Expression of porcine myosin heavy chain 1 gene in Berkshire loins with a high pH 24 value

An important factor in evaluating porcine meat quality is the pH at post-mortem 24 h (pH 24 ). In the current work, we conducted a GeneFishing polymerase chain reaction (PCR)–based screen to identify differentially expressed genes in Berkshire loins with high versus low pH 24 . Total RNAs were extracted from Berkshire loins with high and low pH 24 and subjected to GeneFishing PCR. Four genes were found to be differentially expressed in the high and low pH 24 groups. Semi-quantitative reverse transcription (RT)-PCR confirmed that MYH1 encoding myosin heavy chain 1 was specifically expressed in high pH 24


INTRODUCTION
Porcine meat quality is severely affected by a condition known as PSE, which stands for "pale, soft and exudative," adjectives that describe the resultant poor quality of the meat.This condition, which is very problematic in the pork industry (Barbut et al., 2008), is developed in the muscle as a result of the accelerated glycolysis that occurs during the early post-mortem stage and is also closely related to the rapid initial post-mortem decline in Abbreviations: ACP, Annealing control primer; pH 24 , pH at post-mortem 24 h; ATP, adenosine triphosphate; DEG, differentially expressed gene; MYH1, myosin heavy chain 1; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; PSE, pale, soft and exudative; RT, reverse transcription.
#These authors contributed equally to this work.pH (Bowker et al., 2000).
The mechanism underlying PSE development is thought to be anaerobic glycolysis in muscle tissues (Bowker et al., 2000).Specifically, the circulatory failure caused by exsanguination after slaughter results in lack of the oxygen that is required for aerobic glycolysis, leading to an adenosine triphosphate (ATP) homeostatic imbalance in muscle tissues.To maintain the cellular ATP concentration under anoxic conditions, muscle glycogen is metabolized via anaerobic glycolysis, which is less efficient at generating ATP than aerobic glycolysis.Consequently, levels of glycogen and ATP decrease and the lactic acid, a waste product of the anaerobic glycollysis, accumulates lowering muscle pH (Briskey and Wismer-Pedersen, 1961;Kastenschmidt et al., 1968).In particular, the rapid initial post-mortem decline in pH stimulates the denaturation of muscle proteins, leading to the development of PSE in muscle (Bowker et al., 2000).The pH at post-mortem 24 h (pH 24 ), a common indicator for the evaluation of pork quality, can be used to monitor PSE.It shows high genetic correlations with other meat quality parameters such as drip loss, cooking loss, waterholding capacity and meat tenderness (Nguyen et al., 2006;Duan et al., 2009).In the current study, we first tried to screen the differentially expressed genes (DEGs) in Berkshire breeds with high versus low pH 24 to identify the pH 24 -responsible genes, using annealing control primer (ACP)-based GeneFishing PCR (Kim et al., 2004).From the screen, four porcine genes were discovered to be differentially expressed in high and low pH 24 lines.The MYH1 gene encoding myosin heavy chain 1 was confirmed to be highly expressed in Berkshire loins with excellent meat quality and a high pH 24 value.Collectively, our results provide insight into the genetic relationship between pH 24 and muscle fiber type composition and suggest that MYH1 may be a novel pH 24 -responsible gene.

Animals and meat samples
Three hundred and twenty three Berkshire breeds were bred under same condition (Da-San-Jong-Don Co. Ltd., Namwon, Korea) and then slaughtered in 10 batches when their body weight reached 110 kg.Subsequently, the samples were used for meat quality evaluation, GeneFishing™ PCR and semi-quantitative RT-PCR.

Meat quality evaluation
The analysis was carried out according to AOAC (Association of Official Analytical Chemists) method.The contents of protein, lipid, water and collagen in Berkshire loins were measured by FOSS (Foodscan Near-Infrared Spectrophotometer), and the meat quality parameters such as pH 24 value, water-holding capacity, drip loss and cooking loss were determined by previously described methods (Prevolnik et al., 2009;Tan et al., 2009).

ACP (annealing control primer)-based GeneFishing™ PCR
DEGs were screened by ACP-based PCR method using the GeneFishingTM DEG kits (Seegene, Seoul, South Korea) (Kim et al., 2004).Briefly, second-strand cDNA was synthesized at 50°C during one cycle of first-stage PCR in a final reaction volume of 20 µl containing 3 -5 µl (about 50 ng) of diluted first-strand cDNA, 1 µl of dT-ACP2 (10 µM), 1 µl of 10 µM arbitrary ACP and 10 µl of 2 × Master Mix (Seegene, Seoul, South Korea).The PCR protocol for second-strand synthesis was one cycle at 94°C for 1 min, followed by 50°C for 3 min and 72°C for 1 min.After second-strand DNA Kang et al. 7569 synthesis was completed, the second-stage PCR amplification protocol was 40 cycles of 94°C for 40 s, followed by 65°C for 40 s, 72°C for 40 s and 5 min final extension at 72°C.The amplified PCR products were separated in 2% agarose gel and stained with bromide.

and sequencing
The differentially expressed bands were extracted from the gel by using the GENCLEAN® II Kit (Q-BIO gene, Carlsbad, CA, USA), and directly cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
The cloned plasmids were sequenced with ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Semi-quantitative RT-PCR
The differential expression of DEGs was confirmed by RT-PCR using MYH1-specific primers.The first-strand cDNA was normalized by the pig GAPDH gene.The normalized cDNA was used as a template.The PCR reaction was conducted in a final reaction volume of 20 µl containing 2 -4 µl (about 50 ng) of diluted firststrand cDNA, 1 µl of each MYH1-specific primer (10 µM) (forward, 5'-AGCAGGAGCTGATTGAGACC-3'; reverse, 5'-TTCAGTCTGA AGCTGGGACA-3') and 10 µl of 2× Master Mix (Seegene, Seoul, South Korea).The PCR amplification protocol was an initial 3 min denaturation at 94°C, followed by 20 -25 cycles of 94°C for 40 s, 60°C for 40 s, 72°C for 40 s and a 5 min final extension at 72°C.The amplified PCR products were separated in 2% agarose gel and stained with ethidium bromide.

AND DISCUSSION
Excellent meat quality in Berkshire loins with a high pH 24 value Berkshire breeds are known to have excellent meat quality (Suzuki et al., 2003), and hence their loins were used to identify the DEGs in the loins with high vs. low pH 24 values.To evaluate meat quality, 323 Berkshire breeds were raised under the same conditions (Da-San-Jong-Don Co. Ltd., Namwon, Korea) and then slaughtered in 10 batches when their body weight reached 110 kg.Subsequently, the sample loins were divided into high (pH >5.88) and low (pH <5.88) pH 24 groups and then evaluated for meat quality using standard methods (AOAC: Association of Official Analytical Chemists; Prevolnik et al., 2009;Tan et al., 2009).Mean values for meat quality parameters (pH 24 , water-holding capacity, drip loss and cooking loss) for representative loins with high and low pH 24 are shown in Table 1.Noticeably, most meat with high pH 24 value exhibited a higher waterholding capacity and lower drip and cooking losses than did the meats with low pH 24 value.These data demonstrate that the Berkshire loins with a high pH 24 are meats with excellent quality compared to those of the low pH 24 group and suggest that the high pH 24 value may be used as a meat quality parameter for identifying excellent Berkshire loins.

Screening of differentially expressed genes (DEGs) in Berkshire loins with high vs. low pH 24
Next, to identify the DEGs, the total RNAs extracted from loins with high and low pH 24 were used for GeneFishing PCR (Kim et al., 2004) with a combination of six ACPs.As shown in Figure 1, of the six ACPs, four showed differentially expressed DNA bands, in which two bands increased and two bands decreased in intensity in the high pH 24 loin when compared with the low pH 24 loin.The sizes of the bands varied from 200 to 700 bp (Figure 1).Subsequently, the four differentially expressed DNA bands were purified from the agarose gels and cloned into TOPO TA cloning vectors for sequencing analysis.The National Center for Biotechnology (NCBI) Basic Local Alignment Search Tool (BLAST) searches revealed that the four DEGs were matched with known genes: ACP1 with MYH1 (myosin heavy chain 1; Acc.No. NM 00110495.1),ACP2 with SMPX (small muscle protein, Xlinked; Acc.No. NM_001078687), ACP3 with MYH2b (myosin heavy chain type 2b; Acc.No. NM001123141.1)and ACP4 with HSP40 (heat shock protein 40; Acc.No. AK237314.1).

High level of MYH1 gene expression in Berkshire loins with high pH 24
Noticeably, of the four DEGs, ACP1 and ACP3 encoding myosin heavy chain isoforms, showed significant increases in band intensity in the loin with high pH 24 (Figure 1).A few recent studies have suggested that the myosin heavy chain composition of muscles has an important effect on meat quality in pigs (Chang et al., 2003;Hu et al., 2008;Wimmers et al., 2008).In particular, the ACP1 encoding myosin heavy chain 1 (MYH1) isoform is reported to be a critical factor for muscle growth and meat quality (Chang et al., 2003;Hu et al., 2008), suggesting that porcine MYH1 may be closely associated with the high pH 24 levels found in Berkshire loins of excellent quality.To prove the hypothesis, we sought to determine whether MYH1 is highly expressed in the high pH 24 group (H1 to H10) with excellent meat quality.For this, total RNAs extracted from high (H1 to H10) and low (L1 to L10) pH 24 groups in Table 1 were used for semiquantitative RT-PCR with MYH1-specific primers.As shown in Figure 2, the MYH1 gene was expressed more significantly in the high pH 24 group than in the low pH GeneFishing PCR results for identifying differentially expressed genes in Berkshire loins with high vs. low pH 24 using six arbitrary annealing primers (ACP1 to ACP6).Total RNAs were extracted from Berkshire loins with high and low pH 24 using TRI-Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions and were then subjected to ACP-based PCR using the GeneFishingTM DEG kits (Seegene, Seoul, South Korea) (Kim et al., 2004).Lane M, L and H indicate 100bp size marker, low pH 24 and high pH 24 , respectively.The differentially expressed PCR products are indicated as arrows (numbers 1 to 4). group.Collectively, our data demonstrate that porcine MYH1 is more highly expressed in the high pH 24 group, suggesting that it may be a novel pH 24 -responsible gene.Porcine skeletal muscles are heterogeneous tissues consisting of different types of fibers.Traditionally, they contain three types of muscle fibers: type I (red; slowtwitch), type IIB (white; fast-twitch) and type IIA (red; fasttwitch) (Bowker et al., 2000;Eggert et al., 2002).Type I fibers are generally oxidative, whereas type IIB fibers are glycolytic, meaning that the type I fiber is much less susceptible to accelerated post-mortem glycolysis and PSE development than type IIB (Bowker et al., 2000;Eggert et al., 2002).Type IIA fiber is intermediate between type I and IIB fibers with respect to the postmortem metabolism (Hamalainen and Pette, 1993).Muscle fiber type composition has been reported to have important effects on post-mortem metabolism during the conversion of muscle to meat (Eggert et al., 2002;Lefaucheur et al., 2004).The muscle fiber type composition may also be closely related to the pH 24 value.In this study, the MYH1 gene, which is reported to be a main component for the low-twitch high-oxidative type I fiber (Termin et al., 1989;Gil et al., 2001), was highly expressed in Berkshire groups associated with high pH 24 .This finding suggests that muscles of the high pH 24 groups with excellent meat quality may commonly have a high level of type I fibers composed of myosin heavy chain 1 isoforms.Taken together, our results provide a starting point for the understanding of the genetic relationship between pH and muscle fiber type composition after slaughter.Furthermore, the association between high pH 24 and MYH1 expression may have important industrial applications for recognizing and promoting excellent quality in Berkshire loins.
values for meat quality parameters of high (n = 10) and low (n = 10) pH 24 groups, respectively;e Total average represents mean value for meat quality parameters of 323 Berkshire breeds slaughtered.

Figure 1 .
Figure1.GeneFishing PCR results for identifying differentially expressed genes in Berkshire loins with high vs. low pH 24 using six arbitrary annealing primers (ACP1 to ACP6).Total RNAs were extracted from Berkshire loins with high and low pH 24 using TRI-Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions and were then subjected to ACP-based PCR using the GeneFishingTM DEG kits (Seegene, Seoul, South Korea)(Kim et al., 2004).Lane M, L and H indicate 100bp size marker, low pH 24 and high pH 24 , respectively.The differentially expressed PCR products are indicated as arrows (numbers 1 to 4).

Figure 2 .
Figure 2. Confirmation of the significant expression of MYH1 gene in Berkshire loins with a high pH 24 value.(A) Semi-quantitative RT-PCR analysis of MYH1 transcript level in Berkshire loins with high versus low pH 24 values.For each reverse transcription reaction, total RNAs were extracted from each high pH 24 (H1 to H10) and low pH 24 (L1 to L10) groups; they were then used for first-strand cDNA synthesis and finally subjected to PCR using MYH1-specific primers (forward, 5'-AGCAGGAGCTGATTGAGACC-3'; reverse, 5'-TTCAGTCTGAAGCTGGGACA-3').The GAPDH level was used as an internal control to normalize the amount of cDNA template.The RT-PCR analysis was carried out at least three times, with similar results.(B) Measurement of the MYH1/GAPDH ratio.The relative intensities of the MYH1 and GAPDH bands in (A) were measured with a densitometer.

Table 1 .
Meat quality traits of Berkshire loins