Brain, Vol. 125, No. 6, 1309-1319,
June 2002
© 2002 Guarantors of Brain
Expression of foetal type acetylcholine receptor is restricted to type 1 muscle fibres in human neuromuscular disorders
1 Institute of Pathology,Departments of 2 Neurology and 3 Orthopedics, University of Würzburg, Germany and 4 Neurosciences Group, Department of Clinical Neurology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
Correspondence to: Dr Stefan Gattenlöhner, Institute of Pathology, University of Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany E-mail: stefan.gattenloehner{at}mail.uni-wuerzburg.de
Received September 11, 2001. Revised December 18, 2001. Accepted January 24, 2002.
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Summary |
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In adult muscle, acetylcholine receptors (AChR) are restricted mainly to the motor endplate where the adult isoform (
ß
) is expressed. When skeletal muscle is denervated in animal models, there is atrophy of the muscle and a marked increase in expression of the AChR foetal isoform (ß
) containing a -subunit. Similar changes in AChR expression are thought to occur in human muscle. While the role of denervation in regulating AChR gene expression has been widely studied, it has not been determined whether the transcriptional programmes responsible for defining different fibre types have an impact on the expression of AChR genes. We investigated biopsies from patients with a wide spectrum of neuromuscular diseases for expression of the AChR - and -subunits using RNase protection assays, /-duplex reverse transcriptase polymerase chain reaction, immunohistochemistry for foetal AChR and RNA in situ hybridization. Muscle from all patients with neurogenic disorders and, to a lesser extent, myogenic disorders, exhibited markedly increased transcription of the AChR -subunit but, in contrast to previous animal studies, did not show increased AChR -subunit. Moreover, both immunohistochemistry and RNA in situ hybridization revealed that AChR -subunit hyperexpression occurred exclusively in atrophic type 1 and not in atrophic type 2 muscle fibres, irrespective of the underlying neuromuscular disease. We conclude that up-regulation of the AChR -subunit in human muscle disorders is restricted to type 1 muscle fibres and, therefore, that AChR -subunit expression is controlled by a muscle fibre type-restricted transcriptional programme. The factors influencing expression of this and other functional proteins should be relevant to the understanding and treatment of a range of neuromuscular disorders. Keywords: acetylcholine receptor; muscle; fibre type; denervation; innervation
Abbreviations: AChR= acetylcholine receptors; RT–PCR = reverse transcriptase–polymerase chain reaction
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Introduction |
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The nicotinic acetylcholine receptors (AChR) of skeletal muscle are a pentameric ion channel composed of four different subunits (Changeux, 1992
; Changeux et al., 1992). Many studies on small mammals have shown that during development of the neuromuscular junction, a change from the foetal type (ß) to the adult type (ß) occurs with replacement of the -subunit by the -subunit (Mishina et al., 1986; Witzemann et al., 1987, 1989). A similar change occurs during late gestation in humans (Hesselmans et al., 1993) and, in the adult, the foetal type of the AChR is found mainly on myoid cells in the thymus (Marx et al., 1989; Hara et al., 1993) and in some extraocular muscle fibres (Horton et al., 1993; Kaminski et al., 1996; MacLennan et al., 1997). In other adult human innervated muscles, only extremely low levels of AChR -subunit transcripts have been detected by RNase protection assays or reverse transcriptase–polymerase chain reaction (RT–PCR) (MacLennan et al., 1997; Gattenloehner et al., 1998, 1999) and not by less sensitive Northern blotting (Geuder et al., 1992).
Re-expression of the foetal type occurs after experimental denervation by nerve section or crush (Witzemann et al., 1989). Similarly, in human conditions that result in severe lower motor neurone dysfunction, such as amyotrophic lateral sclerosis (Tsujihata et al., 2001) or diabetic neuropathy (Vincent and Newsom-Davis, 1982), there is increased expression of foetal AChR. However, despite many studies concerning AChR gene expression in rodents (Whiting et al., 1986; Changeux, 1992; Changeux et al., 1992; Witzemann et al., 1989, 1990, 1991), little is known about the factors that control human AChR expression.
In muscle biopsies from patients, histochemistry is used to distinguish type 1 (slow twitch or red) and type 2 (fast twitch or white) fibres (Johnson et al., 1973; Round et al., 1980). Type 1 fibres contain more mitochondria and myoglobin, and utilize aerobic oxidation for their energy requirement, whereas type 2 fibres contain more glycogen and produce energy through the anaerobic pathway. The molecular factors that govern fibre type are not well characterized, but it is thought that distinct pathways are involved in type 1 and type 2 fibres (Olson and Williams, 2000; Buckingham, 2001). Whether the AChR genes are targets of these fibre type-related signalling cascades has not been investigated but, more than 30 years ago, Miledi and Zelena (Miledi and Zelena, 1966) showed that expression of AChR sensitivity along innervated rat muscle fibres was detectable in the ‘slow’ (i.e. type 1) soleus muscle, but not in ‘fast’ (i.e. type 2) muscles. At that time, the distinction between adult and foetal AChRs had not been made.
We have recently shown that AChR -subunit expression can be a useful tool in the diagnosis of rhabdomyosarcomas (Gattenloehner et al., 1998, 1999). Here we applied similar approaches to look for AChR -subunit and foetal AChR expression in muscle biopsies from patients undergoing routine investigations for muscle and nerve disorders. Our results show up-regulation of AChR -subunit expression in many neurogenic and also in some myogenic disorders. Surprisingly, up-regulation was only found in type 1 fibres, irrespective of the underlying disease, and foetal AChR expression was also restricted to atrophic type 1 fibres.
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Materials and methods |
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Biopsies
Seventy-seven muscle biopsies (15 from patients with neurogenic disorders, 23 from patients with non-inflammatory myopathies, eight from patients with inflammatory myopathy and 31 biopsies that were histologically normal) were obtained for frozen sections from patients undergoing routine investigations. All specimens came from the Departments of Surgery, Orthopedics or Neurology of the University of Würzburg (Würzburg, Germany) and were frozen within 15 min after biopsy. After frozen sections had been obtained, the tissue was immediately stored at –80°C for long-term storage. The biopsies were kept on dry ice when being handled. A comparison of RT–PCR data from biopsies taken 3 years apart showed that the storage conditions did not influence relative levels of the AChR
- and -subunit transcripts (data not shown). All patients gave informed consent and ethical approval for the use of human muscle biopsies was received from the University of Würzburg Ethics Committee (contract C-16 of the IZKF). Clinical data and diagnoses are given in Table 1. This information was disclosed to the experimental investigators (S.G. and A.M.) only at the end of the experiments by the participating neurologist (Dr C. Schneider).
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Molecular biology
RNA isolation, cDNA-synthesis and the three-step immunoperoxidase-based immunohistochemistry procedure were performed as previously described (Gattenloehner et al., 1998
; Marx et al., 1989). For the simultaneous amplification of the AChR and AChR mRNA, we used a duplex RT–PCR technique with primers specific for the AChR and AChR as described by Gattenloehner et al. (1999). To quantify the AChR and AChR transcripts, we scanned the ethidium bromide-stained gel of RT–PCR products with an Agfa Scanner Arcus II (Duffenhofer, Würzburg, Germany) and measured the intensity using NIH (MacIntosh) software (FMS Software, Würzburg, Germany) by creating standard areas in which the extinction was calculated (Gattenloehner et al., 1999).
For the RNase protection assay, total RNA was processed according to Pharmingen’s RiboQuant protocol using anti-sense RNA as the probe. For the probes, PCR products were generated using primers specific for the AChR - and -subunits as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AChR upper primer (UP): 5'-AAG CTA CTG TGA GAT CAT CGT-3'; AChR lower primer (LP): 5'-TGA CGA AGT GGT AGG TGA TGT-3'; AChR UP: 5'-ATC TCT GTC ACC TAC TTC CCC-3'; AChR LP: 5'-AAG TGG ATG AGG ATG GCG ACA-3'; GAPDH UP: 5'-CAA CAG CGA CAC CCA CTC CTC-3'; GAPDH LP: 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3'). PCR products were cloned in a pGEM T-vector (Promega, Heidelberg, Germany) and sequenced by the cycle sequencing method using dye terminators and the ABI 373 sequencer (Applied Biosystems, Weiterstadt, Germany), following the manufacturer’s instructions. The predicted length of the transcripts were: GAPDH unprotected, 233 bp, and protected, 156 bp; AChR subunit unprotected, 334 bp, and protected, 244 bp; AChR -subunit unprotected, 306 bp, and protected, 254 bp (because of an internal PstI site) (Fig. 1).
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Histochemical muscle fibre typing
To identify the muscle fibre subtype expressing the
-subunit of the AChR, serial histological sections of muscle biopsies were studied by enzyme histochemistry for ATPase reactions at pH 4.6 and pH 9.4 followed by immunohistochemistry for foetal AChR expression using a monoclonal antibody, mAbM1B8 (Jacobson et al., 1999). Two different sections were stained from each case investigated (see Table 1).
In situ hybridization
The in situ hybridization procedure was as previously described (Czub et al., 1996). Like the immunohistochemical studies, this part of the investigation was performed in a blind fashion, since we were neither aware of the diagnoses nor the immunohistochemical or enzyme histochemical findings. From all cases investigated (see Table 1), four different sections were hybridized [two sections each for short-term (1 week) and long-term (4 weeks) exposure]. For the AChR specific probe, a PCR product was generated using AChR -subunit specific primers: AChR UP: 5'-ATC TCT GTC ACC TAC TTC CCC-3'; AChR LP: 5'-AAG TGG ATG AGG ATG GCG ACA-3'. The PCR product was cloned into the pGEMT vector and sequenced by the cycle sequencing method using dye terminators and the ABI 373 sequencer according to the manufacturer’s instructions.
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Results |
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-Subunit but not -subunit transcripts are increased in chronic neurogenic disordersTo quantify AChR transcription in various human muscle disorders, RNase protection assays were performed on the nine biopsies of sufficient size for at least three independent determinations with both
- and -probes. These biopsies consisted of three from patients with neurogenic disorders and two each from patients with inflammatory myopathies, non-inflammatory myopathies or histologically-normal muscle (control muscle). The muscles from which the biopsies were taken are shown in Table 1.
As shown in Fig. 1, all muscle biopsies, irrespective of the underlying pathology, showed AChR -subunit mRNA expression that was not significantly different from that in the normal biopsies. By contrast, the AChR -subunit was dramatically increased in muscle biopsies derived from patients with neurogenic disorders. When the AChR - and -subunit-specific bands were quantified by measuring their extinction, a 30–100-fold overexpression of the -subunit AChR was shown in muscle biopsies from patients suffering from neurogenic disorders compared with control muscle biopsies (data not shown). However, none of the muscle biopsies derived from inflammatory or non-inflammatory myopathies exhibited significant overexpression of AChR -subunit message by RNase protection assay (e.g. Fig. 1).
Quantitative analysis of AChR -subunit mRNA by AChR /-directed duplex PCR
Application of RNase protection assays is limited by the relatively high quantity of RNA required for the assay, i.e. by biopsy size. To extend the studies to a larger series of muscle biopsies and to increase the sensitivity of AChR -subunit transcript detection, we used a multiplex RT–PCR strategy to detect AChR - and -ubunit message simultaneously (Gattenloehner et al., 1999) and to quantify amplificates of AChR - and -subunit message on gels using the NIH software scanning program (Fig. 2). This technique has been shown to be applicable to small biopsies (<150 ng of extractable RNA) as is typically available from patients with myositis, neurogenic or non-inflammatory myopathies. In a double-blinded study, the / ratios were calculated for muscle biopsies derived from patients with neurogenic disorders (n = 15), non-inflammatory myopathies (n = 23) and inflammatory myopathies (n = 8), and compared with results from histologically-normal muscle biopsies (n = 31). The cases investigated are listed in Table 1 and the results presented in Fig. 3.
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Histologically normal muscle typically exhibited an
/ ratio between 0.06 and 0.3 (mean value ± SD: 0.17 ± 0.068) (Fig. 3). In all muscle biopsies with neurogenic disorders (n = 15), the / ratios were >0.69, and in seven biopsies they were 1.0 or greater (Table 1). The difference between values in neurogenic and normal muscles was highly significant (P < 0.0001, Mann–Whitney U-test).
The / ratios were less elevated in the muscle biopsies from patients with myogenic disorders, but taking the mean + 3SD of the normal biopsies (0.37) as the cut-off, 17 out of 23 of the non-inflammatory myopathies and five out of eight of the inflammatory myopathies had raised values, and as a group each was significantly different (P < 0.0001) from the normal values.
The / ratios did not correlate significantly with the age of the patients (data not shown) or the localization of the muscle biopsy (Table 1).
AChR -subunit expression and muscle fibre type
All muscle biopsies were examined for muscle fibre type atrophy by conventional ATPase enzymhistochemistry. Some biopsies had predominantly type 1 fibre atrophy whereas others showed predominantly type 2 fibre atrophy, or modest degrees of both. There was a striking association between the relative amount of -subunit AChR transcripts and the presence of type 1 fibre atrophy (Table 1). To examine in more detail the distribution of AChRs in atrophic muscle fibres, AChR expression was investigated with a monoclonal antibody, M1B8, specific for the foetal AChR isoform, in patients with neurogenic disorders (n = 10), non-inflammatory myopathy (n = 10), inflammatory myopathy (n = 6) and histologically normal muscle biopsies (n = 10). In Fig. 4, strong positivity is shown in a biopsy from a patient with inclusion body myositis and a / ratio of 0.97 (Case 40) compared with absence of foetal AChR in histologically-normal muscle with an / ratio of 0.14 (Fig. 4A and B).
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In all muscle biopsies with
/ ratios >1.0 (n = 7, see Table 1), immunohistochemistry revealed strong immunoreactivity (+++) in large groups of angulated atrophic fibres and ATPase staining of serial sections showed that all fibres expressing the foetal AChR were atrophic type 1 muscle fibres (Fig. 5). All muscle biopsies with / ratios between 0.67 and 1.0 (n = 13, see Table 1) showed distinct immunoreactivity (++/+) on the surface of scattered single atrophic muscle fibres, all of which also belonged to the type 1 fibre subtype (Fig. 6A). By contrast, there was no reactivity on angulated atrophic type 2 fibres (Fig. 5B and C) or on single rounded and polygonal atrophic type 2 fibres (Fig. 6A and B). Moreover, all muscle biopsies with / ratios <0.67 (n = 6) lacked detectable immunoreactivity for the -subunit of the AChR, irrespective of the presence of fibre type atrophy (Table 1).
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AChR
-subunit expression and increased transcriptionIn situ hybridization was performed on muscle sections, with identification of the fibre types and AChR protein expression on serial sections. In all muscle biopsies tested with
/ ratios >0.67 (n = 10), atrophic, M1B8-positive, muscle fibres (Fig. 7A, above) were positive for -subunit RNA by in situ hybridization (Fig. 7A, below). By contrast, there was no evidence by in situ hybridization for AChR -subunit expression in those muscle biopsies that lacked type 1 fibre atrophy (n = 15, see Table 1)—including six muscle biopsies from histologically normal muscle (Fig. 7B and C)—regardless of the presence of type 2 fibre atrophy.
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Discussion |
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We used molecular and immunological techniques to measure AChR
-subunit expression in muscle biopsies from neurogenic and primary myopathic disorders. Expression of the AChR gene was most marked in chronic neurogenic disorders, but occurred to a lesser extent in biopsies from patients with inflammatory and primary myopathic muscle diseases. Surprisingly, expression of the foetal AChR isoform, identified by immunohistochemistry or by in situ hybridization for the -subunit, was restricted in all cases to type 1 muscle fibres. This striking finding suggests that denervation-induced AChR expression in humans is regulated in a manner which depends on the fibre type.
We find / duplex RT–PCR, which can be performed on small amounts of tissue, a very useful technique for investigation of muscle biopsies. / ratios >1.0 are strongly in favour of a neurogenic disorder, while ratios <0.67 appear to exclude neurogenic disorders. Nevertheless, almost all values in biopsies from inflammatory or non-inflammatory myopathies had increased / ratios compared with histologically normal biopsies. Although the extent of AChR -subunit RNA and protein hyperexpression in these cases was usually much lower than in neurogenic disorders (Fig. 3, Table 1), these results are consistent with clinical and electrophysiological studies that frequently reveal mild to moderate degrees of ‘denervation’ among inflammatory (Figarella-Branger et al., 1992; Heuss et al., 1995) and non-infammatory myopathies (Bradley et al., 1978). The marked increase in AChR -subunit message and foetal AChR protein confirms previous findings in chronically denervated human muscle (Tsay and Schmidt, 1989), but differs from results in rat models of denervation where AChR -subunit transcription is only transiently up-regulated (Witzemann et al., 1991).
An unexpected result was the lack of significant up-regulation of steady state AChR -subunit RNA levels as measured by RNase protection assay or RT–PCR in any of the muscle biopsies studied irrespective of the underlying pathology (Fig. 1). This is in apparent contrast to findings by MacLennan et al. (1997), who showed considerable up-regulation of all AChR subunits in one sample of partially denervated gastrocnemius (the muscle most often biopsied in our neurogenic cases). However, the tissue from their diabetic patient was obtained from an amputated leg, and the effects of the diabetic neuropathy would have been complicated by ischaemia. It is possible that the two combined produce up-regulation of all AChR subunits, whereas during chronic denervation the effects are attenuated (Adams et al., 1995). Two important questions are how the -subunit is up-regulated independently of the -subunit, and how the foetal AChR (2ß) is formed if only the -subunit is up-regulated. An explanation for the latter may be that the and other subunits are usually present in excess; for instance, only in the seven neurogenic cases was there more -subunit than -subunit expression. The former question, and the distribution of -subunit expression along the length of individual muscle fibres, will require further investigation.
Our most striking finding was that the up-regulation of AChR -subunit RNA and foetal AChR was only found on type 1 fibres, as shown by both immunohistochemistry and in situ hybridization. Consequently, states of isolated or predominantly type 2 muscle fibre atrophy, like Becker muscular dystrophy (Fig. 6), were not associated with AChR -subunit hyperexpression (Table 1) as previously reported in muscle atrophy associated with ageing (Lexell and Downham, 1992; Lexell, 1993, 1995) or immobilization (Fisher and Brown, 1998; Ibebunjo and Martyn, 1999). Particularly striking was the lack of AChR -subunit expression by atrophic type 2 fibres side-by-side with AChR -subunit hyperexpresion in atrophic type 1 fibres in severe neurogenic muscle atrophy (Fig. 5), dystrophy (Fig. 6A) and inclusion body myositis (Fig. 4A) that are all known to affect both fibre types (Schroder and Adams, 1968; Askanas et al., 1994). The latter observations, therefore, indicate that transcription of the AChR -subunit gene is not only regulated by innervation-dependent factors, but modified by factors that are involved in muscle fibre type determination. Indeed, our observation that innervation and fibre type can operate in concert to regulate gene expression is not unprecedented. In contrast to the up-regulation of the foetal AChR in type 1 muscle fibres following denervation, it has been reported that denervation selectively down-regulates neurotrophin-4 (NT-4) expression in type 1 muscle fibres (Funakoshi et al., 1995; Wells et al., 1999; Belluardo et al., 2001)
The molecular mechanisms underlying the fibre-type bias of AChR expression are still unclear. However, many differences are beginning to emerge regarding the ontogeny and control of the different fibre types and fibre type specific gene expression. Signalling molecules such as insulin growth factor (IGF), protein kinase C (PKC), calcineurin, p70S6k, NFAT and MEF2 are thought to play a role in inducing type 1 versus type 2 muscle fibre development (Delling et al., 2000; Swoap et al., 2000; Wu et al., 2000), while up-regulation of AML-1 after denervation appears to occur independent of fibre type (Zhu et al., 1994). Although there is still considerable uncertainty about many aspects of the pathways concerned (Olson and Williams, 2000; Buckingham 2001), activation of PKC isoforms appears to be important in development of type 1 fibres (DiMario and Funk, 1999). Conversely, calcium-induced activation of PKC is also thought to be critical for reducing extra-junctional AChR expression after innervation (Montgomery et al., 2000) and lack of calcium-induced PKC activation is thought to up-regulate AChR -subunit gene expression by extra-junctional nuclei when muscle activity is reduced by denervation. Thus, it is likely that factors other than the activity of calcium-dependent PKC isoforms are involved in regulation of genes that define the phenotype of type 1 fibres in denervated muscle. It will be interesting to look at the expression of growth factors, PKC isoforms and the various transcription factors in these human muscle biopsies.
Our results emphasize how little is known about the phenotypic characteristics of the muscle fibres and the factors that influence them in various disease states. They raise the possibility that expression of those proteins involved in the structural integrity of the motor endplate and sarcolemma may be differentially regulated. In Duchenne muscular dystrophy, there is reduced muscle glycolytic activity and in its mouse model, the mdx/utrn-/- double knock-out mouse, which lacks both dystrophin and utrophin, there is a shift towards type 1 fibre types (Rafael et al., 2000). In mdx and the double knock-out mice, the AChR distribution at the neuromuscular junction is patchy with discontinuous AChR staining (Rafael et al., 2000; Minatel et al., 2001). This suggests that in dystrophic or regenerating (DiMario and Funk, 1999) muscles, changes in expression of proteins that are specific to fibre type, the sarcolemma and the neuromuscular junction proteins may go in parallel. Similarly, in the AChR deficiency syndrome caused by AChR -subunit mutations (Engel et al., 1999; Croxen et al., 2001), the AChR -subunit is thought to be crucial for maintaining neuromuscular transmission, but the present study suggests that its expression might be restricted to type 1 fibres. A systematic study of expression of the factors involved in controlling expression of all these proteins could lead to a better understanding of the pathogenesis of the associated disorders. It could also be of relevance to any treatment protocols aimed at up-regulating these or compensatory proteins.
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Acknowledgements |
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We wish to thank Mrs Margrit Bonengel, Mrs Christl Kohaut, Mrs Eva Bachmann and Mr Erwin Schmitt for expert technical assistance. The research reported in this paper was supported by the Sander-Stiftung Grant 99.112.1 and BMBF IZKF Projects C-5 and C-16, 01 KS 9603.
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