*3.2. Enhancement of Myoblast Viability and Di*ff*erentiation by TTR with T4*

To assess the role of TTR and T4 on myoblast viability and differentiation, C2C12 cells were grown with T4 or T4 + TTR protein for two or three days. Cell viability was increased in T4 + TTR protein treated cells compared to that in only T4 treated cells (Figure 2A). The T4 and T3 concentrations were measured in CM and cells. A lower T4 concentration in media with a consequent higher concentration in cells was observed with T4 + TTR treatment than in those with only T4 treatment. The results indicate that TTR outside the cell enhances the transport of T4 to the cell interior in myoblast viability (Figure 2A). Further, cells were cultured in serum-free media with added T4 or T4 + TTR protein for three days to induce differentiation. The T4 + TTR treatment significantly induced myotube formation with elevated mRNA (MYL2) and protein expression of myogenic factors (MYOD and MYL2), RXRγ, and TRα. However, TTR mRNA and protein expression were decreased by TTR + T4 treatment and their expression in exosomes was also reduced from that of only T4-treated cells (Figure 2B). T4 and T3 concentrations were increased by TTR + T4 treatment (Figure 2C). TTR in mouse MSCs was assessed to determine its expression during differentiation. For this, MSCs were incubated with differentiation media for zero or two days. Expression of TTR and myogenic genes or proteins were increased on day 2 compared to that on day 0 (Figure 2D). Next, MSCs were cultured in serum-free conditions supplemented with T4 or T4 + TTR protein for two days to induce differentiation. Similar to the results with C2C12 cells, MSCs exhibited increased myotube formation with elevated thyroid hormone concentration under T4 + TTR treatment (Figure 2E). Interestingly, decreased TTR mRNA was observed in the exosomes following T4 + TTR treatment (Figure 2E). Furthermore, T3 was present in exosomes isolated from serum-free MSCs culture media supplemented with T4 (Figure 2F). These data showed that TTR protein with T4 not only enhanced myoblast proliferation and myogenic differentiation, but also increased MSC differentiation into muscle cells.

**Figure 1.** The role of secreted TTR from cells during myogenic differentiation. Normal and TTR knockdown cells were cultured with serum-free media for two days (**A**,**B**). (**A**) Proteins were isolated from cells, DMEM (control) and cultured media (CM). TTR protein level was analyzed by Western blot. TTR mRNA level in cells by real-time RT-PCR, and protein level in cell culture media of TTRwt and TTRkd by Western blot. Band intensity was measured by using ImageJ. (**B**) TTR mRNA levels in normal cell, exosomes isolated from mouse plasma, media of cultured C2C12 cells (CM) with or without T4 treatment, and TTRwt and TTRkd by RT-PCR. Cells were cultured in 2% FBS or serum-free media supplemented with TTR antibody for two (**C**) or three days (**D**,**E**) for immunoneutralization. (**C**) Myotube formation and fusion index was observed by Giemsa staining. (**D**) Gene expression was observed by real-time RT-PCR. (**E**) T4 and T3 concentration in cells was observed by ELISA. TTRwt indicates cells transfected with scrambled vector. Means ± SD (*n* = 3). \* *p* ≤ 0.05, \*\* *p* ≤ 0.001, \*\*\* *p* ≤ 0.0001.

#### *3.3. Reduction of T4 Concentration Inside Cells and Myoblast Di*ff*erentiation by Bovine Albumin Serum (BSA) Treatment*

For comparative assessment of T4 transport through TTR to the cell interior, C2C12 cells were cultured in serum-free media supplemented with T4 or T4 + BSA protein for two days. Myotube formation and MYOG expression were decreased in BSA-treated cells, while TTR and D2 expressions were increased at the translational level (Figure 3A). Interestingly, elevated TTR in both exosomes (mRNA) and CM (protein) was also observed in BSA-treated cells (Figure 3B). High T4 and T3 concentrations in T4 + BSA supplemented media with subsequent low levels in both hormone concentrations in the cell, under the same conditions, indicated that BSA reduced the transport of T4 to the cell interior (Figure 3C). Furthermore, elevated T4 concentration was observed in T4 + BSA + TTR supplemented cells relative to that in T4 + BSA treated cells (Figure 3D). Interestingly, decreased TTR mRNA was found in exosomes of T4 + BSA + TTR treated cells (Figure 3E). Additionally, T3 was present in exosomes, and there was no difference in T3 concentration in exosomes supplemented with T4, T4 + BSA, or T4 + TTR (Figure 3F). We observed that BSA reduces myotube formation by decreasing T4 transport.

**Figure 2.** Myoblast viability and differentiation by treatment with TTR proteins. (**A**) C2C12 cells were cultured in 10% FBS media supplemented with T4 or T4 + TTR protein for two days. Cell viability was observed by MTT assay. T4 or T3 concentration in cultured media and cells were observed by ELISA. Cells were cultured in serum-free media supplemented with T4 or T4 + TTR protein for three days (**B**,**C**). (**B**) Myotube formation and fusion index by Giemsa staining, mRNA level in cells by real-time RT-PCR, exosomes by RT-PCR, protein expression by Western blot and immunocytochemistry. (**C**) T4 or T3 concentration in cells was observed by ELISA. (**D**) When mouse MSCs reached 100% confluency, media were switched to 2% FBS and cultured for zero and two days. MSC differentiation, TTR mRNA level by real-time RT-PCR and protein expression by Western blot. (**E**) MSCs were cultured in serum-free media supplemented with T4 or T4 + TTR protein for two days. T4 or T3 concentration in cells was observed by ELISA. (**F**) MSCs were cultured with serum-free media supplemented with T4 for two days and exosomes were isolated from cultured media. T3 concentration in cell and exosomes was observed by ELISA. Means ± SD (*n* = 3). \* *p* ≤ 0.05, \*\* *p* ≤ 0.001, \*\*\* *p* ≤ 0.0001.

#### *3.4. TTR Internalization Into Myoblast*

To elucidate TTR internalization to the cell interior, TTR protein or BSA was fluorescently labeled and C2C12 cells were cultured under serum-free conditions supplemented with labeled TTR protein or BSA for one day. Higher fluorescence of labeled TTR protein was evident in the cells treated with labeled TTR than with BSA or in non-treated cells (Figure 4A). TTR overexpression was achieved by transfection with the TTR ORF plasmid and cultured with 10% FBS for two days. Increased cell viability was observed in TTR-overexpressing cells (Figure 4B). Next, TTR-overexpressing cells were cultured with serum-free media for two days. Increased myotube formation with enhanced TTR mRNA/protein expression was observed in TTR-overexpressing cells (Figure 4C). Additionally, elevated concentrations of THs were observed in TTR-overexpressing cells supplemented with T4 (Figure 4C).

**Figure 3.** Myoblast differentiation following BSA treatment. Cells were cultured in serum-free media supplemented with T4 or T4 + BSA for two days (**A**–**E**). (**A**) Myotube formation and fusion index were observed by Giemsa staining. mRNA level was observed by real-time RT-PCR and protein expressions by Western blot and immunocytochemistry. (**B**) TTR mRNA in exosomes of cultured media using RT-PCR and protein level in cultured media by Western blot. (**C**) T4 or T3 concentration in cultured media or cells was observed by ELISA. (**D**,**E**) Cells were cultured with serum-free media supplemented with T4, T4 + BSA, T4 + TTR or T4 + BSA + TTR for two days. T4 or T3 concentration in T4 + BSA or T4 + BSA + TTR treated cells. TTR mRNA in exosomes of cultured media (in T4 + BSA or T4 + BSA + TTR treated cells) using RT-PCR. (**F**) Cells were cultured in serum-free media supplemented with T4 or T4 + BSA or T4 + TTR for two days and exosomes were isolated from each cultured medium. T3 concentration in exosomes. Means ± SD (*n* = 3). \* *p* ≤ 0.05, \*\* *p* ≤ 0.001, \*\*\* *p* ≤ 0.0001.

**Figure 4.** Endocytosis of TTR protein and TTR overexpression effects. (**A**) TTR protein or BSA were labeled with fluorescence and cells were cultured with serum-free media supplemented with labeled TTR protein or BSA for 1 day. Detection of labeled TTR protein and BSA in cells (Red: TTR, Blue: Nucleus). (**B**) TTR overexpression was performed by transfecting with TTR ORF plasmid followed by incubation with 10% FBS for two days. Cell viability was analyzed by MTT assay. (**C**) TTR overexpressing cells were incubated with serum-free media for two days. Myotube formation and fusion index were observed by Giemsa staining, TTR mRNA level by real-time RT-PCR, and protein expression by Western blot and immunocytochemistry. Control or TTR-overexpressing cells were incubated with serum-free media supplemented with T4 for two days. T4 or T3 concentration was measured by ELISA. Means ± SD (*n* = 3). \* *p* ≤ 0.05, \*\* *p* ≤ 0.001, \*\*\* *p* ≤ 0.0001.

#### *3.5. Regulation of RXR*γ *and TR*α *Expression by TTR During Myoblast Di*ff*erentiation*

To determine the role of T4 or TTR on RXRγ and TRα expression, C2C12 cells were grown with or without serum in normal or TTR knockdown cells, and the effects were studied during myoblast differentiation. Increases in mRNA and protein expression of RXRγ and TRα were evident on day 2 compared to the levels on day 0 (Figure 5A). Next, T4 treatment under serum-free conditions stimulated RXRγ expression at both the transcriptional and translational level. However, TRα protein expression was decreased by T4 treatment (Figure 5B). Interestingly, TTR knockdown reduced expression of RXRγ and TRα (Figure 5C). Further, RXRγ and TRα knockdown were performed and followed by culturing with 2% FBS for two days. Myotube formation, myogenic genes and D2 expression were decreased by RXRγ or TRα knockdown, whereas TTR and TRα expressions were increased in RXRγkd cells. Most gene or protein expressions were decreased in TRα knockdown cells (Figure 5D,E). Overall, the above results indicate that expression of RXRγ is controlled by TTR via T4 transportation into the cell during myoblast differentiation.

**Figure 5.** RXRγ and TRα expression during myoblast differentiation. (**A**) Cells were cultured with 2% FBS for two days. RXRγ and TRα expressions using real-time RT-PCR or Western blot. (**B**) Cells were cultured in serum-free media supplemented with T4 for two days. RXRγ and TRα expression by real-time RT-PCR or Western blot. (**C**) RXRγ and TRα expression in TTRkd and TTRwt cells using real-time RT-PCR or Western blot. (**D**) RXRγ knockdown was performed and followed by culture with 2% FBS for two days. Myotube formation and fusion index were observed by Giemsa staining, mRNA expression by real-time RT-PCR, and protein expression by Western blot in RXRγkd and RXRγwt cells. (**E**) TRα knockdown was performed and followed by culture with 2% FBS for two days. Myotube formation and fusion index were observed by Giemsa staining, mRNA expression by real-time RT-PCR, and protein expression by Western blot in TRαkd and TRαwt cells. TTRwt, RXRγwt, or TRαwt indicate cells transfected with the scrambled vector. Means ± SD (*n* = 3). \* *p* ≤ 0.05, \*\* *p* ≤ 0.001, \*\*\* *p* ≤ 0.0001.

#### *3.6. Relationship between TTR and D2 According to Muscle Age*

To determine the effect of muscle age on TTR and D2 expression, mouse muscle at 16- and 26-weeks were collected. Myofiber size (width) and expression of TTR and D2 were decreased in 26-week muscle compared with 16-week muscle (Figure 6A). Interestingly, a decreased TTR level was observed in exosomes isolated from 26-week plasma (Figure 6A). The T3 concentration in 16-week muscle was higher than that in 26-week muscle (Figure 6B). Further, a significant increase in the T4 concentration in the plasma of 26-week mice the was observed, whereas there was no difference in the T3 concentration in the plasma of either age group (Figure 6B). The above findings suggest that expressions of TTR and D2 correlate with the age-dependent differences of muscle.

**Figure 6.** TTR expression and T3 concentration in age-dependent differences of muscle.. Expression of TTR and D2 proteins were analyzed in 16- and 26-week mouse muscles. (**A**) TTR and D2 proteins expression by Immunohistochemistry and Western blot. Exosomes were isolated from 16- and 26-week plasma. TTR mRNA level in cell and exosomes of 16- or 26-week plasma by RT-PCR. (**B**) T3 or T4 concentration in 16- or 26-week muscles or plasma was observed by ELISA. Means ± SD (*n* = 3). \* *p* ≤ 0.05, \*\* *p* ≤ 0.001, \*\*\* *p* ≤ 0.0001.
