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
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ABSTRACT |
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mechanical stimulation; apparent elastic modulus; skeletal muscle cells; nitric oxide; stretch
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.
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MATERIAL AND METHODS |
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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-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 0.030.05 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
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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.
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RESULTS |
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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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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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DISCUSSION |
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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 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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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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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