61-69 (1996) Gustav Fischer Verlag lena' Stuttgart· New York
A~'a 'is'o~k.ita
Nitric oxide synthase I (NOS-I) is deficient in the sarcolemma of striated muscle fibers in patients with Duchenne muscular dystrophy, suggesting an association with dystrophin Zarko Grozdanovic', Georg Gosztonyf and Reinhart Gossrau' I Department of Anatomy, Konigin-Luise-Strafle 15, D-14195 Berlin and 2Department of Neuropathology, Hindenburgdamm 30, D-12200 Berlin, Benjamin Franklin University Clinic, Free University of Berlin, Germany
Accepted 14 September 1995
Summary Previously, we have demonstrated the expression of the brain-type nitric oxide synthase (NOS-I) in the sarcolemmal region of somatic and visceral striated muscle fibers in a variety of mammalian species through the use of enzyme histochemical and immunochemical techniques. Here we report that NOS-I protein and its NADPH diaphorase (NADPHd) activity are co-localized in the sarcolemma of human skeletal muscles. NOS-I immunolabeling and NADPHd activity showed no significant variation between type I and II fibers. In muscle biopsy specimens from patients with Duchenne muscular dystrophy (DMD), both NOS-I protein and activity were absent or markedly reduced. We, therefore, propose that NOS-I is complexed with dystrophin and/or dystrophin-associated proteins, adding a novel member to the sarcolemmal dystrophinglycoprotein complex (DGC). The nature of the NOS-I-DGC link, and its role in skeletal muscle physiology and pathophysiology remain to be elucidated.
Key words: nitric oxide synthase I - NADPH diaphorase sarcolemma - dystrophin - Duchenne muscular dystrophy
skeletal muscle -
Introduction Recent evidence indicates that non-cardiac striated muscles, both somatic and visceral, constitute the richest source of nitric oxide synthase (NOS), the enzyme which promotes the formation of nitric oxide (NO) from L-arginine, in the mammalian body. Nakane et al. (1993, 1994)noted the expression of the human brain NOS (NOS-I) in human skeletal muscle. Balon and Nadler (1994) documented the release of NO from rat isolated muscle preparations at rest. Using light microscopy, Kobzik et al. (1994) discerned NOS-I immunoreactivity in type II fibers of some rat skeletal muscles. We demonstrated immunoreactive NOS-I protein and activity, i. e., NADPH diaphorase (NADPHd) staining, Correspondence to: R. Gossrau
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in type I and II fibers of all somatic and visceral striated muscles from a variety of mammalian species (mouse, rat, gerbil, hamster, guinea-pig, and marmoset; Nakos and Gossrau, 1994; Grozdanovic et al., 1995a). Antibodies directed against NOS-I produced strong sarcolemmal staining of extrafusal muscle fibers; the sarcoplasm was not stained (Kobzik et al., 1994; Grozdanovic et al., 1995a). In addition, NOS-I immunostain and NADPHd activity were co-localized in the surface membrane of intrafusal fibers (Grozdanovic et al., 1995a). In the rat tongue, electron microscopy (Langer et al., 1995) revealed NOS-I immunoreactivity in the longitudinal sarcolemma of extrafusal visceral (see Grozdanovic et al., 1995a) muscle fibers. Most recently, Kobzik et al. (1995) reported anti-endothelial NOS (NOS-III) labeling in the sarcoplasm of rat skeletal muscles, which was not related to a special fiber type but to the activity of mitochondrial succinate dehydrogenase. Physiological data suggest that NO may be involved in the regulation of glucose transport (Balon and Nadler, 1994), force development (Kobzik et al., 1994), and oxidative metabolism (Kobzik et al., 1995). However, both the ultrastructural localization of NOS-III and the precise function(s) of NO remain to be determined. The present study was performed to examine the expression of NOS-I in normal human and Duchenne muscular dystrophy (DMD) muscle using enzyme histochemical and immunochemical techniques. To our best knowledge, we report for the first time that NOS-I protein and activity are present in the sarcolemmal region of normal fibers and that they are both absent or significantly reduced in muscle fibers from DMD patients.
Material and Methods Human muscle specimens. Skeletal muscle biopsy specimens from four DMD patients (6 to 12 years old) were analysed. As normal controls, diagnostic biopsy specimens (four) and samples of surgical material (four; muscles: sternocleidomastoideus, omohyoideus, quadriceps, and gastrocnemius) from patients who had no pathological and/or clinical evidence of neuromuscular disorders were investigated. The biopsies were obtained from the Department of Neuropathology (Head: Prof. Dr. Dr. h. c. mult. J. CervosNavarro) and the surgical samples were obtained from the Department of Otorhinolaryngology (Head: Prof. Dr. H. Scherer) and the Department of Orthopedics (Head: Prof. Dr. U. Weber), Benjamin Franklin University Clinic, Free University of Berlin. Muscle specimens were snap frozen in liquid nitrogen-cooled isopentane and cut in a cryostat at a thickness of 10 urn. The sections were thawed onto poly-lysine-coated glass slides and either fixed in 4010 formaldehyde (30 min at 4°C) or used unfixed. Fixed sections were rinsed in tap water (10 min), then in distilled water (5 min), and air-dried. Enzyme histochemistry. The NADPHd reaction for NOS (EC 1.14.13.39) was performed either according to Scherer-Singler et al. (1983) in the presence or absence of Triton X-IOO and 0.5 mM potassium permanganate to block NOS-unrelated NADPHds (Grozdanovic et al., 1995b), respectively, or according to Nakos and Gossrau (1994) in the presence of 0.5 -1010 formaldehyde in the incubation medium. For muscle fiber typing, myosine adenosine triphosphatase (myosine ATPase; EC 3.6.1.32) was demonstrated by the calcium method, and succinate dehydrogenase (SDH; 1.3.99.1), cytochrome C oxidase (CO; EC 1.9.3.1.) in the absence of HzOz as well as NADH diaphorase (NADHd) with the procedures described by Lojda et al. (1979). The sections were incubated for 30-60 min at 37°C; afterwards, the media were poured off, and the sections were rinsed in tap water, then in distilled water and, finally, mounted in glycerol jelly. Controls were performed with media free of substrate. Immunohistochemistry. Fixed sections were rinsed in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) containing 0.3010 (v/v) Triton X-l00 (15 min), pre-incubated with 2010 normal horse serum (30 min), washed in PBS-Triton (15 min), and incubated with a polyclonal NOS-I antiserum (at a dilution of 1: 1000) for 20 h at room temperature raised against NOS purified from pig brain (Mayer et al., 1990). The sections were washed in PBS-Triton (2x 15 min), incubated with a Cy3-conjugated goat anti-rabbit IgG (Jackson, West Grove, PA, USA) (diluted 1: 100,1 h), washed in PBS-ltiton (2x 15 min), and embedded in glycerol: PBS (1 : 1). Control incubations were performed using non-relevant IgG or PBS instead of primary antiserum.
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Results
Normal human muscles Non-NADPHd enzymes. In all muscles (either biopsy specimens or surgical material) investigated, myosin ATPase, SDH, CO, and NADHd were more active in type I than type II fibers.
Figs. I, 2. In a normal muscle biopsy, NADPH diaphorase activity is exclusivelypresent in the sarcolemmal region (arrows) of type 1 (I) and II (II) fibers. Fig. 1. x250, Fig. 2 x 400.
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NADPHd. NADPHd was present with equal activity in the sarcolemmal region of both type I and II fibers (Figs. 1,2). In addition to sarcolemmal staining, formazan production was rarely observed inside the fibers, especially type I fibers. When the reaction was carried out in the presence of formaldehyde or permanganate, which have turned out to be rather selective inhibitors of non-NOS-associated NADPHds (Nakos and Gossrau, 1994; Grozdanovic et al., 1995b), the reaction product was almost exclusively seen in the fiber surface, leaving the fiber inside almost unstained. By contrast, when Triton X-100 was omitted from the incubation medium, and the reaction was performed on formaldehyde-fixed cryosections, the sarcolemmal staining was markedly reduced, while increased quantities of formazan were detected inside the muscle fibers. NOS immunohistochemistry. NOS-I antiserum gave a consistent, intense, and continuous immunofluorescence staining of the sarcolemmal region, with no obvious differences between type I and II fibers (Fig. 3). No staining of intracellular structures was observed. Muscles from DMD patients Non-NADPHd enzymes. Abnormalities in staining for myosin ATPase, SDH, CO, and NADHd activities were in agreement with the results previously reported in the literature (Goebel, 1989). NADPHd. When the standard staining procedure was employed, i. e., with Triton X-toO but without formaldehyde or permanganate in the incubation medium, the amount of formazan generated in the sarcolemmal area was dramatically reduced or totally diminished (Figs. 4, 5). In muscle fibers which exhibited weak residual activity, discontinuities of sarcolemmal staining were common. Furthermore, small quantities of formazan were occasionally encountered inside some fibers; addition of either formaldehyde or permanganate to the incubation medium, which was applied to fresh cryosections, resulted in reduced (but not deficient) intracellular staining.
Fig. 3. NOS immunofluorescence is seen in the sarcolemmal region (arrows) of type 1 (I) and II (II) fibers. x 400.
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Figs. 4, 5. In a biopsy from a patient with Duchenne muscular dystrophy NADPH diaphorase activity is absent in the region of the sarcolemma (arrows); inside rare muscle fibers NADPH diaphorase is still active (+). Fig. 4 x 250, Fig. 5 x 400.
NOS immunohistochemistry. NOS-I immunostain was absent or drastically deficient in the sarcolemma of most DMD muscle fibers (Fig. 6). Some scattered fibers exhibited focal defects in sarcolemmal staining, which was less intense than in normal fibers.
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Fig. 6. In a muscle biopsy from a patient with Duchenne muscular dystrophy, with the exception of some focal NOS immunoreactivity (small arrows), the enzyme is absent from the sarcolemmal region (arrows); thin arrows = unspecific fluorescence. x 400.
Discussion We have demonstrated that NOS-I and its reductase, i. e., NADPHd, activity are coexpressed in the sarcolemma of normal human skeletal muscles. A strong correlation between NOS-I immunostain and NADPHd activity was obtained, when the incubation medium was supplemented with the fixation aldehyde formaldehyde or the oxidizer permanganate. This is consistent with the previous results from animal studies (Nakos and Gossrau, 1994; Grozdanovic et al., 1995 a, b), providing further support for the concept that these reagents can be used to differentiate between NOS- and non-NOS-associated NADPHds (Blottner et al., 1995). The residual intracellular staining, which persists after formaldehyde or permanganate treatment, can tentatively be attributed to the presence of NOS isozymes other than NOS-I in the myofiber sarcoplasm. Indeed, NOS-II (inducible NOS) and NOS-III (endothelial NOS) have been localized to the sarcoplasm of animal skeletal muscle fibers (Williams et al., 1994; Kobzik et al., 1995). Examination of adjacent sections reacted for myosin ATPase, SDH, CO, and NADHd activities showed that these enzymes were predominantly expressed in type I fibers (Meijer, 1991), while no significant differences were observed for sarcolemmal NOS-I staining between type I and II fibers. This is in marked contrast to laboratory animals and marmoset monkeys, where NOS-I immunostain and NADPHd activity are concentrated in the sarcolemma of type II fibers (Kobzik et aI., 1994; Grozdanovic et aI., 1995 a). In DMD muscle, both NOS-I protein and its activity are absent or dramatically reduced in the sarcolemma. DMD is caused by a deficiency of dystrophin, a rod-shaped cytoskeletal protein located on the cytoplasmic face of the sarcolemma (Hoffman et al. , 1987. 1988; Arahata et al., 1988; Bonilla et al., 1988; Koenig et al., 1988; Watkins et al.,
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1988; Zubrzycka-Gaarn et aI., 1988; Cullen et aI., 1990). Biochemical evidence indicates that dystrophin is anchored to an oligomeric transmembrane-spanning glycoprotein complex, which interacts with the extracellular matrix (Campbell and Kahl, 1989; Ervasti et aI., 1990; Yoshida and Ozawa, 1990; Ervasti and Campbell, 1991; Ervasti et aI., 1991; Ibraghimov-Beskrovnaya et aI., 1992). In DMD muscle, the deficiency of dystrophin in the sarcolemma (Arahata et aI., 1988; Bonilla et aI., 1988; Zubrzycka-Gaarn et aI., 1988) is paralleled by the loss of all the dystrophin-associated proteins (Ervasti et aI., 1990; Ibraghimov-Beskrovnaya et aI., 1992; Ohlendieck et aI., 1993), supporting the hypothesis that the disintegration of the dystrophin-glycoprotein complex (DGC) leads to the disruption of the linkage between the membrane cytoskeleton and the extracellular ma trix and, consequently, muscle fiber necrosis (Ervasti and Campbell, 1993; Matsumura and Campbell, 1993, 1994). The observation that NOS-I is absent or markedly deficient in the DMD muscle sarcolemma points to the existence of a structural link between NOS-I and dystrophin and /or dystrophin-associated proteins. The available evidence indicates that the loss of dystrophin-associated proteins is a characteristic pathogenetic event occurring specifically in dystrophin-deficient DMD muscle (Ervasti and Campbell, 1993; Matsumura and Campbell, 1993, 1994; Ohlendieck et aI., 1993). Along the same line of reasoning, the absence of NOS-I in the sarcolemma of DMD muscle fibers is unlikely to be due to nonspecific membrane alterations secondary to muscle fiber degeneration. Currently, we are trying to throw more light on this problem by investigating the status of NOS-I in patients with Becker muscular dystrophy, the milder allelic form of DMD, and other neuromuscular disorders. Furthermore, it would be intere sting to elucidate the structural interrelationships between NOS-I and DGC. Immunoelectron micro scopic data (Langer et aI., 1995) suggest that NOS-I is presumably an integral sarcolemmal protein. In conclusion, to our best knowledge, our study is the first to show that NOS-I is a constant component of the sarcolemma of normal human skeletal muscles, which is absent in skeletal muscles from all DMD patients investigated. Further studies are needed to determine whether the loss of NOS-I is a direct consequence of the deficiency of dystrophin and, if so, whether this has implications for the pathogenesis of muscle fiber necro sis in DMD.
Note added in proof: After submission of this paper an article by Brenman et ai. (Cell 82, 743 -752, 1995) has been published also showing the association of NOS I with dystrophin in human skeletal muscle.
Acknowledgement We are thankful to Ms Heidrun Richter for technical assistance and Ms Ursula Sauerbier for photographic work.
References Arahata K, Ishiura S, Ishiguro T, Tsukahara T, Suhara Y, Eguchi C, Ishihara T, Nonaka I, Ozawa E, and Sugita H (1988) Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystroph y peptide. Nature 333: 861 - 863 Balon TW, and Nadler JL (1994) Nitric oxide release is present from incubated skeletal muscle prepara tions. J Appl Physiol 77: 2519-2521 Blottner D, Grozdanovic Z, and Gossrau R (1995) Histochemistry of nitr ic oxide synthase in the nervous system. Histochem J 27: 785 - 811
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Bonilla E, Samitt CE, Miranda AF, Hays AP, Salviati G, DiMauro S, Kunkel LM, Hoffman EP, and Rowland LP (1988) Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface. Cell 54: 447 -452 Campbell KP, and Kahl SD (1989) Association of dystrophin and an integral membrane glycoprotein. Nature 345: 259 - 262 Cullen MJ, Walsh J, Nicholson LVB, and Harris JB (1990) Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling. Proc R Soc Lond (Bioi) 240: 197-210 Ervasti JM, and Campbell KP (1991) Membrane organization of the dystrophin-glycoprotein complex. Cell 66: 1121-1131 Ervasti JM, and Campbell KP (1993) Dystrophin-associated glycoproteins: their possible roles in the pathogenesis of Duchenne muscular dystrophy. In: Partridge T (Ed) Molecular and cell biology of muscular dystrophy. Chapman and Hall, London, pp 139- 166 Ervasti JM, Ohlendieck K, Kahl SD, Gaver MG, and Campbell KP (1990) Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 345: 315 - 319 Ervasti JM, Kahl SD, and Campbell KP (1991) Purification of dystrophin from skeletal muscle. J Bioi Chern 266: 9161-9165 Goebel HH (1989) Muskelpathologie. In: Cervos-Navarro J, Ferszt R (Eds) Klinische Neuropathologie. Georg Thieme, Stuttgart, New York, pp 457 -486 Grozdanovic Z, Nakos G, Dahrmann G, Mayer B, and Gossrau R (1995a) Species-independent expression of nitric oxide synthase in the sarcolemma region of visceral and somatic striated muscle fibers. Cell Tissue Res 281: 493 -499 Grozdanovic Z, Nakos G, Christova T, Nikolova Z, Mayer B, and Gossrau R (1995 b) Demonstration of nitric oxide synthase (NOS) in marmosets by NADPH diaphorase (NADPH-d) histochemistry and NOS immunoreactivity. Acta histochem 97: 321-332 Hoffman EP, Brown Jr RH, and Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51: 919 - 928 Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterston R, Brooke M, Specht L, Kupsky W, Chamberlain J, Caskey CT, Shapiro F, and Kunkel LM (1988) Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med 318: 1363-1368 Ibraghimov-Beskrovnaya 0, Ervasti JM, LeveilleCJ, Slaughter CA, Sernett SW, and Campbell KP (1992) Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355: 696 - 702 Kobzik L, Reid MB, Bredt DS, and Stamler JS (1994) Nitric oxide in skeletal muscle. Nature 372: 546-548 Kobzik L, Stringer B, Balligand J-L, Reid MB, and Stamler JS (1995) Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. Biochem Biophys Res Commun 211: 375-381 Koenig M, Monaco AP, and Kunkel LM (1988) The complete sequence of dystrophin predicts a rodshaped cytoskeletal protein. Cell 53: 219 - 228 Langer J-U, Gossrau R, Mayer B, and Grozdanovic Z (1995) Immunoelectron microscopic localization of nitric oxide synthase in striated muscle fibers. Endothelium 3 (Suppl): S 108 Lojda Z, Gossrau R, and Schiebler TH (1979) Enzyme histochemistry. A laboratory manual. Springer, Berlin Heidelberg New York. Matsumura K, and Campbell KP (1993) Deficiency of dystrophin-associated proteins: a common mechanism leading to muscle cell necrosis in severe childhood muscular dystrophies. Neuromusc Disord 3: 109-118 Matsumura K, and Campbell KP (1994) Dystrophin-glycoprotein complex: its role in the molecular pathogenesis of muscular dystrophies. Muscle Nerve 17: 2 -15 Meijer AEFH (1991) The pentose phosphate pathway in skeletal muscle under pathophysiological conditions. Progr Histochem Cytochem 22: 1 - 118 Nakane M, Schmidt HHHW, Pollock JS, Forstermann U, and Murad F (1993) Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett 316: 175-180 Nakane M (1994) Biochemical and molecular biochemical analysis of brain-type nitric oxide synthase. In: Takagi H, Toda N, Hawkins RD (Eds) Nitric oxide: roles in neuronal communication and neurotoxicity. Taniguchi symposia on brain sciences, no. 17. Japan Scientific Societies Press, Tokyo, pp 19-27
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Nakos G, and Gossrau R (1994) When NADPH diaphorase (NADPHd) works in the presence of formaldehyde, the enzyme appears to visualize selectively cells with constitutive nitric oxide synthase (NOS). Acta histochem 96: 335 - 343 Ohlendieck K, Matsumura K, Ionasescu VV, Towbin JA, Bosch EP, Weinstein SL, Sernett SW, and Campbell KP (1993) Duchenne muscular dystrophy: deficiency of dystrophin-associated proteins in the sarcolemma. Neurology 43: 795 - 800 Scherer-Singler U, Vincent SR, Kimura H, and McGeer EG (1983) Demonstration of a unique population of neurons with NADPH diaphorase histochemistry. J Neurosci Meth 9: 229-234 Watkins SC, Hoffman EP, Slayter HS, and Kunkel LM (1988) Immunoelectron microscopic localization of dystrophin in myofibres. Nature 333: 863 - 866 Williams G, Brown T, Becker L, Prager M, and Giroir BP (1994) Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes. Am J Physiol 267: R 1020- R 1025 Yoshida M, and Ozawa E (1990) Glycoprotein complex anchoring dystrophin to sarcolemma. J Biochem 108: 748 -752 Zubrzycka-Gaarn EE, Bulman DE, Karpati G, Burghes AHM, Belfall B, Klamut HJ, Talbot J, Hodges RS, Ray PN, and Worton RG (1988) The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 333: 466-469
A~'a 'is'o~k.ita
Nitric oxide synthase I (NOS-I) is deficient in the sarcolemma of striated muscle fibers in patients with Duchenne muscular dystrophy, suggesting an association with dystrophin Zarko Grozdanovic', Georg Gosztonyf and Reinhart Gossrau' I Department of Anatomy, Konigin-Luise-Strafle 15, D-14195 Berlin and 2Department of Neuropathology, Hindenburgdamm 30, D-12200 Berlin, Benjamin Franklin University Clinic, Free University of Berlin, Germany
Accepted 14 September 1995
Summary Previously, we have demonstrated the expression of the brain-type nitric oxide synthase (NOS-I) in the sarcolemmal region of somatic and visceral striated muscle fibers in a variety of mammalian species through the use of enzyme histochemical and immunochemical techniques. Here we report that NOS-I protein and its NADPH diaphorase (NADPHd) activity are co-localized in the sarcolemma of human skeletal muscles. NOS-I immunolabeling and NADPHd activity showed no significant variation between type I and II fibers. In muscle biopsy specimens from patients with Duchenne muscular dystrophy (DMD), both NOS-I protein and activity were absent or markedly reduced. We, therefore, propose that NOS-I is complexed with dystrophin and/or dystrophin-associated proteins, adding a novel member to the sarcolemmal dystrophinglycoprotein complex (DGC). The nature of the NOS-I-DGC link, and its role in skeletal muscle physiology and pathophysiology remain to be elucidated.
Key words: nitric oxide synthase I - NADPH diaphorase sarcolemma - dystrophin - Duchenne muscular dystrophy
skeletal muscle -
Introduction Recent evidence indicates that non-cardiac striated muscles, both somatic and visceral, constitute the richest source of nitric oxide synthase (NOS), the enzyme which promotes the formation of nitric oxide (NO) from L-arginine, in the mammalian body. Nakane et al. (1993, 1994)noted the expression of the human brain NOS (NOS-I) in human skeletal muscle. Balon and Nadler (1994) documented the release of NO from rat isolated muscle preparations at rest. Using light microscopy, Kobzik et al. (1994) discerned NOS-I immunoreactivity in type II fibers of some rat skeletal muscles. We demonstrated immunoreactive NOS-I protein and activity, i. e., NADPH diaphorase (NADPHd) staining, Correspondence to: R. Gossrau
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in type I and II fibers of all somatic and visceral striated muscles from a variety of mammalian species (mouse, rat, gerbil, hamster, guinea-pig, and marmoset; Nakos and Gossrau, 1994; Grozdanovic et al., 1995a). Antibodies directed against NOS-I produced strong sarcolemmal staining of extrafusal muscle fibers; the sarcoplasm was not stained (Kobzik et al., 1994; Grozdanovic et al., 1995a). In addition, NOS-I immunostain and NADPHd activity were co-localized in the surface membrane of intrafusal fibers (Grozdanovic et al., 1995a). In the rat tongue, electron microscopy (Langer et al., 1995) revealed NOS-I immunoreactivity in the longitudinal sarcolemma of extrafusal visceral (see Grozdanovic et al., 1995a) muscle fibers. Most recently, Kobzik et al. (1995) reported anti-endothelial NOS (NOS-III) labeling in the sarcoplasm of rat skeletal muscles, which was not related to a special fiber type but to the activity of mitochondrial succinate dehydrogenase. Physiological data suggest that NO may be involved in the regulation of glucose transport (Balon and Nadler, 1994), force development (Kobzik et al., 1994), and oxidative metabolism (Kobzik et al., 1995). However, both the ultrastructural localization of NOS-III and the precise function(s) of NO remain to be determined. The present study was performed to examine the expression of NOS-I in normal human and Duchenne muscular dystrophy (DMD) muscle using enzyme histochemical and immunochemical techniques. To our best knowledge, we report for the first time that NOS-I protein and activity are present in the sarcolemmal region of normal fibers and that they are both absent or significantly reduced in muscle fibers from DMD patients.
Material and Methods Human muscle specimens. Skeletal muscle biopsy specimens from four DMD patients (6 to 12 years old) were analysed. As normal controls, diagnostic biopsy specimens (four) and samples of surgical material (four; muscles: sternocleidomastoideus, omohyoideus, quadriceps, and gastrocnemius) from patients who had no pathological and/or clinical evidence of neuromuscular disorders were investigated. The biopsies were obtained from the Department of Neuropathology (Head: Prof. Dr. Dr. h. c. mult. J. CervosNavarro) and the surgical samples were obtained from the Department of Otorhinolaryngology (Head: Prof. Dr. H. Scherer) and the Department of Orthopedics (Head: Prof. Dr. U. Weber), Benjamin Franklin University Clinic, Free University of Berlin. Muscle specimens were snap frozen in liquid nitrogen-cooled isopentane and cut in a cryostat at a thickness of 10 urn. The sections were thawed onto poly-lysine-coated glass slides and either fixed in 4010 formaldehyde (30 min at 4°C) or used unfixed. Fixed sections were rinsed in tap water (10 min), then in distilled water (5 min), and air-dried. Enzyme histochemistry. The NADPHd reaction for NOS (EC 1.14.13.39) was performed either according to Scherer-Singler et al. (1983) in the presence or absence of Triton X-IOO and 0.5 mM potassium permanganate to block NOS-unrelated NADPHds (Grozdanovic et al., 1995b), respectively, or according to Nakos and Gossrau (1994) in the presence of 0.5 -1010 formaldehyde in the incubation medium. For muscle fiber typing, myosine adenosine triphosphatase (myosine ATPase; EC 3.6.1.32) was demonstrated by the calcium method, and succinate dehydrogenase (SDH; 1.3.99.1), cytochrome C oxidase (CO; EC 1.9.3.1.) in the absence of HzOz as well as NADH diaphorase (NADHd) with the procedures described by Lojda et al. (1979). The sections were incubated for 30-60 min at 37°C; afterwards, the media were poured off, and the sections were rinsed in tap water, then in distilled water and, finally, mounted in glycerol jelly. Controls were performed with media free of substrate. Immunohistochemistry. Fixed sections were rinsed in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) containing 0.3010 (v/v) Triton X-l00 (15 min), pre-incubated with 2010 normal horse serum (30 min), washed in PBS-Triton (15 min), and incubated with a polyclonal NOS-I antiserum (at a dilution of 1: 1000) for 20 h at room temperature raised against NOS purified from pig brain (Mayer et al., 1990). The sections were washed in PBS-Triton (2x 15 min), incubated with a Cy3-conjugated goat anti-rabbit IgG (Jackson, West Grove, PA, USA) (diluted 1: 100,1 h), washed in PBS-ltiton (2x 15 min), and embedded in glycerol: PBS (1 : 1). Control incubations were performed using non-relevant IgG or PBS instead of primary antiserum.
NOS-I deficiency in DMD sarcolemma
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Results
Normal human muscles Non-NADPHd enzymes. In all muscles (either biopsy specimens or surgical material) investigated, myosin ATPase, SDH, CO, and NADHd were more active in type I than type II fibers.
Figs. I, 2. In a normal muscle biopsy, NADPH diaphorase activity is exclusivelypresent in the sarcolemmal region (arrows) of type 1 (I) and II (II) fibers. Fig. 1. x250, Fig. 2 x 400.
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NADPHd. NADPHd was present with equal activity in the sarcolemmal region of both type I and II fibers (Figs. 1,2). In addition to sarcolemmal staining, formazan production was rarely observed inside the fibers, especially type I fibers. When the reaction was carried out in the presence of formaldehyde or permanganate, which have turned out to be rather selective inhibitors of non-NOS-associated NADPHds (Nakos and Gossrau, 1994; Grozdanovic et al., 1995b), the reaction product was almost exclusively seen in the fiber surface, leaving the fiber inside almost unstained. By contrast, when Triton X-100 was omitted from the incubation medium, and the reaction was performed on formaldehyde-fixed cryosections, the sarcolemmal staining was markedly reduced, while increased quantities of formazan were detected inside the muscle fibers. NOS immunohistochemistry. NOS-I antiserum gave a consistent, intense, and continuous immunofluorescence staining of the sarcolemmal region, with no obvious differences between type I and II fibers (Fig. 3). No staining of intracellular structures was observed. Muscles from DMD patients Non-NADPHd enzymes. Abnormalities in staining for myosin ATPase, SDH, CO, and NADHd activities were in agreement with the results previously reported in the literature (Goebel, 1989). NADPHd. When the standard staining procedure was employed, i. e., with Triton X-toO but without formaldehyde or permanganate in the incubation medium, the amount of formazan generated in the sarcolemmal area was dramatically reduced or totally diminished (Figs. 4, 5). In muscle fibers which exhibited weak residual activity, discontinuities of sarcolemmal staining were common. Furthermore, small quantities of formazan were occasionally encountered inside some fibers; addition of either formaldehyde or permanganate to the incubation medium, which was applied to fresh cryosections, resulted in reduced (but not deficient) intracellular staining.
Fig. 3. NOS immunofluorescence is seen in the sarcolemmal region (arrows) of type 1 (I) and II (II) fibers. x 400.
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Figs. 4, 5. In a biopsy from a patient with Duchenne muscular dystrophy NADPH diaphorase activity is absent in the region of the sarcolemma (arrows); inside rare muscle fibers NADPH diaphorase is still active (+). Fig. 4 x 250, Fig. 5 x 400.
NOS immunohistochemistry. NOS-I immunostain was absent or drastically deficient in the sarcolemma of most DMD muscle fibers (Fig. 6). Some scattered fibers exhibited focal defects in sarcolemmal staining, which was less intense than in normal fibers.
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Fig. 6. In a muscle biopsy from a patient with Duchenne muscular dystrophy, with the exception of some focal NOS immunoreactivity (small arrows), the enzyme is absent from the sarcolemmal region (arrows); thin arrows = unspecific fluorescence. x 400.
Discussion We have demonstrated that NOS-I and its reductase, i. e., NADPHd, activity are coexpressed in the sarcolemma of normal human skeletal muscles. A strong correlation between NOS-I immunostain and NADPHd activity was obtained, when the incubation medium was supplemented with the fixation aldehyde formaldehyde or the oxidizer permanganate. This is consistent with the previous results from animal studies (Nakos and Gossrau, 1994; Grozdanovic et al., 1995 a, b), providing further support for the concept that these reagents can be used to differentiate between NOS- and non-NOS-associated NADPHds (Blottner et al., 1995). The residual intracellular staining, which persists after formaldehyde or permanganate treatment, can tentatively be attributed to the presence of NOS isozymes other than NOS-I in the myofiber sarcoplasm. Indeed, NOS-II (inducible NOS) and NOS-III (endothelial NOS) have been localized to the sarcoplasm of animal skeletal muscle fibers (Williams et al., 1994; Kobzik et al., 1995). Examination of adjacent sections reacted for myosin ATPase, SDH, CO, and NADHd activities showed that these enzymes were predominantly expressed in type I fibers (Meijer, 1991), while no significant differences were observed for sarcolemmal NOS-I staining between type I and II fibers. This is in marked contrast to laboratory animals and marmoset monkeys, where NOS-I immunostain and NADPHd activity are concentrated in the sarcolemma of type II fibers (Kobzik et aI., 1994; Grozdanovic et aI., 1995 a). In DMD muscle, both NOS-I protein and its activity are absent or dramatically reduced in the sarcolemma. DMD is caused by a deficiency of dystrophin, a rod-shaped cytoskeletal protein located on the cytoplasmic face of the sarcolemma (Hoffman et al. , 1987. 1988; Arahata et al., 1988; Bonilla et al., 1988; Koenig et al., 1988; Watkins et al.,
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1988; Zubrzycka-Gaarn et aI., 1988; Cullen et aI., 1990). Biochemical evidence indicates that dystrophin is anchored to an oligomeric transmembrane-spanning glycoprotein complex, which interacts with the extracellular matrix (Campbell and Kahl, 1989; Ervasti et aI., 1990; Yoshida and Ozawa, 1990; Ervasti and Campbell, 1991; Ervasti et aI., 1991; Ibraghimov-Beskrovnaya et aI., 1992). In DMD muscle, the deficiency of dystrophin in the sarcolemma (Arahata et aI., 1988; Bonilla et aI., 1988; Zubrzycka-Gaarn et aI., 1988) is paralleled by the loss of all the dystrophin-associated proteins (Ervasti et aI., 1990; Ibraghimov-Beskrovnaya et aI., 1992; Ohlendieck et aI., 1993), supporting the hypothesis that the disintegration of the dystrophin-glycoprotein complex (DGC) leads to the disruption of the linkage between the membrane cytoskeleton and the extracellular ma trix and, consequently, muscle fiber necrosis (Ervasti and Campbell, 1993; Matsumura and Campbell, 1993, 1994). The observation that NOS-I is absent or markedly deficient in the DMD muscle sarcolemma points to the existence of a structural link between NOS-I and dystrophin and /or dystrophin-associated proteins. The available evidence indicates that the loss of dystrophin-associated proteins is a characteristic pathogenetic event occurring specifically in dystrophin-deficient DMD muscle (Ervasti and Campbell, 1993; Matsumura and Campbell, 1993, 1994; Ohlendieck et aI., 1993). Along the same line of reasoning, the absence of NOS-I in the sarcolemma of DMD muscle fibers is unlikely to be due to nonspecific membrane alterations secondary to muscle fiber degeneration. Currently, we are trying to throw more light on this problem by investigating the status of NOS-I in patients with Becker muscular dystrophy, the milder allelic form of DMD, and other neuromuscular disorders. Furthermore, it would be intere sting to elucidate the structural interrelationships between NOS-I and DGC. Immunoelectron micro scopic data (Langer et aI., 1995) suggest that NOS-I is presumably an integral sarcolemmal protein. In conclusion, to our best knowledge, our study is the first to show that NOS-I is a constant component of the sarcolemma of normal human skeletal muscles, which is absent in skeletal muscles from all DMD patients investigated. Further studies are needed to determine whether the loss of NOS-I is a direct consequence of the deficiency of dystrophin and, if so, whether this has implications for the pathogenesis of muscle fiber necro sis in DMD.
Note added in proof: After submission of this paper an article by Brenman et ai. (Cell 82, 743 -752, 1995) has been published also showing the association of NOS I with dystrophin in human skeletal muscle.
Acknowledgement We are thankful to Ms Heidrun Richter for technical assistance and Ms Ursula Sauerbier for photographic work.
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