Open Journal of Cell Biology, 2012, 2, ***-***
Published Online September 2012 (http://www.SciRP.org/journal/ojcb)
The Number of Cyclic Stretch Regulates Cellular
Elasticity in C2C12 Myoblasts
Kenji Takemoto, Takeomi Mizutani*, Kazushi Tamura, Kazuki Takeda, Hisashi Haga,
Kazushige Kawabata
Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan
Received ********* 2012
ABSTRACT
Mechanical stimulations have been shown to regulate cellular mechanical properties. However, the stimulation patterns
for effective regulation are as yet unclear. We investigated the effects of application of differing numbers of mechanical
stimulation sets, each set consisting of 8% extension and compression to cells via deformation of cell culture elastic
chamber, on cellular elasticity. Elasticity increased with only a single step-like stretch and with a single step-like stretch
after 1 set of mechanical stimulation, whereas elasticity did not change with a single step-like stretch after 10 sets of
mechanical stimulation. These results indicate that the increase in cellular elasticity with the single step-like stretch
depends on the number of applied mechanical stimulations. Immunofluorescence staining showed that phosphorylation
and dephosphorylation of myosin regulatory light chain (MRLC), which regulates intracellular contractile force and
cellular elasticity, accompanied cellular elasticity changes. These findings suggest that cellular elasticity changes under
cyclic and step-like stretches are mediated by MRLC.
Keywords: Cellular Elasticity; Mechanical Stimulation; Atomic Force Microscopy; Myosin Regulatory Light Chain;
Regulation of Cellular Function
1. Introduction
Cellular responses to external mechanical stimuli, such
as gravity, stretch and shear flow, and ultrasound, play
important roles in expressing cellular physiological func-
tions [1-4]. For example, cardiac or smooth muscle cells
respond to the stretch induced by elevated blood pressure
and alter their mechanical properties to modulate blood
pressure to maintain adequate blood circulation [5]. In
addition, bone cells respond to ultrasound by increasing
cellular growth rate and extracellular matrix secretion [6].
Such controlled cellular growth and matrix secretion is
expected to be available in the clinical field. Thus, cel-
lular response to external mechanical stimuli is not only a
fundamental cellular function, but it is also adaptable for
medical usage. Therefore, investigation of the types of
available mechanical stimulus and their resulting cellular
responses are of great interest in both cell biology and
biophysics.
Mechanical stretch has the ability to change cellular
physiological functions, such as differentiation and cellu-
lar morphology. For example, cyclic stretch (with some
additives) inhibits to differentiate myoblasts into myotu-
bes [7]. In addition, uniaxial cyclic stretch induces cell
orientation perpendicular to the stretch axis [8], although
other stretch patterns like a single step-like stretch do not
induce such effects. In the case of a single step-like
stretch, this mechanical stimulus also keeps cells stre-
tched, i.e., mechanical stimulus continues to be im-
pressed to cells; however, cellular responses to a single
step-like stretch are different from responses to cyclic
stretches. To use mechanical stretch more effectively for
cell regulation, it is important to consider its application
pattern.
Our previous work showed that cellular elasticity
could be regulated by mechanical stretch [9]. Briefly,
application of only a single step-like stretch increased
cellular elasticity, while more than 30 sets of cyclic
stretch before the single step-like stretch inhibited the
increase in cellular elasticity. These results indicate that
adequate patterns of mechanical stretch are available to
regulate cellular elasticity. However, the stretch patterns
for effective regulation of cellular elasticity are not clear
as yet.
The purpose of this study was to determine the number
of cyclic stretches that are sufficient to inhibit the in-
crease in cellular elasticity after the single step-like
stretch. We examined the cellular elasticity response by
changing the number of cyclic stretches from 1 to 10 sets.
*Corresponding author.

K. TAKEMOTO ET AL.
2
In addition, we examined signaling cascades known to be
involved in the regulation of cellular elasticity following
cyclic stretches. Cellular elasticity is known to be related
to actomyosin-based contractile force [10]. As candidates
for regulating contractile force under mechanical stretch,
we examined changes in phosphorylation of myosin re-
gulatory light chain (MRLC) and intracellular Ca2+. Our
results indicated that the number of cyclic stretches
regulated phosphorylation of MRLC and resulting cel-
lular elasticity. We discuss the need to investigate the
relationship between the expected cellular physiological
response and mechanical stimulation parameters, and we
address the regulatory mechanism of cellular elasticity
following mechanical stimulation.
2. Materials and Methods
2.1. Cell Culture
Murine C2C12 skeletal myoblasts obtained from RIKEN
Cell Bank (Tsukuba, Japan) were cultured on plastic
flasks supplied with Dulbecco’s modified Eagle’s medi-
um (DMEM; Sigma-Aldrich, MO, USA), 10% FBS, and
1% antibiotic solution (Sigma-Aldrich) at 37°C in a
humidified atmosphere of 5% CO2.
2.2. Stretch Device and Sample Preparation
Living cells were stretched or contracted via deformation
of a deformable elastic cell culture chamber combined
with a uniaxial stretch device [11]. For cellular elasticity
measurements, an elastic chamber was formed into a
5-mm thick bottom (30 × 30 mm) with a wall height of
20 mm by using transparent silicone rubber (SYLGARD
184; Dow Corning, Michigan, USA). For immunofluore-
scence observation, we used 10 μm of a thin-bottom
elastic chamber and a high magnification lens. To deform
the chamber at a constant velocity and degree, we used a
motor-driven uniaxial slide stage (TAM-401SOMEB-
MDC(15); Sigma Koki, Tokyo, Japan): 2 ends of the
chamber were attached to the stage with handmade steel
clamps (Figures 1(A) and (B)). By using this experi-
mental setup, we applied 8% rectangular-like repetitive
strain or a single step-like strain to cells. One set of
rectangular-like strain consisted of the following 4 steps:
stretch (from 0% to 8% of strain with 200 μm/s velocity);
rest (maintaining the 8% strain for 18 s); compression
(from 8% to 0% of strain with 200 μm/s velocity); rest
(maintaining the 0% of strain for 18 s). Thus, application
of 1 set of rectangular-like strain took 1 min. A set of
rectangular-like strain was defined as 1 set of cyclic
stretch.
The chamber was sterilized by exposure to ultraviolet
light for 6 h, and then the bottom of the chamber was
coated with 20 μg/mL fibronectin (Roche Diagnostics,
Basel, Switzerland) in PBS for 30 min. Cells grown on
Figure 1. Experimental setup for stretching C2C12 myoblasts.
Stretching cells were performed by deformation of a de-
formable elastic cell culture chamber to which cells adhered.
(A) Overhead view of uniaxial stretch device. The elastic
chamber was formed into a bottom of 5 mm or 10 m thick
and a wall of 20 mm height by transparent silicon rubber.
Two ends of this chamber were fixed with handmade steel
clamps; (B) Lateral view in measurement system of cellular
elasticity. The chamber fixed with clamps was combined
with uniaxial slide stage and was deformed at a constant
velocity and degree by motors. Cellular elasticity under uni-
axial stretch was measured by atomic force microscopy
(AFM); (C) Monitor of cellular position by optical micro-
cope. Relative position of cell (dashed line), cellular nucleus
(indicated as N) and cantilever were checked by the optical
image. We adjusted the top of cantilever (center of the AFM
measurement area) onto cellular nucleus. Scale bar indicates
15 m; (D)-(E) Verification of targeted region of elasticity
measurement. We used 15 m area (16 pixels × 16 lines) of
elasticity image (D) for the evaluation of cellular elasticity.
Position of the area was determined by the above procedure.
To verify that the position is on an adequate place (on cellu-
lar nucleus), wide-field elasticity image (60 m, 64 pixels ×
64 lines) for the cell (C) was measured (E). Elasticity distri-
tion in (D) is very similar to distribution in (E), suggesting
that the targeted 15 m area is roughly on celular nucleus.
the plastic flasks were trypsinized and cultivated in the
incubator for 1 day prior to the stretch experiments. A
few hours before the stretch experiments, the cell cul-
ture medium was replaced with 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES)-buffered DMEM
(pH 7.2 - 7.3) with 10% FBS and 1% antibiotic solu-
tion.
2.3. Cellular Elasticity Mapping with Atomic
Force Microscopy
Cellular elasticity mapping was achieved by a combina-
tion of a commercial atomic force microscope (AFM)
and our developed force mapping method [12]. The AFM
(NanoScope IIIa BioScope; Veeco, CA, USA) was
equipped with a piezo translator with a maximum xy scan
range of 100 μm and a z range of 10 μm. We used a
silicon-nitride cantilever (MLCT-AUNM; Veeco, CA,
USA) with the following specifications: a spring constant
of 0.01 N/m; tip geometry, pyramidal shape with an

K. TAKEMOTO ET AL. 3
opening angle of 35˚. The spatial distribution of cellular
elasticity was measured by the force mapping method.
Briefly, the relationship between the force acting on the
cantilever versus distance between the cantilever and
sample (so-called force curve) was taken at each pixel
point in a scan area. The force versus distance curves
were taken typically at 5.0 Hz with a z range of 1500 nm.
The force curves were fit to the Hertzian contact model
for a pyramidal indenter by using the non-linear least-
squares method. The Hertzian contact model predicts the
following relation:

2
0
1
2tan
F
v
F
zz kE



 (1)
where z is the position of the piezo scanner, F is the
loading force, v is the Poisson ratio, and α is the half-
opening angle of the cantilever tip. Here v was assumed
as 0.5 for simplification. Young’s modulus (elasticity) E
was acquired as fit parameters when the force curve data
were fit to the above model.
To monitor minute time scale of cellular elasticity
change, we improved temporal resolution to obtain re-
presentative cellular elasticity at each condition. Con-
ventional force mapping mode for 64 pixels × 64 lines
requires about 30 min to obtain one image. The 64 pixels
× 64 lines measurement has an advantage for comparison
of cellular elasticity maps and immunofluorescent images
[12] because of the better spatial resolution. On the
contrary, it has a weakness in the temporal resolution
to obtain one image. In the present study, we priori-
tized temporal resolution over the spatial resolution by
the reduction of target pixel points to 16 pixels × 16 lines.
A measurement for the 16 pixels × 16 lines takes 2 - 3
min.
To ensure that the targeted measurement region on a
cell is identical during a set of AFM measurements, we
used an inverted optical microscope (TE2000; Nikon,
Tokyo, Japan) and adjusted the center of the target region
onto the cellular nucleus by transference of the cantilever
position (Figures 1(C)-(E)). As a representative value of
cellular elasticity at each condition, we applied averaged
elasticity from the 16 × 16 pixel points for a 15 μm scan
area. To compare cellular elasticity under mechanical
stimulations, we chose cells with initial elasticity of 10 to
12 kPa.
2.4. Intracellular Ca2+ Measurement
Changes in cytosolic Ca2+ concentration ([Ca2+]) in re-
sponse to mechanical stretch were measured using a Ca2+
indicator (Quest Fluo-8™ AM; AAT Bioquest, Inc., CA,
USA). We prepared a Fluo-8 stock solution (50 mM of
Fluo-8 AM esters in DMSO) and stored them at –20°C.
For [Ca2+] measurements, we added 200 μL of DMEM
with 10% FBS and 1% antibiotic solution to 10 μL of the
stock solution. Cells were then incubated with the Fluo-8
AM mixture overnight at 37˚C, and then culture media
was replaced with HEPES-buffered DMEM (pH 7.2 - 7.3)
containing 1.8 mm Ca2+ prior to the mechanical stretch
application. Intracellular Ca2+ signals were measured by
a confocal laser scanning microscope (TCS-SP5 confocal
imaging system; Leica) with a 20× objective lens every
15 s. We recorded the fluorescence intensity over 520 nm
excited by 490 nm. The effects of cyclic stretch on rela-
tive [Ca2+]r were estimated from the following equation:

 
2
after afterbefore before
Ca =
rIB IB

 
 (2)
where Iafter and Ibefore are the intensities after and before
the cyclic stretch, and Bafter and Bbefore are the correspond-
ing background autofluorescence values.
2.5. Immunofluorescence Microscopy and
Quantification of Changes in
Diphosphorylated Myosin Regulatory Light
Chain
Cells were fixed with 1% formaldehyde in PBS for 5 min
and permeabilized with 0.5% Triton-X100 in PBS for 5
min. For staining diphosphorylated MRLC (pp-MRLC),
cells were incubated with rabbit anti-pp-MRLC primary
antibody (dilution 1:250; 3671S; Cell Signaling Techno-
logy, MA, USA) diluted in PBS containing 0.5% skim
milk (SM-PBS) for 2 h, then with Alexa594-conjugated
goat anti-rabbit IgG secondary antibody (dilution 1:500;
Invitrogen) diluted in SM-PBS and AlexaFluor-488 phal-
loidin (dilution 1:200; Invitrogen) for 1 h. The stained
cells were observed using a confocal laser scanning
microscope (C1 confocal imaging system; Nikon) with a
60× objective lens. To quantify the changes in pp-MRLC
under mechanical stretch, we measured the relative
intensity of pp-MRLC. The pp-MRLC intensities on an
F-actin-positive area in a cell were summed using ImageJ
software (NIH, MD, USA). The values from 40 - 50 cells
in each condition were averaged and normalized to the
average under static conditions.
3. Results and Discussion
To determine the number of cyclic stretches that are
sufficient to inhibit the cellular elasticity increase after
the single step-like stretch, we examined the cellular
elasticity response by changing the number of cyclic
stretch sets from 0 to 10. Figure 2(A) shows the cellular
elasticity map on the nucleus with 10 sets of cyclic
stretch and a subsequent single step-like stretch. The
elasticities were averaged and their time course was
plotted (Figure 2(B)). The immediate increase after the
cyclic stretch and the decrease after 20 min were similar
to data previously reported [13]. After these responses,

K. TAKEMOTO ET AL.
4
Figure 2. Representative cellular elastic responses under distinct mechanical stimulations. Myoblasts were cultured on a de-
formable chamber and subjected to 2 1 or 10 sets of mechanical cyclic stretch via deformation of the chamber. Changes in cel-
lular cortex elasticity around the nucleus were evaluated by atomic force microscopy (details are described in Materials and
Methods and Figure 1). (A) Temporal changes of cellular elasticity with 10 sets of cyclic stretch and a subsequent single
step-like stretch. 16 × 16 pixels of the elasticity map were measured every 2 - 3 min. Numbers in these images represent the
relative time (min) from the starting point of the cyclic stretch. The cell was subjected to 10 sets of cyclic stretch from 0 to 10
min and subjected to a single step-like stretch at 40 min (see the time course of applied strain in Figure 2(B)). Scale bar = 5 μm;
(B) Time course of the averaged elasticity of the cell subjected to 10 sets of cyclic stretch. Dotted line denotes applied cyclic
stretch (strain on substrate). Cellular elasticity of the 16 × 16 pixels map was averaged and plotted as a function of time. The
error bar denotes the standard error from the 16 × 16 of elasticity data. There is not a clear distinction in cellular elasticity
between before (indicated as an arrow) and after the single step-like stretch (indicated as an arrowhead); (C) Temporal
changes of cellular elasticity after 1 set of cyclic stretch and a subsequent single step-like stretch. Numbers in these images rep
resent the relative time (min) from the starting point of the cyclic stretch. The cell was subjected to 1 set of cyclic stretch from 0
to 1 min and subjected to a single step-like stretch at 31 min (see the time course of applied strain in Figure 2(D)). Scale bar = 5
μm; (D) Time course of the averaged elasticity of the cell subjected to 1 set of mechanical cyclic stretch. Dotted line denotes
applied cyclic stretch (strain on substrate). Cellular elasticity of the 16 × 16 pixels map was averaged and plotted as a function
of time. The error bar denotes the standard error from the 16 × 16 elasticity data. Cellular elasticity after a single step-like
stretch (indicated as an arrowhead) increased significantly compared to before the single step-like stretch (indicated as an arrow).
the elasticity did not increase despite the application of a
single step-like stretch (arrow and arrowhead in Figure
2(B)). In contrast, with 1 set of cyclic stretch, cellular
elasticity increased in response to the subsequent single
step-like stretch (Figures 2(C) and (D)). To characterize
the cellular elasticity response to a single step-like stretch
after a different number of cyclic stretches, relative
elasticity changes were statistically analyzed using data
from 4 independent experiments (Figure 3(B)). Elasticity
with only a single step-like stretch, 1 or 2 sets of cyclic
stretch followed by a single step-like stretch significantly
increased compared to changes in static conditions. On
the other hand, elasticity changes with 7 or 10 sets of
cyclic stretch followed by a single step-like stretch were
not significantly different than changes under static con-
ditions. Furthermore, the elasticity changes with 10 sets
of cyclic stretch were significantly smaller than changes
with 1 set of cyclic stretch. These results indicate that
both a single step-like stretch, 1 and 2 sets of cyclic
stretch with a single step-like stretch increase cellular
elasticity, while 7 and 10 sets of cyclic stretch inhibit the
increase caused by single step-like stretch. In other words,
cellular elasticity responses with a single step-like stretch
are dependent on the number of cyclic stretches that are
applied prior to the single step-like stretch.
Next, we elucidated the residual effect directly on
cellular elasticity after 10 or 2 sets of cyclic stretch.
Cellular elasticity is known to be in a range [14]. We
observed an increase in elasticity after 10 or 2 sets of
cyclic stretch, which then decreased (Figures 2 and 3). If
elasticity reaches a maximum after the cyclic stretches
and the time constants for the decay in elasticity are
different, elasticity changes on the single step-like stretch
following the cyclic stretch would be different until the
residual effect by the cyclic stretch disappears. To
examine our hypothesis, we measured the time constant
of elasticity decay by the 10 or 2 sets of cyclic stretch
(Figure 4). Cellular elasticity immediately increased
after the application of 10 sets of cyclic stretch and
decreased to a constant value for the following 15 min
(i.e., time constant = 15 min). In the case of 2 sets of
cyclic stretch, the profile of the change was similar to that
of the 10 sets cyclic stretch, although the time constant

K. TAKEMOTO ET AL. 5
Figure 3. Effects of the number of cyclic stretches on the
cellular elasticity response. (A) The schematic drawing de-
scribes the pattern of applied strain to myoblasts. We ap-
plied 1, 2, 7, or 10 sets of cyclic stretch to cells and applied a
single step-like stretch 30 min after the cyclic stretch. The
closing time point of the cyclic stretch is defined as the ori-
gin of the time course (0 min); accordingly, the starting time
point of the single step-like stretch is at 30 min. The legends
are as follows: “static”, condition without any mechanical
stretch; “cyclic stretch”, term from the beginning of the cy-
clic stretch application to just before application of the sin-
gle step-like stretch; “cyclic stretch + single step” and “sin-
gle step,” term after the application of the single step-like
stretch; (B) Cellular elasticity responses under distinct cy-
clic stretches were statistically compared. E0 denotes the
cellular elasticity just before the single step-like stretch ap-
plication, and E1 denotes the cellular elasticity at the begin-
ning of the “cyclic stretch + single step” or “single step”
term. Thus, (E1 − E0)/E0 represents the cellular elasticity
change by the single step-like stretch. The time interval
between E0 and E1 is 9 min. Each data point represents the
mean ± standard error from ten experiments. *Denotes sig-
nificant differences from the “static” state (p < 0.01).
was 25 min. These results imply that there may be a
residual effect on elasticity during the 25 min after the
application of 10 or 2 sets of cyclic stretch. In other words,
30 min is sufficient to eliminate the residual effect on
elasticity by the cyclic stretches. Taken together, our
finding that 10 or 2 sets of cyclic stretch differently affect
the cellular elasticity response induced by the single
step-like stretch is valid because we used 30 min intervals
between the end of the cyclic stretch and the beginning of
the single step-like stretch (Figures 2(B) and (D)).
Figure 4. Confirmation of the quasi-stable state of cellular
elasticity after the cyclic stretch. To elucidate the residual
effect on cellular elasticity caused by the cyclic stretch, the
time course of cellular elasticity changes after 2 or 10 sets of
cyclic stretch was measured. (A) Schematic drawing indi-
cates the pattern of applied strain to cells. The time point at
the end of the cyclic stretch application was defined as the
origin (i.e., 0 min) of the following time course data; (B)
Time course of normalized cellular elasticity after 10 sets of
cyclic stretch. Each elasticity data point was normalized by
the division of the elasticity just before application of the
cyclic stretch. Error bars were calculated as a standard er-
ror from 6 independent experiments. Cellular elasticity im-
mediately increased after 10 sets of cyclic stretch and then
decreased to a quasi-state during the following 20 min; (C)
Time course of normalized cellular elasticity after 2 sets of
cyclic stretch. Normalization of cellular elasticity was per-
formed in the same manner described above. Error bars
were calculated as a standard error from six independent
experiments. Time course of cellular elasticity response to 2
sets of cyclic stretch was similar to that of 10 sets. Hence, the
direct effect on cellular elasticity by mechanical pulses would
be eliminated by 30 min intervals (the end of the term is
indicated as dashed lines in Figures 4(B) and (C)).
To survey molecular mechanisms as to how cyclic
stretch affect the cellular elasticity response by a single
step-like stretch, we focused on phosphorylation of MRLC,
which are thought to contribute to cellular elasticity [15].
First, we examined changes in pp-MRLC with a single
step-like stretch and/or with cyclic stretches. Previous

K. TAKEMOTO ET AL.
6
reports have shown that pp-MRLC increases intracellular
contractile force and cellular elasticity [15], and diphos-
phorylation can be induced by a single step-like stretch
[16]. To quantify changes in pp-MRLC, we stained cells
with anti-pp-MRLC and measured the intensity (Figure
5). The pp-MRLC intensity with only a single step-like
stretch was significantly larger than the intensity under
static conditions. A similar change was seen after the
single step-like stretch following 2 sets of cyclic stretch.
However, the intensity after a single step like stretch fol-
lowing 10 sets of cyclic stretch did not show clear differ-
ences compared to the static conditions. These results
correlate with the cellular elasticity data (Figures 2 and
3). We also verified that there was no residual pp-MRLC
induced by the 2 or 10 sets of cyclic stretch prior to the
single step-like stretch. Taken together, these results
suggest that the cellular elasticity response under mecha-
nical stimulation originates from the pp-MRLC change.
Next, we investigated the contribution of cellular [Ca2+]
to the diphosphorylation of MRLC because many groups
have reported that [Ca2+] increases with cyclic stretch [8],
and MRLC phosphorylation is induced by activation of
myosin light-chain kinase (ML- CK) in a Ca2+-dependent
manner [17]. Our preliminary data showed that [Ca2+]
did not change after1 or 10 sets of cyclic stretch and a
subsequent single step-like stretch (Figure 6). This result
implies that Ca2+ does not contribute to regulation of
elasticity changes.
Figure 5. Changes in diphosphorylation of MRLC with 2 or 10 sets of cyclic stretch. Myoblasts were stained with anti-pp-
MRLC and phalloidin after application of mechanical stimulations. (A) Schematic drawing indicates the pattern of the applied
strains. Cells were fixed and stained at the following 3 time points: “static”, term without mechanical stimulations; “cyclic
stretch”, time point 29 min after cyclic stretch; “cyclic stretch + single step”, time point 38 min after the single step-like stretch;
(B)-(C) A set of fluorescent images after 10 sets (B) and 2 sets (C) of cyclic stretch. Scale bar = 50 μm; (D) Changes in
pp-MRLC intensity were statistically compared. Each data point represents the mean ± standard errors from 40 - 50 cells from
2 independent experiments. *Indicates significant differences (p < 0.01).

K. TAKEMOTO ET AL. 7
Figure 6. Changes in intracellular Ca2+ concentration under
1 or 10 sets of cyclic stretch. Changes in cytosolic Ca2+ con-
centration ([Ca2+]) in response to 1 or 10 sets of cyclic stretch
were measured using a Ca2+ indicator. (A) Temporal
changes in intracellular [Ca2+] under 1 set of cyclic stretch.
Numbers in these images represent the relative time (sec)
from the starting point of the cyclic stretch. The cell was
subjected to 10 sets of cyclic stretch from 0 to 60 sec and
subjected to a single step-like stretch at 1860 sec (see the
time course of applied strain in Figure 6(C)). The scale bar
indicates 100 μm; (B) Definition of cells perfomed time-lapse
observation of [Ca2+] under 1 set of cyclic stretch by num-
bers; (C) Time course of [Ca2+] intensity of cells under 1 set
of cyclic stretch. [Ca2+] did not change under 1 set of cyclic
stretch and a subsequent single step-like stretch; (D) Tempo-
ral changes in intracellular [Ca2+] under 10 sets of cyclic
stretch. Numbers in these images represent the relative time
(sec) from the starting point of the cyclic stretch. The cell
was subjected to 10 sets of cyclic stretch from 0 to 600 sec
and subjected to a single step-like stretch at 2400 sec (see the
time course of applied strain in Figure 6(F)). The scale bar
indicates 100 μm; (E) Definition of cells perfomed time-lapse
observation of [Ca2+] under 10 sets of cyclic stretch by num-
bers; (F) Time course of [Ca2+] intensity of cells under 10
sets of cyclic stretch. [Ca2+] also did not change under 10 sets
of cyclic stretch and a subsequent single step-like stretch.
A variety of mechanical stimuli have the potential to
regulate cellular physiology. With respect to cyclic stretch,
changing the magnitude or frequency rearranges cell
orientation [8,18] and cytoskeletal networks [19], and up-
or down-regulates cell growth rate and protein synthesis
[20]. Moreover, application of a large number (~1000) of
cyclic stretches also affects cell growth [21] and cell
orientation. In the present study, we showed that ap-
plication of even a small number (1 - 10) of cyclic
stretches differently changed the cellular elasticity re-
sponse (Figures 2 and 3) and molecular activity (Figure
5), although cell alignment did not change (Figure 7).
Thus, there may be a relationship between the predicted
cellular physiological response and parameters of the
mechanical stimulations. To regulate cellular functions
more effectively, the parameters of mechanical stimula-
tion should be considered.
To discuss the mechanism as to how cells change their
elasticity after mechanical stretches, changes in the cyto-
skeleton and activity of its regulators should be con-
siderd. Many types of cells are reported to sense me-
chanical stimulation, which are transformed into intra-
cellular signals [22,23]. With respect to regulators of cel-
lular mechanical properties, Ca2+ influx, integrin com-
plex activation, and RhoA activation are known to act as
first messengers [24]. As such, F-actin polymerization,
cellular elasticity change, and focal adhesion (FA) crea-
tion are induced [13,25]. In these processes, MRLC
phosphorylation is thought to be a principal mediator (An
and Hai 2000). Our results showed that MRLC phos-
phorylation and dephosphorylation accompanied cellular
elasticity changes (Figures 2 and 3) and FA area (Figure
8). Taken together, these data indicate cellular elasticity
changes resulting from cyclic and step-like stretches are
likely mediated by MRLC. Furthermore, we hypothesize
that MRLC changes are indendent of Ca2+ because the
Ca2+ time-course was diffent from time-course of pp-
MRLC and elasticity (Figure 6), which is supported by a
previous report indicatingthat influx of Ca2+ by cyclic
stretch occurs in second time scale [8]. To investigate
our hypothesis, experiments using expression of a non-
Figure 7. Effects of 2 or 10 sets of cyclic stretch on the cell
morphology. Cyclic stretch (over 1 Hz of frequency, and
10% of amplitude) induced cell orientation perpendicular to
the stretch axis. In this study, we examined effects of 2 and
10 sets of cyclic stretch on the cell morphology. The closing
time point of cyclic stretch is defined as the origin of the time
course (0 min). The “static” is a condition whithout any me-
chanical stretch. The direction of stretching cells is shown by
a double-headed arrow. The scale bar indicates 100 μm. Cell
orientation did not change after the application of 2 and 10
sets of cyclic stretch.

K. TAKEMOTO ET AL.
8
Figure 8. Changes in focal adhesion (FA) area under 2 or 10 sets of cyclic stretch. Myoblasts were cultured on elastic chamber
and stained with anti-vinculin and phalloidin at a suitable timing after the application of mechanical stimulations. (A) Sche-
matic drawing indicates the pattern of the applied strains. Cells were fixed and stained at the following 3 timings: “static”,
term without any mechanical stimuli; “cyclic stretch”, time point 29 min after cyclic stretch; “cyclic stretch + single step”, time
point 38 min after the single step-like stretch; (B)-(C) A set of fluorescent images under the condition of 10 sets (B) and 2 sets
(C) of cyclic stretch. The scale bar indicates 50 μm; (D) Changes in FA area were statistically compared. To quantify the
changes in FA areas under mechanical stretch, we used following methods. We used an established method [26]. Briefly, whole
areas of a cell were measured by the cell contour which was traced using F-actin image and ImageJ software (NIH, MD, USA),
and the vinculin positive areas in the cell were summed. The degree of FA areas in a cell is defined as following equation:
vin
F-actin
FA Related Area RatioA
A

where Avin is the total areas of vinculin within a cell and AF-acti n is the total areas of F-actin. Each data represent mean ± stan-
dard errors from 40 - 50 cells for two independent experiments. *indicates significant differences (p < 0.01) and **also indicates
significant differences (p < 0.05) [26].
phosphorylatable MRLC [27] and RhoA inhibitors [28]
are necessary.
Although it is still unclear how the different elasticity
responses to single step-like stretch after 2 or 10 sets of
cyclic stretch are induced, we predict that it may be due
to a balance between activation and deactivation of
MRLC. We observed that a single step-like stretch
increased elasticity, and 2 sets of cyclic stretch did not
alter the elasticity response; however, 10 sets of cyclic
stretch inhibited the increase caused by the single stepike

K. TAKEMOTO ET AL. 9
stretch (Figures 2 and 3). As these elasticity changes
were accompanied with di- and de-phosphorylation of
MRLC (Figure 5), proteins that activate (kinases) or
inhibit (phosphatases) the activity of MRLC may be
involved in the elasticity response depending on the
number of cyclic stretches. We hypothesize the following:
net MRLC photion is determined by a balance of activity
between MRLC kinases and phosphatases; differences in
reaction rate between kinases and phosphatases respond-
ing to the cyclic stretch; homeostatic mechanism in kinase
and phosphatase activity. To investigate these hypothezes,
future work will include measuring the time course of
both kinase and phosphatase activity after application of the
cyclic stretches.
4. Acknowledgements
This work is supported by Scientific Research (C) (2157-
0158) to H. H., by Exploratory Research (21654058) to
K. K., and by Grant-in-Aid for Young Scientists (B)
(23770167) to T. M. from the Japan Society for the Pro-
motion of Science.
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