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Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
1Correspondence: Baylor College of Medicine, One Baylor Plaza, Department of Medicine, Pulmonary Section, Suite 520B, Houston, TX 77030, USA. E-mail: boriek{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: dystrophin • skeletal muscle • MAP kinases • AP-1 • mechanotransduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. The 427 kDa cytoskeletal protein dystrophin is localized in the subsarcolemmal region of skeletal and cardiac muscle (4)
. Dystrophin binds to the cytoskeletal actin and to the cytoplasmic tail of transmembrane DGC protein ß-dystroglycan, and thus forms a part of the link from the cytoskeleton to the extracellular matrix (5
, 6)
. Functional inactivation of the dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and in mdx mice (a mouse model of DMD) (7
8
9)
.
Mechanical stress is considered one of the major factors that cause many pathological situations associated with skeletal muscles such as muscle hypertrophy and atrophy (10
11
12)
. Several reports suggest that skeletal muscles and cardiac myocytes that lack dystrophin are more susceptible to muscle injury caused by mechanical stresses (13
14
15)
. Although the primary pathogenetic processes are apoptotic and necrotic muscle cell death, the molecular mechanism(s) that causes the muscle cell death in the absence of dystrophin has remained unknown. Accumulating evidence suggests that the DGC complex has an important signaling role in muscle and is not merely a molecular scaffold serving the mechanical function (16)
. We postulate that the absence of dystrophin causes activation of a pernicious signal transduction pathway leading to the activation of an array of proinflammatory transcription factors and catabolic molecules, which could possibly contribute to muscular dystrophy. Indeed, we have recently shown that the muscle specific activity of nuclear factor kappaB (NF-
B) and the level of NF-B-regulated cytokines such as IL-1ß and TNF-
in muscle fibers from dystrophin-deficient mdx mice begin to increase even before the onset of muscular dystrophy, suggesting a possible deregulation of this signaling pathway (17)
.
Mechanical forces are powerful stimulators of the mitogen-activated protein (MAP) kinase pathways in different cell types (10
, 18
, 19)
. Activation of MAP kinases, therefore, is suggested as an important component of mechanotransduction (mechanical signal transduction), the process by which cells transduce physical force-induced signals into biochemical responses (20
21
22)
. In mammalian cells, three parallel MAP kinase pathways have been described: extracellular signal related kinase (ERK1/2), protein kinase 38 (p38), and c-Jun-N-terminal kinases (JNKs). These kinases act via regulation of the activity of many downstream transcription factors, including activator protein-1 (AP-1), c-myc, Elk1, and CCAAT/enhancer binding protein-ß (C/EBP-ß) (22
23
24
25)
. We recently showed that application of mechanical stress to skeletal muscle fibers leads to the activation of ERK1/2 and AP-1 through two distinct signaling pathways that depend on the direction of the force applied to the muscle sheet (26)
. However, the effects of dystrophin deficiency on the signal transduction pathway leading to activation of MAP kinases in response to mechanical stretch remained unknown.
In the present investigation, we performed a systematic study to understand the exact role of dystrophin in altering the mechanical properties and the underlying biochemical mechanism(s) leading to the activation of MAP kinase signaling pathways. In contrast to published work (27)
on mechanical properties of muscle fibers of mdx mice, we used muscle fibers before the onset of muscle necrosis. Therefore, our study describes mechanical as well as biochemical signaling mechanisms in normal and mdx muscle fibers that are attributed to the loss of dystrophin and not due to the secondary structural and signaling effects that occur during muscle degeneration. Our data show that dystrophin is a load-bearing protein, loss of which increases muscle compliance and causes a higher basal level of activation of ERK1/2 and downstream transcription factor AP-1. The data show that compared with control muscles, there is increased activation of ERK1/2 and AP-1 in response to mechanical stretch in the muscle fibers of mdx mice. Furthermore, our data show that an increased influx of Ca2+ ions as well as greater activity of stretch-activated (SA) channels is responsible for the anomalous activation of ERK1/2 and AP-1 in mdx muscle fibers in response to applied mechanical stretch.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ATP [specific activity, 3000 (111 TBq) Ci/mmol] was obtained from Perkin-Elmer (Boston, MA, USA).
Animals and tissue preparation
Mice (C57BL6/SCSN and DMDMDX/J) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). These mice were housed and fed in stainless steel cages on a 12 h on and 12 h off lighting schedule. The animal facility is a virus-free facility. Experimental protocols were approved by the Animal Protocol Review Committee of the Baylor College of Medicine Animal Program (animal welfare assurance #A-3823-01) and assigned protocol #AN-1727. All procedures were conducted in strict accordance with public health service animal welfare policy. The experiments used a total of 32 dystrophin-deficient mdx and control mice. For the mechanics experiments we used eight C57/BL10 SNJ 3-wk-old control mice (weight: 16.0±4.0 g; age: 33.3±9.0 days) and eight C57/BL10SCSN DMDMDX/J dystrophin-deficient mice (weight: 6.3±1.1 g; age: 20.4±1.2 days). For biochemical assays we used eight C57/BL10 SNJ 3-wk-old control mice (weight: 9.0±0.8 g; age: 16.4±1.2 days) and eight C57/BL10SCSN DMDMDX/J dystrophin-deficient mice (weight: 7.8±1.1 g; age: 17.1±1.2 days). The mice were anesthetized with pentobarbital sodium and the diaphragm muscles were excised. The diaphragm was excised by making an incision in the thoracic cavity and removing the muscle including its insertions on the spine and ribs. The excised muscles were immediately submerged in Krebs-Ringer solution (pH 7.18, containing in mM: 137 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, and 24 NaHCO3) equilibrated with a mixture of 95% O2–5% CO2. To study the effects of Gd3+ ions on ERK1/2 activation, we used HEPES buffer (in mM: 10 HEPES [pH 7.2], 137 NaCl, 5 KCL, 2 CaCl2, 1 MgSO4). The solution was maintained at a temperature of 25°C throughout muscle preparation and the experimental mechanical testing phase of this study. For the mechanics experiments, two pairs of markers (0.2 mm in diameter) made of surgical silk thread were sutured on the surface of the muscle at 1 mm intervals. The marker pairs were placed along two neighboring muscle fibers so that muscle fiber directions were well defined.
In vitro mechanical apparatus
An in vitro muscle mechanical testing system was used to measure passive mechanical properties of the diaphragm muscle from the mdx mice and their controls. The description of this apparatus has been detailed in our previous work (28)
. Briefly, the system consists of two independent orthogonal axes that are driven by stepper motors or micrometers. The system is designed to stretch the tissue at a constant rate in the plane of the muscle sheet. Two small identical alligator clamps (0.2 cm width; Newark Electronics, Newark, NJ, USA) were used to hold the muscle during uniaxial loading either along or transverse to the muscle fibers. For the diaphragm, one clamp was attached to the central tendon and the opposite clamp was attached to the rib cage near the muscle insertion.
Muscle length–tension curves
Passive lengthening and passive shortening cycles were used to characterize passive length–tension relations of the diaphragm. All lengthening-shortening cycles started from the unstressed length of the muscle. We measured the uniaxial length–tension relationship along the fibers and uniaxial length–tension relationship in a direction transverse to the fibers. The strain rates were between 0.1% and 1.0% change in length/s. Stretch ratios are generally insensitive with respect to strain rate as long as the change in strain rate does not exceed an order of 103. Muscle stretching is sensitive to the history of deformation; therefore, we preconditioned the muscle by lengthening and shortening the muscle for 5 cycles from 60% Lo to Lo. After preconditioning, the length–tension curves were reproducible.
Strain calculations
Strains were calculated according to Boriek et al. (26
, 29)
with the following modifications. Coordinates for any three markers are denoted xi and yi (i = 1, 2, 3), where three markers define a triangle in a plane. Subsequently, the four-sutured markers would define four triangles. Displacements of the markers from an unstressed state to stressed states of loading are denoted by ui for displacements along the fibers and by vi for displacement transverse to the fibers. For example, the equation
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Values of the coefficients a2, a3, etc., were used to find the partial derivatives, which were substituted into the following equations defined to calculate strains.
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x denotes strain along the fibers, y denotes strain transverse to the fibers, and xy denotes shear strain in the plane of the muscle sheet. Strains were computed relative to unstressed length. Unstressed length was defined as the length of the markers in the absence of applied forces. For small strains, x and y are equivalent to the fractional change of muscle fiber length relative to the unstressed length in their respective direction. The length–tension curve in Fig. 1
is plotted using extension ratio 
; =1.0 + .
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Ex vivo stretch protocol
Mechanical stretch was applied to the entire costal muscles of the left hemidiaphragm of normal and mdx mice by passively stretching the muscle in the direction of the muscle fibers by applying passive tension of 
0.4 N/cm. This is equivalent to a passive stress of 11 N/cm2. Mechanical stretch of 50% from the unstressed state was achieved in the normal diaphragm. Unstressed length or length of excised muscle is the shortest length of the muscle; it is equivalent to the length of the muscle at a lung volume of total lung capacity. Optimal length is 125% of this unstressed length. Therefore, our stretch of 50% placed the diaphragm at a length equivalent to 120% of optimal muscle length.
Assay of ERK1/2, p38, and FAK
After muscles were stretched for 15 min, samples were washed with phosphate-buffered saline (PBS) and homogenized in lysis buffer A (20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.3% NP-40, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/mL benzamidine, 1 mM dithiothreitol, and 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM ß-glycerophosphate). The protein concentration of the samples was measured using BioRad protein assay reagent. An 80 µg aliquot of protein was resolved on each lane on 10% SDS-PAGE, electrotransferred onto nitrocellulose membrane, and probed with the phospho-specific anti-p44/42 MAPK (Thr202/Tyr204), anti-p38 (Thr180/Tyr182) MAP kinase, or anti-FAK (Tyr576/577) (Cell Signaling Technology, Inc.) raised in rabbits and detected by chemiluminescence (ECL, Amersham Pharmacia). The bands obtained were quantitatively assessed with Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Molecular Dynamics, Sunnyvale, CA, USA). Total amount of these kinases in the cell extracts was determined by using antibodies that bind to both phosphorylated and unphosphorylated proteins.
c-Jun kinase assay
The c-Jun kinase assay was performed by a described method (26
, 30)
. After application of mechanical stretch to the muscles for 15 min, the tissues were washed with 1x PBS and cell extracts were prepared by grinding the tissue in lysis buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.3% Nonidet P-40, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/mL benzamidine, and 1 mM dithiothreitol. Cell extracts (600–700 µg protein/sample) were immunoprecipitated with 0.5 µg of anti-JNK1 antibody for 4–6 h at 4°C. Immune complexes were collected by incubation with protein A Sepharose beads for 1 h at 4°C. The beads were washed three times with lysis buffer, followed by two additional washes with kinase buffer (20 mM HEPES, pH 7.4, 1 mM dithiothreitol, 50 mM NaCl). Kinase assays were performed for 15 min at 30°C with GST-Jun (1-79) as a substrate (2 µg/sample) in 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 µM dithiothreitol, and 10 µCi of [-32P] ATP. Reactions were stopped with the addition of 20 µL of 2x SDS sample buffer, boiled for 5 min, and subjected to 10% SDS-PAGE. GST-Jun-(1-79) was visualized by staining with Coomassie brilliant blue, and the dried gel was exposed to X-ray film to see the radioactive bands of GST-c-Jun (1-79).
Electrophoretic mobility shift assay
To determine AP-1 activation, electrophoretic mobility shift assay (EMSA) was performed as described previously (17
, 31)
. After applying mechanical stretch for 15 min, the muscles were immediately frozen with dry ice and suspended at 1 mg muscle weight per 18 µL of low salt lysis buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin, 0.5 mg/mL benzamidine), followed by mechanical grinding. Cells in the lysis buffer were allowed to swell on ice for 5 min, followed immediately by two cycles of freeze-thaw lysis. Tubes containing the lysed muscle cells were then vortexed vigorously for 10 s, and the lysate was centrifuged for 10 s at 14,000 rpm. The supernatant (cytoplasmic extracts) was removed and saved at –70°C for further biochemical analysis. The nuclear pellet was resuspended in 4 µL of ice-cold high-salt nuclear extraction buffer (20 mM HEPES [pH 7.9], 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin, 0.5 mg/mL benzamidine) per milligram of original muscle weight and incubated on ice for 30 min with intermittent vortexing. Samples were centrifuged for 5 min at 4°C and the supernatant (nuclear extract) was either used immediately or stored at –70°C. The protein content was measured by the method of BioRad (Hercules, CA, USA) protein assay reagent. EMSAs were performed by incubating 14 µg of nuclear extract with 16 fmol of the 32P-end-labeled AP-1 consensus oligonucleotide for 15 min at 37°C. The incubation mixture included 2–3 µg of poly dI·dC in a binding buffer (25 mM HEPES [pH 7.9], 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl). The DNA–protein complex thus formed was separated from free oligonucleotide on 7.5% native polyacrylamide gel using buffer containing 50 mM Tris, 200 mM glycine (pH 8.5), and 1 mM EDTA. The gel was dried, and the radioactive bands were visualized and quantitated by a PhosphorImager (Molecular Dynamics), using ImageQuant software. The specificity of the AP-1 bands were confirmed by using a mutated probes and cold competition assays.
Statistical analysis
All experiments were repeated at least three times unless indicated otherwise. Results are expressed as mean ± SD. Statistical analysis used Student’s t test or ANOVA to compare quantitative data populations with normal distribution and equal variance. A value of P < 0.05 was considered statistically significant unless otherwise specified.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stress–strain relationship of muscle fibers of normal and mdx mice
Since dystrophin is an important component of the DGC, a large complex of membrane-associated proteins critical to the integrity of the skeletal muscle fibers, we first investigated the effects of dystrophin deficiency on muscle mechanical properties. Optimal length (Lo) of normal diaphragm muscles was 125% of unstressed length and dystrophin-deficient diaphragm muscles were 150% of unstressed length. Length–tension relationships of representative diaphragm muscles from 17-day-old mdx and its normal control mouse are shown in Fig. 1
. These relationships are measured in the direction of the muscle fibers and transverse to the fibers. The data demonstrate that during lengthening, there is a slow and continuous increase in tension over the range of imposed strains. At a tension of 8 g/cm, muscle lengths as a fraction of their unstressed length in the direction along the fibers were 151% and 125% in the mdx and control mice, respectively, yielding a compliance ratio of 1.21. At the same level of tension, muscle lengths as a fraction of their unstressed length in the direction transverse to the fibers were 122% and 110% in mdx and control mice, respectively, resulting in a compliance ratio of 1.11. Both curves for the mdx muscle shifted to the right compared with their respective controls. These data demonstrate an increase in muscle compliance and muscle extensibility in the mdx mouse diaphragm compared with normal muscle.
Spurious activation of ERK1/2 in mdx muscle fibers in response to mechanical loading
We recently showed that mechanical stretching of the normal diaphragm muscle fibers activates ERK1/2, JNK1, and p38 MAP kinase (26)
. In the current study, we investigated whether dystrophin deficiency in mdx muscle fibers alters the activation of either of these kinases in response to mechanical stretch. Diaphragm muscle from normal or mdx mice (16-17 days old) were stretched by applying a tension in the direction of the muscle fibers of 0.4 N/cm and the muscle was kept in a stretched state for 15 min. At the end of the stretch protocol, muscles were lysed and subjected to immunoblotting with phospho-ERK1/2 and phospho-p38 MAP kinase antibodies or for immunoprecipitation with anti-JNK1 to determine the activity of JNK1. As shown in Fig. 2
A, the basal level of activation of ERK1/2 as measured by using phospho-specific antibodies was significantly higher in muscle from mdx mice than in corresponding controls. Mechanical stretching of normal or mdx diaphragm muscle resulted in an increased activation of ERK1/2. However, the level of activation of ERK1/2 in dystrophin-deficient muscle fibers was significantly higher in mechanically loaded muscle (Fig. 2A
), even though an identical magnitude of tension was applied to the dystrophin-deficient and control mice. This indicates an aberrant activation of ERK1/2 signaling pathway in mdx diaphragm muscles in response to mechanical loading. The increased activation of ERK1/2 in muscles from mdx mice was not due to a difference in the amount of these kinases, as the total cellular level of ERK1/2 remained the same in control and dystrophin-deficient diaphragm (Fig. 2B
). Although mechanical stretching of either normal or mdx diaphragm resulted in increased activation of JNK1 (Fig. 2C
) and p38 MAP kinase (Fig. 2D
), there was no significant difference in the level of activation of these kinases in response to mechanical stretch. These data suggest that dystrophin deficiency in mdx muscle fibers causes a spurious activation of ERK1/2 in response to mechanical stretch.
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PD98059 inhibits the activation of ERK1/2 in response to mechanical stretch of normal and mdx muscle fibers
PD98059 is a specific inhibitor of the MAP kinase kinase 1/2 (MEK1/2), a kinase that phosphorylates ERK1/2 in the classical ERK1/2 signaling pathway (32
, 33)
. We recently showed that MEK1/2 are involved in stretch-induced ERK1/2 activation in the diaphragm muscle fibers only when stretch is applied along the direction of muscle fibers and not in a transverse direction (26)
. We investigated whether MEK1/2s are involved in mechanical stretch-induced signaling in diaphragm muscle of mdx mice. Diaphragm muscle from 17-day-old normal and mdx mice was pretreated for 30 min with 50 µM PD98059, followed by the application of the same mechanical tension (0.4 N/cm) to normal or dystrophin-deficient diaphragms, resulting in a mechanical stretch of 40% (normal) and 60% (mdx). Either muscle was kept in the stretched state for 15 min, followed by measurement of ERK1/2 activity by immunoblotting. As shown in Fig. 3
, pretreatment of either normal or mdx diaphragm muscle with PD98059 resulted in a complete inhibition of ERK1/2 in response to mechanical stretch. These results thus indicate that ERK1/2 is still the phosphorylation target of MEK1/2 in response to axial mechanical stretch of dystrophin-deficient muscle fibers of mdx mice.
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Involvement of protein-tyrosine kinases (PTK) in mechanical stretch-induced activation of ERK1/2 in mdx muscle fibers
PTK have been shown to play an indispensable role in the activation of ERK1/2 signaling pathway in response to divergent stimuli (26
, 34
, 35)
. Although mostly receptor-associated PTK are involved in the cell signaling in response to growth factors, recent findings indicate that cell signaling in response to mechanical stress involves non-receptor-associated PTK. Focal adhesion kinase (FAK) is one such nonreceptor-associated PTK that has been implicated in the mechanosensing of the cells (36
37
38)
. We investigated the role of PTK in the mechanical stretch-induced activation of ERK1/2 in dystrophin-deficient muscle fibers. Pretreatment of muscles with 50 µM genistein (an inhibitor of PTK) for 30 min reduced the mechanical stretch-induced activation of ERK1/2 in normal as well as dystrophin-deficient muscle fibers (Fig. 4
A). An application of equal magnitude of mechanical tension increased the activity of FAK in normal and mdx muscle fibers; however, the level of activation of FAK in response to mechanical stretch was higher in muscle fibers from mdx mice (Fig. 4B
). These data suggest that mechanosensing proteins such as FAK are more prone to activation in the dystrophin-deficient muscle fibers in response to mechanical stretch.
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Role of Ca2+ in mechanical stretch-induced ERK1/2 activation in mdx muscle fibers
Ca2+ ions mediate a large number of cellular responses by binding to specific intracellular proteins, which may be thought of as Ca2+ receptors (39
, 40)
. Ca2+ ions are also important for muscle cells for excitation-contraction coupling (40)
. We investigated whether Ca2+ mobilization from either intracellular stores or influx of Ca2+ from extracellular source plays a role in stretch-induced activation of ERK1/2 in muscle fiberslacking dystrophin. Diaphragm muscles from either 16- to 17-day-old normal or age-matched mdx mice were treated in a muscle bath with 20 µM TMB-8 (an intracellular Ca2+ antagonist) for 45 min, followed by application of mechanical stretch for 15 min. As shown in Fig. 5
, pretreatment of the muscle with TMB-8 inhibited the activation of ERK1/2 in normal muscles without any significant effect on the activation of ERK1/2 in mdx muscle fibers in response to mechanical stretch. Conversely, removal of Ca2+ ions from the Krebs-Ringer solution blocked only the mechanical stretch-induced activation of ERK1/2 in mdx muscle fibers, with no effect on the normal muscle fibers. These data provide strong evidence that hyperactivation of ERK1/2 in response to mechanical stretch in dystrophin-deficient mdx muscle fibers occurs due to the influx of Ca2+ ions from extracellular source.
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Stretch-activated channels are involved in the activation of ERK1/2 in response to mechanical stretch of mdx muscle fibers
Previous studies from our laboratory along with many other reports have shown that in many cell types, such as smooth muscle and endothelial cells, the mechanical stretch-dependent cellular responses are associated with an increased influx of Ca2+ ions mainly through calcium and/or SA channels (35
, 41
, 42)
. We next investigated the role of these ion channels in the stretch-induced ERK1/2 activation in muscle fibers of mdx mice. Diaphragm muscle from normal or mdx mice was preincubated for 45 min in the presence of nifedipine (an antagonist of the L-type calcium channels), followed by the application of mechanical stretch and measurement of ERK1/2. As shown in Fig. 6
, nifedipine treatment only marginally decreased the mechanical stretch-induced activation of ERK1/2 in normal or mdx muscle fibers. Similar results were obtained with Verapamil, another potent antagonist of calcium channel (data not shown). These data suggest that dystrophin deficiency in mdx mice does not alter the role of L-type calcium channels in mechanical stretch-induced activation of the ERK1/2 signaling pathway.
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The activity of SA channels is blocked by treatment of cells with Gd3+ ions (42)
. To evaluate the role of SA channels in the mechanical stretch-induced activation of ERK1/2, normal or mdx diaphragm muscle was pretreated with 50 µM gadolinium (III) chloride for 45 min, then subjected to mechanical stretching for 15 min. Pretreatment with Gd3+ ions significantly reduced the stretch-induced activation of ERK1/2 in mdx muscle fibers (Fig. 6)
. These data clearly suggest that dystrophin deficiency in muscle fibers of mdx mice leads to the activation of SA channels, which may allow the influx of Ca2+ ions from extracellular sources in response to applied mechanical stretch.
Increased activation of AP-1 transcription factor in mdx muscle fibers in response to mechanical stretch
Activation of MAP kinases generally leads to the activation of many downstream transcription factors (such as AP-1) by either direct or indirect phosphorylation of their subunits (43
44
45)
. Since the activity of ERK1/2 was increased in response to mechanical stretch, we determined the DNA binding activity of AP-1 in normal and mdx diaphragm muscle in response to mechanical stretch. The basal level of AP-1 was higher in mdx muscle fibers than controls (Fig. 7
). Mechanical stretch further increased the activation of AP-1 in normal as well as in mdx muscle fibers. The level of activation of AP-1 in response to mechanical stretch was higher in mdx muscle fibers.
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Mechanical stretch-induced activation of AP-1 is decreased by inhibition of ERK1/2 and removal of Ca2+ ions from medium
We next investigated whether the higher level of activation of ERK1/2 is responsible for the increased activity of AP-1 in mdx muscle fibers. Diaphragm muscle from normal and mdx mice was pretreated for 30 min in the presence of 50 µM PD98059 (a MEK1/2 inhibitor), followed by the application of mechanical stretch for 15 min. The data presented in Fig. 8
show that mechanical stretch-induced AP-1 activation in mdx muscle fibers is decreased significantly by PD98059. Similarly, activation of AP-1 was blocked when muscle fibers were stretched in the absence of Ca2+ ions in Krebs-Ringer solution. These data clearly demonstrate that the higher activation of AP-1 in response to mechanical stretch of mdx muscle fibers is attributed at least in part to the increased ERK1/2 activation and influx of Ca2+ ions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since dystrophin has binding sites for DGC and actin thin filaments, both involved in retaining structural integrity of the fiber (46)
, we can expect dystrophin to contribute to the mechanical properties of skeletal muscle fiber cells. Our study is the first one at the tissue level to demonstrate that dystrophin is a load-bearing protein. Our data demonstrated increase in muscle compliance in the dystrophin-deficient diaphragm muscles compared with controls, both along and transverse to the fiber direction (Fig. 1)
. Consistent with these findings, data by Pasternak et al. (47)
at the single cell level suggested an increase in compliance of dystrophin-deficient myotubes. In contrast, Stedman et al. (27)
showed that muscle fibers from dystrophin-deficient mdx mice are significantly stiffer than their normal age-matched controls. The discrepancy in the results of our present study and those of Stedman et al. (27)
on mechanical properties of diaphragm muscle from mdx mice could best be attributed to the difference in the age of the mice used in the studies. Whereas the Stedman et al. report (27)
used the diaphragm muscle from 1.5-year-old mice, we determined the mechanical properties from 16- to 17-day-old control and mdx mice. It has been shown that at 1.5 years of age, there is a sevenfold increase in collagen content in the mdx diaphragm compared with control mice diaphragm (27)
. Muscular dystrophy in mdx mice starts at the age of 3 wk and peaks at 4–5 wk, followed by regeneration of muscle fibers of hind limb (but not diaphragm), which starts 6–7 wk (27
, 48)
. In mdx adult mice, there is an increased level of vinculin, talin, -actinin, and integrin as well as some elevation in the dystrophin-related protein utrophin. However, Law et al. have shown there is no systematic difference between 2-wk-old mdx mice and age-matched controls with regard to concentrations of the structural proteins vinculin, talin, utrophin, or -actinin (49)
. There is no change in the expression of vinculin, costameres, desmin, and nebulin in 18-day-old mdx mice (50)
, indicating that the absence of dystrophin does not disrupt the cytoskeleton in the prenecrotic state; as a result, using muscles from mdx mice before 3 wk of age would resolve this problem by avoiding muscle injury and the up-regulation of other key cytoskeletal or transmembrane proteins. Therefore, our experiments using 16- to 17-day-old mice better explain the differences of the mechanical properties of mdx and control mice, which are attributable mainly to the absence of dystrophin. Our data clearly demonstrate that dystrophin deficiency leads to loss in muscle stiffness and increased extensibility.
Although the loss of muscle fibers in DMD patients or mdx mice due to mutation and functional inactivation of dystrophin gene has been well known, the molecular mechanisms leading to the pathological state remained enigmatic. It has been suggested that a disruption of Ca2+ homeostasis that occurs in necrotic muscle fibers of DMD human and mdx mice might contribute to the muscle cell death, possibly by the activation of certain calcium-dependent proteases (40
, 51)
. Spencer et al. observed a significant difference in the activity of the calpains, a family of calcium-dependent cysteine proteases, in muscle fibers of normal and mdx mice at different stages of development (52)
. This study showed there was no difference in the activity of calpain I and II in prenecrotic muscle fibers (2 wk), but the activity of these proteases was significantly higher in the mdx mice when necrosis peaks (4 wk) (52)
. Although the study by Spencer et al. (52)
implicates the role of Ca2+ in muscle cell death in mdx mice, the initial signaling events leading to the increased activity of these proteases remained obscure. It is possible that deficiency of dystrophin in muscle fibers could lead to the activation of a signaling pathway that compromises survival mechanism(s) by up-regulating the expression of muscle wasting molecules.
Our data show that the activity of ERK1/2 and AP-1 in muscle fibers from mdx mice in response to equal magnitude of applied mechanical stress is significantly higher in mdx mice than their normal counterpart. On the other hand, there was no significant difference in the magnitude of activity of other MAP kinases such as JNK1 and p38, suggesting that dystrophin deficiency in mdx mice leads to a deregulation of only ERK1/2 among the MAP kinase signaling pathways. Because we used young mice in the prenecrotic state, the increase in the ERK1/2 activity in these fibers is not the result of an inflammatory response that occurs in the necrotic muscle fibers (53)
. We confirmed the absence of necrosis and lymphocytes infiltration in diaphragm muscle from mdx mice at the age of 17–18 days by histological and immunocytochemistry techniques (data not shown). To understand the reasons for aberrant activation of ERK1/2 in mdx muscle fibers, we investigated the role of PTK and MEK1/2 by using their specific pharmacological inhibitors. Our preliminary data showed higher total protein tyrosine phosphorylation in mdx muscle fibers than normal fibers in response to mechanical stretch (data not shown). Treatment of diaphragm muscle fibers with PTK inhibitor genistein reduced the stretch-induced activation of ERK1/2 in normal as well as mdx muscle fibers (Fig. 4A
). The activity of FAK, a nonreceptor PTK involved in cell signaling, was also significantly higher in mdx muscle fibers compared with the controls (Fig. 4B
), indicating that the stretch-induced activation of ERK1/2 in mdx muscle fibers could be a result of the increased activation of PTK such as FAK. Mechanical stretch-induced activation of ERK1/2 in normal or mdx muscle fibers was also completely blocked by pretreatment with MEK1/2 inhibitor PD98059 (Fig. 3)
. These results clearly suggest that PTK and MEK1/2 constitute the mechanical stretch-induced signaling pathway leading to the activation of ERK1/2; the absence of dystrophin did not disrupt this pathway.
In many cell types (including skeletal muscle) that respond to extracellular signals, Ca2+ serves as a second messenger that triggers intracellular responses. Changes in the intracellular Ca2+ concentration are detected by calcium binding proteins, which regulate the activity of a variety of Ca2+-dependent enzyme (39
, 40
, 54)
. The concentration of the Ca2+ ions in the cells is controlled by either influx of Ca2+ through specific calcium channels in the plasma membrane or release of sequestered Ca2+ from intracellular stores such as sarcolemma and mitochondria (55)
. An aberrant calcium homeostasis has been reported in DMD patients and mdx mice (40)
. We observed that removal of Ca2+ from the Krebs ringer solution drastically reduced the mechanical stretch-induced activation of ERK1/2 in dystrophin deficient muscle (Fig. 5)
, which suggests that an increased Ca2+ influx from extracellular source in mdx myotubes might be responsible for the anomalous activation of ERK1/2. Indeed, the Ca2+ being cofactor of many signaling proteins, including protein kinase C, is a strong activator of the Ras/Raf/MEK/ERK pathway (55)
. Removal of Ca2+ from the extracellular medium did not affect the level of activation of ERK1/2 in normal muscle. On the other hand, the inhibition of Ca2+ release from intracellular stores using TMB-8 inhibited the mechanical stretch-induced activation of ERK1/2 in normal muscle (Fig. 5)
. These results clearly indicate that the absence of dystrophin results in increased activity of some calcium channel(s), which allows greater influx of Ca2+ ion. It has been known that lack of functional dystrophin leads to greater mechanical instability of the sarcolemma, rendering dystrophic muscle more susceptible to mechanical stress (13
14
15)
. These reports along with our present data therefore suggest that dystrophin functions not only to stabilize the sarcolemma, but also to stabilize a calcium channel. A deficiency of dystrophin results in the deregulation of such channels, which is more pronounced in response to applied external mechanical stress.
To understand the nature of the calcium channels that might be involved in mechanical stretch-induced Ca2+ influx and the activation of ERK1/2, we investigated the role of L-type calcium channels and SA channels using their specific pharmacological inhibitors nifedipine and gadolinium (III) chloride, respectively. Our data in Fig. 6
suggest that SA channels but not L-type calcium channels are involved in the mechanical stretch-induced activation of ERK1/2 in mdx myotubes. SA channels are considered an important component of the stretch-induced signaling pathways in many cell type (10
, 41
, 56)
. Activation of these channels allows the movement of Na+, K+, and Ca2+ ions across the membrane (10)
. Since the mechanical stretch-induced activation of ERK1/2 in mdx diaphragm muscle was completely reversed in the presence of Gd3+ ions (an antagonist of SA channel), the deregulation of SA channels could be one potential reason for the spurious activation of ERK1/2 in mdx muscle fibers in response to mechanical stretch. Indeed, many patch-clamping studies have confirmed the presence of SA channels in myotubes and have shown that lack of dystrophin in DMD patients causes an increased Ca2+ influx through these mechanosensitive channels (57
58
59)
.
We observed that spurious activation of ERK1/2 in mdx muscle fibers in response to mechanical stretch is associated with the elevated level of activation of the AP-1 transcription factor (Fig. 7)
. Although the exact mechanism of activation of AP-1 in mdx muscle fibers is not known, the increased activation of ERK1/2 could trigger the activation of AP-1. This is supported by data showing that inhibition of ERK1/2 using PD98059 significantly decreased the activation of AP-1 in mdx muscle fibers in response to mechanical stretch (Fig. 8)
. Removal of Ca2+ ions from the medium, which inhibits ERK1/2 activation in mdx muscle fibers (Fig. 5)
, inhibited the stretch-induced activation of AP-1 (Fig. 8)
. Our study is the first to demonstrate a higher level of activation of AP-1 in mdx mice. It is important to recognize that the increased activation of AP-1 was observed in 16- to 17-day-old mdx mice in which the muscle degeneration had not yet started. Since many inflammatory genes contain AP-1 consensus binding sites in their promoter region, the activation of AP-1 in mdx muscle fibers could be a potential trigging mechanism for the initiation and manifestation of muscular dystrophy at later stages of development. In fact, a recent report from our group (17)
showed that levels of TNF- and IL-1ß, both of which contain consensus sequence for AP-1 in their promoter/enhancer region (44
, 45)
, were higher in the mdx mice. The increased activation of AP-1 transcription factors therefore could provide a plausible mechanism for the elevated expression of TNF- and IL-1ß in mdx muscles.
The present study provides novel information regarding the role of dystrophin in mechanotransduction in skeletal muscle fibers. The data clearly demonstrate that in the prenecrotic state, the mdx mouse diaphragm is more compliant than in normal mice. The loss in muscle stiffness could lead to the dysfunction of SA channels, which may allow increased influx of Ca2+ ions. This could be responsible at least in part for the aberrant activation of the ERK1/2 signaling pathway and the downstream transcription factor AP-1. Since in vivo myotubes are always subjected to the mechanical forces, the aberrant regulation of this pathway in response to mechanical stretch could be involved in the muscle pathogenesis.
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ACKNOWLEDGMENTS |
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Received for publication July 25, 2003. Accepted for publication August 8, 2003.
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) and mdx (+), respectively. The abscissa is a ratio of muscle fiber length in the direction of loading at a stressed state compared with its length at an unstressed state. The data show that after mechanical stretch there is a slow and continuous increase in tension over the range of imposed strains. At a tension of 8 g/cm, muscle lengths as a fraction of their unstressed length in the direction along the fibers are 151% and 125% in the mdx and control mice, respectively, yielding a compliance ratio of 1.21. At the same level of tension, muscle lengths as a fraction of their unstressed length in a direction transverse to the fibers are 122% and 110% in mdx and control mice, respectively, resulting in a compliance ratio of 1.11. Both curves for the dystrophin-deficient muscle shift to the right compared with their respective controls. These data suggest that the dystrophin-deficient diaphragm muscle is more compliant than normal diaphragm. Therefore, dystrophin contributes to the mechanical properties of the muscle.
