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Am J Physiol Cell Physiol 290: C411-C419, 2006. First published September 14, 2005; doi:10.1152/ajpcell.00192.2005
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Impact of sarcoglycan complex on mechanical signal transduction in murine skeletal muscle

Elisabeth R. Barton

Department of Anatomy and Cell Biology, School of Dental Medicine, and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 21 April 2005 ; accepted in final form 9 September 2005


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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 REFERENCES
 
Loss of the dystrophin glycoprotein complex (DGC) or a subset of its components can lead to muscular dystrophy. However, the patterns of symptoms differ depending on which proteins are affected. Absence of dystrophin leads to loss of the entire DGC and is associated with susceptibility to contractile injury. In contrast, muscles lacking {gamma}-sarcoglycan ({gamma}-SG) display little mechanical fragility and still develop severe pathology. Animals lacking dystrophin or {gamma}-SG were used to identify DGC components critical for sensing dynamic mechanical load. Extensor digitorum longus muscles from 7-wk-old normal (C57), dystrophin- null (mdx), and {gamma}-SG-null (gsg–/–) mice were subjected to a series of eccentric contractions, after which ERK1/2 phosphorylation levels were determined. At rest, both dystrophic strains had significantly higher ERK1 phosphorylation, and gsg–/– muscle also had heightened ERK2 phosphorylation compared with wild-type controls. Eccentric contractions produced a significant and transient increase in ERK1/2 phosphorylation in normal muscle, whereas the mdx strain displayed no significant proportional change of ERK1/2 phosphorylation after eccentric contraction. Muscles from gsg–/– mice had no significant increase in ERK1 phosphorylation; however, ERK2 phosphorylation was more robust than in C57 controls. The reduction in mechanically induced ERK1 phosphorylation in gsg–/– muscle was not dependent on age or severity of phenotype, because muscle from both young and old (age 20 wk) animals exhibited a reduced response. Immunoprecipitation experiments revealed that {gamma}-SG was phosphorylated in normal muscle after eccentric contractions, indicating that members of the DGC are modified in response to mechanical perturbation. This study provides evidence that the SGs are involved in the transduction of mechanical information in skeletal muscle, potentially unique from the entire DGC.

muscular dystrophy; eccentric contractions; extracellular signal-regulated kinase 1/2

THE MUSCULAR DYSTROPHIES encompass a large group of degenerative muscle disorders in which there can be profound weakness and fragility. A majority of diseases within this family are caused by mutations in genes encoding for proteins of the dystrophin-associated glycoprotein complex (DGC) and lead to the partial or complete absence of the DGC. These include abnormalities in dystrophin, which cause Duchenne and Becker muscular dystrophy, errors in laminin, an extracellular matrix protein, which cause congenital muscular dystrophy, and abnormalities in members of the sarcoglycan (SG) complex, which lead to a subset of limb-girdle muscular dystrophies (LGMD; reviewed in Refs. 11, 29).

The DGC is concentrated at the costameres along the sarcolemma of striated muscle. The complex as a whole links the intracellular components of the cytoskeleton to the extracellular matrix (9). As the muscle generates force, muscle fibers undergo tremendous tensile and shear stress, which is transmitted to the outside of the cell. The DGC is poised at the central site of stress transmission and has been proposed to be critical in maintaining membrane integrity (35). In addition to stabilizing the membrane, the complex also may sense mechanical stress and transmit that "outside-in" information back to the nucleus via signaling proteins associated with the complex.

The SG complex is a subcomplex of the DGC and in skeletal muscle consists of {alpha}-, {beta}-, {gamma}-, and {delta}-SG (4, 20). These proteins form an integral membrane complex in which the short intracellular domains can associate with {alpha}-dystrobrevin (46) and filamin C (FLNC) (43). Each protein possesses a single transmembrane domain (20, 34), and the large extracellular domains have multiple predicted N-glycosylation sites and numerous cysteine residues that are conserved across species (32). The cysteines are thought to be critical for assembly of the complex, because several instances of LGMD arise from mutations that disrupt these cysteines (32). The intracellular regions of {alpha}-, {beta}-, and {gamma}-SG have potential tyrosine phosphorylation sites. In cell culture studies, adhesion gives rise to phosphorylation of each of these SGs, indicating that the SGs can be modified in response to cell attachment (47).

Characterization of mouse models that lack components of the SG complex suggests that there is a separation between muscle contractile fragility and the dystrophic phenotype. Muscles from mdx mice, which harbor a mutation in the dystrophin gene, lack the entire DGC and serve as an animal model for Duchenne muscular dystrophy (DMD) (39), exhibiting membrane fragility already apparent in 6-wk-old animals (35). The general consensus is that a significant component of DMD is due to the loss of sarcolemmal stabilization. In contrast, muscles from mice lacking the {gamma}-SG as the result of gene targeting show severe symptoms of dystrophy, including high serum creatine kinase levels and degenerating/regenerating muscle, yet there is little mechanical impairment in young animals (19). In these mice, the SG subcomplex is disrupted, with the additional loss of {beta}- and {delta}-SG and decrease of {alpha}-SG, yet dystrophin and other components of the DGC remain (21). Therefore, another mechanism besides membrane fragility must contribute to the onset of heightened muscle fiber degeneration in the SG-null mice.

Several studies have demonstrated that contractile activity activates MAPK signaling. In isolated rat muscle, isometric contractions evoked an ~2.5-fold increase in ERK1/2 phosphorylation (22, 37). Also, JNK activity was elevated in human muscle after a single bout of eccentric exercise (37). The response to mechanical perturbations is rapid: within 5 min of the exercise regimen, significant phosphorylation levels were detected (38). At this point, it is unclear which of the membrane complexes described is mediating the response in vivo. However, experiments in cell culture suggest that both integrins and the DGC are involved. The response of cultured myocytes to adhesion (which imposes mechanical stress on the membrane) showed that there was phosphorylation of {alpha}- and {gamma}-SG (47). In the same study, direct activation of integrins also induced SG phosphorylation even in suspended cells, where there was no mechanical load on the cells.

To test whether the SG complex acts as a muscle mechanosensor in vivo, mechanical stress was imposed on skeletal muscles isolated from animal models for DMD and LGMD, and the phosphorylation state of proteins associated with known signaling cascades was monitored after perturbation.


   METHODS
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 METHODS
RESULTS
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Animals and experimental design. All experiments were approved by the University of Pennsylvania's Animal Care and Use Committee. Four groups of mice were utilized for this study: mdx, {gamma}-SG-null (gsg–/–), C57Bl/6, and C57Bl/10 mice. The mdx mouse (C57Bl/10 background strain) harbors a mutation in the dystrophin gene, resulting in a lack of dystrophin and loss of the DGC (39). The gsg–/– mouse lacks {gamma}-SG via gene targeting, resulting in the additional loss of {beta}- and {delta}-SG and decrease of {alpha}-SG (21). The gsg–/– mouse line was backcrossed for 10 generations onto the C57Bl/6 strain. The C57Bl/6 and C57Bl/10 mice were utilized as controls for each strain in this study.

Whole muscle mechanics. Isolated whole muscle mechanics were performed on the extensor digitorum longus (EDL) muscles from 7-wk-old animals as previously described (3). Muscles were removed from the animals after anesthetization with intraperitoneal injection of ketamine/xylazine (80 and 10 mg/kg body wt, respectively), keeping the tendons intact. The muscle tendons were attached to a rigid post and to a lever arm of a servomotor (Cambridge Technologies, Cambridge, MA) in a bath of Ringer solution gas equilibrated with 95% O2-5% CO2 and maintained at 22°C. Optimum length (Lo) of each muscle was defined as the length at which maximal twitch force developed from supramaximal stimulation. A series of eccentric contractions was delivered to the muscle. Each contraction, separated by a period of 5 min, consisted of an 80-Hz, 700-ms pulse delivered via two parallel platinum plate electrodes, where a stretch of 10% Lo was imposed on the muscle in the last 200 ms of the contraction. Figure 1A illustrates the pattern of force generated by a single eccentric contraction. This results in forces approximately two times higher than that achieved with maximum isometric tetanus. After a series of five eccentric contractions, the muscles were removed from the mechanics bath and placed in oxygenated Ringer for 3–90 min. A set of unstimulated muscles was kept in oxygenated Ringer for the duration of the experiment and served as an unstimulated control. At the end of this period, muscles were rapidly frozen in liquid nitrogen and stored at –200°C for subsequent analysis. For each time point, n = 3 muscles were used for each strain.



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  		Fig. 1. Eccentric contraction activates ERK1/2 pathway. A: illustration of force and length traces during eccentric contractions. Lo, optimum length. B: immunoblots of extensor digitorum longus (EDL) muscle homogenates from C57 mice. Muscles underwent a series of 5 eccentric contractions (Stim +) or remained at rest in a bath of Ringer buffer (Stim –). The contractions produced a transient phosphorylation of ERK1/2 (P-ERK1, P-ERK2, top blots). There was no effect of stimulation on total ERK1/2 levels (bottom blots). C: time course of ERK1/2 phosphorylation induced by eccentric contraction. Muscles were rapidly frozen at 3, 30, 45, 60, and 90 min after a series of 5 eccentric contractions (n = 2 for each condition) and homogenized in lysis buffer containing protease and phosphatase inhibitors. ERK1/2 phosphorylation was measured on 10 µg of total protein by immunoblots utilizing antibodies specific for the total and phosphorylated forms of ERK (Cell Signaling Technology). Top: transient phosphorylation of ERK1; bottom, phosphorylation of ERK2. Units reflect the intensity of the immunoblot bands in arbitrary units. *P < 0.05 vs. no stimulation.

 
Immunoblotting for ERK phosphorylation. Homogenized lysates from treated and control muscles were probed with antibodies that recognize the phosphorylated and total forms of ERK1/2 MAPK. Muscles were homogenized in 10 volumes per muscle wet weight of modified lysis buffer [50 mM Tris·HCl, pH 7.4, 1% (wt/vol) Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM NaVO4, 1 mM NaF, and 1 mM EGTA]. Homogenates were centrifuged for 10 min at 10,000 g to pellet debris, and the total protein was measured in the supernatant (Bio-Rad, Hercules, CA). A total of 10–30 µg of protein from each muscle lysate were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were incubated in blocking buffer [5% nonfat dry milk in Tris-buffered saline (TBS) plus 0.1% Tween 20 (5% milk/TTBS)] and then incubated in primary antibody diluted in 5% milk/TTBS overnight at 4°C. Antibodies recognizing phosphorylated ERK1/2 (no. 9101) and total ERK1/2 (no. 9102; Cell Signaling, Beverly, MA) were used. Membranes were then washed in 5% milk/TTBS and incubated with horseradish peroxidase-conjugated secondary antibody. After a series of washes in milk, TTBS, and TBS, protein detection was performed with enhanced chemiluminescence (ECL; PerkinElmer, Boston, MA) and exposure on an imaging system (Kodak 1D, Eastman Kodak, Rochester, NY) and to X-ray film. Analysis of band intensity was performed using image analysis software (Kodak 1D). Multiple exposures of each membrane were used in the analysis to ensure signal linearity. The membranes were stained with Coomassie brilliant blue R-250 after immunoblotting to confirm equal protein loading.

Immunoprecipitation. Equal protein amounts from muscle lysates were pooled, and 100 µg were subjected to immunoprecipitation with anti-phosphorylated tyrosine (P-Tyr-100, no. 9411; Cell Signaling Technology) or anti-{gamma}-SG (NCA-g-SARC; Novocastra, Vector Laboratories, Burlingame, CA). Samples were purified using a Seize immunoprecipitation kit (Pierce, Rockford, IL) and subjected to immunoblotting with P-Tyr antibodies as described in Immunoblotting for ERK phosphorylation.

Statistical analysis. All data are expressed as means ± SE. A one-way analysis of variance was used to compare all variables among groups. A significance level of P < 0.05 was used for all comparisons.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Signal transduction in normal muscle. The response to mechanical stimuli has been demonstrated in a number of different protocols in which changes in stress are imposed to skeletal muscle in both in vivo and ex vivo preparations (26, 30, 31, 37, 47). To establish that the protocol followed in the current study promoted similar responses, the EDL muscles from C57Bl/6 animals were subjected to a series of five eccentric contractions and then pinned at resting length in oxygenated Ringer for 3–90 min poststimulation. An example of immunoblots of muscle lysates probed for phosphorylated ERK1/2 (P-ERK1/2) or total ERK1/2 at the above time points is shown in Fig. 1B. As shown in Fig. 1C, the series of eccentric contractions produced a transient elevation of ERK1/2 phosphorylation without any significant change in total ERK levels. P-ERK1 increased by ~10-fold, and P-ERK2 doubled at 30 min poststimulation. In the nonstimulated muscles, the duration of incubation within the Ringer bath did not affect ERK1/2 phosphorylation. These results show that isolated mouse EDL muscle responds to contraction in a fashion similar to that of rat limb muscles (22, 37) and mouse diaphragm (26) and establish this procedure on the mouse EDL as a viable method for producing contraction-induced signaling cascades.

Resting ERK phosphorylation is elevated in dystrophic muscles. To determine whether muscles from dystrophic mice displayed any differences in ERK1/2 phosphorylation at rest, total and P-ERK1/2 were measured in EDL from young (6–7 wk old) mice (Fig. 2A). P-ERK1 was elevated in gsg–/– and mdx muscles compared with C57Bl/6 controls, and gsg–/– muscles also displayed increased P-ERK2 (Fig. 2B). There was no difference between C57Bl/6 and C57Bl/10 muscles (data not shown). The change was not due to an increase in total ERK levels, because there was no significant difference in the amount of ERK protein in any of the mouse strains (Fig. 2C). Therefore, the basal phosphorylation of ERK is increased in the absence of {gamma}-SG or dystrophin in resting EDL muscle.



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  		Fig. 2. ERK phosphorylation is elevated in dystrophic muscle. A: phosphorylated and total ERK1/2 were measured in homogenates of resting EDL muscles dissected from normal (C57), {gamma}-sarcoglycan ({gamma}-SG)-null (gsg–/–), and dystrophin-null (mdx) mice and normalized to total protein. B: both gsg–/– and mdx muscles exhibited significantly increased P-ERK1 compared with C57 muscles (*P < 0.05), and gsg–/– muscles also had significantly elevated P-ERK2 (*P < 0.05). C: there was no significant difference in total ERK content among any of the mouse strains.

 
Functional profile of EDL muscles. Mechanical measurements were acquired from EDL muscles of all strains and ages (Table 1). Muscles from mdx mice were significantly larger than those from age-matched C57Bl/10 mice. The mdx muscle generated significantly more absolute tetanic force; however, there was no significant difference in specific force in muscles from 7-wk-old animals. At 20 wk of age, the gsg–/– muscles appeared slightly larger than those from C57Bl/6 mice, and force production was diminished. However, neither measurement in gsg–/– EDL muscle was significantly different from that in C57Bl/6.


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  		Table 1. Functional profile of young and old EDL muscles

 
Because the susceptibility to eccentric contraction-induced damage is known to differ among the animal models for muscular dystrophy, the drop in force after five eccentric contractions was compared in the EDL muscles of all mouse strains at 7 wk of age. The results, displayed in Fig. 3A, show that susceptibility to damage does not differ between wild-type EDL muscles and muscles lacking {gamma}-SG, a finding similar to previously published results (19). However, muscles from mdx animals of the same age have significantly greater loss in force after the eccentric contraction protocol, where there was a 16% drop in force production. These results are similar to those previously reported, where susceptibility to damage is apparent in mdx mice as young as 6 wk (35).



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  		Fig. 3. Effects of eccentric contractions in dystrophic muscle. A: susceptibility to eccentric contraction-induced damage. The decrement in force after 5 eccentric contractions was determined in n = 7 EDL muscles from each mouse strain at 7–8 wk of age. There was no significant difference in the drop in force between C57Bl/6 and gsg–/– muscles (P = 0.093). Eccentric contractions caused significant decreases in force in EDL muscles from mdx mice compared with C57Bl/10 muscles (P = 0.011). B: ERK phosphorylation in dystrophic muscle. Muscles from gsg–/– and mdx mice displayed no significant increase in ERK1 phosphorylation at 30 and 60 min after stimulation (top). Both C57Bl/6 and C57Bl/10 muscles had significant increases at both time points compared with no stimulation controls; however, the responses in C57Bl/6 and C57Bl/10 were significantly different from each other. Muscles from mdx mice showed no significant increase in ERK2 phosphorylation at either time point after stimulation, in contrast to all other strains (bottom). Muscles from gsg–/– mice had significantly higher responses to P-ERK2 than C57Bl/6 controls for each time point. Data are presented as proportional changes in phosphorylation compared with no stimulation (means ± SE; for n = 3 muscles per condition) and are normalized to total ERK1/2 content. *P < 0.05 compared with unstimulated muscles of the same strain. {dagger}P < 0.05 compared with strain controls at the same time point. {ddagger}P < 0.05, C57Bl/6 vs. C57Bl/10 at the same time point.

 
Signal transduction in dystrophic muscle. The response of dystrophic muscles to mechanical stimuli was compared with that in normal (C57) muscle. Muscles were subjected to a series of five eccentric contractions and incubated in oxygenated Ringer for either 30 or 60 min (n = 3 muscles for each strain and time point). A set of unstimulated muscles from each strain served as controls. Data are presented as proportional changes in phosphorylation compared with no stimulation and normalized to total ERK1/2 for each sample. Both C57 strains displayed increased P-ERK1/2 at both time points (Fig. 3B). The response and time course varied for P-ERK1, where C57Bl/10 muscles had less phosphorylation 30 min after stimulation and almost three times more P-ERK1 60 min after stimulation (20.2 ± 0.14 vs. 7. 0 ± 0.17 for C57Bl/10 and C57Bl/6, respectively; Fig. 3B, top). Both dystrophic strains had no significant increase in P-ERK1 at either time point. Muscles from mdx mice showed no significant increase in ERK2 phosphorylation at either time point after stimulation, in contrast to all other strains (Fig. 3B, bottom). However, muscles from gsg–/– mice had significantly higher P-ERK2 responses than C57Bl/6 controls for each time point. Therefore, the dystrophic strains differed from wild-type muscle, and from each other, in their response to eccentric contraction.

Impairment to mechanical signal transduction precedes susceptibility to contractile damage. To clarify whether the reduction in ERK1 phosphorylation in gsg–/– muscles was due to the presence of a dystrophic phenotype or to the lack of SGs, the level of P-ERK1 was compared between young (7 wk) and old (20 wk) gsg–/– mice. Figure 4 shows the results of this experiment. Regardless of the amount of force lost during the eccentric contractions (8% in young mice, n = 3; 33% in old mice, n = 3), the proportional increase in P-ERK1 (stimulated/control) was significantly reduced compared with 15.9 in young C57 mouse muscles (n = 4). These measurements show that there was dissociation between the decrease in ERK1 phosphorylation in response to eccentric contraction and the increase in susceptibility to contraction-induced damage due to the accumulated severity of the phenotype. The impaired ERK1 phosphorylation preceded the onset of mechanical fragility.



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  		Fig. 4. Reduction in ERK phosphorylation precedes susceptibility to contractile damage. Muscles from 7-wk-old C57 and gsg–/– mice (n = 3 of each strain) and 20-wk-old gsg–/– mice (n = 4 of each strain) were subjected to eccentric contractions and monitored for ERK1 phosphorylation 30 min after the contraction protocol. No drop in force (filled bars) was incurred by the contractions in the muscles from the young mice, yet at 20 wk of age, the gsg–/– muscles showed a significant drop in force (33 ± 4%). The proportional increase in ERK1 phosphorylation (Stim/Control, open bars) was significantly diminished in both the 7- and 20-wk-old gsg–/– muscles compared with the response measured in C57 muscles (*P < 0.05). {dagger}P < 0.05 for drop in force compared with C57 muscles.

 
{gamma}-SG is phosphorylated during eccentric contraction. Phosphorylation is an important mechanism by which a cell regulates the activation of enzymes and binding affinities between proteins. Modifications of tyrosines on both {alpha}- and {gamma}-SGs have been documented in studies of adherent muscle cell culture studies (47). To test whether there was any change in tyrosine phosphorylation state after eccentric contractions in normal skeletal muscle, immunoprecipitation was performed on lysates from C57 muscles subjected to a series of five eccentric contractions by using an antibody specific for tyrosine phosphorylation (P-Tyr), after which immunoblotting was utilized to identify changes in tyrosine phosphorylation. Changes in intensity were found in four bands, ~120, 91, and 35 kDa and a faint signal at 50 kDa (Fig. 5), suggesting that mechanical perturbation, similar to cell adhesion, has a detectable effect on tyrosine phosphorylation in at least these proteins. To test whether the putative site for phosphorylation on {gamma}-SG (tyrosine 6) was modified, lysates were subjected to immunoprecipitation with anti-{gamma}-SG and then probed with anti-P-Tyr. By 30 min after eccentric stimulation, {gamma}-SG was phosphorylated. Therefore, as in cell culture studies (47), {gamma}-SG is phosphorylated in response to mechanical perturbation.



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  		Fig. 5. Eccentric contraction causes changes in tyrosine phosphorylation. Tyrosine phosphorylation of ~120-, 91-, 50-, and 35-kDa proteins were detected by immunoprecipitation (IP) of lysates from normal muscles subjected to eccentric contraction with phospho-tyrosine antibody (PTyr). Identification of the 35-kDa band was determined to be {gamma}-SG with an antibody specific for {gamma}-SG. Phosphorylation of {gamma}-SG was absent in muscles that had not been subjected to eccentric contraction and appeared at 30 min after the stimulation protocol. WB, Western blotting.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study tested whether signal transduction wrought by mechanical perturbation involves the SG complex. Eccentric contractions of the mouse EDL muscle produced a robust and transient increase in ERK1/2 phosphorylation and also caused the phosphorylation of tyrosines on at least three proteins, one of which was {gamma}-SG. The absence of the entire DGC in mdx EDL muscles, or the SG complex alone in the gsg–/– mice, was associated with a heightened basal ERK1/2 phosphorylation. Furthermore, the proportional increase in ERK phosphorylation following eccentric contractions was different from normal muscle in both dystrophic strains, even though the gsg–/– muscle displayed no significant susceptibility to contractile damage. Muscles from both gsg–/– and mdx mice showed no significant increase in P-ERK1, yet they displayed unique patterns in P-ERK2 after eccentric contraction. The impairment in ERK1 phosphorylation persisted in muscles from older gsg–/– mice, suggesting that the signaling defect preceded any mechanical defect in these muscles. These results suggest that changes in the signaling response to mechanical perturbation may play a role in the onset of the dystrophic phenotype in the gsg–/– mice.

A variety of methods have been used to invoke changes in signal transduction changes in muscle. These include dynamic and static passive stretch in both longitudinal and transverse directions, stimulation, and the combination of both stimulation and stretch (eccentric contractions) in ex vivo and in vivo models (1, 2, 6–8, 14, 17, 24, 26, 37, 38, 47). The changes also have been exhibited in vitro, where either attachment alone or the distortion of a flexible substrate can elicit modifications to key signaling proteins (14, 40, 41, 47). Although the sample preparation and mechanical perturbation protocol could give rise to disparities in the response pattern, it has been demonstrated that MAPK responses in skeletal muscle correlate better with the peak tension generated than with the tension-time integral or the rate of tension development (30). Furthermore, there is general consensus that the ERK pathway is most sensitive to mechanical perturbation. Therefore, the current study used a series of eccentric contractions to produce a maximum change in force and stretch on the muscle and used ERK phosphorylation as an index of mechanical signaling. The force generated with the combined protocol was approximately twice that produced in an isometric contraction of the same preparation and several orders of magnitude greater than that achievable by passive stretch alone.

Several structural characteristics suggest that the SG complex may mediate signals received at the extracellular region, such as the binding of an unknown ligand or an induced conformational change, and transmit them to the intracellular domain. First, the clustering of the cysteine-rich regions suggests interactions between the proteins that are intriguingly similar to those found in receptor molecules such as epidermal growth factor receptor (33). Second, the potential for SG tyrosine phosphorylation opens up potential for the SGs to be involved in mechanical signal transduction. Third, the linkage through {alpha}-dystrobrevin to neuronal nitric oxide synthase (nNOS) (15, 46) sets up another possible pathway by which the SG could sense mechanical load and transmit that information through known signaling proteins. Finally, the interaction of {gamma}- and {delta}-SG to FLNC and thereby to the actin cytoskeleton provides a clear link to the signal transduction cascades associated with this protein (5, 18, 42, 43).

A potential signaling role for SGs is similar and possibly complementary to the integrin complex. In muscle and several other cell types, the integrin complex is essential for adhesion-mediated survival (9, 28, 47). Adult muscle expresses a specific isoform, {alpha}7/{beta}1 heterodimer (44), which is concentrated at the myotendinous junction and at the costameres along the length of the muscle fiber similar to the DGC. Unlike classic receptors, integrins have no inherent kinase or enzymatic activity but, instead, rely on their association with non-receptor protein tyrosine kinases, such as focal adhesion kinase (FAK), to relay information from the extracellular matrix to the cell nucleus. The activation of FAK also affects its association with a vast array of other signaling proteins. These include Grb2, an adapter protein associated with the Ras-ERK/MAP kinase pathway, and p85, which leads to activation of the phosphatidylinositol 3-kinase pathway. It is presumably through these interactions that FAK mediates integrin signaling (36). Recent work has shown that increased expression of {alpha}7-integrin in mdx:utr–/– mice ameliorates the severe dystrophic phenotype in these animals, which lack both dystrophin and utrophin, suggesting that the integrin and dystrophin complexes have overlapping functions and can compensate for the loss of each other (10).

Further evidence of the interaction between the integrins and the SGs has been provided by cell culture studies (47). It was shown that cell adhesion could evoke tyrosine phosphorylation of the SGs and that the phosphorylation was possibly mediated by FAK. In the current set of experiments, phosphorylation of {gamma}-SG was observed 30 min after the series of eccentric contractions. In addition, a band of ~120 kDa was also phosphorylated before the 35-kDa band phosphorylation. Although it is possible that this band is FAK, this was not directly tested in this study. Also not clear at this point is whether ERK and {gamma}-SG phosphorylation are causally related. Both modifications are significantly increased at 30 min after eccentric contraction. Other time points were not assessed for {gamma}-SG phosphorylation. Future studies are warranted in which careful examination is necessary of the time course and potential links between the phosphorylation events that occur after mechanical perturbation.

Former studies have demonstrated that the loss of the entire DGC from murine muscle results in aberrant signaling and a reduction in the response to mechanical strain (25, 27). These studies have established a role for the DGC in mediating information about the mechanical state of the muscle either between fibers or into the nucleus, yet it has proven difficult to determine which of the several components of the complex contribute to this signaling property. DGC function is multifaceted, and it is thought that the numerous symptoms of muscular dystrophy arise from the lack of interaction between members of the DGC. Certainly, the demonstration that the specific loss of {alpha}-dystrobrevin and the displacement of nNOS lead to a dystrophic phenotype implies that the dystrophin complex as a unit can mediate signaling events via nNOS activation (15). The current study has been able to isolate the SGs as players in the mechanical signal transduction process, and displays a unique ERK1/2 response after eccentric contraction. Because the SG complex has been shown to associate with the NH2 terminus of {alpha}-dystrobrevin (46), it is possible that the absence of the SGs affects the manner in which nNOS is activated. The absence of the SG complex in skeletal muscle has been associated with diminished nNOS localization (13), and in cardiac muscle from gsg–/– mice, mislocalization of endothelial NOS has been shown to contribute to the progression of cardiomyopathy (23). Although not directly tested in the current study, it will be important to determine whether changes in ERK phosphorylation that arise in gsg–/– skeletal muscle are associated with impaired nNOS signaling.

The interaction of the SGs with FLNC provides a direct structural link from the SGs to the actin cytoskeleton and to the signal transduction pathways associated with the filamin protein (43), and the experiments described in this report show that the SGs are poised either to mediate or receive mechanical information. It is not clear at this point whether modifications to the SG complex shown in this and former studies (47) are due to mechanical stress sensed first by a protein such as FLNC, which then leads to tyrosine phosphorylation of {gamma}-sarcoglycan, or whether the SGs can directly sense membrane distortion. Furthermore, in this study, there was no investigation into how eccentric contraction might regulate the interaction between the SGs and FLNC or the activity of calpain-3, which can specifically cleave the NH2 terminus of FLNC and inhibit the interaction of FLNC with {gamma}- and {delta}-SG (18). However, the mechanical sensing properties of the SG complex and its associated proteins have become more established as a critical property of the dystrophin complex.

Because the muscles from the mdx and gsg–/– mice displayed different responses to eccentric contraction, the possibility is raised that both complexes possess a signaling role for mechanotransduction. On one hand, both showed no significant P-ERK1 response, suggesting that {gamma}-SG and the proteins lost in this animal model are linked to ERK1. This is because the mdx muscle shares this response and also lacks not only these proteins but also the entire DGC. On the other hand, the P-ERK2 response differs significantly between the two models, suggesting that those proteins missing only in mdx and not in gsg–/– muscle mediate ERK2 phosphorylation. Furthermore, because gsg–/– muscles displayed increased resting P-ERK2 and heightened P-ERK2 after eccentric contraction, it is possible that there is an compensatory response in adaptor proteins or other associated proteins to counteract the loss of P-ERK1 sensitivity. The physiological separation of these signaling responses needs to be determined in future studies.

What would occur in muscles with an intact SG complex but without full-length dystrophin? A transgenic mouse was developed more than a decade ago in which Dp71, a shorter transcript of the dystrophin gene that lacks the NH2-terminal actin binding domain found in full-length dystrophin, was expressed, showing that the localization of the complex was not sufficient to rescue the dystrophic phenotype (12, 16). More recently it was demonstrated that mdx muscles expressing Dp71 displayed similar susceptibility to eccentric contraction damage compared with wild-type mdx (45). The general consensus is that actin binding, and, therefore, a tether to the cytoskeleton, is required for proper function of the DGC, supporting a structural role for this complex. It has yet to be determined if the signaling response in these mice mimics that measured in the mdx mouse.

A working model of the response of skeletal muscle to mechanical perturbation is shown in Fig. 6, which incorporates the observations described in the current study with those from several other investigators. Mechanical perturbation can be caused by stretch or contraction (or both) and leads to distortions in the extracellular matrix or the actin cytoskeleton. These changes are sensed by proteins spanning the sarcolemma, which include both the integrin and SG complexes. The complexes can instigate signaling cascades that include ERK1/2 and are likely to be mediated by FAK. Modification of SG complex members also is caused by mechanical perturbation. In conclusion, this study demonstrates that the SG complex is a critical component of mechanical signal transduction in skeletal muscle. These experiments build on a large body of work that has implicated the DGC as a signaling complex and begin to resolve the differences between mechanical fragility associated with the loss of the entire DGC and mechanical signaling properties inherent in the complex.



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  		Fig. 6. Model for mechanical sensors in skeletal muscle. The dystrophin glycoprotein (DG) complex is poised to detect mechanical changes. Mechanical perturbation, induced by stretch or contraction, or both (large arrows), can cause membrane distortions that are sensed by proteins spanning the sarcolemma, including the integrins and SGs. This leads to activation of the MAPK pathways (represented by ERK) and also phosphorylation of SG complex members. The kinase likely to mediate these changes is focal adhesion kinase (FAK). nNOS, neuronal nitric oxide synthase; {alpha}-DB, {alpha}-dystrobrevin; SYN, syntrophin.

 

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 ABSTRACT
 METHODS
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REFERENCES
 
This study was supported by grants from the Muscular Dystrophy Association and the American Heart Association.


   ACKNOWLEDGMENTS
 
Technical assistance from L. A. Morris is gratefully acknowledged. I thank E. M. McNally for providing encouragement, insight, and the {gamma}-SG C57Bl/6 null mice and H. L. Sweeney and T. S. Khurana for support and many helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. R. Barton, Dept. of Anatomy and Cell Biology, 441A Levy Bldg., 240 S. 40th St., Univ. of Pennsylvania, Philadelphia, PA 19104 (e-mail: erbarton{at}biochem.dental.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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