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 −80°C 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 4°C. The fixed organs were then processed for routine paraffin embedding, and the paraffin-embedded tissue blocks were cut to 6-µm 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 37°C with 5% CO₂. 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 µg/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™ reagent (Invitrogen) according to the manufacturer’s protocol and then stored in diethylpyrocarbonate-treated H₂O at −80°C until used. One microgram of RNA in a reaction mixture with a total volume of 20 µl was primed with oligo (dT)₂₀ primers (Bioneer, Daejeon, Korea) and then reverse transcribed at 42°C for 50 min and 72°C for 15 min. Subsequently, 2 µl 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 SYBR® 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 4°C 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 4′ 6′-diamino-2-phenylindole (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 4°C, after which the protein concentrations were determined by the Bradford method using protein assay dye solution [33]. Total protein (40 µg) heated at 90°C 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 4°C. 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 (22°C–25°C). 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 (2-aminoethylether)-N,N,N′,N′-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 BaCl₂, 10 mM glucose, 0.5 mM MgCl₂, 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 T₄ concentration (DRG International, Inc. Margurg, Germany). Cell lysate and enzyme conjugate were added in a specific antibody-coated 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 µg/mL, Santa Cruz Biotechnology) overnight at 4°C. 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.

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Figure 1. TTR gene expression in different mouse tissues.

A) mRNA expression showed in three different tissues, M (muscle), B (brain) and L (liver), by real time RT-PCR. TTR, STIM1 and Orai1 were found to be expressed in all tissues, while expression of MYOG, MYL2, D2 (Deiodinase 2), Cav1.1 and Cav3.1 genes was specific to individual organs. B) TTR protein detection by immunohistochemistry revealed its presence in skeletal muscles of the forelimb, hind limb and trunk, as well as in the liver. The liver was used as a positive control (n = 3). The p value indicates the statistical significance of the data and different letters indicate significant difference among groups.

https://doi.org/10.1371/journal.pone.0063627.g001

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).

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Figure 2. TTR and MYOG in C2C12 differentiation.

C2C12 cells grown to 70% confluence and serum starved for 2, 4 and 6 days were used for mRNA and protein expression analysis. A) TTR mRNA showed up to a 5 fold difference in expression at day 2–6 upon real time RT-PCR analysis. B) Immunoblot analysis of TTR and ß-actin expression revealed low initial protein expression followed by a gradual increase with time. C) Distribution of TTR expression in cell cytoplasm as observed by immunostaining. The expression level was significantly upregulated at day 6. D) Time course study of mRNA expression of MYOG by real time RT-PCR. The maximum expression level was observed on day 4 of differentiation. E) Total proteins extracted from cells at different time points revealed very low expression of MYOG at the basal level that gradually increased up to day 4, then decreased at day 6. F) Cells differentiated and stained against the antibody of MYOG displayed the highest nuclear staining on day 4. The p value indicates the statistical significance of the data and different letters indicate significant difference among groups.

https://doi.org/10.1371/journal.pone.0063627.g002

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 knock-down was also verified at the protein level by immunostaning and immunoblot analysis (Fig. 3G, 3H).

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Figure 3. TTR and MYOG knockdown and their effect.

TTR knock-down cells (TTR_kd) showed altered mRNA expression of myogenic genes. A) A decrease in mRNA expression was observed on day 6 of transfection in TTR and day 5 in MYOG and MYL2. However Myf5 and MyoD showed little enhancement. Control indicates the time at which cells were transfected (n = 3). B) Immunostaining of cells transfected with either TTR_wd or TTR_kd (day 6). A significant decrease of cytoplasmic TTR protein was observed in TTR_kd cells when compared with TTR_wd cells. C) Similarly, TTR_kd led to decreased nuclear myogenin protein expression. D) Western blot of TTR_kd agrees with the immunostaining results. E) Fusion index was performed with TTR_wd and TTR_kd at day 4. A significant decrease of nuclei fusion in TTR_kd was analyzed as compared to TTR_wd cells. F) mRNA expression of MYOG knock-down in C2C12 and its effect on MYL2 and TTR, which peaked on day 4 of differentiation. MYL2 is affected by MYOG knock-down, while TTR showed no change. G &H) immunostaining and immunoblot analysis verifying myogenin knockdown up to the protein level during differentiation at day 4. The p value indicates the statistical significance of the data and different letters indicate significant difference among groups.

https://doi.org/10.1371/journal.pone.0063627.g003

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 Ca²⁺ channels (VDCCs) during myogenesis, the whole-cell patch-clamp recording of C2C12 cells at different time intervals was performed. Small T-type Ca²⁺ currents were detected in control cells at day 0 (Fig. 4A). However, only ∼54% of the cells expressed measurable T-type Ca²⁺ currents. After five days of differentiation, in addition to T-type current, a distinct L-type Ca²⁺ current was detected (Fig. 4B) in response to 500 ms depolarizing pulses from a holding potential of −80 mV. Fig. 4B shows representative currents recorded at −30 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 − voltage (I–V) relationship for the initial peak Ca²⁺ current in T-type channel (black arrow) at different day of culture. Fig. 4D shows an average I–V relationship for the secondary peak Ca²⁺ current in L-type channel (red arrow) at different day. Mixed T- and L-type currents were recorded over the −30 mV depolarizing pulse from a holding potential. After day 2, the T-type Ca²⁺ current was detected in most of the control cells (∼86%), whereas the L-type Ca²⁺ current was detected in ∼45% of C2C12 cells. The maximum detection rate (100%) of the T-type Ca²⁺ current was attained at day 3 (Fig. 4E). The detection probability of the L-type Ca²⁺ current gradually increased during myogenesis, reaching its maximum at day 5 (Fig. 4 E). Current density through T- and L-type Ca²⁺ channels increased significantly during myogenesis (Fig. 4F, P<0.05). Although the detection probability of the T- and L-type Ca²⁺ current gradually increased during myogenesis, the T- and L-type Ca²⁺ 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 T- and L-type Ca²⁺ channels are functionally expressed in differentiating C2C12 cells.

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Figure 4. L-type and T-type calcium current and expression of Ca²⁺ channel related genes during myogenesis.

Whole-cell patch clamp recordings in response to 500 ms depolarization from a holding potential (HP) of −80 mV stepped to values between −80 mV and 90 mV for 500 ms. Ba²⁺ (10 mM) was used as a charge carrier. A) Superimposition of different traces under the control conditions in response to test pulses from −80 to 40 mV. B) Representative superimposed L/T-type Ca²⁺ current traces after 5 days of culture in differentiation medium (DM). C) Average current − voltage (I–V) relationship for the initial peak Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 −30 mV, HP −80 mV) and the L-type (measured at 0 mV). mRNA expression of Ca²⁺ 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 Ca²⁺ channel blocker (100 µM) 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 ± S.D., n = 3). The p value indicates the statistical significance of the data and different letters indicate significant difference among groups.

https://doi.org/10.1371/journal.pone.0063627.g004

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 µM) 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 L-type Ca²⁺ channels. In the case of TTR_kd cells, a significant decrease in detection probability and the current density was observed (Fig. 5D).

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Figure 5. Effects of TTR silencing on voltage operated Ca²⁺ channels during the myogenesis.

Currents were evoked by 500-ms step pulses in 10 mV increments applied from −80 mV HP (see voltage protocol in Figure 3). Current traces were obtained for every 10 mV step between −80 and 80 mV. A) I–V relationships for T-type Ca²⁺ 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 Ca²⁺ current from TTR_wd (n = 7) and TTR_kd (n = 2). C) Summary of the detection probabilities of L/T-type Ca²⁺ 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 −30 mV, HP −80 mV) and the L-type (measured at 10 mV). E & F) mRNA expression assessed by real-time RT-PCR on Cav1.1, Cav3.1, STIM1 and Orai1 in TTR_wd, TTR_kd or MYOG_kd cells on day 4 of transfection. TTR knock-down resulted in a decreased effect on all calcium channel related genes. MYOG knock-down showed decreased expression of Cav1.1, STIM1 and Orai1. Control indicates day 0 (mean ± S.D., n = 3). p value indicates the statistical significance of the data and different letters indicate significant difference among groups.

https://doi.org/10.1371/journal.pone.0063627.g005

The mRNA expression of T- and L-type Ca²⁺ 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 Ca²⁺ entry related genes such as STIM1, Orai1 and Cav1.1 (Fig. 5F), indicating that MYOG regulates these genes.

Cell Differentiation is also Affected by T₄

Thyroxin (T₄) is an important constituent of FBS used in cell culture experiments. In this study, the effect of T₄ on the expression of genes involved in myogenesis was investigated. TTR and Cav1.1 expression were enhanced by T₄ treatment, while this effect was small in other genes (Fig. 6A). In wild type cells (TTR_wd), wider myotube formation was observed after T₄ treatment. However, there was no apparent change in the morphology of TTR_kd cells with or without T₄ treatment (Fig. 6B). Changes in the T₄ concentration of cells were measured by ELISA (Fig. 6C). A gradual increase in T₄ concentration was observed in TTR_kd cells. In contrast, the concentration of T₄ in TTR_wd cells increased up to day 4 and then decreased at day 6. Interestingly, the intracellular T₄ 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 T₄ concentration can be assumed to be due to the presence of T₄ in cell culture media and its transport across the membrane.

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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 T₄. TTR expression was greatly influenced by T₄ treatment; however, the expression of other genes related to myogenesis and the Ca²⁺ channel were variable. B) The T₄ effect on TTR silencing showed no change on day 6. C) T₄ 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.

https://doi.org/10.1371/journal.pone.0063627.g006

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 voltage-gated 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 up-regulated, 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 Ca²⁺ influx [39], [40], whereas non-excitable cells express VGCC proteins but lack voltage-gated Ca²⁺ 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 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 Ca²⁺entry into skeletal muscle fiber has been known to contribute to calcium 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 Ca²⁺ resulted exclusively from an influx of extracellular Ca²⁺ across the plasma membrane, mostly via L- and N-type VGCCs [45]. Also Ca²⁺ concentrations in myoblast cells upon treatment with the nifedipine, an L-type Ca²⁺ blocker has been found to inhibit the myogenic differentiation [46]. Mibefradil, a T- type Ca²⁺ 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 µM of nifedipine, a concentration that fully inhibits Ca_V1.1, 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 L- types 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 understand this relationship of TTR with excitable and non-excitable cells.

Effect of Thyroid Hormones

Thyroid hormone (T₄) plays an important role in cellular development, differentiation and metabolism [48], [49], [50]. Specifically, T₄ regulates gene expression mediated through thyroid hormone receptors in the nucleus of target cells. TTR is one of the three primary T₄ transport proteins found in serum and has a high binding affinity for T₄ [51]. TTR can both inhibit and enhance T₄ transport across the membrane. To identify the effects of T₄ in TTR expression and myogenesis, the cells were cultured in differentiation medium supplemented with T₄ 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 T₄. However, other genes related to myogenesis including STIM1, Orai1 and Cav3.1 showed almost no change in response to T₄ treatment. When cells transfected with shRNA against TTR (TTR_kd) and vector (TTR_wd) were treated with T₄, no enhancement of myotube formation in TTR_kd cells was observed, while TTR_wd cells treated with T₄ showed a slight increase in myotube size. Moreover, T₄ concentration analysis showed that the T₄ 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 T₄ 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.