Effect of Porcine Akirin2 on Skeletal Myosin Heavy Chain Isoform Expression

Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway.


Introduction
Skeletal muscle is comprised of muscle fibers, whose characteristics affect both lean meat production and meat quality [1,2]. Three main fiber types (slow oxidative type (I), fast oxiditave-glycotic type (IIa), and fast glycotic type (IIb)) can be distinguished according to their myosin ATPase stability after acid or alkali pretreatment [3]. Myosin ATPase is localized to the globular head of the myosin heavy chain (MHC) [4]. Therefore, MHC seems to represent the most appropriate marker for muscle fiber type. Four MHC isoforms of mammalian skeletal muscles are codified by four genes such as slow-twitch oxidative type I (MHCI), and three fast types, namely oxidative type IIa (MHCIIa), oxido-glycolytic type IIx (MHCIIx), and glycolytic type IIb (MHCIIb) [5,6]. Muscle fiber type composition, one of the main factors influencing meat quality, directly affects the muscle color, tenderness, and the content of intramuscular fat (IMF) in farm animals [7]. Improvement in meat quality is an important animal breeding goal, and consumers pay particular attention to meat quality. Regulation of muscle fiber type composition may be advantageous to achieve good meat quality in farm animals. Therefore, it is necessary to identify candidate genes that might contribute to improve meat quality by regulating muscle fiber type composition.
The Akirin2 gene was previously reported to be associated with nuclear factor-κB (NF-κB) and to be involved in immune reactions, embryonic development and skeletal myogenesis [8][9][10][11][12]. The Akirin2 gene has been previously shown to possess expression differences in musculus longissimus muscle between low-marbled and high-marbled steer groups and to be located within genomic region of a quantitative trait locus for marbling (the amount of IMF) [13]. Analysis of single nucleotide polymorphism (SNP) of Akirin2 suggests that it is associated with marbling and may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese black beef cattle [14,15]. A recent study also demonstrated that the SNP of Akirin2 was significantly associated with longissimus muscle area and marbling score in Korean native cattle [16]. These studies suggested that the Akirin2 gene may be associated with the content of IMF and affect the meat quality.
Until now, very little research has been conducted on the function of porcine Akirin2 (pAkirin2). In our previous study, we cloned the pAkirin2 cDNA and examined its tissue distribution [17]. The pAkirin2 cDNA was sub-cloned into prokaryotic expression vector pET28a(+), and target protein was successfully induced to express and was purified as expected [18]. Moreover, the purified recombinant pAkirin2 significantly increased the proliferation of C2C12 cells [18]. In the present study, we examined the pAkirin2 mRNA expression in different types of muscle tissues of pigs and the effect of pAkirin2 on differentiation of C2C12 myoblasts. We also examined the effects of pAkirin2 on expressions of MHC isoform and oxidative muscle fiber genes in C2C12 myotubes.

pAkirin2 mRNA Expression in Different Types of Muscle Tissues of Pigs
The expression of pAkirin2 mRNA in the soleus (SOL) muscle, longissimus lumborum (LL) muscle, and extensor digitorum longus (EDL) muscle of Duroc × Landrace × Yorkshire (DLY) pigs was assessed by real-time quantitative PCR. As shown in Figure 1, the expression of pAkirin2 mRNA was higher in the EDL and LL muscles than in the SOL muscle of pigs.

Effect of pAkirin2 on Myoblast Differentiation
MHC is a marker for later stages of myogenesis. To assess the function of pAkirin2 in myoblast differentiation, we introduced pAkirin2 into C2C12 myoblasts. As shown in Figure 2, overexpression of pAkirin2 increased the number of MHC-positive cells, suggesting that pAkirin2 promoted myoblast differentiation.

Effect of pAkirin2 on MHC Isoform Expression in C2C12 Myotubes
We evaluated the effect of pAkirin2 on MHC isoform expression. As shown in Figure 3, the mRNA expressions of MHCI and MHCIIa were significantly increased, whereas the mRNA expression of MHCIIb was significantly decreased, in C2C12 myotubes transfected with the plasmid pcDNA3.1(+)-pAkirin2.

Effect of pAkirin2 on Oxidative Muscle Fiber Gene Expression in C2C12 Myotubes
To explore the effect of pAkirin2 on oxidative muscle fiber gene expression, we measured the expressions of MEF2C (myocyte enhancer factor-2C), NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), and MCIP1.4 (modulatory calcineurin interacting protein 1 exon 4 isform) by real-time quantitative PCR. The data obtained showed that overexpression of pAkirin2 significantly increased the mRNA expressions of MEF2C, NFATc1 and MCIP1.4 in C2C12 myotubes on day 4 ( Figure 4). On day 8, overexpression of pAkirin2 strongly increased MEF2C mRNA expression but had no significant effect on NFATc1 and MCIP1.4 mRNA expressions (Figure 4).

Discussion
Skeletal muscle is composed of three groups of muscle fibers (slow, fast and intermediate). In animal production industry, specific combinations of fast and slow muscles affect the meat quality [19]. In the present study, we determined the expression of pAkirin2 mRNA in the fast EDL and LL muscles and slow SOL muscle of pigs. We found that pAkirin2 mRNA expression was most abundant in the EDL muscle, followed by the LL muscle, and to a lesser extent in the SOL muscle. Our data suggested that the expression of pAkirin2 was significantly higher in fast muscles than in slow muscles and might be closely related to the expression of muscle fiber type-related genes.
Oxidative fibers (type I and type IIa) seem to be positively related to the color, water-holding capacity and tenderness of meat [20]. Type IIb fibers have a larger diameter than other fiber types and contribute to increase in muscle mass [21]. However, in pigs, higher percentage of type IIb fiber has been shown to be negatively related to pH45 min and positively to drip loss and R-value (adenine/inosine ratio), thereby resulting in reducing the meat quality [20,22]. In this study, we found that MHCI and MHCIIa were upregulated, whereas MHCIIb was downregulated, by pAkirin2 overexpression. Although the result is in contradiction to the finding that pAkirin2 mRNA is higher expressed in fast than in slow muscle of pigs, the reason for this remains unclear. Taken together, these results suggested that the pAkirin2 gene may have an important function in regulating meat quality by affecting fiber type-specific gene expression.
The calcineurin signaling pathway has been implicated in the regulation of slow skeletal muscle fiber gene expression [23,24]. MEF2 and NFAT proteins are the major downstream effectors of calcineurin [23][24][25][26][27]. NFAT is one of the primary cofactors for MEF2 [28]. Elevated calcium signaling is essential for optimal expression of the MHCI via calcineurin/NFAT pathway [24,[29][30][31]. In the present study, overexpression of pAkirin2 increased the expression of transcription factors MEF2C and NFATc1, both involved in the regulation of oxidative muscle fiber genes. It should be noted that activated NFATc1 (dephosphorylated in the nuclear) is known to promote slow fiber type-specific gene expression, whereas inactivated NFATc1 (phosphorylated in the cytoplasma) can also be found in fast muscles [32]. In addition, NFATc1 can interact with MEF2 isoforms in slow fiber type-specific gene expression depending on the promoter context. For example, together with NFATc1, the isoform MEF2D regulates the slow MHCI promoter [33], but the isoform MEF2C is involved in fast MHCIIa and fast MHCIIx promoter activation [34,35]. Because activation of MEF2C at target promoters occurs primarily via phosphorylation, increased expression is not necessary. However, since activation of the calcineurin/NFATc1 signaling pathway is accompanied by the increase of certain NFATc1 mRNA and protein levels [36] and the primers used for NFATc1 in this study include the induced isoforms, the observed increase in NFATc1 mRNA expression by pAkirin2 overexpression can be utilized as an indication for activation of the calcineurin/NFATc1 signaling pathway in this experimental setup. MCIP1.4 is a direct downstream target of the calcineurin/NFAT pathway, which has recently been renamed as regulator of calcineurin 1 (RCAN1) [37]. MCIP1.4 was reported to increase the number of MHCI-expressing slow fibers [38]. Here we also observed that overexpression of pAkirin2 enhanced the expression of MCIP1.4. Together, our results suggest that the role of pAkirin2 in regulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway.

Animals and Tissue Sample Collection
Three 10-week-old female DLY pigs (body weight of 31.27 ± 0.18 kg) were slaughtered in a humane manner according to protocols approved by the Animal Care Advisory Committee of Sichuan Agricultural University under permit No. YYS130125. The SOL, LL and EDL muscles were removed and immediately snap frozen in liquid nitrogen before being stored at −80 °C for RNA isolation.

RNA Isolation and Reverse Transcription
Total RNA was isolated using RNAiso Plus reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. The concentrations of total RNA were quantified using a Beckman DU-800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). cDNA was synthesized from one microgram of total RNA using a PrimeScript ® RT reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer's protocols. The first-strand cDNA was subsequently used as a template for real-time quantitative PCR.

Real-Time Quantitative PCR
Real-time quantitative PCR was performed on a 7900HT Real-time PCR system (384-cell standard block) (Applied Biosystems, Foster, CA, USA) in a final volume of 10 μL. The gene specific primers used are listed in Table 1. The PCR mixture consisted of 1 μL of the first-strand cDNA sample, 1 μL each of forward and reverse primers from 10 μM stocks, 2 μL DEPC-treated water, and 5 μL of SYBR select Master Mix (Applied Biosystems). The initial denaturation step at 95 °C for 10 min was followed by 45 cycles of denaturation for 15 s at 95 °C, and annealing and extension for 30 s at 60 °C. Each primer pair used yielded a single peak in the melting curve and a single band with the expected size in agarose gel. Identities of the PCR products were confirmed by DNA sequencing. Data analysis was performed using the comparative Ct method [39] with GAPDH and/or β-actin as an endogenous control.

Cell Immunofluorescence Assay
Cells were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min and permeabilised with 0.1% Triton X-100 in PBS for 20 min. Cells were then blocked in 5% bovine serum albumin (BSA) for 30 min and incubated with MHC antibody (Santa Cruz Biotechnology; sc-20641, 1:100, Santa Cruz, CA, USA) at 4 °C overnight. The cells were rinsed with PBS and incubated with FITC-conjugated secondary antibody (Santa Cruz Biotechnology; 1:200) for 1 h at room temperature. To stain the nuclei, the cells were incubated in the DNA stain 4'6-diamidino-2-phenylindole (DAPI) for 10 min. Images were captured using a Nikon Eclipse TS100 inverted fluorescence microscope (Nikon, Tokyo, Japan).

Statistical Analysis
All data were expressed as means ± SE (standard error) and were analyzed using SPSS11.5 software (SPSS Inc., Chicago, IL, USA). Group differences were analyzed by ANOVA and the differences between groups were considered to be statistically significant when p < 0.05.

Conclusions
In conclusion, we found that pAkirin2 mRNA expression level is higher in the EDL and LL muscles than in the SOL muscle. Overexpression of pAkirin2 promoted differentiation of C2C12 myoblasts. Moreover, we also provided the evidence that overexpression of pAkirin2 led to up-regulation of MHC isoform (MHCI and MHCIIa) and oxidative muscle fiber gene (MEF2C, NFATc1 and MCIP1.4) expressions. This study contributes to understand the role of Akirin2 in the regulation of muscle fiber types using pig as a model organism and helps to explore the key genes regulating meat quality.