Human Molecular Genetics, 2000, Vol. 9, No. 9 1357-1367
© 2000 Oxford University Press
Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure
Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK and 1Department of Human Genetics, Center for Gene Therapy, University of Michigan Medical School, Ann Arbor, MI 48109-0618, USA
Received 21 January 2000; Revised and Accepted 17 March 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The X-linked muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle. Protein structure predictions, patient mutations, in vitro binding studies and transgenic and knockout mice suggest that dystrophin plays a mechanical role in skeletal muscle, linking the subsarcolemmal cytoskeleton with the extracellular matrix through its direct interaction with the dystrophin-associated protein complex (DAPC). Although a signaling role for dystrophin has been postulated, definitive data have been lacking. To identify potential non-mechanical roles of dystrophin, we tested the ability of various truncated dystrophin transgenes to prevent any of the skeletal muscle abnormalities associated with the double knockout mouse deficient for both dystrophin and the dystrophin-related protein utrophin. We show that restoration of the DAPC with Dp71 does not prevent the structural abnormalities of the post-synaptic membrane or the abnormal oxidative properties of utrophin/dystrophin-deficient muscle. In marked contrast, a dystrophin protein lacking the cysteine-rich domain, which is unable to prevent dystrophy in the mdx mouse, is able to ameliorate these abnormalities in utrophin/dystrophin-deficient mice. These experiments provide the first direct evidence that in addition to a mechanical role and relocalization of the DAPC, dystrophin and utrophin are able to alter both structural and biochemical properties of skeletal muscle. In addition, these mice provide unique insights into skeletal muscle fiber type composition.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The lack of dystrophin in muscle leads to the clinical features of Duchenne muscular dystrophy (DMD), a progressive degenerative muscle disease resulting in death by the early twenties. Dystrophin is a 427 kDa cytoskeletal protein that localizes around the entire sarcolemmal membrane and is concentrated at neuromuscular and myotendinous junctions in skeletal muscle (1).
Mutations in the dystrophin gene are also present in a variety of animal models including the mdx mouse (2,3). Although this mouse shows a skeletal muscle pathology indicative of muscular dystrophy, it does not display any of the clinical signs that affect dystrophin-deficient DMD patients and the mouse has an almost normal lifespan. However, direct studies of this useful animal model and experiments integrating transgenic mouse technology have contributed to the current understanding of dystrophin function.
Dystrophin is comprised of four protein domains (4). The N-terminal domain has been shown by in vitro binding assays and by transgenic mouse experiments to contain actin-binding sites that link dystrophin to the F-actin subsarcolemmal cytoskeleton (5–11). The rod domain contains a series of 24 spectrin-like repeats interrupted by four non-helical ‘hinge’ regions (12). The cysteine-rich domain has been shown by in vitro binding assays and transgenic mouse experiments to bind directly to ß-dystroglycan (13–15). ß-Dystroglycan binds to 
-dystroglycan which binds to the -2 chain of laminin in the extracellular matrix (ECM) (16). The dystroglycans are components of the dystrophin-associated protein complex (DAPC), together with the sarcoglycans, the syntrophins and dystrobrevin (17–19). The C-terminal domain of dystrophin contains the syntrophin-binding site (20–22), but this region is not essential for syntrophin localization in vivo (23). The membrane localization of both the transmembrane sarcoglycans and ß-dystroglycan and the cytoplasmic syntrophins and dystrobrevin are dependent upon the presence of dystrophin at the membrane. In DMD and mdx muscle, the entire protein complex is lost from the muscle membrane (24,25). In the past several years, other forms of muscular dystrophy have been shown to be caused by mutations in genes encoding four of the sarcoglycans (reviewed in refs 17,18), suggesting that any perturbation of this protein complex will result in a similar muscle pathology.
Protein sequence analyses together with physiological function studies support a role for dystrophin in a mechanical link between the F-actin cytoskeleton and the ECM through its direct interaction with the DAPC (16,26,27). Protein structure predictions and studies of recombinant protein fragments suggest that the rod domain of dystrophin confers flexibility and elasticity on the molecule (28–30). Measurements of normalized force generation of muscles from mdx mice show a reduction of ~50% compared with control littermates (27).
In vivo evidence of the importance of the connection between the actin cytoskeleton and the DAPC comes from transgenic mouse experiments. A dystrophin transgene deleted for the majority of the rod domain (
17–48) overexpressed in mdx skeletal muscle is sufficient for localization of the DAPC to the membrane, prevention of muscle degeneration and nearly normal muscle force generation (31,32). This dystrophin molecule is missing 46% of its coding sequence and is based on a mutation in a patient with an extremely mild form of Becker muscular dystrophy (33). These data suggest that the majority of the rod domain is not critical for dystrophin mechanical function. The expression of a dystrophin transgene (Dp71) comprising only the cysteine-rich and C-terminal domains is sufficient for localization of the DAPC to the muscle membrane in mdx mice, but is unable to prevent any of the muscle pathology (34,35). These data suggest that for a dystrophin protein to be mechanically functional, it must restore the link between actin and the DAPC and that restoration of the DAPC alone is not sufficient.
Another transgenic mouse experiment tested the importance of DAPC localization. Consecutive regions throughout the cysteine-rich and C-terminal domains were deleted from an otherwise full-length dystrophin protein and expressed from transgenes in mdx skeletal muscle. These experiments confirmed in vitro evidence that exons 64–70 of dystrophin are necessary for binding to ß-dystroglycan and showed that this binding is required for the localization of the sarcoglycans to the membrane (14). Dystrophin proteins deleted for the cysteine-rich domain were unable to restore the DAPC to the membrane and were also unable to prevent any of the muscle pathology. These transgenic experiments provide the first direct in vivo evidence that both the N-terminal actin-binding domains and the predicted DAPC-binding domains were needed for normal dystrophin function and to prevent a dystrophic pathology and further supported a mechanical role for dystrophin.
Two other hypotheses for how the lack of dystrophin leads to muscle degeneration include the leaky membrane theory and the calcium theory. However, neither of these mechanisms appear to be inherent to dystrophic muscle and may represent secondary events associated with muscle degeneration (reviewed in ref. 36).
Utrophin is a paralog of dystrophin that is localized exclusively in the neuromuscular junction (NMJ) and myotendinous junction of adult mammalian skeletal muscle (37,38). When utrophin is overexpressed from a human skeletal actin promoter in transgenic mdx skeletal muscle and localizes around the entire sarcolemmal membrane it is able to compensate for dystrophin deficiency and prevent muscle pathology (39). Mice homozygous for a targeted knockout mutation of utrophin do not show any signs of muscular dystrophy, but only display a very mild myasthenia (40,41). However, when utrophin deficiency is combined with the dystrophin-deficient mdx mouse, a much more severe progressive muscular dystrophy is produced, suggesting that dystrophin and utrophin play synergistic roles in skeletal muscle (42,43). Utrophin/dystrophin-deficient double knockout (dko) mice show all of the clinical features of DMD, including short stature, joint contractures, labored breathing and death by 20 weeks of age. The fact that dko mice die despite the fact that their skeletal muscle degeneration is similar to that of mdx mice suggests that dystrophin and utrophin have additional functions besides providing a ‘mechanical’ link.
dko muscle has a complete lack of folding of its post-synaptic NMJ membranes (42,43) and expresses various proteins in a pattern similar to developing muscle (42,44). Evaluation of physiological function showed the dko soleus to be resistant to fatigue relative to control, utrophin-deficient and mdx soleus muscles (45). In addition, analysis of myosin heavy chain (MHC) composition showed that there was a shift towards slower MHC isoforms in dko soleus, extensor digitorum longus (EDL) and diaphragm muscles compared with these muscles from mdx littermates (45). These data suggest that the utrophin/dystrophin-deficient mouse has an alteration in oxidative/glycolytic muscle fiber ratios, but do not provide direct evidence of this situation.
We therefore used this utrophin/dystrophin-deficient (dko) mouse as a null background to investigate non-mechanical roles of dystrophin. We assayed a number of morphological and biochemical features in dko mice expressing one of three classes of truncated dystrophin transgenes. Expression of a rod-domain-deleted dystrophin (17–48) was used to determine whether this mechanically functional protein could ameliorate the entire range of abnormalities of the dko mouse. The Dp71 transgene was used to examine whether restoration of the DAPC was sufficient to ameliorate any of the abnormalities. Full-length dystrophin deleted for only the cysteine-rich domain (Cys or Cys-2) was used to investigate whether a dystrophin that was unable to restore the DAPC could ameliorate any of the abnormalities. The results presented here suggest that in addition to a mechanical role in physiological function and relocalization of the DAPC, dystrophin and the dystrophin-related protein utrophin are able to alter biochemical properties of skeletal muscle and structural properties of the post-synaptic membrane.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have previously reported several abnormalities characteristic of the dystrophin/utrophin-deficient dko mouse (42) and have more recently shown that these abnormalities are caused by the deficiency of full-length utrophin and dystrophin in skeletal muscle only (44). To understand why the lack of both dystrophin and utrophin, but not the absence of either individually, leads to the severe clinical features of the dko mouse, we decided to delineate further phenotypic differences between mdx and dko skeletal muscle. The most intriguing differences that we observed previously were associated with the ultrastructure of the post-synaptic membrane (PSM) and with abnormal expression of MHCs (42) and their effect on the physiological function of dko skeletal muscles (45).
Utrophin/dystrophin-deficient muscle has abnormal motor endplate topology
Previous studies have shown that dko skeletal muscle displays a complete absence of PSM folding. To analyze a large number of NMJs in multiple mouse mutants we utilized a fluorescent method to label acetylcholine receptors (AchRs) in thick, longitudinal sections of skeletal muscle. Using this technique, we were able to view the en face topology of motor endplates of all of the NMJs present in a single plane of an entire longitudinally sectioned muscle. Tibialis anterior (TA) and quadriceps muscles from wild-type C57Bl/10 mice had a pattern of AChR staining that was smooth and continuous in appearance (Fig. 1a and b). Although TA and quadriceps from mdx mice had some PSMs with a normal continuous pattern of AChR staining (Fig. 1c), 79% of PSMs in quadriceps and 68% of those in TA showed a discontinuous pattern of AChR staining, defined by two or more discrete synaptic boutons (Fig. 1d). In dko muscles 94% of PSMs in TA and 93% in quadriceps were discontinuous (Fig. 1e). In addition, the only continuous staining patterns were much smaller in area than in wild-type or mdx muscles (Fig. 1f). These results are similar to previous studies analyzing a limited number of NMJs using electron microscopy, thus supporting the use of this approach to study the structure of NMJs (46).
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Utrophin/dystrophin-deficient muscle lacks glycolytic fibers
We have previously shown that dko soleus is resistant to fatigue relative to mdx soleus muscle and that dko EDL, diaphragm and soleus muscles display a shift in MHC composition towards ‘slower’ isoforms (45). However, immunofluorescence analysis of MHC isoforms in dko muscle is of limited value in defining fiber type composition since, from 2 weeks of age, several MHC isoforms are co-expressed within many fibers (42,45). To directly evaluate the oxidative/glycolytic state of dko muscles, we stained cross-sections of TA, quadriceps, soleus, EDL and diaphragm muscles of wild-type, mdx and dko mice for the mitochondrial enzyme NADH-tetrazolium reductase. Sections of quadriceps (Fig. 2), TA and EDL (data not shown) muscles from wild-type (Fig. 2a) and mdx (Fig. 2b) mice had a characteristic ‘patchwork’ pattern of staining with a mixture of stained oxidative and unstained glycolytic fibers. Gastrocnemius muscles from wild-type and mdx mice have small regions containing a mixture of oxidative and glycolytic fibers, but consist mostly of unstained glycolytic fibers. In striking contrast, sections of TA, quadriceps, EDL and gastrocnemius muscles from dko mice displayed a uniform pattern of staining throughout all muscle sections (Fig. 2c and data not shown), confirming that all fibers from these muscles were capable of oxidative metabolism and further suggesting that no exclusively glycolytic fibers are present. Since mdx and dko muscle show the same level of regenerating fibers (42,45), this assay is comparing equivalent samples. Since almost all fibers in the diaphragm and soleus of normal mice are oxidative, these tissues are uninformative in this analysis.
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We also performed ATPase histochemical staining on sections from mdx and dko soleus to examine the myosin ATPase composition of these muscles. Type I fibers are defined by an alkaline-labile/acid-resistant ATPase activity. Type II fibers are defined by an acid-labile/alkaline-resistant ATPase activity. In wild-type and mdx muscle each fiber has only a single ATPase activity and stains only after one of the two separate pH incubations. In contrast, dko soleus has a significant proportion of fibers that stain after both acid and alkali incubations (data not shown), indicating an abnormality of this parameter of fiber type.
A non-mechanically functional dystrophin is able to ameliorate NMJ and fiber type abnormalities in dko skeletal muscle
To explore whether any of the NMJ or fiber type abnormalities in dko muscle could be corrected by expression of mutant forms of dystrophin, we crossed two transgenic lines that express truncated dystrophins onto the dko background. The two lines used, Cys and Cys-2, express transgenes deleted for portions of the dystrophin cysteine-rich domain (Fig. 3). These proteins both localize to the muscle sarcolemma, yet are unable to stabilize the dystroglycan and sarcoglycan subcomplexes (14). Due to this disruption in the link between the cytoskeleton and the ECM, the Cys proteins are unable to prevent the dystrophic pathology of mdx skeletal muscle and are not ‘mechanically functional’ dystrophin molecules.
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Muscles from
Cys/dko mice display a dystrophic pathology (Fig. 4) and percentages of centrally nucleated fibers similar to those seen in Cys/mdx littermates. To investigate whether any parameters of dko skeletal muscle pathology were ameliorated by the Cys transgene, the percentages of continuous and discontinuous patterns of AChRs were determined. Over 40% of PSMs in TA and almost 50% of PSMs in quadriceps showed a continuous pattern of localization with less than two isolated synaptic boutons (Fig. 6e). These proportions are significantly different ( = 0.05) compared with the results obtained from dko muscles (Figs 5 and 6e and f), indicating that the Cys dystrophin protein is able to restore a normal topology at the PSM.
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To investigate whether
Cys dystrophin has any effect on the oxidative/glycolytic metabolism of dko muscle, we analyzed the pattern of NADH-tetrazolium reductase staining in Cys/dko and Cys-2/dko muscles. Sections of both TA and quadriceps (Fig. 7c and d) muscles from these mice showed the presence of glycolytic fibers and a pattern of stained and unstained fibers similar to that observed in mdx muscles (Fig. 2b). These data indicate that the Cys and Cys-2 dystrophin proteins are able to maintain a normal fiber type distribution in dko muscle despite an inability to prevent any of the gross morphological features of muscular dystrophy.
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Restoration of the DAPC with a C-terminal dystrophin protein does not prevent any dko skeletal muscle abnormalities
Since an otherwise full-length cysteine-rich-deleted dystrophin protein unable to bind and stabilize either dystroglycan or the sarcoglycans was able to support normal NMJ structure and fiber type, we next asked whether simple stabilization of other portions of the DAPC would rescue these abnormalities of dko skeletal muscle. We therefore crossed dko mice with transgenic mdx mice expressing the Dp71 isoform of dystrophin. The Dp71 transgene consists of only the cysteine-rich and C-terminal domains of dystrophin (Fig. 3) and has been shown to be sufficient for localization of the entire DAPC to the sarcolemma, but is unable to prevent any of the dystrophic pathology of mdx skeletal muscle (34).
Although the Dp71 dystrophin protein localizes to the sarcolemma and is able to restore the DAPC to the membrane of dko muscle fibers (data not shown), it is unable to ameliorate the muscle degeneration seen in the dko mouse (Fig. 4). The percentage of centrally nucleated fibers in the TA and quadriceps muscles of the Dp71/dko mouse is identical to that seen in the muscles of mdx littermates (data not shown).
To investigate whether other parameters of dko skeletal muscle pathology were ameliorated in these mice, Dp71/dko TA and quadriceps muscles were stained to assay the distribution of AChRs at the PSMs. Dp71/dko muscles showed a percentage of endplates with continuous synaptic boutons (Fig. 6c and d) not statistically different (at the 95% confidence level) from that seen in dko muscle (Fig. 5). These data demonstrate that neither the Dp71 dystrophin protein nor stable expression of the DAPC by itself is able to ameliorate this abnormality in the dko mouse.
To investigate whether the Dp71 transgene has any effect on the oxidative/glycolytic metabolism of dko muscle, we analyzed the pattern of NADH-tetrazolium reductase staining in TA and quadriceps muscles of Dp71/dko mice. These muscles showed a uniform pattern of staining identical to that seen in dko muscles (Fig. 7), suggesting that all muscles fibers are capable of oxidative metabolism and that the Dp71 protein is unable to ameliorate this abnormality. Muscles from Dp71/mdx mice have a normal pattern of oxidative and glycolytic fibers (data not shown), indicating that neither the Dp71 protein nor the DAPC is able to affect fiber type composition.
A rod domain-deleted dystrophin protein prevents all abnormalities of dko skeletal muscle
To test whether the region of the rod domain encoded by exons 17–48 is necessary for the phenotypic amelioration achieved with the Cys transgenes, transgenic/mdx mice lacking exons 17–48 were crossed onto the dko background. The 17–48 transgene lacks the region of the rod domain of dystrophin between hinges 2 and 3 (Fig. 3). This dystrophin protein contains all N-terminal actin-binding sites and complete cysteine-rich and C-terminal domains and has been shown to restore the mechanical function of mdx muscle to nearly wild-type levels (31). The 17–48 transgene is localized uniformly at high levels around the sarcolemma in all skeletal muscles (i.e. diaphragm, EDL, quadriceps, soleus and TA) of 17–48/dko mice analyzed. In quadriceps, the transgene product is present at ~10-fold higher levels than that of endogenous dystrophin in wild-type muscle. The expression of this transgene was able to prevent all of the clinical signs of the dko mouse until at least 18 months of age. Muscle degeneration in 17–48 mice was nearly absent in all of the muscles analyzed (Fig. 4) and the percentage of centrally nucleated fibers was identical to that of 17–48/mdx littermates (data not shown).
To investigate whether the abnormal PSM topography of dko muscles was also prevented by expression of the transgene in 17–48/dko muscles, localization of the AChRs in TA and quadriceps muscles were viewed en face. PSMs of both 17–48/dko muscles and 17–48/mdx muscles showed nearly wild-type levels (Fig. 5) of continuous junctions (Fig. 6a and b), demonstrating that this truncated dystrophin protein is capable of preventing this abnormality.
To determine whether the prevention of all of the above parameters correlates with the restoration of normal oxidative/glycolytic metabolism, sections from TA and quadriceps muscles were analyzed for the presence of NADH-tetrazolium reductase activity. 17–48/dko muscles showed a normal pattern of stained oxidative and unstained glycolytic fibers (Fig. 7a). These data indicate that all of the abnormalities of the dko mouse are prevented by this rod domain-deleted protein. In addition, we have previously described a transgenic/dko mouse that is ‘rescued’ due to expression of a rod domain-deleted utrophin transgene based on the 17–48 dystrophin deletion (44). Both the PSM topology and NADH staining (data not shown) are identical to those seen in the dystrophin 17–48/dko mouse, suggesting that either a mechanically functional dystrophin or utrophin is capable of preventing all of the abnormalities of the dko mouse and that the region encoded by exons 17–48 is not critical for this improvement.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The studies described in this manuscript show that mice deficient in both utrophin and dystrophin have alterations in skeletal muscle fiber types and abnormalities of PSM topography that are worse than in mdx littermates. Surprisingly, a truncated dystrophin protein with defective binding to dystroglycan is able to ameliorate these phenotypic features of the dko pathology. These data show that dystrophin has a role in skeletal muscle that is not directly dependent on the mechanical link between F-actin and the dystroglycan/sarcoglycan subcomplexes of the DAPC.
Although the dystrophin-deficient mdx mouse displays progressive muscle degeneration, these mice show no gross phenotypic abnormality and live up to 2.5 years of age; nearly as long as wild-type mice (J.S. Chamberlain, unpublished data). Utrophin deficiency alone causes only a very mild myasthenia and these mice also have a normal lifespan (40,41; unpublished data). When utrophin is also absent from the mdx mouse, gross clinical features comparable to human DMD and premature death result (42,43). The discontinuous junctions observed in mdx muscle have been suggested to be due to degeneration of the muscle fibers (46–48). Our previous analyses have shown that the muscle degeneration and regeneration that occur in dko and mdx mice are equivalent, as determined by the percentages of centrally nucleated fibers and by the uptake of small molecular weight dyes (42,43). Therefore, since nearly all PSMs in dko mice are discontinuous, degeneration alone cannot account for this observation. Dystrophin has been suggested to be an integral member of the cytoskeleton which as a whole is critical for the aggregation of ion channels and transmitter receptors (reviewed in ref. 49). In this model, the absence of dystrophin leads to abnormal AChR–cytoskeleton interactions. More recently, it has been directly demonstrated that dystrophin is required for organizing large AChR aggregates from smaller clusters during muscle regeneration (50). It is possible that in mdx muscle utrophin might compensate for this role and that our observations of AChR clustering in the dko mouse are due to a total loss of this interaction.
The physiological function parameter that showed the most significant differences between dko muscles and those from their mdx littermates was alterations in fiber type, in that dko muscles appeared to be ‘slower’ or more oxidative. MHCs are the major determinant of myosin ATPase activity, which in turn determine the maximal velocity of muscle contraction (vmax). Slow fibers normally express Type I MHC, have large numbers of mitochondria and an oxidative metabolism, have an acid-resistant myosin ATPase activity and are red in appearance due to high concentrations of myoglobin. Fibers with fast velocities of contraction have an acid-labile myosin ATPase activity (Type II fibers) and are comprised of one of three different isoforms of MHC with relative vmax IIb > IIx > IIa. Type IIb fibers are exclusively glycolytic and are white in appearance due to the absence of myoglobin. Types IIa and IIx are capable of both oxidative and glycolytic metabolism.
Although the first physiological studies suggesting that different muscles had different velocities of contraction were carried out over 30 years ago (51) and the MHC isoforms of the contractile apparatus have been identified (reviewed in ref. 52), the basis for skeletal muscle fiber types is still not well understood. Early experiments demonstrated that a change in the electrical stimulation pattern to a muscle can alter the velocity of contraction with which it responds. For instance, if a slow muscle is re-innervated with a nerve that formerly innervated a fast muscle, the slow muscle becomes fast (53). However, changes resulting from a particular stimulus differ between species (reviewed in 54). A stimulation protocol of a fast muscle that leads to an increase in slower fibers in the rabbit and rat does not cause overt changes in the ATPase fiber types of mouse fast-twitch muscle. However, this stimulation protocol does lead to an increase in the succinate dehydrogenase and NADH-tetrazolium reductase activities (54).
Until recently there were no clues to the molecular factors that linked electrical stimulation through the NMJ to the change in physiological function of the muscle and all of the parameters that define a slow or fast muscle. Recently, calcineurin was identified as having the capacity to selectively activate the promoters of the slow fiber-specific proteins troponin I slow (TnIs) and myoglobin in vitro (55). Surprisingly, another recent experiment shows that mice with a targeted disruption of myoglobin exhibit normal exercise capacity and a normal ventilatory response to low oxygen levels. Heart and soleus muscles from these animals are depigmented, but function normally in standard assays of muscle performance in vitro across a range of work conditions and oxygen availability (56). These conclusions indicate that a complete understanding of the requirements for oxidative metabolism in skeletal muscle fibers remains elusive.
Muscle fibers from DMD patients lack the order and predictable relationship of fiber type characteristics present in normal adult muscle (57). DMD muscle shows a selective loss of NADH light fibers and an increase in NADH dark fibers consistent with those observed in dko muscle. Prolonged contraction times and an increase in mitochondrial enzymes have been found in DMD muscle (58) and, together with the above histological data, suggest a switch from glycolytic to oxidative metabolism in muscles from DMD patients. A 31P magnetic resonance spectroscopy study has shown that skeletal muscle from Becker muscular dystrophy patients was less acidic than controls after prolonged exercise, also suggesting a defective glycolytic activity in dystrophin-deficient muscle (59). Although they are not an equivalent genetic model, these data suggest more similarities between dko and DMD muscle.
The data presented in this manuscript show that a rod-domain-deleted dystrophin that is able to interact with both the subsarcolemmal actin cytoskeleton and the DAPC complex is able to prevent essentially all of the skeletal muscle abnormalities associated with the dko mouse. Since this mechanically functional dystrophin transgene is expressed at high levels in all skeletal muscles of the dko mouse, it is also able to prevent all of the clinical features and premature death of this mouse. These data are the same as those observed with expression of an analogous utrophin transgene (44). These combined data suggest that expression of either a truncated dystrophin or utrophin uniformly around the skeletal muscle membrane is able to compensate for the absence of both full-length proteins.
It has previously been shown that dystrophin must interact with both F-actin and the DAPC for it to link the cytoskeleton and the ECM and prevent muscle degeneration and reduced force generating capacity of the mdx mouse (14,27,31,34). However, the degree of muscle degeneration in the dko mouse is not sufficiently different from mdx muscle to explain the severe clinical features. A critical question is whether the loss of small amounts of utrophin present at the membrane in mdx muscle might compensate to a small extent for dystrophin loss, providing a mechanical link in the absence of dystrophin, or whether dystrophin and utrophin play a non-mechanical role in skeletal muscle. Cys and Cys-2 are nearly full-length dystrophin proteins deleted for consecutive exons that together encode the critical region of the ß-dystroglycan-binding site (14). Neither of these dystrophin proteins is able to restore the dystroglycans or sarcoglycans to the muscle membrane in mdx or dko muscle (14; data not shown). However, this mechanically non-functional protein is able to ameliorate both the post-synaptic membrane topographical abnormality and the fiber type abnormality of dko skeletal muscle. The Dp71 transgene, which comprises the cysteine-rich and C-terminal domains of dystrophin and corresponds to the major isoform of dystrophin expressed in non-muscle tissues, was unable to prevent any of the abnormalities of the dko mouse. These data suggest that the DAPC, including the dystroglycans, sarcoglycans and syntrophins, do not play a role in the amelioration of these abnormalities.
Since the Dp71 protein encodes exons 63–79 and is unable to ameliorate dko abnormalities, and the absence of exons 17–48 does not interfere with correction of these phenotypes, either sequence upstream of exon 17 or between exons 48 and 63 must encode the dystrophin region responsible for the improvement in dko abnormalities produced by the Cys transgenic protein. This amelioration may be due to dystrophin sequence itself or to an as yet unidentified protein that binds to one of these regions. The remainder of the abnormalities in Cys/dko muscle are similar to those seen in mdx mice and may be due to the degenerative process of the muscle itself, resulting from the loss of mechanical function of dystrophin.
It is not clear from the data presented here whether dystrophin and utrophin have a direct effect on fiber type characteristics or whether their role in maintaining the structure of the PSM indirectly affects fiber type, since the two parameters are always correlated. However, these data are the first to suggest a non-mechanical role for the very large dystrophin and utrophin cytoskeletal proteins. These experiments also provide insight into the complicated process of fiber type determination and suggest the need for a further understanding of the signaling process between the NMJ and the skeletal muscle fiber.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse breeding and genotyping
mdx mice heterozygous for a utrophin knockout mutation (utrn+/–; mdx) were mated with mdx mice positive for one of the four transgenes. Progeny of the genotype utrn+/–; mdx who were also positive for the transgene (Tg/utrn+/–; mdx) were mated to each other to produce Tg/utrn–/–; mdx (abbreviated Tg/dko, where Tg is Dp71,
Cys, Cys-2 or 17–48). These transgenic lines correspond to the previously published lines as follows: Dp71 = MCA-1 (34); Cys = 64–67; Cys-2 = 68–70 (14); 17–48 = 11956 CVBA3" (31). Tg/utrn+/+; mdx littermates were used as controls. Cys-2 data were excluded from some analyses because of the low numbers of Tg/dko mice obtained from that line. However, the Cys-2/dko data appeared equivalent to the data obtained for larger numbers of Cys/dko mice. All progeny resulting from these crosses were genotyped for their utrophin knockout allele status and the presence of each transgene by PCR as previously described (14,31,34,42).
Skeletal muscle morphology and central nuclei counts
Ten-week-old mice were killed and tissues excised, embedded in OCT mounting medium (BDH, Poole, UK) and frozen in liquid-nitrogen-cooled isopentane. Frozen sections (8 µm) were cut on a Bright cryostat (Hacker Lab, Fairfield, NJ) and used for either NADH or hematoxylin and eosin staining as described below. One slide of each muscle sample was stained with hematoxylin and eosin as standard. Fibers containing centralized nuclei were divided by the total number of fibers within a field to obtain the percentage of centrally nucleated fibers. At least 500 fibers were counted from each pair of muscles obtained from three mice of each genotype.
NADH staining
Unfixed frozen sections (8 µm) were incubated for 30 min at 37°C in 0.2 M Tris, pH 7.4, 1.5 mM NADH and 1.5 mM Nitroblue Tetrazolium (all from Sigma, St Louis, MO) (60). After incubation, slides were rinsed in 30, 60, 90, 60 and 30% acetone for 2 min each and then mounted in Aquamount (BDH). Slides were viewed with a Leica DMRE microscope and photographed using a Leica DMLD photomicrograph system.
Bungarotoxin staining and statistical analysis
Tibialis anterior and quadriceps muscles from 10-week-old mice were excised, fixed in 1% paraformaldehyde in potassium phosphate-buffered saline (KPBS) for 1 h at room temperature and then placed in a 20% solution of sucrose in KPBS as a cryoprotectant for 1 h or more. Muscles were then covered in OCT mounting medium and placed flat on cork discs. The cork disc containing the muscle sample was then submerged in a liquid phase of isopentane cooled in liquid nitrogen until frozen. Frozen muscles were then cut into 30 µm longitudinal sections, placed onto positively charged microscope slides (Superfrost Plus; BDH) and stored at –80°C.
Immediately before staining, sections were thawed, allowed to reach room temperature and then equilibrated in KPBS for 5 min. Slides were then washed gently in 0.1 M glycine in KPBS for 1 h on a rocker, extracted with 0.5% Triton X-100 in KPBS on ice and rinsed in KPBS for 10 min at room temperature. Sections were then blocked for 1 h at room temperature in 1% gelatin and then incubated for 3 h in 5 ng/µl Alexa 488-conjugated -bungarotoxin (Molecular Probes, Eugene, OR). Slides were then washed three times for 15 min in KPBS + 0.1% Tween-20 and finally mounted in Vectashield (Vector Laboratories, Burlingame, CA) and coverslipped. Slides were viewed with a Leica DMRE microscope and Sensys digital camera (Photometrics Ltd, Munchen, Germany) using Q-FISH software.
All PSMs stained for AChR were scored as either ‘continuous’ or ‘discontinuous’. Discontinuous AChR clusters were defined as those with two or more discrete synaptic boutons. All scorable PSMs from each section were counted. Since different numbers of PSMs were counted for each sample, no standard error could be calculated.
To determine if there are differences between the genotypes, ANOVA was used. In the ANOVA, the arcsine transformation of the proportion of continuous PSMs was used to stabilize the variance. In the event that the ANOVA detected a significant difference between genotypes, Fisher’s least significant difference test was employed to test all pairwise comparisons at the = 0.05 level.
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ACKNOWLEDGEMENTS |
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We would like to thank Pam Goodman of the Biostatistics Program at The Ohio State University for carrying out the statistical analysis of the data and William Miller for reading the manuscript. This work was supported by the Muscular Dystrophy Association, the Association Française Contre les Myopathies, the Medical Research Council and the Muscular Dystrophy Campagin of Great Britain. J.A.R. was supported by a Burroughs Wellcome Fund Hitchings-Elion Fellowship.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Molecular and Cellular Biochemistry, 410 Hamilton Hall, The Ohio State University, College of Medicine, 1645 Neil Avenue, Columbus, OH 43210, USA. Tel: +1 614 292 7043; Fax: +1 614 292 4118; Email: rafael.1@osu.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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