FNDC5 expression closely correlates with muscle fiber types in porcine longissimus dorsi muscle and regulates myosin heavy chains (MyHCs) mRNA expression in C2C12 cells

Animal feeding, muscle sampling, FNDC5 expression, and MyHC mRNA composition analysis

We selected 16 LJH (a crossed pigs containing Chinese native pig genotypes) and DLY (Duroc × Landrace × Yorkshire) weanling castrated pigs (eight pigs each hybrid combination), fed in Lvjia Yuan Livestock Industry Co., Ltd, Zhejiang Province, China. All animal feeding management and slaughtering processes were approved by the Laboratory Animal Management Committee of Zhejiang Academy of Agricultural Sciences in China (No. 2018110). At final body weights of 100–110 kg, these pigs were transferred to an abattoir by ordinary commercial truck (journey time ∼2.5 h) according to the commercial standard slaughtering process in China. The longissimus dorsi (LD) muscle located in the last 3–4 thoracic vertebrae was immediately collected after slaughter.

The total RNA in muscle was isolated using the Trizol method, and cDNA was synthesized using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR (qPCR) was performed for the FNDC5 mRNA level, with the 18s rRNA gene as the reference gene. The FNDC5 primer sequences were as follows: F-5′-tgcaggccatccattcag-3′, R-5′-ccacagagaccacga-3′, generating a 182 bp amplicon (Cai et al., 2017); 18s primer sequences were as follows: F-5′-cccacggaatcgagaaagag-3′, R-5′-ttgacggaagggcacca-3′(Men et al., 2017).The relative quantification of FNDC5 mRNA was calculated using the 2 ^−ΔΔCT method (Livak & Schmittgen, 2002).

According to the description in Men et al. (2017), the MyHC mRNA composition was analyzed using qPCR. The relative ratio of each MyHC-type mRNA was calculated as the corresponding copy number per mg of muscle sample divided by the sum of four MyHC-types mRNA, multiplied by 100.

Culture, differentiation, and transfection of C2C12 myoblast cells

According to Zhou et al. (2013), C2C12 cells (an immortalized mouse myoblast cell line from the Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences, Shanghai, China) were cultured and induced to differentiate into myotubes in Dulbecco’s modified Eagle’s medium (DMEM) with 2% horse serum. Mycoplasma detection on C2C12 myoblast cells was carried out using a MycAway™ -Color One-Step Mycoplasma Detection Kit by YEASEN Biotech Co. Ltd., Shanghai city, China.

The small interfering RNA targeting FNDC5 (si-592), the native oligonucleotide fragments (3′-uucuccgaacgugucacgutt-5′, 3′-acgugacacguucggagaatt-5′) for siRNA transfection, and the eukaryotic expression vector overexpressing FNDC5 were supplied by GenePharma Biotechnology Co. Ltd., Shanghai city, China, according to the coding region sequence of FNDC5 (NM_027402.4). The si-592 sequences included 3′-gccaguaugauaugaucaat-5′and 3′-uugaugaugauaucauacuggctt-5′and were transfected into C2C12 cells twice at day 0 and day 2 post inductive differentiation, respectively. The FNDC5 eukaryotic expression vector was constructed using plasmid pcDNA3.1(+), and transfected into C2C12 cells once at day 0 post inductive differentiation.

The transfection experiment was grouped as follows: nuclease-free water as the blank group, transfection reagent as the mock group, pcDNA3.1(+) plasmid the as vector-NC group, native oligonucleotide fragments as the si-NC group, si-592 fragment as FNDC5(-) group, and pcDNA3.1(+)-FNDC5 vector as FNDC5(+) group. According to the instructions of GP-RNA-mate kit supplied by GenePharma Biotechnology Co. Ltd, 0.4 mL of each transfection mixture was added into each well of a 6-well plate with 1.6 mL of fresh serum-free DMEM medium, and incubated at 37 °C for 5 h. After 4 days of incubation, the cultured cells were harvested for RNA or protein determination.

ZLN005 or irisin treatment on C2C12 myoblasts

ZLN005, a small molecule activator of PGC-1α, was purchased from Cayman Chemical Company (Ann Arbor city, Michigan, USA). C2C12 cells (70–80% confluent) were induced in differentiation medium (DMEM+2% horse serum) containing various concentrations of ZLN005 (0, 5, and 10 µmol/mL) for 4 days and then collected. For the ZLN005-10 group, some cells were transfected with si-592, and harvested for RNA extraction. Recombinant human irisin protein was purchased from Pepro Tech Inc. Rocky Hill, NJ, USA. C2C12 cells were incubated in differentiation medium for 4 days, cultured in medium containing 0, 20, or 200 ng/mL irisin for 5 h, and then harvested for RNA extraction and metabolic enzyme activity assays.

Quantitative real-time PCR (qPCR) and western blotting of cells

The RNA extraction, cDNA synthesis, and qPCR for C2C12 cells were performed similarly to the above descriptions for muscle tissue. All data were normalized against the mRNA expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Determinations were performed in triplicate. The primer sequences in Table 1 were designed and synthesized by GenePharma Biotechnology Co. Ltd, Shanghai city, China.

Total protein extraction, SDS-PAGE gel electrophoresis, and western blotting for cells were performed according to Cai et al. (2017). The FNDC5 protein was separated using 12% SDS electrophoresis, transferred onto a cellulose acetate membrane, and then incubated with recombinant rabbit anti-FNDC5 monoclonal antibodies (ab174833, Abcam, Cambridge, UK), or a monoclonal anti- β-Actin antibody produced in mouse (A5441, Merck Life Science Co., Ltd, Shanghai city, China. Chemiluminesence detection was performed on an FR-1800 Luminescent and Fluorescent Biological Image Analysis System of Furi company, Shanghai city, China. The scanned images were processed and analyzed using Gel-Pro analyzer software from Media Cybernetics, Rockville, MD, USA. The relative content of FNDC5 protein in a sample was calculated through the ratio of the gray value to that of the internal reference actin.

Detection of metabolic enzyme activity in cells

The harvested cell cultures were homogenized in one mL of phosphate buffered saline (pH 7.4), and then centrifuged at 1,000 rpm for 10 min at 4 °C. The supernatant was collected to detect the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and creatine kinase (CK), and protein concentration using the standard commercial kits from Nanjing Jiancheng Biochemical Institute, Nanjing city, China.. Enzyme activities were expressed as U/mg protein.

Statistical analysis

All data are presented as the mean ±  standard deviation (SD) and all statistical analysis programs were operated in SPSS 16.0 (IBM Corp., Armonk, NY, USA). Data from porcine muscle tissue were analyzed using the t-test (P < 0.05) between LJH and DLY pig groups. Data from the cell experiments were analyzed using one-way analysis of variance and Duncan’s test (P < 0.05) among multiple treatment groups.

Knockdown or up expression of FNDC5 gene in C2C12 myoblast cells

As shown in Fig. 2, the expression levels of FNDC5 mRNA and protein were not significantly different among the blank, mock, si-NC, and vector-NC groups (P >  0.05). In the FNDC5(-)group, the expression levels of FNDC5 mRNA and protein were decreased significantly compared with those in the blank group by si-592 (P <  0.05). In the FNDC5(+) group, the expression levels of FNDC5 mRNA and protein were increased significantly compared those in the blank group by pcDNA3.1(+)-FNDC5 vector (P <  0.05).

Effects of down or up regulating FNDC5 expression on MyHCs mRNA levels in C2C12 myoblast cells

As shown in Fig. 3, MyHCI, MyHCIIx and MyHCIIb mRNA levels were not affected significantly in the FNDC5 (-) or FNDC5 (+)group (P >  0.05). MyHCIIa mRNA expression in the FNDC5 (-) group were decreased significantly compared with that in other groups (P <  0.05), and increased significantly in the FNDC5 (+) group compared that in other groups (P <  0.05).

Effects of FNDC5 knockdown expression on PGC1α-FNDC5-MyHCs mRNA expression under ZLN005 in C2C12 myoblast cells

As shown in Fig. 4, compared with the ZLN005-0 group, the ZLN005-5 and ZLN005-10 groups showed significantly the increased expressive levels of PGC1α, FNDC5, MyHCI and MyHCIIa mRNA (P < 0.05). Compared with that in the ZLN005-10 group, ZLN005-10 + si592 group showed significantly the decreased expressive levels of FNDC5 and MyHCIIa mRNA (P <  0.05).

Effects of adding irisin on PGC1α-FNDC5-MyHCs mRNA expression in C2C12 myoblast cells

As shown in Fig. 5, PGC1α , FNDC5, MyHCI, and MyHCIIa mRNA expressions were significantly upregulated with the increasing irisin addition (P <  0.05).The Irisin-200 group also showed significantly increased MyHCIIx mRNA expression compared with that in the irisin-0 group (P < 0.05).

Effects of adding irisin on metabolic enzyme activities and other downstream gene expression levels in C2C12 myoblast cells

As shown in Fig. 6, the enzyme activities of SDH, MDH and CK, and the mRNA expression levels of IL15 (interleukin 15), VEGF (vascular endothelial growth factor), NRF1 (nuclear respiratory factor 1), and TFAM (transcription factor A, mitochondrial) increased with increasing irisin addition in C2C12 myotubes (P < 0.05). The activity of LDH was significantly lower in the irisin-20 and irisin-200 groups than that in the Irisin-0 group (P < 0.05).