Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells

Jingying Sarah Zhang,2 William E. Kraus,1 and George A. Truskey2

1Department of Medicine and 2Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708-0281

Submitted 13 January 2004 ; accepted in final form 15 March 2004


    ABSTRACT
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C2C12 myotubes were subjected to a single 10% step stretch for 0–4 days. The apparent elastic modulus of the cells, Eapp, was subsequently determined by atomic force microscopy. Static stretch led to significant increases (P < 0.01) in Eapp beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor L-arginine onto stretched cells further enhanced Eapp, NOS activity, and nNOS expression, whereas the presence of the NO inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) reversed the effects of mechanical stimulation and of L-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with L-NAME showed a significant decrease in Eapp, whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in Eapp. Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis.

mechanical stimulation; apparent elastic modulus; skeletal muscle cells; nitric oxide; stretch


RECENT EVIDENCE INDICATES that neuronal nitric oxide synthase (nNOS) protein and mRNA levels in skeletal muscle are regulated by mechanical activity (9, 38, 39). For instance, nNOS activity is greatly enhanced up to sevenfold by passive stretch (3, 22, 37). Accumulating evidence has shown that NO is a mechanogenic transducer and effector, which involves a great variety of mechanisms in response to mechanical activity, leading to regulation of cell growth (33), differentiation (19), migration (26), and apoptosis (21). Tidball et al. (38) demonstrated that C2C12 muscle-derived NO stimulated by cyclic stretch is a positive regulator of two main focal contact proteins, talin and vinculin, at the transcriptional level through a cGMP-dependent protein kinase pathway. These two proteins, talin and vinculin, which are physically associated, may interact with nNOS directly or indirectly via dystrophin (5, 30). Vinculin is an important cellular protein that mediates cell spreading, elasticity, and maintenance (14–16). For example, upregulation of vinculin by cyclic stretch and static stretch results in more stable focal adhesions, well-spread cells, and moderately immobile cells, whereas downregulation or blockade of vinculin expression leads to smaller adhesions and more motile cells (13, 32). Talin, on the other hand, shares binding sites with actin, vinculin, and focal adhesion kinase (FAK), and these proteins are responsive to mechanical stimuli. When upregulated by mechanical stimuli, these proteins may stabilize the cytoskeleton via the linkage with actin filaments, thus enabling cells to bear more stress. In addition, Tidball and Koh (23, 24) observed that NO produced by skeletal muscle cells subjected to either static stretch or cyclic stretch regulated sarcomere structure and calpain-activated cytoskeleton degradation, which are important for the mechanical properties of these cells. Furthermore, Smith et al. (35) and Tidball and Koh (23) demonstrated that endogenous NO is closely involved in inducing hypertrophy of skeletal muscle fibers by decreasing protein degradation and increasing protein synthesis.

Although these studies suggest that stretch-induced NO may modulate mechanical properties of the muscle cells by stabilizing focal contacts, the effects of stretch-induced NO on the intrinsic mechanical behavior of skeletal muscle still remain unknown. The goals of this study were to test the hypotheses that 1) NO causes an increase in the mechanical properties of differentiating myoblasts, and 2) NO affects the mechanical properties by inhibiting calpain-mediated proteolysis of focal contact proteins. We exposed skeletal muscle cells to a single 10% static stretch for as long as 4 days and examined the relationship among externally applied stretch, focal contact protein levels, and mechanical properties of the skeletal muscle cells.


    MATERIAL AND METHODS
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Cell culture. Murine C2C12 muscle cells were used (American Type Culture Collection, Rockville, MD) in all experiments. Cell culture wells made from Silastic membrane (0.01-in. thick, 3.2 x 1.5 cm; Specialty Manufacturing, Hemlock, MI) were assembled into holders with the use of adhesive silicone (type A; Nusil Technology, Carpinteria, CA) as previously described (11) and then treated with 100 µg/ml growth factor-reduced Matrigel (GFR; Becton Dickinson, Bedford, MA) for 1 h at 37°C. The cells were plated in the GFR Matrigel-coated well at a density of 1.5 x 105 cells/well in 2.0 ml of growth medium with Dulbecco's modified Eagle’s medium (DMEM; GIBCO BRL, Grand Island, NY), 10% newborn calf serum (HyClone Laboratories, Logan, UT), chicken embryo extract (GIBCO BRL), and 0.5% gentamicin (GIBCO BRL) at 37°C and 5% CO2. The growth medium was changed every day until the cells reached 90% confluence. The growth medium was then switched to a differentiation medium with low growth factors, consisting of DMEM supplemented with 10% horse serum (Intergen, Purchase, NY) and 0.5% gentamicin to promote differentiation of myoblasts into myotubes.

Mechanical loading. Immediately after growth medium was changed to differentiation medium, C2C12 cells were exposed to a 10% static step stretch for a 4-day period with or without 2 mM L-arginine (Sigma, St. Louis, MO) and N{omega}-nitro-L-arginine methyl ester (L-NAME; Sigma) as indicated for each experiment. The property of the Silastic membrane wells has been well characterized and demonstrated a Poisson ratio of 0.5 (11). Unstretched sister cultures grown in the same wells served as controls.

Modulation of NO activity. To assess the role of NO in the mechanical behavior of the skeletal muscle cells, we performed two sets of experiments. In the first experiment, stretched or unstretched cells were treated with or without 2 mM L-arginine for 4 days to increase NOS activity. In addition, 100 µM L-NAME was added. In the second experiment, unloaded cells and statically stretched cells without any other treatment were cultured for 4 days. The cells at day 4 were then incubated with either 100 µM L-NAME or 20 mM calpain inhibitor I (A.G. Scientific, San Diego, CA) for 4 h to confirm the contribution of NO to the mechanical properties of the cultured cells.

Western blot analysis. At the end of an experiment, cultured C2C12 cells were lysed in 0.5% SDS, 0.6% Nonidet P-40 (NP-40), 0.15 M NaCl, 1 mM EDTA, 10 mM Tris, pH 7.9, and antiproteases (10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, and 0.5 mM PMSF) for 30 min on ice. Nuclear and insoluble debris were removed by centrifuging for 5 min at 12,000 rpm. Solubilized protein (15 µg for vinculin, talin, or desmin or 50 µg for nNOS) was loaded in each lane of an 8% (wt/vol) SDS-PAGE gel. Protein concentrations were determined by using the bicinchoninic acid assay (BCA; Sigma). Proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and then the membrane was blocked with 5% nonfat dry milk in TBST (Tris-buffered saline with 0.1% Tween 20) overnight at 4°C. After transfer, membranes were incubated for 1 h at room temperature with the following primary monoclonal antibodies in blocking buffer: anti-talin (1:400; Sigma), anti-vinculin (1:400; Sigma), anti-nNOS (1:200; Sigma), and anti-desmin (1:400; Pharmingen, Lexington, KY). After three washes with TBST for 30 min each, membranes were incubated with horseradish peroxidase-conjugated IgG anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at the ratio of 1:5,000 for 45 min. Proteins were detected by enhanced chemiluminescence reagents (Amersham, Piscataway, NJ) on films. Developed films were scanned and subjected to densitometry using NIH Image (version 1.62). Protein level was determined by Western blot analysis, quantified by densitometry, and expressed as intensity in arbitrary units. Three independent experiments were performed for each condition. Coomassie blue staining was performed to assess the efficiency of protein transfer. Kaleidoscope prestained standards (Bio-Rad, Hercules, CA) were used as protein markers.

NO assay. NO production was determined with the Griess colorimetric reagent (BIOXYTECH NOS assay kit; OxisResearch, Portland, OR). Briefly, the medium was collected every 24 h, and the concentration of released NO was calculated by measuring the absorbance at 540 nm of the nitrite and nitrate accumulated in 2 ml of culture medium in each well. Duplicate samples were assayed for each trial of each condition in these experiments.

Atomic force microscopy. A Bioscope atomic force microscope (AFM) mounted with an oxide-sharpened silicon nitride cantilever (Digital Instruments, Santa Barbara, CA) with a cone angle of 35° and spring constants of 0.03–0.05 N/m was used according to the protocol previously described (12). Briefly, before the experiment, the spring constant was calculated and the sensitivity was measured by using the software Nanoscope 5.03 (version r3). Force-indentation curves were collected. The elastic modulus of the cells was obtained by using the following equation to calculate the cone tip shape as

(1)
where F is the applied loading force, k is the spring constant of the AFM cantilever, d is the deflection of the cantilever, Eapp is the apparent elastic modulus of the sample (hereafter referred to as Eapp in text), {delta} is the indentation depth, z is the piezo height, and {Omega} is the function of the cantilever geometry. For the conical tip we used, {Omega} was calculated as

(2)
where {alpha} = 35° is the angle of the cone tip (Digital Instruments) and {nu} is the Poisson ratio. A value of {nu} = 0.5 was used. The elastic modulus was determined by calculating the slope of the force vs. the square of the indentation from the raw data. To determine the viscous contribution to the elastic modulus, hysteresis was quantified by calculating the area from force vs. indentation extension and retraction (28). AFM was performed with indentations for which the finite thickness of the cell did not affect the apparent modulus (28). For all of the experiments, fully fused multinucleated myocytes were chosen for measurement of their mechanical properties, and single nucleated myoblasts were not examined.

Statistics analysis. All values are expressed as means ± SD unless otherwise noted. Data from the experiment on short-term exposure to L-NAME were analyzed by one-way analysis of variance (ANOVA) using InStat 2.0, whereas the remaining data were analyzed by two-way ANOVA using StatView 5.0. Tukey's post hoc test was applied to determine the significance of differences between groups when ANOVA indicated that significant interactions were found. Correlation between nNOS expression and NOS activity as well as correlation between nNOS expression and Eapp were examined using StatView 5.0.


    RESULTS
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Effects of mechanical loading and NO modulation on Eapp of skeletal muscle cells. We first tested the hypotheses that 1) static stretch affects the mechanical properties of the skeletal muscle cells and 2) the effects of stretch on the mechanical properties are mediated by NO. AFM was used to measure Eapp and hysteresis of the multinucleated myocytes. As shown in Fig. 1A, mechanical loading for 24 h did not affect Eapp of the cells compared with control cells. However, by differentiation day 2, Eapp for statically stretched cells increased to 8.3 ± 1.6 kPa (P < 0.01) and continued to increase until day 4 at 14.3 ± 2.4 kPa (P < 0.001), relative to control cells. Treating stretched cells with L-arginine caused Eapp to increase relative to controls, from 9.7 ± 1.9 kPa on day 2 (P < 0.01) to 17.3 ± 2.3 kPa at day 4 (P < 0.001). Stretched cells with and without L-arginine were not significantly different until day 3 (P < 0.05) and day 4 (P < 0.001). The enhanced Eapp resulted from mechanical loading, and L-arginine was completely abolished by the NO inhibitor L-NAME, with no significant difference between control groups until day 4 (17%, P < 0.05), suggesting that the effects of mechanical stimulation are mainly mediated by NO.



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Fig. 1. A: time course of apparent elastic modulus (Eapp) of skeletal muscle cells in response to mechanical stimulation and NO modulation by the NO donor L-arginine (L-Arg; 2 mM) and the NO inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 100 µM). Myotubes were subjected to 10% passive static stretch for 4 days. B: effects of static stretch and addition of L-Arg and L-NAME on normalized hysteresis. Values are means ± SD of 12–23 cells from 3 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control. ##P < 0.01 and ###P < 0.001, static stretch with L-Arg vs. static stretch alone. xxxP < 0.001, static stretch with both L-Arg and L-NAME vs. static stretch with L-Arg alone.

 
Unlike the effect of stretch and L-arginine on Eapp, static stretch and the addition of L-arginine exhibited a significant effect on hysteresis only on differentiation day 1, relative to control cells (Fig. 1B). Hysteresis was consistently ~17% of the input energy.

Recent studies have shown that NO regulates calpain-initiated cytoskeletal degradation (18, 23, 29). Inhibition of the Ca2+-dependent protease calpain results in more stabilized focal adhesions of cells (6). Thus, to examine whether the effect of NO on mechanical behavior of muscle cells was due to inhibition of calpain-mediated proteolysis of the cytoskeleton, we measured the Eapp on unloaded and loaded multinucleated myocytes cultured for 4 h, followed by treatment with L-NAME in either the absence or presence of calpain inhibitor for 4 h (Fig. 2A). After 4-h exposure to L-NAME (100 µM), Eapp decreased significantly for both control (P < 0.001) and stretched cells (P < 0.001). The inhibitory effect was reversed by addition of calpain inhibitor I (20 µM), with no apparent difference between controls. No significant change of hysteresis was detected (Fig. 2B). Together, these results suggest that NO modulates the mechanical properties of the skeletal muscle cells in part by inhibiting calpain-activated proteolysis of the cell cytoskeleton.



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Fig. 2. A: effects of L-NAME and calpain inhibitor I on Eapp of skeletal muscle cells. Cells were treated with L-NAME (100 µM) in the absence or presence of calpain inhibitor I (20 µM) for 4 h on differentiation day 4. B: effects of L-NAME and calpain inhibitor on hysteresis. Values are means ± SD of 14–16 cells from 3 experiments. ***P < 0.001 and ###P < 0.001 compared with respective controls.

 
Effects of mechanical loading on NOS activity and on NOS protein expression. To determine whether NOS activity correlated with modulation of the mechanical properties of the skeletal muscle cells, we determined NOS activity for the same conditions tested in Fig. 1 (Fig. 3). The unloaded cells exhibited an increase in NO throughout 4 days of differentiation, from 0.13 ± 0.03 µM on day 1 to 0.49 ± 0.05 µM on day 4 (Fig. 3A). Static stretch and the addition of L-arginine induced a substantial increase in NOS activity from 0.49 ± 0.05 µM (P < 0.001) and 0.94 ± 0.09 µM (P < 0.001) on day 1 to 1.33 ± 0.11 µM (P < 0.001) and 1.71 ± 0.1 µM (P < 0.001) on day 4, respectively. All three groups showed pronounced NO production from day 3 to day 4 that paralleled changes in the observed significant increases of Eapp on day 3 and day 4. This finding is consistent with other reports that NOS increases during differentiation (17, 37). However, the stimulatory effect of L-arginine on NO production in the presence of the NO inhibitor L-NAME abolished the effects of L-arginine and brought the NO level back to the baseline value of approximately 0.07 µM throughout (P < 0.01).



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Fig. 3. NO production and neuronal NO synthase (nNOS) expression of cultured C2C12 cells treated as described in Fig. 1. A: NO activity was determined as nitrite and nitrate accumulation in culture media from differentiation day 1 to differentiation day 4 in the absence or presence of L-Arg with or without L-NAME. Values are means ± SD. Each condition is represented as the average of data from 7–9 cells from 3 experiments. B: representative Western blot of nNOS protein expression level on shift day 2 in statically stretched cells (lane 2) or after the addition of L-Arg (lane 3) showed a significant increase compared with control cells (lane 1). No significant difference was found in the presence of L-NAME-treated cells (lane 4). C: nNOS expression level was determined by Western blot analysis and quantified by densitometry. Values are means ± SD; n = 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control.

 
To assess the correlation between NOS activity and its protein content, we examined the protein level of nNOS as well. As revealed in Fig. 3, B and C, static stretch led to a significant increase in nNOS protein on days 2, 3, and 4 that was correlated with pronounced production of NO and a considerable increase in Eapp (Fig. 4, A and B), implying that NO plays an important role in mediating mechanical properties of the skeletal muscle cells. The addition of L-arginine increased the expression of nNOS on all days studied, relative to controls.



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Fig. 4. A: correlation of nNOS protein expression and mechanical properties. Eapp demonstrates a linear dependence with nNOS expression level (R2 = 0.8863, P < 0.001). B: correlation of nNOS protein expression and NO production. NO release shows a linear dependence with nNOS expression level (R2 = 0.8817, P < 0.001). Control and treatment groups were used.

 
Effects of mechanical loading and NO modulation on levels of talin, vinculin, and desmin. To assess the contribution of NO to the mechanical properties, we performed Western blot analysis to measure the protein expression levels of the focal contact proteins talin and vinculin after static stretch. As shown in Fig. 5A, static stretch led to an increase in expression of the intact 225-kDa talin of 1.7- to 3.6-fold (P < 0.05). The addition of L-arginine significantly increased expression of the intact 225-kDa talin up to 4.3-fold (P < 0.01). In contrast, addition of L-NAME (100 µM) led to decreased expression of talin to a level that was not different from that of unloaded cells. This finding suggests that NO mediates Eapp in part by increasing accumulation of talin synthesis.



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Fig. 5. Protein levels of talin, vinculin, and desmin in C2C12 cells subjected to a 10% step-increased strain in the absence or presence of L-Arg with or without L-NAME. Total cell extracts were obtained at 1, 2, 3, and 4 days. Protein expression was analyzed by Western blot, and the band intensities were quantified by densitometry. A: levels of intact talin were significantly increased on all days studied for the statically stretched cells in both the absence and presence of L-Arg (2 mM). Addition of L-NAME (100 µM) abolished the upregulatory effect. B: representative Western blot of talin protein expression level on shift day 2. Top bands are intact 225-kDa talin, and bottom bands are degraded 195-kDa talin. Static stretching of cells (lane 2) and the addition of L-Arg (lane 3) resulted in significant decreases in the ratio of degraded talin compared with controls (lane 1). The presence of L-NAME (lane 4) reversed the effect of inhibition of talin degradation. C: the ratio of intact 225-kDa talin vs. degraded 190-kDa talin. D: vinculin levels. E: desmin levels. All experimental conditions were the same as described in Fig. 1. Values are means ± SD; n = 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control. ##P < 0.01, static stretch with both L-Arg and L-NAME vs. static stretch with L-Arg alone.

 
NO plays an important role in preventing calpain-activated proteolysis of the cytoskeleton. Talin is responsive to mechanical stimuli and can be stabilized by an NO donor that prevents calpain-activated-proteolysis of the cytoskeleton. Thus talin may serve as a linker to stabilize the cytoskeleton (17, 18, 23). We examined the effect of NO on stabilization of the cytoskeleton protein talin (Fig. 5, B and C). Intact talin has a molecular mass of 225 kDa, but this can be degraded to 190 and 47 kDa, as demonstrated in Fig. 5B. The ratio of the intact 225-kDa talin was compared with that of degraded 190-kDa talin. A significant increase in intact talin was detected only for the loaded cells on days 1 and 2 (P < 0.05). Similarly, the presence of L-arginine led to an increase in the ratio of intact talin to total talin, especially on day 1 (P < 0.001), day 2 (P < 0.001), and day 3 (P < 0.05), confirming our hypothesis that NO acts, at least in part, by restraining the activity of calpain-initiated proteolysis of talin. Vinculin, another very important focal contact protein that is regulated by mechanical loading, exhibited a significant increase on day 3 and day 4 for both statically stretched cells and L-arginine-treated stretched cells (Fig. 5D). This trend was correlated with the increase of elastic moduli of the skeletal muscle cells. The change of protein synthesis of talin and vinculin was correlated with data obtained from AFM analysis and NO production.

The intermediate filament desmin may also be involved in regulating the mechanical behavior of the skeletal muscle cells (7). Thus desmin was also examined by Western blot analysis, as shown in Fig. 5E. A significant increase of desmin level compared with control cells occurred only at day 2. The addition of L-arginine caused a considerable increase on days 2, 3, and 4 (P < 0.05).


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Mechanical stretch is perceived by the muscle cells through the ECM-focal contacts complex (8, 13, 32), resulting in modified protein expression to meet the imposed functional demands. The mechanism by which this occurs is incompletely understood. Recent studies have suggested that mechanical signals are transduced in part by endogenous NO, which may influence mRNA stability and mRNA translation (37, 38). In the current study, we demonstrated for the first time the link between the mechanical properties of the cells and NO-mediated protein expression. These findings support our hypothesis that endogenous NO affects the Eapp of the skeletal muscle cells by increasing accumulation of the focal contact proteins talin and vinculin and by inhibiting the calpain-induced proteolysis of cytoskeleton proteins. The increased protein synthesis and decreased protein degradation induced by stretch and the induction of NO levels with L-arginine thus reinforce the cytoskeleton of the cells, leading to the increase in Eapp. The reinforced cytoskeleton also enables the cells to resist passive deformation, thus preventing the cells from damage when a strain is imposed.

The level of NO production after stretch as observed in this study is consistent with other reports, although no direct comparison can be made because cell types, strain magnitude, and duration of strain were different among studies (25, 38). Neuronal NOS enzyme (nNOS) is the most abundant isoform in skeletal muscle, and its protein expression level significantly increases during the formation of multinucleated skeletal muscle cells in culture (34). NO released by control cells during the course of differentiation, as shown in Fig. 3A, demonstrated a time-dependent manner, indicating that NOS activity is regulated by muscle development. Lee et al. (25) reported that the peak activity of NOS was detected when multinucleated differentiated myocytes (myotubes) were formed. This finding is consistent with our data showing that the pronounced release of NO coincided with the formation of myotubes on days 3 and 4. We found that NO production was well correlated with nNOS expression as revealed in Fig. 4 (P < 0.05), suggesting that NO release is attributable to the activity of nNOS enzyme in the long term. Although endothelial NOS (eNOS, type III) expressed in skeletal muscle is responsive to mechanical stimulation as well, eNOS isoform is expressed at a low level relative to nNOS in skeletal muscle (2, 20).

Our data show that the expression of talin and vinculin after mechanical loading is positively regulated by NOS activity (Fig. 5). These findings indicate that mechanical stimulation stabilizes the cytoskeleton by concomitant interaction between integrin and NOS-mediated signaling pathway to fulfill the structural and functional needs of the cells. Talin and vinculin may, in concert, contribute to the increased Eapp. In this scenario, talin may initially serve as a key structural protein to aid in stabilizing the cytoskeleton by activating the integrin-mediated adhesion at the initial stage when cells start to attach and spread. Vinculin, on the other hand, may be not only a structural protein but also a functional protein. Goldmann et al. (15) demonstrated that vinculin-deficient cells exhibit lower stiffness compared with wild type, as measured by AFM. Although static stretch and the addition of L-arginine led to an increase in desmin, the magnitude of increase in desmin level was significantly less than that of talin and vinculin levels, suggesting that the major change of elastic modulus resulted from talin and vinculin.

nNOS is localized at the sarcolemma and is associated with the cytoskeleton-dystrophin complex through {alpha}1-syntrophins (27). We observed that static stretch and the addition of L-arginine led to significant increases in the protein level of nNOS. There are at least two plausible mechanisms through which nNOS protein can be upregulated by stretch. First, NO may act through cGMP-regulated signaling pathway, as illustrated in Fig. 6. NO is the most potent activator of soluble guanylate cyclase, which catalyzes cGMP from GTP. cGMP then activates downstream signaling molecules that are involved in a large number of physiological processes in cells. For instance, Ca2+ channels such as ryanodine receptor Ca2+ release channel (RyR1) is regulated by cGMP in skeletal muscle cells (27). In addition, the critical role of Ca2+/calmodulin in NOS activity implies that NO may be implicated in the Ca2+ signaling pathway. However, there are mixed reports about the effects of NO on opening probability of RyR1 channel in skeletal muscle. The contrary observation may be due to biphasic effects of NO, such that Ca2+ release from the sarcoplasmic reticulum (SR) through RyR1 channel is inhibited at lower concentrations of NO, whereas it is stimulated at relatively higher concentrations (1, 4). In support of this suggestion, 1 mM sodium nitroprusside activates Ca2+ release in skeletal muscle, which in turn activates nNOS expression and Ca2+-involved signaling pathways (4). The increased intracellular Ca2+ level from the SR leads to an increase in nNOS gene expression by activating nNOS gene promoter (31). However, regulation of RyR1 activity is complex, and other factors may exert effects on open probability of RyR1 as well. For instance, RyR1 is responsive to NO and reactive oxidant species (36). On the other hand, the increased intracellular Ca2+ is not solely dependent on SR. Tidball et al. (37) found that Ca2+ influx was required for NO release, suggesting that alternative sources such as mechanosensitive ion channels are also involved in regulating nNOS expression. Ye et al. (40) showed that NO released by endothelial cells was partially attenuated in Ca2+-free buffer.



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Fig. 6. Schematic presentation of integrin- and nNOS-mediated signaling events that are activated by stretch. DGC, dystrophin-associated glycoprotein complex; PIN, protein inhibitor of nNOS; FAK, focal adhesion kinase.

 
An alternative mechanism of NO regulation of nNOS protein levels is via the dystrophin-associated glycoprotein complex (DGC), which maintains the integrity of the cell membrane. This complex is associated with a number of signaling components, such as voltage-gated sodium ion channels, growth factor receptor-bound protein 2 (grb2), caveolin 3, and nNOS. DGC proteins participate in the transduction of mechanical signals through the linkage between the cytoskeleton and the matrix via laminin binding. A recent study found that muscle atrophy caused a decrease in the proteins of the DGC, a significant decrease in RhoA, and weakened cytoskeletons of the muscle cells, suggesting that this complex is involved in RhoA signaling (10). This signaling pathway, activated by integrin clustering following stretch, regulates the formation of actin-myosin complex, stress fiber, and focal adhesions. The direct association of small GTPases and DGC proteins further supports this suggestion (10). Moreover, significant loss of nNOS in the skeletal muscle of dystrophin-deficient patients is consistent with the regulatory effect of DGC on nNOS (9). Thus DGC may alter nNOS gene expression by Ca2+ signaling through the RhoA pathway (Fig. 6). Caveolin-3 and the protein inhibitor of nNOS (PIN) serve as regulatory proteins of nNOS. It is also likely that DGC exerts regulatory effect through the signaling components to which it binds. However, the essential question of which mechanism prevails under physiological condition still remains.

In summary, we found that mechanical stimulation increases the apparent elastic modulus of skeletal muscle cells, which is mediated by endogenous NO that increases accumulation of vinculin and talin, thereby further stabilizing focal contacts to resist mechanical stress. NO may also contribute to the mechanical behavior of skeletal muscle by inhibiting the calpain-activated breakdown of cytoskeleton proteins. Specifically, NO prevents talin degradation, which may serve as a critical component of the cytoskeleton stabilization. This study elucidates the role of an important signaling molecule, NO, in the mechanical behavior of skeletal muscle. This mechanism may have profound implications for muscle functions in pathological states such as chronic heart failure, characterized by skeletal muscle weakness and atrophy. These and future findings may point the way toward preventive and therapeutic treatments for these disease states.


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We acknowledge support from National Aeronautics and Space Administration Grants NAG-910 and NGT5-50266 and from a grant by Medtronic.


    ACKNOWLEDGMENTS
 
We thank Dorothy Slentz for invaluable discussion.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. A. Truskey, Dept. of Biomedical Engineering, Box 90281, Duke Univ., Durham, NC 27708-0281 (E-mail: gtruskey{at}duke.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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