|Home | About | Journals | Submit | Contact Us | Français|
Sarcoglycans are a group of single-pass transmembrane glycoproteins. In striated muscle, sarcoglycans interact with dystrophin and other dystrophin-associated proteins (DAPs) to form the dystrophin-associated glycoprotein complex (DGC). The DGC protects the sarcolemma from contraction-induced injury. Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations. In the absence of dystrophin, the DGC is disassembled from the sarcolemma. This initiates a chain reaction of muscle degeneration, necrosis, inflammation and fibrosis. In contrast to human patients, dystrophin-null mdx mice are only mildly affected. Enhanced muscle regeneration and the up-regulation of utrophin and integrin are thought to protect mdx muscle. Interestingly, trace amounts of sarcoglycans and other DAPs can be detected at the mdx sarcolemma. It is currently unclear whether sub-physiological sarcoglycan expression also contributes to the mild phenotype in mdx mice. To answer this question, we generated δ-sarcoglycan/dystrophin double knockout mice (δ-Dko) in which residual sarcoglycans were completely eliminated from the sarcolemma. Interestingly, utrophin levels were further increased in these mice. However, enhanced utrophin expression did not mitigate disease. The clinical manifestation of δ-Dko mice was worse than that of mdx mice. They showed characteristic dystrophic signs, body emaciation and more macrophage infiltration. Their lifespan was reduced by 60%. Furthermore, δ-Dko muscle generated significantly less absolute muscle force and became more susceptible to contraction-induced injury. Our results suggest that sub-physiological sarcoglycan expression plays a critical role in ameliorating muscle disease in mdx mice. We speculate that low-level sarcoglycan expression may represent a useful strategy to palliate DMD.
The dystrophin gene is the first human disease gene identified by the reverse genetics approach (1). Mutations in the dystrophin gene lead to Duchenne muscular dystrophy (DMD), the most common form of lethal muscle disease. In normal muscle, dystrophin and dystrophin-associated proteins (DAPs) form the multimeric dystrophin-associated glycoprotein complex (DGC) to maintain the sarcolemmal stability. Within the DGC, DAPs are organized into three distinctive subcomplexes including the dystroglycan subcomplex, sarcoglycan–sarcospan subcomplex and cytoplasmic subcomplex (2). The dystroglycan subcomplex is composed of α- and β-dystroglycans. Dystrophin and the dystroglycan subcomplex constitute the physical link between laminin-2 in the extracellular matrix and the F-actin cytoskeleton. On one hand, dystrophin binds to F-actin through its N-terminal and rod domains. On the other hand, dystrophin binds to the transmembrane protein β-dystroglycan through its cysteine-rich domain. β-dystroglycan is connected to laminin-2 through α-dystroglycan. The sarcoglycan–sarcospan subcomplex is composed of α-, β-, γ- and δ-sarcoglycans and sarcospan. This subcomplex is thought to stabilize α- and β-dystroglycan interaction as well as dystrophin and β-dystroglycan interaction (3). The cytosolic subcomplex includes dystrobrevin and syntrophin. These are important scaffolding proteins involved in signal transduction.
The pathogenic mechanism(s) of DMD are not completely understood. Currently, mechanical and/or signaling defects are the prevailing hypotheses. According to these models, the absence of dystrophin destabilizes the DGC. Consequently, the physical link across the sarcolemma is interrupted and the DGC-mediated signal transduction pathways are also impaired. As a result, muscle cells become highly susceptible to contraction-induced injury. Eventually, muscle cells undergo necrosis and are replaced by connective tissues.
Interestingly, the clinical consequences of dystrophin gene mutations vary dramatically among species. The absence of dystrophin leads to severe muscular dystrophy and premature death in human patients. However, dystrophin-deficient mdx mice are only mildly affected. Despite histological signs of muscle degeneration, necrosis and inflammation, most of the body muscles are efficiently regenerated in adult mdx mice. The only exception is the diaphragm which shows prominent fibrosis and severe force deficit as seen in DMD patients (4). Overall, adult mdx mice show little weakness. Characteristic dystrophic changes (such as muscle wasting and dilated cardiomyopathy) are seen only in very old mdx mice (≥20-month-old) (5,6). Another significant difference is the life span. The life span of DMD patients is shortened by ~60–70%. Compared with wild-type mice, the life span of mdx mice is only reduced by ~20% (6).
In the majority of DMD patients, the phenotype correlates well with the genotype (7–10). In-frame mutations often lead to mild Becker type muscular dystrophy while out-of-frame mutations in general result in severe Duchenne-type muscular dystrophy (7–10). However, exceptions to the reading frame rule have been found in 5–10% DMD patients and up to 30% BMD patients (8,11,12). The phenotypic variations among human patients and between humans and mice suggest the existence of genetic modifiers that may compensate for dystrophin deficiency (13). A better understanding of these genetic modifies/compensatory mechanisms may bring in new perspectives on therapy development.
Currently, the best understood compensatory mechanisms include utrophin and integrin up-regulation and enhanced muscle regeneration. Utrophin is a structural and functional homologue of dystrophin. Similar to dystrophin, utrophin can also stabilize the sarcolemma by organizing DAPs into the utrophin-associated glycoprotein complex (UGC) (14). Utrophin is selectively expressed in the neuromuscular junction in normal mice but it is up-regulated throughout the sarcolemma in mdx mice (15). Removing utrophin from mdx mice results in severe dystrophic phenotype (16,17). Increasing utrophin expression by genetic and/or pharmacological approaches ameliorates muscle disease (18–20).
α7β1 integrin is another major laminin receptor in muscle (21,22). In both DMD patients and mdx mice, α7β1 integrin expression is increased (23). Transgenic over-expression of α7 integrin rescues muscular dystrophy in utrophin/dystrophin double knockout mice while α7 integrin/dystrophin double knockout mice show early onset muscular dystrophy and premature death (24–26).
Besides compensatory protein up-regulation, enhanced muscle regeneration also contributes to the mild disease in mdx mice. This is demonstrated by muscle hypertrophy and the prevalence of centrally located nuclei in mdx skeletal muscle. Most importantly, blocking regeneration by irradiation or inactivating the myogenic transcription factor MyoD leads to severe myopathy (27–29).
It is currently unknown whether these are the only factors contributing to the benign phenotype in mdx mice. It has been shown that the DAPs are not completely eliminated from the mdx sarcolemma (30). However, restoring the DAPs to normal levels does not ameliorate muscle disease (31–34). The biological role of residual DAPs in mdx mice is yet to be elucidated. We recently demonstrated that a seemingly insignificant level of dystrophin expression (~5%) helped preserving muscle force in dystrophin-deficient mice (35). Here, we hypothesized that low-level sarcoglycan expression may represent a yet unrecognized modifier in DMD pathogenesis. We further hypothesized that a complete elimination of the sarcoglycan complex (SGC) from mdx muscle may aggravate muscle disease.
To test this hypothesis, we generated δ-sarcoglycan/dystrophin double knockout mice (δ-Dko) by crossing mdx mice with δ-sarcoglycan knockout (δSG KO) mice. Immunostaining and western blot confirmed the complete removal of the sarcolemmal sarcoglycans in δ-Dko mice. Interestingly, the utrophin level was further up-regulated. Nevertheless, clinical symptoms and muscle pathology were exacerbated in δ-Dko mice and muscle strength was further deteriorated. Taken together, we have demonstrated for the first time that the residual SGC may play a critical role in DMD pathogenesis. A complete elimination of the SGC is associated with deleterious consequences in mdx mice.
δSG KO mice were originally generated on a mixed genetic background (36). These mice were subsequently bred to the C57Bl/6 background at The Jackson Laboratory (http://jaxmice.jax.org/strain/004582.html). To compare with the C57Bl/10 (BL10) background mdx mice, we backcrossed the Jackson δSG KO mice with BL10 mice for three generations and obtained N3 δSG KO mice. These mice were 87.5% on the BL10 background and 22.5% on the BL6 background (Fig. 1A). δ-Dko mice were generated by crossing mdx mice with the N3 generation δSG KO mice. The resulting δ-Dko mice were 93.8% on the BL10 background and 6.2% on the BL6 background. The genotype of different mouse lines was confirmed by PCR (Fig. 1B).
Kyphosis and hind limb joint contractures are the characteristic clinical presentations in muscular dystrophy but are not seen in young adult mdx mice and δ-sarcoglycan deficient mice. Interestingly, these dystrophic signs were evident in age and sex matched δ-Dko mice (Fig. 2A). While DMD patients are often emaciated, the body weight of young mdx mice was higher than that of normal mice (Fig. 2B) (37). Consistent with clinical findings in patients, δ-Dko mouse body weight was significantly lower than that of age and sex matched normal mice and single gene deficient mice (Fig. 2B).
According to the latest survival studies (38,39), DMD patients only live ~30–40% of the normal life span. In contrast, mdx mice can live up to ~80% of the normal life span (Table 1) (6). Eliminating utrophin from mdx mice dramatically reduces the life span to ~10% of normal (Table 1) (16,17,40). Interestingly, the median survival of δ-Dko mice reached ~40% of that of normal mice (Table 1) (Figs 2C and D). Furthermore, it seemed the fertility was not affected in δ-Dko mice (Table 2).
The clinical presentation was also confirmed by microscopic studies. On HE staining, mdx, δSG KO and δ-Dko mice all showed muscle degeneration/regeneration, variable fiber size and inflammation. However, δ-Dko mouse muscle appeared to be worse than those from the other two (Fig. 3A). Subsequent morphometric quantification confirmed this observation. While all three dystrophic strains showed elevated levels of central nucleation, the percentage of the centrally nucleated myofibers in δ-Dko and mdx muscles was significantly higher than that in δSG KO muscle (Fig. 3B). The most striking difference was seen in macrophage infiltration. The macrophage number was doubled in δ-Dko muscle (Fig. 3C). Nevertheless, we did not see a dramatic difference in muscle fibrosis (Supplementary Material, Fig. S1). Neither did we see further deterioration of the sarcolemmal integrity by Evans blue dye (EBD) uptake assay and serum creatine kinase measurement (Fig. 3D, Supplementary Material, Fig. S2).
Next, we examined muscle strength in the extensor digitorium longus (EDL) muscle (Fig. 4). Consistent with previous reports (reviewed in 41), muscle cross-sectional area (CSA) normalized specific twitch and tetanic forces were significantly reduced in mdx and δSG KO mice (Fig. 4A). Yet, absolute twitch and tetanic forces were maintained at wild-type levels in these mice as a consequence of increased muscle weight and CSA (Fig. 4B, Table 3). In contrast, both specific and absolute muscle forces were significantly reduced in δ-Dko mice (Fig. 4B). Specifically, the specific force of δ-Dko mice reached only 40, 65 and 50% of BL10, mdx and δSG KO mice, respectively. The absolute force of δ-Dko mice was ~60% of the other three strains (BL10, mdx and δSG KO).
To further characterize the physiological property of δ-Dko mouse muscle, we applied 10 rounds of eccentric contraction. During these contractions, the EDL muscle was intentionally stretched at the peak of the tetanic force to create a higher mechanical stress. This assay allows a more accurate judgment on how strong a muscle can protect itself from contraction-induced injury.
BL10 muscle force was minimally affected by repeated eccentric contractions (Fig. 4C). Mdx and δSG KO muscles showed progressive force reduction, especially in the first four rounds of eccentric contraction. Interestingly, these two strains displayed similar levels of force deficiency over the entire eccentric contraction protocol (Fig. 4C). In contrast, δ-Dko muscle was much more sensitive to lengthening contraction damage. Compared with single gene deficient mice, the levels of force reduction were doubled in the first two rounds of eccentric contraction in δ-Dko mice (Fig. 4C).
Genetic ablation of δ-sarcoglycan compromises the assembly of the SGC at the sarcolemma (36). Besides δ-sarcoglycan, other three sarcoglycans should be eliminated from the microsomal preparation but not necessarily from whole muscle lysate in δ-sarcoglycan deficient mice. We first confirmed the lack of δ-sarcoglycan expression in δ-sarcoglycan deficient mice (Fig. 5). α-sarcoglycan levels were not altered in whole muscle lysates in any strain but were reduced in mdx microsomal preparation and eliminated in δSG KO and δ-Dko microsomal preparations (Fig. 5) (30,36,42). β-sarcoglycan levels were reduced in mdx microsomal preparation (Fig. 5B). γ-sarcoglycan levels were reduced in both whole muscle lysate and the microsomal preparation from mdx mice (Fig. 5) (30). The lack of δ-sarcoglycan expression in whole muscle lysate was confirmed in δSG KO and δ-Dko mice (Fig. 5A). Importantly, the entire SGC disappeared in the microsomal preparations obtained from δSG KO and δ-Dko mice (Fig. 5B) (36).
Besides the SGC, we also examined other DAPs. α-dystroglycan expression was decreased in whole muscle lysates and the microsomal preparations in mdx, δSG KO and δ-Dko mice (Fig. 5). An ~80–90% reduction of β-dystroglycan was observed in whole muscle lysates from mdx and δ-Dko mice (Fig. 5A). However, β-dystroglycan in whole muscle lysate was only reduced by ~10% in δSG KO mice (Fig. 5A) (43). Interestingly, total cellular syntrophin and dystrobrevin levels were not altered in either single or double knockout mice (Fig. 5A) (42,44–46).
As expected, dystrophin was not detected in mdx and δ-Dko mice (Fig. 5). However dystrophin expression in δSG KO mice was comparable to that of BL10 mice (Fig. 5). Utrophin levels were not altered in δSG KO mice but were significantly increased in mdx mice (Fig. 5, Supplementary Material, Fig. S3) (42,47). Surprisingly, utrophin expression was further increased in δ-Dko mice. On average, it was approximately 3-fold higher than that of normal mice (Fig. 5, Supplementary Material, Fig. S3).
To further confirm immunoblot results, we examined the DGC by immunofluorescence staining. Dystrophin was detected in all myofibers in BL10 and δSG KO mice (Fig. 6A). In mdx and δ-Dko muscle, dystrophin was only observed in rare revertant fibers (Fig. 6A). The SGC was enriched at the neuromuscular junctions in BL10 mice (Fig. 6B). In contrast to the complete absence of sarcolemmal staining in δSG KO and δ-Dko muscles, uniform low-level sarcoglycan expression was observed in mdx muscle except for revertant fibers. In these revertant fibers, the SGC was restored to the wild-type levels (Fig. 6B). Consistent with immunoblot results (Fig. 5A), we only observed nominal β-dystroglycan expression in mdx and δ-Dko mice (Fig. 6C). The loss of δ-sarcoglycan alone did not change dystrobrevin and syntrophin expression (Figs 5A and and6C).6C). However, membrane-associated syntrophin and dystrobrevin were greatly reduced in mdx and δ-Dko mice on immunostaining (Fig. 6C) (30,42). Consistent with immunoblot results (Fig. 5), enhanced utrophin expression was observed at the sarcolemma of both mdx and δ-Dko mice (Fig. 6A). Further, utrophin staining in δ-Dko muscle appeared stronger (Fig. 6A).
The structural integrity of the sarcolemma is largely dependent on the level and the intactness of the DGC. Dystrophin deficiency and subsequent DGC disassembly lead to DMD, a fatal muscle disease in human but not in mice. Among all the DGC components, the SGC stands out as the only subcomplex that dose not bind to dystrophin. Sub-physiological levels of the SGC have been detected in mdx mice (30). However, its biological implication is not completely understood. Here we tested the hypothesis that sub-physiological SGC expression could contribute to the mild phenotype in mdx mice.
The SGC is assembled from a core composed of β- and δ-sarcoglycan. Subsequent addition of γ- and then α-sarcoglycan completes the tetrameric complex (3,48,49). Genetic ablation experiments suggest that the membrane localization of individual sarcoglycan depends on each other. When the α- or γ-sarcoglycan gene is inactivated, other sarcoglycans are still detectable on the sarcolemma. However, the entire SGC is lost in the absence of β- or δ-sarcoglycan (3,48,50,51). Crossing mdx mice with β- or δ-sarcoglycan knockout mice will allow us to completely eliminate the residual SGC from the mdx sarcolemma. In this study, we generated δ-Dko mice by crossing mdx mice with the commercially available δSG KO mice (Fig. 1). δ-Dko mice were identical to mdx mice except for the complete absence of the SGC at the sarcolemma (Figs 1, ,5,5, ,66).
Removing the remaining SGC from mdx mice induced dystrophic symptoms and a further reduction of the life span (Fig. 2). We also observed more inflammatory cell infiltration in δ-Dko muscle (Fig. 3C). Physiology assay revealed not only specific force reduction, but more relevantly, a significant reduction of the absolute force in δ-Dko muscle (Fig. 4A and B). In addition, δ-Dko muscle was more easily injured during eccentric contraction (Fig. 4C). Nevertheless, myofiber regeneration was comparable to that of mdx mice (Fig. 3B) and the levels of muscle fibrosis and sarcolemmal integrity were similar to those of single knockout mice (Fig. 3D, Supplementary Material, Figs S1 and S2).
We examined dystrophin, utrophin and DAP expression in single and double knockout mice. Interestingly, the levels of the dystroglycan and syntrophin/dystrobrevin subcomplexes appeared to be similar between mdx and δ-Dko mice (Figs 5 and and6).6). However, utrophin expression was further enhanced in δ-Dko muscle (Figs 5 and and6,6, Supplementary Material, Fig. S3). The boosted utrophin up-regulation may likely reflect an increased effort of muscle cells to try to compensate for a severer disease. It is interesting to note that the amount of utrophin at the δ-Dko sarcolemma has reached the levels thought to be protective in mdx mice (Supplementary Material, Fig. S3) (15,52).
The biological function of the SGC is only partially elucidated. Supra-physiological expression of α-sarcoglycan is not toxic but γ-sarcoglycan over-expression leads to severe muscle disease (53,54). Dystrophin domain structure also modulates SGC function. In the presence of the full-length dystrophin protein, the SGC help maintain membrane stability by strengthening β-dystroglycan/dystrophin and/or β-dystroglycan/α-dystroglycan interactions (3). However, an intact SGC provides minimal protection in the presence of the shorter N-terminal domain truncated dystrophin isoforms such as Dp71 and Dp116 (31–34,55,56). The results presented here shed new light on our understanding of SGC function and suggest that sub-physiological SGC expression plays an essential role in maintaining the mild phenotype in mdx mice.
Phenotypic variations have been reported in DMD patients who carry the same mutation. In one extreme case, Winnard et al. (57) described three patients who were mildly, moderately and severely affected, respectively. Interestingly, all three patients had exactly the same exon 3–7 deletion which disrupted the reading frame (57). Similar findings have also been reported by Beggs et al. (10). More recently, a patient with undetectable dystrophin expression was found to have a mild clinical course and a Golden Retriever muscular dystrophy dog was found to have normal strength despite the absence of dystrophin (58,59). Collectively, these studies suggest the existence of genetic modifiers in muscular dystrophy (13). Because of the structure and/or function similarity, utrophin and integrin have been considered as the most obvious genetic modifiers for DMD. However, changes in utrophin and integrin alone cannot explain all clinical cases (60,61). In this regard, recent studies have identified additional modifiers such as myostatin, insulin-like growth factor and neuronal nitric oxide synthase (13). Our results suggest that residual SGC expression could represent another important, yet unappreciated modifier in DMD.
Studies in sarcoglycan knockout mice and limb-girdle muscular dystrophy patients also support the therapeutic relevance of sub-physiological level sarcoglycan expression. Inactivating the γ-sarcoglycan gene diminishes but does not eliminate other SGC components. Muscles from γ-sarcoglycan deficient mice show pronounced histopathology but specific forces are normal and they are also resistant to contraction-induced injury (62,63). LGMD 2D is caused by α-sarcoglycan gene mutation. Interestingly there is a strong correlation between the remaining α-sarcoglycan protein level and clinical severity. Patients with residual expression often present with a milder phenotype (64–68).
It is currently unclear how residual sarcoglycan expression protects muscle. There are several possibilities. First, it has been shown that the SGC directly binds to α-dystrobrevin, a signaling molecule in the cytosolic subcomplex of the DGC/UGC (69). In normal muscle, α-dystrobrevin can also be recruited to the DGC via a direct interaction with the dystrophin C-terminal domain. The loss of both dystrophin and the SGC may compromise membrane localization of α-dystrobrevin in δ-Dko mice (Fig. 6C). Since α-dystrobrevin null muscle exhibits extensive degeneration and necrosis (40,70), it is possible that a reduction of the sarcolemmal α-dystrobrevin level may have contributed to the severe phenotype in δ-Dko mice. Despite an intriguing hypothesis, this cannot explain all our observations. The total amount of cellular dystrobrevin was not reduced (Fig. 5A) (42). Furthermore, the sarcolemmal dystrobrevin levels were quite comparable between mdx and δ-Dko mice (Fig. 6C). We believe that the lack of a further reduction of sarcolemmal α-dystrobrevin in δ-Dko muscle is likely due to an enhanced utrophin up-regulation (Figs 5 and and6A).6A). Similar to dystrophin, the utrophin C-terminal domain also binds α-dystrobrevin. In addition to protein expression data, the aggravated force deficit in δ-Dko muscle (Fig. 4) does not support the α-dystrobrevin hypothesis either. It has been shown that α-dystrobrevin deficiency is not accompanied with muscle force reduction (70).
Another possibility is filamin-C. Filamins are F-actin cross-linking proteins involved in signal transduction (71,72). Filamin-C is specifically expressed in striated muscles and it interacts with γ- and δ-sarcoglycan (73). The filamin C gene mutation leads to an autosomal dominant myopathy (74). Filamin-C deficient mice show severe myogenesis defects and die soon after birth (75). Interestingly, membrane bound filamin-C level is markedly increased in DMD and LMGD 2C (γ-sarcoglycan deficient) patients as well as γ- and δ-sarcoglycan knockout mice and mdx mice (73,76). It is possible that filamin C levels could have been altered in δ-Dko mice. Future studies on filamin C expression may shed light on our observations.
Taken together, our results suggest that residual sarcoglycan expression represents an important compensatory mechanism in mdx mice. We speculate that similar mechanisms may exist in human patients. We further speculate that the differences in residual sarcoglycan levels may help explain phenotypic variances in some patients. Finally, our findings raise the possibility of sub-physiological sarcoglycan expression as a therapeutic strategy to ameliorate muscle disease in severe DMD patients.
All animal experiments were approved by the Animal Care and Use Committee of the University of Missouri and were in accordance with NIH guidelines. The breeding pairs for BL10 (C57BL/10SnJ), mdx (C57BL10ScSn-Dmdmdx/J) and BL6 background δSG KO (δ-sarcoglycan null B6.129-Sgcdtm1Mcn/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The experimental δSG KO mice were obtained after three generations of backcrossing of the Jackson Laboratory δSG KO mice with BL10 mice (Fig. 1A). The experimental δ-Dko mice were generated by crossing mdx with the N3 generation δSG KO mice (Fig. 1A). The genotype of the δ-sarcoglycan locus was determined using a protocol provided by The Jackson Laboratory (http://jaxmice.jax.org/pub-cgi/protocols). The mdx allele was genotyped using the amplification-resistant mutation system PCR assay (77). All experimental mice were housed in a specific pathogen-free facility and were kept under a 12 h light (25 lux)/12 h dark cycle with free access to food and water.
Whole muscle lysate was prepared from the tibialis anterior (TA) muscle according to our previously published protocol (77). Briefly, freshly dissected muscle was homogenized in a buffer (15 µl buffer/mg muscle) containing 10% sodium dodecyl sulfate (SDS), 5 mm ethylenediaminetetraacetic acid (EDTA), 62.5 mm Tris, pH 6.8 and 1% protease inhibitor (Roche, Indianapolis, IN). Crude lysate was boiled for 2 min and vigorously votexed for 30 s. Whole muscle lysate was then collected after spinning for 2 min at 14 000 rpm (Eppendorf centrifuge, model 5417C). Protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA) and 150 µg protein/lane was loaded in whole muscle lysate western blot. Dystrophin was detected with a mouse monoclonal antibody against the dystrophin C-terminal domain (Dys-2, 1:100; clone Dy8/6C5, IgG1; Novocastra, Newcastle, UK). Utrophin was detected with a mouse monoclonal antibody against utrophin amino acid residues 768–874 (#610896, 1:200; clone 55, IgG1; BD Biosciences, San Diego, CA). α-Dystroglycan was detected with a mouse monoclonal antibody (#05–593, 1:1000; clone IIH6C4, IgM; Millipore, Temecula, CA). β-Dystroglycan was detected with a mouse monoclonal antibody against the β-dystroglycan C-terminus (NCL-b-DG, 1:100; clone 43DAG1/8D5, IgG2a; Novocastra). α-Sarcoglycan was detected with a mouse monoclonal antibody against α-sarcoglycan amino acid residues 217–289 (VP-A105; 1:1000; clone Ad1/20A6, IgG1; Vector Laboratories, Burlingame, CA). γ-Sarcoglycan was detected with a mouse monoclonal antibody against γ-sarcoglycan amino acid residues 167–178 (VP-G803; 1:1000; clone 35DAG/21B5, IgG2b; Vector Laboratories). δ-Sarcoglycan was detected with a rabbit polyclonal antibody against δ-sarcoglycan amino acid residues 206–260 (sc28281, 1:200; clone H-55; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Syntrophin was detected with a pan-syntrophin mouse monoclonal antibody that recognized the syntrophin PDZ domain (ab11425, 1:2000; clone 1351, IgG1; Abcam, Cambridge, MA). Dystrobrevin was detected with a mouse monoclonal antibody against dystrobrevin amino acid residues 249–403 (#610766, 1:1000; clone 23, IgG1; BD Biosciences, San Diego, CA). As a loading control, membrane was also probed with an anti-α-tubulin antibody (1:3000; clone B-5-1-2; Sigma, St Louis, MO).
The microsome enriched membrane fraction was prepared according to a published protocol with modifications (78,79). Briefly, the freshly isolated quadriceps muscle was homogenized in a buffer (10 µl buffer/mg muscle) containing 20 mm sodium pyrophosphate, 20 mm sodium phosphate monohydrate, 1 mm MgC12, 303 mm sucrose, 0.5 mm EDTA, pH 7.0 and 1% protease inhibitor (Roche). The crude lysate was spun at 14 000g for 15 min at 4°C. The supernatant was spun at 100 000g for 40 min at 4°C. The pellet was resuspended in a buffer (7.5 µl buffer/mg muscle) containing 600 mm KCl, 303 mm sucrose, 20 mm Tris–HCl, pH 7.4 and 1% protease inhibitor (Roche). The microsome enriched pellet was obtained after spinning at 100 000g for 40 min at 4°C. The final microsomal preparation was resuspended in a buffer containing 303 mm sucrose, 20 mm Tris–maleate, pH 7.0. Protein concentration was determined using a protein assay kit (Bio-Rad) and 60 µg protein/lane was used in microsomal preparation western blot. Dystrophin, utrophin, α-dystroglycan, α-Sarcoglycan, γ-Sarcoglycan and δ-Sarcoglycan were detected using the antibodies described earlier. β-Sarcoglycan was revealed with a mouse monoclonal antibody against the β-sarcoglycan N-terminus (NCL-b-SARC, 1:250; clone 5B1, IgG1; Novocastra).
Immunostaining was performed essentially as we described before (80,81). Dystrophin was examined with Dys-2 (1:30; Novocastra). Utrophin was examined with a mouse monoclonal antibody against the utrophin N-terminal domain (VP-U579, 1:20; clone DRP3/20C5, IgG1; Vector Laboratories, Burlingame, CA). β-Dystroglycan was revealed with NCL-b-DG (1:50; Novocastra). α-Sarcoglycan was detected with VP-A105 (1:50; Vector Laboratories). β-Sarcoglycan was revealed with NCL-b-SARC (1:50; Novocastra). γ-Sarcoglycan was detected with VP-G803 (1:50; Vector Laboratories). δ-Sarcoglycan was detected with sc28281 (1:50; Santa Cruz Biotechnology Inc.). Dystrobrevin was revealed with a mouse monoclonal antibody (#610766, 1:200; clone 23, IgG1; BD Biosciences, San Diego, CA). Syntrophin was revealed with the ab11425 antibody (1:200; Abcam).
General histopathology was examined by standard hematoxylin–eosin (HE) staining. The percentage of centrally nucleated myofibers was quantified in HE stained cross-sections of the TA muscle as we described before (82). Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used for immunohistochemical detection of macrophages. Mouse macrophage was recognized with a rat anti-mouse F4/18 antibody (#RM2920, 1:200; clone CI:A3-1, IgG2b; Caltag Laboratories, Burlingame, CA). Macrophage infiltration was also confirmed by non-specific esterase staining according to a previously published protocol (35,81,83). The number of macrophage was quantified on digitized images. Morphometric quantification was performed on at least three cross-sections located at the proximal, middle and distal portions for each muscle sample. EBD uptake assay was performed according to a previously published protocol (35,81).
Muscle force was measured on a 305B dual-mode servomotor transducer (Aurora Scientific, Inc., Aurora, ON, Canada) as described before (35,82). A LabView-based DMC program (Version 3.12, Aurora Scientific, Inc.) was used to control the servomotors and to acquire the data. Length and force data were analyzed using a LabView-based DMA program (Version 3.12, Aurora Scientific, Inc.).
The EDL muscle was isolated as described (82). After mounting in a jacket organ bath in oxygenated Ringer's solution, the optimal muscle length (Lo) was determined based on the muscle length at which the maximal twitch force was elicited. At first, all the myofibers were activated with three 500 ms tetanic stimulations at 150 Hz. After 2 min resting, the absolute twitch force was measured. After 3 min resting, the absolute tetanic force was determined with stimulation at 80, 120 and 150 Hz, respectively. The muscle was allowed for 1-min rest between each tetanic contraction. At the end of 150 Hz stimulation, the muscle length at Lo was recorded with an electronic digital caliper (±0.01 mm; Control Co., Friendswood, TX). The specific muscle force was obtained after normalizing the absolute muscle force to muscle CSA (kN/m2). Muscle CSA was calculated according to the formula, CSA = (muscle mass, in g)/[(1.06 g/cm3) × (optimal fiber length, in cm)]. 1.06 g/cm3 is muscle density. The optimal fiber length was calculated as 0.44 × Lo. 0.44 represents the ratio of fiber length to optimal muscle length (Lf/Lo) for the EDL muscle.
After tetanic force measurement, the EDL muscle was subjected to 10 cycles of eccentric lengthening contraction (82). In each cycle, the muscle was stimulated for 700 ms. The muscle was lengthened by 10% Lo at 0.5 Lo/s in the last 200 ms. The muscle was allowed for 2-min rest between each cycle. The maximal isometric tetanic force developed during the first 500 ms of stimulation of the first cycle was designed as the baseline tension (100%). The percentage of tetanic force loss at each cycle was determined according to the following formula: Force drop %= (F1−Fn)/F1, where F1 was the tetanic force obtained during the first cycle, and Fn represented the tetanic force obtained during the nth cycle.
Data are presented as mean ± standard error of mean (s.e.m.). Statistical analysis was performed with the SPSS software (SPSS, Chicago, IL) and the Prism 4 software (GraphPad, San Diego, CA). For multiple group comparison, statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc analysis. Difference was considered significant when P < 0.05. For survival study, statistical significance was determined by Mantel-Cox log-rank test. The Bonferroni corrected threshold was used to set the significance level.
Conflict of Interest statement. None declared.
This work was supported by grants from the National Institutes of Health AR-49419, AR-57209 and NS-62934 (D.D.) and the Muscular Dystrophy Association (D.D.).