α7β1-Integrin links laminin in the extracellular matrix with the cell cytoskeleton and therein mediates transduction of mechanical forces into chemical signals. Muscle contraction and stretching ex vivo result in activation of intracellular signaling molecules that are integral to postexercise injury responses. Because α7β1-integrin stabilizes muscle and provides communication between the matrix and cytoskeleton, the role of this integrin in exercise-induced cell signaling and skeletal muscle damage was assessed in wild-type and transgenic mice overexpressing the α7BX2 chain. We report here that increasing α7β1-integrin inhibits phosphorylation of molecules associated with muscle damage, including the mitogen-activated protein kinases (JNK, p38, and ERK), following downhill running. Likewise, activation of molecules associated with hypertrophy (AKT, mTOR, and p70S6k) was diminished in mice overexpressing integrin. While exercise resulted in Evans blue dye-positive fibers, an index of muscle damage, increased integrin protected mice from injury. Moreover, exercise leads to an increase in α7β1 protein. These experiments provide the first evidence that α7β1-integrin is a negative regulator of mechanotransduction in vivo and provides resistance to exercise-induced muscle damage.
- downhill running exercise
- MAP kinase
the α7β1 integrin is a glycoprotein that links laminin in the extracellular matrix with actin in the cytoskeleton (40). This integrin is localized in the sarcolemma of skeletal muscle and concentrated at neuromuscular and myotendinous junctions and costameres (2, 8, 27). Splicing of α7 transcripts produces alternative cytoplasmic (α7A and α7B) and extracellular domain variants (i.e., X2) and thereby contributes to functional diversity of this integrin (8, 41). The α7BX2 chain is the most abundant isoform in adult skeletal muscle (8). The cytoplasmic domain of the α7B isoform is composed of 77 amino acids, the largest of all integrin α-chains. Homologous sequences in the α7B cytoplasmic domain suggest it has a potential for modulating signaling, including serine/threonine protein kinase and tyrosine phosphatase activity (41). Mutation of the single tyrosine residue in this domain inhibits the normal negative regulation of localization of the integrin with agrin-induced acetylcholine receptor clusters (9).
Patients with Duchenne muscular dystrophy, and mdx mice that also lack dystrophin, exhibit increased amounts of α7β1-integrin (17). The α7BX2 isoform protein is also increased in response to muscle injury (22) and increasing amounts of this integrin maintains junctional integrity and ameliorates the development of disease in an animal model of severe muscular dystrophy (10, 11). Mutations in the α7 gene (ITGA7) cause human congenital myopathy (16). These results suggest that the α7β1-integrin has a protective effect in maintaining muscle structure and function.
Mitogen-activated protein (MAP) kinase signaling is divided into four pathways that include the c-Jun NH2-terminal kinase (JNK), the p38 kinase, the extracellular signal-regulated kinases 1 and 2 (ERK1/2), and ERK5 cascades. MAP kinases are activated in response to exercise and contraction (15, 28, 38) and are crucial for numerous physiological processes including cell proliferation, differentiation, inflammation, apoptosis, hypertrophy, and gene transcription (13, 14, 25). A strong correlation between peak tension and JNK and ERK phosphorylation in situ indicates these MAP kinases are particularly responsive to mechanical stress (28). Muscle lengthening, exercise, and stretch rapidly and transiently increase JNK and p38 phosphorylation (5, 6) and result in muscle damage (32, 33). JNK is also activated in mdx mice and may contribute to pathogenesis (24). These results suggest a direct relationship between the extent of MAP kinase activity and muscle damage.
Integrins also respond to mechanical forces in vitro and activate MAP kinase signaling (23, 39). However, it is not known whether integrin is an upstream modulator of signaling in response to mechanical stimulation in vivo. To examine mechanotransduction by integrin in vivo and further test the hypothesis that α7β1-integrin has a protective effect in maintaining muscle structure and function, we have investigated the role of the α7β1-integrin in modulating signaling and providing protection from exercise-induced damage in skeletal muscle in wild-type mice and transgenic animals expressing eightfold more α7BX2. Our results indicate that the α7β1-integrin is a negative regulator of signaling and protects against exercise-induced muscle damage.
MATERIALS AND METHODS
Muscle creatine kinase-intron-α7BX2 construct.
A chimeric intron, composed of the 5′ donor site from the first intron of the human β-globin gene and the branch site and a 3′ acceptor site from the human immunoglobulin heavy chain variable region, was amplified from the pCI mammalian expression vector (Promega) and subcloned between the muscle creatine kinase (MCK) promoter and rat α7BX2 cDNA to stabilize the transgene transcript. The sequence of the construct was verified and expression was confirmed by transfecting C2C12 myoblasts. The exogenous rat integrin was detected by immunofluorescence in myotubes using O26 antibody (11).
Production of transgenic mice.
The MCK-intron-α7BX2 construct, encoding the rat α7BX2 isoform, was gel purified. Transgenic mice were produced in a SJ6/C57BL6 background at the University of Illinois Transgenic Animal Facility as described (11). Twenty positive founder lines were identified and four lines were found to overexpress the rat α7BX2 integrin protein in skeletal muscle two-, four-, and eightfold compared with wild-type controls. The line expressing eightfold more integrin was used to detect changes in signaling. Where indicated, additional lines were used to assess muscle damage. Genotypes were determined by PCR analysis (primers: MCK1, 5′-CAAGCTGCACGCCTGGGTCC-3′ and AATII, 5′-GGCACCCATGACGTCCAGATTGAAG-3′) as described by Burkin et al. (11).
Animals and downhill running exercise.
Protocols for animal use were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign. The animals were fasted overnight and experiments were conducted at approximately the same time of day. Five-week-old female wild-type and transgenic mice remained at rest (basal) or were run on a treadmill (Exer-6M, Columbus Instruments) in a downhill running decline of 20°. Mice used for cell signaling analyses were run for 30 min at 15 m/min after a 1-min warmup at 10 m/min. Mice were euthanized via cervical dislocation immediately after exercise or 3 h postexercise. The gastrocnemius-soleus complexes were rapidly dissected and frozen in liquid nitrogen. For studies examining muscle damage and integrin protein after exercise, mice were run at 17 m/min for 30 min after the same warmup period. These mice were euthanized 24 h after exercise, and their gastrocnemius-soleus complexes were rapidly dissected, frozen in liquid nitrogen-cooled isopentane, and stored at −80°C. Wild-type and transgenic mice not subjected to downhill running were otherwise treated identically as those that were exercised.
Preparation of skeletal muscle lysates.
For signaling analyses, gastrocnemius-soleus complexes were homogenized in ice-cold lysis buffer composed of 2% Triton X-100, 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 7.5), 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1/200 protease inhibitor cocktail III (Calbiochem), and 1 mM PMSF. Homogenates were rotated 30 min at 4°C and centrifuged for 5 min at 10,000 g. This process was repeated once, the supernatants were pooled, and protein concentrations were determined with the use of the Bradford assay (Bio-Rad). To determine α7 protein levels, gastrocnemius-soleus complexes were extracted in SDS buffer containing 10% SDS, 100 mM Tris·HCl (pH 8.0), 10 mM EDTA, and 10% glycerol. Homogenates were boiled for 3 min, supernatants were collected, and protein concentrations were determined (A280).
Equal amounts of protein (100 μg) were separated by SDS-PAGE using 6, 8, and 10% acrylamide gels and transferred to nitrocellulose membranes. Equal protein loading was verified by Ponceau S staining. Membranes were blocked in Tris-buffered saline (pH 7.8) containing 5% BSA and membranes were incubated overnight with primary antibody (1:500 phospho-p70S6k, 1:1,000 for all others). Polyclonal antibodies against signaling molecules were obtained from Cell Signaling Technology. Polyclonal rabbit anti-α7CDB-347 antiserum that recognizes the α7B cytoplasmic domain was used to confirm α7 levels (41). Membranes were probed with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000; Jackson Laboratories) and bands were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech).
Assessment of myofiber damage.
Wild-type and transgenic mice were injected with Evans blue dye (50 μl/10 g body wt ip) 90 min before downhill running exercise began. Twenty-four hours postexercise, gastrocnemius-soleus complexes were dissected and frozen as described above. Frozen 7-μm-thick sections were prepared (3 sections per sample) and a total of 50 fields were observed per animal with the use of fluorescence microscopy (×40 objective, Excitation570/Emission640 filters; Leica DMRXA2). The mean numbers of Evans blue-positive fibers in 50 fields are given.
Transgenic expression of rat α7-integrin was detected in gastrocnemius muscle as described (11). In brief, 8-μm-thick sections were fixed in acetone and blocked with PBS containing 10% horse serum. Endogenous mouse immunoglobulin was blocked with 70 μg/ml goat anti-mouse monovalent Fab fragments (Jackson ImmunoResearch). Rat α7 was localized with the use of 5 μg/ml purified O26 monoclonal antibody and FITC-labeled donkey anti-mouse (1:100; Jackson ImmunoResearch), and detected by immunofluorescence microscopy (Leica DMRXA2 microscope; ×40 objective; Excitation480/Emission527 filters).
All averaged data are presented as the means ± SE. Comparisons of signaling and damage between wild-type and transgenic mice, basal and exercised, were performed by one-way ANOVA, followed by Tukey’s post hoc analysis (SigmaStat). An unpaired t-test was used to compare basal and postexercise increases in α7 protein. Differences were considered significant at P < 0.05.
Characterization of mice overexpressing α7BX2 integrin.
Transgenic mice that express the sequence encoding the rat α7BX2 integrin chain under control of the muscle creatine kinase promoter were generated as described in materials and methods. α7BX2 is the predominant α7 chain isoform expressed in adult skeletal muscle. Quantitation of integrin by Western blot analysis demonstrated that the three strains of mice used in these experiments produced 2-, 4-, and 8-fold more α7B chain than their wild-type counterparts (Fig. 1A). Immunofluorescence microscopy, using a monoclonal antibody that detects the rat α7 chain, shows that the integrin encoded by the rat transgene is localized to the sarcolemma (Fig. 1B). No outwardly discernable differences (weight, mobility, and vigor) were noted between the transgenic and the control animals. Except where indicated, mice expressing an eightfold increase in integrin were used in these experiments. Mice not subject to downhill running were otherwise treated identically as those that were.
Overexpression of α7BX2 integrin inhibits phosphorylation of MAP kinase signaling molecules.
Exercise that induces muscle damage results in the rapid phosphorylation and activation of molecules in the MAPK signaling cascades, including JNK, p38, and ERK. As shown in Fig. 2A, phosphorylation of JNK1, p38, and ERK1/2 was increased ∼80% in wild-type mice immediately after a single bout of downhill running exercise compared with mice that were not exercised. In contrast, phosphorylation of the MAP kinases was not increased immediately after exercise in mice overexpressing α7BX2 integrin.
Phosphorylation of the MAP kinases is usually transient (5, 6), and stress-induced phosphorylation of the JNK1, p38, and ERK1/2 kinases is back to basal levels by 3 h postexercise in wild-type mice (Fig. 2B). Phosphorylation of JNK1 and ERK1/2 is similarly decreased 3 h postexercise in transgenic mice. However, increased integrin appears to delay exercised induced phosphorylation of p38: a 2.7-fold increase in the phosphorylation of p38 was observed 3 h after exercise in α7 transgenic mice. This delay in exercise-induced phosphorylation of p38 suggests that muscle is responsive to mechanical forces and that increased integrin transiently inhibits p38 activation immediately postexercise. Total MAP kinase levels remained at basal levels and unchanged in wild-type and α7-transgenic mice immediately or 3 h postexercise (data not shown).
Overexpression of α7BX2 integrin inhibits phosphorylation of AKT, mTOR, and p70S6k.
To determine whether the attenuation of signaling observed in the MAP kinases was restricted to this family of molecules, we examined the phosphorylation and thus the activation of protein kinase B (AKT), the mammalian target of rapamycin (mTOR) and the ribosomal S6 kinase (p70S6k) in response to downhill running. These molecules are activated by muscle lengthening and stretching ex vivo (18, 19, 35, 38) and they are important in initiating protein synthesis and hypertrophy in skeletal muscle (4). Phosphorylation of AKT and mTOR were increased 3.7- and 3.8-fold, respectively, in wild-type mice 3 h postexercise compared with animals that were not exercised (Fig. 3, A and B). As observed with the MAP kinases, phosphorylation of AKT and mTOR was not increased upon exercise in the mice with enhanced α7β1-integrin levels. Likewise, phosphorylation of p70S6k was increased 2.7-fold in wild-type mice immediately after exercise, and no increase was observed in α7 transgenic animals (Fig. 2C). Total amounts of AKT, mTOR, and p70S6k protein remained unchanged in wild-type and transgenic mice in the basal state or after exercise. Thus the α7β1-integrin also inhibits signaling that is normally activated by exercise and that leads to protein synthesis and hypertrophy.
Overexpression of α7BX2 integrin prevents muscle damage.
Activation of JNK has been correlated with the extent of injury induced by eccentric exercise (5). The lack of activation of the stress-activated protein kinases in the α7 transgenic mice suggests enhanced levels of the integrin may protect against exercise-induced muscle damage. To examine this hypothesis, the uptake of Evans blue dye was used to determine whether the sarcolemma of wild-type and transgenic mice were differentially compromised by exercise. Evans blue dye binds to albumin and this complex is taken up in muscle with compromised membrane integrity (29). Considerable dye uptake was observed in wild-type gastrocnemius muscle 24 h postexercise (Fig. 4). In contrast, limited injury was evident in mice overexpressing the α7BX2 integrin. The number of Evans blue-positive fibers was increased in the wild-type-exercised group, whereas few positive fibers were detected in mice overexpressing α7BX2 integrin.
To determine whether the extent of fiber damage was modulated in proportion to the amount of integrin, similar studies were done using mice overexpressing twofold and fourfold α7BX2 integrin compared with the eightfold level of expression in the mice used in the above experiments. Prevention of damage was the same in all lines of transgenic mice with enhanced integrin levels (Fig. 4). Differences in basal values between wild-type and transgenic mice were not significant.
An approximate twofold increase in α7β1-integrin occurs in patients with Duchenne muscular dystrophy and in mdx mice that also lack dystrophin (17). This additional integrin may compensate in part for the lack of the dystrophin complex and thereby modulate the degree and progression of disease. Thus increasing integrin appears to be a normal compensatory mechanism that ameliorates disease-related muscle injury. Therefore, does exercise-induced injury also promote an increase in the amount of integrin in skeletal muscle?
As shown in Fig. 5, there is a 70% increase in α7 protein in wild-type skeletal muscle within 24 h of a single bout of downhill running exercise, and this increase largely persists for up to 1 wk. Increasing integrin appears to be a mechanism by which muscle is protected from stress-related damage as well as from disease-related injury.
We demonstrate here that α7β1-integrin attenuates the phosphorylation of signaling molecules activated by downhill running exercise, stabilizes the sarcolemmal membrane, and prevents exercise-induced muscle damage in skeletal muscle. Transgenic overexpression of the integrin suppresses activation of the MAP kinases and molecules stimulated by muscle lengthening and/or contraction, including AKT, mTOR, and p70S6k. Exercise also enhances the expression of integrin protein in wild-type mice. This report represents the first evidence that α7β1-integrin prevents exercise-induced muscle damage and affords protection from mechanical stress in vivo.
The α7β1-integrin provides a mechanism through which forces can be transmitted between laminin on the outside and the cytoskeleton within cells. Integrins have also been proposed to be sensors of tensile strain at the cell surface, relaying mechanical information to molecules important for signal transduction (21). Release of growth factors does not appear to fully account for stretch-induced activation of signaling ex vivo or in vitro (18, 19) and integrins provide an additional link to signaling molecules. Although we anticipated enhanced activation of signaling in mice expressing eightfold more α7β1-integrin, these mice showed minimal or no signaling responses after an intense bout of eccentric running exercise compared with wild-type mice. One exception to this was the unexpected increase in p38 phosphorylation at 3 h postexercise. Although p38 is activated in response to contraction and stretch (6, 18), p38 phosphorylation is virtually unaffected by an actomyosin ATPase inhibitor (N-benzyl-p-toluene sulfonamide) that reduces force development by 95% (12). Thus p38 may be activated by a factor(s) associated with contraction and not by the initial mechanical stimuli or forces created by actin-myosin cross-bridge formation. The delayed activation of p38 in the α7-integrin transgenic mice used in this study also suggests that eccentric exercise may activate p38 secondarily in the presence of increased integrin.
Inhibition of signaling on integrin overexpression occurred not only in the MAP kinases immediately after exercise, but also in molecules associated with the regulation of mechanical load-induced hypertrophy. This loss of activation is most striking in p70S6k, where a 2.3-fold increase was observed immediately after exercise in wild-type mice, but no phosphorylation was detected in the transgenic animals. The immediate increase in p70S6k phosphorylation after exercise reported herein contrasts with previous reports of a several-hour delay in p70S6k activation (3). Upstream regulators, AKT and mTOR, are also believed to be activated before initiating direct p70S6k phosphorylation. Recent reports, however, suggest that p70S6k activity is not dependent on AKT phosphorylation following stretch (18) and immediate increases in p70S6k are found in C2C12 cells in response to multiaxial, but not uniaxial strain (19). Therefore, the temporal pattern of phosphorylation observed in this study may be specific to the forces incurred during downhill running.
Although activation of p70S6k positively regulates hypertrophy (3), downhill running exercise does not appear to promote growth (7). This may be explained by the increase in integrin expression that occurs after downhill running and its negative regulatory impact on activation. Studies examining repeated bouts of exercise will clarify the influence of the α7β1-integrin on p70S6k signaling. The constitutive activation of c-Raf-1 kinase, an upstream positive regulator of ERK, in α7-null mice underscores the regulation of the α7β1-integrin on kinase signaling cascades (37).
The loss of signaling observed following eccentric exercise in the α7-integrin transgenic animals may be mediated by several mechanisms. Increased laminin-myofiber-actin attachment increases muscle “stiffness” following eccentric exercise (36) and results in decreased mechanosensitivity. Thus increased adhesion of muscle cells to the extracellular matrix may result in muscle stiffness and desensitization to mechanical signals. Alternatively, integrin may mediate activation of phosphatases, leading to rapid downregulation of signaling. For example, epidermal growth factor receptor signaling is promoted by α1β1-integrin activation of a ubiquitous tyrosine phosphatase (30).
Attenuation of cellular signaling has also been documented in responses to aging (26, 34, 35, 42). The phosphorylation of JNK, p38, and ERK1/2 is decreased in response to knee extensor resistance exercise in old vs. young men, whereas total protein content was not different between the two groups (42). Contraction-mediated increases in mTOR, p70S6k, and ERK1/2 also have been demonstrated to be lower in old rats compared with young rats in situ (35). In contrast, JNK, p38, and p70S6k signaling is not compromised in extensor digitorum longus muscles from old mice after passive stretch ex vivo (20). Differences in the models employed (in vivo and in situ vs. ex vivo) may account for these different results. Regardless, it will be interesting to determine whether integrins are “upstream” regulators of age-related decreases in mechanosensitivity.
Increases in integrin may also modulate the severity of skeletal muscle diseases. An approximate twofold increase in α7β1-integrin occurs in patients with Duchenne muscular dystrophy and in mdx mice that also lack dystrophin (17). This additional integrin may compensate in part for the lack of the dystrophin complex and thereby modulate the degree and progression of disease. Increased α7β1-integrin is also found in δ- and γ-sarcoglycan-deficient mice, the models for type 2D and 2F human limb girdle muscular dystrophies, respectively (1). Moreover, mice that lack both utrophin and dystrophin (mdx/utrn−/−) develop a severe form of muscular dystrophy, and the transgenic induction of an additional twofold increase in α7β1-integrin ameliorates development of pathology in these animals (10, 11). Increases in α7-integrin mRNA and protein also occur in skeletal muscle in response to transection injury (22). The lack of stress-activated protein kinase signaling in the transgenic mice in our study and the reports of increased α7β1-integrin with disease and injury prompted us to evaluate muscle damage in mice overexpressing the α7-integrin. Interestingly, exercise-induced injury in this study promoted a 70% increase in α7B protein in wild-type skeletal muscle within 24 h of a single bout of downhill running exercise. Thus increasing integrin appears to be a mechanism by which skeletal muscle may be protected from exercise-related damage as well as from disease related injury and perhaps aging.
This work was supported by National Institute on Aging Grant AG-14632 and a grant from the Muscular Dystrophy Association.
We thank E. Chaney and J. Mulligan for technical assistance and G. Q. Wallace for helpful discussions.
Present address for D. J. Burkin: Department of Pharmacology, University of Nevada School of Medicine, 1664 North Virginia Ave., Reno, NV 89557.
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