Protein Expression Landscape Defines the Differentiation Potential

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Protein expression landscape defines the differentiation potential specificity of adipogenic and myogenic precursors in the skeletal muscle Kai Qiu, Xin Zhang, Liqi WANG, Ning Jiao, Doudou Xu, and Jingdong Yin J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00530 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018

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Journal of Proteome Research

Protein expression landscape defines the differentiation potential specificity of adipogenic and myogenic precursors in the skeletal muscle

Kai Qiu,† Xin Zhang,† Liqi Wang,† Ning Jiao,† Doudou Xu,† and Jingdong Yin*†

†

State Key Lab of Animal Nutrition & Ministry of Agriculture Feed Industry Centre,

College of Animal Science & Technology, China Agricultural University, Beijing 100193, China *E-mail: [email protected]. Tel: 8610-62733590-1404; Fax: 8610-62733688.

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ABSTRACT: The balance of adipogenic and myogenic differentiation of skeletal muscle-derived mesenchymal stem cells is particularly important in muscle development and intramuscular fat deposition. This study aimed to explore the differential regulation between adipogenic and myogenic precursors by comparative analysis of their global proteome expression profile. Adipogenic and myogenic precursors isolated from neonatal porcine longissimus dorsi muscle by the preplate method were verified for their unique and distinct differentiation potential under myogenic or adipogenic induction. A total of 433 differentially expressed proteins (DEP) (P < 0.05 and FC > 1.20 or < 0.83) between adipogenic and myogenic precursors were detected via a tandem mass tag (TMT)-coupled LC-MS/MS approach, including 339 up-regulated and 94 down-regulated proteins in myogenic precursors compared with adipogenic precursors. Based on functional annotation and enrichment analysis of 433 DEP, adipogenic and myogenic precursors showed significantly different metabolic pattern of energy substances and differential regulations of gene expression, cell structure & development, ion homeostasis, and cell motility & migration. Three pathways including PPAR signaling pathway, phosphatidylinositol signaling system, and autophagy signaling pathway, which was differentially regulated between adipogenic and myogenic precursors, was also discovered to play crucial roles in cell differentiation. In conclusion, these differentiated regulation patterns between the two cell subsets of mesenchymal precursors together defines their differentiation potential specificity. KEY WORDS: Intramuscular fat, Cell differentiation, Cell structure, Ion homeostasis, Cell migration.


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INTRODUCTION Almost half of total human meat consumption in the world was provided by pig industry.1,2 Skeletal muscle, accounting for 20%~50% of body weight, is the primary meat production tissue of pigs.3 Intensive genetic selection for lean growth has dramatically reduced the intramuscular fat (IMF) content, which impairs the eating quality of meat.4,5 IMF, mainly located between muscle fiber bundles, is a polygenic trait in livestock species.6,7 In order to increase IMF content, proteomics and transcriptomics were widely used in recent years to reveal the key markers associating with the mechanism(s) responsible for differential IMF deposition between high- and low-IMF breeds of livestock.8-10 However, few studies were conducted to evaluate adipogenic and myogenic differentiation potential of precursor cells in skeletal muscle to illustrate the underlying mechanisms of IMF deposition. During skeletal muscle development, mesenchymal stem cells (MSCs) first diverge to either myogenic progenitor cells or adipogenic-fibrogenic progenitor cells.11 Fetal myogenic progenitors further develop into muscle fibers and satellite cells,12,13 while fetal adipogenic-fibrogenic progenitors develop into stromal-vascular fraction of mature skeletal muscle in which reside adipocytes, fibroblasts, and resident progenitor cells.14-16 Therefore, both of myocytes and adipocytes in skeletal muscle derived from mesenchymal progenitors. The dysdifferentiation of mesenchymal progenitors which turning them into cells with an adipocyte phenotype has been postulated to be responsible for the accumulation of IMF.17 Skeletal muscle provides a balanced

environment for coexistence

of

both

myogenic

precursors

and

adipogenic/fibrogenic precursors.18 The competitive balance between myogenesis and adipogenesis during the development of skeletal muscle is in direct relation to the content of IMF.11 Therefore, an overall understanding of the mechanisms responsible 3 ACS Paragon Plus Environment


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for adipogenic and myogenic differentiation of skeletal muscle-derived precursors will help to design nutritional or breeding methods to manipulate muscle development, IMF deposition, and fatty acid composition. The lineage commitment of MSCs were directed by several factors, including cytokines, cellular components, adhesion molecules, ion channels, integrins, and transcriptional regulators.19-21 In addition, adipogenesis from multipotent precursors was also regulated by a series of cellular mechanisms including hedgehog, Wingless and Int (Wnt)/β-catenin, bone morphogenesis protein (BMP) mediated signaling pathways, and AMP-activated protein kinase.22 In this study, the protein expression profiles of adipogenic and myogenic precursors isolated from neonatal porcine skeletal muscle by the preplate method were compared by proteomic to reveal the distinct regulation of cell differentiation. EXPERIMENTAL PROCEDURES Ethics Statement All procedures conducted in the present study were approved by the Institutional Animal Care and Use Committee of China Agriculture University (ID: SKLAB-B-2010-003). Animal and Skeletal Muscle Tissue Three newborn Yorkshire boars within 3-day-old (BW = 1.36 ± 0.23 kg) were purchased from the Beijing Pig Breeding Center. After euthanized, all pigs were disinfected by 75% ethanol and slaughtered. The longissimus dorsi muscle weighting about 1.5 g over the last lib was isolated, and immediately rinsed in 75% ethanol for 3 s, then temporarily kept in PBS (Hyclone, Thermo Scientific, OH) containing penicillin (100 U/mL) and streptomycin (100 mg/mL). 4 ACS Paragon Plus Environment

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Cell Isolation and Culture According to the preplate technique established by our lab

23

, cell isolation was

conducted immediately in a biological safety cabinet (HF-1100, Heal Force, China). Briefly, the skeletal muscle was made into single cell suspension by being minced and serial enzymatic dissociation including 1 hour in 0.17% protease (Sigma-Aldrich, MO) and 1 hour in 0.15% collagenase-type XI solution (Sigma-Aldrich, MO). After filtered, centrifugation, and then resuspended, the cell suspension was cultured at 37 °C and 5% CO2 in a collagen I-coated dish (Sigma-Aldrich, MO) containing DMEM/F12 (Hyclone, Thermo Scientific, OH) complemented with 10% FBS (Gibco-BRL, CA), 2 mM glutamine (Gibco-BRL, CA), and 5 ng/ml bFGF (basic fibroblast growth factor, PEPTECH, MA). After 2 hours, non-adherent cells contained in the supernatant were transferred to another dish for a period of 72 hours. Then, non-adherent cells contained in the supernatant were removed. The first (0~2 h) and second (2~74 h) sets of adherent cells were named as adipogenic (Adi) and myogenic (Myo) precursors, respectively. Myogenic precursors were then purified by removing rapidly adhering cells which adhering to a new collagen I-coated dish within 2 hours. Adipogenic and Myogenic Differentiation Both adipogenic and myogenic precursors were subjected to induce adipogenic and myogenic differentiation. As reaching 100% confluence, cells was induced for adipogenic differentiation in adipogenic differentiation medium (DMEM/HIGH GLUCOSE supplemented with 10% FBS, l µM dexamethasone, 0.5 mM 1-methyl-3-isobutylmethyl-xanthine, and 10µg/ml insulin) for 3 days, then adipogenic differentiation medium was replaced with adipogenic maintained medium (DMEM/HIGH GLUCOSE complemented with 10% FBS and 10 µg/ml insulin) for another 5 days. Oil red O staining was performed to assess the efficiency of 5 ACS Paragon Plus Environment


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adipogenic differentiation. As reaching 80%~90% confluence, cells were induced for myogenic differentiation by low serum medium (DMEM/F12 supplemented with 2% horse serum) for 5 days. Myotubes were identified by myosin staining (anti-myosin, 1:300, CST, MA) according to the protocol of immunofluorescence assay. Except for horse serum from Hyclone Ltd, other reagents used in induction medium were purchase from Sigma-Aldrich. Quantitative Real-Time PCR Analysis Total RNA samples were extracted from both adipogenic and myogenic precursors using HiPure Total RNA Mini Kit (Magen, China), and then were reverse-transcribed into cDNA using a PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Synthesized cDNA of each sample was used for RT-qPCR analysis by employing a quantitative real-time PCR kit (Takara, Japan) in an ABI 7,500 Real-Time PCR system (Applied Biosystems, CA) according to standard procedures. All samples were measured in triplicate. The primers of selected genes were listed in Supplementary Table S1. GAPDH was used as an internal control. Relative gene expression level was calculated by 2−∆∆Ct method. Sample Preparation and Tandem Mass Tag (TMT) Labeling Cell samples were scraped from dishes and incubated with lysis buffer (0.5% SDS, 8M urea) and a Halt protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL) in the ratio of 100:1. Cell suspensions were then grind for 2 min with high-throughput tissue grinding machine (Fielda-650D, Jiangsu Wave Field Intelligent Technologies Inc, Wuxi, China) and incubated for 30 min on ice. After centrifuged for 30 min at 12,000 × g at 4 °C, supernatants were collected to test the protein concentration by using a BCA Protein Assay Kit (Huaxingbio Science, Beijing, China). 6 ACS Paragon Plus Environment

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Protein digestion was performed according to the standard procedure and the resulting peptide mixtures were labeled using the 6-plex TMT reagent (Art.No.90111, Thermo Fisher, MA) according to the manufacturer’s instructions. Briefly, protein samples (80 µg) were dissolved in 100 µL TEAB (100 mM) and then treated with 10 mM tris (2-carboxyethyl) phosphine (TCEP) for 1 h at 37 °C to reduce disulfides. Afterward, the alkylation of samples reacted in 40 mM iodoacetamide for 40 min at room temperature in the dark. The alkylated samples were mixed with about six-fold volume of ice-cold acetone and kept in -20 °C for 4 h, and then the acetone was removed by centrifugation at 10,000 × g for 20 min. The precipitates were resuspended in 100 µL TEAB (100 mM) and digested by trypsin (substrate/enzyme ratio 40:1, w/w) at 37 °C overnight, and then a final concentration of 1% trifluoroacetic acid was added to stop the trypsin digestion. The resulting peptide mixtures were labeled with one of the 6-plex TMT reagent as (Adi-1)-127N, (Adi-2)-127C, (Adi-3)-128N, (Myo-1)-128C, (Myo-2)-129N, and (Myo-3)-129C. Finally, all the labeled samples were combined together at equal amounts, vacuum dried and stored at -80 °C until LC–MS/MS analysis. HPLC Fractionation and LC-MS/MS Analysis Samples were resuspended with loading buffer (5 mM Ammonium hydroxide solution containing 2% ACN, pH = 10), and fractionated by high pH reversed-phase liquid chromatography (HPLC, ACQUITY Ultra Performance LC, Waters, MA) to increase proteomic depth. As a result, a total of 10 fractions were obtained by pooling two of 20 fractions collected from the sample. Each fraction (2 µg of protein) was injected into the C18-reversed phase column (75 µm × 25 cm, Thermo Fisher, MA) in solvent A (2% ACN and 0.1% FA) and separated by a Thermo Scientific Easy nanoLC 1200 with a linear gradient of solvent B (80% ACN and 0.1% FA): 0-1 min, 0-5% B; 1–63 7 ACS Paragon Plus Environment


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min, 5-23% B; 63-88 min, 23-48% B; 88-89 min, 48-100% B; 89-95 min, 100% B; 95-100 min, 100-0% B; 100-120 min, 0% B. The column flow was maintained at a flow rate of 300 nL/min. Q-Exactive mass spectrometer (Thermo Fisher, MA) was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. The full-scan MS spectra (m/z 350-1,300) were collected with a mass resolution of 70K and followed by 20 sequential high energy collisional dissociations (HCD) for MS/MS scans with a resolution of 35K. Every microscan was recorded using dynamic exclusion of 18 seconds. Sequence Database Searching and Protein Identification MS/MS data were analyzed for protein identifications using Protein Discoverer TM Software

2.1

against

uniprot-proteome-UP000008227-Susscrofa

(Pig)-26103s-

20170226 fasta. The highest score for a given peptide mass (Best match to that predicted in the database) was used to identify parent proteins. The parameters for protein searching were listed in Supplementary Table S2. Peptide spectral matches were validated based on Q-values at a 1% false discovery rate (FDR). Paired t-test was used to reveal the difference of protein expression profile between adipogenic and myogenic precursors. Specifically, Adi-1, Adi-2, and Adi-3 were considered as the control of Myo-1, Myo-2, and Myo-3, respectively. Fold-Change (FC) > 1.20 or < 0.83 and P < 0.05 were set as the threshold for differentially expressed proteins (DEP) between Adi and Myo. Bioinformatics Analysis GO and KEGG enrichment analysis for the DEP were performed to identify their prevalence in Cellular components, Molecular functions, Biological processes, and Signaling pathways by matching Blast2GO software or KEGG database to the NCBI database. Fisher Exact test was used to identify the significantly enriched GO terms or 8 ACS Paragon Plus Environment

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KEGG pathways in the DEP compared to the proteomic background. P < 0.05 was considered as the threshold for significant GO or KEGG enrichment. Western Blot The relative abundances of proteins selected for the technical validation of proteomics were determined by Western Blot. Equal amounts of protein (20 µg), together with a pre-stained protein ladder (Thermo Fisher, MA), were electrophoresed on SDS polyacrylamide gels. Then proteins were electrotransferred to a polyvinylidene difluoride membrane (Millipore, MA) and blocked for 1 h in 5% non-fat dry milk at room temperature in Tris-Buffered saline and Tween-20 (TBST; 20 mmol/L Tris-Cl, 150 mmol/L NaCl, 0.05 % Tween 20, pH 7.4). The transfer efficiency was assessed by gel staining with Coomassie Blue. Samples were incubated with corresponding primary antibodies for 2 h at 25°C or overnight at 4°C against GAPDH, BAG2, CACYBP, CCNH, CRYAB, DPT, FABP5, and VCL (1:500 dilution, CUSABIO, China). After being washed with TBST (pH 7.4), the membranes were incubated with DyLightTM 800-labeled secondary antibodies (1:10,000 dilution, KPL, MD) for 1 h. Band densities were detected with the Odyssey Clx (LI-COR, NE) and quantified using an AlphaImager 2200 (Alpha Innotec, CA). Statistical Analysis Relative mRNA expression and relative protein abundance was analyzed by Mann-Whitney U test using SPSS software (v20). Data are presented as means ± SEMs. P ≤ 0.05 was considered as the criterion for statistical significance. RESULTS Differentiation Characteristics of Adipogenic and Myogenic Precursors On the basis of selective adhesion to collagen-coated tissue culture plates, adipogenic and myogenic precursors were isolated from porcine skeletal muscle (Fig. 1A). 9 ACS Paragon Plus Environment


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Cellular morphology of adipogenic and myogenic precursors under the microscope was shown in Fig. 1B. Differentiation potential of the adipogenic and myogenic precursors was verified by tracing their differentiation fate in myogenic and adipogenic inductions, respectively. Satellite cell marker Pax7 (paired domain transcription factor 7) and myogenic-specific genes such as MyoD1 (myoblast determination protein 1), MyoG (myogenin) and Mymk (myomaker, myoblast fusion factor) were highly expressed in myogenic precursors (Fig. 1C). Upon myogenic induction, myogenic precursors committed to multi-nuclei Myotubes. However, no myogenic activity was observed in adipogenic precursors (Fig. 1D). On the other hand, adipogenic precursors showed high mRNA expression of genes encoding C/EBPα (CCAAT enhancer-binding proteins), LPL (adipocyte-specific genes including lipoprotein lipase), and ADD1 (sterol regulatory element binding transcription factor 1) (Fig.1C). Under the adipogenic induction, adipogenic precursors differentiated into mature adipocytes, adopting a round shape, accumulating lipid (as shown by Oil-red O stained), while no adipogenic activity was observed in myogenic precursors (Fig. 1D). Quantitative Mapping of the Proteome between Adipogenic and Myogenic Precursors In the current study, three pigs were used for the isolation of adipogenic and myogenic precursors. Using the TMT labeling (Fig. 2A), a total of 6,199 proteins were identified and quantitated (Fig. 2B). Among them, 433 DEP (Supplementary Table S3) were detected including 339 up-regulated and 94 down-regulated ones in myogenic precursors, compared with adipogenic precursors (Fig. 2B). The relative expression of DEP between adipogenic and myogenic precursors was visualized in heat-map (Fig. 2C). 10 ACS Paragon Plus Environment

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To validate the results of TMT analysis, Western Blot was conducted to determine the relative abundances of seven proteins randomly selected from the DEP, including BCL2 associated athanogene 2 (BAG2), calcyclin binding protein (CACYBP), cyclin H (CCNH), crystallin alpha B (CRYAB), dermatopontin (DPT), fatty acid binding protein 5 (FABP5), and vinculin (VCL). As shown in Fig. 3, the results of Western Blot showed a high consistency for the data of TMT analysis. Top Ten Up-Regulated or Down-Regulated DEP The top 10 up-regulated or down-regulated proteins were listed in Table 1. Up to five of the top 10 up-regulated DEP functions in cell division cycle and cell proliferation, including breast cancer anti-estrogen resistance 3, S100 calcium binding protein A2, USH1 protein network component harmonin binding protein 1, S100 calcium binding protein A4, and adrenoceptor alpha 1B. Besides, three of them are enzymes, namely sphingomyelin phosphodiesterase acid like 3B involved in fat catabolism, protein N-terminal asparagine amidohydrolase functioning in a step-wise process of protein degradation, and guanine deaminase playing a role in microtubule assembly. Among the top 10 down-regulated DEP, tuftelin and zinc finger protein 503 are involed in neuronal development and differentiation. Solute carrier family 29 member 1 and zinc finger protein 451 functions in DNA synthesis and repair. Solute carrier family 38 member 5 and epidermal fatty acid-binding protein are involved in amino acid and fat acid uptake, respectively. Dermatopontin participates in cell-matrix interactions and matrix assembly. Fibrillin 2 involves in elastic fiber assembly as a component of connective tissue microfibrils. Bioinformatics Exploration of DEP via GO Analysis Gene ontology (GO) enrichment analysis was conducted with 433 DEP. In GO analysis, we significantly enriched 28 cellular components terms, 47 molecular 11 ACS Paragon Plus Environment


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functions terms, and 81 biological processes terms (P < 0.05). As shown in Supplementary Table S4, DEP were mainly involved into four kinds of cellular components including vacuole, lysosome, plasma membrane, and muscle fiber. On the basis of molecular function, DEP can be mainly classified into five categories, including cell structure & development, ion channel activity, transcription and translation, ATP production, and hydrolase & peptidase activity (Fig. 4). Adipogenic and myogenic precursors possessed distinct metabolism pattern of carbohydrate, protein, and fat, because 11 biological processes related to carbohydrate metabolism, 20 related to protein metabolism, and 15 related to fat metabolism were significantly enriched with DEP (Supplementary Table S5). Beyond that, DEP also mainly took part in biological processes including gene expression regulation, muscle development, ion homeostasis, and cell migration (Fig. 5). Based on the results of GO analysis, the functions of DEP were mainly divided into four categories including gene expression regulation, cell structure & muscle development, ion homeostasis, and cell migration (Supplementary Table S6). In addition, representative DEP interactions among the four categories were shown in Fig. 6. DEAD-box helicase 3 X-linked (DDX3X) involved in both gene expression regulation and muscle development. Solute carrier family 9 member 3 regulator 1 (SLC9A3R1) kept cell migration and ion homeostasis connect together. Cell structure and

development

connected

with

ion

homeostasis

via

ectonucleotide

pyrophosphatase/ phosphodiesterase 1 (ENPP1), FK506 binding protein 1A (FKBP1A), and ryanodine receptor 1(RYR1). Cell structure & development was closed related to cell migration by nine DEP, including vinculin (VCL), breast carcinoma amplified sequence 3 (BCAS3), nexilin F-actin binding protein (NEXN), coronin, actin binding protein 1B (CORO1B), plexin A2 (PLXNA2), semaphorin 3A 12 ACS Paragon Plus Environment

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(SEMA3A), hexosaminidase subunit alpha (HEXA), Wnt family member 5A (WNT5A), and glucosidase alpha, acid (GAA). Two crucial and central proteins which enabled cell structure & development, ion homeostasis, and cell migration to connect together were hexosaminidase subunit beta (HEXB) and apolipoprotein E (APOE). Bioinformatics Exploration of DEP via KEGG Analysis In Table 2, five signal pathways were significantly enriched (P < 0.05) by matching 433 DEP to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and NCBI BLAST databases, namely as PPAR signaling pathway, lysosome, protein digestion and absorption, mineral absorption, and phosphatidylinositol signaling system. Three DEP related to β-oxidation participated in PPAR signaling pathway, including long-chain specific acyl-CoA dehydrogenase, mitochondrial (ACADL), fatty acid-binding protein 3 (FABP3), and FABP5. A total of 11 DEP with catalytic activity were involved in lysosome pathway and up-regulated in myogenic precursors, including cathepsin B, alpha-N-acetylgalactosaminidase, GM2 ganglioside activator, scavenger receptor class B member 2, Pro-cathepsin H, cathepsin C, tripeptidyl peptidase 1, N-acetylgalactosamine-6-sulfatase, ceroid-lipofuscinosis, neuronal 5, cathepsin Z, and N-acetyl-alpha-glucosaminidase. In the pathway of protein digestion and absorption, two peptidases were up-regulated in myogenic precursors, namely dipeptidyl peptidase 4 and prolylcarboxypeptidase. Three DEP including STEAP2 metalloreductase (STEAP2), cytochrome b reductase 1, and heme oxygenase 1 functioned in the pathway of mineral absorption. In addition, phosphatidylinositol signaling system was significantly enriched by three up-regulated DEP in myogenic precursors,

namely

inositol

1,4,5-trisphosphate

receptor

type

3

(ITPR3),

diacylglycerol kinase alpha (DGKA), and myotubularin 1 (MTM1). 13 ACS Paragon Plus Environment


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DISCUSSION The deposition of intramuscular fat including extramyocellular and intramyocellular fat not only closely associates with human diseases,24-26 but also affects meat quality in livestock production.27 Most of intramuscular lipids were generated in adipocytes,28 therefore, the balance between adipogenic and myogenic differentiation in skeletal muscle determines the content of intramuscular fat. It is widely accepted that adipogenic and myogenic precursors in skeletal muscle are derived from a common origin MSCs.29 However, mechanisms that balance adipogenic or myogenic differentiation potential of the precursors in skeletal muscle remain unclear. Recently, there are a few of proteomic studies concerning MSCs, such as revealing tissue-specific proteomic dynastic in porcine MSC populations derived from different tissues,30 functionally enriching proteins belonging to extracellular vesicles released by porcine adipose tissue-derived MSCs,31 and delineating secreted proteins from MSCs during adipogenic differentiation.32 However, based on our knowledge, there are no reports previously focused on proteomic profile of adipogenic/myogenic precursors with mesenchymal origin underpinning their specific differentiation potential. In this study, differences of protein expression pattern between adipogenic and myogenic precursors were detected by proteomics to reveal the regulatory mechanism underlying cell differentiation. Owing to the limited knowledge of cell surface markers of muscle-derived cells and available commercialized antibodies for pigs, preplate technique, a surface marker-independent method, was established by our lab to isolate porcine skeletal muscle-derived adipogenic and myogenic precursors, both of which were demonstrated to be MSCs-derived lineage.23 It was certificated again in this study that adipogenic and myogenic precursors have distinct differentiation fate and cannot 14 ACS Paragon Plus Environment

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convert their commitment into opposing lineages. The expression pattern of total protein defined significant differences between adipogenic and myogenic precursors, which together decides the distinct differentiation fate in a very intricate way. Compared with adipogenic precursors, 339 proteins were up-regulated and 94 down-regulated in myogenic precursors. Bioinformatics exploration revealed that DEP were mainly involved in gene transcription & translation, cell structure & development, ion homeostasis, cell migration, and autophagy, of which distinct regulations were deduced to paly important roles in cell specific differentiation potential. Four molecular functions and five biological processes related to gene expression regulation were enriched with 21 DEP through GO analysis, which indicated that gene expression pattern was mediated differentially between adipogenic and myogenic precursors. Five DEP among them were up-regulated in myogenic precursors, namely Wnt family member 5A, sirtuin 2, eukaryotic translation initiation factor 4A2, microtubule associated cell migration factor, and DExH-box helicase 57. Wnt5A, a member of the Wnt family, can induce nucleus assemblage by combining with β-Catenin, and then regulate the expression of related genes.33 In addition, Wnt5A can interact with E-cadherin and directly affect cell motility.34 Sirtuin 2 involved in the regulation of ribosomal DNA recombination, gene silencing, DNA repair and chromosome stabilizing.35 Obviously, eukaryotic translation initiation factor 4A2 and DExH-box helicase 57 are both involved in gene expression regulation.36,37 Microtubule associated cell migration factor is a cytoskeletal protein that regulates the activation of Cdc42 and affects cell migration.38 Six helicases involved in gene transcription was up-regulated in adipogenic precursors, including four DEAD-box helicases (DDX27/47/3X/18), DEAH-box helicase 8, DExD-box helicase 21, and 15 ACS Paragon Plus Environment


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BRCA1 interacting protein C-terminal helicase 1. Five ribosomal proteins involved in mRNA translation were up-regulated in adipogenic precursors, namely NMD3 ribosome export adaptor, pescadillo ribosomal biogenesis factor 1, ribosomal protein L7 like 1, NHP2 ribonucleoprotein, and ribosome production factor 2 homolog. In addition, nucleophosmin 1 was also up-regulated in adipogenic precursors. In summary, adipogenic and myogenic precursors showed great differences in gene expression regulation, depending on which, we speculate that differential regulation of gene expression mediated by these DEP has very important significance on the orientation of cell differentiation. However, the specific mechanism underlying the gene expression regulation needs further study. Eight molecular functions and 11 biological processes related to cell structure and development were enriched with 47 DEP via GO analysis, which indicated that differences might exist in cell structure and development between adipogenic and myogenic precursors. Actin and myosin, components of muscle fiber, were well demonstrated to be abundantly expressed during the late stage of myogenic differentiation.39 In this study, six kinds of actin or myosin were up-regulated in myogenic precursors, including capping actin protein, nexilin F-actin binding protein, phosphatase and actin regulator 4, xin actin binding repeat containing 1, tropomyosin 2, and unconventional myosin-XVIIIa. Beyond that, myopalladin, synemin, vinculin, caldesmon 1, and neurofibromin 2 related to cell structure and development were up-regulated in myogenic precursors. Myopalladin, located in Z-line and I-band of muscle fiber, was closely related to muscle strength and development,40 and its mutation would result in restrictive myopathy.41 Synemin, one of the seventy intermediate filaments, protects cells by forming a filamentous network structure in cytoplasm41 and plays an important role in myogenic differentiation by combining 16 ACS Paragon Plus Environment

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with actin and signaling proteins,42 whose knockout would bring out muscle disease.43 Vinculin were necessary for the function of many focal adhesions (FA), such as stability and reinforcement FA, and promoted extracellular signal perception44 and actin cytoskeleton regulation.45 Vinculin enhanced cell migration rate and tightly related to development and function maintain of muscle.46 Caldesmon 1, like myosin, is the a marker protein for the maturation and differentiation of smooth muscle.47 Neurofibromin 2 can trigger the myogenicity of cells,48 which is essential for skeletal muscle growth and development.49 In addition, two insulin-like growth factor-binding proteins, namely IGFBP2 or IGFBP5, were also up-regulated in myogenic precursors. Both of IGFBP2 and IGFBP5 could promote muscle cell differentiation and muscle development by combining with IGF-Ⅱ.50 Obviously, myogenic precursors show the prototypes of myocytes in the aspect of cell structure and development, which keep themselves different from adipogenic precursors. Therefore, it could be deduced that the specific regulation pattern in cell structure and development plays a vital role in the differentiation potential of myogenic precursors. As to the structure and development of adipogenic precursors, transferrin receptor (CD71) was highly expressed. CD71, a membrane protein, is a surface antigen of mesenchymal cells and responsible for transferring iron ions to the cytoplasm.51 Studies have founded that CD71 shows high expression in both resting and differentiated state of adipogenic cells,52 but only at the late stage of myogenic differentiation of myogenic cells.53 Apolipoprotein E and very low density lipoprotein receptor are also up-regulated in adipogenic precursors. Both of them highly expressed in the process of adipogenic differentiation and mature adipocytes.54,55 FSHD region gene 1, a kind of muscular dystrophy proteins, was up-regulated in adipogenic precursors. This is consistent with previous reports that FSHD region gene 17 ACS Paragon Plus Environment


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1 inhibited myogenetic differentiation of cells.56 Consequently, those proteins above related to cell structure and development jointly gives adipogenic precursors the capability of adipogenic differentiation and lipid deposition. Based on the results of GO and KEGG analysis, four molecular functions, 10 biological processes and one signal pathway related to cellular ion homeostasis, such as cation channel activity, ion homeostasis, ion transport, and mineral absorption, were enriched with 33 DEP, including 26 up-regulated and seven down-regulated ones in myogenic precursors. Therefore, significant differences in the regulation of cellular ion homeostasis between adipogenic and myogenic precursors were observed. Acid environment formed by active transportation of H+ via ATP6V1 is an important step for many biological processes, such as zymogen activation, receptor-mediated endocytosis, proton gradient formation of synaptic vesicle, and maintaining acid environment of lysosome.57 ATP6V1 protein family are extensively involved in the regulation of cell differentiation58 and could regulate the permutation of actin filament.59 In the present study, three H+ transporter proteins were up-regulated in myogenic precursors, including ATPase H+ transporting V1 subunit A, ATPase H+ transporting V1 subunit B2, and ATPase H+ transporting V1 subunit H. Therefore, the acidification of cytoplasm or intracellular organelles was more active in myogenic precursors than adipogenic precursors, which maybe partly responsible for their distinct differentiation potential. Ryanodine receptor 1 (Ryr1) and ITPR3, belonging to transmembrane proteins of sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER), were the main Ca2+ channels of SR/ER, in charge of Ca2+ releasing into the cytoplasm.60 Calcium voltage-gated channel auxiliary subunit alpha2 delta 1 (CACNA2D1), a transmembrane protein of cell membrane, could accept electric potential variation, and then release Ca2+ into the cytoplasm from extracellular 18 ACS Paragon Plus Environment

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matrix.61 In this study, Ryr1, ITPR3, and CACNA2D1 were up-regulated in myogenic precursors, which could be used to further explain that the concentration of cytoplasmic Ca2+ in myogenic precursors was higher than adipogenic precursors.23 In addition, the close relationship between the expression or mutations of CACNA2D1 and meat color and backfat thickness of animal62 maybe build by the regulation of Ca2+ concentration. Other ion transporters up-regulated in myogenic precursors were also detected, such as anoctamin 10 (ANO10), STEAP2, antioxidant 1 copper chaperone (ATOX1), solute carrier family 38 member 10 (SLC38A10), SLC4A8, and SLC9A3R1. ANO10, a transmembrane protein, was Ca2+ activated Cl- releasing channel.63 STEAP2 could promote cell uptake of Fe2+ and Cu2+,64 and regulate cell proliferation and differentiation.65 SLC9A3R1, located in the cell membrane, was responsible for the exchange of Na+ and H+ between intracellular and extracellular matrix.66 In the present study, four members of SLC family were up-regulated in adipogenic precursors, including SLC30A1, SLC39A1, SLC1A5, and SLC38A5. SLC30A1, also called Zn2+ transporter, played an important role in maintaining cell homeostasis.67 SLC39A1 participated in the biological processes such as endocytosis.68 In summary, cellular ion homeostasis especially H+ and Ca2+, was differentially controlled between adipogenic and myogenic precursors. Therefore, the interaction between the regulation of cellular ion homeostasis and the potential of cell differentiation was acknowledged in the present study. Cell types and differentiation state could be reliably distinguished by their motility behaviors.69 The motility and migration of stem cell is closely related to the its differentiation potential, and the enhancement of mobility tended to promote cell differentiation.70 In this study, nine biological processes related to cell motility and 19 ACS Paragon Plus Environment


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migration, such as regulation of cell migration, locomotor rhythm, and locomotory behavior, were enriched with 34 DEP. Thirty proteins of them were up-regulated in myogenic precursors, such as fibroblast activation protein alpha (FAP), microtubule associated cell migration factor (BCAS3), phosphatase and actin regulator 4 (PHACTR4), nexilin F-actin binding protein (NEXN) and coronin 1B. FAP promoted cell migration via the PI3K and sonic hedgehog pathways.71 BCAS3 was a cytoskeletal protein that controls directional cell migration.38 PHACTR4 regulated directional migration through integrin signaling and cofilin activity.72 NEXN, a filamentous actin-binding protein, functioned in cell adhesion and migration.73 The type I subclass of coronins, a family of actin-binding proteins, regulates various actin-dependent cellular processes, including migration.74 Depletion of coronin 1B significantly compromised myosin accumulation and stability at junctions.75 Only four proteins related to cell migration were up-regulated in adipogenic precursors including APOE, activin A receptor type 1, ETS proto-oncogene 1 transcription factor, and heat shock protein family A (Hsp70) member 5. APOE binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A.76 Activin receptor-like kinase 1 inhibits human microvascular endothelial cell migration.77 Therefore, it could be speculated that motility and migration was significantly enhanced in myogenic precursors, compared with adipogenic precursors. This was also reflected by the strength of ATP production in myogenic precursors, because acyl-CoA dehydrogenase long chain and two proton transfer-related proteins (cytochrome b reductase 1 and proton myo-inositol cotransporter) were up-regulated in myogenic precursors. In this study, six signaling pathways were differentially regulated between adipogenic

and

myogenic

precursors.

In

autophagy,

double-membrane 20

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autophagosomes envelop intracellular components and then fuse with lysosomes to form autolysosomes, which degrade their contents to regenerate nutrients.78 Programmed

autophagy

triggers

metabolic

reprogramming

during

cellular

differentiation.79 As many as 11 proteins with catalytic activity related to lysosome were up-regulated in myogenic precursors, which indicated autophagy signaling pathway was more active in myogenic precursors than adipogenic precursors. PPAR signaling pathway was significantly enriched, whose key role was widely demonstrated in the regulation of MSCs differentiation, especially the promotion of adipogenic differentiation.80 The DEP participated in PPAR signaling pathway were FABP3 and FABP5, both of which were up-regulated in adipogenic precursors. However, ACADL catalyzing the oxidative decomposition of fatty acids81 was down-regulated in adipogenic precursors. Thus, it suggested that PPAR signaling pathway was more active in adipogenic precursors than myogenic precursors, which is consistent with previous report that PPAR family were crucial in adipogenic differentiation.82 Activation of phosphatidylinositol signaling system could promote myogenic differentiation, because all three DEP related to it were up-regulated in myogenic precursors, including ITPR3, DGKA, and MTM1. In conclusion, porcine skeletal muscle-derived adipogenic and myogenic precursors isolated by preplate technique have distinct differentiation potential. Quantitative proteomics reveals that the two cell subsets of mesenchymal precursors show significantly different metabolic pattern of energy substances and variant regulations of gene expression, cell structure & development, ion homeostasis, and cell motility & migration. PPAR signaling pathway, phosphatidylinositol signaling system, and autophagy signaling pathway, which was differentially regulated between adipogenic and myogenic precursors, was also discovered to play crucial roles in cell 21 ACS Paragon Plus Environment


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differentiation. These differentiated regulation patterns between adipogenic and myogenic precursors together defines their differentiation potential specificity. ASSOCIATED CONTENT Supporting Information Supplementary Table S1. The primers used for RT-qPCR of selected genes. Supplementary Table S2. Proteome Discoverer Database Search Parameters. Supplementary Table S3. The list of differentially expressed proteins between adipogenic and myogenic precursors. Supplementary Table S4. Cell components enriched by differentially expressed proteins via GO analysis. Supplementary Table S5. The significantly enriched biological processes related to the metabolism of three main nutrients (carbohydrate, protein, and fat) via GO analysis. Supplementary Table S6. Differentially expressed proteins participated in gene expression, cell structure & muscle development, ion homeostasis, and cell migration. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: 8610-62733590-1404; Fax: 8610-62733688. Author Contributions J. D. Y. was responsible for the project development and designed this study; X. Z., L. Q. W., D. D. X., and K. Q. conducted the cell isolation and culturing; N. J., X. Z., and K. Q. performed protein extraction, TMT proteomics, and bioinformatics analysis; K. Q. organized the experimental data and drafted the manuscript. All authors contributed, commented, and approved the final content of the manuscript. Notes The authors declare that they have no competing financial interests. ACKNOWLEDGMENTS 22 ACS Paragon Plus Environment

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This study was financially supported by the National Natural Science Foundation of China (Grant No. 31790412 and Grant No. 31672431) and National key research and development program of China (Grant No. 2018YFD0500402). Figure Legends Figure 1. Obtainning and identification of adipogenic (Adi) and myogenic (Myo) precursors derived from porcine skeletal muscle (n= 3). (A) The schematic diagram of cell isolation. (B) Morphology of Adi and Myo at Passage 3. The magnification of microscope is (4×10). (C) RT-qPCR analysis of adipogenesis and myogenesis-related gene expression between Adi and Myo. * represents significant difference between two groups (P < 0.05). (D) The potential of cell differentiation. Adi and Myo were strained with oil-red O after adipogenic differentiation induction. Myosin expression of Adi and Myo after myogenic differentiation was examined by immunofluorescence. The magnification of microscope is (10×10). Figure 2. Quantitative proteomics analysis of adipogenic (Adi) and myogenic (Myo) precursors (n=3). (A) Technical process of proteomics via a tandem mass tag (TMT)-coupled LC-MS/MS approach. (B) Overview of proteins identified by proteomics. Meating the criterion of P < 0.05 and fold change (FC, Myo/Adi) < 0.83, the proteins were consided to be down-regulated in Myo, compaired with Adi. Similarly, the up-regulated proteins in Myo were determinated by P < 0.05 and FC > 1.20. Blue and red triangles represent the down-regulated and up-regulated proteins respectively. Black dots represent proteins, whose expression was not significant changed between Adi and Myo. (C) Hierarchical clustering of differentially expressed proteins between Adi and Myo. Heatmap were generated by MeV (4.9.0). Figure 3. Validation of the proteomics data (n=3). (A-G) Relative abundance of proteins between adipogenic (Adi) and myogenic (Myo) precursors analyzed by 23 ACS Paragon Plus Environment


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Western Blot. (H) Relative expression of proteins analyzed by TMT proteomics. BAG2, BCL2 associated athanogene 2; CACYBP, calcyclin binding protein; CCNH, cyclin H; CRYAB, crystallin alpha B; DPT, dermatopontin; FABP5, fatty acid binding protein 5; VCL, vinculin. (H) Relative protein expression of BAG2, CACYBP, CCNH, CRYAB, DPT, FABP5, and VCL in protemics data. FC, fold change (Myo/Adi).* represents significant difference between two groups (P < 0.05). Figure 4. Classification of Molecular Function terms enriched by differentially expressed proteins (DEP) between adipogenic and myogenic precursors via GO analysis. Pillars with same color in the histogram represent one of the categories, including cell structure & development, ion channel activity, transcription & translation, ATP production, and hydrolase & peptidase activity. The figure at the top of pillar represents the number of DEP involved in the Molecular Function term. Figure 5. Classification of Biological Process terms enriched by differentially expressed proteins (DEP) between adipogenic and myogenic precursors via GO analysis. Pillars with same color in the histogram represent one of the categories, including muscle development, cell migration and motility, gene expression, and ion homeostasis. The figure at the top of pillar represents the number of DEP involved in the Biological Process term. Figure 6. The interaction of differentially expressed proteins (DEP) between adipogenic (Adi) and myogenic (Myo) precursors involved in gene expression, cell structure & development, ion homeostasis, and cell migration. The yellow nodes of proteins represent one of molecular function or biological process enriched by GO analysis with DEP. The red and green circles represent proteins up-regulated and down-regulated in Myo, respectively. Networks were visualized by Cytoscape (v3.2.1) 24 ACS Paragon Plus Environment

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Table 1. The Top 10 Up-regulated and Down-regulated Proteins in Myogenic Precursors (Myo) Compared with Adipogenic Precursors (Adi) (n = 3) Protein name

Gene name

Fold change (Myo/Adi)

P-value

BCAR3

2.85

0.016

SMPDL3B

2.78

0.034

Alpha-crystallin B chain

CRYAB

2.63

0.029

S100 calcium binding protein A2

S100A2

2.51

0.047

USH1 protein network component harmonin

USHBP1

2.29

0.017

S100 calcium binding protein A4

S100A4

2.14

0.048

Protein N-terminal asparagine amidohydrolase

NTAN1

2.03

0.004

Leucine rich repeat containing 20

LRRC20

2.01

0.015

Adrenoceptor alpha 1B

ADRA1B

1.96

0.010

GDA

1.93

0.032

HSPA5

0.72

0.030

SLC29A1

0.70

0.004

Tuftelin

TUFT1

0.69

0.042

Zinc finger protein 451

ZNF451

0.68

0.040

Solute carrier family 38 member 5

SLC38A5

0.67

0.037

Dermatopontin

DPT

0.66

0.016

BCL2 associated athanogene 2

BAG2

0.66

0.038

Zinc finger protein 503

ZNF503

0.64

0.004

Epidermal fatty acid-binding protein

FABP5

0.62

0.025

Fibrillin 2

FBN2

0.56

0.004

Up-regulated proteins Breast cancer anti-estrogen resistance 3 Sphingomyelin phosphodiesterase acid like 3B

binding protein 1

Guanine deaminase Down-regulated proteins 78 kda glucose-regulated protein Solute carrier family 29 member 1



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Table 2. KEGG Signaling Pathways Significantly Enriched by Differentially Expressed Proteins between Adipogenic (Adi) and Myogenic (Myo) Precursors (n = 3) Protein name

Gene name

Fold change (Myo/Adi)

P-value

PPAR signaling pathway Long-chain specific acyl-coa dehydrogenase,

ACADL

1.28

0.045

mitochondrial Fatty acid-binding protein 3

FABP3

0.77

0.009

Epidermal fatty acid-binding protein

FABP5

0.62

0.025

Lysosome Cathepsin B

CTSB

1.65

0.007

Alpha-N-acetylgalactosaminidase

NAGA

1.59

0.005

GM2 ganglioside activator

GM2A

1.56

0.039

Scavenger receptor class B member 2

SCARB2

1.55

0.002

Pro-cathepsin H

CTSH

1.50

0.035

Cathepsin C

CTSC

1.37

0.009

Tripeptidyl peptidase 1

TPP1

1.28

0.020

N-acetylgalactosamine-6-sulfatase

GALNS

1.27

0.032

Ceroid-lipofuscinosis, neuronal 5

CLN5

1.26

0.036

Cathepsin Z

CTSZ

1.25

0.031

N-acetyl-alpha-glucosaminidase

NAGLU

1.22

0.015

Protein digestion and absorption Dipeptidyl peptidase 4

DPP4

1.71

0.010

Prolylcarboxypeptidase

PRCP

1.23

0.006

Mineral absorption STEAP2 metalloreductase

STEAP2

1.40

0.015

Cytochrome b reductase 1

CYBRD1

1.22

0.008

Heme oxygenase 1

HMOX1

0.77

0.022

Inositol 1,4,5-trisphosphate receptor type 3

ITPR3

1.50

0.010

Diacylglycerol kinase alpha

DGKA

1.28

0.020

Myotubularin 1

MTM1

1.24

1.20. Blue and red triangles represent the downregulated and up-regulated proteins respectively. Black dots represent proteins, whose expression was not significant changed between Adi and Myo. (C) Hierarchical clustering of differentially expressed proteins between Adi and Myo. Heatmap were generated by MeV (4.9.0). 126x124mm (300 x 300 DPI)



Figure 3. Validation of the proteomics data (n=3). (A-G) Relative abundance of proteins between adipogenic (Adi) and myogenic (Myo) precursors analyzed by Western Blot. (H) Relative expression of proteins analyzed by TMT proteomics. BAG2, BCL2 associated athanogene 2; CACYBP, calcyclin binding protein; CCNH, cyclin H; CRYAB, crystallin alpha B; DPT, dermatopontin; FABP5, fatty acid binding protein 5; VCL, vinculin. (H) Relative protein expression of BAG2, CACYBP, CCNH, CRYAB, DPT, FABP5, and VCL in protemics data. FC, fold change (Myo/Adi).* represents significant difference between two groups (P < 0.05). 173x111mm (300 x 300 DPI)


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Figure 4. Classification of Molecular Function terms enriched by differentially expressed proteins (DEP) between adipogenic and myogenic precursors via GO analysis. Pillars with same color in the histogram represent one of the categories, including cell structure & development, ion channel activity, transcription & translation, ATP production, and hydrolase & peptidase activity. The figure at the top of pillar represents the number of DEP involved in the Molecular Function term. 119x81mm (300 x 300 DPI)



Figure 5. Classification of Biological Process terms enriched by differentially expressed proteins (DEP) between adipogenic and myogenic precursors via GO analysis. Pillars with same color in the histogram represent one of the categories, including muscle development, cell migration and motility, gene expression, and ion homeostasis. The figure at the top of pillar represents the number of DEP involved in the Biological Process term. 108x68mm (300 x 300 DPI)


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Figure 6. The interaction of differentially expressed proteins (DEP) between adipogenic (Adi) and myogenic (Myo) precursors involved in gene expression, cell structure & development, ion homeostasis, and cell migration. The yellow nodes of proteins represent one of molecular function or biological process enriched by GO analysis with DEP. The red and green circles represent proteins up-regulated and down-regulated in Myo, respectively. Networks were visualized by Cytoscape (v3.2.1) 150x130mm (300 x 300 DPI)


Protein Expression Landscape Defines the Differentiation Potential

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