Quantification of myosin heavy chain isoform in porcine muscle using an enzyme-linked immunosorbent assay

Meat Science 56 (2000) 261±269

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Quanti®cation of myosin heavy chain isoform in porcine muscle using an enzyme-linked immunosorbent assay $

F.F.S Depreux a, C.S. Okamura b, D.R Swartz c, A.L Grant d, A.M. Brandstetter e, D.E. Gerrard d,* a Cardiovascular Research Center, Massachusetts General Hospital-East, Charlestown, MA 02129, USA Department of Veterinary Medicine and Surgery, University of Missouri-Columbia, Columbia, MO 65211, USA c Department of Anatomy, Indiana University Medical School, Indianapolis, IN 46202, USA d Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA e Krannert Institute of Cardiology, Indiana University Medical School, Indianapolis, IN 46202, USA

b

Received 27 January 2000; received in revised form 27 April 2000; accepted 27 April 2000

Abstract The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) to quantify the relative abundance of the myosin heavy chain (MyHC) isoforms in porcine muscle. Longissimus muscle samples were taken from halothane positive (HAL+) and negative (HALÿ) pigs and subjected to ELISA using newly generated and commercially available myosin monclonal antibodies (mAbs). Muscle of HAL+ pigs possessed less type I (P <0.01) and IIA (P<0.1), and more type IIB MyHC (P <0.01) than muscle of HAL- pigs. Abundance of IIX MyHC content was negatively correlated (P < 0.0001) to the amount of IIB MyHC in porcine muscle. These data show indirect ELISA can be used to detect genotype di€erences in muscle MyHC content, and it provides a rapid, sensitive method for determining muscle ®ber type in porcine skeletal muscle. Furthermore, these data suggest that the proportion of glycolytic muscle ®bers increases at the expense of oxidative ®bers. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Myosin; Muscle ®ber type; ELISA

1. Introduction Heavy muscled pigs o€er the pork industry great potential for increasing the eciency of meat production. Unfortunately, a higher frequency of pale, soft, and exudative (PSE) meat is often associated with aggressive muscling in pigs. Although not completely understood, PSE meat is thought to result from postmortem protein denaturation caused by increased muscle glycolysis when carcass temperatures are elevated. Changes in glycolytic potential of muscle are associated with shifts in abundance of various muscle ®ber types. Muscle ®ber types (reviewed by Karlsson, Klont & Fernandez, 1999) are classically determined by inherent ATPase activity $ Purdue University Agricultural Research Programs Journal Paper No. 16,208. * Corresponding author. Tel.: +1-765-494-8280; fax: +1-765-4946816. E-mail address: [email protected] (D.E. Gerrard).

associated with various myosin heavy chain (MyHC) isoforms. To date, there are four major adult MyHCs (Schiano & Reggiani, 1994); type I (slow-twitch, oxidative metabolism), type IIA (fast-twitch, oxidative metabolism), type IIB (fast twitch, glycolytic metabolism) and type IIX(D) (fast-twitch, intermediate metabolism). Although it is possible to ®nd muscle ®bers expressing multiple MyHCs (termed IIC ®bers), the bulk of muscle ®bers contain a homogeneous population of MyHC and are classi®ed according to the predominate MyHC. Because of the unique structure and function of myosin, a great deal of homology exists across the MyHC isoforms. E€orts to monitor changes in muscle ®ber type have led to the development of a myriad of antibodies against various MyHCs of several species. Therefore, we chose to develop and assemble monoclonal antibodies to the various porcine MyHCs and develop an indirect enzyme-linked immunosorbent assay for quantifying the relative di€erences in MyHC content in the longissimus muscle of pigs.

0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00051-6

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2. Materials and methods

2.3. Monoclonal antibody production

2.1. Animals

Five female Balb C mice were injected twice (Harlow & Lane, 1988), intraperitoneally with 100 mg crude MyHC with complete Freund's adjuvant (Sigma Chemical). A third injection was made containing 100 mg crude MyHC with incomplete Freund's adjuvant (Sigma Chemical). Mouse sera were monitored for antibody production by indirect enzyme-linked immunosorbent assays (ELISA) using horseradish peroxidase conjugated goat anti-mouse antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Mice containing high antibody titers were sacri®ced, and hybridoma were constructed using standard protocols (Kohler, Hentartner & Schulman, 1978; Kohler & Milstein, 1975). Hybridoma were screened using ELISA and immunocytochemistry (Harlow & Lane, 1988). Candidate clones were repeatedly subcloned (three times) to verify authenticity. Resulting monoclonals were isotyped using a IsoStripTM Mouse Monoclonal Antibody Kit (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA). Ascites were produced (Harlow & Lane, 1988) from positive clones by injecting 2  10b hybridoma cells in mice intraperitoneally. After harvesting, ascites was diluted (1:1) with glycerol for long term storage at ÿ20 C.

Five halothane positive (HAL+) and ®ve halothane negative (HALÿ) pigs (115 5 kg) were processed according to industry procedures. Longissiumus muscle (LM) samples opposite the 12th and 13th rib were taken from pigs immediately post-exsanguination. Samples were snap frozen in isopentane cooled in liquid nitrogen or directly frozen in liquid nitrogen and stored at ÿ80 C. At 45 min post-mortem, pH of the LM opposite the 10th rib was recorded (pH45). At 24 h post-mortem, carcasses were ribbed between the 10th and 11th rib, and objective muscle color and ®rmness scores were collected according to National Pork Producers Council standards (NPPC, 1991). Samples from the LM at the 10th rib were removed and water-holding capacity (drip loss) was determined using established methodologies (Rasmussen & Stou€er, 1996). 2.2. Myosin extraction and crude protein preparation Because muscle of neonatal pigs are enriched for oxidative muscle ®bers (Lefaucheur, Ho€man, Okamura, Gerrard, Rubinstein & Kelly, 1997), two 21 day neonatal pigs were euthanized according to Purdue University Animal Care and Use Committee guidelines. Longissimus muscle samples opposite the last rib were collected from 21 day neonatal pigs. Samples were powdered in liquid nitrogen and subjected to a high ionic strength extraction bu€er (0.3 M KC1, 0.1 M KH2PO4, 50 mM K2HPO4, 10 mM EDTA, pH 6.5) (BaÈr & Pette, 1988). In addition, liver samples were collected from neonatal pigs and processed similar to that outlined for muscle samples and were used as negative controls. Extracted samples were diluted to 50% with glycerol and stored at ÿ20 C. For crude type II MyHC preparation, approximately 200 mg of total protein was subjected to SDS-polyacrylamide gel electrophoresis according to the procedures of Talmadge and Roy (1993) using a mini-gel apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Strips of each gel were removed and stained with Coomassie brilliant blue. These gel slices were placed along side the remaining unstained gel and used as landmarks for excising a strip at about 200 kD containing type II MyHC isoform. Gel strips containing MyHC were placed in dialysis tubing (Pharmacia & Upjohn Diagnostics, Kalamazoo, MI, USA) with 2 ml PBS bu€er (1 mM Na2HPO4, 1.6 mM NaH2PO4, 0.15 M NaCl) and electrophoresed at 250 V for 150 min at 4 C. Crude type II MyHC preparations were dialyzed overnight at 4 C against PBS. The remaining extracts were quanti®ed for total protein (BCA; Sigma Chemical Co., St. Louis, MO, USA) and stored at ÿ80 C in 50% glycerol.

2.4. Indirect ELISA Protein content of myosin extractions was determined by the BCA method. Indirect ELISA protocols were adapted from Doumit, Lonergan, Arbona, Killefer and Koohmaraie (1996). Seven mg of total protein was diluted in binding bu€er (0.05 M Tris, pH 8.5) and coated onto 96-well ELISA plates (Corning Inc., Acton, MA, USA) overnight at room temperature. Wells were then washed using washing bu€er (WB; 0.05 M 2-hydroxyethylpiperazine 2-ethanesulfonic acid (HEPES), 0.15 M NaCl, 0.05% Tween-20, 0.05% NaN3, pH 7.4). Nonspeci®c binding sites were saturated using a blocking solution [0.05 M HEPES, 3% non-fat dried milk (NFDM), 0.1% NaN3, pH 7.4] for 20 min at room temperature. After washing (WB), primary monoclonal antibodies were diluted (Table 1) in incubation bu€er (TB; 0.05 M HEPES, variable NaC1 concentrations; Table 1), 0.3% NFDM, 0.05% Tween-20, 0.05% NaN3, pH 7.4) and incubated as described in Table 1. After washing (WB), diluted (1:5000) secondary anti-mouse (IgG or IgM) antibodies (Jackson Immunoresearch Laboratories) conjugated to alkaline phosphate (AP) were incubated for 1 h at 37 C in secondary incubation bu€er (0.05 M HEPES, 0.15 M NaC1, 0.3% NFDM, 0.05% Tween-20, 0.05% NaN3, pH 7.4). After washing (WB), p-nitrophenyl phosphate solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) was added and incubated for 30 min at room temperature. The reaction was stopped by the addition of 5% EDTA. Absorbance

F.F.S. Depreux et al. / Meat Science 56 (2000) 261±269 Table 1 Optimal incubation conditions for immunocytochemistry Clone

NaCl (M)

Ab dilution

T ( C)

Duration (h)

A4.840 6B8 BF-F3 SC-71

0.6 0.6 0.3 0.15

1:100 1:1500 1:100 1:100

37 37 4 37

1 1 24 1

at 405 nm was measured using CERES UV.900 HDi (Biotek Instrument Ltd., Herefordshire, UK) plate reader. Lefaucheur, Ho€man, Gerrard, Okamura, Rubinstein & Kelly (1998) showed that the deep (RST) and super®cial (WST) portions of semitendinosus are considerably di€erent regarding muscle ®ber type composition. The RST has more slow ®bers; whereas, the WST has more type IIB ®bers. Therefore, these two muscle portions were used to optimize conditions for indirect ELISA. The RST was used for the optimizing the conditions for evaluating the amounts of type I, IIA and IIX MyHC isoforms, whereas WST was used to optimize conditions for IIB MyHC isoform abundance. Experiments were performed to optimize primary and secondary antibodies dilutions by serial dilution of antibodies and standards. Optimal primary mAb dilutions and conditions were determined when slopes of the standard serial dilution reached their maximal value. Resulting ELISA absorbance values were adjusted for background (liver immunoreactivity) and normalized to MyHC isoform values derived from MyHC isoform contents found in the deep (for type I, IIA and IIX antibodies) and super®cial (for type IIB antibodies) semitendinosus muscle. 2.5. Myo®brillar preparation and immunochemistry Myo®brils were isolated using procedures of Swartz, Greaser and Marsh (1993). Brie¯y, strips from rabbit and porcine psoas major and bovine cutaneous trunci muscles were collected immediately after slaughter and placed in rigor bu€er (RB; 10 mM imidazole, 75 mM KCl, 2 mM EGTA, 2 mM MgCl2, 2 mM NaN3, pH 7.2). Each muscle sample was further reduced in size and homogenized. The homogenate was centrifuged, rehomogenized and diluted in 10 vol. of RB. Samples were ®ltered, re-centrifuged and suspended in 10 vol. RB containing 0.05% Triton X-100 under mild agitation. The ®nal myo®bril pellet was suspended in RB made to 50% glycerol (v:v) and 1 mM dithiothreitol and stored at ÿ20 C. Immunolocalization of MyHC in the myo®brillar preparation was adapted from Swartz et al., 1993 Aliquots of myo®brils were incubated 20 min at room temperature with dilute (1:20) 6B8 primary mAb in BSA/RB bu€er (1 mg bovine serum albumin/ml RB). After washing and centrifugation at 13,500  g for 8±10 s, ¯uorscein isothiocyanate (FITC)-conjugated anti-

263

mouse secondary antibodies (Jackson Immunoresearch Laboratories) were diluted (1:2500) in BSA/RB bu€er and incubated 20 min with myo®brils at room temperature in the dark. After washing and centrifugation, myo®brillar pellets were re-suspended in 10 vol. of BSA/ RB bu€er, spread on coverslips, and ®xed 10 min in 3% formaldehyde in RB at room temperature. Coverslips were mounted using mounting media (70% glygerol, 75 mM KCL, 10 mM Tris (pH 8.5), 2 mM EGTA, 2 mM MgC12, 2 mM NaN3, 1±2 mg/ml p-phenylenediamine) and evaluated using ¯uorescence microscopy. 2.6. Western blotting Procedures used for muscle protein puri®cation and digestion are described in Swartz et al. (1993). Western blotting of whole and proteolytically-digested (trypsin and papain) myosin subfragments were performed as described by Fritz, Swartz and Greaser (1989). Rabbit myosin was puri®ed and proteolytically fragmented. Ten mg of protein were separated by SDS-PAGE, then transferred to polyvinylidene di¯uoride membranes (PVDF-Plus, MSI, Westboro, MA, USA) in 25 mM Tris, 192 mM glycine, 15% methanol, 0.1% SDS and 0.01% 2-mercaptoethanol. After transfer, membranes were washed and blocked. After PBS washing, the primary antibody was diluted (1:100) in PBS containing 0.1% Tween-20 and 0.1% BSA, and it was incubated with membranes for 1 h at room temperature. After PBS washing, membranes were incubated at room temperature with horseradish peroxidaseconjugated anti-mouse antibodies (Jackson Immunoresearch Laboratories) diluted (1:1000) in PBS containing 0.1% Tween-20, 0.1% BSA. After PBS washing, color was developed using 0.6 mg of 3,30 -diaminobenzidine/ ml and 0.015% hydrogen peroxide. Membranes were dried and scanned on a ¯atbed scanner. 2.7. In situ hybridization Localized MyHC expression was determined using in situ hybridization procedures reported by Lefaucheur et al. (1998). Ten-mm muscle cross-sections were cut, ®xed, dehydrated and treated with proteinase K. Sections were hybridized overnight with adult MyHC isoform antisense riboprobes (Lefaucher et al., 1998). Slides were washed, treated with RNAase and washed. Slides were air-dried, dipped in Kodak NTB-2 nuclear track emulsion (Eastman Kodak Co., Rochester, NY) and exposed for 5±10 days in light-tight boxes with desiccant at 4 C. After photographic development, slides were analyzed using dark ®eld microscopy. 2.8. Immunohistochemistry Characterization of the 6B8, A4.840 (Hughes & Blau, 1992), BF-F3 (Schiano et al., 1989), SC-71 (Bottinelli,

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Schiano & Reggiani, 1991) mAbs was performed on serial cross-sections adjacent to those subjected to in situ hybridization. LM muscle sections were blocked 30 min with normal goat serum in HEPES bu€er (0.05 M HEPES, 0.15 M NaCl, 0.05% NaN3, pH 7.4) at 37 C. Slides were washed with HEPES bu€er, and primary antibodies were incubated at various times, temperatures and concentrations of NaCl in HEPES bu€er (Table 2). Slides were washed in HEPES bu€er and incubated 1 h at 37 C with FITC-conjugated goat antimouse or FITC-conjugated donkey anti-mouse antibodies (Jackson Immunoresearch Laboratories) diluted (1:100) in HEPES bu€er. After washing with HEPES bu€er, coverslips were placed on slides and slides were evaluated using a Nikon FX microscope (Nikon Inc., Melville, NY, USA) equipped with ¯uorescence microscopy. Images from ®ve randomly selected ®elds per slide were digitized and evaluated using image processing software (IP lab version 3.2.4, Scanalytics, Inc., Fairfax, VA). The area of each muscle ®ber type (MyHC) was calculated and expressed as a percentage of the total muscle ®ber area within the ®eld. 2.9. Statistical analysis Normalized MyHC abundance values were subjected to an ANOVA procedure of the statistical analysis system (SAS, 1985) with genotype as the main e€ect. Means were separated using Student Neuman±Keuls test (SAS). Relative abundance of MyHC content calculated from digitized images of muscle cross-sections were regressed (SAS) against MyHC estimates determined by ELISA.

(Fig. 1 b and f). A large number of type 6B8-positive ®bers was located near the center of muscle ®ber fascicles with type I muscle ®bers (Fig. 1 a and e). Immunoreactivity was not detected using pre-immune serum. Porcine muscle is unique because the distribution of ®ber types in porcine muscle is tightly controlled; type I and IIA ®bers are located in the center of muscle fascicles with concentric circles of type IIX(D) and IIB ®bers arranged toward the outer limits of each fascicle (Lefaucheur, Edom, Ecolan & Butler-Brown, 1995). These data are consistent with the unique structure and distribution of muscle ®bers in porcine muscle and show that the 6B8 mAb localizes to muscle ®bers expressing predominately type IIA MyHC. Because our goal was to generate myosin-speci®c probes, we further documented the speci®city of our clones using rabbit, bovine and porcine myo®brils. The 6B8 mAbs localized to the A-band of rabbit (arrows, Fig. 2) and porcine myo®brils but did not react with bovine myo®brils. The A-band of a sarcomere includes myosin; however, other non-myosin proteins reside within this area as well and could lead to ambiguous conclusions. Using gel electrophoresis (Fig. 3a), whole and digested rabbit myosin was separated and western blotting (Fig. 3b) revealed that the 6B8 mAb speci®cally reacted with puri®ed myosin, heavy meromyosin and myosin S1 (Fig. 3b). However, immunoreactivity to the rod portion of the myosin was not observed. Given that immunoreactivity was not observed in the rod portion of myosin protein, we concluded that the mAbs from

3. Results and discussion 3.1. Characterization of mAbs Optimal incubation conditions (salt concentration, antibody dilution, temperature and duration) for each antibody used for immunocytochemistry are shown in Table 1. Clonal analyses of more than 400 hybridoma clones yielded one positive MyHC clone, 6B8. Immunocytochemistry revealed that 100% of ®bers expressing type IIA MyHC mRNA immunoreacted with the 6B8 mAbs Table 2 Optimal incubation conditions for enzyme-linked immunosorbent assays Clone

NaCl (M)

Ab dilution

T ( C)

Duration (h)

A4.840 6B8 BF-F3 SC-71

0.6 0.6 0.3 0.15

1:200 1:2000 1:200 1:200

4 37 4 37

2 1 2 1

Fig. 1. Muscle ®ber type determination by in situ hybridization (left column) and immunocytochemistry (right column). Antisense riboprobes to type I (a), IIA (b), IIX/D (c) and IIB MyHC (d) mRNAs were used. Corresponding antibodies against type I (e), IIA (f), IIA/X/D (g) and IIB (h) MyHCs were used on adjacent muscle sections. Magni®cation equals 250. Arrows denote location of reference ®ber.

F.F.S. Depreux et al. / Meat Science 56 (2000) 261±269

Fig. 2. Immuno¯uorescence labelling of rabbit myo®brils using the 6B8 monoclonal antibody. Arrows denote approximate location of Aband.

265

the 6B8 clone recognize an epitope in the S1 portion of myosin. Furthermore, immunoreactivity was not observed when 6B8 mAbs were used on bovine myo®brils. These data suggest that the 6B8 mAbs recognize type IIA MyHC in a species- and ®ber-speci®c manner. Immuno¯uorescent staining of muscle ®bers using commercially available MyHC antibodies showed that all recognize di€erent muscle ®ber types in sectioned porcine muscle (Fig. 1). Again, more oxidative ®bers (type I and IIA) were located toward the center of fascicles, whereas those with a more glycolytic metabolism (type IIX and B) reside at the periphery. The mAb SC-71, however, reacted to some ®bers expressing IIA MyHC mRNAs (Fig. 1 b, c and g). Reactivity to type IIA ®bers is not surprising because the SC-71 clone was originally designated as IIA MyHC-speci¯c mAb in rat muscle (Bottinelli et al., 1991). However, immunohistological and in situ hybridization data in general showed that SC71 mAbs can be used e€ectively as indirect biochemical probes for detecting IIX(D) ®bers in pig muscle. Fast IIB and IIX ®bers constitute about 75% of the total muscle ®ber population in porcine longissimus muscle (Lefaucheur et al., 1995). Within IIB and IIX ®ber populations, several ®bers co-express both IIB and IIX proteins and reveal a mismatch between the type of MyHC protein and mRNA present (Fig. 1 c, d, g and h). The exact reason for co-localization of MyHCs is not known but probably involves transition from one ®ber type to another (Andersen & Schiano, 1997). Pette and Vrbova (1992) showed that skeletal muscle ®ber plasticity follows a well-de®ned pathway (I$IIA$IIX$IIB); therefore, it remains intuitive that as muscle ®bers change from one type to another, at least two di€erent MyHCs types are present, especially during the earliest transition periods. This co-localization of di€erent MyHC is frequently observed in muscle ®bers of rodents (Andersen & Schiano; Rosser, Wick, Waldbilling & Bandman, 1996), especially in the growing animal (Butler-Brown & Whalen, 1984; Larsson, Birdal, Campione & Schiano, 1993). Curiously, the bulk of co-localization of MyHC isoforms was observed among the IIB and IIX ®bers (Fig. 1 c, d, g and h) raising the possibility that substantial transition exists between these two ®ber types in the longissimus muscle of pigs shortly before market weight. This observation is intriguing given that protein turnover rates favor more glycolytic ®ber types (Dadoune, Terquem & Alfonsi, 1978). If true, it is possible that changes in whole body growth eciency may be related to changes in muscle ®ber type. Additional studies to investigate fully this association may be warranted. 3.2. ELISA optimization

Fig. 3. SDS-PAGE (a) and Western blotting [(b), using the 6B8 antibody] of rabbit myo®brils and myosin.

Optimal dilution of the primary 6B8 antibody was approximately 1:2000 (Table 2 and Fig. 4a) for 1 h

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(Table 2 and Fig. 4b). At this dilution, optimal dilution of secondary alkaline phosphatase-labeled polyclonal antibodies was 1:5000 (Fig. 4c). Optimal incubation for the secondary Abs was also 1 h at 37 C (Fig. 4d). A similar strategy was employed for optimizing conditions of other commercially available MyHC mAbs (data not shown). Results of these experiments are summarized in Table 2. Optimal well coating was 3.5, 0.5, 7 and 3.5 mg of total myo®brillar protein for RST, WST and LM, for type I, IIA, SC-71 (IIX) and IIB mAbs, respectively (Fig. 5 a±e). Reducing background levels below an optical density of 0.1 unit was not possible when using the BF-F3 (IIB) and A4-840 (I) mAb clones even when blocking conditions were changed. Inter-assay coecients of variation of normalized LM samples (®ve replications) were 5.92, 4.26, 6.93 and 8.04% for 7 mg of total protein for type I, IIA, IIX and IIB MyHC mAbs, respectively. The intra-assay coecients of variation were less than 5% for all MyHC mAbs (six replications).

Correlation analyses between the relative amount of MyHC determined from ELISA and immunocytochemistry methods resulted in correlation coecients of +0.9773, +0.8537, +0.8185 and ÿ0.5051 for type I, IIA, IIB and IIX-speci®c mAbs, respectively. The negative correlation observed with SC-71 mAb probably is related to di€erences in the anity constant (K) of antibody for both the IIA and IIX MyHC isoforms. When the SC-71 mAb was used for immunocytochemistry, the relative intensity of the ¯uorescence was different between type IIA and IIX ®bers suggesting the K was di€erent for type IIA and IIX MyHC epitopes. Because muscle ®bers with lower levels of ¯uorescence were not di€erentiated from those with higher ¯uorescence during muscle ®ber analyses, many type IIA ®bers were likely misidenti®ed as type IIX ®bers when using the SC-71 mAb. Conversely, when the SC-71 mAb was used for ELISA, the relative di€erences detected between samples are likely more re¯ective of the true quantitative di€erences in type IIX MyHC content. If the K of the SC-71 mAbs is lower for IIA than for the

Fig. 4. Optimization of primary antibody dilution (a), primary antibody incubation time (b), secondary antibody dilution (c) and secondary antibody incubation time (d) for the 6B8 antibody in ELISA.

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267

IIX MyHC isoform, then the content of IIX MyHC is underestimated in the RST because of the abundance of type IIA MyHC. This muscle (RST) has 6.8 times more IIA MyHC content than LM as determined by ELISA but only 2.5 times more when evaluated using immunohistochemistry (25 versus 10%). When using classical histochemical methods (ATPase activity) for determining muscle ®ber type, the former more accurately re¯ects the real di€erence between these two divergent muscles (Lefaucheur et al., 1995). Therefore, use of the SC-71 (IIX) mAb should be restricted to comparing the amount of IIX within a given muscle across animals, whereas the other mAbs used in this study can be used to determine relative di€erences in MyHC isoform content among muscles as well as animals. 3.3. MyHC isoform content and genotype Halothane positive pigs used in this study yielded pale, soft and exudative meat postmortem (EsseÂn-Gustavsson, KarlstroÈm & LundstroÈm, 1992; Monin et al, 1999; Pommier & Houde, 1993). Table 3 shows the meat from Hal+ pigs had greater (P <0.01) water loss, and lower (P < 0.01) color and ®rmness NPPC scores. Hal+ pigs have a greater proportion of glycolytic muscle ®bers (EsseÂn-Gustavsson et al.; Fiedler, Ender, Wicke, Von Lengerken & Meyer, 1999). To distinguish between di€erent subpopulations of MyHC isoforms, indirect ELISA was employed. The relative abundance of type I and IIA MyHCs was greater (P <0.01) in Halÿ pigs, whereas the relative abundance of IIB MyHC was greater (P < 0.01) in Hal+ pigs (Fig. 6). Curiously, the amount of type IIA MyHC in porcine muscle was only correlated to the amount of type I MyHC and not IIB and IIX (SC-71) MyHC. Conversely, the amount of IIB MyHC content was negatively correlated (P<0.0001) to type IIX MyHC content (Table 4). These observations further support the use of the SC-7 1 mAb for determining IIX MyHC content in the LM muscle using ELISA because if the SC-71 mAb cross-reacts with both type IIA and IIX MyHC isforms, we would have expected a relationship between type IIA and IIX MyHC isoform content. This was not the case and most likely is because there are fewer type IIA Table 3 Means and standard errors of meat quality characteristics from HAL+ and HALÿ pigsa Variable

Fig. 5. Optimal muscle protein binding of ELISA. Raw data (a) were normalized (b) against the optical densities for myosin extracted from the red (RST) or white (WST) semitendinosus muscles. Type I (c), IIA (b), IIX (d) and IIB (e) abundance values where normalized to the RST (b,c and d) and WST (e), respectively.

pH45 Drip loss (%) Color Firmness a

Hal+ a

5.93 5.50a 2.70a 2.60a

Halÿ

S.E.

b

0.10 0.58 0.22 0.22

6.36 3.17b 3.60b 3.60b

Means bearing di€erent letters within the same row di€er (P < 0.01).

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classical means of muscle ®ber typing, and should facilitate further the study of the relationships between muscle ®ber type, growth and meat quality attributes. Acknowledgements

Fig. 6. Relative abundance of each adult myosin heavy chain isoform [I, IIA, IIX(D) and IIB] in the longissimus muscle of halothane postive (Hal+) and halothane negative (Halÿ) pigs. Genetic line comparisons bearing letters a, b, and c di€er (P <0.1, 0.05 and 0.01, respectively).

Table 4 Correlation coecients among muscle MyHC isoform contents Variable

Type IIA

Type IIX

Type IIB

Type I Type IIA Type IIX

0.76555b

0.61163a 0.43684

ÿ0.63l28a ÿ0.46076 ÿ0.92820c

a b c

P < 0.05. P <0.01. P <0001.

muscle ®bers in the LM of pigs (Lefaucheur et al., 1995) and therefore, there was less chance of cross-reactivity by the SC-71 mAb. In addition, the observations that type I MyHC was correlated with IIA and IIX MyHC content and type I and IIB MyHC content was negatively correlated is consistent with Pette and VrbovaÂ's (1992) model of muscle ®ber switching as previously discussed. In this model, increases in glycolytic muscle ®bers occur at the expense of more oxidative and less glycolytic ®bers. Collectively, these data show that an ELISA can be con®gured to evaluate muscle ®ber types in porcine muscle and suggest that muscle of Hal+ pigs have less IIX MyHC and greater IIB MyHC compared to muscle of Halÿ pigs. As a result, increases in IIB ®ber content in the muscle of pigs is likely responsible for the increases in the potential glycolytic metabolism of white muscles (Fiedler et al., 1999) and may participate in generating an adverse muscle environment that is responsible for PSE development. Furthermore, a porcine-speci®c IIA mAb has been developed and characterized to a point where it is suitable for immunohistological, western blotting and ELISA. Additional monoclonal antibodies for each adult porcine skeletal muscle MyHC isoform are commercially available that can be used for indirect ELISA to quantify the relative amount of MyHC isoform in di€erent types of porcine skeletal muscle. This biochemical tool is less cumbersome and more rapid than

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