Transthyretin Is a Key Regulator of Myoblast Differentiation

Transthyretin (TTR) is a known carrier protein for thyroxine (T4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd) did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.


Introduction
Myogenesis is the formation of multinucleated myofiber with a contractile capacity from muscle satellite cells (MSCs). Myogenesis involves cell cycle arrest, myogenic activation, cell alignment, multiple rounds of cell fusion and an increase in size with peripheral localization of the nuclei [1]. This process is highly regulated and involves growth factors, cytoskeletal proteins, and muscle specific transcription factors such as myogenin (MYOG) and myocyte enhancer factor-2 (MEF2) which are regulated by an increase in cytosolic Ca 2+ concentration [2], [3], [4], [5], [6].
Increased intracellular calcium activates intracellular protease, calpains, calcineurine and serine-threonine phosphatase, which plays a critical role in cell migration and fusion to myotube formation [7], [8], [9], [10]. Kubo Y. [11] proposed that T-type voltage-gated calcium channel (VGCC) and inward rectifier K + current increase the basal intracellular Ca 2+ level, which may be essential to the initial stages of mesodermal stem cell differentiation. Ca 2+ through T-type VGCC also contributes to other differentiation processes including neural differentiation [12] and neuroendocrine differentiation of prostate cancer cells [13]. Moreover, there is a great deal of evidence that Ca 2+ influx through T-type VGCCs results in signalling that affects the expression of genes involved in cell proliferation, programmed cell death, and neuronal differentiation [14], [15], [16], [17], [18], [19], [20]. Similarly, L-type VGCC like Cav1.1 is also present in skeletal muscle [21]. In addition to VGCC, human myoblasts can generate Ca 2+ signals by Ca 2+ release from inositol 1,4,5triphosphate-sensitive Ca 2+ stores followed by entry through store operated calcium (SOCE) channels [3]. STIM1and Orai1 are essential component of store-operated Ca 2+ entry (SOCE) that is evoked in response to a fall in Ca 2+ in the endoplasmic reticulum. STIM1is a calcium sensor in endoplasmic reticulum and Orai1 in the plasma membrane [22], [23].
Transthyretin (TTR), which exists in tetrameric form is a carrier protein for thyroxine (T 4 ) and retinol-binding protein in the blood [24], [25], [26]. The liver and choroid plexus of the brain are major organs responsible for the synthesis of TTR in plasma and cerebrospinal fluid, respectively. In addition to the liver and brain, mRNA expression of TTR has been reported in the skeletal muscle of rats [27]. TTR gene knock-out mice increased neuropeptide Y, suggesting that TTR is critical in nervous system [28]. RNA interference targeting TTR in mammalian cells has been found to increase the initial efficacy of neural prosthetic devices before insertion [29]. We recently reported that TTR is induced in bovine primary MSC differentiation [30]. Herein, we investigated the role of TTR during myogenesis in C2C12. Silencing of TTR demonstrated the inability of cell alignment before fusion, leading to the formation of impaired myotubes.

Mouse Tissues
In this study, 6 or 18 weeks old male C57BL/6 mice were used for RNA isolation. Briefly, four week old mice were obtained from Daehan Biolink (Eumseong, Korea) and housed four per cage in a temperature-controlled room with a 12 hr light/12 hr darkness cycle. Throughout the study period, animals were allowed free access to standard rodent chow containing 4.0% (wt/wt) total fat (Rodent NIH-31 Open Formula Auto, Zeigler Bros., Inc., Gardners, PA, USA) and water. At 6 and 18 weeks of age, mice were anesthetized with sodium pentobarbital and exsanguinated. Tissue samples were then collected, quickly frozen in liquid nitrogen, and stored at 280uC until processed for RNA extraction. For immunohistochemistry, mice were anesthetized by intraperitoneal injection of tribromoethanol (Avertin, 250 mg/ kg, Sigma Aldrich CA, USA) for transcardial perfusion with PBS (phosphate buffered saline) to remove the blood. The animals were then perfusion fixed with 10% neutral buffered formalin, after which solid organs and skeletal muscles from the trunk and extremities were removed and post-fixed in the same fixative overnight at 4uC. The fixed organs were then processed for routine paraffin embedding, and the paraffin-embedded tissue blocks were cut to 6-mm thick sections for immunohistochemistry. The experimental protocols for the care and use of laboratory animals were approved by the Institutional Animal Care and Use Committee of Konkuk University.

Cell Culture
C2C12 cells, a murine myoblast cell line, were cultured in DMEM (Dulbecco's modified Eagle's medium; HyClone Laboratories, Logan, UT) supplemented with 10% FBS (fetal bovine serum, HyClone Laboratories) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37uC with 5% CO 2 . For differentiation, cells grown to 70% confluence were switched to differentiation media (DMEM with 2% FBS) and then cultured for 0, 2, 4, and 6 days, during which time the medium was changed every two days. Cells were treated with T4 (50 ng/ml) for 4 and 6 days. C2C12 cells were kindly provided by Korean Cell Line Bank, Republic of Korea.
TTR and MYOG Knock-down C2C12 cells grown in 6-well plates to 30% confluence were transfected with 1 ng of vector, TTR and MYOG shRNA construct per well using transfection reagent and transfection medium (Santa Cruz Biotechnology, CA, USA). After 3 days, the cells were treated with 2 mg/mL Puromyocin (Santa Cruz Biotechnology) for selection. Selected cells were grown upto 70% confluence before switching to differentiation media. Sequences of shRNA constructs are provided in Table S1.

Fusion Index
Fusion index was analyzed as previously described [31], [32]. Cell nuclei were stained with Giemsa G250 (Sigma Aldrich) and pictures were captured randomly at three different spots. Further, the number of nuclei in myotubes and the total number of nuclei in cell were counted in each field. Fusion index was calculated as the percentage of total nuclei incorporated in myotubes vs. total number of nuclei.

RNA Extraction and Real Time RT-PCR Analysis
Total RNA was extracted from the cells and tissues using Trizol TM reagent (Invitrogen) according to the manufacturer's protocol and then stored in diethylpyrocarbonate-treated H 2 O at 280uC until used. One microgram of RNA in a reaction mixture with a total volume of 20 ml was primed with oligo (dT) 20 primers (Bioneer, Daejeon, Korea) and then reverse transcribed at 42uC for 50 min and 72uC for 15 min. Subsequently, 2 ml of cDNA product and 10 pmoles of each gene-specific primer were used to perform PCR, which was conducted using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Power SYBRH Green PCR Master Mix (Applied Biosystems) was used as the fluorescence source. Primers were designed with the Primer 3 software (http://frodo.wi.mit.edu) using the sequence information listed at the National Center for Biotechnology Information. The detailed information about primer sequences are provided as Table S2.

Immunocytochemistry
C2C12 cells grown in a covered glass-bottom dish were treated with differentiation medium and stained for TTR, MYOG, Cav1.1, and Cav3.1 protein following 0, 2, 4 and 6 days of incubation. Briefly, cells were rinsed with PBS and fixed with 4% formaldehyde, after which they were permeabilized with 0.2% Triton X-100 (Sigma Aldrich). The cells were then incubated with primary antibody (rabbit polyclonal IgG TTR (1:50), mouse monoclonal IgG MYOG (1:50), mouse monoclonal IgG Cav1.1 (1:50), and rabbit polyclonal IgG Cav3.1 (1:50), Santa Cruz Biotechnology) at 4uC in a humid environment overnight. Secondary antibody (1:100; Alexa Fluor 488 goat anti-rabbit and goat anti-mouse SFX kit; Molecular Probes, Eugene, OR, USA) was then applied for 1 hr at room temperature. The samples were rinsed with PBS, after which the nuclei were counterstained with 49 69-diamino-2phenylindole (DAPI; Sigma-Aldrich). Pictures were taken using a fluorescent microscope equipped with a digital camera (Nikon). Detail information about clone names of monoclonal antibodies are provided as Table S1.

Western Blot Analysis
Cells treated with differentiation media and cultured for different time periods were subjected to Western blot analysis. Briefly, after incubation, cells were washed with ice-cold PBS and lysed in RIPA lysis buffer containing protease inhibitor cocktail (Thermo Scientific, NH, USA). Total protein was isolated by centrifugation of the lysate at 13000 rpm for 10 min at 4uC, after which the protein concentrations were determined by the Bradford method using protein assay dye solution [33]. Total protein (40 mg) heated at 90uC for 3 min with ß-mercaptoethanol (Sigma-Aldrich) was electrophoresed in 10% SDS-polyacrylamide gel and then transferred to PVDF membrane (Millipore, MA, USA). The blots were subsequently blocked with 5% skim milk in TBST for an hour and then incubated overnight with TTR (1:500), MYOG (1:1000) or ß-actin antibody (1:2000) (Santa Cruz Biotechnology) diluted with 3% skim milk in TBS at 4uC. Blots were next washed in TBST and incubated with horseradish peroxidase conjugated secondary antibody for an hour at room temperature, after which they were washed as described above and developed using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific).

Patch-clamp Analysis
Cells were transferred into a bath mounted on the stage of an inverted fluorescence microscope (Ti-U; Nikon Instruments, Inc., Melville, NY, USA). The bath (approximately 0.20 mL) was superfused at 5 mL/min, and voltage clamp experiments were performed at room temperature (22uC-25uC). Patch pipettes with a free-tip resistance of approximately 2.5 MOhm were connected to the head stage of a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Inc., Sunnyvale, CA, USA). The pCLAMP software v.10.2 and Digidata 1440 (Molecular Devices, Inc.) were used to acquire data and apply command pulses. Whole-cell currents were recorded at 10 kHz and low-pass filtered at 5 kHz. Current traces were stored and analyzed using Clampfit v.10.2 and Origin v. 8.0 (Microcal Inc, Northampton, MA, USA). For comparison of whole-cell currents between cells, the current amplitudes were normalized to the membrane area measured by electrical capacitance. The maximum absolute value of the current obtained (in pA) was divided by the cell capacitance (in pF). The pipette solution for the whole-cell patch clamp contained 100 mM Cs-aspartate, 32 mM CsCl, 10 mM ethylene glycol-bis (2aminoethylether)-N,N,N9,N9-tetraacetic acid (EGTA), 5 mM Mg-ATP, and 10 mM HEPES at pH 7.2 (titrated with CsOH). The bath solution for whole-cell recording of the VGCC contained 120 mM NaCl, 5 mM CsCl, 10 mM TEA-Cl, 10 mM BaCl 2 , 10 mM glucose, 0.5 mM MgCl 2 , and 10 mM HEPES at pH 7.4 (titrated with NaOH).

ELISA
Cell lysates extracted from cells differentiated for different lengths of time were used for ELISA to measure the T 4 concentration (DRG International, Inc. Margurg, Germany). Cell lysate and enzyme conjugate were added in a specific antibodycoated microtiter and then incubated for 1 hr at room temperature. The mixture was subsequently removed and washed to remove the unbound samples. After addition of the substrate solution for 20 min, the reaction was terminated by adding stop solution and the color intensity was measured using a spectrophotometer at 450 nm (Tecan Group Ltd. Switzerland).

Immunohistochemistry
TTR expression in mouse tissues was evaluated by immunohistochemistry using a TTR antibody (Santa Cruz Biotechnology). Briefly, paraffin-embedded tissue sections were deparaffinized, hydrated, and then quenched for endogenous peroxidase activity. The sections were blocked with 5% goat serum in PBS, after which they were incubated with the TTR antibody (2 mg/mL, Santa Cruz Biotechnology) overnight at 4uC. The sections were incubated with biotinylated anti-goat IgG (Vector, CA, USA) and subsequently with horseradish peroxidase-conjugated streptavidin (Vector). Positive signals were visualized by adding diaminobenzidine and hydrogen peroxide as substrates. A negative control experiment was also carried out by omitting the primary antibody. Stained sections were counterstained with methyl green and then dehydrated, mounted, and examined using a light microscope.

Statistical Analysis
The normalized expression means were compared using Tukey's Studentized Range (HSD) to identify significant differences in gene expression. A nominal p-value of less than 0.05 was considered to be statistically significant. Real time RT-PCR data were analyzed by one-way ANOVA using PROC GLM in SAS package ver. 9.0 (SAS Institute, Cary, NC, USA).

TTR Expression and Localization in Mouse Tissues
TTR mRNA expression was observed in liver, muscle and brain tissues of different aged mice. The mRNA expression of the TTR gene was higher in 18 week old mice than 6 week old mice in muscle and liver tissues, with higher values being observed in liver tissue. The myogenesis marker genes, MYOG and MYL2 (Myosin light chain 2), being marker genes were expressed in higher levels and at earlier stages in muscle tissues. Moreover, Cav1.1, STIM1 (Stromal interaction molecule 1) and Orai (Calcium release-activated calcium channel protein 1) were found to be highly expressed in muscle tissues (Fig. 1A). All the skeletal muscles stained for TTR, including forelimb, hind limb and trunk muscles, showed higher levels of TTR expression (Fig. 1B). Although the TTR expression was observed within the cytoplasm, but the intensity was not even throughout the muscle fibers.

TTR is Expressed during Myoblast Differentiation
To investigate the role of TTR during C2C12 myoblast differentiation into myotubes, cells were cultured and differentiated at different time points, after which their mRNA expression was analyzed by real time RT-PCR. The data after normalization revealed a more than 4 fold increase in the expression level of TTR at day 2, which remained almost constant to day 6 ( Fig. 2A). The expression level of proteins was verified by Western blot and immunostaining (Fig. 2B, 2C). The cells were confirmed to be in the stage of myotube formation by checking the mRNA and protein expression of MYOG at different time points during cell differentiation (Fig. 2D, 2E, 2F).

TTR Knock-down Affects Myotube Formation
To assess the previously unrecognized role of TTR during myogenesis, cells were transfected either with 1 ng of TTR shRNA (TTR kd ) or vector GFP (TTR wd ). As shown in Fig. 3A, TTR kd significantly reduced mRNA expression of the genes involved in myogenesis at terminal differentiating stages, such as MYOG and MYL2. Myf5 and MyoD, which are known to be involved in earlier stages of myogenesis, were unaffected at day 4. However, expression of MyoD was reduced significantly upto 75% when checked at day 2 (Fig. S1). shRNA transfection against TTR prevented the formation of myotubes as well as the decrease in the cytoplasmic distribution pattern of TTR and nuclear expression of MYOG protein (Fig. 3B, 3C: inset). The expression pattern obtained using immunoblot showed decreased levels of protein in TTR kd cells (Fig. 3D). Fusion index calculated at day 4 after TTR kd also agrees with the above observation, showing approximately 50% reduction as compared to TTR wd (Fig. 3E). In contrast, MYOG knock-down during differentiation did not have any effect on TTR mRNA expression (Fig. 3F). MYOG knockdown was also verified at the protein level by immunostaning and immunoblot analysis (Fig. 3G, 3H).

Voltage-gated Calcium Currents and Calcium-dependent Gene Expression
Calcium plays a critical role in multiple steps associated with myotube formation. In this study, to identify the functional expression of voltage-dependent Ca 2+ channels (VDCCs) during myogenesis, the whole-cell patch-clamp recording of C2C12 cells at different time intervals was performed. Small T-type Ca 2+ currents were detected in control cells at day 0 (Fig. 4A). However, only ,54% of the cells expressed measurable T-type Ca 2+ currents. After five days of differentiation, in addition to T-type current, a distinct L-type Ca 2+ current was detected (Fig. 4B) in response to 500 ms depolarizing pulses from a holding potential of 280 mV. Fig. 4B shows representative currents recorded at 230 mV (red line), where the T-type current shows the maximum curve (black arrow), and at 10 mV (blue line) where the L-type current shows a peak current amplitude (red arrow). Fig. 4C and 4D are the individual graphs obtained from the superimposed L/ T type calcium currents data (Fig. 4B). Fig. 4C shows average current 2 voltage (I-V) relationship for the initial peak Ca 2+ current in T-type channel (black arrow) at different day of culture. Fig. 4D shows an average I-V relationship for the secondary peak Ca 2+ current in L-type channel (red arrow) at different day. Mixed T-and L-type currents were recorded over the 230 mV depolarizing pulse from a holding potential. After day 2, the Ttype Ca 2+ current was detected in most of the control cells (,86%), whereas the L-type Ca 2+ current was detected in ,45% of C2C12 cells. The maximum detection rate (100%) of the T-type Ca 2+ current was attained at day 3 (Fig. 4E). The detection probability of the L-type Ca 2+ current gradually increased during myogenesis, reaching its maximum at day 5 (Fig. 4 E). Current density through T-and L-type Ca 2+ channels increased significantly during myogenesis (Fig. 4F, P,0.05). Although the detection probability of the T-and L-type Ca 2+ current gradually increased during myogenesis, the T-and L-type Ca 2+ current densities did not show a gradual increase (Fig. 4C, 4D). The size of the myotubes was significantly higher in older cultures; however, the current densities of each type of channel showed a transient increase at a specific point and increased only slightly during myogenesis. These electrophysiological data indicate that both Tand L-type Ca 2+ channels are functionally expressed in differentiating C2C12 cells.
Cav1.1, Cav3.1 and Orai1 mRNA showed a gradual increase in expression up to day 4 (Fig. 4G). In contrast, STIM1 peaked at day 2. Localization of protein by immunostaining revealed a similar expression pattern (Fig. 4H). Moreover, the L-type calcium channel was blocked with nifedipine (100 mM) for 4 days during myogenesis and considerable reduction in myotube formation was observed (Fig. 4I). Cav1.1 and MYOG mRNAs expression were significantly reduced by blocker, while TTR was unaffected (Fig. 4J).

TTR Controls the Calcium Channel
It was essential to identify the elements responsible for increased expression rates of T-and L-type Ca 2+ channels during myogenesis and confirm if there was any relationship between the TTR and VGCCs. No specific pharmacological blocker of TTR was available at the time of the study; therefore, TTR-specific shRNA was transfected into C2C12 cells. The prevention of the regulation of these Ca 2+ channels was validated by the whole-cell patch-clamp recordings (Fig. 5A, 5B). As shown in Fig. 5C, the detection probability of T-and L-type Ca 2+ channels was significantly reduced by TTR kd at four days after switching to the differentiation medium. The current density between TTR wd and TTR kd cells was compared among cells expressing T-and Ltype Ca 2+ channels. In the case of TTR kd cells, a significant decrease in detection probability and the current density was observed (Fig. 5D).
The mRNA expression of T-and L-type Ca 2+ channel subunits (Fig. 5E) along with the STIM1 and Orai1 was found to be highly influenced by TTR silencing. The reduction in expression of Cav1.1 in TTR kd cells, demonstrating its direct involvement in myogenesis. In addition, MYOG silencing was also found to affect Ca 2+ entry related genes such as STIM1, Orai1 and Cav1.1 (Fig. 5F), indicating that MYOG regulates these genes.

Cell Differentiation is also Affected by T 4
Thyroxin (T 4 ) is an important constituent of FBS used in cell culture experiments. In this study, the effect of T 4 on the expression of genes involved in myogenesis was investigated. TTR and Cav1.1 expression were enhanced by T 4 treatment, while this effect was small in other genes (Fig. 6A). In wild type cells (TTR wd ), wider myotube formation was observed after T 4 treatment. However, there was no apparent change in the morphology of TTR kd cells with or without T 4 treatment (Fig. 6B). Changes in the T 4 concentration of cells were measured by ELISA (Fig. 6C). A gradual increase in T 4 concentration was observed in TTR kd cells. In contrast, the concentration of T 4 in TTR wd cells increased up to day 4 and then decreased at day 6. Interestingly, the intracellular T 4 concentration of TTR wd cells was significantly higher than that of TTR kd cells at day 4, when C2C12 cells showed peak myotube formation. The increase in intracellular T 4 concentration can be assumed to be due to the presence of T 4 in cell culture media and its transport across the membrane.

TTR-interaction during Myoblast Differentiation
TTR, which is known as a carrier protein for thyroxin and retinol binding protein, has been shown to play a critical role in homeostasis of the nervous system [34]. We previously identified TTR as one of the genes highly up-regulated in different depots of bovine muscle tissue during myogenesis [30]. This was a novel finding, as the protein had previously been reported as a systematic precursor to deposition and amyloid fiber formation. Immunohistochemical analysis was used to detect the presence of TTR protein in skeletal muscles of the forelimb, hind limb and trunk, as well as in the liver (control). The presence was further confirmed by mRNA expression of TTR in different muscle depots (Fig. 1). This study describes the role of TTR as a key factor in myoblast differentiation. In this study, shRNA that silenced TTR, was used to investigate (i) inhibition of myotube formation based on MYOG and MYL2 mRNA expression (ii) changes in mRNA expression of the calcium channel related genes, STIM1, Orai1, Cav1.1 and Cav3.1, and (iii) time dependent occurrence of voltagegated calcium currents during myogenesis. Our results are in accordance with Mock et al [35], in which they demonstrate the decrease in muscle mass of a TTR null mouse.
To investigate the function of TTR in myogenesis, C2C12 cells were transfected with shRNA against TTR and observed for changes in cell morphology and gene expression. Microscopic observations revealed a reduction in myotube formation in TTR silenced cells when compared with wild type (wd) cells. These findings were supported by decrease in TTR mRNA and protein expression observed by real time RT-PCR and immunostaining, respectively. Muscle differentiation is accompanied by specific alterations in the pattern of muscle specific gene expressions [36], particularly those of two groups of transcription factors, the MyoD family (including Myf5, MyoD, MYOG) and the MEF2 family. To confirm this, the mRNA expression of these genes was compared in TTR kd and TTR wd cells. Myf5 was unaltered, whereas variation in the expression of MYOD, MYOG and MYL2 was observed. Myf5 and MyoD are known to exist in the early stages of myoblast differentiation [37], while MYOG is expressed throughout myotube formation and MYL2 is expressed at a later stage. As shown in Fig. 2 and 3, TTR silencing affects the expression of MYOG, which can be assumed to be a result of the early interference of TTR during myogenesis. The effect of TTR on MYL2 also confirms its interference during myogenesis. The time course study revealed that the expression of MYOG and MYL2 was highest on day 4, after the culture medium was replaced with differentiation medium. To confirm the role of TTR in myogenesis via involvement of the calcium channel(s), the mRNA expression of STIM1, Orai1, and VGCCs was studied in detail. Darbelly et al. [38] reported that STIM1 and Orai1-dependent store operated calcium entry (SOCE) plays a crucial role in the regulation of myogenesis in human myoblasts. Specifically, they found a correlation between the amplitude of SOCE and MYOG/MEF2 expression and reported that SOCE was the limiting factor in the signaling cascade that controls the fate of myoblasts. However, SOCE amplitude is regulated by STIM1 and Orai1. In the time course study, Orai1, Cav1.1, and Cav3.1 were found to be upregulated, with their expression peaking at day 4. However, the expression of these genes was down-regulated in TTR kd cells when compared to TTR wd SOCE and VGCC calcium influx functions in reciprocal mechanism pathways. Nevertheless, excitable cells have been found to express SOCE proteins, but contribute little to Ca 2+ influx [39], [40], whereas non-excitable cells express VGCC proteins but lack voltage-gated Ca 2+ currents [41], [42]. The decrease in mRNA expression of SOCE genes, namely STIM1 and Orai1, and the mRNA expression of VGCC genes, namely Cav1.1 and Cav3.1, draws attention to the crucial role played by TTR during myogenesis. These results suggest that TTR initiates myogenesis in both excitable and non-excitable cells.

Effect of myogenin Silencing
Most studies conducted to investigate the process of differentiation have focused on the expression of MYOG, a marker gene involved in myogenesis. In the present study, C2C12 cells were . C) Average current 2 voltage (I-V) relationship for the initial peak Ca 2+ current (T-type, black arrow) at 0 (n = 12), 2 (n = 19), 3 (n = 19), and 5 (n = 20) days after culture in DM. D) Average I-V relationship for the secondary peak Ca 2+ current (L-type, red arrow) at 2 (n = 10), 3 (n = 13), and 5 (n = 19) days after culture in DM. E) Summary of the detection probabilities of L/T-type Ca 2+ current from the patch clamp recording after culture in DM. Note that both the T-and L-type detection rate gradually increases through the time course. F) Histograms of the current density of the T-type calcium current (measured using a test pulse at 230 mV, HP 280 mV) and the L-type (measured at 0 mV). mRNA expression of Ca 2+ channel related genes was analyzed by real-time RT-PCR. G) Up-regulated mRNA expression of calcium channel related genes during myogenesis at different time points. Cav1.1 (L-type), Cav3.1 (T-type) and Orai1 showed maximum expression on day 4, whereas STIM1 on day 2 represents the variation in calcium homeostasis at given time intervals. H) Immunofluorescence labeling indicates the distribution of Cav1.1 and Cav3.1 protein in the cytoplasm on days 0, 4 and 6. The highest expression of Cav1.1 and Cav3.1 protein was observed on day 4 and 6, respectively. I) Cells were treated with nifedipine as a L-type Ca 2+ channel blocker (100 mM) during differentiation A change in morphology and decrease in myotube formation was observed. J) Effect of nifedipine on mRNA expression of TTR, Cav1.1 and MYOG. Nifedipine reduced Cav1.1 and MYOG expression, whereas TTR was unaffected (mean 6 S.D., n = 3). The p value indicates the statistical significance of the data and different letters indicate significant difference among groups. doi:10.1371/journal.pone.0063627.g004 transfected with shRNA against MYOG and an expected change in cell morphology and decrease in myotube formation was observed. While MYOG wd cells showed well-established myotubes, no myotubes were observed in the MYOG kd cells. mRNA analysis revealed a decrease in the expression of MYOG and MYL2; however, TTR remained almost unaffected, indicating that TTR may remain upstream of MYOG during differentiation. Surprisingly, real time RT-PCR analysis revealed a decrease in the mRNA expression of STIM1, Orai1 and Cav1.1, while Cav3.1 showed increased expression. These findings indicate that MYOG acts during the initial stage of myotube formation. While studying the ability of mononucleated myocytes to synthesize DNA during myogenesis, Andrés. [43] found that the induction of MYOG protein precedes that of p21. DNA synthesis revealed that MYOG is expressed in the premitotic state of myocytes and p21, a marker of the postmitotic state of myocytes. The decrease in expression of STIM1, Orai1 and Cav1.1 involved in myogenesis due to silencing of MYOG can be assumed to occur in conjunction with the early expression of MYOG. Immunocytochemical analysis revealed that less MYOG was present in the nuclei. However, real time RT-PCR analysis revealed that TTR was unaffected, while STIM1 and related genes were greatly influenced by MYOG silencing. It is speculated that: i) TTR is upstream to MYOG and ii) MYOG either directly (such as through gene regulation by attachment to the STIM1 promoter) or indirectly (by some unknown pathway) induces the STIM1 expression, which subsequently works through Orai1 and/or iii) the early expression of MYOG had a direct effect on VGCCs to influence myotube formation. The expression of TTR and its silencing effect on MYOG, STIM1, Orai1 and VGCC related genes suggests that TTR is the key regulator during myogenesis and activate the MYOG transcription factor through some unknown mechanism. However, a detailed investigation is needed to determine exactly how TTR affects MYOG and how MYOG silencing affects the mRNA expression of STIM1, Orai1 and Cav1.1.

TTR Influences VGCCs
Calcium signaling plays an important role in many cellular processes including cell growth and differentiation, and Ca 2+ entry into skeletal muscle fiber has been known to contribute to calcium Figure 5. Effects of TTR silencing on voltage operated Ca 2+ channels during the myogenesis. Currents were evoked by 500-ms step pulses in 10 mV increments applied from 280 mV HP (see voltage protocol in Figure 3). Current traces were obtained for every 10 mV step between 280 and 80 mV. A) I-V relationships for T-type Ca 2+ current from TTR wd (n = 25) and TTR kd (n = 21). The magnitude of each current was measured on day 4 after culture in DM. B) I-V relationships for L-type Ca 2+ current from TTR wd (n = 7) and TTR kd (n = 2). C) Summary of the detection probabilities of L/Ttype Ca 2+ current between TTR wd and TTR kd at day 4 after culture in DM. Note that the detection probability was significantly decreased in TTR kd . D) Histograms of the current density of the T-type calcium current (measured using a test pulse at 230 mV, HP 280 mV) and the L-type (measured at signaling. It has been suggested that T-type calcium current (I Ca,T ) in developing muscles is involved in pacemaker-like activity while L-type calcium current (I Ca,L ) could serve as an early contraction triggering mechanism and/or initially to fill and maintain the intracellular calcium stores [44]. In this study, we revealed a role of VGCC during C2C12 cell differentiation. Using TTR kd , we found that TTR regulates the amplitude of VGCC during C2C12 cell differentiation. The effect of TTR kd was observed in all cells tested, regardless of the time spent in cultures. It has been reported that the TTR-induced increase in Ca 2+ resulted exclusively from an influx of extracellular Ca 2+ across the plasma membrane, mostly via L-and N-type VGCCs [45]. Also Ca 2+ concentrations in myoblast cells upon treatment with the nifedipine, an L-type Ca 2+ blocker has been found to inhibit the myogenic differentiation [46]. Mibefradil, a T-type Ca 2+ blocker had been found responsible for 57% inhibition of cell fusion. However, mibefradil blocks I K(DR) , I (h-eag), and I K(IR) in addition to I ca(T). Since it is known that blocking 50% of I K(IR) reduces cell fusion by only 25%, it was speculated that the 32% inhibition in cell fusion by mibefradil was either an isolated or combined effect of I K(DR) , I (h-eag), or I ca(T) [47]. In the present study, I ca(T) was detected before the concentration of the serum was reduced. However, the detection rate increased and reached 100% in fused cells at day 3. The I Ca(L) current was not detected in the early stage, but there was a drastic increase in cells with I Ca(L) on day 2, after which the myocytes started to fuse. A gradual increase in the detection rate was found thereafter. Chronic application of 100 mM of nifedipine, a concentration that fully inhibits Ca V 1.1, sharply affected the C2C12 myoblast fusion. RT-PCR experiments revealed that the expression of Cav1.1 and MYOG decreased, while that of TTR remained unaffected. Repeating patch clamping experiments with cells transfected with vector (TTR wd ) and shRNA against TTR (TTR kd ) revealed a decrease in the detection rate of both T-and Ltypes in TTR kd , indicating that TTR plays a clear role in initiation of and during the cell differentiation process. It has been reported that SOCE in skeletal muscle requires both STIM1 and Orai1, and SOCE and VGCC represent two distinct and independent molecular entities [39]. However, in this study, the silencing of TTR and MYOG was found to lead to a decrease in both SOCE proteins (STIM1 and Orai1) and Cav1.1. These results demonstrate the crucial role of TTR during myogenesis in both excitable (voltage-gated calcium entry) and non-excitable (store-operated calcium channel) cells. Accordingly, a detailed study is required to Figure 6. Thyroid hormone effect on myogenesis. A. mRNA expression of the TTR gene and genes associated with the calcium channel and myogenesis was assessed by real-time RT-PCR at day 4 and 6 of differentiation with 50 ng/mL T 4 . TTR expression was greatly influenced by T 4 treatment; however, the expression of other genes related to myogenesis and the Ca 2+ channel were variable. B) The T 4 effect on TTR silencing showed no change on day 6. C) T 4 concentration measured by ELISA in the extracts of TTR wd and TTR kd cells, respectively, at different days of differentiation. Control indicates the time at which proliferative media was replaced with differentiation medium. p value indicates the statistical significance of the data and different letters indicate significant difference among groups. doi:10.1371/journal.pone.0063627.g006 understand this relationship of TTR with excitable and nonexcitable cells.

Effect of Thyroid Hormones
Thyroid hormone (T 4 ) plays an important role in cellular development, differentiation and metabolism [48], [49], [50]. Specifically, T 4 regulates gene expression mediated through thyroid hormone receptors in the nucleus of target cells. TTR is one of the three primary T 4 transport proteins found in serum and has a high binding affinity for T 4 [51]. TTR can both inhibit and enhance T 4 transport across the membrane. To identify the effects of T 4 in TTR expression and myogenesis, the cells were cultured in differentiation medium supplemented with T 4 hormone. Based on the mRNA expression of different genes involved in myogenesis, TTR was highly expressed and showed a four fold difference in response to treatment with 50 ng/mL T 4 . However, other genes related to myogenesis including STIM1, Orai1 and Cav3.1 showed almost no change in response to T 4 treatment. When cells transfected with shRNA against TTR (TTR kd ) and vector (TTR wd ) were treated with T 4 , no enhancement of myotube formation in TTR kd cells was observed, while TTR wd cells treated with T 4 showed a slight increase in myotube size. Moreover, T 4 concentration analysis showed that the T 4 in cells was higher on day 4, at which the expression of most genes involved in myogenesis peaked. It could be attributed to the fact that the thyroid hormone is considered as positive regulator of muscle development [52]. This result indicates a direct or indirect role of TTR in uptaking T 4 during myogenesis.
In summary, our work demonstrates a robust inhibition of myoblast differentiation in response to TTR silencing in C2C12 cells. The findings presented herein indicate that TTR initiates cell differentiation either by influencing the early stage expression of the transcription factor MYOG or by directly influencing the SOCE proteins STIM1 and Orai1 as well as the VGCC proteins Cav1.1 and Cav3.1. The effects of TTR on two distinct calcium influx pathways indicate that TTR is an essential entity that initiates the process of myogenesis, regardless of whether the cells are excitable or non-excitable in terms of calcium influx. Figure S1 MyoD expression in TTR knock-down cells. A) Decreased cell alignment was seen in TTR knock-down cells (TTR kd ) as compared to TTR wild type cells (TTR wd ) on day two as seen under phage contrast microscope. B) TTR kd showed reduced mRNA expression of both TTR and MyoD as compared to TTR wd by real-time PCR on day 2 in C2C12 during myogenesis. p value indicates the statistical significance of data and different letters indicate significant difference among groups. (TIF)