Uncoupling Protein 3 Promotes the Myogenic Differentiation of Type IIb Myotubes in C2C12 Cells

Abstract

Uncoupling protein 3 (Ucp3) is an important transporter within mitochondria and is mainly expressed in skeletal muscle, brown adipose tissue and the myocardium. However, the effects of Ucp3 on myogenic differentiation are still unclear. This study evaluated the effects of Ucp3 on myogenic differentiation, myofiber type and energy metabolism in C2C12 cells. Gain- and loss-of-function studies revealed that Ucp3 could increase the number of myotubes and promote the myogenic differentiation of C2C12 cells. Furthermore, Ucp3 promoted the expression of the type IIb myofiber marker gene myosin heavy chain 4 (Myh4) and decreased the expression of the type I myofiber marker gene myosin heavy chain 7 (Myh7). In addition, energy metabolism related to the expression of PPARG coactivator 1 alpha (Pgc1-α), ATP synthase, H⁺ transportation, mitochondrial F1 complex, alpha subunit 1 (Atp5a1), lactate dehydrogenase A (Ldha) and lactate dehydrogenase B (Ldhb) increased with Ucp3 overexpression. Ucp3 could promote the myogenic differentiation of type IIb myotubes and accelerate energy metabolism in C2C12 cells. This study can provide the theoretical basis for understanding the role of Ucp3 in energy metabolism.

Keywords: C2C12 cells; Ucp3; energy metabolism; myogenic differentiation; types of myotubes.

Figure 1

Expression characteristics of Ucp3 in myogenic differentiation of C2C12 cells. (A,B) Timecourse of Ucp3 and Myog mRNA expression during myogenic differentiation of C2C12 cells. C2C12 cells were differentiated into myoblasts using DMEM, 2% horse serum and 1% double antibody. Bar graphs with the same superscript letters indicate no significant difference (p > 0.05), while those with different superscript letters indicate significant differences (p < 0.05).

Figure 2

Ucp3 overexpression promotes myogenic differentiation in C2C12 cells. (A) qRT–PCR was used to test the Ucp3 overexpression efficiency in C2C12 cells. (B) The expression of Myog gene after affecting the Ucp3 overexpression. (C) Expression changes of Ucp3 and myogenic factor Myog at protein level. (D) Expression changes in Myhc gene after overexpression of Ucp3, as determined using qRT–PCR. (E,F) Myotube formation was detected using immunofluorescence and quantified positive cell statistics. **: p < 0.01.

Figure 3

Ucp3 silencing slowed myogenesis of C2C12 cells. (A) Expression level of Ucp3 was determined using qRT–PCR following shRNA knockdown. (B) mRNA levels of Myog knockdown, as detected using qRT–PCR. (C) Western blot was used to test Ucp3 and myogenic factor Myog. (D) The level of Myhc mRNA after knockdown of Ucp3. (E,F) Myotube formation was detected using immunofluorescence and quantified positive cell statistics. **: p < 0.01.

Figure 4

Active Ucp3 could motivate the muscle-fiber-type transformation from type I to IIb. (A) qRT–PCR was used to test the expression of oxidized muscle fiber marker gene Myh7. (B,C) Immunofluorescence staining was used to identify the positive signal of Myh7. (D) The level of colytic muscle fiber marker gene Myh4 in C2C12 cells. (E,F) Immunofluorescence staining was used to identify the positive signal of Myh4. *: p < 0.05, **: p < 0.01.

Figure 5

Ucp3 knockdown increased the muscle-fiber-type transformation from type IIb to I. (A) Expression changes of the oxidized muscle fiber marker gene Myh7 were determined using qRT–PCR following inhibitor of Ucp3. (B,C) Myotube formation was detected using immunofluorescence under the same conditions and quantified using positive cell statistics. (D) Expression changes of the colytic muscle fiber marker gene Myh4 using qRT–PCR, as specified in the legend. (E,F) Positive signal identification assay carried out in the same way as in panel. *: p < 0.05, **: p < 0.01.

Figure 6

Ucp3 had a positive regulatory effect on energy metabolism in C2C12 cells. (A–C) Relative levels of genes associated with energy metabolism due to overexpression of Ucp3 in C2C12 cells. (D–F) Quantitative real-time PCR analysis of gene expression levels associated with energy metabolism by silencing Ucp3 gene. *: p < 0.05, **: p < 0.01.