Transthyretin: A Transporter Protein Essential for Proliferation of Myoblast in the Myogenic Program

Abstract

Irregularities in the cellular uptake of thyroid hormones significantly affect muscle development and regeneration. Herein, we report indispensable role of transthyretin (TTR) in maintaining cellular thyroxine level. TTR was found to enhance recruitment of muscle satellite cells to the site of injury, thereby regulating muscle regeneration. Fluorescence-activated cell sorting (FACS) and immunofluorescence analysis of TTRwt (TTR wild type) and TTRkd (TTR knock-down) cells revealed that TTR controlled cell cycle progression by affecting the expression of Cyclin A2. Deiodinase 2 (D2) mediated increases in triiodothyronine levels were found to regulate the expression of myogenic marker, myogenin (MYOG). Moreover, use of a coumarin derivative (CD) revealed a significant reduction in cellular thyroxine, thereby indicating that TTR play a role in the transport of thyroxine. Taken together, these findings suggest that TTR mediated transport of thyroxine represents a survival mechanism necessary for the myogenic program. The results of this study will be highly useful to the strategic development of novel therapeutics to combat muscular dystrophies.

Keywords: differentiation; myoblast; proliferation; transthyretin.

Figure 1

Relationships between muscle injury and thyroid hormone (T3 and free T4) concentrations. Tissue and plasma samples of mice subjected to cardiotoxin (CTX) injection were analyzed for the expressions of TTR and of the thyroid hormones, T3 and free T4. (A,B) Estimation of the concentrations of free T4 and T3 at early time points in plasma and muscle samples by ELISA. Concentrations of T3 and free T4 are measured as amounts (pg or ng) per gram of muscle tissue; (C) Transverse section of muscles after Hematoxylin–Eosin (H&E, 400×) and immunostaining with PAX7, TTR, or deiodinase 2 (D2) antibodies. The upper and bottom panel show non-injected (control) and CTX injected muscle, respectively. p-Values indicate statistical significance of the data (mean ± S.D., n = 3, **: p < 0.001, ***: p < 0.0001).

Figure 2

Effect of TTR knock-down on cell migration. Scratch testing was used to examine differences in cell migration. (A) TTR protein expression of control and scratched cells in the presence of T4 (red: TTR; blue: nucleus); (B) Difference in T3 and free T4 concentrations in normal and scratched samples at 12 or 24 h after T4 supplementation; (C) Analysis of scratched scrambled vector (control: TTR_wt) and TTR shRNA transfected cells (TTR_kd) conducted by staining with TTR antibody followed by 4′,6′-diamino-2-phenylindole (DAPI) nuclear staining (green: TTR; blue: nucleus); (D) Differences between the migration patterns of TTR_wt and TTR_kd cells in the presence or absence of T4; (E) Estimation of free T4 and T3 hormone levels in scratched and non-scratched cells in TTR_wt and TTR_kd cell cultures with or without T4 supplementation; (F) TTR and D2 protein expression was analyzed by Western blotting of TTR_wt and TTR_kd cells supplemented with T4. p-Values indicate statistical significance of the data (mean ± S.D., n = 3, *: p < 0.05, **: p < 0.001, ***: p < 0.0001).

Figure 3

TTR expression during cell proliferation. TTR and CyclinA2 expressions during cell cycle progression of C2C12 cells: (A) difference in growth patterns of TTR_wt and TTR_kd cells as determined by FACS; (B,C) TTR and CyclinA2 expression in TTR_wt and TTR_kd cells by RT-PCR or Western blot; and (D) confirmation of expressional differences of TTR or CyclinA2 by immunocytochemistry (green: TTR; red: CyclinA2; Blue: Nucleus). p-Values indicate statistical significance (mean ± S.D., n = 3, *: p < 0.05, **: p < 0.001).

Figure 4

Estimation of T3 and free T4 concentrations with serum free media in C2C12 cells. C2C12 cells were incubated with serum free media for two days. (A) Difference in T3 and T4 concentrations in the presence or absence of T4 in serum free media; (B) Fusion indices determined on culture Day 2 with or without T4 supplementation. Expressions of TTR and MYOG as determined by RT-PCR and Western blot; (C) Differential expression of TTR, MYOG, and D2 in TTR_kd cells by RT-PCR and Western blot; (D) Analysis of T4 concentrations in TTR_kd and TTR_wt cells in the presence of T4. p-Values indicate statistical significance (mean ± S.D., n = 3, *: p < 0.05, **: p < 0.001, ***: p < 0.0001).

Figure 5

Effect of coumarin derivative (CD) on T4 to TTR binding. C2C12 cells grown in DMEM supplemented with 10% or 2% FBS were washed and incubated in serum-free media for 12 h. (A) Pictures showing fluorescence attributed to CD to TTR binding in C2C12 cells during growth in proliferation and differentiation media; (B) Giemsa staining showing myotube differences between C2C12 cells grown in differentiation media with or without CD (1 μM); (C) Graph showing CD intensity as fold differences in TTR_wt and TTR_kd cells; (D) CD intensity was measured in serum free media containing different concentrations of T4. p-Values indicate statistical significance (mean ± S.D., n = 3, *: p < 0.05, **: p < 0.001).