Intramuscular preadipocytes impede differentiation and promote lipid deposition of muscle satellite cells in chickens

Isolation of chicken muscle satellite cells and intramuscular preadipocytes

The 7-day-old Beijing-You chickens were obtained from the experimental farm of the IAS (CAAS, Beijing, China). Primary cells were isolated as described previously with a slightly modification [15]. Chickens were stunned by electrical stunning (120 mA, 50 Hz) and then slaughtered with a quick, single cut to the throat. After sprayed with 75% ethanol, the pectoralis major muscle was isolated. The muscle was minced to 1 mm³ segments and digested for 60 min with 0.1% w/v type I collagenase (Gibco, Grand Island, NY). After terminating digestion with complete medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco), 10% fetal bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (Gibco), the cell suspension was centrifuged at 350 g for 10 min. The top layer containing mature adipocytes and the bottom pellet containing MSC were separately collected. The mature adipocyte layer was inoculated into a 25-cm² cell culture flask containing complete medium. The flask was incubated inverted for 6 d enabling adipocytes to attach to the upper surface and dedifferentiate, and subsequently re-inverted for another 6 d to allow the adherent cells to proliferate.

The cell pellet, containing MSC, was resuspended in complete medium, as above, filtered through 100- and 40-μm sterile sieves, and then cultured in complete medium in 100-mm dishes. After cells were incubated for 2 h, the medium containing unattached cells was transferred to a new dish, and purified MSC were obtained.

All cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C. After reaching 80% confluence, both cell types were passaged with 0.25% trypsin-EDTA (Gibco), and passage 3 cells were used for further experiments.

Co-culture of muscle satellite cells and intramuscular preadipocytes

Cells were co-cultured using Transwell plates with 0.4-μm membrane inserts (Corning Inc., Kennebunk, ME). The MSC (2 × 10⁵ per well) were plated in the lower wells of the 6-well plates, and IMPA (1 × 10⁵ per well) were seeded into the upper inserts, initially in separate plates, i.e., not in a co-culture configuration. After both cell types reached confluence, the upper inserts containing IMPA were then transferred to the lower wells containing MSC to establish the co-cultures. The mono-cultured MSC served as controls. Following co-culture or mono-culture for 2 or 4 d, MSC were harvested for further analysis. Comparisons between the two culture arrangements were made with 3 repetitions to allow for statistical evaluation.

Immunofluorescence

Myosin heavy chain (MHC) was used as a marker of MSC differentiation. After washing 3 times with PBS, the MSC were fixed in 4% paraformaldehyde for 20 min, permeabilized in 0.25% Triton X-100 for 15 min, and then blocked with 10% goat serum (CWBio, Beijing, China) for 30 min. Subsequently, the cells were incubated with primary anti-MHC hybridoma supernatant (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA) overnight at 4 °C. After thorough washing, the cells were incubated in the dark for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (1:100, CWBio, Beijing, China). Fluorescence signals of MSC in the lower chambers were detected and photographed by confocal fluorescence microscopy (Nikon TE-2000-E, Tokyo, Japan) [16].

Oil red O staining

After 4 d of co-culture or mono-culture, the MSC were washed 3 times with PBS, fixed in 4% paraformaldehyde for 15 min, and then stained with a 0.5% solution of Oil Red O in 60% isopropanol for 1 h at room temperature. The cells were washed 3 times with PBS, and the stained fat droplets in the MSC were visualized by light microscopy and photographed [17]. Isopropanol (100%) was incubated for 10 min to extract Oil Red O from the cells and then the extracts were transferred to a 96-well-plate. The OD₅₁₀ was measured photometrically with a microplate reader (Molecular Devices, SpectraMax M2, San Jose, CA) [18].

RNA isolation and sequencing

After co-culture or mono-culture for 2 d, MSC were lysed by TRIzol reagent (Invitrogen Corp., Carlsbad, CA) and total RNA was extracted according to the manufacturer’ protocol. The RNA concentration and integrity were assessed using a 2100 Bioanalyzer and RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA) separately. RNA samples with an A260/A280 ratio between 1.8 and 2.0 and a RNA Integrity Number > 8.0 were used for RNA sequencing and quantitative real-time PCR (qPCR). Based on ultra-high throughput sequencing (HiSeq 2500; Illumina, San Diego, CA), RNA-sequencing was performed by Annoroad Genomics (Beijing, China).

Bioinformatics analysis of RNA-seq

Sequence adapters and low quality reads (read quality < 30) were removed using Trimmomatic (v0.32) [19]. Quality control checks on raw sequence data were performed with FastQC. Then, sequencing reads were mapped to the chicken reference genome [Ensembl Galgal4 (GCA_000002315.2)] using the HISAT(v2.0.4) program. To calculate the expression quantity of each transcript, alignment results were analyzed by the Cufflinks (v2.0.2) program. The FPKM (fragments per kilobase of transcript per million mapped fragments) method was used to quantify gene expression. Analyses of differential expression of transcripts were performed with edgeR (v2.2.5). Genes with a FDR value of less than 1% and fold-change ≥2 were considered to be differentially expressed genes (DEG).

Principal component analysis (PCA) was performed to identify the variability and repeatability of samples. A volcano plot was used to visualize the overall distribution of differentially expressed genes. Gene ontology (GO) analysis of DEG was performed using DAVID functional annotation clustering [20]. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed by KOBAS 3.0 (http://kobas.cbi.pku.edu.cn). Cytoscape was used to depict a pathway network of enriched pathways.

The significance level for GO terms and the KEGG pathway was set with the P-value < 0.05.

Quantitative real-time PCR

The specific primers (Additional file 1: Table S1) were designed using Primer Premier 6.0 software, and were subsequently synthesized by the Sangon Biotech Corp (Shanghai, China). After reverse transcription, qPCR was performed using a SYBR Fast qPCR Master Mix (KAPA, Wilmington, MA).

The qPCR mixture contained 10 μL of 2× iQ™ SYBR Green Supermix, 0.5 μL (10 mM) of each primer and 20 ng of cDNA, with ddH₂O added to 20 μL. Samples were amplified using the real-time PCR Detection System ABI 7500 (Applied Biosystems, Shanghai, China). The PCR cycle parameters were as follows: 95 °C for 3 min, then 40 cycles of 95 °C for 3 s, and 60 °C for 34 s. The amplification procedure was performed with 3 repetitions for each sample. The 2^-ΔΔCt method was used to calculate the relative abundance of transcripts [21].

Statistical analysis

All data are presented as the mean ± standard deviation (SD), for 3 replicates from each experiment. Statistically significant differences between the 2 culture conditions were tested by independent-samples t-tests using SAS 9.2 software. P < 0.05 (*) or P < 0.01 (**) was considered to be significant. All figures were constructed using GraphPad Prism version 5.02 (GraphPad Software Inc., La Jolla, CA).

Differential expression analysis of mono-cultured and co-cultured MSC

A total of 1653 known DEG, of which 922 were upregulated and 731 were downregulated, were identified in MSC that were co-cultured in the presence of IMPA (|log2 FC| ≥ 1 and FDR < 0.01) (Fig. 1a and Additional file 2: Table S2).

To evaluate the consistency and variance of the 6 samples, PCA (based on all gene transcripts) and hierarchical clustering (based on all DEG) were performed. PCA demonstrated that the co-cultured and mono-cultured MSC were distinctly clustered (Fig 1b), and this was supported by the hierarchical clustering result (Fig. 1c), verifying acceptable repeatability in each and obvious difference between the culture conditions.

Accuracy of the RNA-seq data was examined by qPCR of 20 randomly chosen DEG and correlation between the 2 methods. Fold changes of the DEG were significantly correlated over a wide range (r = 0.96, P < 0.01) (Fig. 1d), confirming reliability of the RNA-seq results.

Functional enrichment analysis of the DEG

The function of the known DEG was examined by GO enrichment analysis. A total of 75 Biological Process (BP) terms were significantly enriched (P < 0.05) (Additional file 3: Table S3). These BP terms were mainly associated with muscle development, lipid metabolism, ossification, angiogenesis, cell activities (cell growth, adhesion, migration, and differentiation), regulation of ion transmembrane transport, protein phosphorylation, and signal transduction (P < 0.05). The DEG related to muscle development and lipid deposition were examined further. There were 48 DEG related to muscle differentiation and development, including the key genes MYOD1, MYOG, PAX7, and TMEM8C (Additional file 4: Table S4). The 44 DEG related to lipid deposition included the key genes CD36, FABP4, ACSBG2, CYP7A1 and PLIN2 (Additional file 5: Table S5).

After KEGG pathway analysis, 20 pathways were significantly enriched (Fig. 2 and Additional file 6: Table S6). Consistent with the GO results, several of these pathways were related to muscle development and contraction, including MAPK signaling, Adrenergic signaling in cardiomyocytes, Cardiac muscle contraction, Vascular smooth muscle contraction, and Calcium signaling pathway. Many of the pathways were involved in lipid deposition, including PPAR signaling, Glycosphingolipid biosynthesis, and ABC transporters. In addition, six enriched pathways were related to cell junction, including extracellular matrix (ECM)-receptor interaction, Focal adhesion, Cell adhesion molecules (CAMs), Gap junction, Cytokine-cytokine receptor interaction, and Regulation of actin cytoskeleton.

IMPA constrain differentiation of MSC

To explore this negative influence of IMPA on MSC differentiation, transcript abundance of 5 key genes involved in muscle differentiation was quantified by qPCR. In addition, immunofluorescence was used to explore MHC (the major marker of terminal myogenic differentiation, encoded by MYH1E) to directly assess differentiation of MSC. Compared with mono-cultured MSC, the abundance of MYOD1, MYH1E, PAX7, TMEM8C, and MYOG transcripts was significantly downregulated in co-cultured MSC on day 2 (Fig. 3a). Immunofluorescence results showed that MHC-positive cells were also reduced in number in co-cultured MSC on day 2 (Fig. 3b). These results confirmed that IMPA suppressed differentiation of the MSC.

IMPA promote lipid accumulation in MSC

This influence of IMPA on lipid deposition in MSC was examined by qPCR of transcripts of 5 key genes involved in lipid deposition in MSC that were co-cultured for 2 days. The transcript abundance of FABP4, CD36, APOA1, DISP3, and ADIPOQ was all significantly upregulated in co-cultured MSC on day 2 (Fig. 4a). Staining with Oil Red O on day 4 demonstrated that there were more lipid droplets in co-cultured MSC, which further confirmed that IMPA promoted lipid deposition in MSC (Fig. 4b).

Pathways related to differentiation and lipid deposition in MSC

As differentiation of muscle cells and their lipid deposition are complex processes, the underlying molecular mechanisms may involve interactions between multiple signaling pathways.

The pathway network of interconnected signals was constructed on the basis of the KEGG pathway database. As shown in Fig. 5a, the calcium signaling pathway and MAPK signaling pathway were the two most interactive pathways. Calcium signaling tightly interacted with pathways related to muscle contraction, and MAPK signaling interplayed with cell junction related pathways. The MAPK signaling pathway functioned downstream of calcium signaling. Thus, MAPK signaling may be the key pathway involved in the present study.

The MAPK signaling pathway contains 4 distinct cascades, ERK1/2, JNK/MAPK, p38-MAPK and ERK5, all of which have been demonstrated to be involved in skeletal myogenesis [22]. The data obtained here indicate that DEG were seldom enriched in the p38-MAPK and ERK5 pathways, and just a few DEG were enriched in the ERK1/2 pathway (Fig. 5b). In contrast, many DEG were enriched in JNK/MAPK signaling, including Rac2, MAP3K8, ARR3, the core effector JNK (MAPK10), and its downstream effector c-JUN (JUN), all indicating that the JNK/MAPK signaling pathway may be the key pathway influencing differentiation of MSC.

Apart from muscle differentiation, MAPK signaling is also involved in lipid metabolism [23, 24]. PPAR signaling occurred downstream of MAPK signaling, and numerous critical genes including CD36, FABP4, ADIPOQ, APOA1, CYP7A1, PLIN2, and SLC27A1 were enriched in this pathway, consistent with PPAR signaling being the key pathway regulating lipid deposition in MSC.

Pathways related to cell junctions (ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, and gap junction) might form a regulatory network with the MAPK signaling pathway and contribute to the differentiation of MSC and their lipid deposition.