Single-nucleus transcriptomics reveal the cytological mechanism of conjugated linoleic acids in regulating intramuscular fat deposition

Conjugated linoleic acids (CLAs) can serve as a nutritional intervention to regulate quality, function and fat infiltration in skeletal muscles but the specific cytological mechanisms are still unknown. Here, we applied single-nucleus RNA-sequencing (snRNA-seq) to characterize the cytological mechanism of CLAs regulates fat infiltration in skeletal muscles based on pig models. We investigated the regulatory effects of CLAs on cell populations and molecular characteristics in pig muscles and found CLAs could promote the transformation of fast glycolytic myofibers into slow oxidative myofibers. We also observed three subpopulations including SCD+/DGAT2+, FABP5+/SIAH1+, and PDE4D+/PDE7B+ subclusters in adipocytes and CLAs could increase the percentage of SCD+/DGAT2+ adipocytes. RNA velocity analysis showed FABP5+/SIAH1+ and PDE4D+/PDE7B+ adipocytes could differentiate into SCD+/DGAT2+ adipocytes. We further verified the differentiated trajectory of mature adipocytes and identified PDE4D+/PDE7B+ adipocytes could differentiate into SCD+/DGAT2+ and FABP5+/SIAH1+ adipocytes by using high IMF content Laiwu pig models. The cell-cell communication analysis identified the interaction network between adipocytes and other subclusters such as fibro/adipogenic progenitors (FAPs). Pseudotemporal trajectory analysis and RNA velocity analysis also showed FAPs could differentiate into PDE4D+/PDE7B+ preadipocytes and we discovered the differentiated trajectory of preadipocytes into mature adipocytes. Besides, we found CLAs could promote FAPs differentiate into SCD+/DGAT2+ adipocytes via inhibiting c-Jun N-terminal kinase (JNK) signalling pathway in vitro. This study provides a foundation for regulating fat infiltration in skeletal muscles by using nutritional strategies and provides potential opportunities to serve pig as an animal model to study human fat infiltrated diseases.


Introduction
Meat is one of the most important sources of animal protein for humans, and its quality is associated with human health.Recently, due to the development of economic levels and the improvement of living standards, people are seeking for 'less but better' meat (Sahlin & Trewern, 2022).Intramuscular fat (IMF) deposition is a key factor positively related to meat quality traits and lipo-nutritional values of meat, such as flavor, tenderness, and juiciness (Hausman, Basu, Du, Fernyhough-Culver, & Dodson, 2014;W. Yi, Huang, Wang, & Shan, 2023).Besides, fat infiltration in skeletal muscle (also known as myosteatosis) is the pathologic fat accumulation in skeletal muscle with poor metabolic and musculoskeletal health, it always accompanied by the decline of muscle quality and function (Biltz et al., 2020;Jiang, Marriott, & Maly, 2019).Myosteatosis is now considered as a common feature of ageing and is also related to some diseases (Wang, Valencak, & Shan, 2024).However, the occurrence mechanism and cell sources of fat accumulation in skeletal muscle is very complicated.Recently, with the rapid development of multi-omics including single-cell RNA sequencing (scRNA-seq) (Tabula Muris et al., 2018), single-nucleus RNA-seq (snRNA-seq) (Petrany et al., 2020), and spatial transcriptomics (ST) (Jin et al., 2021), more and more cell types have been found to contribute to lipid deposition in skeletal muscle including myogenic cells (e.g., satellite cells (SCs) (Asakura, Komaki, & Rudnicki, 2001) and myogenic factor 5 (Myf5) + mesenchymal stem cells (MSCs) (Yin et al., 2013)) and non-myogenic cells (e.g., fibro/adipogenic progenitors (FAPs) (Uezumi, Fukada, Yamamoto, Takeda, & Tsuchida, 2010), fibroblasts, myeloid-derived cells(Z.Xu et al., 2020), pericytes (Farrington-Rock et al., 2004), endothelial cells (ECs) (Lang et al., 2008), PW1 + /Pax7 -interstitial cells (PICs) (Mitchell et al., 2010), and side population cells (SPs) (Tamaki et al., 2002)) based on animal models.Hence, more and more researches have been focusing on exploring the regulatory mechanism of myosteatosis at the cytological levels.Besides, fat infiltration in skeletal muscle is regulated by many influential triggers, including ageing, metabolic and nonmetabolic diseases, disuse and inactivity, and muscle injury (Wang et al., 2024).Many genes and signaling pathways participate in the formation and regulation of fat infiltration in skeletal muscle (Biferali et al., 2021;Wosczyna et al., 2021).However, the specific mechanism of nutrients regulates fat infiltration in muscle is still unknown.
Nutritional regulation strategy is one of the most vital strategies to regulate lipid accumulation in skeletal muscle based on some animal models and clinic trials, including vitamins (Gilsanz, Kremer, Mo, Wren, & Kremer, 2010;L. Zhao et al., 2020), conjugated linoleic acid (CLAs) (van Vliet, Fappi, Reeds, & Mittendorfer, 2020), linseed (Wei et al., 2016), plant extract (You et al., 2023), and so on.Hence, exploring the potential nutritional strategies to regulate fat accumulation in skeletal muscle is valuable for animal production and human health.Pigs are not only an important source of animal protein in the human diet but also serve as a valuable model for human medical biology due to their similar physiological structure in terms of size, metabolic characteristics, and cardiovascular system (Groenen et al., 2012;Lunney et al., 2021).Chinese local pig species could be used as excellent animal models to study the mechanism of lipid deposition due to they have better meat quality and high IMF content.A high IMF content always results in better juiciness, tenderness, and flavor of pork.There are different myogenesis potential in neonatal skeletal muscle between Laiwu pigs and Duroc pigs(D.D. Xu et al., 2023).Our previous study has revealed the cell heterogeneity and transcriptional dynamics of lipid deposition in skeletal muscle of Laiwu pigs and we found high IMF content Laiwu pigs had lower muscle fiber diameter (Wang, Zhao, et al., 2023).Heigai pig is a model of Chinese indigenous pig breeds, which has advantages including high farrowing rate, good pork quality, and strong disease resistance(Liyi Wang et al., 2022).Specially, we have found CLAs can improve meat quality especially increase IMF content both in lean type pig breeds and fat type pig breeds (Wang, Huang, Wang, & Shan, 2021;L. Wang et al., 2022).However, although many studies have discussed the effects of CLA on IMF deposition, there are still gaps in the cytological mechanism of CLAs in regulating lipid deposition in skeletal muscle.
Here, we present a snRNA-seq dataset collected from longissimus dorsi muscle (LDM) of Heigai pigs after feeding CLAs supplement to allow for analysing heterogeneity of transcriptional states in muscles.We investigated the regulatory effects of CLAs on the muscle fiber type transformation and IMF deposition.We also identified the differentiation trajectories of three subclusters in adipocytes based on Heigai pig models and high IMF content Laiwu pig models.Based on the pseudotemporal trajectories analysis, we found CLAs could promote FAPs differentiate into SCD + /DGAT2 + adipocytes via regulating mitogen-activated protein kinase (MAPK) signalling pathway.This study paves a way to regulate lipid accumulation in muscles by using nutritional strategies and provides theoretical basis on using pig as an animal model to study human muscle-related diseases.

CLAs changed cell populations and transcriptional dynamics in LDM
Our previous study discovered CLAs improved IMF content in LDM of Heigai pigs(L.Wang et al., 2022), here, we also found CLAs significantly increased TG content but significantly decreased TC content in LDM of pigs (Figure .1A).
Meanwhile, immunofluorescence staining results showed more lipid droplets in the LDM of the CLAs group (Figure .1B).To investigate the changes of cell heterogeneity in pig muscles after CLAs treatment at the cellular level, we performed snRNA-seq from LDM tissue of Heigai pigs using the 10× Genomics Chromium platform (Figure . 1C).First, after Cell Ranger analyses, the estimated number of cells, fraction of reads in cells, mean reads per cell, median genes per cell and median UMI counts per cell in LDM were showed in Supplementary Fig. S1a.We obtained 25507 cells from 2 individual libraries, comprising 10835 cells from CON group and 11705 cells from CLA group for the downstream analysis after the quality control of snRNA-seq data (Figure . S1B).Based on the Seurat package, we used Uniform Manifold Approximation and Projection (UMAP) plots to show the different subclusters (Figure . 1D).We identified 8 different clusters in two groups of pig muscles, including myofibers (CAPN3), FAPs/fibroblasts (PDGFRA), ECs (CD34), adipocytes (PPARG), immune cells (PTPRC), muscle satellite cells (MuSCs) (PAX7), myeloid derived cells (MRC1) and pericytes (RGS5) using the expression of marker genes (Figure . 1E).Next, the percentage of these cell types showed differences in different group (Figure . 1F).Compared with the CON group, the FAPs/fibroblasts (3.39% vs. 8.31%), ECs (1.26% vs. 2.94%), adipocytes (1.74% vs. 2.37%), myeloid derived cells (0.93% vs. 2.17%) and pericytes (0.45% vs. 0.7%) had a higher proportion in the CLA group.However, CLA decreased the proportion of myofibers (90.46% vs. 81.1%).The top 10 most differentially expressed genes (DEGs) were showed in the heatmap and the bar plot showed the top 3 KEGG enrichment of DEGs between the 8 different cell types in LDM (Figure .1G).Besides, violin plots displayed CLAs upregulated the expression of mature adipocyte master genes including ADIPOQ, FABP4, PLIN1, and LIPE, adipogenic marker genes including PPARG, PPARA, CEBPA, and CEBPB, and lipid metabolism-related genes including LPL, ELOVL4, ACAA2 and HACD2 in pig muscles (Figures.S1C-E).These results indicated the cell types in LDM of Heigai pigs had significant differences after feeding CLAs which might induce the alterations in lipid deposition.
These data indicated the differentiated trajectory of mature adipocytes in muscle of pigs and the percentage of SCD + /DGAT2 + and FABP5 + /SIAH1 + subclusters was higher in HLW group.

Transcriptional dynamics of glycerophospholipid metabolism in high IMF deposition pigs
Our previous studies have discovered the changes of glycerophospholipid metabolism in muscles after CLA treatment(L.Wang et al., 2022) and in high IMF content Laiwu pigs (Wang, Zhao, et al., 2023).To further investigate the transcriptional dynamics of glycerophospholipid metabolism in high IMF deposition pigs, we then compare the gene program in Heigai pigs and Laiwu pigs.After CLA treatment, the snRNA-seq dataset the expression of genes involved in the glycerophospholipid metabolism in different groups and subclusters was displayed slight differences in (Figures . S4A-C).Also, in Laiwu pigs, there are differences in gene program involved in the glycerophospholipid metabolism between two groups (Figures.S4D-F).Interestingly, we found LCLAT1 was enriched in PDE4D + /PDE7B + subcluster and AGPAT3 and AGPAT5 was enriched in SCD + /DGAT2 + subcluster (Figures. S5B and S5E).We also discovered the increase of diglycerides and phosphatidylinositols and the decrease of phosphatidic acids and phosphatidylethanolamines in high IMF deposition pigs might due to the changes of AGPAT3, AGPAT4, AGPAT5, CEPT1, and CDIPT1 (Figures.S5C and S5F).These data revealed the significant differences in lipid composition and distribution in LDM of high IMF deposition pigs might be due to the different expression levels of glycerophospholipid metabolism-related genes.

FAPs/fibroblasts
To further explore the association between adipocytes nuclei and other cell clusters, we applied cell-cell communication analysis by using CellPhoneDB on 8 cell types in muscles of Heigai pigs and found adipocytes mainly interacted with ECs, 37.06%) and PDE4D + /PDE7B + (4.11% vs. 2.18%) was higher than that in CON group but the proportion of in Fibroblasts (60.76% vs. 25.49%) was lower ( These data indicated that CLAs may promote the directed differentiation of FAPs into SCD + /DGAT2 + subclusters via inhibiting JNK signaling pathway.

Discussion
CLAs can serve as a nutritional intervention to regulate lipid deposition in skeletal muscle of human according to clinic trials (van Vliet et al., 2020).In animal production, CLAs could regulate meat quality in pigs and cattle, especially improve IMF content (L.Wang et al., 2022;Zhang et al., 2016).These studies pointed out that the CLAs plays a vital role in regulating fat infiltration in skeletal muscle.However, to date, the cellular mechanism of CLAs regulates lipid deposition has not been studied.Here, we utilized the 10x Genomics platform to identify the cell heterogeneity and transcriptional changes in muscles after CLAs treatment based on pig models.This study revealed the effects of CLAs on cell populations and molecular characteristics of muscles and highlighted the cytological mechanism of CLAs regulates pork quality in skeletal muscle.
CLAs is always found in ruminant animals and dairy products, it is a class of positional and geometric isomers of linoleic acids with a conjugated double bond.
CLAs not only has anti-cancer, anti-hypertension, anti-adipogenic, and anti-diabetic effects, but also can improve muscle function and decrease body fat percentage.For example, adding 3.2 g/day CLAs significantly increased muscle mass in higher body fat percentage Chinese adults (Chang et al., 2020).After 0.9 g/day CLAs supplementation, body weight variation and muscle mass significantly increased and body fat percentage variation decreased in student athletes (Terasawa, Okamoto, Nakada, & Masuda, 2017).LC-MS metabolomics results discovered CLAs changed 57 metabolites which enriched in lipids/lipid-like molecules in plasma of humans (He et al., 2022).However, another study found that for sedentary older adults, CLAs had no significant influence on muscle anabolic effects (van Vliet et al., 2020).Therefore, D. Xu et al., 2023;Z. Xu et al., 2020).We also found CLAs improved TG content and increased the percentage of adipocytes in LDM.Previous study demonstrated there are three subclusters in adipocytes and the formation and deposition of IMF mainly relied on DGAT2 + /SCD + adipocytes and FABP5 + /SIAH1 + adipocytes (Wang, Zhao, et al., 2023).Specially, we found CLAs enhanced the percentage of SCD + /DGAT2 + subclusters.These indicated CLAs improve IMF deposition might through increasing SCD + /DGAT2 + subpopulations of adipocytes.Our findings could provide a foundation for using nutritional strategies to increase pork quality especially IMF deposition.NMJ are responsible for formation and maintenance of the synaptic apparatus and previous studies have identified these cell populations in murine skeletal muscles by using snRNA-seq (Dos Santos et al., 2020;Petrany et al., 2020).We also identified MTJ and NMJ cell populations in LDM of Laiwu pigs (Wang, Zhao, et al., 2023).
These results indicate it also exist MTJ and NMJ cell populations in porcine skeletal muscles.In our previous study, we also found the percentage of type IIa myofibers had an increased tendency and type IIb myofibers had a decreased tendency in high IMF content Laiwu pigs (Wang, Zhao, et al., 2023).These results suggest that IMF content is closely related to muscle fiber type.Besides, slow muscle fibers always called slow-twitch oxidative muscle fibers like I myofibers have higher activities in mitochondrial oxidative metabolic enzymes and myoglobin while fast muscle fibers always called fast-twitch glycolytic muscle fibers like II myofibers have higher levels of glycolytic enzymes and glycogen (Schiaffino & Reggiani, 2011).Similarly, we also found I myofibers enriched in metabolic pathways, oxidative phosphorylation, and thermogenesis.In recent years, studies have focused on exploring the influences of CLAs on regulating muscle fiber type.In commercial pigs, the MyHC I mRNA abundance were improved in LDM of the CLAs group (Men, Deng, Xu, Tao, & Qi, 2013).In mice, t10, c12-CLAs, but not c9, t11-CLAs can increase oxidative skeletal muscle fiber type in gastrocnemius muscle and C2C12 myoblasts (Duan et al., 2021).
CLAs have been found to prevent sarcopenia by maintaining redox balance during aging, actively regulating mitochondrial adaptation, improve muscle metabolism, and inducing hypertrophy of type IIX myofibers after endurance exercise (Barone et al., 2017;Chen, Yang, & Park, 2018).Also, we discovered CLAs enhanced the percentage of I and IIA myofibers but reduced the percentage of IIB myofibers.
Previous study has found PPARγ coactivator-1α (PGC1α) serves a valuable role in skeletal muscle metabolism and is a master regulator of oxidative phosphorylation genes and could regulate muscle fiber type transformation (Handschin & Spiegelman, 2011).In our study, the PGC1α expression was also increased after CLA treatment in myofiber.These results suggested CLAs can promote glycolytic skeletal muscle fiber types switching into oxidative skeletal muscle fiber types through upregulating PGC1α expression.
Numerous studies have discovered that the cell sources of IMF cells and found several cell subsets lead to the ectopic IMF formation and deposition including SCs, Myf5 + MSCs, FAPs, ECs, pericytes, Fibroblasts, myeloid-derived cells, SPs, and PICs (Sciorati, Clementi, Manfredi, & Rovere-Querini, 2015;Z. Xu et al., 2020).In this study, we found CLAs increased the percentage of preadipocytes such as FAPs, ECs, myeloid-derived cells, and pericytes.Importantly, FAPs are the major source of IMF cells (Joe et al., 2010;Uezumi et al., 2010) and our previous study also verified the adipogenic capacity of FAPs in 2D and 3D culture models (Wang, Zhao, et al., 2023).
Xu et al. found that FAPs serve as a cellular interaction hub in skeletal muscle of pigs(D.D. Xu et al., 2023).We used pseudotemporal trajectory and RNA velocity analysis combined with in vitro study to investigate that FAPs could first differentiate into PDE4D + /PDE7B + adipocytes and then differentiate into DGAT2 + / SCD + and FABP5 + /SIAH1 + adipocytes.However, the regulatory mechanism of FAPs directional differentiation still needs to be further explored.In vitro studies demonstrated trans-10, cis-12 CLAs inhibited skeletal muscle differentiation in C2C12 cells and inhibited 3T3-L1 adipocyte adipogenesis (Hommelberg et al., 2010;Yeganeh, Taylor, Poole, Tworek, & Zahradka, 2016).However, the influences of CLAs on regulating the adipogenic differentiation of FAPs are still unclear.In this study, we found CLAs facilitated FAPs differentiating into SCD + /DGAT2 + adipocytes.Previous studies have found mice orally treated with CLAs mixture upregulated Scd1 expression in muscle (Parra, Serra, & Palou, 2012).Besides, SCD1 expression is modulated by mTOR signaling pathway in cancer cells(J.M. Yi, Zhu, Wu, Thompson, & Jiang, 2020;S. H. Zhao et al., 2021).Moreover, MAPK signaling pathway were enriched in adipogenic differentiation of FAPs after CLAs treatment.Previous studies have discovered JNK had negative effects on regulating the adipogenic differentiation of human mesenchymal stem cells (Jang et al., 2015) and FAPs could prevent skeletal muscle regeneration after muscle injury by ST2/JNK signaling pathways (Yamakawa et al., 2023).In this study, based on JNK signing pathway activator treatment and in vitro experiment, we found CLAs may promote FAPs directed differentiation into SCD + /DGAT2 + adipocytes via inhibiting JNK signaling pathway.These results may provide new targets for treating human fat infiltrated diseases by nutritional strategies.
However, we did not further explore the mechanistic action and the downstream transcriptional regulators need to be discussed.
In a word, we provide detailed insights into the cytological mechanism of CLAs regulates fat infiltration in skeletal muscles based on pig models via using snRNA-seq.
We analysed the effects of CLAs on the cell heterogeneity and transcriptional dynamics in pig muscles and discovered CLAs could promote glycolytic muscle fiber types switching into oxidative muscle fiber types through regulating PGC1α.We also identified the differentiation trajectories of adipocytes and FAPs.Our data also demonstrated CLAs could promote FAPs differentiate into DGAT2 + /SCD + adipocytes via inhibiting JNK signalling pathway.This study provides a new way of developing nutritional strategies to combat myosteatosis and other muscle-related diseases and also offers potential opportunities to promote the utilization of pigs as animal models to study human diseases.

Animals and samples
The Zhejiang University Animal Care and Use Committee approved all procedures and housing (ZJU20170466).56 Heigai pigs (average body weight: 85.58 ± 10.39 kg) were divided randomly into CON group (added 1% soyabean oil) and CLA group (added 1% CLAs) for 40 days (5 days pre-feeding period and 35 days formal test period) and the nutritional levels and the feeding process as we previously reported(L.Wang et al., 2022;Wang, Zhang, Huang, Zhou, & Shan, 2023).At the end of experiment, we collected LDM from the right side of the carcass for subsequent immunofluorescence staining, biochemical assay, and snRNA-seq analyses.For Laiwu pigs, based on the determination of IMF content in Laiwu pigs, we divided them into two groups: HLW group and low IMF content Laiwu pigs (LLW) group.
The two most representative samples from each group were selected for later snRNA-seq and datasets generated from muscle of high IMF content pig samples were downloaded from the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA011059; https://ngdc.cncb.ac.cn/gsa) as we previously discussed (Wang, Zhao, et al., 2023).

Triglycerides (TG) and total cholesterol (TC) determination
The contents of TG and TC in LDM were determined by commercial kits (TG, E1025-105; TC, E1015-50) bought from Beijing APPLYGEN Gene Technology Co., LTD.

Immunofluorescence staining
The paraffin section was dewaxed and immersed in pre-heated sodium citrate, then placed in a microwave oven and heated for 15 min to perform antigen retrieval.
Fixed the sections that cooled to room temperature in 4% paraformaldehyde for 10 min, followed by 10 min permeated with 0.5% Triton-X100and 1 h blocked with SnRNA-seq results were demultiplexed and converted to FASTQ format by using Illumina bcl2fastq software and followingly processed by the Cell Ranger.Then, the Seurat packages was used to analysis the cell Ranger output.After the quality control, 22540 cells were obtained.DoubletFinder package was used to remove doublets and Harmony package was used to performed batch correction of data integration between samples.We further used Seurat, UMAP, the FindAllMarkers function to visualize the data, find clusters, and select marker genes.Monocle 2 package was used to perform trajectory analysis and model differentiation trajectories.
RNA velocity analysis was independently performed in FAPs by SAMTools and the Velocyto (Bergen, Lange, Peidli, Wolf, & Theis, 2020).CellPhoneDB package was used to cell communication analysis and make further speculations about potential cellular interaction mechanisms.

Primary FAPs isolation, magnetic cell sorting and cell culture
Primary FAPs isolation were performed as previously described (Wang, Zhao, et al., 2023).Briefly, a piece of muscle from a 3-day-old piglet was minced, and added 5 times the volume of 0.2% collagenase type I, then digested at 37 °C for 1 hour.,After filtering and centrifuging, adding red blood cell lysate to split for 5 min at 4 °C followed by incubated with a Dead Cell Removal Kit at room temperature for 15 min.
Then adding CD140a antibody, incubating and centrifugating.Next, added 20 μL antibiotin microbeads, incubation and centrifugation.After passing through the magnetic column, the cells on the adsorption column were PDGFRα + cells.For FAPs adipogenic differentiation, when the cells confluence reached 90%, the10% FBS growth medium was replaced with induction medium after 4 days, the medium was changed to differentiation mediumand cultured for another 4 days until the adipocytes were mature.

Nile Red staining
Rinse cultured FAPs with 1 x PBS 3 times, discard PBS, fix FAPs with 4% formaldehyde for 15 min, repeat the rinsing step, and then add Nile Red solution (1:500 for lipid droplet staining) and DAPI (1:500 for nuclei staining) for 5 min.
Sealed cell with glycerol and used fluorescent microscope to capture images.

Total RNA extraction and quantitative real-time PCR (qPCR)
Total RNA extraction and qPCR were conducted as described before (Shan et al., 2016).Briefly, total RNA of FAPs were extracted by using TRIzol and the Spectrophotometer and a ReverAid First Strand cDNA Synthesis Kit were used to measure the purity and concentration of total RNA and reversed RNA samples.qPCR was performed by using Applied Biosystems StepOnePlus Real-Time PCR System with Hieff qPCR SYBR® Green Master Mix and gene-specific primers (Supplementary Table 1).Relative changes in gene expression were analysed using the 2 -ΔΔCT method and normalized using 18S ribosomal RNA as an internal control.

Protein extraction and western blotting
Protein extraction and western blotting were carried out as mentioned previously (Shan et al., 2016).In brief, total proteins were isolated from cells or tissues with RIPA buffer.After measuring the concentrations, proteins were separated using SDS-PAGE and subsequently transferred to a polyvinylidene fluoride membrane (PVDF, Millipore Corporation).Then blocked PVDF membrane with blocking buffer (5% fat-free milk) for 1 h and incubated with primary antibodies overnight at 4 °C.

Statistical analysis
GraphPad 3D).Meanwhile, immunofluorescence results showed more SCD1 + adipocytes in the LDM of the CLAs group (Figure.3E).The top 10 most DEGs were displayed in the heatmap and the bar plot showed the top 3 KEGG enrichment of DEGs between the 3 subclusters in LDM (Figure.3F).To explore the effects of CLAs on differentiated trajectory of adipocytes, we carried out the RNA velocity analysis of adipocytes (Figure.S2C).RNA velocity results showed the differentiated trajectory of mature adipocytes in muscles of Heigai pigs (Figure.3G).Transcriptional dynamics of PDE4D and CAPN3 were showed in Figure.S2D based on RNA velocity analysis.These results indicated in mature adipocytes, PDE4D + /PDE7B + adipocytes and FABP5 + /SIAH1 + adipocytes could differentiate into SCD + /DGAT2 + adipocytes (Figure.3H).

FAPs
/fibroblasts, MuSCs, and pericytes (Figure.5A).In addition, dot plot represented stronger communication from adipocytes to other subclusters through LRP6, FGFR1, and COL4A2 pathways in LDM of Heigai pigs (Figure.5C).Next, we also applied cell-cell communication analysis by using CellPhoneDB on 9 cell types in muscles of Laiwu pigs and found adipocytes mainly interacted with SPs, FAPs/fibroblasts, ECs, and pericytes (Figure.5B).Also, dot plot showed stronger communication from adipocytes to other subclusters through COL6A3, LAMC1, and THBS1 pathways in LDM of Laiwu pigs (Figure.5D).These results suggested adipocytes has tight association with FAPs/fibroblasts.Characterization of FAPs after CLAs treatment through clustering and pseudotime analysisPrevious studies have demonstrated that FAPs could differentiate into mature adipocytes and are the main cell sources of IMF cells(Joe et al., 2010;Uezumi et al., 2010).To further investigate the effects of CLAs on occurrence mechanism of IMF deposition, we then performed subcluster and pseudotemporal trajectory analysis on FAPs/fibroblasts.First, UMAP plots displayed the cell distribution in different subpopulations of FAPs/fibroblasts (Figure.6A) and we identified 3 subpopulations in FAPs/fibroblasts based on the expression of marker genes, including FAPs (PDGFRA), Fibroblasts (COL1A1), and PDE4D + /PDE7B + subclusters (PDE4D and PDE7B) (Figure.6B).In CLA group, we found the proportion of FAPs (70.40% vs. Figure.6C).The top 10 most DEGs between the 3 subpopulations were showed in the heatmap and the bar plot displayed the significant enrichment of the signalling pathways in muscles by using KEGG enrichment analyses and we also found calcium and cGMP-PKG signalling pathway were enriched in PDE4D + /PDE7B + subclusters (Figure.6D).Besides, dot plot showed CLAs upregulated the expression of preadipocytes-related genes including CD38 and CD34, adipogenic master genes including ADIPOQ, FABP4, PLIN1, and LIPE, mature adipocyte marker genes including CEBPA, and lipid metabolism-related genes including LPL, ELOVL4, ACAA2 and HACD2 in FAPs/Fibroblasts (Figure.S5A).To further explore FAPs' differentiated trajectory, we applied a pseudotemporal trajectory analysis and RNA velocity analysis of FAPs/Fibroblasts.According to the results, we found FAPs could differentiate into PDE4D + /PDE7B + and Fibroblasts subpopulations (Figures.6Eand S5C).The pseudotemporal heatmap also displayed transcriptional dynamics including TIMP3 and THBS1 at Point 1 (Figure.6F).UMAP plots also showed transcriptional dynamics of marker genes in three subpopulations such as ITGA5, HSPH1, and ETF1 (Figures.S5B-E).To further identify the differentiated trajectory of IMF cells, we isolated primary FAPs from pigs and found the expression of FABP4, ADIPOQ, SCD, and DGAT2 was significantly increased but PDE4D expression was significantly downregulated during adipogenic differentiation in vitro (Figure.6G).Besides, the expression of FABP5 and SIAH1 was first significantly increased then significantly decreased during adipogenic differentiation (Figure.6G).Hence, FAPs may first differentiate into PDE4D + /PDE7B + preadipocytes and then differentiate into PDE4D + /PDE7B + adipocytes (Figure.6H).These results displayed the differentiated trajectory of preadipocytes into mature adipocytes in pig muscles and CLAs might influence this process then affect IMF deposition.CLAs promoted FAPs directed differentiation into SCD + /DGAT2 + subclustersTo further explore the cytological mechanism of CLAs regulating IMF deposition, we next investigated the regulatory effects of CLAs on the differentiation trajectory of FAPs differentiate into adipocytes.First, to explore the role of CLAs in the adipogenic differentiation of FAPs, we isolated primary FAPs from piglets and induced these adipogenic differentiation.Nile Red staining and OD490 results revealed CLAs can promote the adipogenic differentiation of FAPs after adipogenic differentiation for 8 days in vitro (Figures.7A-B).Besides, the mRNA expression of SCD, SIAH1 and adipogenic genes, including FABP4, PPARG, and FASN were significantly upregulated but PDE4D was significantly downregulated (Figure.7C).The protein levels of FABP4 and SCD1 were significantly upregulated and PDE4D were significantly downregulated (Figure.7D).In addition, the marker genes' expression at key points of adipogenic differentiation such as ADIPOQ, ELOVL6, ACACA, ARBB1, NEB, and MYBPC1 were significantly upregulated and THBS1 and TIMP3 were significantly downregulated (Figure.S6A).Interestingly, we next found MAPK signaling pathway were enriched in adipocytes nuclei especially SCD + /DGAT2 + subcluster (Figure.7E).The expression of MAPK signaling pathway including ERK, c-Jun N-terminal kinase (JNK), and p38 signaling pathway related genes were changed in muscle nuclei (Figures.7F and S6B-C).Furthermore, we observed that the protein levels of JNK phosphorylation were significantly decreased after CLA treatment during FAPs adipogenic differentiation (Figure.7G).Hence, we next used JNK activator Anisomysin to explore the regulatory mechanism (Figure.7H).Oil Red O staining result showed that after 24h 20 nM Anisomysin treatment, the increased adipogenic differentiation of FAPs by CLA were significantly inhibited (Figure.7I).Also, CLA + Anisomysin group had the lower OD 490 value compared with CLA group (Figure.7J).Nile Red staining result also showed that Anisomysin significantly inhibited the enhanced lipid droplets in FAPs after CLA treatment (Figure.7K).MAP2K4 expression significantly upregulated and the expression of FABP4 and SCD significantly decrease after Anisomysin treatment (Figure.7L).
the specific influences of CLAs on skeletal muscle is still disputed and the function on lipid deposition in human skeletal muscle needs further investigation.In animal models, many studies have demonstrated the important effects of CLAs on regulating fat accumulation in skeletal muscles.In porcine models, our foregoing studies have discovered that adding CLAs into the pig diet could significantly increase IMF contents in LDM of lean pig breeds and Heigai pigs(Wang et al., 2021;L. Wang et al., 2022).Zhang et al(Zhang et al., 2016)found 2% dietary CLAs significantly increased IMF deposition and reduced subcutaneous fat deposition in cattle.However, in mouse model, 0.5% mixed isomer CLAs did not lead to lipid accumulation in muscle of mice(M, S, & M, 2013).Hence, CLAs supplementation positively affect IMF deposition in muscles of pigs and ruminants but the effects of CLAs may have species-specific.In our study, we identified 8 cell types in skeletal muscles of Heigai pigs, including myofibers, FAPs/fibroblasts, ECs, adipocytes, immune cells, MuSCs, myeloid derived cells and pericytes.Recently, a variety of single cell studies have been performed on mouse/human skeletal muscles and they also identified some cell populations such as myofibers, FAPs/fibroblasts, ECs, adipocytes, immune cells, MuSCs, myeloid derived cells, tenocytes, SPs, and pericytes(Petrany et al., 2020; D. However, the specific function of CLAs on fat infiltration and deposition of skeletal muscle in people and rodents needs further study.Skeletal muscle contains slow and fast muscle fibers and there are four major muscle fiber types in mice, including slow muscle fibers with type I muscle fibers (Myh7), fast muscle fibers with type IIA muscle fibers (Myh2), type IIX muscle fibers (Myh1), and type IIB muscle fibers (Myh4)(Dos Santos et al., 2020;Petrany et al., 2020).In pigs, illustrated there are three myofiber composition including type I, type IIA, and type IIB in skeletal muscles by ST technology(Jin et al., 2021).In this study, we identified six different subpopulations in myofibers including I myofibers, IIA myofibers, IIX myofibers, IIB myofibers, MTJ, and NMJ and IIB myofibers had the highest percentage in pig muscles.MTJ are known to exhibit structural specialization;