1University of Illinois at Chicago, School of Kinesiology, Chicago, Illinois; and 2Georgia Institute of Technology, School of Applied Physiology, Atlanta, Georgia
Submitted 27 April 2004 ; accepted in final form 9 September 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
growth; hypertrophy; muscle; strain; tension
Defining the mechanical environment within a single muscle cell is technically very difficult, if not impossible, in vivo. This is because of numerous anatomical factors that include characteristics such as fiber orientation within the muscle (i.e., angle of pinnation), interactions between the muscle fiber and its own extracellular matrix/basal lamina, and the mechanical impact that results from the complex recruitment of motor units so that an activated muscle fiber from one motor unit will be contracting next to an inactive fiber from a different, nonrecruited motor unit. All of these factors contribute to both the magnitude and the type of forces experienced by a single muscle fiber within the tissue in vivo. Thus, to study mechanotransduction-mechanosensory capacities of muscle cells, it is necessary to apply clearly defined mechanical stimuli, and for this we rely on the use of in vitro mechanical stimulators (stretch devices). These devices can be used within the confines of a cell culture environment, and the mechanical stimuli that are delivered to the culture membrane are easily controlled and can be readily defined.
In vitro stretch devices can generally be grouped into one of two classes, uniaxial or multiaxial. The major difference between a uniaxial and multiaxial stretch device is the type of mechanical deformation delivered to the elastic culture membrane. In this study, we utilized these two types of devices in an attempt to identify whether muscle cells have the fundamental ability to differentiate between different types of mechanical forces. Alterations in well-known signaling molecules were evaluated as a means to discern whether differential mechanotransduction-mechanosensory information was being processed. The molecules evaluated included the extracellular signal-regulated kinase (ERK), protein kinase B (PKB/Akt), and the 70-kDa S6 kinase (p70S6k). These signaling molecules were selected because previous reports have shown them to respond to mechanical stimuli and because they all have been implicated in the regulation of mechanical load-induced hypertrophy (1, 36).
The findings from these studies demonstrate that both uniaxial and multiaxial stretch induce an increase in PKB/Akt and ERK phosphorylation but that only multiaxial stretch induces an increase in p70S6k phosphorylation. Evidence is provided suggesting that the unique activation of p70S6k is a result of myotubes sensing multiaxial vs. uniaxial tensile strains and is not due to differences in surface area deformation or shear stress. In addition, our findings demonstrate that disrupting the actin cytoskeleton inhibits the multiaxial stretch-induced phosphorylation of p70S6k, whereas pharmacological inhibitors that have been reported to block signaling to p70S6k after growth factor stimulation did not (6, 11, 12, 25). These findings suggest that a novel signaling pathway is used by multiaxial stretch to induce phosphorylation of p70S6k. It is also important to note that although disruption of the actin cytoskeleton blocked the multiaxial stretch-induced phosphorylation of p70S6k, it had no effect on the signaling to PKB/Akt, leading to the additional conclusion that mechanical stimuli can activate distinct mechanosensory-mechanotransduction pathways and that the activation of these pathways is specific to the types of mechanical forces applied.
![]() |
MATERIAL AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
C2C12 skeletal myotube cultures were grown by plating C2C12 myoblasts on type I collagen-coated Bioflex membranes (Flexcell International, Hillsborough, NC) at a density of 1.5 x 104 cells/cm2 as previously described (17). Myoblasts were maintained in low-glucose DMEM supplemented with antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin; Sigma) and 10% FBS. Upon confluence (
48 h), cells were switched to DMEM supplemented with antibiotics and 10% HS to promote differentiation. Cells were maintained in this medium for 24 h and then switched back to the 10% FBS medium for an additional 56 days, after which distinct multinucleated myotubes were present. Media were changed every 48 h, and fresh medium was added 18 h before the initiation of mechanical stretch. Three hours before the initiation of stretch, culture plates were removed from the incubator, rapidly placed on the uniaxial or multiaxial stretch device, and returned to the incubator.
Mechanical stretch. Two separate devices were used to stretch the C2C12 myotube cultures. The uniaxial stretch device consisted of a slight modification of the device described by Clark et al. (5). This is a motor-driven system that applies uniaxial stretch to a rectangle-shaped membrane. The sides of the membrane are unconstrained, so there is minor compression transverse to the direction of applied stretch. This is a feature of incompressibility and is very similar to what would happen if an isolated cell were stretched on a single axis. The multiaxial device (FX-3000; Flexcell International, Hillsborough NC) is a vacuum-operated system that produces nonuniform multiaxial (radial and circumferential) tensile strains with very little compression on a circle-shaped membrane (7). The airflow through the stretch plates is unrestricted, which diminishes the potential for internal pressure gradients in the cell culture plates. This is important because it allows the rate of deformation of the membrane to be a true reflection and a result of the rate of pressure change via the vacuum system.
The stretch programs employed in both devices were designed to produce cyclic 15% membrane stretch, cyclic 11% increases in membrane surface area (SA) or cyclic 24%
SA. These changes were produced at a frequency of 1 Hz in a triangular wave form. Stretch cycles were sustained for 45 s followed by 15 s of rest. This pattern was repeated for a total of 10 min.
The SA produced by the uniaxial stretch device was calculated from previously generated finite analysis (5). These data indicate that a 15% and 32% stretch along the major axis of the membrane produces an 11% and 24%
SA, respectively. The calculations for
SA produced by the multiaxial stretch device were based on spherical deflection geometry in which
SA is represented by a spherical cap (described below) (2). These calculations indicate that 7% and 15% membrane stretch produce an 11% and 24%
SA, respectively.
![]() |
![]() |
![]() |
![]() |
![]() |
Application of fluid shear stress.
Skeletal myotubes grown on type I collagen-coated Bioflex membranes were subjected to 10 min of fluid shear with the use of an orbital shaker in the incubator (Armalab, Bethesda, MD) as described by Kraiss et al. (23). This technique produces nonuniform laminar shear with the majority of cells being exposed to near-maximal (peak) shear stress (max), which can be calculated as
![]() |
Western blot analysis.
Skeletal myotubes were collected in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet NP-40, 1 mM -glycerol phosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF) and measured for protein concentration with the DC protein assay kit. From the myotube homogenate, samples containing 1060 µg of protein were dissolved in Laemmli buffer and subjected to electrophoretic separation by SDS-PAGE on 7.5% acrylamide gels as previously described (16). After electrophoretic separation, proteins were transferred to a PVDF membrane, blocked in 5% blotto [5% powdered milk in TBST (Tris-buffered saline, 1% Tween 20)] for 3 h followed by an overnight incubation at 4°C with primary antibody. After overnight incubation, membranes were washed for 30 min in TBST and then probed with anti-rabbit antibody for 45 min at room temperature. After another 30 min of washing in TBST, the blots were developed with ECL. Once the appropriate image was captured, membranes were stained with Coomassie blue to verify equal loading in all lanes. Densitometric measurements were carried out using the FluorSmax Imager with QuantityOne software (Bio-Rad). It should be noted that PKB/Akt and p70S6k contain multiple phosphorylation sites and that the phosphospecific antibodies against these proteins often reveal a doublet or triplet banding pattern (see Figs. 17). The doublet and triplet banding pattern likely reflects changes in mobility that result from different phosphorylation states of the protein; thus, when images were quantified, all of the doublet and triplet bands were counted as a single entity. The phosphospecific ERK antibody reveals two distinct bands that represent ERK1 (44 kDa) and ERK2 (42 kDa). For quantification, both ERK1 and ERK2 were each counted as a single entity.
|
|
Pharmacological inhibitors. Before initiation of stretch, culture medium was replaced with DMEM containing 10% FBS and vehicle (DMSO) or 10 µM cytochalasin D, 500 µM gadolinium(III) chloride, 250 µM genistein, 50 µM PD-98059, 20 µM BIM, or 100 µM LY-294002. The concentration for each of the inhibitors employed was based on previously published work and testing that preceded the stretch experiments (8, 21, 24, 29). After a 45-min preincubation, myotubes were subjected to 10-min cyclic 15% multiaxial stretch as described above and then collected for Western blot analysis.
Conditioned media. C2C12 myotubes grown on type I collagen-coated Bioflex membranes were placed in serum-free DMEM (serum starved) for 90 min, followed by the addition of fresh serum-free medium immediately before the 10 min of 15% cyclic (1 Hz) multiaxial stretch or static conditions were initiated. The conditioned medium from static or stretched myotubes was immediately removed and placed on a new set of serum-starved myotubes for 10 min. Myotubes were subjected to Western blot analysis as described above.
Statistical analysis.
All values are expressed as means ± SE. Statistical significance was determined by ANOVA followed by Student-Newman-Keuls post hoc analysis. Differences between groups were considered significant if P 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine whether the differences in signaling events between uniaxial and multiaxial stretch were the result of differences in the time course of activation, we assessed alterations in the phosphorylation of PKB/Akt, ERK, and p70S6k after 60 min of 15% cyclic (1 Hz) stretch. The results from these experiments indicated that PKB/Akt serine-473 phosphorylation was not elevated after 60 min or either uniaxial or multiaxial stretch (Fig. 1D). Furthermore, at 60 min, both uniaxial and multiaxial stretch produced a similar change in ERK phosphorylation (Fig. 1E), whereas only multiaxial stretch produced a significant increase in p70S6k threonine/serine-421/424 phosphorylation (Fig. 1F).
Stretch-sensitive signaling events are not responsive to fluid shear stress.
The initial results of this study suggested that the multiaxial stretch-specific signaling events were the result of differences in the mechanical forces delivered through the elastic culture membrane. However, cyclic stretch produces motions in the overlying culture medium that, in turn, exert reactive forces (fluid shear stress) on the myotube culture. A variety of cell types are sensitive to fluid shear stress, and signaling events characteristic of this response include increases in ERK and p70S6k phosphorylation (19, 23). This effect is cell-type specific and dependent on the magnitude of fluid shear stress applied; for example, an increase in the magnitude of fluid shear stress produces an increase in the magnitude of phosphorylation (19, 23). In an effort to establish whether any of the signaling events induced by cyclic stretch were a result of fluid shear stress, myotube cultures were subjected to 10 min of 2 dyn/cm2 peak fluid shear stress on an orbital rotary shaker at 1 Hz frequency. This magnitude of fluid shear stress is up to fourfold that produced during cyclic 1 Hz, 7% stretch (11% SA) on the multiaxial device (3). Subjecting the myotubes to fluid shear stress did not alter PKB/Akt, ERK, or p70S6k phosphorylation (Fig. 2, AC). These results suggest that the signaling events induced by cyclic stretch evolved independently of fluid shear stress and therefore were the result of the mechanical forces delivered through the elastic culture membrane. However, it should be noted that the cellular response to shear stress depends on both the magnitude of shear stress and the type of shear stress (static vs. pulsatile). The orbital rotary shaker used in this study produces static flow with a constant rate of fluid shear stress, whereas cyclic stretch produces pulsatile shear stress, and therefore we cannot fully rule out the possibility that shear stress may have contributed to the signaling events produced by cyclic stretch.
|
The increase in PKB/Akt phosphorylation after both 11% and 24% cyclic SA was similar on both devices (Fig. 3, A and D). Cyclic 11%
SA induced similar increases in ERK phosphorylation on the uniaxial and multiaxial devices (Fig. 3B). However, cyclic 24%
SA elicited a greater increase in ERK phosphorylation on the multiaxial compared with the uniaxial device (Fig. 3E; P
0.05). Only stretch on the multiaxial device produced an increase in p70S6k phosphorylation, and this effect was observed after both 11% and 24%
SA (Fig. 3, C and F). Thus, despite normalizing for
SA, the differences in signaling events produced by the uniaxial and multiaxial devices were still evident, with the induction of p70S6k phosphorylation remaining specific to stretch on the multiaxial device. These data demonstrate that the ability of C2C12 myotubes to distinguish between the different types of mechanical forces that produced uniaxial and multiaxial stretch was not dependent on differences in
SA produced by a given amount of stretch.
|
|
Multiaxial stretch-induced phosphorylation of p70S6k and PKB/Akt is stimulated by distinct mechanosensory-mechanotransduction pathways.
Our data indicate that multiaxial stretch-specific signaling events were induced by mechanical forces delivered through the elastic culture membrane and that these mechanical forces were not due to differences in SA or localized regions of large tensile strain. To determine which mechanisms might be involved in the multiaxial stretch-specific signaling, we evaluated changes in p70S6k phosphorylation in myotube cultures that had been incubated with a well-known compound that disrupts the actin cytoskeleton, cytochalasin D. Disruption of the actin cytoskeleton (Fig. 5, A and B) completely blocked the multiaxial stretch-induced increase in p70S6k phosphorylation (Fig. 5C). However, disruption of the cytoskeleton had no effect on the multiaxial stretch-induced phosphorylation of PKB/Akt (Fig. 5D) or ERK (data not shown). These data demonstrate that signaling to p70S6k and PKB/Akt occurs through stimulation of distinct mechanosensory-mechanotransduction pathways and that the actin cytoskeleton is a vital component of the pathway that imparts multiaxial stretch-specific signaling to p70S6k.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanical forces produced during cyclic stretch include those delivered through the elastic culture membrane as well as fluid shear stress. Fluid shear results from motions in the overlying cell culture medium that, in turn, impose reactive forces (i.e., fluid shear stress) on the cells in culture. Endothelial cells are a well-known example of a cell type that is very sensitive to fluid shear forces. In particular, application of fluid shear forces on endothelial cells promotes signaling to p70S6k (23). In contrast, the results with C2C12 myotubes demonstrated that application of fluid shear did not result in signaling changes in any of the molecules studied. This lack of a signaling response indicates that 1) the signaling responses produced by either uniaxial or multiaxial stretch resulted primarily from changes in the mechanical forces delivered through the elastic culture membrane, and 2) the unique signaling responses in the multiaxial vs. the uniaxial stretch system likely resulted from the different mechanical forces associated with the multiaxial stretch. In addition, the observation that C2C12 myotubes did not exhibit signaling responses similar to those of endothelial cells when exposed to fluid shear stress demonstrated that mechanosensory-mechanotransduction systems exhibit cell-type specificity, and this may be associated with the unique functional demands of the cells in their tissue environment.
Further experiments were conducted to discern whether the unique response of p70S6k to multiaxial stretch was the result of differences in the magnitude of SA (Fig. 3) or differences in localized regions of large strain (Fig. 4). These experiments demonstrated that the multiaxial stretch-specific signaling events were not a result of differences in
SA or localized regions of large tensile strain, and thus we propose that the multiaxial stretch-specific signaling events resulted from an intrinsic capacity of myotubes to distinguish between uniaxial and multiaxial tensile strains.
To understand how myotubes might distinguish between the different types of stretch, it is important to have a conceptual sense of the cellular architecture associated with mechanotransduction systems. Recent reports have stated that much of the cell's signal transduction machinery is physically immobilized to the cytoskeleton and is spatially integrated within the focal adhesion complex (FAC) (18, 30). Force-dependent alterations in the spatial organization of FAC signaling proteins or force-dependent changes in protein conformation could expose new binding sites and, in turn, activate signaling cascades. The cytochalasin D experiments performed in this study demonstrated that disruption of the cytoskeleton blocked the multiaxial stretch-induced signaling to p70S6k but had no effect on signaling to PKB/Akt, a molecule activated by both uniaxial and multiaxial stretch. These results are significant because they indicate that p70S6k and PKB/Akt are activated by distinct mechanosensory-mechanotransduction pathways and that the activation of these pathways is specific to the types of mechanical forces applied. In addition, they indicate that the actin cytoskeleton is a vital component of the mechanotransduction system that imparts the multiaxial stretch-specific signaling to p70S6k. A conceptual model of an actin cytoskeleton-linked, multiaxial stretch-specific mechanosensor is described in Fig. 8.
|
The results of this study highlight the concept of specificity in mechanotransduction. Although this concept is only beginning to be appreciated, there are some studies that have specifically addressed it. For example, differential activation of fibronectin mRNA expression and small GTPase Rac activity can be induced by tensile vs. compressive strains (20, 27); activation of G proteins is dependent on the rate of strain (13); and induction of VEGF mRNA is dependent on the magnitude of strain (34). Most similar to this study, Lee et al. (27) reported that 3% surface area deformations produced by uniaxial stretch produced an approximately 3-fold increase in fibronectin mRNA expression, whereas 3% surface deformations produced by equibiaxial stretch produced only an approximately 1.5-fold increase in fibronectin mRNA expression. The quantitatively different responses of fibronectin mRNA to uniaxial and equibiaxial stretch are very similar to the quantitatively different responses of ERK phosphorylation that we observed in response to uniaxial and multiaxial stretch. The results of this study are consistent with the examples described above and provide further evidence that cells can differentially sense specific mechanical signals that in turn elicit specific intracellular responses.
Although it is apparent that specific types of mechanical forces can induce specific cellular responses, it is not clear how this specificity arises. One possibility is that cells contain several distinct mechanosensors that are activated in response to specific mechanical stimuli. Evidence of distinct mechanosensors is lacking, but there are studies that suggest they exist. For example, MacKenna et al. (28) have shown that stretch induces JNK1 and ERK2 activation. The stretch-induced ERK2 activation is blocked with a combination of anti-4 and -
5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, whereas stretch-induced activation of JNK1 is not blocked by these inhibitors (28). These results indicate that the mechanotransduction pathways for JNK1 and ERK2 are distinct and may involve the activation of different mechanosensors.
A recent study by Kumar et al. (24) also provides evidence that distinct mechanosensors may be contributing to results in which different types of mechanical forces activate different mechanosensory-mechanotransduction pathways. In this study, strain was applied either along the longitudinal axis of the muscle fibers or transverse to the fibers, and the result was an induction of ERK phosphorylation through different upstream pathways. This work deserves particular attention, because uniaxial stretch has been used previously to induce alignment of fusing myotubes with the longitudinal axis of the membrane (38). One consideration with the results from this study may be that uniaxial stretch, applied along the longitudinal axis, activates the common ERK cascade, as described in the study by Kumar et al. (24), whereas multiaxial stretch, being simultaneously longitudinal and transverse, represents activation of both the longitudinal and the transverse ERK cascade. In this study, the myotubes subjected to uniaxial stretch were randomly aligned, resulting in cells being subjected to a distribution of longitudinal and transverse stretch, and this feature discounts the possibility that orientation of stretch contributed to the observed differences between uniaxial and multiaxial stretch. Thus these results provide further evidence that different mechanical forces activate distinct mechanosensory-mechanotransduction pathways; whether the specificity arises through the activation of unique mechanosensors remains to be determined.
To date, the complexity of the in vivo cellular environment has limited our ability to observe specificity in mechanotransduction. However, the results of this study first clearly show that muscle cells can differentiate between uniaxial and multiaxial cyclic stretch and, second, demonstrate that specificity is a fundamental property of the mechanotransduction machinery. With the development of new analytical techniques, the ability to characterize and/or model the mechanical environment in vivo is rapidly advancing. These models are providing new insights into how the mechanical environment is altered during the onset of pathological adaptations such as cardiac hypertrophy following a myocardial infarct and atherosclerosis (10, 26). Combining this information with the use of well-defined in vitro studies will greatly accelerate our understanding of how mechanical information is converted in specific physiological and pathological adaptations.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bottlang PM, Brown TD, and Banes AJ. Hyperelastic constitutive properties of polydimethyl siloxane cell culture membranes. In: Advances in Bioengineering. New York: ASME, 1993, vol. 26, p. 607609.
3. Brown TD, Bottlang M, Pedersen DR, and Banes AJ. Loading paradigmsintentional and unintentionalfor cell culture mechanostimulus. Am J Med Sci 316: 162168, 1998.[CrossRef][ISI][Medline]
4. Carabello BA. Concentric versus eccentric remodeling. J Card Fail 8: S258S263, 2002.[CrossRef][ISI][Medline]
5. Clark CB, Burkholder TJ, and Frangos JA. Uniaxial strain system to investigate strain rate regulation in vitro. Rev Sci Instrum 72: 24152422, 2001.[CrossRef][ISI]
6. Dickenson JM. Stimulation of protein kinase B and p70 S6 kinase by the histamine H1 receptor in DDT1MF-2 smooth muscle cells. Br J Pharmacol 135: 19671976, 2002.
7. Gilbert JA, Weinhold PS, Banes AJ, Link GW, and Jones GL. Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. J Biomech 27: 11691177, 1994.[ISI][Medline]
8. Goel HL and Dey CS. Insulin stimulates spreading of skeletal muscle cells involving the activation of focal adhesion kinase, phosphatidylinositol 3-kinase and extracellular signal regulated kinases. J Cell Physiol 193: 187198, 2002.[CrossRef][ISI][Medline]
9. Goldspink G, Scutt A, Loughna PT, Wells DJ, Jaenicke T, and Gerlach GF. Gene expression in skeletal muscle in response to stretch and force generation. Am J Physiol Regul Integr Comp Physiol 262: R356R363, 1992.
10. Goubergrits L, Affeld K, Fernandez-Britto J, and Falcon L. Atherosclerosis and flow in carotid arteries with authentic geometries. Biorheology 39: 519524, 2002.[ISI][Medline]
11. Govindarajan G, Eble DM, Lucchesi PA, and Samarel AM. Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells. Circ Res 87: 710716, 2000.
12. Graves LM, He Y, Lambert J, Hunter D, Li X, and Earp HS. An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells. J Biol Chem 272: 19201928, 1997.
13. Gudi SR, Lee AA, Clark CB, and Frangos JA. Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac fibroblasts. Am J Physiol Cell Physiol 274: C1424C1428, 1998.
14. Hannan KM, Thomas G, and Pearson RB. Activation of S6K1 (p70 ribosomal protein S6 kinase 1) requires an initial calcium-dependent priming event involving formation of a high-molecular-mass signalling complex. Biochem J 370: 469477, 2003.[CrossRef][ISI][Medline]
15. Henderson JH and Carter DR. Mechanical induction in limb morphogenesis: the role of growth-generated strains and pressures. Bone 31: 645653, 2002.[CrossRef][ISI][Medline]
16. Hornberger TA, Hunter RB, Kandarian SC, and Esser KA. Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats. Am J Physiol Cell Physiol 281: C179C187, 2001.
17. Hornberger TA, Stuppard R, Conley KE, Fedele MJ, Fiorotto ML, Chin ER, and Esser KA. Mechanical stimuli regulate rapamycin-sensitive signaling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochem J 380: 795804, 2004.[CrossRef][ISI][Medline]
18. Ingber DE, Dike L, Hansen L, Karp S, Liley H, Maniotis A, McNamee H, Mooney D, Plopper G, and Sims J. Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. Int Rev Cytol 150: 173224, 1994.[ISI][Medline]
19. Jo H, Sipos K, Go YM, Law R, Rong J, and McDonald JM. Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and G/
-dependent signaling pathways. J Biol Chem 272: 13951401, 1997.
20. Katsumi A, Milanini J, Kiosses WB, del Pozo MA, Kaunas R, Chien S, Hahn KM, and Schwartz MA. Effects of cell tension on the small GTPase Rac. J Cell Biol 158: 153164, 2002.
21. Kim HP, Roe JH, Chock PB, and Yim MB. Transcriptional activation of the human manganese superoxide dismutase gene mediated by tetradecanoylphorbol acetate. J Biol Chem 274: 3745537460, 1999.
22. Ko KS and McCulloch CA. Intercellular mechanotransduction: cellular circuits that coordinate tissue responses to mechanical loading. Biochem Biophys Res Commun 285: 10771083, 2001.[CrossRef][ISI][Medline]
23. Kraiss LW, Weyrich AS, Alto NM, Dixon DA, Ennis TM, Modur V, McIntyre TM, Prescott SM, and Zimmerman GA. Fluid flow activates a regulator of translation, p70/p85 S6 kinase, in human endothelial cells. Am J Physiol Heart Circ Physiol 278: H1537H1544, 2000.
24. Kumar A, Chaudhry I, Reid MB, and Boriek AM. Distinct signaling pathways are activated in response to mechanical stress applied axially and transversely to skeletal muscle fibers. J Biol Chem 277: 4649346503, 2002.
25. Laser M, Kasi VS, Hamawaki M, Cooper GT, Kerr CM, and Kuppuswamy D. Differential activation of p70 and p85 S6 kinase isoforms during cardiac hypertrophy in the adult mammal. J Biol Chem 273: 2461024619, 1998.
26. Latimer DC, Roth BJ, and Parker KK. Analytical model for predicting mechanotransduction effects in engineered cardiac tissue. Tissue Eng 9: 283289, 2003.[CrossRef][ISI][Medline]
27. Lee AA, Delhaas T, McCulloch AD, and Villarreal FJ. Differential responses of adult cardiac fibroblasts to in vitro biaxial strain patterns. J Mol Cell Cardiol 31: 18331843, 1999.[CrossRef][ISI][Medline]
28. MacKenna DA, Dolfi F, Vuori K, and Ruoslahti E. Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts. J Clin Invest 101: 301310, 1998.
29. Nakamura TY, Iwata Y, Sampaolesi M, Hanada H, Saito N, Artman M, Coetzee WA, and Shigekawa M. Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters. Am J Physiol Cell Physiol 281: C690C699, 2001.
30. Plopper GE, McNamee HP, Dike LE, Bojanowski K, and Ingber DE. Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol Biol Cell 6: 13491365, 1995.[Abstract]
31. Pullen N and Thomas G. The modular phosphorylation and activation of p70s6k. FEBS Lett 410: 7882, 1997.[CrossRef][ISI][Medline]
32. Russell B, Motlagh D, and Ashley WW. Form follows function: how muscle shape is regulated by work. J Appl Physiol 88: 11271132, 2000.
33. Shioi T, McMullen JR, Tarnavski O, Converso K, Sherwood MC, Manning WJ, and Izumo S. Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation 107: 16641670, 2003.
34. Suzuma I, Suzuma K, Ueki K, Hata Y, Feener EP, King GL, and Aiello LP. Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)- but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways. J Biol Chem 277: 10471057, 2002.
35. Thomas G and Hall MN. TOR signalling and control of cell growth. Curr Opin Cell Biol 9: 782787, 1997.[CrossRef][ISI][Medline]
36. Ueyama T, Kawashima S, Sakoda T, Rikitake Y, Ishida T, Kawai M, Yamashita T, Ishido S, Hotta H, and Yokoyama M. Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy. J Mol Cell Cardiol 32: 947960, 2000.[CrossRef][ISI][Medline]
37. Vandenburgh H and Kaufman S. In vitro model for stretch-induced hypertrophy of skeletal muscle. Science 203: 265268, 1979.[ISI][Medline]
38. Vandenburgh HH and Karlisch P. Longitudinal growth of skeletal myotubes in vitro in a new horizontal mechanical cell stimulator. In Vitro Cell Dev Biol 25: 607616, 1989.[ISI][Medline]