Mechanical stimulation increases proliferation, differentiation and protein expression in culture: Stimulation effects are substrate dependent

Mechanical stimulation increases proliferation, differentiation and protein expression in culture: Stimulation effects are substrate dependent

ARTICLE IN PRESS Journal of Biomechanics 40 (2007) 3354–3362 www.elsevier.com/locate/jbiomech www.JBiomech.com Mechanical stimulation increases prol...

228KB Sizes 0 Downloads 19 Views

ARTICLE IN PRESS

Journal of Biomechanics 40 (2007) 3354–3362 www.elsevier.com/locate/jbiomech www.JBiomech.com

Mechanical stimulation increases proliferation, differentiation and protein expression in culture: Stimulation effects are substrate dependent Alberto Grossia, Kavita Yadava, Moira A. Lawsona,b, a

Department of Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark The LMC Centre for Advanced Food Imaging, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark

b

Accepted 8 May 2007

Abstract Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects various signal transduction pathways, gene sequences and protein synthesis. One indication of which cells are competent to undergo the fusion process is their expression of two proteins, Myo-D and myogenin. The mechanism by which the cells are able to to regulate Myo-D and myogenin is poorly understood. In the present work, we investigate the role of mechanical loading, through specific receptors to intracellular matrix proteins such as laminin and fibronectin, in both Myo-D and myogenin expression in C2C12 cells. We propose to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C2C12 cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin recepotors plays a role in myoblast differentiation and fusion. r 2007 Elsevier Ltd. All rights reserved. Keywords: Myoblast; Fusion; Mechanical stimulation; Muscle; Development; Myogenin; Myo-D; Integrin

1. Introduction Mechanical loading and deformation play an important role in the physiology of a variety of tissues and several studies, in both animal and cell culture models, reported a large list of cellular responses (Kessler et al., 2001). Mechanical stimulation leads to proliferation and differentiation of osteoblast (Duncan and Turner, 1995), chondrocyte (Deschner et al., 2003), fibroblast and cardiomyocyte (Deschner et al., 2003; Ruwhof and van der, 2000), and myoblast (Simpson et al., 1994). Two Corresponding author. Current address: Wageningen Center for Food Science, Agrotechnology and Food Innovations, P.O. Box 17, 6700 AA Wageningen, The Netherlands. Tel.: +31 317483005. E-mail address: [email protected] (M.A. Lawson).

0021-9290/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2007.05.007

different kind of mechanical forces were defined, those transmitted to the cells from the extracellular matrix (ECM) and those generated within the cytoskeleton of individual cells and exerted to their ECM adhesions (Ingber, 1997). Integrins are the main membrane receptors that connect the cytoskeleton to the ECM and relay physical or mechanical signals from the surrounding environment into the interior of the cell (Calderwood et al., 2000). This role is explicated by the heterodimeric nature of integrin receptors that consist of two subunits, a and b. Both subunits are transmembrane glycoproteins with relatively large extracellular domains and a cytoplasmatic tails that bind the cytoskeleton network (Clark and Brugge, 1995). When mechanical forces are applied on integrin receptors, several mechano-sensor proteins are recruited on the

ARTICLE IN PRESS A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

2. Results 2.1. Mechanical stimulation of myoblasts via laminin receptors leads to increased cell proliferation At time 0, prior to cell stimulation and viewed using a 20  objective lens, all cultures had a seeding density of

approximately 100 cells per field. After magnetic stimulation for 24 h with laminin coated beads, approximately 340 cells could be seen per field (CBML, Fig. 1a), while samples which contained no beads (C) and subjected to the magnetic field (MF) (CM) had significantly fewer cells (Po0.001) (Fig. 1a). A small numerical but non-significant increase (Po 0.2) was seen in samples in which laminin coated beads had been added (CBL, Fig. 1a). Therefore, although the binding of ligand to laminin receptors may lead to a small increase in cell proliferation, mechanical stimulation via laminin receptors has a stronger influence on this process. Cells mechanically stimulated via fibronectin receptors (CBMF) showed a greater increase in cell proliferation then laminin, but this increase was equal to that seen in cultures containing fibronectin coated beads without mechanical stimulation (CBF, Fig. 1b). These results support the theory that different ECM molecules can play antagonistic roles during myoblasts proliferation and that mechanical stimuli are an important

Number of cells per field

400



350 300 250 200 150 100 50 0 C

CBML

CM

CBL

CM

CBF

600 Number of cells per field

membrane to form focal adhesions (FA) structures (Fluck et al., 1999), after which signaling pathways are activated (Wang et al., 2001). This activation generates a process known as mechano-transduction (Huang et al., 2004). It is also reported that, in mechano-sensitive tissues, signals transmitted to FA through integrin receptors lead to an activation of the MAP kinase pathway leading to the phosphorylation of ERK1/2. The activation of this pathway through integrin receptors serves to transmit the proliferation or differentiation response (Ueyama et al., 2000). Insights into the molecular events that regulate muscle differentiation have been provided by studies on a family of skeletal muscle specific transcription factors: the Myo-D family. Members of this family include Myo-D, Myogenin, Myf5, and MRF4/Myf6/Herculin (Braun et al., 1989, 1990; Pinney et al., 1988; Wright et al., 1989). These myogenic regulators factors act at different stages during muscle development. Both Myo-D and Myf5 are predominantly involved in myoblast determination and cell cycle regulation, while myogenin is active during myotube formation. MRF4/Myf6/Herculin is essential during the later stages of myofibrillogenesis (Hasty et al., 1993; Nabeshima et al., 1993; Rudnicki and Jaenisch, 1995; Kitzmann et al., 1998). It has been reported that cells expressing Myo-D and myogenin are fusion competent since they are able to actively fuse to form multinucleated myotubes (Sabourin and Rudnicki, 2000). Anatomical studies describe a gradual transition occurring in the ECM surrounding muscle cells during the early stages of myogenesis, from a matrix rich in interstitial molecules, like fibronectin, to one enriched in basal lamina components, like laminin (Godfrey et al., 1988; Godfrey and Gradall, 1998). The antagonistic roles on the myogenic differentiation of fibronectin and laminin were well established indicating the first one as an inhibitor and the second as an ‘‘enhancer’’ of the myogenic phenotype (von der and Ocalan, 1989). To understand the role of mechanical stimulation through specific cellular receptors on myogenic differentiation, we adapted a method previously developed, which employs ferromagnetic microbeads (Yuge and Kataoka, 2000; Yuge et al., 2003). Using this method, we investigated the role of mechanical stimuli through specific laminin and fibronectin binding integrins in the proliferation and differentiation of myoblasts. The myogenic response to this stimulus was studied by monitoring the expression pattern of Myo-D and myogenin, the ratio of ERK1/2 phosphorylation and the changes in myotube development.

3355



500 400 300 200 100 0 C

CBMF

Fig. 1. Proliferation of C2C12 cells is effected by both chemical and mechanical stimulation. Cells were stimulated with: (a) laminin or (b) fibronectin and stained with DAPI. The number of nuclei per field was detected. Cells mechanically stimulated with laminin coated beads (CBML) proliferated more rapidly than C, CM, CBL samples. Cells mechanically and chemically stimulated with fibronectin (CBMF, CBF) proliferated more rapidly than C and CM, but this increase was not affected by mechanical stimulation. Error bars—7SE and ‘*’ indicates results significantly different.

ARTICLE IN PRESS A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

3356



14

CPK Activity(MIunits/μg)

CPK Activity (MIunits/μg)

16 12 10 8 6 4 2

16 14 12 10 8 6 4 2 0

0 C

CBML

CM

CBL

C

CBMF

CM

CBF

Fig. 2. The activity of CPK increases after mechanical stimulation through laminin receptors: (a) cells cultures mechanically stimulated with laminin coated beads showed a significantly (Po0.001) higher CPK activity than C, CM and CBL samples; and (b) CPK activity was unaffected by fibronectin chemical or mechanical stimulation. Error bars—7SE and ‘*’ indicates results significantly different.

Laminin

Fibronectin

Myo-D

Myo-D CBML

CBMF

CBF



2.5

C

Band Intensity

Band Intensity

3

CBL

2 1.5 1 0.5 0 CBML

CBL

CBMF

Laminin

CM

3 2.5 2 1.5 1 0.5 0

CBF

C

CM

Fibronectin

Laminin

Fibronectin

Myogenin CBML

1.5

CBMF

C

CBF

CM

2



Band Intensity

Band Intensity

2

CBL

1 0.5 0

1.5 1 0.5 0

CBML

CBL

CBMF

CBF

C

CM

Fig. 3. The level of Myo-D and myogenin expression increases after mechanical stimulation through laminin receptors. Protein extracts obtained from cells, after different treatment (Table 1), were resolved in 8–12% polyacrylamide gel and transferred onto a PVDF membrane. Blots were developed with specific monoclonal antibodies as described in Materials and Methods. The apparent density of the bands was estimated by scanning with a video densitometer. Error bars—7SE and ‘*’ indicates results significantly different.

regulator of cell proliferation only when applied to specific integrin isoform. 2.2. Cells mechanically stimulated with laminin coated beads show increased differentiation The activity of creatine phosphokinase (CPK) increases as myoblasts differentiate into myotubes (Al-Khalili et al.,

2004). Cells that have been stimulated using laminin coated beads (CBML) had a significantly higher CPK activity (Po0.001) than any of the other test conditions studied (Fig. 2a). No differences in CPK activity were detected between cells samples stimulated with fibronectin (Fig. 2b). These results indicate that mechanical stimulation of myoblasts through laminin but not fibronectin receptors can significantly stimulate myoblast differentiation.

ARTICLE IN PRESS A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

2.3. Mechanical stimulation via laminin receptors leads to increases in Myo-D and myogenin expression Mechanical stimulation via laminin receptors on the cells surface induces an increase in the expression of the myogenic regulatory factors myogenin and Myo-D (Fig. 3a, b, Table 1). No difference was seen in cells chemically stimulated with laminin or fibronectin, or with cells mechanically stimulated through the fibronectin receptor, as the expression of myogenin and Myo-D in

Table 1 Experimental categories

CBML

C2C12 cells C2C12 cells exposed to C2C12 cells with beads C2C12 cells with beads C2C12 cells with beads 0.5 mT magnetic field C2C12 cells with beads 0.5 mT magnetic field

0.5 mT magnetic field coated with fibronectin coated with laminin coated with fibronectin exposed to coated with laminin exposed to

Laminin

these cells was indistinguishable from the control groups. Samples that had been pretreated with a blocking antibody to b1 integrin and then mechanically stimulated with laminin coated beads did not show an increase in Myo-D or myogenin expression (data not shown). These results demonstrate that the effect of mechanical stimulation on myogenin and Myo-D expression is substrate dependent and occurs through integrin receptors. 2.4. Mechanical stimulation via laminin receptors leads to increases in ERK1/2 phosphorylation Integrins are positioned to be a primary sensor for relaying chemical or physical signals from the surrounding environment into the interior of the cells, activating signal transduction pathways (Calderwood, 2004). Chemical and mechanical stimuli through laminin and fibronectin receptors did not change the expression level or ERK1/2 in myoblasts (Fig. 4a), mechanical signals via laminin receptors increased the phosphorylation of these signaling proteins two-fold (Fig. 4b). No differences were shown in the level of ERK1/2 phosphorylation when myoblasts were stimulated with fibronectin (Fig. 4b). This indicates that mechanical stimuli are important regulators of myoblast

Categories

C CM CBF CBL CBMF

3357

Fibronectin

ERK 1/2 CBL

CBMF

CBF

25 20 15 10 5 0

CM

C

Band Intensity

Band Intensity

CBML

25 20 15 10 5 0

CBML

CBL

CBMF

CBF

Laminin

CM

C Fibronectin

P-ERK 1/2

10

CBL

CBMF



CBF

C

Band Intensity

Band Intensity

CBML

5 0

CM

10 5 0

CBML

CBL

CBMF

CBF

C

CM

Fig. 4. Mechanical strain on laminin receptors induces an increase in ERK1/2 phosphorylation. Cell lysates were analyzed by Western blot with antibody directed against: (a) ERK1/2 protein; and (b) phosphorylated ERK1/2. The apparent density of the bands was estimated by scanning with a video densitometer. Error bars—7SE and ‘*’ indicates results significantly different.

ARTICLE IN PRESS A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

3358

differentiation only when applied through specific membrane receptors. 2.5. Mechanical stimulation with laminin coated beads induces the formation of smaller but numerous myotubes C2C12 cells are able to fuse and form myotubes in vitro after switching the cells from nutrient rich media containing fetal calf serum to nutrient poor medium containing horse serum. The size and frequency of myotube formation can be visualized and quantified by staining cell membranes and nuclei. When cell cultures are mechanically stimulated through their laminin receptors, the resultant myotubes contain significantly (Po0.001) fewer nuclei than cultures simply exposed to the laminin coated beads (Fig. 5a). Although the myotubes formed after mechanical stimulation are small, the number of myotubes formed are significantly increased (Po0.001) (Fig. 5b). Few if any Laminin

Fibronectin

12 ∗

Nuclei per Myotube

10 8 6 4 2 0

CBML CBL Laminin

CBMF CBF Fibronectin

180

Number of Myotubes

160



140 120 100 80 60 40 20 0 CBML

CBL

CBMF

CBF

Fig. 5. Mechanical stimulation through laminin receptors leads to an increase in small myotube formation. (a) The number of nuclei per myotube; and (b) the total number of myotubes, in cultures of cells mechanically stimulated (CBML, CBMF) as compared to chemically stimulated samples (CBL, CBF). Stimulation with laminin coated beads causes an increase in the number of myotubes formed, but the resultant myotubes are significantly smaller. Few if any myotubes are seen when cultures are stimulated with fibronectin coated beads. Error bars—7SE and ‘*’ indicates results significantly different than controls.

myotubes can be seen in cultures stimulated with fibronectin coated beads. No significant differences were seen in the numbers of nuclei found in myotubes exposed to mechanical stimulation via the fibronectin receptor as compared to myotubes generated by exposure to fibronectin coated beads but having no force applied. Therefore, mechanical stimulation via laminin but not fibronectin receptors has a significant effect on myotube formation in vitro. 3. Discussion Several methods have been developed to study the cellular response to mechanical signals, including membrane strain studies and, more recently, magnetic microbeads. The first device can deliver two main classes of mechanical deformations to the cell culture population: uniaxial or multiaxial (Hornberger et al., 2005). While the utilization of magnetic microbeads allowed greater control of the applied forces and the targeting of specific membrane receptors (Glogauer and Ferrier, 1998). In the present study, in order to examine whether there is a different mechanical response on laminin and fibronectin receptors, we adapted the method, previously developed, (Yuge and Kataoka, 2000; Yuge et al., 2003) which employs magnetic microbeads. Numerous anatomical factors, like fiber orientation and interaction between the muscle fiber and different ECM components, make it very difficult if not impossible to define the mechanical environment within a single muscle cell. For these reasons and in order to add knowledge in the field of mechanotransduction induced by magnetic microbeads stimulation a MF of 0.05 T was chosen (Yuge and Kataoka, 2000; Yuge et al., 2003). The aim of this study was to determine how chemical and physical stimuli trough specific transmembrane integrin receptors affect proliferation and differentiation of myoblasts. The preliminary studies showed that mechanical stimulation via laminin-1 receptors leads to an increase to cell proliferation when compared to cell only chemically stimulated. In contrast, mechanically and chemically stimulated cells via fibronectin receptors showed similar increases in proliferation, both eliciting a greater response in proliferation as compared to cells mechanically stimulated via laminin-1 receptors. These results clearly indicate that while mechanical stimulation is an important regulator of cell proliferation when applied to specific laminin-1 ligands, chemical signals via fibronectin receptors induce the highest level of cell proliferation. Differentiation leading to myoblast fusion is also strongly affected by signals from the ECM. Although previous studies have shown that chemical stimuli through laminin binding integrins caused increases in differentiation, this was not seen in this study. This may have been due to the shorter incubation times of cells in the presence of laminin than used in some of the previous work (Lawson and Purslow, 2000). We demonstrate that the degree of myoblast differentiation can be increased in the presence of

ARTICLE IN PRESS A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

mechanical stimuli. Differentiation was higher in C2C12 mechanically stimulated through laminin receptors compared to the other samples. It is clear from these results that differentiation is increased upon stimulation through laminin binding surface receptors. It is widely reported that myogenesis is under the control of members of the myogenic regulatory factors (MRFs), Myo-D, myogenin, Myf5, Myf6 and that the concomitant expression of Myo-D and myogenin promotes the fusion of myoblasts into myotubes (Pinney et al., 1988; Wright et al., 1989; Braun et al., 1989, 1990). In this study we show that only when C2C12 cells were mechanically stimulated through laminin receptors was it possible to detect a significant increase of Myo-D and myogenin. Chemical signaling through the laminin receptors was insufficient to cause an increase in the expression of these regulatory factors. Neither chemical nor mechanical signals through fibronectin receptors increased expression of myogenin or Myo-D. Therefore, our results show that mechanotransduction events affecting myoblasts fusion potential occur when mechanical stimuli are specifically applied to lamininbinding integrins and not to other receptors. Changes in protein expression due to integrin-mediated signaling would be expected to occur after activation of the MAP Kinase pathway. Cytoskeletal responses to integrin ligation lead to aggregation of integrins and recruitment and activation of mechano-sensor proteins like focal adhesion kinase (FAK) (Schlaepfer et al., 1999) and low molecular weight GTPase (Meyer et al., 2000). These are known to initiate signal transduction pathways that regulate activation of MAP kinase (Yamazaki et al., 1999) ultimately leading to phosphorylation of ERK1/2 phosphorylation (Laboureau et al., 2004) which in turn contributes to the activation of various genes. As we have shown protein expression increases due to mechanical stimulation via laminin receptors, we would expect that these signals also alter the degree of ERK1/2 phosphorylation in the cells. As seen with the expression of Myo-D and myogenin, there is a similar increase in ERK1/2 phosphorylation in cells mechanically stimulated through laminin receptors. Stimulation through fibronectin receptors showed no changes in ERK1/2 phosphorylation. This result is in agreement with a previous study which demonstrated that a6b1 integrin specific receptors for laminin-1 may promote exit from the cell cycle in myoblasts while a5b1 integrin specific receptors for fibronectin induce proliferation because of their different role in activating mechano-sensor protein after stimulation (Sastry et al., 1996). In order to support our thesis and to detect changes in myotube formation, C2C12 cells which had been chemically and mechanically stimulated were incubated an additional 48 h and number of myotubes and number of nuclei per myotubes were determined. We showed that laminin chemical stimulated cells present a significant increase of number of nuclei per myotubes while laminin mechanical stimulated cells significantly tend to form a higher number

3359

of myotubes. This confirms our thesis that mechanical stimulation through laminin receptors accelerates the differentiation process and promotes cells fusion, forming a higher number of myotubes but smaller in size. This is probably due to the higher number of fusion competent cells in the population, and the higher probability that cells will be able to fuse with myoblasts near by, instead of fusing into an already existing myotube. We showed that this model can be used to investigate the role of chemical and mechanical stimulation trough specific integrin receptors during myogenesis. Major changes occur in the ECM composition during embryo development. Evidence of a transition from an ECM rich in interstitial molecules, such as fibronectin, to one enriched in basal lamina components, like laminin, during early myogenesis has been described (Godfrey and Gradall, 1998). A number of integrin receptors are expressed by myoblasts, but it is well documented that laminin (Laminin 1) bind to the a6b1 and a7b1 integrin receptors while fibronectin bind to a5b1 integrin receptors (Givant-Horwitz et al., 2005). Molecular and histological studies presented evidence that the expression of these integrin receptors change during the course of myogenesis (Cachaco et al., 2005), underling the fundamental role of ECM components in development, which is mediated, indeed, by specific integrin receptors. In this study we have shown that increases in mechanical forces trough laminin receptors, in vitro, cause the formation of numerous myotubes, each made up of fewer fused cells. We believed that this is due to the fact that more cells are fusion competent (i.e. express both Myo-D and myogenin). Further study should be address to understand how mechanical stimuli activate signaling pathways and promote transcription of specific gene sequences. 4. Experimental procedures 4.1. Cell culture A mouse myoblast cell line, C2C12 (ATCC, Manassas, VA, USA) was seeded in 25 cm2 cell culture flask and maintained in Dulbecco modified Eagle medium (D-5523, Sigma, St. Louis MO) containing 10% foetal bovine serum and 100,000 units/l media of penicillin and 100 mg/l media of streptomycin in an atmosphere of 5% CO2 at 37 1C. Cells were allowed to proliferate to 75% confluence before splitting. All cell cultures were used before the 10th passage. 4.2. Antibodies and probes The following antibodies and probes were used as described: Anti Myogenin (M-255, Santa Cruz Biotechnology), Anti Myo-D (Ab-1 5.8A, Neomarkers, Fremont CA), Anti ERK1/2 and Anti phosphor ERK1/2 (Invitrogen, San Francisco, CA). Horseradish peroxidase-conjugated sheep and donkey affinity-purified antibody to mouse

ARTICLE IN PRESS 3360

A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

and rabbit IgG, respectively, (Amersham Biosciences), Fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO), and 40 ,6-Diamidino-2-phenylindole (DAPI) (Sigma, St. Louis MO). Laminin-1 and Fibronectin (Sigma, St Louis, MO).

The magnet was placed 10 mm over the monolayer of cells during the stimulation period.

4.3. Preparation of protein coated magnetic beads

On laminin or fibronectin coated surface 2  105 cells/ml were plated and stimulated as described. After 24 h, the cells were fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI. The results shown are from five separate experiments, where a total of 10 images of each preparation were obtained as above. The numbers of nuclei per field were obtained digitally using Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). This experiment was repeated five times on different days.

For 2-D magnetic stimulation experiments, encapsulated super-paramagnetic microbeads (EMI- 100/40) made of polystyrene, having diameter 1 mm with a ferrite content of 36–45% were used. 0.1 ml of 10% magnetic bead solution was diluted 1:10 in 0.1 M sodium acetate buffer (pH 5.4) for laminin and 0.1 M borate buffer (pH 9.5) for fibronectin. The solution was centrifuged at 2000 rpm for 5 min to remove any surfactant used for preservation in the bead solution. The supernatant was then removed and the beads were dispersed in 1 ml of 0.1 M sodium acetate/0.1 M borate buffer after sonication. To coat the particles, 1 ml of the specified protein solution (fibronectin from rat plasma 100 mg/ml; laminin from basement membrane of Engelbreth-Holm Swarm mouse sarcoma 10 mg/ml, Sigma, St Louis, MO) was added to the bead suspension and the mixture was stirred at 60–100 rpm for 2 h at RT. The concentrations used for each substrate coated were previously determined (Lawson and Purslow, 2001), to allow for a uniform coating over the surface. The mixture was then centrifuged at 2000 rpm for 5 min, the supernatant was removed and the pellet was re-suspended in 1 ml of 0.1 M sodium acetate buffer (pH 5.2)/0.1 M borate buffer (pH 9.5) containing 1% (w/v) BSA to reduce nonspecific binding and self-aggregation of the beads.

4.5. Proliferation assays

4.6. Differentiation/creatine-phosophokinase activity assay Cells were cultured and stimulated as described above. After stimulation for 24 h, the cells were scraped from the surface of the culture dish, homogenized in PBS and the resultant suspension was centrifuged to remove any cellular debris. Protein determination was carried out using the BCA protein determination kit (Pierce, Rockford, Ill.). As per manufacturer’s instructions, 0.1 ml of the matrix homogenate was added to one vial of the CPK assay kit (Sigma, St. Louis, MO) that had been diluted with 3 ml distilled water. After 5 min a baseline reading at 340 nm was recorded; 5 min later a second 340 nm reading was taken. The difference between the final and initial readings was used to calculate the CPK activity in the samples. The procedure was repeated on four different days; five replicates were prepared each single day.

4.4. Magnetic bead stimulation 4.7. Protein extraction procedure The procedure for magnetic stimulation of cell populations has been extensively described by Glogauer et al. (1998). Briefly, C2C12 cells (1  106 cells/ml) were incubated overnight on protein fibronectin (100 mg/ml) or laminin (10 mg/ml)) coated coverslip dishes in order to ensure that receptors on all cell surfaces that may be subjected to mechano-forces were exposed to the desired substrate. The concentrations used for each substrate coated were previously determined by Lawson and Purslow (2000) to allow for a uniform coating over the glass surface. After coating the cultures were rinsed with PBS and 50 ml of the magnetic bead solution was added. Cultures were incubated statically with the beads for 30 min at 37 1C to allow the beads to attach to the surface of the cells, rinsed with PBS to remove any unattached microbeads and then incubated in Hepes-buffered DMEM (Sigma, St. Louis, MO). Cultures to be stimulated were placed on a heating plate maintained at 37 1C under an electromagnet for a period of 6 h. Control cultures were kept in an incubator at 37 1C during that period. A MF of 0.5 mT was generated by an electromagnet (Power Generator 0–30 V, 0.1–100 Hz; Elcanic A/S, Denmark). The magnet produced alternating MF at frequency of 1 Hz.

After different treatment C2C12 cells were washed twice with cold PBS, scraped (mechanically detached), and transferred to microcentrifuge tubes. Cell suspension was centrifuged at 1000g for 5 min, and cell pellet was resuspended in lysis buffer (in 150 mM NaCl, 20 mM Tris, pH 7.4, 1% Triton X-100 detergent surfactant and a protease inhibitor cocktail was added; Complete from Roche Applied Science). Rapidly, the new cell suspension was frozen using liquid nitrogen, and then thawed; this step was repeated three times. To reduce viscosity, the cell fraction was sonicated three times for 10 s at 0 1C. The homogenized protein solution was centrifuge for 20 min at 20,000g at 4 1C, the supernatant was collected and protein determination was performed with BCA protein assay kit (PIERCE, Rockford, IL). 4.8. Western blotting analysis Equal volumes of samples were diluted in 4X LDS sample buffer with 50 mM DTT, were electrophoresed on 8–12% SDS-polyacrylamide gel under reducing conditions and transferred to polyvinylidene difluoride (PVDF)

ARTICLE IN PRESS A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

membranes (0.2 mm pore size; Invitrogen); using a dry transfer method. The membranes were blocked in 5% nonfat dried milk in Tris-buffered saline/Tween-20 (TBS-T; 20 mM Tris, pH 7.5, 150 mM NaCl, 0.3% Tween-20) for 1 h at room temperature. Immunoblotting was performed using rabbit polyclonal affinity-purified antibody (1:2000) and mouse monoclonal affinity-purified antibody (1:2000) to Myo-D, myogenin, ERK1/2 and phospho ERK1/2, respectively, for 16 h at 4 1C in 1% non-fat dried milk in TBS-T. The membrane was then washed three times in TBS-T and incubated with a 1:2000 horseradish peroxidaseconjugated sheep and donkey affinity-purified antibody to mouse and rabbit IgG, respectively, (Amersham Biosciences), in TBS-T for 1 h at room temperature. After three washes in TBS-T, proteins were visualized using the enhanced chemiluminescent system (ECL-plus, Amersham Biosciences). Immunoreactive bands were detected using a scanner, and densitometric values were analyzed with Phoretix 1D 2003.02 (Nonlinear Dynamics, Newcastle, UK) 4.9. Statistical analysis For all assays, three or more separate experiments were performed. Means7SE were calculated. Comparisons between two groups were analyzed by unpaired t-tests, or when multiple comparisons were made, ANOVA was performed. In all instances, statistical significance was set at Po0.05. Conflict of interest None References Al-Khalili, L., Kramer, D., Wretenberg, P., Krook, A., 2004. Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylation. Acta Physiologica Scandinavica 180, 395–403. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E., Arnold, H.H., 1989. A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. EMBO Journal 8, 701–709. Braun, T., Bober, E., Winter, B., Rosenthal, N., Arnold, H.H., 1990. Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12. EMBO Journal 9, 821–831. Cachaco, A.S., Pereira, C.S., Pardal, R.G., Bajanca, F., Thorsteinsdottir, S., 2005. Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse. Developmental Dynamics 232, 1069–1078. Calderwood, D.A., 2004. Integrin activation. Journal of Cell Science 117, 657–666. Calderwood, D.A., Shattil, S.J., Ginsberg, M.H., 2000. Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling. Journal of Biological Chemistry 275, 22607–22610. Clark, E.A., Brugge, J.S., 1995. Integrins and signal transduction pathways: the road taken. Science 268, 233–239. Deschner, J., Hofman, C.R., Piesco, N.P., Agarwal, S., 2003. Signal transduction by mechanical strain in chondrocytes. Current Opinion in Clinical Nutrition and Metabolism Care 6, 289–293.

3361

Duncan, R.L., Turner, C.H., 1995. Mechanotransduction and the functional response of bone to mechanical strain. Calcified Tissue International 57, 344–358. Fluck, M., Carson, J.A., Gordon, S.E., Ziemiecki, A., Booth, F.W., 1999. Focal adhesion proteins FAK and paxillin increase in hypertrophied skeletal muscle. American Journal of Physiology 277, C152–C162. Givant-Horwitz, V., Davidson, B., Reich, R., 2005. Laminin-induced signaling in tumor cells. Cancer Letters 223, 1–10. Glogauer, M., Ferrier, J., 1998. A new method for application of force to cells via ferric oxide beads. Pflugers Archiv 435, 320–327. Godfrey, E.W., Gradall, K.S., 1998. Basal lamina molecules are concentrated in myogenic regions of the mouse limb bud. Anatomy and Embryology (Berlin) 198, 481–486. Godfrey, E.W., Siebenlist, R.E., Wallskog, P.A., Walters, L.M., Bolender, D.L., Yorde, D.E., 1988. Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles. Developmental Biology 130, 471–486. Hasty, P., Bradley, A., Morris, J.H., Edmondson, D.G., Venuti, J.M., Olson, E.N., Klein, W.H., 1993. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364, 501–506. Hornberger, T.A., Armstrong, D.D., Koh, T.J., Burkholder, T.J., Esser, K.A., 2005. Intracellular signaling specificity in response to uniaxial vs. multiaxial stretch: implications for mechanotransduction. American Journal of Physiology—Cell Physiology 288, C185–C194. Huang, H., Kamm, R.D., Lee, R.T., 2004. Cell mechanics and mechanotransduction: pathways, probes, and physiology. American Journal of Physiology—Cell Physiology 287, C1–C11. Ingber, D.E., 1997. Tensegrity: the architectural basis of cellular mechanotransduction. Annual Review of Physiology 59, 575–599. Kessler, D., Dethlefsen, S., Haase, I., Plomann, M., Hirche, F., Krieg, T., Eckes, B., 2001. Fibroblasts in mechanically stressed collagen lattices assume a ‘‘synthetic’’ phenotype. Journal of Biological Chemistry 276, 36575–36585. Kitzmann, M., Carnac, G., Vandromme, M., Primig, M., Lamb, N.J., Fernandez, A., 1998. The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. Journal of Cell Biology 142, 1447–1459. Laboureau, J., Dubertret, L., Lebreton-De, C.C., Coulomb, B., 2004. ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoA. Experimental Dermatology 13, 70–77. Lawson, M.A., Purslow, P.P., 2000. Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific. Cells Tissues Organs 167, 130–137. Lawson, M.A., Purslow, P.P., 2001. Development of components of the extracellular matrix, basal lamina and sarcomere in chick quadriceps and pectoralis muscles. British Poultry Science 42, 315–320. Meyer, C.J., Alenghat, F.J., Rim, P., Fong, J.H., Fabry, B., Ingber, D.E., 2000. Mechanical control of cyclic AMP signalling and gene transcription through integrins. Nature Cell Biology 2, 666–668. Nabeshima, Y., Hanaoka, K., Hayasaka, M., Esumi, E., Li, S., Nonaka, I., Nabeshima, Y., 1993. Myogenin gene disruption results in perinatal lethality because of severe muscle defect. Nature 364, 532–535. Pinney, D.F., Pearson-White, S.H., Konieczny, S.F., Latham, K.E., Emerson Jr., C.P., 1988. Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway. Cell 53, 781–793. Rudnicki, M.A., Jaenisch, R., 1995. The MyoD family of transcription factors and skeletal myogenesis. Bioessays 17, 203–209. Ruwhof, C., van der, L.A., 2000. Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways. Cardiovascular Research 47, 23–37. Sabourin, L.A., Rudnicki, M.A., 2000. The molecular regulation of myogenesis. Clinical Genetics 57, 16–25. Sastry, S.K., Lakonishok, M., Thomas, D.A., Muschler, J., Horwitz, A.F., 1996. Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. Journal of Cell Biology 133, 169–184.

ARTICLE IN PRESS 3362

A. Grossi et al. / Journal of Biomechanics 40 (2007) 3354–3362

Schlaepfer, D.D., Hauck, C.R., Sieg, D.J., 1999. Signaling through focal adhesion kinase. Progress in Biophysics and Molecular Biology 71, 435–478. Simpson, D.G., Carver, W., Borg, T.K., Terracio, L., 1994. Role of mechanical stimulation in the establishment and maintenance of muscle cell differentiation. International Review of Cytology 150, 69–94. Ueyama, T., Kawashima, S., Sakoda, T., Rikitake, Y., Ishida, T., Kawai, M., Yamashita, T., Ishido, S., Hotta, H., Yokoyama, M., 2000. Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy. Journal of Molecular and Cellular Cardiology 32, 947–960. von der, M.K., Ocalan, M., 1989. Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 40, 150–157. Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase

(MAPK) activation. Biochemistry and Biophysics Research. Communications 288, 356–361. Wright, W.E., Sassoon, D.A., Lin, V.K., 1989. Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56, 607–617. Yamazaki, T., Komuro, I., Shiojima, I., Yazaki, Y., 1999. The molecular mechanism of cardiac hypertrophy and failure. Annals of the New York Academy of Sciences 874, 38–48. Yuge, L., Kataoka, K., 2000. Differentiation of myoblasts is accelerated in culture in a magnetic field. In Vitro Cellular and Developmental Biology—Animal 36, 383–386. Yuge, L., Okubo, A., Miyashita, T., Kumagai, T., Nikawa, T., Takeda, S., Kanno, M., Urabe, Y., Sugiyama, M., Kataoka, K., 2003. Physical stress by magnetic force accelerates differentiation of human osteoblasts. Biochemistry and Biophysics Research. Communications 311, 32–38.