Article |
Address correspondence to Shin'ichi Takeda, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, 187-8502 Kodaira, Tokyo, Japan. Tel.: 81-42-346-1720. Fax: 81-42-346-1750. E-mail: takeda{at}ncnp.go.jp
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Abstract |
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Key Words: 1-syntrophin; skeletal muscle; hypertrophy; regeneration; neuromuscular junction
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Introduction |
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Results |
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Hypertrophy and extensive fiber splitting of 1syn-/- regenerating TA muscles at 28 wk after cardiotoxin treatment
From 2 wk after injection, regenerating TA muscles of 1syn-/- mice were much larger than regenerating wild-type TA muscles (Fig. 1 a). The relative weight of regenerating
1syn-/- TA muscles was much increased at 2, 4, 8, and 12 wk after cardiotoxin injection compared with those of wild-type muscles. The relative weight of the untreated TA muscle to body weight of
1syn-/- and wild-type mice was constant, regardless of sex or age.
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Discussion |
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MHC composition in the regenerating 1syn-/- muscle
Interestingly, the cardiotoxin-injected mutant mice showed a marked deficit in exercise capacity at 7 d compared with the treated wild-type mice. Reduced exercise capacity has also been shown in mdx mice by the wire net test. The decreased capacity in regenerating 1-syntrophin knockout mice can be, at least partly, explained by the alteration of MHC composition. In the mutant regenerating muscles, the temporal increase of MHC I and IIa has been considerably inhibited and results in a relative increase of MHC IIb expression. A relative decrease of slow components at this period may affect the stamina of mutant mice in the test.
In mdx muscle, the reduced contractile force has been explained by the decreases in the proportion of type IIx and increases in type I MHC (Coirault et al., 1999). However, 1-syntrophin knockout mice did not exhibit altered MHC composition at 48 wk after the cardiotoxin injection. Actually, we observed predominant hypertrophy of type IIb fibers at the period by immunohistochemistry, whereas glycerol SDS-PAGE did not detect a significant increase of MHC IIb. This suggests that the hypertrophied mutant type IIb fibers contain considerable amounts of MHC IIx, since MHC IIx transcripts are abundant in histochemical type IIb fibers (Smerdu et al., 1994). Therefore, the composition of MHC cannot explain the reduced contractile force in the regeneration period of mutant mice.
Possible mechanisms of the reduction of contractile force in the regenerating 1syn-/- muscle
In mdx muscle, deficiency in exercise capacity, reduced contractile force production, and muscle hypertrophy (Coulton et al., 1988; Carter et al., 1995) can be related to the occurrence of many cycles of muscle degeneration and regeneration, membrane fragility, or reduced amount of myofibrils. In contrast, regenerating 1syn-/- TA muscles show no evidence of muscle degeneration, fibrosis, or loss of myofibers (Kameya et al., 1999). In addition, our examination showed no evidence of delay in maturation of myofibers. Therefore, we speculate that abnormal regeneration of syntrophin-null muscle is mainly caused by the lack of functional molecules that interact with
1-syntrophin. We have shown previously that two molecules are lacking from the sarcolemma of
1syn-/- muscle in vivo. First, nNOS was completely absent from the sarcolemma in
1syn-/- muscle (Kameya et al., 1999). Could nNOS be the molecule responsible for the reduction of contractile force in
1syn-/- muscle? NO is widely accepted as a versatile regulator of muscle functions through the cGMP pathway (Kobzik et al., 1994). It has been suggested that NO generated by nNOS at the sarcolemma regulates contraction-stimulated glucose uptake (Roberts et al., 1997) or increases local blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction (Thomas et al., 1998). Recently, NO has been reported to protect dystrophin-deficient mdx muscle from degeneration when expressed at a high level (Wehling et al., 2001). Moreover, NO can modulate the forcefrequency relationship in skeletal muscle (Stamler and Meissner, 2001). However, at least in in vitro studies, NO depletion results in an increase of contractile force (Stamler and Meissner, 2001), whereas
1syn-/- muscle showed the opposite response in the regeneration process. Thus, the loss of nNOS from the sarcolemma may not be directly responsible for the decline in contractile force of the regenerating muscle in the mutant mice. We have shown already that
1-syntrophin plays a major role in recruiting a water channel, AQP4, to the sarcolemma in fast twitch muscle fibers (Yokota et al., 2000). VGSC (Schultz et al., 1998; Gee et al., 1998), stress-activated protein kinase 3 (Hasegawa et al., 1999), and phosphatidylinositol 4,5-bisphosphate (Chockalingam et al., 1999) can also bind to
1-syntrophin. Therefore, it is possible that one or more of these molecules are responsible for the abnormal regeneration, although there is no direct evidence for the involvement of these proteins in contractile force production.
The alternate explanation for this phenomenon is that aberrant NMJs formation in the 1syn-/- mice may affect the force generation of regenerating muscle. Interestingly, a specific force deficit caused not only by atrophy but also by other unknown mechanism(s) exists in skeletal muscle after denervation (Kalliainen et al., 2002).
Finally, proteins involved in excitation-contraction coupling might be responsible for this reduction of contractile property in the mutant regenerating muscle. However, the protein responsible for the deficit in contractile force generation in the mutant mice remains to be determined.
Aberrant formation of NMJs in regenerating TA muscle of 1syn-/-
Our results and a previous report (Adams et al., 2000) demonstrated that 1-syntrophin plays an important role in formation of highly organized NMJs both in the developmental process and in muscle regeneration. Since
1-syntrophin is a PDZ protein without any known catalytic domain, the molecules play their roles in NMJ formation by sorting several structural or signaling molecules at the synaptic membrane. Adams et al. (2000) reported that utrophin was severely down-regulated at the NMJs in their
1syn-/- mice, suggesting that
1-syntrophin is an important regulator of utrophin expression at NMJs. It is reasonable to conclude that aberrant NMJs in
1syn-/- muscle are due to the deficiency of utrophin, since utrophin-null muscle showed similar aberrant NMJs to those of
1syn-/- mice (Deconinck et al., 1997; Grady et al., 1997). However, our results showed that the level of utrophin expression is not reduced at the NMJs in the absence of
1-syntrophin in either nonregeneration or regeneration (Fig. 7). This discrepancy in utrophin expression might be derived from a difference in the targeting strategy of the
1-syntrophin gene. We inserted an NEO gene in the second exon encoding the PDZ domain (Kameya et al., 1999) and later detected a trace of truncated, PDZ-less
1-syntrophin at NMJs (Fig. 8). Ahn and Kunkel (1995) reported that the COOH-terminal fragment conserved among the three syntrophin homologues is sufficient to interact with utrophin. Therefore, it is possible that PDZ-less
1-syntrophin at NMJs recruits utrophin at the sarcolemma. Thus, the most prominent feature of NMJs in
1syn-/- muscle, shallow gutters, is not explained by the lack of utrophin expression. At the same time, we should point out that AChR is not colocalized with utrophin/dystrophin-associated proteins at the NMJs in
1syn-/- muscle on confocal microscopic analysis (Fig. 7). Therefore,
1-syntrophin might play a role in formation of highly organized NMJs by targeting functional molecules other than utrophin at the synaptic membrane.
1-Syntrophin might interact with other PDZ proteins such as membrane-associated guanylate kinase with inverted domain organization-1 (Strochlic et al., 2001), or the molecule might interact with another class of proteins, one which has a consensus sequence in its COOH-terminal (S/T/V-X-V-COOH) to interact with the PDZ domain, such as ErbB4 (Zhu et al., 1995) or muscle-specific receptor tyrosine kinase (Torres et al., 1998), at NMJs. Aberrant NMJs and deficits in exercise capacity in
1syn-/- mice were observed from an early stage of regeneration. Moreover, the temporal increase of slow muscle components early in the regenerating process might be regulated by the influence of slow nerves. Therefore, aberrant NMJs may be partly responsible for the deficit in exercise capacity of
1syn-/- mice.
The role of 1-syntrophin in skeletal muscle development and regeneration
Is aberrant NMJ formation related to muscle hypertrophy and/or reduced contractile force in the regeneration process? Mutant skeletal muscle showed neither hypertrophy nor reduced contractile force in the developmental stage without cardiotoxin treatment, although they do have aberrant NMJs. However, in regeneration hypertrophy becomes apparent 2 wk after toxin treatment, and the shallower synaptic gutter and enlargement of NMJs became more apparent at 48 wk after cardiotoxin treatment in 1syn-/- muscles (unpublished data). This disparity between developmental stage and regeneration process in
1syn-/- mice may be caused by the difference of expression patterns of
1-syntrophin and its associated factors between developmental and regeneration processes. AQP4 is abundantly expressed in the neonatal stage without accompanying expression of
1-syntrophin (Yokota et al., 2000), but it is not expressed until later in the regeneration process as shown in this study. AQP4 may not be related directly to hypertrophy and reduced contractile force in the regeneration process of
1syn-/- mice, but other molecules can show the same pattern of expression and explain the peculiar phenomenon. Accordingly,
1-syntrophin and its related molecules may have a particular role in regeneration, rather than in development, of skeletal muscle.
Muscular dystrophy and muscle hypertrophy
The primary absence of dystrophin is accompanied by a secondary deficiency of several dystrophin-associated molecules, including - and ß-dystroglycans, sarcoglycans, sarcospan, syntrophins, and dystrobrevins, from the sarcolemma (Ozawa et al., 1995). Therefore, molecular dissection of associated molecules by gene targeting is a powerful tool to clarify the molecular pathogenesis of muscular dystrophy. In particular, the expression of dystrophin-associated proteins other than nNOS is well preserved in
1syn-/- mice; therefore, the mutant mice are a good model to examine the functions of
1-syntrophin and its own downstream elements both in physiological and pathological conditions. In the present study, we clearly demonstrated that
1-syntrophin plays an important role in muscle regeneration: in the absence of
1-syntrophin, regenerating muscle showed a marked decrease in the exercise capacity and contractile force and an increase in muscle hypertrophy and aberrant NMJ formation. DMD patients and all dystrophin-deficient models (dogs, cats, and mice) pass through an early phase of muscle hypertrophy to some extent (Partridge, 1991). The deficit caused by the lack of
1-syntrophin may largely account for the phenomenon found in the early regeneration stage of DMD.
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Materials and methods |
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Cardiotoxin injection and tissue preparation
0.1 ml of 10 µM cardiotoxin (Wako Pure Chemical Industries) in 0.9% saline was injected directly into the right TA muscle with a 27-gauge needle under ether anesthesia. Mice were killed by cervical dislocation, and the cardiotoxin-injected TA muscles (right) and noninjected contralateral TA muscles (left) were removed for analysis at 1, 3, and 5 d, and 1, 2, 4, 8, 12, and 24 wk after injection. Body weight and wet muscle weight (TA) were measured. Several of the muscles were frozen in isopentane cooled by liquid nitrogen for histological, immunohistochemical analysis, and the other muscles were frozen directly in liquid nitrogen for RNA isolation, and stored at -80°C.
Hematoxylin and eosin staining
10-µm cryosections were cut in the middle part of the muscle belly to obtain the largest CSA, placed on poly-L-lysinecoated slides, air dried, and stained with hematoxylin and eosin. The sections were viewed and photographed using an HC-2500 digital camera systemTM (Fuji Photo Film Co., Ltd.).
Single muscle fiber isolation
We isolated single muscle fibers according to the method described previously (Rosenblatt et al., 1995) with a simple modification. In brief, the TA muscle was rinsed in PBS, put into a Petri dish containing 0.5% type I collagenase (Worthington Biochemical Corp.) in DME (Invitrogen), and incubated at 37°C for 1.53 h.
Immunohistochemical analysis
Antibodies.
Monoclonal antibodies BF-B6, BA-D5, or BF-F3 obtained from DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen Abt. Menschliche und Tierische Zellkulturen) detect neonatal type MHC, MHC I, or MHC IIb, respectively. Monoclonal antibody SC-71 (DSM) strongly labels MHC IIa and labels MHC IIx to a lesser extent. We concluded that the fibers negative for BA-D5, BF-F3, and SC-71 antibodies were type IIx. The following polyclonal antibodies were used for immunofluorescence: anti1-syntrophin (Asahi Techno Glass Co., Ltd.), anti-myogenin (Santa Cruz Biotechnology, Inc.), antiMyf-5 (Santa Cruz Biotechnology, Inc.), antiMyf-6 (Santa Cruz Biotechnology, Inc.), anti-MyoD (Santa Cruz Biotechnology, Inc.), antiutrophin (Imamura and Ozawa, 1998), anticaveolin-3 (Asahi Techno Glass Co., Ltd.), anti-NOS1 (Santa Cruz Biotechnology, Inc.), antisodium channel (III-IV Linker Region) (Upstate Biotechnology), and anti
-dystrobrevin-1 and -2 (Yoshida et al., 2000). Human ß1-syntrophin (195378 a.a.) was fused to GST in the pGEX vector (Amersham Biosciences) and maltose-binding protein in the pMAL-c2 vector (New England Biolabs, Inc.). The GST fusion ß1-syntrophin protein was used as an antigen. Obtained rabbit antiserum was purified with the affinity column coupled with the maltose-binding proteinß1-syntrophin fusion protein. The last 43 amino acids of mouse AQP4 were fused to GST in the pGET-1
T vector (Amersham Biosciences), and the recombinant proteins were purified and used to obtain rabbit polyclonal antibodies.
Immunofluorescence.
Acetone-fixed cryosections (6 µm) were blocked with 5% goat serum and 2% BSA in PBS and then incubated with primary antibody at 4°C overnight. 4% paraformaldehyde-fixed single muscle fibers were blocked with 20% goat serum in PBS and then incubated with a primary antibody in 0.35% carrageenan in PBS at 4°C overnight. FITC-conjugated antirabbit goat antibody (Biosource International) or Alexa 488labeled goat antirabbit IgG (H+L) (Molecular Probes) was used as the secondary antibody. To identify NMJs, AChRs were detected with Alexa 594labeled -BgTx (Molecular Probes). The sections were viewed and photographed by a laser scanning microscope, FLUOVIEWTM (Olympus). Z serial images were collected from whole-mount single fiber samples with a 63x oil objective using TCSSPTM (Leica). A single projected image was created by overlaying each set of z series images.
Northern blot analysis of IGF-1
A First-Strand cDNA Synthesis kitTM (Amersham Biosciences) was used to synthesize first strand cDNA from total RNA isolated from TA muscles of wild-type mice. IGF-1 cDNA was amplified by PCR using two oligonucleotide primers (5'-GTCTTCACACCTCTTCTACC-3' and 5'-CCTTCTGAGTCTTGGGCATGTCAG-3'). A 320-base pair PCR product covering exon 3 and part of exon 4 of the IGF-1 gene was cloned into a pCRR 2.1 vector (Invitrogen) and confirmed by sequencing using a DNA analysis system LIC-4200L-2TM (LI-COR, Inc.). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was amplified using RT-PCR Control Amplimer SetTM (CLONTECH Laboratories, Inc.) and cloned as described above. The cDNA fragment was digested with EcoRI (TaKaRa Shuzo Co., Ltd.) and labeled with 32P using a Random Primer DNA Labeling kit version 2TM (TaKaRa Shuzo Co., Ltd.). Total RNA was prepared from TA muscles by using RNAzolTM B (TEL-TEST, Inc.). 30 µg of total RNA was electrophoresed on a 1.0% denaturing agarose-formaldehyde gel, transferred to Hybond N+ nylon membraneTM (Amersham Biosciences) and heated at 80°C for 2 h. The hybridization was performed using 32P-labeled DNA probes. Prehybridization (30 min) and hybridization (overnight) were performed at 42°C in ULTRAhybTM hybridization buffer (Ambion). Washing was performed for 2 x 5 min at 42°C with 2 x SSC and 0.1% SDS, 2 x 15 min at 42°C with 0.1 x SSC and 0.1% SDS. The hybridized probe was detected and quantified using the Bio-Imaging analyzer BAS-2500TM (Fuji Photo Film Co., Ltd.).
Measurement of fiber CSA
The CSA of each fiber classified by the expression of MHC was measured using a Mac SCOPETM (Mitani Co.). We measured fiber CSA on the sections from three muscles in each group (noninjected and cardiotoxin-injected wild-type and 1syn-/- mice). A total of 200 fiber profiles were measured in the predetermined area of TA muscles. All fiber profiles traced in each group of muscles were pooled and plotted for their size distribution according to percent frequency.
Wire net holding test
The untreated male mice of 8-wk-old wild-type (n = 3) and 1syn-/- (n = 3) or the mice at 7 d8 wk after cardiotoxin injection into TA muscles of hind legs of 8-wk-old wild-type (n = 13) and
1syn-/- (n = 8) were examined. The mice were placed on a fine wire net. Then, the net was slowly turned over. The time until the mouse fell off was measured up to a maximum time of 300 s.
MHC isoform separation
The muscles frozen in isopentane cooled by liquid nitrogen were homogenized and extracted on ice for 60 min in 4 vol of buffer (pH 6.5) as described previously (Butler-Browne and Whalen, 1984). MHC separation on polyacrylamide gels containing 30% glycerol was performed according to the methods described previously (Agbulut et al., 1996) with simple modification. In brief, mini gels were made in the mini protein III Dual slab cell system (Bio-Rad Laboratories Inc.). 0.5 µg of total protein was run on each well. The upper buffer contained 10 mM of 2-mercaptoethanol. During electrophoresis, the temperature of the buffer was maintained at 5°C. After migration, the gels were silver stained using 2D-Silver Stain II "Daiichi" (Daiichi Pure Chemicals Co., Ltd.). The image was scanned and then analyzed using Lumi-Imager F1 software (Hoffmann-La Roche, Inc.).
Muscle physiology
Tetanic force and isometric twitch force of TA muscles were measured as described previously (Xiao et al., 2000). The entire TA was removed with its tibial origin intact, and the distal portion of the TA tendon and its origin were secured with a 50 silk suture. The TA was mounted in a vertical tissue chamber and was connected to a force transducer UL-50GR (Microtech) and length servosystem (Shimazu). Electrical stimulation using a SEN3301 (Nihon Kohden) was applied through a pair of platinum wires placed on both sides of the muscle in physiological soft solution (150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 5 mM Hepes, pH 7.4, 0.02 mM D-tubocurarine). The TA was positioned midway between the two electrodes. Muscle fiber length was adjusted incrementally by using a micropositioner until peak isometric twitch force (Pt) responses were obtained (i.e., optimal fiber length [L0]). The dependence of force generation on the rate of stimulation and maximum tetanic force (P0) was assessed by use of a range of stimulation frequencies (20, 50, 75, and 100 pulses per second) delivered in 500-ms duration trains with 2 min intervening between each train. After these measurements, the stimulated muscle was dried and weighed after tendon and bone attachments were removed. All forces were normalized for a dried CSA, the latter estimated on the basis of the following formula: dried muscle weight (in milligrams)/[L0 (in millimeters) x 1.056 (in milligrams per cubic millimeter)]. The estimated CSA was used to determine specific twitch forces (Pt/CSA) and specific tetanic forces (P0/CSA) of the muscles.
Statistical analysis
The relative weight of muscle, the number of myofibers and the expression of IGF-1 mRNA, the holding time on the wire net holding test, and the levels of MHC protein isoforms between wild-type and 1syn-/-, noninjected and cardiotoxin-injected muscle were compared using Student's t test. CSA distributions and mean fiber CSA were compared using F test and Student's or Welch's t test. P < 0.05 was considered statistically significant.
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Footnotes |
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* Abbreviations used in this paper:
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Acknowledgments |
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This work was supported by grants for Health Science Research for the Center of Excellence program, Human Frontier Science Program, Research on Nervous and Mental Disorders (10B, 13-B) from the Ministry of Health and Welfare, and Grant-in Aids for Scientific Research (11170264) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Submitted: 15 April 2002
Revised: 2 August 2002
Accepted: 5 August 2002
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