A Novel tRF-Lys-TTT-012 in Qingyu Pigs Mediates the Conversion of Muscle Fibers from Fast-Twitch to Slow-Twitch Type

Abstract

Skeletal muscle, the largest organ within the animal body, consists of multiple muscle fiber types. The distribution of these fiber types significantly impacts both athletic performance and the quality of meat. Growing evidence has demonstrated that transfer RNA (tRNA)-derived small RNAs (tsRNAs) are not merely byproducts of tRNA metabolism but also participate in multiple cellular metabolic processes. However, the role of tsRNAs in skeletal muscle fiber type transition remains elusive. In this study, a total of 403 differentially expressed tsRNAs were identified through small RNA sequencing in psoas major muscle (PM) and latissimus dorsi muscle (LD), among which 220 tsRNAs including tRF-Lys-TTT-012 were upregulated in psoas major muscle. Functional studies in C2C12 and PK15 cells demonstrated that it inhibited the proliferative capacity of C2C12 cells while promoting myogenic differentiation, increased the proportion of slow muscle fibers after differentiation, and drove muscle fiber type transition toward slow fibers. Additionally, tRF-Lys-TTT-012 enhanced mitochondrial number and function, potentially linking to the promotion of slow fiber characteristics. Collectively, tRF-Lys-TTT-012 may serve as a promising marker for slow muscle fibers and uncover a novel potential target for skeletal muscle fiber type transition toward the slow fiber phenotype.

Keywords: mitochondria; skeletal muscle fiber type; tsRNA.

Figure 1

Analysis of the tsRNA composition in LD and PMs. (A) There are significant differences between the LD and PM tissues in pigs, and tsRNA sequencing was performed to analyze them. HE staining of cross-sections from LD and PM tissues. (B) Quantitative comparison of the cross-sectional area of muscle fibers in HE-stained sections. (C) Immunofluorescence staining of MYHC I and MYHC IIb in LD and PM tissues. Scale bar represents 100 μm. All data were presented as mean ± SEM. The symbol * denotes a significance level of * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

The overview of tRFs and tiRNA expression in the LD and PMs. (A) The principal component analysis (PCA) of tRFs and tiRNAs expressions in LD and PM samples. (B) Venn diagram of tRFs and tiRNAs expression in muscle samples from both groups. (C) Distribution of different types of tRF and tiRNA in two groups of muscle samples. (D) Stacked plot of all isoforms of tRFs and tiRNAs clustered by tRNA anticodons. The x-axis represents tRNAs with the same anticodon, while the y-axis shows the number of all isoforms of tRFs and tiRNAs originating from the respective anticodon tRNA. (E) The clustering heatmap of differentially expressed tsRNAs in samples of the LD and PM. (F) Seed sequence characteristics of differentially expressed tsRNAs.

Figure 3

Enrichment analysis of the top 10 tsRNAs in the LD and PM of QingYu pigs. (A) The top 10 signaling pathways enriched with the target genes of the top 10 tsRNAs in PM. We have highlighted the key terms in red for emphasis. (B) The top 10 BP enriched with the target genes of the top 10 tsRNAs in PM. We have highlighted the key terms in red for emphasis. (C) The top 10 MF enriched with the target genes of the top 10 tsRNAs in PM. (D) The top 10 signaling pathways enriched with the target genes of the top 10 tsRNAs in LD. (E) The top 10 BP enriched with the target genes of the top 10 tsRNAs in LD. We have highlighted the key terms in red for emphasis. (F) The top 10 MF enriched with the target genes of the top 10 tsRNAs in LD.

Figure 4

tRF-Lys-TTT-012 highly expressed in PM. (A) Volcano plot of differentially expressed tsRNAs in the LD and PMs. Lys-TTT-012 is highlighted in the plot. tRFs and tiRNAs are represented by red dots (indicating upregulation) or blue dots (indicating downregulation) above the dashed line, while gray dots represent tsRNAs with no significant differential expression. (B) The relative quantification of fast- and slow-twitch muscle fiber markers in the LD and PM of Duroc x Landrace x Yorkshire (DLY) pigs, Qingyu (QY) pigs, and Chenghua (CH) pigs was performed using qRT-PCR. (C) The quantification of tRF-Lys-TTT-012 was conducted in two muscle tissues from three pig species. (D) The shear site map of tRF-Lys-TTT-012 derived from tRNA-Lys-TTT. All data were presented as mean ± SEM. The symbol * denotes a significance level of * p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates no significance.

Figure 5

Enrichment analysis of target genes of tRF-Lys-TTT-012. (A) The top 20 signaling pathways enriched with the target genes of tRF-Lys-TTT-012. (B) The top 20 BP enriched with the target genes of tRF-Lys-TTT-012. (C) The top 20 CC enriched with the target genes of tRF-Lys-TTT-012. (D) The top 20 MF enriched with the target genes of tRF-Lys-TTT-012. We have highlighted the key terms in red for emphasis.

Figure 6

Overexpression tRF-Lys-TTT-012 inhibits proliferation of C2C12 myoblasts in vitro. (A) The transfection efficacy of tRF-Lys-TTT-012 mimics was validated by qRT-PCR. (B) The relative mRNA expression of cell proliferation-related genes after overexpression of tRF-Lys-TTT-012 in C2C12 cells. (C) CCK-8 assay was used to detect the proliferative activity of C2C12 cells transfected with tRF-Lys-TTT-012 mimics. (D) EDU assay was conducted to assess the effect of transfected tRF-Lys-TTT-012 mimics on the proliferation of C2C12 cells. Scale: 1 bar represents 100 μm. (E) Histogram showing quantitative results from EDU assay. ImageJ software counted the proportion of EDU-positive cells. (F,G) Flow cytometry results showed the effect of tRF-Lys-TTT-012 overexpression on the cell cycle of C2C12 myoblasts. All data were presented as Mean ± SEM. The symbol * denotes a significance level of p < 0.05, ** p < 0.01, *** p < 0.001. The relative mRNA levels are normalized to β-actin. NC, negative control; Mimics, tRF-Lys-TTT-012 mimics.

Figure 7

Overexpression of tRF-Lys-TTT-012 promotes the differentiation of C2C12 myoblasts and the transition to slow muscle fibers in vitro. (A) The effect of tRF-Lys-TTT-012 overexpression on the relative mRNA expression of myofiber type-related marker genes during C2C12 cell differentiation. (B) Histogram showing quantitative results of multinucleated myotubes in immunofluorescence assay. (C) Immunofluorescence staining of MYHC with anti-MYH7 antibody in C2C12 cells 6 days after differentiation. Scale: 1 bar represents 100 μm or 200 μm. The immunofluorescence images for Myh7 quantities were analyzed using ImageJ software. (D) The effect of tRF-Lys-TTT-012 overexpression on the relative mRNA expression of myofiber type marker genes during C2C12 cell differentiation. (E) The effect of tRF-Lys-TTT-012 overexpression on the relative mRNA expression of troponin marker genes during C2C12 cell differentiation. (F) The effect of tRF-Lys-TTT-012 overexpression on the relative protein expression of troponin marker genes during C2C12 cell differentiation. (G) Histogram showing quantitative results of the relative protein expression of troponin marker genes. All data were presented as Mean ± SEM. The symbol * denotes a significance level of p < 0.05, ** p < 0.01, ns indicates no significance. The relative mRNA levels are normalized to β-actin. NC, Negative control; Mimics, tRF-Lys-TTT-012 mimics.

Figure 8

Overexpression of tRF-Lys-TTT-012 increases mitochondrial content and promotes mitochondrial function in vitro. (A) The effect of mitochondrial copy number after overexpression of Lys-TTT-012. (B) The relative mRNA expression of PGC-1α after tRF-Lys-TTT-012 overexpression in C2C12 cells. (C,D) The change in protein levels of PGC-1α after overexpression of tRF-Lys-TTT-012. (E) Immunofluorescence analysis depicting the effect of tRF-Lys-TTT-012 overexpression on fluorescence intensity related to mitochondrial reactive oxygen species in PK15 cells. (F) Quantification of fluorescence intensity corresponding to mitochondrial reactive oxygen species staining in PK15 cells with tRF-Lys-TTT-012 overexpression using the ImageJ software. (G) Mitochondrial fluorescence staining showing the impact of tRF-Lys-TTT-012 overexpression on mitochondrial content within PK15 cells. (H) Quantification of mitochondrial content using the ImageJ software based on fluorescence intensity in PK15 cells with tRF-Lys-TTT-012 overexpression. All data were presented as Mean ± SEM. The symbol * denotes a significance level of p < 0.05, ** p < 0.01, *** p < 0.001. The relative mRNA levels are normalized to β-actin. NC, negative control; Mimics, tRF-Lys-TTT-012 mimcis.