Am J Physiol Cell Physiol 302: C1741–C1750, 2012.
First published March 28, 2012; doi:10.1152/ajpcell.00068.2012.
Calcium influx through a possible coupling of cation channels impacts
skeletal muscle satellite cell activation in response to mechanical stretch
Minako Hara,1* Kuniko Tabata,1* Takahiro Suzuki,1 Mai-Khoi Q. Do,1 Wataru Mizunoya,1
Mako Nakamura,2 Shotaro Nishimura,1 Shoji Tabata,1 Yoshihide Ikeuchi,1 Kenji Sunagawa,3
Judy E. Anderson,4 Ronald E. Allen,5 and Ryuichi Tatsumi1
1
Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Hakozaki,
Fukuoka, Japan; 2Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, Japan; 3Department of Cardiovascular
Medicine, Graduate School of Medicine, Kyushu University, Maedashi, Fukuoka, Japan; 4Department of Biological Sciences,
Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada; and 5Muscle Biology Group, Department of
Animal Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, Arizona
Submitted 5 March 2012; accepted in final form 26 March 2012
calcium ion influx; mechanosensitive channel; muscle regeneration;
satellite cells; stretch-activation; voltage-gated channel
SATELLITE CELLS, a population of resident myogenic stem cells
positioned between the basal lamina and the sarcolemma of
* M. Hara and K. Tabata contributed equally to this work.
Address for reprint requests and other correspondence: R. Tatsumi, Dept. of
Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu Univ., Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan (e-mail:
[email protected]).
http://www.ajpcell.org
postnatal skeletal muscle fibers, are normally found in a mitotically and metabolically quiescent (or near-dormant) protracted G1 state (also referred to as G0) in adult muscles. When
muscle is injured, overused, or mechanically stretched, these
cells are activated to enter the cell cycle, proliferate to produce
large numbers of myoblast progeny, differentiate, and fuse
with existing muscle fibers or form new fibers. Through this
process, many myonuclei are accumulated in the tissue and are
responsible for increasing protein synthesis and providing the
potential to enhance muscle growth (hypertrophy and hyperplasia) and regeneration. Consequently, satellite cell activation
is an initial and crucial step in the process of postnatal myogenesis (reviewed in Refs. 12, 28, 45, 50, 66) and may be
triggered by mechanical perturbation of satellite cells and
muscle fibers. However, the detailed mechanism is not fully
elucidated.
Of all growth factors studied thus far, including fibroblast
growth factors (FGFs), insulin-like growth factor-1 (IGF-1),
platelet-derived growth factor BB (PDGF-BB), transforming
growth factor-␤s (TGF-␤1 and -2), and epidermal growth
factor (EGF), hepatocyte growth factor (HGF) is the only
mitogen with an established ability to stimulate quiescent
satellite cells to enter the cell cycle early in primary culture
assay and in vivo (1, 58; reviewed in Refs. 15, 38, 72), even
though Nagata et al. (40) first demonstrated that sphingosine1-phosphate (S1P), a bioactive sphingolipid metabolite, induces satellite cells to cycle and very recently Sassoli et al. (48)
showed that S1P promotes satellite cell renewal and differentiation in damaged muscle. HGF is a heparin-binding protein
localized in the extracellular domain of uninjured skeletal
muscle fibers by possible association with glycosaminoglycan
chains of proteoglycans, and its predominant form is the active
disulfide-linked heterodimer of a 60-kDa ␣-chain and a 30-kDa
␤-chain (62). The intracellular signaling receptor for HGF is
the c-met proto-oncogene; its message and protein have been
found in quiescent and activated satellite cells (1, 19, 58). Thus
release of HGF from its sequestration in the matrix and
subsequent presentation to the receptor c-met may be a critical
aspect of the activation of quiescent satellite cells. Recently,
Wozniak and Anderson (73) provided a novel insight that HGF
released from the matrix may induce c-met RNA expression as
an immediate-early gene within 30 min in response to muscle
fiber stretch and thus enhance HGF-c-met signaling in the
satellite cell activation process.
0363-6143/12 Copyright © 2012 the American Physiological Society
C1741
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Hara M, Tabata K, Suzuki T, Do MQ, Mizunoya W, Nakamura M,
Nishimura S, Tabata S, Ikeuchi Y, Sunagawa K, Anderson JE, Allen
RE, Tatsumi R. Calcium influx through a possible coupling of cation
channels impacts skeletal muscle satellite cell activation in response to
mechanical stretch. Am J Physiol Cell Physiol 302: C1741–C1750, 2012.
First published March 28, 2012; doi:10.1152/ajpcell.00068.2012.—When
skeletal muscle is stretched or injured, satellite cells, resident myogenic
stem cells positioned beneath the basal lamina of mature muscle fibers,
are activated to enter the cell cycle. This signaling pathway is a cascade
of events including calcium-calmodulin formation, nitric oxide (NO)
radical production by NO synthase, matrix metalloproteinase activation,
release of hepatocyte growth factor (HGF) from the extracellular matrix,
and presentation of HGF to the receptor c-met, as demonstrated by assays
of primary cultures and in vivo experiments. Here, we add evidence that
two ion channels, the mechanosensitive cation channel (MS channel) and
the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic
stretch in satellite cell cultures. When applied to 1-h stretch cultures with
individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride,
Gd), satellite cell activation and upstream HGF release were abolished, as
revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent
with a maximum at 0.1 ␮M (GsMTx-4), 10 ␮M (nifedipine), or 100 ␮M
(Gd) and canceled by addition of HGF to the culture media; a potent inhibitor
for transient-type VGC channels (NNC55–0396, 100 ␮M) did not show any
significant inhibitory effect. The stretch response was also abolished when
calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the
significance of extracellular free calcium ions in our present activation model.
Finally, cation/calcium channel dependencies were further documented by
calcium-imaging analyses on stretched cells; results clearly demonstrated that
calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA.
Therefore, these results provide an additional insight that calcium ions may
flow in through L-VGC channels by possible coupling with adjacent MS
channel gating that promotes the local depolarization of cell membranes to
initiate the satellite cell activation cascade.
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CALCIUM INFLUX INTO SATELLITE CELLS
verse tubules act as a voltage sensor in excitation-contraction
coupling (44, 56, 57, 70; reviewed by Refs. 14, 21).
In the present paper, we examined the effect of a calcium
chelator, EGTA, and selective inhibitors for MS channels
(GsMTx-4) and VGC channels (nifedipine and NNC55– 0396)
with and without cyclic stretch of satellite cell primary cultures
on HGF release from the matrix and satellite cell activation.
We provide evidence that extracellular calcium-ion influx
through a possible coupling of MS and L-VGC channels may
be responsible for the mechanosensing machinery of quiescent
satellite cells.
MATERIALS AND METHODS
Materials. Dulbecco’s modified Eagle’s medium (DMEM, lowglucose type, 31600 – 034), normal horse serum (HS, 16050 –122), antibiotic-antimycotic (15240 – 062), and gentamicin (15710 – 064) were purchased from Invitrogen (Grand Island, NY). Poly-L-lysine (P9155),
bovine plasma fibronectin (F1141), protease type XIV (P5147), and
5-bromo-2=-deoxyuridine (BrdU, B5002) were obtained from Sigma (St.
Louis, MO). Recombinant mouse HGF (2207-HG) was purchased from
R&D Systems (Minneapolis, MN). Cation-channel inhibitors, nifedipine
[141– 05783, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], gadolinium chloride (078 – 02661) and
NNC55– 0396 (2268, (1S,2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2naphthalenyl cyclopropanecarboxylate dihydrochloride) were obtained
from Wako Pure Chemical Industries (Osaka, Japan), and GsMTx-4
(4393-s, synthetic peptide of the spider venom toxin) was from Peptide
Institute (Osaka, Japan).
Fluo 3-AM {F026, 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo9-xanthenyl) phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N=,N=tetraacetic acid, pentaacetoxymethyl ester} was obtained from Dojindo Laboratories (Kumamoto, Japan). EGTA [15214, O,O=-bis(2aminoethyl)ethyleneglycol- N,N,N=,N=-tetraacetic acid] was from
Nacalai Tesque (Kyoto, Japan). Calcium ionophore A23187 (21186)
and nonionic copolymer surfactant Pluronic F-127 (P2443) were
purchased from Sigma.
The following materials were obtained for protein expression
analysis: D3 mouse monoclonal anti-desmin and G3G4 mouse monoclonal anti-BrdU antibodies from the Developmental Studies Hybridoma Bank (Iowa City, IA); goat polyclonal anti-human HGF antibody
(AB-294-NA) and goat polyclonal anti-c-met antibodies (AF527)
from R&D Systems; AC-15 mouse monoclonal anti-␤-actin antibody
(ab6276) from abcom (Cambridge, MA); horseradish peroxidase
(HRP)-conjugated AffiniPure donkey Anti-goat IgG (705– 036-147)
from Jackson ImmunoResearch Laboratories (West Grove, PA); affinity-purified biotinylated horse anti-mouse IgG (BA-2000) and the
HRP-labeled avidin kit (PK-6100) from Vector Laboratories (Burlingame, CA); affinity-purified HRP-conjugated goat anti-mouse IgG
(A-4416) and 3,3=-diaminobenzidine (DAB, D5637) from Sigma;
HRP-labeled rabbit anti-goat IgG [414331, Histofine Simple Stain Rat
MAX-PO (G)] and normal rabbit serum (426051) from Nichirei
BioScience (Tokyo, Japan); Amersham enhanced chemiluminescence
(ECL) detection kit (PRN2106), nitrocellulose membrane (Hybond
ECL, RPN2020D), and Hyperfilm ECL (28 –9068) from GE healthcare (Little Chalfont, UK); MagicMark XP molecular weight standards (LC5602) from Invitrogen; and CanGetSignal immunoreaction
solutions (NKB-101) from Toyobo (Osaka, Japan). For the mRNA
expression analysis, an RNeasy Micro kit (74004) and QIAshredder
homogenizer spin column (79654) from Qiagen (Hilden, Germany),
SuperScript III reverse transcriptase (18080 – 044) from Invitrogen,
Oligo(dT) primer (H09876) from Roche (Mannheim, Germany), ExTaq DNA polymerase (RR001A) from Takara Bio (Otsu, Japan),
agarose-LE (01157) from Nacalai Tesque (Kyoto, Japan), and Gel
Red (41000) from Biotium (Hayward, CA) were additionally used.
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In a series of previous reports, we employed a FlexerCell
system (Flexcell International, Hillsborough, NC) to apply
cyclic stretch to isolated rat satellite cells and found that
mechanical stretch triggers satellite cell activation by rapidly
releasing HGF from its tethering in the extracellular matrix and
its subsequent presentation to c-met (59, 60, 63, 65; reviewed
in Ref. 66). This phenomenon is relevant to satellite cells in
living muscle as revealed by an in vivo muscle-stretch model
(64). The most important observation from these previous
experiments was that the release of HGF was completely
dependent on nitric oxide (NO) radical synthesis by NO synthases (NOS) from L-arginine of the substrate (60, 61). Anderson (4) first pointed to production of NO as a key signal
responsible for satellite cell hypertrophy and detachment from
the adjacent fiber following crush injury; those results were
later verified in in vitro cultures of isolated muscle fibers and
their associated satellite cells (5, 6, 71–73; reviewed in Refs. 7,
8). In our subsequent studies, Yamada et al. (75, 76) demonstrated
that matrix metalloproteinases (MMPs), a large family of zincdependent endopeptidases that collectively degrade one or more
extracellular matrix constituents, specifically MMP2, mediate
HGF release, possibly by shedding proteoglycan-core proteins in
response to the NO radical (reviewed in Refs. 66, 68).
In an effort to understand the events upstream from NOradical production, recent studies showed that NO-radical/
MMP-mediated HGF release and the resulting activation of
satellite cells are blocked if calmodulin activity is abolished by
the specific inhibitors, calmidazolium, W-13, and W-12; the
same report showed that the activation cascade can be turned
on when calcium ionophores A23187 or ionomycin are simply
added to unstretched control cultures (67). Therefore, the influx
of calcium ions and their binding to calmodulin are thought to
be involved in the segment of the activation pathway between
sensing the mechanical stimulus and synthesis of the NO
radicals, consistent with the observation that the calciumcalmodulin complex associates with constitutive NOS (cNOS:
i.e., neuronal and endothelial NOS proteins) to activate NOS
enzyme activity, as shown in neuronal and endothelial cells
(43, 69). However, it is still unclear how quiescent satellite
cells sense mechanical stimuli for transduction into an elevation of the intracellular calcium-ion concentration at the most
upstream step of the satellite cell activation cascade.
Experiments in this study were designed to test the hypothesis that mechanosensitive cation channels (MS channels) are
involved in the mechanotransduction system. MS channels are
mechanosensing machinery proteins and are widely distributed
from single-celled bacteria to animal and plant cells. A variety
of experimental approaches revealed that MS channels respond
to a variety of mechanical stimuli, including shear stress,
gravity, osmotic pressure, and stretch, by opening nanoscale
protein pores that are selectively permeable to cations (mainly
calcium and sodium) entering into the cytosol down their steep
electrochemical gradients (22, 42; reviewed in Refs. 3, 9, 27,
31, 33). Therefore, MS-channel gating may transduce physical
stimuli into electrical signals by generating localized depolarization of the cell membranes (through changes in membrane
voltage). This depolarization potentially enables the activation
of voltage-gated calcium channels (VGC channels). Indeed,
calcium signaling has a central role in regulating activity of
many cell types, including skeletal muscle fibers in which the
long-lasting-type of VGC channels (L-VGC channels) in trans-
CALCIUM INFLUX INTO SATELLITE CELLS
Fig. 1. A: significance of extracellular calcium ions in stretch-activation of
satellite cells in vitro. B: satellite cells from adult rat skeletal muscle were
stretched in culture (black bars) for 1 h beginning at 24-h postplating in
FlexerCell FX-2000 System [25% stretch at 12-s intervals, as originally
optimized in Tatsumi et al. (59; modified with permission from Elsevier) and
shown in A again] in the presence (bar d) or absence (bar c) of 1.8 mM EGTA
in DMEM-10% normal horse serum (pH 7.2), followed by an assay to detect
activation using bromodeoxyuridine (BrdU) at 48-h postplating. This experiment also included the following control cultures: bar a, unstretched culture;
bar b, 2-h stretch culture (equivalent to our regular period of stretch, originally
described in Ref. 59); bar e, stretched culture receiving 1.8 mM EGTA for
24 – 48 h postplating; bar f, positive control culture supplemented with 2.5
ng/ml recombinant mouse hepatocyte growth factor (HGF) for 24 – 48 h; bar
g, HGF culture receiving 1.8 mM EGTA for 24 – 48 h; bars h and i, control
cultures with 1.8 mM EGTA for 24 –25 h and 24 – 48 h, respectively. Bars
depict means and SE for 4 cultures per treatment. **Treatment mean was
significantly different from the mean of positive control cultures (bar f) at P ⬍
0.01. NS, no significant difference (P ⬎ 0.05) in the activation index.
SDS-sample buffer (2% SDS, 5% ␤-mercaptoethanol, 10% glycerol,
0.01% bromophenol blue, and 62.5 mM Tris·HCl buffer, pH 6.8), and
stored at ⫺80°C until use.
In vitro activation assay. Cultures were pulse-labeled with 10 ␮M
BrdU in DMEM-10% HS for the final 2 h from 46 to 48 h postplating,
followed by immunocytochemistry for detection of BrdU using a
G3G4 anti-BrdU monoclonal antibody [1:100 dilution in 0.1% bovine
serum albumin (BSA) in phosphate-buffered saline (PBS)] and a
HRP-conjugated anti-mouse IgG antibody (1:500 dilution) according
to Tatsumi et al. (58). The percentage of BrdU-labeled cells was used
as an indicator of activation and entry into the cell cycle.
Immunoblotting and ECL. Mouse recombinant HGF and conditioned media from 1-h stretch cultures with or without a cationchannel inhibitor or EGTA were applied to 10% polyacrylamide gels
for electrophoresis under reducing conditions (35) and transferred to
nitrocellulose membranes (58). The blots were blocked with 10%
powdered milk in 0.1% polyethylene sorbitan monolaurate (Tween
20)-Tris buffered saline (TTBS) prior to incubation with goat polyclonal anti-HGF antibody (1:500 dilution in 1% powdered milk-TTBS
additionally containing 0.05% sodium azide) overnight, and subse-
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Animal care and use. All experiments involving animals were
conducted according to institutional guidelines and with the approval
of Kyushu University Institutional Review Board, in collaboration
with investigators at the University of Arizona and the University of
Manitoba.
Satellite cell isolation and primary culture. Satellite cells were
isolated from muscle in the hindlimb and back of 9-mo-old male
Sprague-Dawley rats according to Allen et al. (2) with a slight
modification (65). Briefly, muscle from the upper hindlimb and back
were excised, trimmed of fat and connective tissue, minced with
scissors, and digested for 1 h at 37°C with 1.25 mg/ml protease type
XIV. Cells were separated from muscle-fiber fragments and tissue
debris by differential centrifugation and filtration through nylon cell
strainers (100-␮m and 40-␮m mesh size) prior to a final centrifugation
step at 1,500 g for 3 min, and then plated on poly-L-lysine and
fibronectin-coated dishes in DMEM containing 10% horse serum
(HS), 1% antibiotic-antimycotic mixture, and 0.5% gentamicin
(DMEM-10% HS, pH 7.2). Cultures were maintained in a humidified
atmosphere of 5% CO2 at 37°C. In addition, companion satellite cell
cultures, prepared at the same time, were immunostained for the
presence of desmin at 30 h after plating, using a D3 monoclonal
anti-desmin antibody, biotinylated anti-mouse IgG antibody, and
HRP-labeled avidin, to determine the percentage of myogenic cells
present; cultures with less than 95% DAB-positive cells were not used
for experiments.
Satellite cell cultures on Bioflex-Amino silicone-bottom plates
(BF-3001A, Flexcell International) were treated with one of the
cation-channel inhibitors (GsMTx-4, nifedipine, gadolinium chloride,
or NNC55– 0396) or a highly selective calcium chelator EGTA (1.8
mM) for 30 min in DMEM-10% HS. GsMTx-4, nifedipine, and NNC55–
0396 are highly selective peptide/chemical-inhibitors, and are therefore
intensely useful for the study of cellular excitability and intracellular
calcium-ion signaling and for characterizing effects of therapeutically
beneficial drugs. Although detailed inhibitory mechanisms of these
compounds still remain unclear, GsMTx-4 selectively inhibits MSchannel activity when applied to the extracellular face of the cell
membrane. It does this by increasing the membrane tension required
for activation, suggesting that GsMTx-4 acts as a gating modifier (54).
By comparison, nifedipine binds directly to inactive L-VGC channels,
which stabilizes their inactive conformation. NNC55– 0396 may bind
to transmembrane or intracellular domains of the channels to exert
their inhibitory effects specifically on transient-type VGC channels
(T-VGC channels) (29; reviewed in Ref. 53). Cells were then subjected to a cyclic-stretch environment [optimized previously at 25%
stretch at 12-s intervals (59); see Fig. 1A for more detail] in a
vacuum-operated cyclic strain-providing instrument FlexerCell FX2000 System (Flexcell International) for 1 h starting at 24-h postplating, as described with each experiment in RESULTS. In the present
study, the stretch periods were all set to 1 h [half the duration from our
original report by Tatsumi et al. (59)] to fit calcium-imaging experiments that compare the calcium-influx activity between stretch cultures (in the presence or absence of cation-channel inhibitors or
EGTA) and unstretched control cultures in the same set of experiments within a limited time period. The stretch-activation activity of
satellite cells after 1 h was not significantly different from that in
2-h-stretch cultures, as revealed by the BrdU-incorporation assay
described below (see Fig. 1B, compare bars b and c)
Conditioned medium. In experiments in which samples of conditioned media from stretch cultures were assayed by Western blotting
for the presence of HGF protein released from the cells, cultures were
washed with serum-free DMEM at 24-h postplating, and treatments
were imposed for as short as 1 h in the presence of serum-free DMEM
(pH 7.2). The culture medium was supplemented with one of the
following for additional treatments: the cation-channel inhibitor
GsMTx-4 (0.1 ␮M), nifedipine (10 ␮M), or EGTA (1.8 mM) 30 min
prior to the addition of cyclic stretch. Conditioned medium from each
treatment was collected, centrifuged for 4 min at 1,300 g, treated with
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CALCIUM INFLUX INTO SATELLITE CELLS
or three times to verify the reproducibility of results, and, in most
cases, one rat was used for each experiment.
RESULTS
It is now clear that, in attempting to construct a cascade
pathway that translates mechanical perturbation of quiescent
satellite cells or muscle fibers into an activation signal, calcium-calmodulin formation is required for NOS activation that
produces the NO radical from L-arginine and which subsequently releases HGF from extracellular tethering (for review
see Ref. 66). Therefore, the purpose of this study was to clarify
a mechanism that responds to mechanical perturbation of
stretch by increasing intracellular calcium-ion concentrations
in satellite cell cultures.
The first experiment was designed to examine the significance of extracellular calcium-ion influx in our stretch-activation model of satellite cells in vitro (Fig. 1). Satellite cells were
prepared from adult rat skeletal muscles and applied to cyclicstretch cultures (25% stretch at 12-s intervals as shown in Fig.
1A) for 1 h beginning at 24-h postplating in DMEM-10% HS
(pH 7.2) with or without 1.8 mM EGTA (calcium chelator that
does not enter into the cytosol), followed by the BrdU-incorporation assay at 48-h postplating (Fig. 1B). This concentration
of EGTA decreases free calcium ion concentrations in the
culture media (1.8 mM) to about 10 ␮M and without diminishing cell viability, even through the last 24-h period, as
monitored by effects on cell density and BrdU incorporation at
the 48-h time point of the assay (bar e and bar i); therefore
EGTA was applied to the pilot experiments to determine the
dependence of activation on the level of extracellular calcium.
Results clearly showed that EGTA treatment abolished stretchactivation (bar d) down to a level equivalent to that in unstretched control cultures without (bar a) and with 1.8 mM
EGTA (bar h). The 1-h cyclic-stretch cultures (bar c) showed
activating activity comparable to cultures receiving 2.5 ng/ml
HGF for 24 h from 24-h postplating in the absence or presence
of 1.8 mM EGTA (bars f and g, respectively), thereby serving
as important controls for other treatment groups in this assay.
Together these results provide evidence that extracellular calcium ions are essential in stretch-induced activation of satellite
cells in primary culture.
This issue was further examined by studying the effect of
cation-channel inhibitors on stretch-activation of satellite cells
(Fig. 2A). In this experiment, stretch was applied to satellite
cell cultures for 1 h beginning 24-h postplating in the presence
of an increasing concentration of one of the selective inhibitors
for MS channels, L-VGC channels and T-type VGC channels
(0.01–1 ␮M GsMTx-4, 0.1–10 ␮M nifedipine, and 1–100 ␮M
NNC55– 0396, respectively) or a less specific classic inhibitor
for MS channels (0.1–100 ␮M gadolinium chloride), and then
subsequently assayed for satellite cell activation at 48-h postplating as described previously. The T-VGC channel inhibitor
NNC55– 0396 (open square) did not show a significant effect
on activation in the range examined here or in another report
(29, 55). Exposures to one of the other three cation-channel
inhibitors all abolished stretch-stimulated activation in a dosedependent manner. GsMTx-4 (closed circle) was the most
powerful of the three inhibitors, with blocking at an optimal
concentration of 0.1 ␮M vs. 10 ␮M for nifedipine (closed
square) and 100 ␮M for Gd (open circle). None of these
cation-channel inhibitors diminished cell viability, again as
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quently treated with HRP-labeled donkey anti-goat IgG antibody at
1:10,000 dilution for 1 h, followed by ECL detection on Amersham
Hyperfilm ECL X-ray film. Internal ␤-actin in cell lysates was
visualized with mouse monoclonal anti-␤-actin antibody (1:1,000
dilution in CanGetSignal solution 1, overnight) and with biotinylated
horse anti-mouse IgG (1:5,000 dilution in CanGetSignal solution 2,
for 1 h) and HRP-labeled avidin (1:500 dilution in TTBS, for 30 min).
Calcium imaging. Satellite cell cultures on a silicon membrane
(11– 004-006, Flexcell International) were loaded with 5 ␮M fluo
3-AM in 0.2% Pluronic F-127 for 1 h in the dark at 24-h postplating,
and then rinsed with PBS and left to equilibrate in DMEM-10% HS
(pH 7.2) for 60 min at room temperature. Cultures were then exposed
to two cycles of stretch in the presence of 1.8 mM EGTA or a
cation-channel inhibitor (0.1 ␮M GsMTx-4 or 10 ␮M nifedipine, each
added another 30 min prior to the stretch stimulation) under the
StageFlexer Jr. system equipped with a 18.5-mm cylindrical loading
post; positive-control cells received calcium ionophore A23187 (3
␮M) in the media, according to Tatsumi et al. (67). Calcium images
were monitored at intervals of 3 s under a Nikon ECLIPSE 80i
fluorescence microscope system (Tokyo, Japan) with an excitation
wavelength of 488 nm and a maximum emission wavelength of 526
nm. Fluorescence intensity was assigned from blue to red in colorcoded images by the installed Nikon computer software. After imaging, cells were fixed for 10 min in cold methanol containing 0.1%
H2O2 and blocked with 5% normal rabbit serum containing 0.6%
H2O2 in PBS for 20 min. Blocking was followed by immunostaining
for the presence of c-met using R&D Systems goat polyclonal primary
antibody (1:100 dilution in PBS containing 5% normal rabbit serum)
and Histofine Simple Stain HRP-labeled anti-goat IgG secondary
antibody to determine if the calcium-imaged cells were myogenic.
Reverse transcription-polymerase chain reaction (RT-PCR). Total
RNA was purified from cultured satellite cells using an RNeasy Micro
kit according to the manufacturer’s recommendation. cDNA was
synthesized from total RNA by a reverse-transcriptase SuperScript III
using oligo(dT) primer. PCR was performed using ExTaq DNA
polymerase on the mRNA expression of ␣1S, ␣1C, ␣1D, and ␣1F
subunits of L-VGC channels (accession no. NM_053873, NM_
012517, NM_017298, and NM_053701, respectively) and transient
receptor potential canonical channel (TRPC)-1 and -6 (accession no.
NM_053558 and NM_053559, respectively). The intron-spanning
primer sets designed by Primer3 software were as follows: for rat ␣1S
subunit, forward 5=-ACATCTTTGTGGGCTTCGTC-3=, reverse 5=TCCGACTGGTTGTAATGCTG-3=, annealing temperature 62°C,
amplicon 259 nucleotides (nt); for rat ␣1C subunit, forward 5=CTCGAAGTTGGGAGAACAGC-3=, reverse 5=-GACGAAACCCACGAAGATGT-3=, annealing temperature 60°C, amplicon 239 nt; for
rat ␣1D subunit, forward 5=-GCAGCACTATGAGCAATCCA-3=, reverse 5=-CGGAAAAGACGGAAAAAGGT-3=, annealing temperature 60°C, amplicon 249 nt; for rat ␣1F subunit, forward 5=-GCTGCTCAGTAAGGGTGAGG-3=, reverse 5=-GTCCTGAAGAGCCACTTTGC-3=, annealing temperature 62°C, amplicon 154 nt; for rat
TRPC1, forward 5=-CGGCAGAATCATTCACACAC-3=, reverse 5=CCAACCCTTCATACCACAGC-3=, annealing temperature 57°C,
amplicon 215 nt; for rat TRPC6, forward 5=-TGAAATTCCTCGTGGTCCTC-3=, reverse 5=-GAGCTTGGTGCCTTCAAATC-3=, annealing temperature 57°C, amplicon 197 nt. The annealing temperatures
and cycle numbers were optimized so that amplification reactions
were within the linear range. The PCR products were visualized by
1.5% agarose gel electrophoresis with 0.01% Gel Red reagent.
Statistical analysis. ANOVA procedures were employed to analyze
experimental results using the general linear model procedures of
SRISTAT2 for Windows software (Social Survey Research Information, Tokyo, Japan). Least-squares means for each treatment were
separated, based on least significant differences. Data were represented as means and SE for four cultures per treatment, and statistically significant differences from the mean of control cultures at P ⬍
0.05 and P⬍ 0.01 were indicated. Each experiment was repeated two
CALCIUM INFLUX INTO SATELLITE CELLS
C1745
monitored at 48 h by cell density and compared with the BrdU
labeling of control cultures receiving cation-channel inhibitors
(bars h–j), and by results showing that addition of HGF (2.5
ng/ml) to individual-inhibitor cultures (bars d– g) stimulated
satellite cell activation to a level equivalent to HGF (bar c) and
stretch (bar b). These observations, therefore, indicated that
both MS and L-VGC channels are essential in the stretch
activation of satellite cells and may be mediating the influx of
extracellular calcium ions in response to mechanical stretch.
Also, as shown in Fig. 2B, the implication of MS and L-VGC
channels in the activation cascade was further emphasized by
Western blot detection of the 60-kDa ␣-chain of HGF in
conditioned media (serum-free DMEM, pH 7.2); media were
collected from the 1-h stretch cultures that had received the
optimal dose of GsMTx-4 (0.1 ␮M), nifedipine (10 ␮M), or
EGTA (1.8 mM) and assayed for HGF. Results qualitatively
demonstrated that the cyclic stretch culture stimulates HGF
release (consistent with our original results) (lane m), and even
in the presence of stretch, GsMTx-4 (lane n), nifedipine (lane
o), and EGTA (lane p) diminish release of HGF with an
apparently similar potency. This indicates that HGF release
from extracellular matrix depends on the upstream MS- and
L-VGC-channel activities. In fact, the messages of ␣1S and ␣1C
subunits of L-VGC channels were detected by RT-PCR analysis for satellite cells at 24-h postplating, which is the time at
which the stretch treatment was applied in the present and our
previous experiments (65, 75–77) (Fig. 3A). Because L-VGCchannel proteins are now classified into Cav1.1, Cav1.2, Cav1.3,
and Cav1.4 according to their corresponding ␣1-subunits, ␣1S,
␣1C, ␣1D, and ␣1F (34, 36), our PCR results indicate that Cav1.1
and Cav1.2 may exist in quiescent satellite cells and be responsible for calcium ion influx by coupling with MS-channel
gating that senses mechanical perturbation of satellite cells.
Finally in a series of extracellular calcium ion dependency
experiments, calcium ion influx was documented by analysis of
calcium imaging studies for two-cycle stretched satellite cells
at 24-h postplating in the presence or absence of cation-channel
inhibitors (Fig. 4). Calcium ionophore treatment (3 ␮M
A23187, without stretch or treatment with channel inhibitors),
stimulated increase in intracellular calcium ion concentrations,
consistent with our previous result (67) and therefore served as
a positive control within this assay (row A). Cyclic stretch also
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Fig. 2. Dose-dependent effect of cation-channel inhibitors on stretch-activation of satellite cells and upstream
HGF release. A: satellite cells were subjected to an
environment of cyclic stretch for 1 h beginning at 24-h
postplating in the presence of various concentrations of
individual cation-channel inhibitors, GsMTx-4 (), nifedipine (), gadolinium chloride (Œ), and NNC55–
0396 (□), and assayed for BrdU incorporation at 48-h
postplating. This experiment also included control cultures as follows: bar a, unstretched culture; bar b, 1-h
stretch culture (positive control); bars c– g, control
cultures receiving 2.5 ng/ml HGF at 24 – 48 h postplating along with none, 0.1 ␮M GsMTx-4, 10 ␮M nifedipine, 100 ␮M gadolinium chloride, and 100 ␮M
NNC55– 0396, respectively; bars h–j, unstretched cultures with individual cation-channel inhibitors (each at
the optimal concentration). Data points and bars depict
means and SE of 4 cultures per treatment. Treatment
mean was significantly different from the mean of
positive control cultures (bar b): *P ⬍ 0.05, **P ⬍
0.01. B: a typical data set of ECL-Western blotting of
HGF in 2 or 3 independent experiments; conditioned
media (serum-free DMEM) from 1-h stretch cultures
with none (lane m), 0.1 ␮M GsMTx-4 (lane n), 10 ␮M
nifedipine (lane o), or 1.8 mM EGTA (lane p), were
analyzed for HGF release from the extracellular tethering by ECL-Western blotting standardized with internal
␤-actin in cell lysates that were harvested after stretch.
Lane q, unstretch medium; lane k, molecular weight
standards (STD); lane l, mouse recombinant HGF (12
ng) to show the band at 60-kDa ␣-chain for active HGF
(top row) and 42-kDa ␤-actin in mouse C2C12 myoblasts (bottom row) as positive controls (PC); lane r,
negative controls (NC) of stretch medium (top row) and
whole cell lysate (bottom row) blotted without the
primary antibody and with secondary antibody.
C1746
CALCIUM INFLUX INTO SATELLITE CELLS
affects calcium ion influx as shown in row B, in which the
fluorescence intensity reached a maximum at 30 – 60 s poststretch and the calcium signal maintained a plateau within at
least 210 s (far-right image) and typically 300 s of the maximum recording time point of the present analysis (image not
shown), and that response was almost abolished to reach a
level comparable to 1.8 mM EGTA-treated cultures when
stretch cultures received 0.1 ␮M GsMTx-4 or 10 ␮M nifedipine (rows C and D, respectively). These observations provide
direct evidence that both MS and L-VGC channels are essential
in mediating the extracellular calcium ion influx in response to
mechanical stretch in skeletal muscle satellite cells in culture.
DISCUSSION
The importance of satellite cell proliferation activity in
skeletal muscle repair and hypertrophy has been appreciated
for a considerable time. It is well documented that one of the
earliest events, triggering the activation of quiescent satellite
cells, is initiated by mechanical or chemical stimuli and enables satellite cells to migrate and enter the cell proliferation
cycle. Bischoff (11) was among the first to explore the bio-
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Fig. 3. Detection of mRNA for the L-type voltage-gated calcium channel
(L-VGC channel) and transient receptor potential canonical channels 1 and 6
(TRPC1, TRPC6) in satellite cells. Satellite cell cultures were maintained in
DMEM-10% HS and assayed for mRNA expression of ␣1S, ␣1C, ␣1D, and ␣1F
subunits of the L-VGC channel (A) and TRPC1 and TRPC6 (B) at 24-h
postplating by RT-PCR. Bands were visualized with Gel-Red staining after
agarose gel electrophoresis. NC, negative controls (satellite cell mRNA without reverse transcription); SCs, satellite cells; PC, positive controls (rat skeletal
muscle for ␣1S and TRPC1, cardiac muscle for ␣1C, brain for ␣1D and TRPC6,
and eye for ␣1F).
chemical link between satellite cell activation and mechanical
perturbation of muscle tissue. His classical experiments used a
crushed-muscle extract, which he found could stimulate satellite cells associated with isolated fibers to synthesize DNA and
divide. Johnson and Allen (30) later reported that the same
crushed-muscle extract could stimulate adult rat satellite cells
in culture to enter the cell cycle earlier than adult rat cultures
in control medium. Shortening of the lag phase observed with
freshly isolated adult satellite cells has since been used as an
assay for satellite cell activation (1, 2, 58), and this assay was
employed in experiments that first demonstrated that HGF was
unique in its ability to activate satellite cell division in vitro (1).
In subsequent experiments, the active agent in crushed-muscle
extract was shown to be HGF, which was present in uninjured
muscle and located primarily in the extracellular matrix (58,
62). In further experiments, Tatsumi et al. (59 – 61, 64) showed
that satellite cells were stimulated to enter the cell cycle when
subjected to mechanical stretch in primary culture and living
muscle. We also demonstrated that satellite cell activation was
due to rapid release of HGF from its extracellular tethering and
the subsequent presentation to the receptor c-met, and later
verified that HGF release is dependent on calcium-calmodulin
formation and the downstream NO radical-mediated activation
of matrix metalloproteinases (MMPs) (47, 67, 75, 76).
These observations suggest that the stretch-activation of
satellite cells is a cascade of sequential events including (in
order) an influx of calcium ions and their binding to calmodulin, NO radical production by cNOS, MMP activation, HGF
release from the matrix, and presentation of HGF to the
signaling receptor c-met (see a review of Ref. 66). In this
model, a complex of HGF and proteoglycan extracellular
domain would be shed from the matrix and presented to the
receptor c-met. In fact, HGF associated with heparan sulfate
moieties has a greater affinity for c-met relative to HGF alone,
and therefore, has enhanced HGF signaling activity (20). Such
a complex has been detected in PBS extracts from crushed
muscle and intact muscle incubated in the NO donor, sodium
nitroprusside (SNP) (Allen et al., unpublished data), supporting
the above insight. In related experiments by Wozniak and
Anderson (73), released HGF may induce c-met expression as
an immediate-early gene, and thus also enhance HGF-c-met
signaling in the satellite cell activation process. They additionally observed that the level of satellite cell activation was
increased in unstretched fibers from mdx and nNOS-knockout
mice and in unstretched normal fibers by treatment with a
competitive NOS-inhibitor, NG-nitro-L-arginine methyl ester
(L-NAME), suggesting that a NO-independent HGF/c-met signaling pathway, and possibly other signaling pathways, control
activation in muscle deficient in dystrophin and nNOS (73).
It has not been determined how quiescent satellite cells sense
mechanical stimuli to generate biochemical signals that elevate
intracellular calcium ion concentrations; therefore, in the present work, the involvement of MS and VGC channels in the
mechanotransduction system was examined. When satellite
cell cultures were incubated with the MS-channel inhibitor
GsMTx-4, the L-VGC channel inhibitor nifedipine, or a less
specific inhibitor, gadolinium chloride, stretch-induced HGF
release from the matrix and the resulting cell activation were
abolished completely in a dose-dependent manner. This was
revealed by Western blotting of conditioned media and a
BrdU-incorporation assay (Fig. 2). By contrast, the T-VGC-
CALCIUM INFLUX INTO SATELLITE CELLS
C1747
channel inhibitor NNC55– 0396 did not have inhibitory effects
on the activation index. Addition of the calcium-chelator
EGTA to culture media also inhibited the stretch-activation
response and HGF release, indicating the significance of extracellular calcium ions in the activation cascade (Figs. 1 and
2B). This means that free calcium ions stored in mitochondria
may now be excluded from a role in this activation pathway.
Calcium-imaging analyses verified these results, and therefore
provide direct evidence that both MS and L-VGC channels are
essential in calcium ion influx in response to mechanical
stretch and that calcium ions may flow in through L-VGC
channels (Fig. 4). A possible explanation for these observations
can be addressed by a possible model of MS/L-VGC-channel
coupling, in which the MS channel responds to mechanical
stimuli by gating cations; this would result in local depolarization of the plasma membrane, a signal that is then quickly
sensed by L-VGC channels (Cav1.1, Cav1.2) that allow a significant amount of calcium ions to permeate the cell and lead
to calcium-calmodulin formation (see Fig. 5B for details). In
the nifedipine treatment, in which MS channels are expected to
retain biological activity to gate-selective cations, significant
calcium influx was not observed, suggesting that MS channels
concerned in the activation pathway allow entry of cations
other than calcium ions and/or gate an undetectable level of
calcium ions that is not sufficient to activate calmodulin-NOS
signaling directly but is enough to depolarize the cell membrane.
Considering the possible implication of MS channels in the
mechanosensing mechanism of satellite cell activation, it is
worth noting the valuable study by Formigli et al. (25), which
provided the first experimental evidence that the transient
receptor potential canonical channel 1 (TRPC1) represents an
essential component of stretch-activated cation channel signaling in the mouse C2C12 myoblast cell line, as assayed by
whole cell patch-clamp and atomic force microscopic pulling.
That study indicated that TRPC1 plays a crucial role in mecha-
notransduction during regulation of the early phases of myoblast differentiation. TRPCs (TRPC1–7) are a family of cation
channels that assemble as homo- or heterotetramers to form
voltage-independent, nonselective cation- (Na⫹ and Ca2⫹) permeable pores (16, 46). Although numerous studies have shown
Fig. 5. A possible mechanism of satellite cell activation in response to
mechanical stimuli. A: Ca-CaM, calcium-calmodulin complex; L-Arg, L-arginine; NOS, NO synthase; MMP2, matrix metalloproteinase 2. [Reproduced
from Tatsumi and Allen (66) with some modifications with permission from
John Wiley and Sons. Copyright 2008.] B: calcium ion influx mechanism was
examined in the present study and the model was schematically presented.
Steps 1 and 2: cations stream into the cell through mechanosensitive cation
channels (MS channel) to induce local depolarization of the cell membrane
(changes in membrane potential); step 3: depolarization promotes gating of
adjacent L-type voltage-gated calcium-ion channels (L-VGC channel), and the
result is calcium ion influx that promotes formation of the Ca-CaM complex
that initiates NOS activity.
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Fig. 4. Effect of cation-channel inhibitors and EGTA on the increase in intracellular calcium ion concentration in response to stretch. Calcium-imaging analysis
was conducted under the StageFlexer Jr. system using fluo 3-loaded satellite cells that were treated with individual cation-channel inhibitors (0.1 ␮M GsMTx-4,
10 ␮M nifedipine) for 30 min just prior to adding 2 cycles of stretch at 24-h postplating. Row ⫹EGTA, cells were incubated with 1.8 mM EGTA in DMEM-10%
HS just prior to the addition of the stretch. Companion cells were treated with 3 ␮M A23187 (a calcium ionophore) and served as a positive control (row A23187)
(67). Fluorescence intensity was assigned from blue to red in color-coded images; the normal resting free calcium-ion level was represented by blue, and gradual
increases were demonstrated by a change to yellow and then red. After calcium imaging, cells were visualized by immunostaining to confirm the presence of
c-met, a marker molecule for myogenic cells in our primary cell cultures (DAB-stained images not shown).
C1748
CALCIUM INFLUX INTO SATELLITE CELLS
pendent on either TRPC signaling or the activation cascade
concerned here.
In summary, the present experiments bridge the gap between
mechanical stretch stimuli and the increase in intracellular
calcium-ion concentration by demonstrating that inhibition of
MS channels and L-VGC channels in vitro, prior to mechanical
stretch, prevents the influx of extracellular calcium ions, HGF
release from the matrix, and reentry of adult satellite cells into
the cell cycle. The physiological significance of the MS channel gating-initiated pathway described using cultured satellite
cells remains to be verified in muscle fibers, as does the crucial
role of calcium-calmodulin formation in the NO-radical production by cNOS that was described recently in isolated
satellite cells in culture (67). Nonetheless, the current report
demonstrated that in isolated satellite cells, a functional coupling of MS-channel and L-VGC-channel gating plays the
central role in mechanosensing machinery that instigates the
activation of satellite cells.
ACKNOWLEDGMENTS
We are grateful to T. Tamura of Nikon Instech for technical assistance with
the calcium imaging. The G3G4 mouse anti-BrdU monoclonal antibody
developed by S. J. Kaufman and D3 anti-desmin monoclonal antibody developed by D. A. Fischman were obtained from the Developmental Studies
Hybridoma Bank, developed under the auspices of the NICHD and maintained
by The University of Iowa, Dept. of Biological Sciences, Iowa City, IA 52242.
GRANTS
This work was supported by Grants-in-Aid for Scientific Research (B)
19380152 and 22380145 and by Invitation Fellowship Programs for Research
in Japan from the Japan Society for the Promotion of Science (JSPS) (all to R.
Tatsumi) and by a research grant of the Graduate School Student Projects
Academic Challenge 2010 from the Entrepreneurship Center of Kyushu
University (to M. Hara). The research was also supported by funds from the
Arizona Agriculture Experiment Station and grants from the US Dept. of
Agriculture National Research Initiative Competitive Grant 2005-3520615255 and Muscular Dystrophy Association Grant MDA3685 (all to R. E.
Allen), and by funds from the Canadian Space Agency 9F007-52237-001-SR
and the Natural Sciences and Engineering Research Council 171302 (to J. E.
Anderson). M.-K. Q. Do received a scholarship from the Ministry of Education, Culture, Sports, Science and Technology-Japan (MEXT) during the
course of this research.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.H., K.T., T.S., M.-K.Q.D., W.M., M.N., S.N., and
R.T. performed experiments; M.H., K.T., T.S., M.-K.Q.D., W.M., M.N., S.N.,
S.T., J.E.A., R.E.A., and R.T. analyzed data; M.H., K.T., T.S., M.-K.Q.D.,
W.M., M.N., S.N., S.T., Y.I., K.S., J.E.A., R.E.A., and R.T. interpreted results
of experiments; M.H., K.T., and R.T. prepared figures; M.H. and R.T. drafted
manuscript; M.H., J.E.A., R.E.A., and R.T. edited and revised manuscript;
M.H., K.T., T.S., M.-K.Q.D., W.M., M.N., S.N., S.T., Y.I., K.S., J.E.A.,
R.E.A., and R.T. approved final version of manuscript; K.T., J.E.A., R.E.A.,
and R.T. conception and design of research.
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CALCIUM INFLUX INTO SATELLITE CELLS