A role for calcium-calmodulin in regulating nitric oxide

Am J Physiol Cell Physiol 296: C922–C929, 2009. First published January 21, 2009; doi:10.1152/ajpcell.00471.2008.

A role for calcium-calmodulin in regulating nitric oxide production during skeletal muscle satellite cell activation Ryuichi Tatsumi,1 Adam L. Wuollet,2 Kuniko Tabata,1 Shotaro Nishimura,3 Shoji Tabata,3 Wataru Mizunoya,1 Yoshihide Ikeuchi,1 and Ronald E. Allen2 1

Department of Bioscience and Biotechnology and 3Department of Animal Science, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan; 2Muscle Biology Group, Department of Animal Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, Arizona Submitted 12 September 2008; accepted in final form 19 January 2009

muscle regeneration; stretch-activation SKELETAL MUSCLE SATELLITE cells are resident myogenic stem cells normally found in a quiescent state in adult skeletal muscles. When muscle is injured, overused, or mechanically stretched, these cells are activated to enter the cell cycle, divide, differentiate, and fuse with muscle fibers to repair damaged regions and to enhance hypertrophy of muscle fibers (reviewed in Refs. 7, 10, 17). Therefore, mechanical changes in muscle can initiate events that lead to satellite cell activation, although the mechanism has not been clearly delineated. Of all growth factors studied thus far, only hepatocyte growth factor (HGF) provides a signal that can activate quiescent satellite cells in primary culture and in vivo (1, 20). HGF is a heparin-binding protein localized in the extracellular domain of uninjured skeletal muscle fibers, and its predominant form is the active heterodimer of the 60-kDa ␣-chain and

Address for reprint requests and other correspondence: R. E. Allen, Muscle Biology Group, Dept. of Animal Sciences, Coll. of Agriculture and Life Sciences, Univ. of Arizona, Shantz Blvd. 204, PO Box 210038, Tucson, AZ 85721-0038 (e-mail: [email protected]). C922

30-kDa ␤-chain (25). The intracellular signaling receptor for HGF is the c-met proto-oncogene, and its message and protein have been found in quiescent and activated satellite cells (1, 8, 20). Thus release of HGF from its sequestration and subsequent presentation to the c-met receptor may be a critical aspect of the activation of quiescent satellite cells. In previous works, we employed a FlexerCell system (Flexcell International, McKeesport, PA) to apply cyclic stretch to isolated rat satellite cells and found that mechanical stretch triggers satellite cell activation by releasing HGF from its tethering in the extracellular matrix (21, 23, 26, 28). This phenomenon is relevant to satellite cells in living skeletal muscle as revealed by an in vivo muscle stretch model (16, 27). These experiments also show that release of HGF is blocked if nitric oxide synthase (NOS) is inhibited by NG-nitro-L-arginine methyl ester (L-NAME), and HGF can be liberated when unstretched satellite cells are incubated in sodium nitroprusside (SNP), a NO donor. Therefore, there is a NO-dependent step in the release of HGF in response to mechanical perturbation of muscle tissues (reviewed in Ref. 29). Recent studies on the downstream events revealed that matrix metalloproteinases (MMPs), at least MMP2, mediate HGF release from extracellular matrix, possibly by shedding proteoglycan core proteins in response to NO (33, 34). In this case, a complex of HGF and proteoglycan extracellular domain would be released from the matrix and presented to the c-met receptor. In fact, HGF associated with heparan sulfate moieties has a greater affinity for the c-met receptor relative to HGF alone; therefore, it has enhanced HGF signaling activity (9), and such a complex has been detected in PBS extracts from SNP-treated muscle (Allen R, Liu X, Tatsumi R, unpublished data). These observations support the above insight. In an effort to understand the events upstream from NO production, experiments in this study were designed to test the hypothesis that calcium ions and calmodulin are involved in NOS activation in satellite cells. The calcium-calmodulin complex is thought to associate with constitutive NOS (cNOS: neuronal and endothelial NOSs) to activate the enzyme activity as shown in neuronal and endothelial cells (15, 30). However, it is still unclear whether quiescent satellite cells express functional calmodulin and can respond to calcium ions at an early time point after plating (12 h postplating), which is the time at which the stretch or SNP treatment was applied to the cells in our previous experiments (21, 23, 24, 26). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Tatsumi R, Wuollet AL, Tabata K, Nishimura S, Tabata S, Mizunoya W, Ikeuchi Y, Allen RE. A role for calcium-calmodulin in regulating nitric oxide production during skeletal muscle satellite cell activation. Am J Physiol Cell Physiol 296: C922–C929, 2009. First published January 21, 2009; doi:10.1152/ajpcell.00471.2008.— When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor NG-nitro-L-arginine methyl ester hydrochloride but not by its less inactive enantiomer NG-nitro-Darginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis.

SATELLITE CELL CALMODULIN

In the present paper, we examined the effect of calcium ionophores with/without calmodulin inhibitors or L-NAME on HGF release from the matrix and activation of satellite cells and provide evidence that calcium-calmodulin plays a possible role to link mechanical perturbation and NOS activation. MATERIALS AND METHODS

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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 plated on poly-L-lysine and fibronectin-coated dishes in DMEM containing 10% HS, 1% antibioticantimycotic mixture, and 0.5% gentamicin (DMEM-10% HS, pH 7.2). Cultures were maintained for 12 h in a humidified atmosphere of 5% CO2 at 37°C. In addition, companion satellite cell cultures were immunostained for the presence of desmin at 30 h after being plated using a D3 monoclonal anti-desmin antibody, biotinylated anti-mouse IgG antibody, and HRPO-labeled avidin to determine the percentage of myogenic cells present. Cultures with less than 95% DAB-stained cells were not used for experiments. Conditioned medium. In experiments in which conditioned media from cultures receiving a calcium ionophore (0 –3 ␮M A23187 or ionomycin) were assayed for their activation activities, cultures were washed with serum-free DMEM at 12 h postplating and treatments were imposed for as short as 2 h in the presence of serum-free DMEM (pH 7.2). The culture medium additionally received a NOS competitive inhibitor L-NAME (10 ␮M), its less inactive enantiomer DNAME (10 ␮M), or a calmodulin inhibitor (0 – 0.3 ␮M calmidazolium, 0 –300 ␮M W-13 or W-12) 30 min before the addition of 3 ␮M A23187. Conditioned medium from each treatment was removed, centrifuged for 4 min at 1,300 g, and frozen at ⫺30°C until use. Calcium ionophore-conditioned media were dialyzed against DMEM for 2 h at 4°C using a Slide-A-Lyzer cassette (10 k molecular weight cut-off; Pierce, Rockford, IL) to remove the ionophore. In the case of immunoneutralization experiments, anti-HGF-neutralizing antibody (2 ␮g/ml) with or without HGF (20 ng/ml) was added to selected treatment-conditioned media for 2 h before the activation assay. HS and antibiotics were added to each treatment medium to prepare DMEM-10% HS immediately before feeding to untreated cultures, which were then maintained for the 24-h period from 12 to 36 h postplating and assayed for satellite cell activation. In vitro activation assay. Cultures were pulse labeled with 10 ␮M BrdU in DMEM-10% HS for the final 2 h from 34 to 36 h postplating, followed by immunocytochemistry for detection of BrdU using a G3G4 anti-BrdU monoclonal antibody (1:100 dilution in 0.1% BSA in PBS) and a HRPO-conjugated anti-mouse IgG antibody (1:500 dilution) according to Tatsumi et al. (20). The percentage of BrdU-labeled cells was used as an indicator of activation and entry into the cell cycle. Immunoblotting and ECL. Calmodulin and 12-h cultured satellite cell lysate were subjected to SDS-PAGE on linear 10 –20% polyacrylamide gradient gels under reducing conditions (14). Separated proteins were transferred to Immun-Blot PVDF membranes at 2.6 V/cm of distance between electrodes for 3 h using an electrode buffer of 25 mM K-phosphate (KP) buffer, pH 7.0. The blots were incubated in 0.2% glutaraldehyde-KP buffer for 30 min (11) and then blocked with 10% powdered milk in 0.1% polyethylene sorbitan monolaurate (Tween 20)-Tris buffered saline (TTBS) before incubation with monoclonal anti-calmodulin antibody (1:500 dilution in 1% powdered milk-TTBS additionally containing 0.05% NaN3) overnight. Membranes were subsequently treated with biotinylated anti-mouse IgG antibody at 1:1,000 dilution for 1 h, then with HRPO-labeled avidin at a 1:500 dilution in TTBS for 30 min, followed by ECL detection onto Kodak X-OMAT-AR X-ray films. In Western blotting of HGF in conditioned media from 2-h cultures receiving a calcium ionophore with/without a calmodulin inhibitor or L-NAME, the media were applied to 10% PAGE and transferred to nitrocellulose membranes at 8.3 V/cm of distance between electrodes for 5 h using an electrode buffer of 25 mM Tris, 0.192 M glycine, 0.1% SDS, and 20% ethanol. The blots were blocked with 10% milk in TTBS before incubation with polyclonal anti-HGF antibody (1:500 dilution in 1% milk-TTBS) for 3 h, subsequently treated with biotinylated anti-goat IgG antibody at 1:10,000 dilution for 45 min, then treated with HRPO-avidin at a 1:1,000 dilution in TTBS for 20 min, followed by ECL detection onto the X-ray films.

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Materials. DMEM, normal horse serum (HS), antibiotic-antimycotic, and gentamicin were purchased from Life Technology (Grand Island, NY). Poly-L-lysine, bovine plasma fibronectin, protease type XIV, and 5-bromo-2⬘-deoxyuridine (BrdU) were obtained from Sigma (St. Louis, MO). Bovine brain calmodulin (P-2277, 95% purity) and 2D1 mouse monoclonal anti-calmodulin antibody (C-6167) were obtained from Sigma. Rabbit polyclonal anti-c-met (m-Met SP260) and goat polyclonal anti-c-met antibodies (AF527) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and R&D Systems (Minneapolis, MN), respectively, and D3 mouse monoclonal anti-desmin and G3G4 mouse monoclonal anti-BrdU antibodies were obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Recombinant human HGF (294-HG), recombinant mouse c-met/Fc chimera (527-ME), biotinylated goat polyclonal anti-human HGF antibody (BAF-294), goat polyclonal anti-human HGF antibody (AB-294-NA), horseradish peroxidase (HRPO)-streptavidin (4800-30-06), and substrate reagent pack (DY-999: tetramethyl benzidine and stabilized hydrogen peroxide) were purchased from R&D Systems. Affinitypurified biotinylated horse anti-mouse IgG (BA-2000) and rabbit anti-goat IgG (BA-5000), FITC-labeled avidin DCS (A-2011), and HRPO-labeled avidin kit (PK-6100) were purchased from Vector Laboratories (Burlingame, CA). Tetramethylrhodamine isothiocyanate (TRITC)-labeled sheep anti-rabbit IgG (55667) was obtained from Cappel Research (Durham, NC). Affinity-purified HRPOconjugated goat anti-mouse IgG (A-4416), 3,3⬘-diaminobenzidine (DAB), and a nonionic copolymer surfactant Pluronic F-127 (P2443) were purchased from Sigma. HRPO-labeled rabbit anti-goat IgG (414331), Histofine Simple Stain Rat MAX-PO (G), and normal rabbit serum (426051) were obtained from Nichirei BioScience (Tokyo, Japan). Quin 2, fluo-3-AM, and EGTA were obtained from Dojindo Laboratories (Kumamoto, Japan). Calcium ionophores A23187 (21186) and ionomycin calcium salt (10634), calmodulin inhibitors calmidazolium [C3930, 1-[Bis(4chlorophenyl)methyl]-3-[2,4-dichloro-b-(2,4-dichlorobcalmodulin inhibitors calmidazolium [C3930, 1-[Bis(4-enzyloxy)phenethyl]imidazolium chloride], W-13 [A0666, N-(4-Aminobutyl)-5chloro-2-naphthalenesulfonamide hydrochloride], and W-12 [A3168, N-(4-Aminobutyl)-2-naphthalenesulfonamide hydrochloride] were purchased from Sigma. L-NAME (483125) and NG-nitro-D-arginine methyl ester hydrochloride (D-NAME, 483124) were obtained from Calbiochem-Novabiochem (La Jolla, CA). Enhanced chemiluminescence (ECL) detection kit (PRN2106) and nitrocellulose membranes (Hybond ECL, RPN2020D) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Polyvinylidene difluoride (PVDF) membranes (Immun-Blot, 162-0174; Sequi-Blot, 162-0186) and biotinylated molecular weight standards (161-0319) were purchased from Bio-Rad Laboratories (Hercules, CA). X-OMAT-AR X-ray film (166-0760) was obtained from Eastman Kodak (Rochester, NY). Animal care and use. Experiments were conducted according to institutional guideline and with the approval of Kyushu University Institutional Review Board and of the University of Arizona Institutional Animal Care and Use Committee. Satellite cell isolation and primary culture. Satellite cells were isolated from muscle groups from the hind limb and back of 9-mo-old male Sprague-Dawley rats according to Allen et al. (2). Briefly, muscle groups from the upper hind limb and back were excised, trimmed of fat and connective tissue, hand minced with scissors, and

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spectrophotometer set to 450 nm and 562 nm. The subtraction of readings at 562 nm from the readings at 450 nm corrects for optical imperfection in the plate and average of the triplicate measurements for individual HGF standards, and samples were obtained. The c-met capturing procedure yielded a good correlation coefficient (r2, typically over 0.990) between HGF concentration standards and absorbance 450 –562 nm in a wide range examined (see Fig. 6A). It is a suitable application to HGF associated with extracellular segments of proteoglycans, i.e., partial core proteins shedded with glycosaminoglycan moieties, which has been detected in PBS extracts from NO donor-treated muscle (Allen R, Liu X, and Tatsumi R, unpublished data), and may be potentially limited to access to the capturing monoclonal antibody immobilized in usual sandwich ELISA systems because of a steric hindrance of its large associates. Statistical analysis. Analysis of variance procedures were employed to analyze experimental results using general linear model procedures of SRISTAT2 for Windows software (Social Survey Research Information, Tokyo, Japan). Least-squares means for each treatment were separated on the basis of the least significant differences. Data were represented as means ⫾ SE for four cultures per treatment, and statistically significant differences from control culture means at P ⬍ 0.01 were indicated by double asterisks. Each experiment was repeated two or three times to verify the reproducibility of results, and, in most cases, one rat was used for each experiment. RESULTS

In previous experiments, it was demonstrated with isolated satellite cells that entry into the cell cycle is accelerated in vitro by treatment with HGF (1), NO donors (3, 5, 23–25), and by mechanical stretch (21, 28, 31, 32). It has been shown subsequently that these stimuli activate quiescent satellite cells in vivo (27) through the release of HGF from the extracellular matrix and its interaction with the c-met receptor. In attempting to construct a pathway for translating mechanical perturbation of the cell or muscle fiber into an activation signal, it is now clear that NO synthesis is required for HGF release and satellite cell activation. It is also well established that activity of all three isoforms of NOS are stimulated by interaction with calcium-calmodulin. Therefore, experiments were designed to determine whether an influx of extracellular calcium ions can stimulate satellite activation by binding calmodulin, stimulating NOS activity, and subsequently releasing HGF. In the original work on mechanical stretch on in vitro satellite cell activation, stretch was applied at 12 h postplating to determine whether the activation process could be triggered prematurely. The same approach was taken in this set of experiments. Figure 1 presents results from the first experiment showing that conditioned media from satellite cells treated with calcium ionophores, ionomycin and A23187, can stimulate dose-dependent activation of untreated satellite cells, as measured by BrdU labeling index (Fig. 1A). In this experiment, satellite cells were cultured for 12 h and then fed serum-free DMEM in the presence of various concentrations of calcium ionophores, A23187 or ionomycin, for 2 h without stretching. Conditioned media were dialyzed against DMEM for 2 h to remove calcium ionophores and then subsequently assayed for satellite cell activation as described previously. Control DMEM, which was added with each ionophore (3 ␮M) followed by dialysis under the same conditions (Fig. 1, data points a and a⬘), did not show activating activity relative to control culture (Fig. 1A, data point c), therefore serving as an important control for other treatment groups within this assay. As seen in Fig. 1A, ionophore-conditioned media stimulated sig-

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Immunolocalization. Twelve-hour satellite cell cultures were fixed for 10 min in cold methanol-0.1% H2O2, incubated for 10 min in 0.1% Triton X-100-PBS, blocked with 5% goat serum in PBS for 30 min, and incubated overnight in a mixture of primary antibodies to calmodulin and c-met (1:100 dilution in 0.1% BSA-PBS). Cells were subsequently incubated with biotinylated anti-mouse secondary antibody (1:10,000 dilution) and then with TRITC-labeled anti-rabbit secondary antibody (1:5,000 dilution) for 1.5 h, followed by incubation for 1.5 h in FITC-avidin DCS (1:500 dilution in PBS). Fluorescence was monitored using Nikon Diaphot TMD300 fluorescent microscope and documented on Fujichrome PROVIA 400F films. Calcium binding assay. Calmodulin and the 12-h cultured satellite cell lysate were applied to 10 –20% polyacrylamide gradient gels; separated proteins were transferred onto Bio-Rad Sequi-Blot PVDF membranes and subjected to a fluorescence assay using a quinoline calcium indicator quin 2 (19, 22). Briefly, the blots including omission of the glutaraldehyde fixation were incubated in a solution containing 60 mM KCl, 5 mM MgCl2, 1 mM CaCl2, and 10 mM imidazole-HCl buffer, pH 6.8 for 1 h, and then rinsed with 20% ethanol for 6 min. The membrane was incubated with 1 mM quin 2 for 1 h in a dark box, followed by thorough washing with deionized water. To determine the reversibility of calcium binding, the calcium-treated blot was incubated with the solution additionally containing 5 mM EGTA for 2 h, followed by the quin 2 treatment. Fluorescent patterns were visualized by illumination with UV light at 365 nm (UVL-56; UVP, San Gabriel, CA) and photographed through a green filter (MC PO1, Kenko, Tokyo) with Fuji Neopan 1600 Super Presto films, which were then sensitized as equivalent to the exposure index of 3200 in the development process. Calcium imaging. Twelve-hour satellite cell cultures in a glassbottom dish (11-004-006; AGC Techno Glass, Funabashi, Japan) were loaded with 5 ␮M fluo-3-AM in 0.2% Pluronic F-127 for 1 h at 17°C in the dark and then rinsed with PBS and left to equilibrate in PBS for 1 h in a humidified atmosphere of 5% CO2 at 37°C followed by addition of 3 ␮M A23187 to DMEM-10% HS medium; control cells received 1.8 mM EGTA, a highly selective calcium chelator, in the media just before the A23187 stimulation. Calcium images were monitored at intervals of 1 s under the Nikon RCM-8000 confocal laser scanning microscope system including argon-ion laser unit (BeamLok models 20657S and 3980; Spectra-Physics, Mountain View, CA), Nikon Diaphot TMD300 microscope and control unit, with an excitation wavelength of 488 nm and a maximum emission wavelength of 526 nm according to Tabata et al. (18). Fluorescence density was assigned from blue to purple in color-coded images by a Nikon computer software installed and documented on Fujichrome PROVIA 400X films with film recorder FR-2000 (Nippon Avionics, Tokyo, Japan). After the imaging, cells were fixed for 10 min in cold methanol-0.1% H2O2 and blocked with 5% normal rabbit serum-0.6% H2O2 in PBS for 20 min, followed by immunostaining for the presence of c-met using the primary antibody (1:100 dilution in 5% normal rabbit serum-PBS) and HRPO-labeled anti-goat IgG secondary antibody to determine whether the calcium-imaged cells are myogenic. C-met-capturing sandwich ELISA. Costar E.I.A. flat-bottom plates (3690, 500 ng IgG/cm2 of binding capacity) were coated with recombinant mouse c-met/Fc chimera by transfer 50 ␮l/well of the protein (1 ␮g/ml in sterile PBS) overnight at room temperature and then blocked with 1% BSA, 5% sucrose, and 0.05% NaN3 in sterile PBS for 2 h. Conditioned media from 2-h ionophore-cultures and human recombinant HGF standards (0.1–5.0 ng/ml in 0.1% BSA-PBS) were incubated for 2 h at 37°C, followed by fixation with cold 3.7% paraformaldehyde-PBS for 5 min and incubation with the blocking solution overnight at 4°C. Plates were subsequently treated with biotinylated polyclonal anti-HGF antibody (1:500 dilution with 0.1% BSA in 0.05% TTBS) for 3 h, then with HRPO-streptavidin (1:1,600 dilution) for 20 min at room temperature, followed by tetramethyl benzidine colorization and the optical density measurements using a


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nificant increase in BrdU incorporation relative to controlconditioned medium with the maximum (3 ␮M) being roughly equivalent to the positive control culture receiving 2.5 ng/ml HGF (Fig. 1A, point b). Figure 1B displays the effect of anti-HGF neutralizing antibody on the activating activity of 3 ␮M ionophore-conditioned media. The ionophore media plus anti-HGF antibody (Fig. 1B, point f) did not stimulate activation relative to the control medium (Fig. 1B, point d), and the addition of HGF negated the neutralizing effect of anti-HGF (Fig. 1B, point g). Control antibody (goat IgG) did not diminish activating activity of the ionophore media (Fig. 1B, point h). These results indicate that the 2-h ionophore treatment causes the release of HGF into culture media and activates satellite AJP-Cell Physiol • VOL

cells, similar to the effect of mechanical stretch or NO donor treatments. Finally, in a series of calcium ion dependency experiments, calcium influx was documented by calcium imaging analysis on the 3 ␮M A23187-treated satellite cells at 12 h postplating (Fig. 1C). The response was almost abolished when EGTA was added to the medium to diminish the extracellular free calcium ions (about 20 ␮M), indicating the significance of extracellular calcium ions in our present activation model. The putative pathway from calcium ions to HGF release is hypothesized to involve calcium-calmodulin. Figure 2 demonstrates that functional calmodulin is present at 12 h postplating, which is the time at which the stretch treatment was applied in

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Fig. 1. Calcium ionophore treatment generates satellite cell activation activity in conditioned medium. A: serum-free DMEM, pH 7.2, was used to prepare conditioned media from satellite cell cultures that were maintained for 2 h beginning at 12 h postplating in the presence of various concentrations of calcium ionophore A23187 (E) or ionomycin (F). The media were dialyzed against DMEM for 2 h to remove calcium ionophore and then evaluated for the activating activity by in vitro activation assay on 36-h cultured satellite cells along with the following control cultures: DMEM plus 3 ␮M A23187 followed by dialysis against DMEM for 2 h (a), DMEM plus 3 ␮M ionomycin followed by dialysis against DMEM for 2 h (a⬘), positive control with 2.5 ng/ml hepatocyte growth factor (HGF) (b); negative control with fresh DMEM (c). B: effects on satellite cell activation by the following ionophore-conditioned medium treatments: medium from calcium ionophore-untreated culture (d), 3 ␮M calcium ionophore media (e), 3 ␮M calcium ionophore media plus 2 ␮g/ml anti-HGF neutralizing antibody (f), 3 ␮M calcium ionophore media plus 2 ␮g/ml anti-HGF neutralizing antibody plus 20 ng/ml HGF added back after the antibody treatment (g), 3 ␮M calcium ionophore media plus 2 ␮g/ml control antibody (h). Data points and bars depict means ⫾ SE for 4 cultures per treatment. **Treatment mean was significantly different from control medium mean (P ⬍ 0.01). C: time course of increase in intracellular calcium ion concentrations in 12-h satellite cells in response to 3 ␮M A23187; calcium-imaging was conducted for a spindle-shaped cell (top) and a round-shaped cell (middle). EGTA companion cells were incubated with 1.8 mM EGTA in DMEM-10% HS just before the addition of the calcium ionophore (bottom). Fluorescence intensity was assigned from blue to purple 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 purple. Arabic numerals inserted in each panel indicate seconds after the A23187 addition. After the calcium imaging, cells were immunostained for the presence of c-met, a marker molecule for myogenic cells in our primary cell cultures (far right, DAB-stained image). BrdU, 5-bromo-2⬘-deoxyuridine.

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our previous experiments (21, 23, 24, 26). Figure 2A demonstrates an immunoblot of 12-h cultured satellite cell lysate and bovine brain calmodulin (positive control); a band corresponding to 17-kDa calmodulin was clearly detected in the satellite cell lysate, and mixture of the cell lysate and the positive control presented a single band of calmodulin. The presence of calmodulin was further investigated at 12 h postplating by double-labeling immunolocalization of calmodulin and c-met, a marker protein for myogenic cells (Fig. 2B). Calmodulin was detected in c-met-positive satellite cells (Fig. 2B, panels a-c/a⬘-c⬘) and c-met-negative fibroblast-like cells (Fig. 2B, panels d/d⬘). In companion cultures that were single stained for each protein, the percentage of calmodulin-positive, c-met-positive, and desmin-positive cells at 12 h in culture were 98.6 ⫾ 0.06, 97.1 ⫾ 0.87 and 95.4 ⫾ 0.53% (means ⫾ SE), respectively. Together, these data provide strong evidence that satellite cells express calmodulin protein at 12 h postplating. The issue was further examined by assessing the calcium binding activity of calmodulin in the 12-h cultured satellite cells in a fluorescence binding assay (Fig. 2C). On the blot of the cell lysate, a band corresponding to calmodulin was intensely fluorescent, which indicates calcium binding, and the response was greatly reduced by washing the calcium-treated membrane with EGTA, indicating that binding of calcium ions is reversible. In addition, there were two faintly fluorescent bands seen just above and under calmodulin in the cell lysate; 19-kDa-positive protein might be expected to be a calciumbinding subunit B of calcineurin, a calmodulin-dependent protein phosphatase; the 14-kDa band is unidentified. AJP-Cell Physiol • VOL

Having established the presence of functional calmodulin in cultured satellite cells at this early time in the activation process, the role of calmodulin in mediating calcium ioninduced HGF release was examined. Figure 3 describes experiments in which conditioned media were collected from satellite cell cultures that were maintained for 30 min beginning 12 h postplating in the presence of increasing concentrations of calmodulin-specific inhibitors (0 –300 nM calmidazolium, 0 –300 ␮M W-13, or 0 –300 ␮M W-12) and followed by addition of 3 ␮M A23187 for the next 2 h. Conditioned media were dialyzed for 2 h against DMEM to remove ionophore, and activating activity was determined in vitro by evaluating BrdU incorporation in 36-h cultured satellite cells as shown in Fig. 1. Calmodulin inhibitors abolished the ionophore-stimulated activating activity in a concentration-dependent manner; calmidazolium was the most powerful of the three inhibitors with an optimal concentration range of 60 –90 nM vs. 30 –90 ␮M for W-13 and 90 –300 ␮M for W-12. These observations indicated that calmodulin is mediating the calcium-induced activation of cells. The next experiment was conducted to determine whether calcium-calmodulin activity is upstream of NO synthesis as hypothesized. In the experiment presented in Fig. 4, satellite cells were cultured for 12 h and then fed serum-free DMEM for the next 2 h with 3 ␮M A23187 in the presence of a NOS inhibitor L-NAME (10 ␮M) or its less active enantiomer D-NAME (10 ␮M). The concentration of L-NAME was optimized in our previous experiments in which inhibition of NOS activity, HGF release, and satellite cell activation have been all

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Fig. 2. Detection of functional calmodulin in satellite cells at 12 h in culture. A: satellite cell lysate (13,000 cells) at 12 h postplating was analyzed for the presence of calmodulin by enhanced chemiluminescence (ECL)-Western blotting. STD, biotinylated molecular weight standards; calmodulin, from bovine brain (15 ng); MIX, mixture of calmodulin (5 ng) and the cell lysate (13,000 cells); CNT, blot of the cell lysate without primary antibody; CBB, stained with Coomassie brilliant blue R-250. B: cells were double stained with antibodies to calmodulin (green, a–d) and c-met (red, a⬘– d⬘). Fluorescence images demonstrate the presence of calmodulin in c-met-positive cells. Control panel (e/e⬘) presents control cells without primary antibodies and with secondary antibodies and FITC-avidin DCS. Percentages of positive cells were shown by the means ⫾ SE for 4 cultures of each treatment. Note the c-met-negative fibroblast-like cell (fb) in d/d⬘. C: satellite cell lysate (51,000 cells) were assayed for calcium binding to calmodulin by a fluorescence detection method using quin 2; 3 positive bands were detected, which were indicated by asterisks on the right side of the lane. Lane a, treated with Ca2⫹; lane b, washed with 5 mM EGTA after Ca2⫹-treatment. Note that adult chicken breast muscle myofibrils (24 ␮g) showed a good fluorescent band of troponin-C along with the other negative proteins (Posi.Cont.); this result is consistent with that from Tatsumi et al. (19) and serves as an important control for other lanes within this assay.


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media (Fig. 6). Figure 6B illustrates a dose-dependent increase in HGF release into conditioned medium in response to ionomycin and A23187, and Fig. 6C shows the inhibition of A23187-induced HGF release by calmodulin inhibitors, calmidazolium, W-12, and W-13. Finally, release of HGF in response to A23187 is shown to be inhibited by the NOS inhibitor L-NAME but not by the inactive D-NAME. Therefore, the release of HGF correlates with generation of activation activity into conditioned medium by cultured quiescent satellite cells in response to an influx of calcium ions. DISCUSSION

demonstrated (23, 24). Conditioned media were dialyzed against DMEM for 2 h and then tested for satellite cell activation as described previously. Conditioned medium from D-NAME-treated cultures (Fig. 4, bar d) stimulated activation to a level equivalent to the positive control culture receiving 2.5 ng/ml HGF (Fig. 4, bar e) and the A23187-conditioned medium ( Fig. 4, bar b), but conditioned medium from A23187-treated cells in the presence of L-NAME (Fig. 4, bar c) did not generate satellite cell-activating activity relative to the negative control culture (Fig. 4, bar f). These results indicated that NOS mediates the generation of activating activity that is initiated by an influx of extracellular calcium ions. In the previous experiments, satellite cell-activating activity is generated in response to calcium ionophore treatment and is inhibited by calmodulin inhibitors and NOS inhibition. Figures 5 and 6 verify that release of HGF into treatment medium is consistent with observed activation effects of the conditioned media. Western blot detection of the 60-kDa ␣-chain of HGF in conditioned media qualitatively demonstrates that ionomycin and A23187 both stimulate HGF release into medium (Fig. 5, lanes b and c), and, in the presence of A23187, the three calmodulin inhibitors inhibit release of HGF in the apparent same order of potency as their inhibition of activation in vitro. Calmidazolium is most potent, and W-12 is least (Fig. 5, lanes d, e, f). The effect of NOS inhibition of ionophoreinduced HGF release also agreed with biological activity in that HGF release from A23187-treated cells was inhibited by the potent NOS inhibitor L-NAME but not by D-NAME (Fig. 5, lanes g and h, respectively). These results are displayed quantitatively using a sandwich ELISA of HGF in the conditioned AJP-Cell Physiol • VOL

Fig. 4. Calcium-induced activation of satellite cells is abolished by altering nitric oxide (NO) metabolism. Conditioned media (serum-free DMEM, pH 7.2) were collected from satellite cell cultures that were maintained for 2 h beginning at 12 h postplating in the presence of 3 ␮M calcium ionophore A23187 and 10 ␮M NG-nitro-L-arginine methyl ester (L-NAME) (c) or NGnitro-D-arginine methyl ester hydrochloride (D-NAME) (d). The media were dialyzed against DMEM for 2 h and then evaluated for the activating activity by in vitro activation assay on 36-h cultured satellite cells along with the following control cultures: medium from A23187-untreated culture (a), 3 ␮M A23187 medium (b), positive control with 2.5 ng/ml HGF (e), and negative control with fresh DMEM (f). Bars represent means ⫾ SE for 4 cultures per treatment; **treatment mean was significantly different from control medium mean (a) at P ⬍ 0.01.

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Fig. 3. Calcium-induced activation of satellite cells is abolished by calmodulin inhibitors. Conditioned media (serum-free DMEM, pH 7.2) were collected from satellite cell cultures that were maintained for 2 h beginning at 12 h postplating in the presence of 3 ␮M calcium ionophore A23187 and various concentrations of a calmodulin inhibitor, i.e., calmidazolium (䊐), W-12 (E), or W-13 (F). The media were dialyzed against DMEM for 2 h and then evaluated for the activating activity by in vitro activation assay on 36-h cultured satellite cells. Data points depict means ⫾ SE for 4 cultures per treatment; **treatment mean was significantly different (at P ⬍ 0.01) from control medium mean (a) not receiving A23187 or calmodulin inhibitors.

At this time, the mechanism of extracellular calcium influx in response to mechanical stretch has not been established but may involve the contribution of stretch-gated calcium channels such as an eukaryotic calcium-permeable stretch-activated cation channel (Mid1) that has been identified in yeast Saccharomyces cerevisiae (12, 13). Alternatively, calcium may be entering via small breaches of the cell membrane that rapidly fuse closed. Preliminary results indicate that extracellular calcium influx is responsible for stretch-induced satellite cell activation in vitro as opposed to calcium release from internal stores (Tatsumi R, unpublished observations). The other question that remains unanswered as we attempt to translate these observations to a physiological setting is whether quiescent satellite cells generate the intracellular signal responsible for HGF release from extracellular matrix, or

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whether the muscle fiber detects and generates the signal responsible for HGF release from the extracellular matrix. NO-mediated HGF release has been demonstrated in isolated muscle fibers in vitro (5, 32; reviewed in Refs. 4, 6), in isolated muscle (25) and in living muscle in vivo (27), and it seems plausible that the released HGF may be coming from the muscle fiber matrix, which also surrounds the satellite cell.

ACKNOWLEDGMENTS We are grateful to Chila Dudas of the Department of Nutritional Sciences, University of Arizona for the technical assistance with the HGF-ELISA. The helpful discussions with Mark Morales were invaluable. The mouse anti-BrdU monoclonal antibody developed by S. J. Kaufman and 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, Department of Biological Sciences, Iowa City, IA. GRANTS This work was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science and by research funds from Ito

Fig. 6. Sandwich ELISA of HGF in conditioned media. Conditioned media from experiments of Figs. 2– 4 were analyzed for HGF concentration by c-met-capturing sandwich ELISA (B–D) standardized with 0.1–5.0 ng/ml human recombinant HGF (A). D: control culture without A23187 (a); A23187 medium (b); A23187 plus 10 ␮M L-NAME (c); A23187 plus 10 ␮M D-NAME (d). Data points and bars represent means ⫾ SE for 4 cultures per treatment; **treatment mean was significantly different from each control medium mean at P ⬍ 0.01. AJP-Cell Physiol • VOL

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Fig. 5. Calcium-induced HGF release from satellite cells is affected by altering calmodulin and NO synthase (NOS) activities. Selected treatmentconditioned media from experiments of Figs. 2– 4 were analyzed for the presence of HGF by ECL-western blotting. Each lane represents proteins from a constant number of cultured satellite cells. STD, biotinylated molecular weight standards; lane a, control medium without calcium ionophore treatment; lane b, 3 ␮M ionomycin medium; lane c, 3 ␮M A23187 medium; lanes d– h, 3 ␮M A23187 plus 90 nM calmidazolium, 90 ␮M W-13, 90 ␮M W-12, 10 ␮M L-NAME, and 10 ␮M D-NAME, respectively. CNT, control blot of 3 ␮M A23187 medium without anti-HGF primary antibody.

However, the interesting question is whether the signaling pathway leading to release of HGF is the same for the muscle fiber as the calcium ion-induced pathway we have partially described in the cultured satellite cell model. It may be that, under physiological conditions, HGF is released from muscle fibers and satellite cells. At this point, we can only say that satellite cells are capable of releasing and responding to HGF when stretched. The answer awaits experiments similar to those reported herein using isolated fibers or fiber bundles. Muscle fiber damage, focal damage, or extensive damage that results in fiber death are characterized by an elevation in intracellular calcium ions, and, with nNOS in close apposition to the sarcolemma, a burst of NO that could be synthesized would diffuse into the extracellular realm. Anderson was first to present this model (3), and the assumption was that NO is being generated primarily by the fiber. The physiological significance of the calcium-stimulated pathway described using cultured satellite cells remains to be verified in muscle fibers, and so does the role of MMP2 in release of HGF that has been described recently in isolated satellite cells in culture (33, 34). Nonetheless, it has been demonstrated that, in isolated satellite cells, an influx of calcium ions can bind calmodulin, activate NOS, and cause the release of HGF, resulting in activation.

SATELLITE CELL CALMODULIN Foundation and the Research Grant for Young Investigator of Faculty of Agriculture, Kyushu University (all to R. Tatsumi). The research was also supported by funds from the Arizona Agriculture Experiment Station and grants from the US Department of Agriculture National Research Initiative Competitive Grant 2005-35206-15255 and Muscular Dystrophy Association Grant MDA3685 (all to R. E. Allen).

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