SnRNA-seq identifies distinct cell populations after CLAs treatment in pig muscles.

(A) TG and TC content of LDM tissues in control and CLAs groups (n=5). (B) LDM tissues stained with the adipogenic marker perilipin (red), muscle fiber marker MyHC (green) and DAPI (blue) in different groups. Scale bars, 200 and 100 μm, respectively. (C) Scheme of the experimental design for snRNA-seq on different muscles. (D) UMAP visualization of all of the isolated single nuclei from Heigai pig muscles colored by cluster identity. (E) UMAP and violin plot displaying the expression of selected marker genes for each cluster in pigs. (F) Nuclear proportion in each cluster in pig muscles of control and CLAs groups. Each cluster is color-coded. (G) Left, heatmap showing the top 10 most differentially expressed genes between cell types identified. Right, KEGG enrichment for marker genes of each cell type in muscles. Each lane represents a subcluster. Error bars represent SEM. * P < 0.05, ** P < 0.01, two-tailed Student’s t-test.

Cell and transcriptional heterogeneity in myofibers.

(A) UMAP plot showing six subclusters of the isolated single nuclei from the control and CLAs muscles. (B) UMAP and violin plot displaying the expression of selected marker genes for each subcluster. (C) Cell proportion in each subcluster in different groups. Each cluster is colour-coded. (D) Violin plot showing the expression of myofiber type marker genes (MYH7, MYH2, MYH1 and MYH4), myofiber type transformation-related genes (PPARGC1A, and STK11), oxidation-related genes (COX5A, COX5B, and COX8A), and glycolysis-related genes (PFKM, HK2, and LDHC) after CLAs treatment. (E) The mRNA expression of myofiber type related genes in LDM muscles after different treatment (n=6). (F) Heatmap representing the top 10 most differently expressed genes between cell subclusters identified. (G) KEGG enrichment for marker genes of each cell type in myofibers. I, type I myonuclei; IIa, type IIa myonuclei; IIx, type IIx myonuclei; IIb, type IIb myonuclei; MTJ, myotendinous junction nuclei; NMJ, neuromuscular junction nuclei. Error bars represent SEM. *P < 0.05, two-tailed Student’s t-test.

Clustering and transcriptional dynamics of adipocytes.

(A) UMAP plot displaying the isolated single nuclei in three subclusters of adipocytes. (B) Bar plot displaying the cell amounts in each subcluster in different groups. (C) Dot plot showing the expression of three subcluster marker genes in muscle nuclei of Heigai pigs. (D) The mRNA expression of three subcluster marker genes in LDM muscles after different treatment (n=6). (E) LDM tissues stained with the adipogenic marker perilipin (red), muscle fiber marker MyHC (green), SCD1 (pink), and DAPI (blue) in different groups. Scale bars, 100 μm. (F) Left, heatmap showing the top 10 most differentially expressed genes between cell types identified. Right, KEGG enrichment for marker genes of each cell type in muscles. (G) Unsupervised pseudotime trajectory of the three subtypes of adipocytes by RNA velocity analysis. Trajectory is colored by cell subtypes. The arrow indicates the direction of cell pseudo-temporal differentiation. (H) Scheme of the differentiation trajectories in mature adipocytes. Error bars represent SEM. *P < 0.05, ** P < 0.01, two-tailed Student’s t-test.

Pseudotemporal and differentiated trajectories of adipocytes in high IMF content Laiwu pig muscles.

(A) Scheme of the experimental design for snRNA-seq on adipocytes of high IMF content Laiwu pig muscles. (B) Cell proportion of adipocytes subclusters in HLW and LLW groups. Each cluster is color-coded. (C) LDM tissues stained with the adipogenic marker perilipin (red), muscle fiber marker MyHC (green), SCD1 (pink), and DAPI (blue) in HLW and LLW groups. Scale bars, 100 μm. (D-E) Pseudotime ordering of all of adipocytes of subcluster DGAT2+/SCD+, FABP5+/SIAH1+, and PDE4D+/PDE7B+. Each dot represents one nucleus (color-coded by its identity), and each branch represents one cell state. Pseudotime is shown colored in a gradient from dark to light blue, and the start of pseudotime is indicated. Activation of the PDE4D+/PDE7B+ cluster can lead to DGAT2+/SCD+ and FABP5+/SIAH1+ fate. (F) Unsupervised pseudotime trajectory of the three subtypes of adipocytes by RNA velocity analysis. Trajectory is colored by cell subtypes. The arrow indicates the direction of cell pseudo-temporal differentiation. (G) Dot plot showing the expression of preadipocytes and mature adipocytes-related genes in different subclusters. (H) Scheme of the differentiation trajectories in mature adipocytes of Laiwu pigs. (I) Violin plot showing the expression of three subcluster marker genes in different groups.

Cell-cell communication analysis of adipocytes in pig muscles.

(A) Cell-cell communication analysis showed the network between adipocytes and other clusters in muscles of Heigai pigs. (B) Cell-cell communication analysis showed the network between adipocytes and other clusters in muscles of Laiwu pigs. (C) Dotplot representing the gene expression and significance of the receptor-ligand relationship in different cell population in muscles of Heigai pigs. (D) Dotplot representing the gene expression and significance of the receptor-ligand relationship in different cell population in muscles of Laiwu pigs. The larger the circle, the smaller the P value of the relationship in the corresponding cell population, the more significant it is.

Clustering and pseudotemporal trajectories of FAPs.

(A) UMAP plot showing three subclusters of the isolated single nuclei from control and CLAs muscle. (B) Violin plot displaying the expression of selected marker genes for each subcluster. (C) Cell proportion in each subcluster in different group. Each cluster is color-coded. (D) Left, heatmap showing the top 10 most differentially expressed genes between cell types identified. Right, KEGG enrichment for marker genes of each cell type in muscles. (E) Pseudotime ordering of all of the FAP/fibroblast of subcluster FAPs, Fibroblasts, and PDE4D+/PDE7B+. Each dot represents one nucleus (color-coded by its identity), and each branch represents one cell state. Pseudotime is shown colored in a gradient from dark to light blue, and the start of pseudotime is indicated. Activation of the FAP cluster can lead to fibroblast fate or PDE4D+/PDE7B+ fate. (F) Pseudotemporal heatmap showing gene expression dynamics for significant marker genes. Genes (rows) were clustered into three modules, and cells (columns) were ordered according to pseudotime in different groups. (G) The expression of adipogenesis and three subcluster marker genes in differentiated FAPs in different differentiation stage (n=6). (H) Scheme of the differentiation trajectories of preadipocytes into mature adipocytes. Error bars represent SEM. *P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student’s t-test.

The cytological mechanism of CLAs regulates FAPs differentiation.

(A) Dfferentiated FAPs stained with Nile Red (red) and DAPI (blue) in different groups. Scale bars, 200 and 100 μm, respectively. (B) OD490 levels of total lipids in differentiated FAPs after different treatment (n=4). (C) The mRNA expression of three subcluster marker genes and adipogenic marker genes in differentiated FAPs after different treatment (n=5). (D) Protein levels of FABP4, SCD1, and PDE4D were detected by western blot. (E) MAPK signalling pathway enrichment in different cells. (F) Dot plot showing the expression of MAPK signalling pathway related genes after CLA treatment in FAPs/Fibroblasts. (G) Protein levels of P-JNK and JNK were detected by western blot. (H) Scheme of CLAs regulating the differentiation trajectories of FAPs into mature adipocytes. (I) Dfferentiated FAPs stained with Oil Red O in different groups after treating with 20 nM Anisomysin. (J) OD490 levels of total lipids in differentiated FAPs after different treatment (n=4). (K) Dfferentiated FAPs stained with Nile Red (red) and DAPI (blue) after 20 nM Anisomysin treatment. Scale bars, 200 μm. (L) The mRNA expression of adipogenic related genes in differentiated FAPs after different treatment (n=4). Error bars represent SEM. *P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student’s t-test and one-way ANOVA analysis. Letters represent statistical significance. Lowercase letters represent P < 0.05 and uppercase letters represent P < 0.01.