- Email: [email protected]
In situ identification of neuronal nitric oxide synthase (NOS-I) mRNA in mouse and rat skeletal muscle
Neuroscience Letters 246 (1998) 77–80
In situ identification of neuronal nitric oxide synthase (NOS-I) mRNA in mouse and rat skeletal muscle Gabriele Lu¨ck a, Ilse Oberba¨umer b, Dieter Blottner a ,* a
Department of Anatomy, Neurobiology Section, University Hospital Benjamin Franklin, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 15, D-14195 Berlin-Dahlem, Germany b Department of Anatomy and Cell Biology, Humboldt University Hospital (Charite´), Spandauer Damm 130, D-14050 Berlin-Charlottenburg, Germany Received 23 February 1998; received in revised form 11 March 1998; accepted 11 March 1998
Abstract Skeletal muscle provides a major source of the signaling molecule nitric oxide (NO) however in situ identification of NOsynthase (NOS) mRNA has not been verified. We have used NOS-I (neuronal NOS) probes prepared from plasmid DNA by reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA transcripts in skeletal muscle cells and myofibers of rat and mouse. Mouse C2C12 myoblasts and myotubes reveal strong cytosolic in situ hybridization (ISH) signals in vitro. In adult animals, ISH signals are detectable in striated myofibers at subsarcolemmal and perinuclear regions whilst the myofibrillar compartment is devoid of signals. Expression of NOS-I mRNA in fusion-competent myoblasts suggests that the NOS/NO system is of relevance to myogenic differentiation. Compartmentalization of NOS-I mRNA may reflect spatiofunctional actions between NOS message and protein and the putative subcellular NO targets. 1998 Elsevier Science Ireland Ltd.
Keywords: Nitric oxide synthase type-1; Skeletal muscle development; Neuromuscular system; Myoblasts; Myotubes; Polymerase chain reaction; Nitric oxide synthase gene expression
Nitric oxide synthase (NOS) occurs in at least three isoforms, classified according to the major types of cells expressing these enzymes as neuronal (NOS-I), macrophage (NOS-II) and endothelial type (NOS-III) [15]. The neuronal isoform (NOS-I) has recently been discovered in skeletal muscle and may be involved in regulating aspects of development and contractility of striated and cardiac muscle [8,11,13]. In skeletal muscle, NOS-I protein and its associated reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity are confined to sarcolemmal structures revealing typical staining-patterns (‘rings’) around cross-sectioned myofibers [11], possibly reflecting the molecular association between NOS-I and the subsarcolemmal dystrophin complex via the ‘linkers’ syntrophin (a 58 kDa cytoskeletal protein [1]), the 95-kDa * Corresponding author. Tel.: +49 30 8383843; fax: +49 30 8383996; e-mail: [email protected]; internet: http://www.ukbf.fuberlin.de/anatomie
postsynaptic density protein (PSD-95) and the socalled GLGF/dystrophin binding domain of the extended N-terminus of NOS-I [6]. The messenger RNA for NOS-I has been found in skeletal muscle preparations at quantitative amounts that exceed those in the human brain tissue [14]. While the cellular localization pattern of NOS mRNA transcripts is well known in defined neuronal populations, e.g. in brain [5], spinal cord [19] or peripheral ganglia [18], mRNA expression and localization in myogenic cells or adult myofibers are less well documented. Thus, we have generated NOS-I riboprobes for in situ hybridization (ISH) for localization of NOS-I mRNA in differentiated myoblast cultures and mature rat skeletal muscle fibers. Mouse C2C12 myoblasts were grown on 2% polyornithine-coated chamber slides (Nunc, Naperville, IL, USA) in proliferation medium supplemented with 10% of fetal calf serum (FCS; GIBCO) and 2% of chick embryo extract (Life Technologies) for 48 h at 37°C. Myotube fusion was induced by fusion medium (2% normal horse serum, GIBCO) as previously described [4]. Prefixed
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00237- 7
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G. Lu¨ck et al. / Neuroscience Letters 246 (1998) 77–80
slide-cultures (4% neutrally-buffered paraformaldehyde, 30 min at 4°C) were rinsed with diethyl pyrocarbonate-treated water, and processed according to a previously described non-radioactive in situ hybridization protocol with digoxigenin (DIG)-labeled riboprobes [19]. The DIG-labeled riboprobes were generated from plasmid-DNA (pDNA) by RT-PCR (Perkin-Elmer PCR system 2400) with the following primers: CAC ACA GGA AAC AGC TAT GAC CAT (primer I); GTC ACG ACG TTG TAA AAC GAC GGC (primer II). PCR was generated for 25 cycles by repeating denaturation at 98°C for 5 min; each cycle involved heating at 98°C for 20 s, annealing at 62°C for 30 s and extension at 77°C for 55 s in the presence of vent–Polymerase (New England Biolabs). Amplified DNA was transcribed with T7/SP6 (sense) or T3 (antisense) RNA polymerase (Boehringer-Mannheim, Germany) in the presence of DIG-11dUTP from a DIG-RNA labeling kit (incubation time: 2 h at 37°C, stopped with 0.2 M EDTA, pH 8.0), followed by time-controlled RNA-probe hydrolysis with 0.1 M Na2HCO3 at 60°C to gain about 200-bp fragments suitable for ISH. The concentrations of the DIG-labeled riboprobes (antisense or sense) were 5–10 ng/ml of hybridization buffer mixture containing 50% deionized formamide, 2× standard saline citrate (SSC), denatured herring sperm DNA (100 mg/ ml), tRNA (100 mg/ml), bovine serum albumin (1 mg/ml), 0.2 M dithiothreitol and 10% dextran sulfate. After prehybridization of sections (50% formamide in 2× SSC, 50°C for
30 min), hybridization was performed for 20 h at 50°C in a moist chamber followed by post-hybridization stringency washing at 40°C with 2× SSC (30 min), 0.75× SSC/50% formamide (60 min), 0.2× SSC/formamide (30 min). Visualization of hybridized DIG-probes was done with alkalinephosphate coupled anti-DIG fab-fragments (diluted 1:500) from a labeling-kit (Boehringer Mannheim, Germany) and development in the dark (for up to 5 days). All chemicals were of molecular grade (Sigma) and only autoclaved RNAse-free glassware and buffers were used. For NOS-I ISH in rat or mouse muscle tissue, 10 mm thick cryosections of 4% neutrally buffered paraformaldehyde (PFA)-fixed and cryoprotected (15% sucrose) muscle preparations from upper limb (deltoideus, biceps and triceps muscle) were thaw-mounted on sterile RNAse-free and silanized glass slides, postfixed with 4% PFA, and hybridized with DIG-riboprobes for NOS-I according to the ISH protocol described. Proliferating mononucleated myoblasts reveal strong NOS-I mRNA ISH-signals in their cytosol (Fig. 1a) just before they have reached confluence after 1 to 3 days in vitro (d.i.v). The hybridization protocol with sense probes in parallel cultures shows negative results (Fig. 1b). In fusioned polynucleated myotubes (3–5 d.i.v.) strong ISHsignals are detectable in elongated myotubes (Fig. 1c). The sense control gives negative results (Fig. 1d). We conclude that in the non-innervated differentiated C2C12 myogenic
Fig. 1. NOS-I mRNA in situ hybridization (ISH) in differentiated C2C12 myoblasts. (a) Antisense probe: mononucleated single myoblasts reveal strong ISH-signals in their cytosol (13 days in vitro). The myonuclei are spared from ISH-signals. (b) Sense control in parallel cultures is negative. (c) Antisense probe: strong ISH-signals are also detected in sarcoplasm of fusioned elongated myotubes with longitudinally oriented myonuclei (3–5 days in vitro). Bundles of contractile myofibrils are not present at this stage. (d) Sense control. The ISH-signal is lacking in parallel myotube cultures. Scale bar, (a,b) 15 mm, (c,d) 100 mm.
G. Lu¨ck et al. / Neuroscience Letters 246 (1998) 77–80
cell line NOS-I mRNA is markedly expressed in the cytosol of both myoblasts and myotubes. In mature myofibers of adult rat or mouse skeletal muscle tissue, the NOS-I hybridization signals are detected at subsarcolemmal areas as seen in individual cross-sectioned myofibers (Fig. 2a). The center area with bundles of contractile myofibrils (i.e. myofibrillar compartment) is devoid of ISH-signals. ISH-signals are lacking in sense control experiments (Fig. 2b). In the cross-sectioned planes the myonuclei are not readily seen as they are located in longitudinal bands beneath the sarcolemma along the myofiber length. The population of myonuclei is clearly visible in longitudinally-cut myofibers (Fig. 2c–e). Here, strong ISH-signals are detectable in the perinuclear and subsarcolemmal sarcoplasm along the length of the myofiber (Fig. 2c,d; rat and mouse deltoideus muscle). The myofibrillar
79
compartment is devoid of signals. In parallel cryosections from identical muscle, the sense control (Fig. 2f) is negative. In addition to the known sarcolemmal distribution of NOS-I protein [4,5,11] and expression of NOS mRNA in skeletal muscle homogenates by biochemical or quantitative DNA/RNA analysis [14], we provide evidence for the cellular distribution and localization of NOS-I mRNA in differentiating skeletal muscle cells and adult myofibers by in situ hybridization. Release of NO from incubated skeletal muscle fiber preparations has previously been shown [3]. The strong cytosolic expression of NOS-I mRNA in differentiated myoblasts is in accordance with recent findings of Ca2+dependent increase of NOS-activity levels, thus NO has been proposed a ‘fusion signal’ [13]. All three isoforms of the NOS-I–III proteins colocalize in C2-myoblasts and
Fig. 2. NOS-I mRNA in situ hybridization (ISH) in adult rat and mouse skeletal muscle (deltoideus). (a) Antisense probe. In cross-sectioned myofibers NOS-I mRNA ISH-signals are detected subsarcolemmal (arrowheads) and are almost lacking in the center area of contractile myofibrils (i.e. myofibrillar compartment). (b) Control sections with the sense probe are ISH negative. (c,d,e) Antisense probes. (c) Rat deltoideus muscle. In longitudinally-cut myofibers, NOS-I mRNA signals are detected subsarcolemmal (double arrowheads) and in the perinuclear sarcoplasm around myonuclei (arrowheads). (d,e) Mouse deltoideus muscle. Sarcolemmal and perinuclear NOS-I mRNA localization patterns are detected in tangentially-cut muscle fibers (arrowheads show myonuclei in (d,e)) or in horizontally-cut myofibers (e). The myofibrillar compartment is devoid of ISH-signal (arrows in (d,e)). (f) The sense control is ISH-negative. Scale bar, 100 mm (f).
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myotubes while the meaning of such an isoform heterogeneity and regulated NO formation in muscle biology is still enigmatic [4]. NOS-II and NOS-III isoforms have been localized in skeletal muscle from guinea pig suggesting some speciesspecific differences [2,8]. Besides sarcolemmal NOS-I, NOS-II and NOS-III colocalize in the sarcoplasm of cardiac myocytes [2,16]. These examples testify to the occurrence of an isoform-specific subcellular localization of musclederived NOS as shown by e.g. sarcolemma associated NOS-I [11], cytosolic NOS-II [2] or the mitochondrial relationship of NOS-III [12] which appear to be coexpressed by striated muscle cells and/or cardiac myocytes, the functional implications of which should be subject to further investigation. Compartmentalization of mRNAs (i.e. subsarcolemmal vs. perinuclear or myofibrillar compartment) as shown for acetylcholine esterase (AChE) or -receptor (AChR) mRNA in subsets of cultured muscle cells or subsynaptic nuclei, is well known [7]. Non-fusioned C2C12 cells or non-innervated myotubes show more uniformly distributed AChE or AChR mRNA [9] similar to the NOS mRNA pattern detected here. In the adult myofiber, the NOS-I mRNA is restricted to subsarcolemmal and perinuclear sarcoplasm along the fiber length and thus appears to be encoded by the majority of its myonuclei. Perinuclear NOS mRNA localization likely reflects some spatial functions of NOS message vs. cytoplasmic and/or sarcolemmal NOS protein in terms of protein targeting by posttranslational modification as well as nuclear gene control via extrinsic/intrinsic signals [10]. NOS-I molecules contain a GLGF-motif at its N-terminus that targets NOS-I via the linkers PSD-95 and syntrophin to the sarcolemmal dystrophin-complex [6]. An alternatively spliced variant of NOS-I, termed NOSm, has been characterized in differentiated myoblasts and in human striated and cardiac muscle [17]. Whether both NOSm or NOS-I proteins and their relevant mRNAs are localized in the same or different compartments of a muscle cell remains to be elucidated. Motoneurons in the ventral spinal cord however lack NOS-I mRNA and protein expression [20] suggesting retrograde modulatory actions of muscle-derived NO for presynaptic somatic motoneurons in neuromuscular functions. We thank Renate Gießler and Frauke Ku¨hl for excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft (Bl 259/3-1 and 3-2). [1] Adams, M.E., Butler, M.H., Dwyer, T.M., Peters, M.F., Murnane, A.A. and Froehner, S.C., Two forms of mouse syntrophin, a 58 kd dystrophin-associated protein, that differ in primary structure and tissue distribution, Neuron, 11 (1993) 531–540. [2] Balligand, J.L., Kobzik, L., Han, X., Kaye, D.M., Belhassen, L., O’Hara, D.S., Kelly, R.A., Smith, T.W. and Michel, T., Nitric oxide-dependent parasympathetic signalling is due to activation of constitutive endothelial (type-III) nitric oxide synthase in cardiac myocytes, J. Biol. Chem., 270 (1995) 14582–14586.
[3] Balon, T.W. and Nadler, J.L., Nitric oxide release is present from incubated skeletal muscle preparations, J. Appl. Physiol., 77 (1994) 2519–2521. [4] Blottner, D. and Lu¨ck, G., Nitric oxide synthase (NOS) in mouse skeletal muscle development and differentiated myoblasts, Cell Tissue Res., (1998) in press. [5] Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615– 624. [6] Brenman, J.E., Chao, C.S., Gee, S.H., McGee, A.W., Crayer, S.E., Santillano, D.R., Wu, Z., Huang, F., Xia, H., Peters, M.F., Froehner, S.C. and Bredt, D.S., Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and al-syntrophin mediated by PDZ-domains, Cell, 84 (1996) 757–767. [7] Bursztajn, S., Berman, S.A. and Gilbert, W., Differential expression of acetylcholine receptor mRNA in nuclei of cultured muscle cells, Proc. Natl. Acad. Sci. USA, 86 (1989) 2928–2932. [8] Fo¨rstermann, U., Inducible NO-synthase II and neuronal NOsynthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function, FASEB J., 10 (1996) 1614–1620. [9] Grubic, Z., Komel, R., Walker, W.F. and Miranda, A.F., Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcholinesterase and its mRNA in cultures of human muscle, Neuron, 14 (1995) 317–327. [10] Hall, Z.W. and Ralston, E., Nuclear domains in muscle cells, Cell, 59 (1989) 771–772. [11] Kobzik, L., Reid, M.B., Bredt, D.S. and Stamler, J.S., Nitric oxide in skeletal muscle, Nature, 372 (1994) 546–548. [12] Kobzik, L., Stringer, B., Balligand, J.L., Reid, M.B. and Starnler, J.S., Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships, Biochem. Biophys. Res. Commun., 211 (1995) 375–381. [13] Lee, K.H., Baek, M.Y., Moon, K.Y., Song, W.K., Chung, C.H., Ha, D.B. and Kang, M.S., Nitric oxide as a messenger molecule for myoblast fusion, J. Biol. Chem., 269 (1994) 14371–14374. [14] Nakane, M., Schmidt, H.H.W., Pollock, J., Fo¨rstermann, U. and Murad, F., Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle, FEBS Lett., 316 (1993) 175– 180. [15] Schmidt, H.H.W. and Walter, U., NO at work, Cell, 78 (1994) 919–925. [16] Seki, T., Hagiwara, H., Naruse, K., Kadowaki, M., Kashiwagi, M., Demura, H., Hirose, S. and Naruse, M., In situ identification of messenger RNA of endothelial type nitric oxide synthase in rat cardiac myocytes, Biochem. Biophys. Res. Commun., 218 (1996) 601–605. [17] Silvagno, F., Xia, H. and Bredt, D.S., Neuronal nitric oxide synthase-m, an alternatively spliced isoform expressed in differentiated skeletal muscle, J. Biol. Chem., 271 (1996) 11204– 11208. [18] Verge, V.M.K., Xu, Z., Xu, X.-J., Wiesenfeld-Hallin, Z. and Ho¨kfelt, T., Marked increase in nitric oxide mRNA in rat dorsal root ganglia after peripheral axotomy: in situ hybridization and functional studies, Proc. Natl. Acad. Sci. USA, 89 (1992) 11617–11621. [19] Vogel, M., Lu¨ck, G., Bachmann, S. and Blottner, D., NOS type-l mRNA expression and protein localization in spinal autonomic neurons, NeuroReport, 8 (1997) 3389–3393. [20] Wu, W., Liuzzi, F.J., Schinco, F.P., Depto, A.S., Li, Y., Mong, J.A., Dawson, T.M. and Snyder, S.H., Neuronal nitric oxide synthase is induced in spinal neurons by traumatic injury, Neuroscience, 61 (1994) 719–726.
In situ identification of neuronal nitric oxide synthase (NOS-I) mRNA in mouse and rat skeletal muscle Gabriele Lu¨ck a, Ilse Oberba¨umer b, Dieter Blottner a ,* a
Department of Anatomy, Neurobiology Section, University Hospital Benjamin Franklin, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 15, D-14195 Berlin-Dahlem, Germany b Department of Anatomy and Cell Biology, Humboldt University Hospital (Charite´), Spandauer Damm 130, D-14050 Berlin-Charlottenburg, Germany Received 23 February 1998; received in revised form 11 March 1998; accepted 11 March 1998
Abstract Skeletal muscle provides a major source of the signaling molecule nitric oxide (NO) however in situ identification of NOsynthase (NOS) mRNA has not been verified. We have used NOS-I (neuronal NOS) probes prepared from plasmid DNA by reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA transcripts in skeletal muscle cells and myofibers of rat and mouse. Mouse C2C12 myoblasts and myotubes reveal strong cytosolic in situ hybridization (ISH) signals in vitro. In adult animals, ISH signals are detectable in striated myofibers at subsarcolemmal and perinuclear regions whilst the myofibrillar compartment is devoid of signals. Expression of NOS-I mRNA in fusion-competent myoblasts suggests that the NOS/NO system is of relevance to myogenic differentiation. Compartmentalization of NOS-I mRNA may reflect spatiofunctional actions between NOS message and protein and the putative subcellular NO targets. 1998 Elsevier Science Ireland Ltd.
Keywords: Nitric oxide synthase type-1; Skeletal muscle development; Neuromuscular system; Myoblasts; Myotubes; Polymerase chain reaction; Nitric oxide synthase gene expression
Nitric oxide synthase (NOS) occurs in at least three isoforms, classified according to the major types of cells expressing these enzymes as neuronal (NOS-I), macrophage (NOS-II) and endothelial type (NOS-III) [15]. The neuronal isoform (NOS-I) has recently been discovered in skeletal muscle and may be involved in regulating aspects of development and contractility of striated and cardiac muscle [8,11,13]. In skeletal muscle, NOS-I protein and its associated reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity are confined to sarcolemmal structures revealing typical staining-patterns (‘rings’) around cross-sectioned myofibers [11], possibly reflecting the molecular association between NOS-I and the subsarcolemmal dystrophin complex via the ‘linkers’ syntrophin (a 58 kDa cytoskeletal protein [1]), the 95-kDa * Corresponding author. Tel.: +49 30 8383843; fax: +49 30 8383996; e-mail: [email protected]; internet: http://www.ukbf.fuberlin.de/anatomie
postsynaptic density protein (PSD-95) and the socalled GLGF/dystrophin binding domain of the extended N-terminus of NOS-I [6]. The messenger RNA for NOS-I has been found in skeletal muscle preparations at quantitative amounts that exceed those in the human brain tissue [14]. While the cellular localization pattern of NOS mRNA transcripts is well known in defined neuronal populations, e.g. in brain [5], spinal cord [19] or peripheral ganglia [18], mRNA expression and localization in myogenic cells or adult myofibers are less well documented. Thus, we have generated NOS-I riboprobes for in situ hybridization (ISH) for localization of NOS-I mRNA in differentiated myoblast cultures and mature rat skeletal muscle fibers. Mouse C2C12 myoblasts were grown on 2% polyornithine-coated chamber slides (Nunc, Naperville, IL, USA) in proliferation medium supplemented with 10% of fetal calf serum (FCS; GIBCO) and 2% of chick embryo extract (Life Technologies) for 48 h at 37°C. Myotube fusion was induced by fusion medium (2% normal horse serum, GIBCO) as previously described [4]. Prefixed
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00237- 7
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slide-cultures (4% neutrally-buffered paraformaldehyde, 30 min at 4°C) were rinsed with diethyl pyrocarbonate-treated water, and processed according to a previously described non-radioactive in situ hybridization protocol with digoxigenin (DIG)-labeled riboprobes [19]. The DIG-labeled riboprobes were generated from plasmid-DNA (pDNA) by RT-PCR (Perkin-Elmer PCR system 2400) with the following primers: CAC ACA GGA AAC AGC TAT GAC CAT (primer I); GTC ACG ACG TTG TAA AAC GAC GGC (primer II). PCR was generated for 25 cycles by repeating denaturation at 98°C for 5 min; each cycle involved heating at 98°C for 20 s, annealing at 62°C for 30 s and extension at 77°C for 55 s in the presence of vent–Polymerase (New England Biolabs). Amplified DNA was transcribed with T7/SP6 (sense) or T3 (antisense) RNA polymerase (Boehringer-Mannheim, Germany) in the presence of DIG-11dUTP from a DIG-RNA labeling kit (incubation time: 2 h at 37°C, stopped with 0.2 M EDTA, pH 8.0), followed by time-controlled RNA-probe hydrolysis with 0.1 M Na2HCO3 at 60°C to gain about 200-bp fragments suitable for ISH. The concentrations of the DIG-labeled riboprobes (antisense or sense) were 5–10 ng/ml of hybridization buffer mixture containing 50% deionized formamide, 2× standard saline citrate (SSC), denatured herring sperm DNA (100 mg/ ml), tRNA (100 mg/ml), bovine serum albumin (1 mg/ml), 0.2 M dithiothreitol and 10% dextran sulfate. After prehybridization of sections (50% formamide in 2× SSC, 50°C for
30 min), hybridization was performed for 20 h at 50°C in a moist chamber followed by post-hybridization stringency washing at 40°C with 2× SSC (30 min), 0.75× SSC/50% formamide (60 min), 0.2× SSC/formamide (30 min). Visualization of hybridized DIG-probes was done with alkalinephosphate coupled anti-DIG fab-fragments (diluted 1:500) from a labeling-kit (Boehringer Mannheim, Germany) and development in the dark (for up to 5 days). All chemicals were of molecular grade (Sigma) and only autoclaved RNAse-free glassware and buffers were used. For NOS-I ISH in rat or mouse muscle tissue, 10 mm thick cryosections of 4% neutrally buffered paraformaldehyde (PFA)-fixed and cryoprotected (15% sucrose) muscle preparations from upper limb (deltoideus, biceps and triceps muscle) were thaw-mounted on sterile RNAse-free and silanized glass slides, postfixed with 4% PFA, and hybridized with DIG-riboprobes for NOS-I according to the ISH protocol described. Proliferating mononucleated myoblasts reveal strong NOS-I mRNA ISH-signals in their cytosol (Fig. 1a) just before they have reached confluence after 1 to 3 days in vitro (d.i.v). The hybridization protocol with sense probes in parallel cultures shows negative results (Fig. 1b). In fusioned polynucleated myotubes (3–5 d.i.v.) strong ISHsignals are detectable in elongated myotubes (Fig. 1c). The sense control gives negative results (Fig. 1d). We conclude that in the non-innervated differentiated C2C12 myogenic
Fig. 1. NOS-I mRNA in situ hybridization (ISH) in differentiated C2C12 myoblasts. (a) Antisense probe: mononucleated single myoblasts reveal strong ISH-signals in their cytosol (13 days in vitro). The myonuclei are spared from ISH-signals. (b) Sense control in parallel cultures is negative. (c) Antisense probe: strong ISH-signals are also detected in sarcoplasm of fusioned elongated myotubes with longitudinally oriented myonuclei (3–5 days in vitro). Bundles of contractile myofibrils are not present at this stage. (d) Sense control. The ISH-signal is lacking in parallel myotube cultures. Scale bar, (a,b) 15 mm, (c,d) 100 mm.
G. Lu¨ck et al. / Neuroscience Letters 246 (1998) 77–80
cell line NOS-I mRNA is markedly expressed in the cytosol of both myoblasts and myotubes. In mature myofibers of adult rat or mouse skeletal muscle tissue, the NOS-I hybridization signals are detected at subsarcolemmal areas as seen in individual cross-sectioned myofibers (Fig. 2a). The center area with bundles of contractile myofibrils (i.e. myofibrillar compartment) is devoid of ISH-signals. ISH-signals are lacking in sense control experiments (Fig. 2b). In the cross-sectioned planes the myonuclei are not readily seen as they are located in longitudinal bands beneath the sarcolemma along the myofiber length. The population of myonuclei is clearly visible in longitudinally-cut myofibers (Fig. 2c–e). Here, strong ISH-signals are detectable in the perinuclear and subsarcolemmal sarcoplasm along the length of the myofiber (Fig. 2c,d; rat and mouse deltoideus muscle). The myofibrillar
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compartment is devoid of signals. In parallel cryosections from identical muscle, the sense control (Fig. 2f) is negative. In addition to the known sarcolemmal distribution of NOS-I protein [4,5,11] and expression of NOS mRNA in skeletal muscle homogenates by biochemical or quantitative DNA/RNA analysis [14], we provide evidence for the cellular distribution and localization of NOS-I mRNA in differentiating skeletal muscle cells and adult myofibers by in situ hybridization. Release of NO from incubated skeletal muscle fiber preparations has previously been shown [3]. The strong cytosolic expression of NOS-I mRNA in differentiated myoblasts is in accordance with recent findings of Ca2+dependent increase of NOS-activity levels, thus NO has been proposed a ‘fusion signal’ [13]. All three isoforms of the NOS-I–III proteins colocalize in C2-myoblasts and
Fig. 2. NOS-I mRNA in situ hybridization (ISH) in adult rat and mouse skeletal muscle (deltoideus). (a) Antisense probe. In cross-sectioned myofibers NOS-I mRNA ISH-signals are detected subsarcolemmal (arrowheads) and are almost lacking in the center area of contractile myofibrils (i.e. myofibrillar compartment). (b) Control sections with the sense probe are ISH negative. (c,d,e) Antisense probes. (c) Rat deltoideus muscle. In longitudinally-cut myofibers, NOS-I mRNA signals are detected subsarcolemmal (double arrowheads) and in the perinuclear sarcoplasm around myonuclei (arrowheads). (d,e) Mouse deltoideus muscle. Sarcolemmal and perinuclear NOS-I mRNA localization patterns are detected in tangentially-cut muscle fibers (arrowheads show myonuclei in (d,e)) or in horizontally-cut myofibers (e). The myofibrillar compartment is devoid of ISH-signal (arrows in (d,e)). (f) The sense control is ISH-negative. Scale bar, 100 mm (f).
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myotubes while the meaning of such an isoform heterogeneity and regulated NO formation in muscle biology is still enigmatic [4]. NOS-II and NOS-III isoforms have been localized in skeletal muscle from guinea pig suggesting some speciesspecific differences [2,8]. Besides sarcolemmal NOS-I, NOS-II and NOS-III colocalize in the sarcoplasm of cardiac myocytes [2,16]. These examples testify to the occurrence of an isoform-specific subcellular localization of musclederived NOS as shown by e.g. sarcolemma associated NOS-I [11], cytosolic NOS-II [2] or the mitochondrial relationship of NOS-III [12] which appear to be coexpressed by striated muscle cells and/or cardiac myocytes, the functional implications of which should be subject to further investigation. Compartmentalization of mRNAs (i.e. subsarcolemmal vs. perinuclear or myofibrillar compartment) as shown for acetylcholine esterase (AChE) or -receptor (AChR) mRNA in subsets of cultured muscle cells or subsynaptic nuclei, is well known [7]. Non-fusioned C2C12 cells or non-innervated myotubes show more uniformly distributed AChE or AChR mRNA [9] similar to the NOS mRNA pattern detected here. In the adult myofiber, the NOS-I mRNA is restricted to subsarcolemmal and perinuclear sarcoplasm along the fiber length and thus appears to be encoded by the majority of its myonuclei. Perinuclear NOS mRNA localization likely reflects some spatial functions of NOS message vs. cytoplasmic and/or sarcolemmal NOS protein in terms of protein targeting by posttranslational modification as well as nuclear gene control via extrinsic/intrinsic signals [10]. NOS-I molecules contain a GLGF-motif at its N-terminus that targets NOS-I via the linkers PSD-95 and syntrophin to the sarcolemmal dystrophin-complex [6]. An alternatively spliced variant of NOS-I, termed NOSm, has been characterized in differentiated myoblasts and in human striated and cardiac muscle [17]. Whether both NOSm or NOS-I proteins and their relevant mRNAs are localized in the same or different compartments of a muscle cell remains to be elucidated. Motoneurons in the ventral spinal cord however lack NOS-I mRNA and protein expression [20] suggesting retrograde modulatory actions of muscle-derived NO for presynaptic somatic motoneurons in neuromuscular functions. We thank Renate Gießler and Frauke Ku¨hl for excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft (Bl 259/3-1 and 3-2). [1] Adams, M.E., Butler, M.H., Dwyer, T.M., Peters, M.F., Murnane, A.A. and Froehner, S.C., Two forms of mouse syntrophin, a 58 kd dystrophin-associated protein, that differ in primary structure and tissue distribution, Neuron, 11 (1993) 531–540. [2] Balligand, J.L., Kobzik, L., Han, X., Kaye, D.M., Belhassen, L., O’Hara, D.S., Kelly, R.A., Smith, T.W. and Michel, T., Nitric oxide-dependent parasympathetic signalling is due to activation of constitutive endothelial (type-III) nitric oxide synthase in cardiac myocytes, J. Biol. Chem., 270 (1995) 14582–14586.
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