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 C(2)C(12) myotubes were subjected to a single 10% step stretch for 0-4 days. The apparent elastic modulus of the cells, E(app), was subsequently determined by atomic force microscopy. Static stretch led to significant increases (P < 0.01) in E(app) 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 E(app), 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 E(app), 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 E(app). 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.