Skeletal muscle fiber types can contribute in part to affecting pork quality parameters. Biceps femoris (Bf) (fast muscle or white muscle) and Soleus (Sol) (slow muscle or red muscle) are two typical skeletal muscles characterized by obvious muscle fiber type differences in pigs. However, the critical proteins and potential regulatory mechanisms regulating porcine skeletal muscle fibers have yet to be clearly defined. In this study, the isobaric Tag for Relative and Absolute Quantification (iTRAQ)-based proteome was used to identify the key proteins affecting the skeletal muscle fiber types with Bf and Sol, by integrating the previous transcriptome data, while function enrichment analysis and a protein–protein interaction (PPI) network were utilized to explore the potential regulatory mechanisms of skeletal muscle fibers. A total of 126 differentially abundant proteins (DAPs) between the Bf and Sol were identified, and 12 genes were found to be overlapping between differentially expressed genes (DEGs) and DAPs, which are the critical proteins regulating the formation of skeletal muscle fibers. Functional enrichment and PPI analysis showed that the DAPs were mainly involved in the skeletal-muscle-associated structural proteins, mitochondria and energy metabolism, tricarboxylic acid cycle, fatty acid metabolism, and kinase activity, suggesting that PPI networks including DAPs are the main regulatory network affecting muscle fiber formation. Overall, these data provide valuable information for understanding the molecular mechanism underlying the formation and conversion of muscle fiber types, and provide potential markers for the evaluation of meat quality.

Alterations in skeletal muscle fibers are closely linked to human diseases, yet the mechanisms by which DNA methylation drives muscle fiber diversity remain unclear. Here, we performed whole-genome bisulfite sequencing (WGBS) to generate single-base-resolution DNA methylomes of fast-twitch Biceps Femoris (Bf) and slow-twitch Soleus (Sol) muscles and integrated transcriptomes to reveal associations between DNA methylation and gene expression. The global methylation level in skeletal muscle averaged 4.22%, with context-specific rates of 70.0% (CG), 1.16% (CHG), and 1.12% (CHH). The distribution of methylation levels of the mCs in CG and CHG contexts was similar but distinct from that in the CHH context. Furthermore, 152,286 DMCs and 51,079 DMRs were identified between Bf and Sol muscles. Bf showed higher global methylation (4.54%) than Sol (3.90%), and distinct differences in DNA methylation occurred throughout the genomic regions and gene functional element regions. CG-context DMCs and DMRs were mainly located in CpG islands and introns, while CHG and CHH DMCs and DMRs were exon-enriched. DMR-associated genes were significantly enriched in pathways related to muscle growth and remodeling and disease-related pathways. Moreover, five DMR-linked differentially expressed genes—HSPB6, ATP2A2, TNNI1, HOXA11, and PTPN3—were identified as critical candidate regulators of muscle fiber diversity. In addition, a context-dependent (CG, CHG, CHH) relationship between DNA methylation patterns and gene expression was observed across different genomic regions in the two muscle types. Our findings highlight the essential role of DNA methylation in skeletal muscle fiber diversity and identify potential epigenetic targets and pathways.