Function of the Porcine TRPC1 Gene in Myogenesis and Muscle Growth

Abstract

In animals, muscle growth is a quantitative trait controlled by multiple genes. Previously, we showed that the transient receptor potential channel 1 (TRPC1) gene was differentially expressed in muscle tissues between pig breeds with divergent growth traits base on RNA-seq. Here, we characterized TRPC1 expression profiles in different tissues and pig breeds and showed that TRPC1 was highly expressed in the muscle. We found two single nucleotide polymorphisms (SNPs) (C-1763T and C-1604T) in TRPC1 that could affect the promoter region activity and regulate pig growth rate. Functionally, we used RNAi and overexpression to illustrate that TRPC1 promotes myoblast proliferation, migration, differentiation, fusion, and muscle hypertrophy while inhibiting muscle degradation. These processes may be mediated by the activation of Wnt signaling pathways. Altogether, our results revealed that TRPC1 might promote muscle growth and development and plays a key role in Wnt-mediated myogenesis.

Keywords: TRPC1; muscle growth; myogenesis; porcine.

Figure 1

TRPC1 is involved in the growth of pigs. TRPC1 expression in different tissues of TP pigs at the embryonic stage by SqRT-PCR (A), WB (B,D), and qRT-PCR (C). The TRPC1 mRNA (E) and protein (F) expression levels in the LD of three pig breeds. LD, longissimus dorsi; BF, back fat; TP, Tibetan pig (n = 6), WJ, Wujin pig (n = 6), YY, Yorkshire (n = 6). Each bar represents the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 2

SNP sites and promoter activity analysis. (A) The sequencing chromatogram at the sites of two SNPs, C-1604T and C-1763T. The arrows indicate the SNP site. (B) Dual-luciferase analysis for promoter activity. Control refers to C2C12 cells co-transfected with PGL3-basic and PRL-TK. CC and TT represent CC and TT homozygous individuals whose promoters are connected to PGL3-basic and co-transfected with PRL-TK cells, respectively. Each bar represents the mean ± SD. ** P < 0.01.

Figure 3

TRPC1 promoted myoblast proliferation and migration. (A) Interference efficiency detection of synthetic siRNA fragments. (B) Efficiency detection of plasmid overexpression. (C,D) CCK8 assay of proliferated myoblasts transfected with RNAi or overexpression fragments. (E,F) EdU staining for proliferated cells following si-TRPC1 or pcDNA3.1-TRPC1 transfection. Nuclei were stained with DAPI, n = 3 in each group, scale bar = 400 μm. (G,H) The mRNA expression levels of proliferation-related genes. (I) Wound healing migration assay of C2C12 myoblasts. Some cells were scraped off with a pipette tip to obtain an acellular area. Twenty-four hours later, cells migrated into the acellular area were stained and counted. Cells were migrated in control conditions, Scale bar = 400 μm. (J) Transwell test for myoblasts. Purple represents migrated cells, n = 3 in each group, scale bar = 200 μm. NC: negative control. The data represent the means ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4

TRPC1 promoted cell fusion, differentiation, and muscle hypertrophy but inhibited muscle degradation. (A) qRT-PCR results showed the expression profiles of the TRPC1 gene during differentiation. MyHC is the myogenic differentiation marker gene. (B) Schematic of cell differentiation time. (C) The protein level of TRPC1 and differentiation marker MyoG. (D,E) The mRNA expression of differentiation marker genes MyoD, MyoG, and MyHC quantified by qRT-PCR. (F,G) Immunofluorescence staining for MyHC protein in control or pcDNA3.1-TRPC1/si-TRPC1-treated myoblasts cultured for 4 days in differentiation medium. MyHC protein expression is shown in red, and nuclei in blue (DAPI). White scale bar = 400 μm. (H,I) Myoblast fusion analysis by immunofluorescence staining for MyHC, and the arrows represent the multinucleated myotubes, white scale bar = 200 μm. (J,K) The mRNA expression of fusion marker genes quantified by qRT-PCR. (L,M) The mRNA expression of muscle degradation markers (Atrogin1, Bmp4, Murf, and Foxo3) and muscle hypertrophy genes (Fst and Nog) quantified by qRT-PCR (n = 3, respectively). NC: negative control. The data represent the means ± SD of three independent experiments. N.S.: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5

Regulation of TRPC1 in myogenesis. The mRNA expression of Wnt-related genes in C2C12 cells in which TRPC1 was overexpressed (A) or knocked down (B), respectively. NC: negative control. The data represent the means ± SD of three independent experiments. N.S.: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.