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(The FASEB Journal. 2005;19:1986-1997.)
© 2005 FASEB

Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase-3 activity

Nia Wedhas*, Henry J. Klamut*,{dagger}, Charu Dogra*, Apurva K. Srivastava*,{dagger}, Subburaman Mohan*,{dagger},{ddagger} and Ashok Kumar*,{dagger},1

* Molecular Genetics Division, Musculoskeletal Disease Center, Jerry L. Pettis VA Medical Center, Loma Linda, California, USA;
{dagger} Department of Medicine, and
{ddagger} Department of Biochemistry, Loma Linda University, Loma Linda, California, USA

1Correspondence: Molecular Genetics Division, Musculoskeletal Disease Center, Jerry L. Pettis VA Medical Center, 11201 Benton St. (151), Loma Linda, CA 92354, USA. E-mail: Ashok.Kumar2{at}med.va.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanosensitive cation channels (MSC) are ubiquitous in eukaryotic cell types. However, the physiological functions of MSC in several tissues remain in question. In this study we have investigated the role of MSC in skeletal myogenesis. Treatment of C2C12 myoblasts with gadolinium ions (MSC blocker) inhibited myotube formation and the myogenic index in differentiation medium (DM). The enzymatic activity of creatine kinase (CK) and the expression of myosin heavy chain-fast twitch (MyHCf) in C2C12 cultures were also blocked in response to gadolinium. Treatment of C2C12 myoblasts with gadolinium ions did not affect the expression of either cyclin A or cyclin D1 in DM. Other inhibitors of MSC such as streptomycin and GsTMx-4 also suppressed the expression of CK and MyHCf in C2C12 cultures. The inhibitory effect of gadolinium ions on myogenic differentiation was reversible and independent of myogenic cell type. Real-time-polymerase chain reaction analysis revealed that inhibition of MSC decreases the expression of myogenic transcription factors MyoD, myogenin, and Myf-5. Furthermore, the activity of skeletal {alpha}-actin promoter was suppressed on MSC blockade. Treatment of C2C12 myoblasts with gadolinium ions prevented differentiation-associated cell death and inhibited the cleavage of poly (ADP-ribose) polymerase and activation of caspase-3. On the other hand, delivery of active caspase-3 protein to C2C12 myoblasts reversed the inhibitory effect of gadolinium ions on myogenesis. Our data suggest that inhibition of MSC suppresses myogenic differentiation by inhibiting the caspase-3 activity and the expression of myogenic regulatory factors.—Wedhas, N., Klamut, H. J., Dogra, C., Srivastava, A. K., Mohan, S., Kumar, A. Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase-3 activity.


Key Words: mechanosensitive cation channels • gadolinium • myogenesis • C2C12 • caspase-3 • apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MECHANOSENSITIVE CATION CHANNELS (MSC) have been described in a wide variety of cells in many different organisms ranging from bacteria to mammals. MSC allow the passage of ions like Na+, K+, Mg2+, and Ca2+ and participate in several physiological processes such as touch and pain sensation, salt and fluid balance, blood pressure control, cell volume regulation, and turgor control (1) . Activation of MSC may represent an important transduction mechanism that converts mechanical forces exerted on the cell membrane into electrical and biochemical signals in physiological processes (2) . Recent reports from our group and others have demonstrated that MSC activate second messengers and modulate gene expression in mammalian cells (3 4 5) . Abnormal regulation of MSC may contribute to the pathogenesis of several diseases including muscular dystrophy, cardiac arrhythmias, and glioma (6) .

Skeletal muscle formation or myogenesis is a complex and highly regulated process that involves the determination of multipotential mesodermal cells to give rise to myoblasts, exit of these myoblasts from the cell cycle, and their differentiation into muscle fibers (7 , 8) . Myogenesis is regulated by the sequential expression of myogenic regulatory factors (MRFs), a group of basic helix-loop-helix (bHLH) transcription factors that include MyoD, Myf-5, myogenin, and MRF4 (9 , 10) . MyoD and Myf-5 are the primary MRFs required for the formation, proliferation, and survival of myoblasts whereas myogenin and MRF-4 act late during myogenesis and activate expression of important muscle-specific genes, such as myosin heavy chain and creatine kinase (CK) (11 , 12) .

Skeletal muscle differentiation clearly requires the coordination of multiple signaling pathways that regulate cell cycle withdrawal and specify myogenesis (i.e., activate MRFs). A promyogenic role has been assigned to mitogen-activated protein kinase (MAPK) pathways, particularly p38 MAPK (13 14 15) , the phosphoinositide 3-kinase/Akt (16 17 18) , and the calcineurin-NFATc3 (nuclear factor of activated T-cells cytoplasmic 3)-dependent pathways (19) .

Some earlier studies demonstrated that embryonic muscle precursor cells undergo temporally regulated disintegration, a process later referred to as programmed cell death or apoptosis (reviewed in ref 20 ). Subsequent in vitro studies further revealed that a significant fraction of myoblasts undergo apoptosis during the differentiation of C2C12 myoblasts, whereas the differentiated C2C12 myotubes are relatively resistant to apoptosis (21 , 22) . It was recently shown that the activity of caspase-3, a key apoptotic serine protease, plays an important role during myogenic differentiation. Genetic deletion of caspase-3 in mice and in vitro treatment of myogenic cells with caspase-3 inhibitor suppressed myoblast fusion and myotube formation (23 , 24) .

Although MSC have been reported in cultured primary myoblasts/myotubes (25 26 27 28 29) and myoblastic cell line C2C12 (30 31 32) , the role of MSC in myogenic differentiation remains unknown. During myogenic differentiation myoblasts undergo striking morphological changes: they elongate, align with their neighbors, and finally fuse to form multinucleated myotubes. We hypothesize that the morphological changes during myogenesis impose mechanical strain on the myoblast membrane leading to the activation of MSC and subsequent transport of the cations required for myogenesis through these channels. Indeed, it has been reported that a calcium influx into myoblasts before differentiation occurs through gadolinium-sensitive MSC (32 , 33) . Using blockers of MSC such as gadolinium, streptomycin, and GsTMx-4 peptide, we report here that the activity of MSC is required for myotube formation and the expression of muscle-specific genes. Inhibition of MSC decreases the expression of MRFs required for the induction of the myogenic program. Our data also suggest that inhibition of MSC suppresses myogenesis by inhibiting the activation of caspase-3 and differentiation-associated apoptosis in myogenic cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Ham’s F-12 nutrient mixture, Dulbecco’s modified Eagle’s medium (DMEM), and LipofectAMINE2000 were obtained from Invitrogen (Carlsbad, CA, USA). Antibody against cyclin A and cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal ß-actin antibody, streptomycin sulfate, gadolinium (III) chloride, protease inhibitors cocktail, horse serum, and fetal bovine serum (FBS) were from Sigma Chemical Co. (St. Louis, MO, USA). Cleaved poly (ADP-ribose) polymerase (PARP) (Asp214) antibody (mouse-specific) was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse monoclonal MF20 antibody specific to myosin heavy chain-fast twitch (MyHCf) protein was obtained from the Developmental Studies Hybridoma Bank of University of Iowa. GsTMx-4 peptide was obtained from Peptides International Inc. (Louisville, KY, USA). EnzChek Caspase-3 assay kit and Alexa Fluor 546 goat anti-mouse antibody were purchased from Molecular Probes (Eugene, OR, USA). Recombinant human active caspase-3 protein was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Pro-JectTM protein transfection reagent kit was from Pierce (Rockford, IL, USA). CK assay kit was obtained from Stanbio Laboratory (Boerne, TX, USA). Luciferase activity assay kit was purchased from Promega (Madison, WI, USA). Dr. Lei Wei of Baylor College of Medicine (Houston, TX, USA) provided SK-Luc plasmid.

Myogenic cell lines
C2C12 and L6 myoblastic cell lines were obtained from American Type Culture Collection (Rockville, MD, USA). The cells were grown at 37°C in a CO2 incubator in DMEM containing 10% FBS. The differentiation in C2C12 myoblasts was induced by replacing the medium with differentiation medium (2% heat-inactivated horse serum in DMEM). On the other hand, the differentiation of L6 myoblast was induced by incubation in DMEM containing 2% FBS. The medium of the cells was replaced with fresh differentiation medium (DM) after every 48 h during differentiation. All culture media were also supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin.

Primary myoblast cultures
Neonatal mice (3 to 5 days old) were killed by rapid exsanguinations using methods approved by the Institutional Animal Care and Use Committee of Loma Linda VA Medical Center. Diaphragm and limb skeletal muscles were excised, minced in minimum volume of phosphate-buffered saline (PBS), and enzymatically dissociated in 20 mL dissociation buffer (PBS with 0.1% collagenase and 100 µg/mL DNase) for 30 min at 37°C with intermittent vortexing. The slurry was mixed with an equal volume of growth medium (DMEM with 10% FBS) and allowed to stand at room temperature for 10 min. The supernatant (containing dissociated cells) was collected and the pellet (undigested tissue) was discarded. The dissociated cells were centrifuged at 3000 rpm for 2 min and resuspended in 1.082 g/mL Percoll (Amersham Biosciences, Arlington Heights, IL, USA) for purification through a density gradient (1.050, 1.060, and 1.082 g/mL) by centrifugation at 3300 rpm for 30 min at 20°C. The Percoll gradient was made in a buffer containing 6.8 g/L NaCl, 0.4 g/L KCl, 0.1 g/L MgSO4, 1.5 g/L NaH2PO4, 1.0 g/L dextrose, and 4.76 g/L HEPES (pH 7.3). The band containing myocytes at the interface between 1.060 and 1.082 g/mL Percoll layers was collected. The cells were washed twice with growth medium (GM) and finally resuspended in DF20 medium (one volume Ham’s F-12 nutrient mixture and one volume DMEM supplemented with 20% FBS). Isolated cells were then serially preplated to yield a pure population (>98%) of primary myocyte cultures as confirmed by immunostaining with either desmin or MyoD antibody. The primary myocytes were grown in DF20 medium at 37°C in the presence of 5% CO2. To induce differentiation, the medium was changed to DM (2% heat inactivated horse serum in DMEM) when the myocytes were more then 85% confluent.

Myogenic index determination
As a morphological parameter of muscle differentiation, the myogenic index was defined as the number of nuclei residing in the cells containing three or more nuclei divided by total number of nuclei in hematoxylin stained cells. Cells were washed twice in PBS, fixed with 3.7% formaldehyde in PBS for 10 min, and permeabilized with 0.1% Triton-X 100 in PBS for 5 min. The cells were then stained with hematoxylin for 20 s, followed by washing in running water. The distribution of nuclei in myoblasts and myotubes was measured by counting the nuclei at least at 10 different locations selected randomly using an inverted microscope with counting grid (Olympus, Japan).

Immunofluorescence
The expression of myosin heavy chain fast twitch (MyHCf) was examined by an immunocytochemical method. Briefly, C2C12 myoblasts were grown in a 24-well plate and allowed to differentiate into myotubes. The cells were fixed with 3.7% paraformaldehyde followed by permeabilization with 0.1% Triton-X-100 as described above. After three washes (3 min each) in PBS, the cells were blocked with 1% bovine serum albumin in PBS for 1 h, then incubated with MF-20 antibody (specific to MyHCf protein) at 1:100 dilutions in PBS for 2 h at room temperature. The cells were washed in PBS, incubated with goat anti-mouse IgG-Alexa 546 at a 1:100 dilution for 1 h, and counterstained for nuclei with DAPI for 5 min. Stained cells were analyzed under a fluorescence microscope (Olympus IX 70). Pictures were captured using Olympus MagnaFire Digital Camera and software.

CK assay
CK activity was measured to assess myogenic differentiation biochemically. After appropriate treatments and at the end of the incubation period, cells were washed twice in cold PBS and lysed in lysis buffer (50 Tris-Cl [pH8.0], 200 mM NaCl, 50 mM NaF, 1 mM DTT, 0.3% IPEGAL). Lysates were centrifuged for 4 min at 16,000 g and the supernatant collected was either used immediately for CK assay or stored at -80°C for future use. The protein content in the samples was measured using BioRad protein assay reagent. CK activity was measured using a spectophotometric-based kit (Stanbio Laboratory, Boerne, TX, USA). Specific activity of CK was calculated after correction for total protein and defined as units per milligram of protein (U/mg).

Quantitative real-time polymerase chain reaction (QRT-PCR)
A Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used to extract RNA from cells. Any contaminating DNA was removed using DNA-freeTM kit from Ambion (Ambion, Austin, TX, USA). Quality and quantity of RNA were analyzed using Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) and NanoDrop instrumentation (NanoDrop Technologies, Wilmington, DE, USA). Quantitation of mRNA expression was carried out according to the manufacturer’s instructions (Stratagene, La Jolla, CA, USA) using the SYBR Green method on a 7900 Sequence Detection system from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). Briefly, purified RNA (1 µg) was used to synthesize the first strand cDNA by reverse transcription system using Ambion’s oligo-dT primer and Qiagen’s Omniscript reverse transcriptase according to the manufacturer’s instructions. The first-strand cDNA reaction (0.5 µL) was subjected to real-time PCR amplification using gene-specific primers. The primers were designed according to ABI primer express instructions using Vector NTI software and purchased from Eurogentec (Eurogentec North America, San Diego, CA, USA). The sequences of primers used are as follows:

5'-GGTGAGTCGAAACACGGATCAT-3' (reverse);

Myf-5: 5'-TGAAGGATGGACATGACGGACG-3' (forward) and

5'-TTGTGTGCTCCGAAGGCTGCTA-3' (reverse);

Myogenin: 5'-CATCCAGTACATTGAGCGCCTA-3' (forward) and

5'-GAGCAAATGATCTCCTGGGTTG-3' (reverse);

GAPDH:5'-ATGACAATGAATACGGCTACAGCAA-3'(forward)and

5'-GCAGCGAACTTTATTGATGGTATT-3' (reverse).

Approximately 25 µL of reaction volume was used for the real-time PCR assay that consisted of 2x (12.5 µL) Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA, USA), 400 nM of primers (0.5 µL each from the stock), 11 µL of water, and 0.5 µL of template. The thermal conditions consisted of an initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min, and a final step melting curve of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. All reactions were carried out in triplicate to reduce variation. The data were analyzed using SDS software, version 2.0, and the results were exported to Microsoft Excel for further analysis. Data normalization was accomplished using the endogenous control (GAPDH) and the normalized values were subjected to a 2-{Delta}{Delta}Ct formula to calculate the fold change between the control and experiment groups. The formula and its derivations were obtained from the ABI Prism 7900 Sequence Detection System user guide.

Western blot
Immunoblotting for MyHCf, cyclin A, cyclin D1, PARP, and ß-actin was performed as described (34 , 35) .

Cell viability assay
Cellular viability/proliferation was measured either using AlamarBlue dye, which measures the metabolic activity of live cells, or by counting the total number of viable cells using a counting chamber and Trypan blue dye exclusion as described (36 , 37) . Cells were seeded into 24-well plates and treated with test substances. After an appropriate period of incubation, media of the cells was removed and 500 µL of 10% AlamarBlue (BioSource International, Camarillo, CA, USA) diluted in phenol red free DMEM was added. Fluorescence was determined 2 h later using a fluorescent plate reader (Fluorolite 1000; Dynex Technologies, Chantilly, VA, USA). AlamarBlue was evaluated using the optimal excitation and emission wavelengths of 546 and 590 nm, respectively.

Caspase-3 activity assay
The activity of caspase-3 was determined using EnzChek Caspase-3 assay kit from Molecular Probes. Briefly, 100 µg proteins (in 50 µL lysis buffer) were added to the 50 µL reaction buffer containing 50 µM Z-DEVD-R110 substrate. Samples were incubated at room temperature for 4 h and the enzyme-catalyzed release of R100 was measured using the optimal excitation and emission wavelengths of 496 and 520 nm, respectively.

Transfection assays
C2C12 myoblasts were grown in a 6-well plate to 40–60% confluence and transfected with SK-Luc (2 µg/well) using LipofectAMINE2000 (Invitrogen). Transfection efficiency was controlled by cotransfection of myoblasts with pSV-ß-galactosidase (0.1 µg/well). When >90% confluent, the cells were differentiated by changing medium to DM in the presence of increasing concentrations of gadolinium for 4 days. Specimens were processed for luciferase and ß-galactosidase expression using the luciferase and ß-galactosidase assay systems with reporter lysis buffer as per the manufacturer’s instructions (Promega, Madison, WI, USA). Luciferase measurements were made using a luminometer (Analytic Scientific Instrumentation, Model 3010). In another experiment designed to understand the involvement of caspase-3 in myogenic differentiation, activated caspase-3 or ß-galactosidase protein (10 ng) (R&D Systems, Inc.) was transfected into subconfluent C2C12 myoblasts using Pro-JectTM protein delivery reagent following the protocol suggested by the manufacturer (Pierce, Rockford, IL, USA).

Statistical analysis
All experiments were repeated at least three times unless otherwise indicated. Results are expressed as mean ± standard deviation (SD). The Student’s t test or ANOVA was used to compare quantitative data populations with normal distributions and equal variance. A value of P <0.05 was considered statistically significant unless otherwise specified.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Numerous patch clamping and biochemical studies have shown that MSC can be blocked by the trivalent cation gadolinium, the cationic antibiotic streptomycin, and a recently identified peptide GsTMx-4. Using these reagents, we have investigated the role of MSC in myogenic differentiation. The concentrations of gadolinium, streptomycin, and GsTMx-4 used in this study have been reported to effectively block MSC (38 39 40) .

Gadolinium inhibits myotube formation in C2C12 cultures
To investigate the role of MSC in myogenic differentiation, we analyzed the effects of gadolinium ions on myoblast fusion. C2C12 myoblasts were incubated in DM for 96 h in the presence of 100 µM gadolinium ions. A phase contrast microscopy was used for myotube formation whereas a fluorescence microscopy was used to study MyHCf protein expression. Myotube formation and the expression of MyHCf in C2C12 cultures were substantially reduced in the presence of gadolinium ions (Fig. 1 A). We also calculated the myogenic index, i.e., the fraction of total nuclei residing in cells containing ≥3 nuclei. As shown in Fig. 1B , treatment of C2C12 myoblasts with gadolinium ions significantly decreased the myogenic index compared with the untreated myoblasts incubated in DM alone. These data suggest that inhibition of MSC prevents myoblast fusion in vitro.



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  		Figure 1. Inhibition of MSC inhibits myotube formation in C2C12 cultures. C2C12 myoblasts were incubated in differentiation medium (DM) in the presence or absence of 100 µM gadolinium ions for 96 h. A) Images of gross morphology (upper panel), and double labeling with MF-20 (middle panel) and DAPI nuclear dye (bottom panel) demonstrate that gadolinium inhibits myotube formation in C2C12 cultures in response to DM. B) The myogenic index (percentage of total nuclei associated with myotubes) was also significantly lower in the gadolinium-treated C2C12 cultures compared with C2C12 cultures incubated in DM alone (*P<0.01). GM, growth medium; DM, differentiation medium.

Inhibition of MSC inhibits CK activity and MyHCf expression in C2C12 cultures
Because CK and MyHCf are important biochemical markers for muscle differentiation (41) , we next studied the effect of MSC inhibition on the levels of CK and MyHCf in differentiating myoblasts. C2C12 myoblasts were incubated in DM in the presence of increasing concentration of gadolinium ions for 96 h. The enzymatic activity of CK was measured using a commercially available kit whereas the expression of MyHCf was determined by immunoblotting with MF20 antibody. Treatment of C2C12 myoblasts with gadolinium ions significantly inhibited the CK activity and the expression of MyHCf protein in a dose-dependent manner (Fig. 2 A, B). There was no effect on CK activity when sham-treated lysates were incubated with equimolar concentration of gadolinium ions (data not shown).



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  		Figure 2. Gadolinium inhibits CK activity and MyHCf expression in C2C12 cultures. C2C12 myoblasts were incubated in DM for 96 h with increasing concentrations of gadolinium ions. The data presented here show that gadolinium ions inhibited A) the specific activity (expressed as U/mg protein) of CK (*P<0.01) and B) the expression of MyHCf in a dose-dependent manner (upper panel) without affecting the expression of an unrelated protein ß-actin (lower panel). Treatment of C2C12 myoblasts with 100 µM gadolinium ions also inhibited C) the specific activity of CK (*P<0.05) and D) the expression of MyHCf without affecting the expression of either cyclin A, cyclin D1 (middle panels), or ß-actin (lower panel) after incubation in DM. GM, growth medium; DM, differentiation medium.

In another set of experiments, we monitored the effects of gadolinium treatment on the expression of MyHCf and CK activity in C2C12 cultures at different time points after the induction of differentiation. Although CK activity in gadolinium-treated C2C12 cultures was lower than control cultures at all time points, a significant difference in CK activity was observed only after 48 h (Fig. 2C ). On the other hand, expression levels of MyHCf were lower in gadolinium-treated C2C12 cultures at all time points studied (Fig. 2D , upper panel).

To examine whether gadolinium treatment inhibits myogenic differentiation by augmenting cellular proliferation, we investigated the effect of gadolinium ions on the expression of cyclin A and cyclin D1. Proliferating C2C12 myoblasts (0 h) expressed high levels of both cyclin A and cyclin D1. Incubation of C2C12 in DM significantly reduced the cellular levels of cyclin A and cyclin D1. However, no significant differences in cyclin A or cyclin D1 levels were observed between control and gadolinium-treated C2C12 cultures in response to DM (Fig. 2D , middle panels).

We also studied the effects of streptomycin and GsTMx-4 peptide (other known blockers of MSC) on the differentiation of C2C12 myoblasts. Treatment of C2C12 myoblasts with streptomycin inhibited CK activity and the expression of MyHCf in a dose-dependent manner (Fig. 3 A, B). GsTMx-4 also inhibited CK activity (Fig. 3C ) and the expression of MyHCf (Fig. 3D ) in C2C12 cultures. However, the effect was partial compared with either gadolinium ions or streptomycin.



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  		Figure 3. Effect of streptomycin and GsTMx-4 peptide on differentiation of C2C12 myoblasts. C2C12 myoblasts were incubated in DM with the indicated concentration of either streptomycin or GsTMx-4 peptide for 96 h and CK activity and MyHCf expression were measured. The data presented here show that streptomycin inhibited the A) CK activity (*P<0.01) and B) the MyHCf expression in a dose-dependent manner. Only marginal decreases in C) CK activity and D) MyHCf expression were observed in response to GsTMx-4 treatment.

Inhibitory effect of gadolinium ions on myogenic differentiation is reversible and independent of myogenic cell type
C2C12 myoblasts were incubated in DM with 100 µM gadolinium ions. After 48 h, the cells were washed with PBS and incubated in DM with or without gadolinium ions. As shown in Fig. 4 A, removal of gadolinium ions after 48 h significantly restored myogenic differentiation as evidenced by an increase in CK activity.



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  		Figure 4. Inhibitory effect of Gadolinium ions is reversible and independent of myogenic cells. A) C2C12 myoblasts were first incubated in DM with gadolinium ions (100 µM). After 48 h, the cells were washed and incubated again for 48 h in DM either without or with 100 µM Gd3+. At the end of the incubation, CK activity was measured. The data show that removal gadolinium ions after 48 h of treatment significantly restored CK activity in C2C12 cultures (*P<0.01). B) Mice primary myoblasts and L6 myoblasts were incubated in DM with 100 µM gadolinium ions for 96 and 144 h, respectively. At the end of the incubation, the cells were lysed and CK activity was measured. The data show that gadolinium treatment significantly inhibited CK activity in primary myoblasts (*P<0.05) and L6 myoblasts (#P<0.01) cultures. DM, differentiation medium.

To understand whether the inhibition of myogenic differentiation by gadolinium ions was specific to C2C12 myoblasts, we investigated the effect of gadolinium ions on the differentiation of mouse primary myoblasts and L6 myoblasts. As shown in Fig. 4B , gadolinium treatment also significantly blocked the differentiation of primary mouse myoblasts and L6 myoblasts.

Gadolinium inhibits the expression of MyoD, myogenin, and Myf-5
Since the activation of MRFs is a prerequisite for myogenesis, we next investigated the effect of blocking MSC on the expression of MyoD, myogenin and Myf-5. C2C12 myoblasts were incubated in DM alone or with 100 µM gadolinium ions. The expression of MyoD, myogenin, and Myf-5 mRNA was measured using QRT-PCR. A sharp increase in the mRNA levels of MyoD and myogenin but not Myf-5 was observed upon incubation of C2C12 myoblasts in the DM (data not shown). However, the expression levels of either MyoD or myogenin or Myf-5 were suppressed in response to gadolinium treatment (Fig. 5 ). These data suggest that MSC regulate myogenic differentiation by influencing the MRF transcript levels.



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  		Figure 5. Effect of gadolinium ions on the expression of Myf-5, myogenin, and MyoD. C2C12 myoblasts were incubated without or with 100 µM gadolinium ions for 36 h. The expression of Myf-5, myogenin, and MyoD was studied using QRT-PCR as described in Materials and Methods. The data presented here show that expression of MyoD, Myf-5, and myogenin was significantly decreased in response to gadolinium treatment of C2C12 myoblasts (*P<0.05).

Gadolinium inhibits skeletal {alpha}-actin promoter activity
To assess whether inhibition of MSC also modulates the expression of structural genes that are normally up-regulated during myoblast differentiation, the effects of gadolinium treatment on {alpha}-actin promoter activity was studied. C2C12 myoblasts were transfected with SK-Luc, a luciferase expression vector driven by the skeletal {alpha}-actin promoter (42 , 43) . The transfected cells were allowed to differentiate for 96 h in DM containing increasing concentrations of gadolinium ions. The cells were then lysed and assayed for luciferase activity. Incubation of C2C12 myoblasts in DM led to expression of high amounts of luciferase. However, treatment of C2C12 myoblasts with gadolinium significantly inhibited the luciferase expression in a dose-dependent manner (Fig. 6 ). There was no change in luciferase activity when sham-treated cell lysates were incubated with different concentrations (10–100 µM) of gadolinium ions (data not shown).



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  		Figure 6. MSC blockade inhibits skeletal {alpha}-actin promoter activity. C2C12 myoblasts were transfected with SK-Luc, a luciferase reporter vector driven by the skeletal {alpha}-actin promoter, along with pSV-ß-galactosidase as described in Materials and Methods. Transfected cells were differentiated in presence of increasing concentration of gadolinium ions for 96 h. At the end of the incubation, the cells were lysed and processed for luciferase activity assay. The light units (normalized to protein) as measure of luciferase activity are presented. The data show that treatment of C2C12 myoblasts with gadolinium ions inhibited the activity of skeletal {alpha}-actin promoter in a dose-dependent manner (*P<0.05).

Gadolinium inhibits the differentiation-associated cell death in C2C12 cultures
Myogenic differentiation precedes severe apoptosis of myoblasts. This process is mediated at least in part by a family of serine proteases called caspases. We investigated the effect of MSC blockade using gadolinium ions on the viability of C2C12 myoblasts in response to reduced serum. C2C12 myoblasts were incubated with gadolinium ions in either GM or DM. After 24 h the viability of the cells was measured using AlamarBlue dye (measures metabolic activity of the cells) or by counting the total number of viable cells using Trypan blue dye exclusion methods. Gadolinium ions did not affect the viability of C2C12 myoblasts in GM (Fig. 7 A, B). On the other hand, treatment with gadolinium ions significantly increased the viability of C2C12 myoblasts in DM (Fig. 7A, B ). The level of cleaved PARP, an important marker of apoptosis, was also decreased in gadolinium-treated C2C12 myoblasts (Fig. 7C ). These data thus suggest that inhibition of MSC prevents cell death in response to reduced serum.



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  		Figure 7. MSC blockade prevents differentiation-associated cell death in C2C12 myoblasts. C2C12 myoblasts cultured in 24-well plates (50,000/well) were incubated in either GM or DM without or with 100 µM Gd3+ ions for 24 h. A) Cellular viability was measured using AlamarBlue dye. A significant decrease in viability was observed in C2C12 cultures incubated in DM alone compared with those incubated in GM alone (*P<0.01). Treatment of C2C12 myoblasts with Gd3+ had no significant effect in GM, but a significant increase in cellular viability was observed upon treatment of myoblast with Gd3+ ions in DM (#P<0.05). B) Number of viable cells were also significantly higher in Gd3+-treated C2C12 cultures compared with untreated C2C12 cultures incubated in DM (#P<0.01). C) A representative immunoblot presented here and quantitative analysis from 3 independent experiments show that the level of cleaved PARA was significantly lower in Gd3+-treated C2C12 cultures compared with untreated controls incubated in DM.

Caspase-3 is involved in the inhibition of C2C12 differentiation on MSC blockade
A recent report suggests that activation of caspase-3 is required for the differentiation of myoblasts into myotubes (23) . We measured the enzymatic activity of caspase-3 in gadolinium-treated C2C12 myoblasts incubated in DM for 24 h. Treatment of myoblasts with gadolinium significantly inhibited the activation of caspase-3 (Fig. 8 A). On the other hand, treatment of sham lysates with equimolar gadolinium ions did not affect the caspase-3 activity (data not shown). We next investigated whether delivery of active caspase-3 protein can overcome the inhibitory effect of gadolinium ions on myogenic differentiation. C2C12 myoblasts were transfected with either active caspase-3 or ß-galactosidase protein. Transfection of recombinant caspase-3 protein resulted in 3.2 ± 0.63-fold increase in CK activity measured after 12 h. Transfected cells were allowed to differentiate in the presence or absence of gadolinium ions in DM for 96 h. Transfection of caspase-3 protein significantly increased the CK activity in gadolinium-treated C2C12 cultures (Fig. 8B ). On the other hand, there was no significant difference in the cellular viability between ß-galactosidase and caspase-3 transfected C2C12 myoblast cultures incubated with gadolinium in DM after 24 h (negative data not shown).



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  		Figure 8. Role of caspase-3 in inhibition of C2C12 myoblast differentiation on MSC blockade. A) C2C12 myoblasts were incubated in either GM or DM without or with 100 µM Gd3+ ions for 24 h and the activation of caspase-3 was measured. The activity of caspase-3 was significantly lower in C2C12 cultures (adherent and floating cells) incubated with Gd3+ compared with those incubated in DM alone (*P<0.05). B) Transfection of active caspase-3 but not ß-galactosidase significantly increased CK activity in Gd3+-treated C2C12 cultures measured after 96 h of incubation in DM (*P<0.05). The data are representative of three independent experiments done at least in triplicate. DM, differentiation medium; GM, growth medium.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mechanosensitive ion channels (MSC) have been described in both prokaryotes and eukaryotes and are implicated in numerous cellular processes (2 , 44) . Despite a wealth of electrophysiological information about them, molecular characterization and elucidation of the role of MSC in mechanosensory transduction in eukaryotes have been bafflingly slow compared with the progress made in our understanding of prokaryotic MSC.

The physiological functions of MSC have been studied using their nonselective blockers, such as gadolinium ions and streptomycin, which block many types of mechano-gated channels in submillimolar concentrations irrespective of their origin, conductance, or selectivity (45 , 46) . Since muscle formation precedes hyperpolarization of the myoblast membrane, a form of mechanical stress, we hypothesized that MSC are activated in myoblasts upon induction of differentiation and that these channels may play a role in myogenesis. An earlier report suggested that gadolinium, a widely used inhibitor of MSC, inhibits phloretin-induced precocious fusion of chick embryonic myoblasts in normal growth medium (26) . Parallel to this published report (26) , we observed that myotube formation in C2C12 cultures was significantly reduced in the presence of gadolinium ions upon induction of differentiation (Fig. 1A ). The quantitative estimation of the distribution of nuclei in myoblasts and myotubes (myogenic index) further confirmed that gadolinium prevents myotube formation in C2C12 cultures (Fig. 1B ). Our immunohistochemical data using MF-20 antibody showed that expression of MyHCf is significantly inhibited in gadolinium-treated C2C12 cultures (Fig. 1A , middle panel). Since the expression of MyHCf is increased during myogenic differentiation, these data indicate that the reduced number of myotubes in gadolinium-treated C2C12 cultures is the result of the inhibition of myogenesis.

The morphological data of myotube formation was confirmed at the biochemical level by measuring the activity of CK and studying the expression of MyHCf in the presence of MSC inhibitors. As expected, a dose- and time-dependent inhibition in both CK activity and the MyHCf expression were seen in the presence of gadolinium ions in C2C12 cultures (Fig. 2) . It is important to recognize that gadolinium ions have high affinity to bind to free bicarbonate and phosphate ions present in several physiological solutions (47) . However, in culture medium (including DMEM used in this study), the phosphate and bicarbonate ions exist as protonated anions that have very low affinity for gadolinium ions (47) . Nevertheless, the possibility that some gadolinium ions are neutralized in the culture medium due to their binding to phosphate and bicarbonate anions cannot be ruled out.

Several cytokines and growth factors inhibit myogenic differentiation by inducing the proliferation of myoblasts (48 49 50) . Recently, we reported that cyclic mechanical strain prevents myogenesis by augmenting the proliferation of myoblasts and inhibiting their withdrawal from the cell cycle (37) . The inhibition of myogenesis on MSC blockade seems to be independent of the cell cycle withdrawal because there was no significant difference in the level of either cyclin D1 or cyclin A in gadolinium-treated C2C12 myoblasts compared with the control myoblasts incubated in DM alone (Fig. 2D , middle panels).

Although most studies of MSC have been performed using their nonselective blockers, GsTMx-4, a peptide isolated from the venom of the spider Grammostola spatulata, has recently been suggested as a specific blocker of MSC currents in mammalian cells (51) . We observed that GsTMx-4 reduced the expression of MyHCf and CK activity in differentiating C2C12 myoblasts (Fig. 3B ). However, GsTMx-4 did not completely block the myogenic differentiation. On the other hand, streptomycin, another inhibitor of MSC, inhibited the differentiation of C2C12 myoblasts (Fig. 3A ). Why GsTMx-4 did not completely suppress the myogenesis similar to gadolinium or streptomycin remains in question, but two possibilities can be discussed. First, in the absence of the exact identity and number of MSC present in mammalian cells, it is possible that GsTMx-4 blocks the activity of only a select few MSC that might not be involved in myogenesis. Second, a recent study showed that GsTMx-4 decreases the inward mechanosensitive single-channel currents but has no effect on outward currents (52) . The myogenic process, on the other hand, might require both inward and outward movement of the cations through MSC.

We also observed that the inhibitory effect of the MSC blockade was reversible. Removal of gadolinium ions from the cultures significantly restored the differentiation of C2C12 myoblasts (Fig. 4A ). Our data are consistent with published reports that suggest that gadolinium-mediated inhibition of MSC is reversible (45) . Furthermore, our data suggest that inhibition of myogenic differentiation was not specific to C2C12 myoblasts. Similar inhibition in CK activity was also observed in primary myoblasts and L6 myoblast cultures upon treatment with gadolinium ions in DM (Fig. 4B ).

Skeletal muscle differentiation can be regulated by at least three possible mechanisms, which include alteration in protein degradation, mRNA stabilization, or gene transcription. Although evidence exists to support the first two mechanisms (48 , 53 , 54) , modulation in gene expression appears to be most important during differentiation. This occurs via the activation of various phosphatases and kinases, which in turn alter the activity of downstream regulatory factors such as MEF2, myf-5, myogenin, MyoD, serum response factor, and nuclear factor of activated T cells (55 56 57) . Since inhibition of MSC decreased the transcripts of MyoD, myf-5, and myogenin (Fig. 6) and also suppressed skeletal-{alpha} actin promoter activity in myoblasts (Fig. 7) , our data suggest that the decreased myogenesis on MSC blockade is the result of decreased transcription of MRFs.

A striking observation of the present investigation is that the blockade of MSC using gadolinium prevented myogenic cell death in response to reduced serum (Fig. 7A, B ). The inhibition of cell death on gadolinium treatment was associated with reduced cleavage of PARP (Fig. 7C ), suggesting that the MSC blockade prevents differentiation-associated apoptosis in myoblasts. It is now well established that the acquisition of apoptosis resistance by myogenic precursors is a prerequisite for their development. Accumulating evidence also suggests that the induction of differentiation and apoptosis in the myogenic lineage may use overlapping cellular mechanisms. Several phenotypic changes such as 1) actin fiber disassembly/reorganization; 2) increased activation of matrix metalloproteinases; and 3) the activation of myosin light chain kinase are common during myoblast differentiation and are apoptotic features of membrane blebbing (ref 23 and references therein).

It has been shown that {alpha}B-crystallin, a small heat-shock protein, suppresses myogenic differentiation-associated apoptosis by inhibiting caspase-3 activation (58) . On the other hand, Fernando et al. have demonstrated that whereas myotube formation and the expression of differentiation markers were drastically reduced in caspase-3–/– myoblasts, there was no difference in either the level of cleaved PARP or the number of annexin V-positive cells between wild-type and caspase-3–/– myoblasts in DM (23) . Our data indicate that MSC blockade using gadolinium suppresses both the differentiation-associated apoptosis (Fig. 7) and the activation of caspase-3 (Fig. 8A ) in C2C12 myoblasts. However, these two events (apoptosis and caspase-3 activity) seem to be independently regulated by MSC blockade. Delivery of active caspase-3 protein to myoblasts significantly increased the myogenic differentiation in gadolinium-treated C2C12 cultures (Fig. 8B ) but had no effect on either cellular viability or PARP cleavage (negative data not shown). Our results thus suggest that the activation of MSC during myogenesis is required for both the ablation of differentiation-incompetent myoblasts and the induction of the myogenic program in apoptosis-resistant myoblasts.

Although our study clearly suggests the role of MSC in myogenic differentiation, the nature of the ions transported through these channels remains in question. The signaling pathways activated in response to MSC activation and that lead to the induction of the myogenic program remain to be investigated. Among various cations, the mobilization of Ca2+ ions through MSC could be essential for the induction of myogenesis. Indeed, Park et al. have reported that Ca2+ influx into myoblasts before differentiation occurs only through the gadolinium-sensitive stretch-activated ion channels (33) . Ca2+ ions mediate a large number of cellular responses by binding to specific intracellular proteins, which may be considered Ca2+ receptors (59 , 60) . Ca2+ ions increase the activity of the calcineurin-NFATc3 signaling pathway, which promotes myogenic differentiation (19 , 61) . Furthermore, elevated levels of cytosolic Ca2+ ions have been found to be sufficient in inducing caspase-3 activity (62) . In summary, this study provides a novel piece of information regarding the role of MSC in myogenic differentiation.


   ACKNOWLEDGMENTS
 
We thank Dr. Lei Wei of Baylor College of Medicine (Houston, TX, USA) for providing the SK-Luc construct. This study was supported by a research grant from Muscular Dystrophy Association to A.K. This work was performed at facilities provided by the Jerry L. Pettis Memorial VA Medical Center in Loma Linda, CA, USA.

Received for publication April 20, 2005. Accepted for publication August 4, 2005.


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 DISCUSSION
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