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J. Anim. Sci. 2004. 82:1931-1941
© 2004 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Myosin heavy chain composition of different skeletal muscles in Large White and Meishan pigs1

L. Lefaucheur*,2, D. Milan{dagger}, P. Ecolan* and C. Le Callennec*

* Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche sur le Veau et le Porc (UMRVP), 35590 Saint-Gilles, France; and and {dagger} INRA, Laboratoire de Génétique Cellulaire, 31326 Castanet-Tolosan, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Four major sarcomeric myosin heavy chains (MyHC) (i.e., I, IIa, IIx, and IIb) are expressed in pig skeletal muscle during postnatal development. The objective of the current study was to compare MyHC composition at mRNA and protein levels in LM, a fast-twitch glycolytic muscle, and rhomboideus (RM), a mixed slow- and fast-twitch oxido-glycolytic muscle, between two pig breeds exhibiting dramatic differences in postnatal muscle growth and meat quality. Eight Large White (LW) and eight Meishan (MS) females were fed under the same standard conditions, and slaughtered at an average BW of 62 kg (131 and 142 d in LW and MS pigs, respectively). In addition to conventional fiber typing by histoenzymology, MyHC composition was analyzed by combining immunocytochemistry, in situ hybridization, and a newly developed real-time PCR assay. Enzyme activities of lactate dehydrogenase, citrate synthase, and ß-hydroxy-acyl-CoA-dehydrogenase were used as markers of glycolytic, oxidative and ß-oxidation capacities, respectively. Results showed that conventional fiber typing in three classes by histoenzymology was insufficient in LM. For the first time, four monoclonal antibodies specific of each MyHC isoform, working in immunocytochemistry, were used. Our results are consistent with the sequential I{leftrightarrow}IIa{leftrightarrow}IIx{leftrightarrow}IIb MyHC transition rule. Breed effect on MyHC composition differed between muscle types. In LM of MS pigs, a shift from IIb to IIx, and to a lesser extent, to IIa, occurred without affecting type I MyHC. In RM, where IIb is absent, a shift from IIx to type I occurred, with a slight decrease in the IIa isoform. Effects were very similar at the mRNA and protein levels, suggesting a transcriptional regulation. In both muscles, MS pigs exhibited a decrease in the relative fiber type specific expression of the fastest isoform (i.e., IIb in LM and IIx in RM). The shift toward a slower phenotype in MS pigs was consistent with a less glycolytic and more oxidative metabolism, potentially using more lipids as fuel. A dramatic increase in cross-sectional area of type I fibers in RM (+27%) and a decrease in that of the fastest IIb fibers in LM (–25%) were observed in MS pigs. Overall, interpretation of earlier data regarding muscle fiber type has been flawed by inaccurate fiber typing in most pig skeletal muscles.

Key Words: Breeds • Enzyme Activity • Muscle Fibers • Myosin • Pigs • Polymerase Chain Reaction


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Eight isoforms of myosin heavy chains (MyHC), each encoded by a separate gene, are located in two clusters in skeletal muscle of mammals (Weiss et al., 1999Go; Shrager et al., 2000Go). In the pig, one cluster contains the {alpha} and type I (or ß) MyHC on chromosome 7, whereas the other one contains the embryonic, IIa, IIx, IIb, neonatal, and extraocular MyHC on chromosome 12 (Davoli et al., 2002Go). In postnatal growing pigs, only types I, IIa, IIx, and IIb MyHC are expressed in skeletal muscle (Lefaucheur et al., 2002Go). Maximum shortening velocity increases from type I to IIa, IIx, and IIb (Pellegrino et al., 2003Go), and under normal physiological conditions, MyHC transition follows an obligatory pathway in the rank order I{leftrightarrow}IIa{leftrightarrow}IIx{leftrightarrow}IIb (Schiaffino and Reggiani, 1996Go; Pette and Staron, 2000Go).

Most studies classify fibers in three types by histochemistry (Brooke and Kaiser, 1970Go; Ashmore and Doerr, 1971Go) and do not take into account the existence of four MyHC isoforms. The Meishan (MS) pigs exhibit lower growth rate, poorer feed efficiency, and lower lean meat content than conventional Western pig breeds (Bidanel et al., 1990Go; Bonneau et al., 1990Go; White et al., 1995Go), but sensory quality of their meat is superior (Touraille et al., 1989Go; Suzuki et al., 1991Go). Surprisingly, no difference in conventional fiber type composition between adult Large White (LW) and MS pigs was observed in LM (Bonneau et al., 1990Go).

Thus, the objective of the current study was to compare the MyHC profile at the mRNA and protein levels in two different skeletal muscle between pure MS and LW pigs. Hereafter, capital and lower case letters are used to denote fiber types according to the conventional mATPase histochemical technique (I, IIA, IIB; Brooke and Kaiser, 1970Go) and MyHC isoforms (I, IIa, IIx, IIb), respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals and Muscle Sampling
Pigs were from an INRA (Institut National de la Recherche Agronomique) experimental herd of Le Magneraud (Surgères, France). They were reared in compliance with French regulations for care and use of animals in research. Eight LW and eight MS females were fed under the same standard conditions until they reached an average BW of 62 kg, corresponding to 131 and 142 d of age in LW and MS animals, respectively. Pigs were housed in two separate pens according to breed and were given ad libitum access to a standard diet (17% CP, 1.5% crude fat, 4.5% crude fiber, 6.8% ash, 0.85% lysine, and 3,091 Mcal of ME/kg, as-fed basis) from 25 kg of live weight onward. The pigs were transported in the afternoon for approximately 3 h to the experimental abattoir of INRA at Saint-Gilles, France, and allowed to rest overnight with ad libitum access to water, but without food. The following morning, pigs were slaughtered by electrical stunning and exsanguination, in compliance with French national regulations for commercial slaughtering.

Two morphologically and functionally distinct skeletal muscles were selected: the LM adjacent to the last rib level, a predominantly fast-twitch glycolytic muscle; and the tubular portion of rhomboideus (RM), a postural mixed slow- and fast-twitch oxidative muscle. Within 30 min of slaughter, muscle samples were taken, mounted on tongue depressors, frozen in 2-methylbutane (isopentane) prechilled with liquid nitrogen, and stored at –70°C until further processing.

Histological Analyses
Histoenzymology, in situ hybridization, and immunocytochemistry were performed on 10-µm-thick serial transverse sections cut on a cryostat at –20°C, as previously described (Lefaucheur et al., 2002Go). Briefly, a section was stained using conventional mATPase histochemistry after preincubation at pH 4.35 to identify types I, IIA, and IIB fibers (Brooke and Kaiser, 1970Go). A serial section was processed for succino-dehydrogenase (SDH) to classify fibers according to their oxidative metabolism (Nachlas et al., 1957Go). In situ hybridizations were performed using 35S-UTP as the labeled nucleotide (New England Nuclear, Boston, MA). The slow/I MyHC riboprobe was a 136-bp fragment including the last 3' 19 bp of the translated region and the entire 3' untranslated region (3'-UTR). The fast/IIa probe was a 134-bp fragment comprising the full length of the 3'-UTR. The fast/IIx and IIb MyHC probes corresponded to the 3' half portion of the 3'-UTR and were 43- and 53-bp fragments, respectively. For immunocytochemistry, NLC-MHCs, 6B8, and BFF3 monoclonal antibodies (mAb) specific for type I, IIa, and IIb MyHC in pig skeletal muscle were used (Lefaucheur et al., 2002Go). Additionally, a new mAb named 8F4 and produced by Biocytex (Marseilles, France) in collaboration with INRA was tested by immunocytochemistry. The abundance of IIx and IIb fibers, as identified by in situ hybridization or immunocytochemistry, made them difficult to localize as individual fibers. Therefore, fiber type composition was expressed as the percentage of area reactive with each riboprobe or mAb. Four random fields of 0.6 mm2 each were analyzed per section. Additionally, the combination of SDH staining with simultaneous immunocytochemical detections of type I (NLC-MHCs mAb) and IIx (8F4 mAb) MyHC on the same section (Fazarinc et al., 1995Go) was realized, which allowed for the distinction of four fiber types (i.e., pure type I, IIa, and IIb fibers) and an heterogeneous population of IIx-containing fibers (IIax + pure IIx + IIxb). This combined staining was used to measure myofiber cross-sectional area from approximately 150 fibers of each type.

Real-Time PCR
Total RNA was isolated from the LM and RM using the guanidium thiocyanate method (Chomczynski and Sacchi, 1987Go) and stored at –70°C. Following treatment with DNase (DNA-free kit, Ambion, Austin, TX), 3.5 µg of RNA was retrotranscribed to cDNA (First-strand cDNA synthesis kit, Amersham-Pharmacia Biotech, Buckinghamshire, U.K.) in a final volume of 15 µL using either 40 pmol of MyHC specific primer (identical to the reverse primer used in the real-time PCR), or 0.2 µg of random hexadeoxy-nucleotides. For unknown reasons, previous tests using mixtures of different pig muscles showed that MyHC specific retrotranscription gave results that were more consistent with the predicted proportions of each MyHC than with random retrotranscription (data not shown). Therefore, the proportion of each MyHC mRNA within a muscle was determined from the MyHC specific retrotranscription. However, the level of expression normalized to 18S (Eukaryotic 18S ribosomal RNA VIC MGB kit, Applera Corp., Foster City, CA) of the most abundant isoform in each muscle (i.e., type I in RM, and IIb in LM) was also determined using the random retrotranscription. Subsequently, the level of expression of the other isoforms normalized to 18S was calculated from their respective proportions determined from the MyHC specific retrotranscription. The real-time PCR was performed on the polymorphic actin-binding site corresponding to the loop 2 spanning from nucleotide 1,795 to 2,031 of the pig IIa MyHC (AB025260) (Chikuni et al., 2001Go). Primers and TaqMan MGB probes (Figure 1Go) were designed using the Primer Express software (Version 1, PE Applied Biosystems, Tucson, AZ). Importantly, the forward and reverse primers were identical for all four MyHC (I, IIa, IIx, and IIb), thus avoiding any difference in primer hybridization between isoforms. In contrast, TaqMan minor groove binder probes labeled with 6-carboxyfluorosceine were specific of each MyHC, and exhibited identical Tm (69°C). The TaqMan probes corresponded to the complementary strand to contain more C than G to follow the manufacturer’s recommendations. All TaqMan PCR assays were performed in triplicate within 3 d in separate wells on 96-well optical plates using an ABI Prism 7000 sequence detection system (PE Applied Biosystems). Amplification efficiency for each MyHC was determined by plotting threshold cycle (Ct) as y-axis vs. log (initial cDNA) sequentially diluted 10-fold (x-axis). Efficiencies varied from 0.94 to 0.96, indicating comparable PCR amplification efficiencies (e = 0.95), which facilitate quantitative comparisons between isoforms (according to manufacturer’s instructions). The formula (1 + 0.95){Delta}Ct was used to quantify the ratio between two MyHC, where {delta}Ct was the difference of Ct between the two MyHC. For a given sample, all four MyHC were measured in triplicate within the same plate, and a common sample was repeated on all plates to estimate the experimental variability. Thus, the intra- and interplate CV were 0.88 and 1.72%, respectively.



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  		Figure 1. Aligned nucleotide sequences of porcine myosin heavy chain (MyHC) I, IIa, IIx, and IIb regions used for real-time PCR. Sequences were obtained from GenBank (U75316, AB025260, AB025262, and AB025261, respectively). Stars and dashes indicate identical nucleotides and spaces for alignment, respectively. Junctions between exons 16, 17, and 18 are indicated. The forward and reverse primes are in bold, and the TaqMan MGB probes are in bold and underlined.

 
Metabolic Enzyme Activities
Activities of lactate dehydrogenase (LDH, E.C. 1.1.1.2.7), citrate synthase (CS, E.C. 1.1.3.7), and ß-hydroxy-acyl-CoA dehydrogenase (HAD, E.C. 1.1.1.35) were used as markers of glycolytic metabolism, global oxidative capacity (tricarboxylic cycle), and lipid ß-oxidation potential, respectively. Enzyme activities were measured (µmol of degraded substrate•min–1•g of fresh muscle–1) on LM and RM as described previously (Lebret et al., 1999Go).

Statistical Analyses
The data were analyzed by the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). Because LM and RM were chosen to exhibit drastically different contractile and metabolic characteristics, the effects of breed were analyzed within each muscle type, and residual standard deviations within each muscle are reported in tables. Because pigs were housed in two separate pens according to breed, the effects of breed and pen were confounded; however, a pen effect on fiber typing is very unlikely according to available data. Body weight was included as a covariate to analyze myofiber cross-sectional area, and least squares means were reported. Additionally, the breed x muscle type interactions were analyzed by ANOVA using a model including the effects of breed, muscle type, animal within breed, and the breed x muscle type interaction. The statistical significance of the breed x muscle type interaction was tested against the general residual error of the model, and reported in the last column of each table. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Histological Characteristics
The conventional mATPase staining after preincubation at pH 4.35 allowed the distinction of three fiber types (i.e., black type I, unstained type IIA, and gray type IIB fibers; Figure 2AGo), as defined by Brooke and Kaiser (1970)Go. Whereas all type I and IIA fibers were highly oxidative in both muscles, IIB fibers exhibited either a weak or no SDH staining in LM (Figure 2BGo), and an intermediate staining in RM (data not shown). Breed effect on type I, IIA, and IIB numerical percentages varied between muscles, as shown by significant interactions between breed and muscle effects (Table 1Go, last column). Thus, no difference between breeds was observed in LM, whereas more type I and fewer type IIB fibers were present in RM of MS vs. LW animals.



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  		Figure 2. Serial sections of longissimus muscle from a Large White pig at 62 kg BW (131 d of age). Histochemical demonstration of mATPase after preincubation at pH 4.35 (A) and succino-dehydrogenase (SDH) activity (B). Other sections were processed for immunostaining with type I (C), IIa (E), IIx (G), and IIb (I) monoclonal antibodies, and probed by in situ hybridization for myosin heavy chain (MyHC) I (D), IIa (F), IIx (H), and IIb (J). Letters denote corresponding type I, IIa, IIx, and IIb fibers on the serial sections.

 

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  		Table 1. Histological characteristics of longissimus and rhomboideus muscles in Large White (LW) and Meishan (MS) breeds
 
The new 8F4 mAb recognized some conventional IIB fibers (Figure 2GGo), which were not reactive with the type I (Figure 2CGo), IIa (Figure 2EGo) and IIb (Figure 2IGo) mAb. Moreover, these fibers were labeled by the IIx riboprobe (Figure 2HGo) and unstained by the type I, IIa, and IIb riboprobes (Figure 2D, F, and JGo, respectively). Therefore, although showing a weak staining, the 8F4 mAb was identified as being specific of the pig IIx MyHC by immunocytochemistry in the present experiment. All conventional IIB fibers of RM were reactive with 8F4 and not reactive with the anti-IIb BFF3 mAb (data not shown), demonstrating that RM only contained type I, IIa, and IIx MyHC. Similar results were obtained at the mRNA level by in situ hybridization in RM (data not shown). Strong interactions between the effects of breed and muscle type on the percentage area stained by each mAb were observed (Table 1Go). In LM of MS pigs, a marked decrease in IIb MyHC to the benefit of IIx, and to a lesser extent, IIa, was observed, whereas type I MyHC was unaffected. In RM of MS pigs, a decrease in IIx MyHC to the advantage of type I occurred, whereas type IIa was not statistically affected. Similar changes were observed for the percentage of area reactive with each riboprobe (Table 1Go). The expression of the IIx and IIb MyHC at the protein and mRNA levels is illustrated in LM of a LW (Figure 2G, H, I, JGo) and MS (Figure 3A, B, C, DGo) pig whose fiber type compositions were close to the average composition of each breed. Type IIb is the prominent isoform in the LW, whereas type IIx is prominent in the MS breed. It is also noteworthy that fibers containing the IIb protein in LM of MS pigs (Figure 3CGo) were weakly labeled with the IIb riboprobe (Figure 3DGo).



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  		Figure 3. Serial sections of longissimus muscle from a Meishan pig at 60 kg BW (142 d of age). Sections were processed for immunostaining with type IIx (A) and IIb (C) monoclonal antibodies, and probed by in situ hybridization for myosin heavy chain (MyHC) IIx (B) and IIb (D). Letters denote corresponding type I, IIa, IIx, and IIxb fibers on the serial sections.

 
On average, myofibers were smaller in the LM of MS pigs compared with LW pigs, whereas the opposite occurred in RM, explaining the strong breed x muscle type interaction (Table 1Go). Type I fibers were larger in RM than in LM (P < 0.01), in particular in MS pigs. Their cross-sectional area was not influenced by breed in LM, but was dramatically increased in RM of MS animals. Pure type IIa fibers were larger in RM than in LM in both breeds, and no breed effect was observed in either muscles. Type IIx containing fibers (i.e., IIax, IIx, and IIbx fibers) were smaller in RM than in LM, and their cross-sectional area was slightly decreased in MS pigs. Pure IIb fibers were only observed in LM, and represented 58.9 ± 4.4% in LW and 25.2 ± 24.3% in MS pigs. Their cross-sectional area was drastically decreased in MS animals (–25%, P < 0.01).

Real-Time PCR
The TaqMan minor groove binder real-time PCR was performed from the MyHC specific retrotranscription to determine the percentage of each MyHC mRNA, and from the random retrotranscription to determine the normalized expression to 18S of type I MyHC in RM, and type IIb MyHC in LM. Very strong breed x muscle type interactions were observed for the percentage of each MyHC isoform (Table 2Go). In LM of MS pigs, a marked decrease in IIb MyHC proportion (62.9 vs. 17.1% in LW and MS, respectively) to the benefit of IIx (23.4 vs. 61.1%), and to a lesser extent, IIa (7.5 vs. 13.4%), was observed, whereas type I MyHC was not significantly affected. In RM of MS pigs, a dramatic decrease in IIx (24.1 vs. 3.8%), and to a lesser extent, in IIa (29.2 vs. 21.9%), occurred, to the advantage of type I MyHC (46.7 vs. 74.4%). Type IIb MyHC was not detected in RM of either breed. A striking difference between LW and MS breeds was observed for the IIb/IIx ratio in LM (2.69 vs. 0.28, respectively), and the I:IIx ratio in RM (1.94 vs. 19.56, respectively).


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  		Table 2. Real time PCR determination of myosin heavy-chain mRNA isoforms of longissimus and rhomboideus muscles in Large White (LW) and Meishan (MS) breeds
 
When normalized to 18S, total MyHC mRNA levels (I + IIa + IIx + IIb) were not significantly influenced by either breed or muscle type (Table 2Go), suggesting that the decrease in the level of expression of one isoform is balanced by the increased expression of one or several other isoforms. The normalized expression of type I MyHC tended to be higher in both muscles of MS pigs, but without reaching statistical significance. In contrast, strong differences between breeds, very similar to those observed for the proportions, occurred for the fast IIa, IIx, and IIb MyHC.

As previously used by Da Costa et al. (2002)Go in porcine skeletal muscle, the relative MyHC mRNA level per unit of cross-sectional fiber area, termed "relative fiber type expression," was calculated by dividing the MyHC mRNA level normalized to 18S with the percentage of area labeled by the corresponding riboprobe. An elevated relative fiber type expression implies a raised steady-state mRNA level for a given MyHC. The relative fiber type expression showed a high variability within muscle type and breed for each MyHC (CV = 53.6% ± 11.3, mean ± SE). The only significant difference between MyHC was a decreased relative fiber type expression of the IIb MyHC in LM of MS pigs. Differences between breeds only involved the fastest isoform in each muscle (i.e., a decreased relative fiber type expression of IIb [–60%] in LM, and of IIx [–48%] in RM; Table 2Go).

Metabolic Enzyme Activities
Meishan animals exhibited lower LDH and higher CS and HAD activities than LW in both muscles (Table 3Go). The LDH:CS ratio, indicative of the glycolytic vs. global oxidative metabolism, was drastically reduced in LM (–38%) and RM (–44%) of MS pigs. In contrast, the HAD:CS ratio, a marker of the relative importance of lipid ß-oxidation vs. the global oxidative capacity, was increased in both muscles of MS animals.


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  		Table 3. Muscle metabolic enzyme activities of longissimus and rhomboideus muscles in Large White (LW) and Meishan (MS) breedsa
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
The immunocytochemical staining with the mAb NCL-MHCs, 6B8, and BFF3 confirmed previous results in the pig (Lefaucheur et al., 2002Go), demonstrating their specificity against type I, IIa, and IIb MyHC, respectively. Until now, immunocytochemistry was limited by the lack of a specific IIx MyHC mAb. Even though antibodies specific for all three fast type II MyHC isoforms have been recently described in rat, cat, rabbit, and marsupials (Lucas et al., 2000Go), they are not commercially available and have not been tested in pig skeletal muscle fibers. In the current study, 8F4 showed a weak but consistent specific reactivity with the IIx MyHC by immunocytochemistry. This specificity could not be further analyzed by Western blots in the current study because 1) pig IIa, IIx, and IIb MyHC could not be physically separated by high-resolution sodium dodecyl sulfate gels as in laboratory animals (Talmadge and Roy, 1993Go); 2) no skeletal muscle pure in type IIa, IIx or IIb is available in the pig; and 3) the specificity of mAb can sometimes be different under denaturing or nondenaturing conditions. Also, the quantification of full MyHC polymorphism by ELISA, as described by Depreux et al. (2000)Go, suffered from drawbacks in our laboratory, likely because of alterations to mAb specificity under these conditions. Thus, some limitations still exist to fully and repeatedly quantify MyHC polymorphism at the protein level in pig skeletal muscle. However, the availability of monospecific mAb against type I, IIa, IIx, and IIb MyHC working in immunocytochemistry in the current study constituted invaluable tools for comparing MyHC expression between LW and MS pigs by immunocytochemistry at the protein level and analyzing the correspondence between the protein and the corresponding mRNA by in situ hybridization on serial sections.

Only one study compared the expression of type I, IIa, IIx and IIb MyHC in LM between MS and Landrace pigs by using conventional real-time PCR coupled with agarose gel electrophoresis (Tanabe et al., 1999Go). However, this technique tends to suffer from a number of drawbacks, such as variation in PCR amplification efficiency among primer sets and the inability to measure accumulation of PCR products accurately in real time. Such deficiencies have been largely overcome in the current study by using the same set of primers for all four MyHC isoforms coupled to the specific detection of each isoform with specific TaqMan MGB probes in real-time PCR assays.

Previous results suggested that the conventional enzyme histochemical classification in types I, IIA, and IIB needed to be revised in pigs to take into account for the existence of three adult fast-type II MyHC (Lefaucheur et al., 1998Go, 2002Go). Using this conventional classification, no difference of fiber type composition between LW and MS pigs was observed in LM in the present experiment, although proportion of MyHC differed drastically between breeds, in particular for IIb and IIx MyHC. These observations definitively show that the classification in types I, IIA, and IIB is insufficient to type myofibers in pig LM. Attempts to identify all four fiber types has been performed by combining mATPase with metabolic enzyme reactions (Larzul et al., 1997Go; Brocks et al., 2000Go; Gil et al., 2001Go); however, the separation of two groups of IIB fibers was only indirectly related with their MyHC isoform composition.

As expected, strong differences between muscle types were observed for MyHC composition. In LW pigs, the most striking difference between muscles concerned MyHC I and IIb, as a result of a dramatic shift of MyHC expression toward the slow type I in RM, whereas IIb was by far the predominant isoform in LM (62.9% by PCR). This is much higher than the 13% reported by Tanabe et al. (1999)Go in LM of Landrace pigs. More likely, this discrepancy could be due to defectiveness of the conventional PCR used by Tanabe et al. (1999)Go to accurately quantify the different MyHC, but could also be related to the genotypes used. In the present experiment, determination of MyHC composition at the mRNA level by real-time PCR and in situ hybridization, and at the protein level by immunocytology, gave very similar results, suggesting that expression of MyHC isoforms was mostly transcriptionally regulated, in accordance with previous data (Cox and Buckingham, 1992Go). Also in agreement with previous results (Lefaucheur et al., 2002Go), the four isoforms (I, IIa, IIx, and IIb) were expressed at the mRNA and protein levels in LM, whereas no IIb was detected by real-time PCR and immunocytochemistry in RM. Thus, contrary to the previous assumption that type IIb MyHC, the isoform exhibiting the fastest speed of contraction, was only expressed in fast moving small mammals (Pette and Staron, 2000Go), present results confirm the high expression of the IIb MyHC isoform in pig LM.

The current study indicates clearly that proportion of the different MyHC isoforms differed drastically between LW and MS breeds, and that breed effect differed between LM and RM, as shown by the highly significant interactions often observed between breed and muscle type. In LM of MS pigs, a shift from IIb to IIx and, to a lesser extent, IIa occurred, without any change in type I MyHC proportion. In RM, where IIb is absent, a shift from IIx to type I was observed, with a slight decrease in the proportion of the intermediary isoform IIa, which was visible by real-time PCR. Breed effects mostly affected the IIx:IIb ratio in LM, and the I:IIx ratio in RM. These changes were all consistent with the "nearest neighbor" rule based on the sequential MyHC isoform transition: I{leftrightarrow}IIa{leftrightarrow}IIx{leftrightarrow}IIb (Schiaffino and Reggiani, 1996Go; Pette and Staron, 2000Go). Thus, in both muscles, MS induced a shift to a slower phenotype (left side), and the differential effect of breed according to muscle type resulted from different initial MyHC profiles in LM and RM (i.e., mostly IIb in LM, and IIx in RM). Once more, the effects of breed on MyHC proportions were very similar at the mRNA and protein levels, confirming a transcriptional regulation of MyHC expression. Interestingly, the cumulative percentage of area reactive for the four MyHC in LM was higher in MS than LW animals, for both mRNA (146 vs. 119%) and protein (140 vs. 115%), suggesting that coexpression of more than one MyHC isoform in a fiber seemed more widespread in LM of MS than LW pigs. No such difference was observed in RM.

By combining the results of real-time PCR and in situ hybridization, we found that relative fiber type expression of the fastest isoform in each muscle (i.e., IIb in LM, and IIx in RM) was lower in MS than LW pigs. These decreased steady-state mRNA levels are consistent with the shift toward a slower phenotype in MS animals. The markedly decreased relative fiber type expression of IIb MyHC in LM of MS pigs is consistent with the weak labeling generally observed with the IIb riboprobe in these samples. Otherwise, no obvious difference of relative fiber type expression was observed between the different MyHC. This contrasts with other data showing a consistently higher relative fiber type expression for IIa MyHC in different pig skeletal muscles (Da Costa et al., 2002Go). Because of their high relative fiber type expression, these authors concluded that MyHC IIa fibers could be a major determinant of overall muscle function and fiber type switching under modulating conditions. Our results do not support this hypothesis.

The postnatal growth of muscle tissue is dependent on the total number of fibers and muscle fiber cross-sectional area and length. It has been reported that total number of fibers is definitively fixed at approximately d 90 of gestation in pigs (Wigmore and Stickland, 1983Go), and that it is lower in MS than in LW pigs (Bonneau et al., 1990Go), which helps to explain the lower postnatal muscle growth capacity of MS pigs, in particular of large glycolytic muscles such as LM (White et al., 1995Go). Moreover, the present experiment showed a strong effect of breed on mean cross-sectional area of myofibers, and more interestingly, that this effect was highly dependent on fiber and muscle types. In LM, which is mostly composed of large fast IIb glycolytic fibers in LW pigs (58.9 ± 4.4%), MS pigs exhibited a dramatic decrease in the cross-sectional area of pure IIb fibers (–25%) in addition to a reduction of their percentage (25.2 ± 24.3%, data not shown) and, to a lesser extent, of the cross-sectional area of IIx MyHC-containing fibers (–17%), whereas type I and IIa fibers were unaffected. This selective postnatal decreased hypertrophy of the fastest fibers likely contributes to the lower postnatal muscle growth of the most glycolytic muscles in MS pigs. A strong positive genetic correlation between lean tissue growth rate and fiber mean cross-sectional area has also been reported in LM within the LW breed (Larzul et al., 1997Go). It is noteworthy that the selective reduction in cross-sectional area of IIb and IIx MyHC-containing fibers in LM of MS pigs fits with the sequential I{leftrightarrow}IIa{leftrightarrow}IIx{leftrightarrow}IIb MyHC transition rule, with the fastest IIb fibers being the most affected. A higher sensitivity of fast-twitch glycolytic fibers to cross-sectional area reduction has already been observed in other situations, such as muscle loss in the lactating sow (Lefaucheur, 1990Go) or in glucocorticoid-treated rats (Kelly et al., 1986Go). Opposite results on mean cross-sectional area of myofibers were observed in RM, a muscle mostly composed of slow type I fibers (56.1 ± 6.8% in LW pigs). Indeed, MS pigs exhibited a dramatic increase in the cross-sectional area of type I fibers (+27%) in addition to an increase in their percentage (66.1 ± 4.1), whereas the cross-sectional areas of fast IIa and IIx fibers were not significantly affected. The opposite effects of breed on myofiber cross-sectional area in LM and RM may be related to their different functions at the origin of their different fiber type compositions. Thus, LM is involved in providing a propulsive voluntary rapid thrust to the hind limb, whereas RM is involved in posture by supporting the head.

Comparisons between wild and domestic pigs (Rahelic and Puac, 1981Go; Weiler et al., 1995Go) suggest that intensive selection for lean muscle growth in modern pigs induced a shift in muscle metabolism toward a more glycolytic and less oxidative fiber type. Our results support this hypothesis in that MS pigs exhibited a less glycolytic and more oxidative metabolism, as well as a higher capacity to use lipids as an energetic substrate; the higher HAD:CS in MS pigs also suggests a qualitative change of mitochondria. Because oxidative metabolism decreases in the rank order I, IIa, IIx, IIb (Lefaucheur et al., 2002Go), these metabolic changes are consistent with the shift of MyHC profile toward a slower type in both muscles in MS pigs. A higher proportion of IIb MyHC has also been reported in pigs carrying the mutated halothane gene (Depreux et al., 2002Go). An interpretation of all these results is that a shift toward a more glycolytic and less oxidative metabolism, and a faster contractile type would be associated with leaner pigs, better feed efficiency, and higher growth rate. However, this conclusion is not always supported by other experiments. Indeed, Nostvold et al. (1979)Go showed that increasing average daily gain and decreasing backfat thickness during eight generations of selection decreased proportion of fast glycolytic fibers in pig LM. A negative genetic correlation between percentage of fast glycolytic fibers and lean percentage has also been reported in LM within the LW breed (Larzul et al., 1997Go). Finally, Henckel et al. (1997)Go found positive phenotypic correlations between lean meat content, CS activity and capillarity in LM of Danish Landrace and LW pigs. Therefore, the specific relationship between MyHC polymorphism, in particular the ratio of IIx:IIb MyHC, and growth performance remains to be established.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
The availability of four myosin heavy-chain monospecific monoclonal antibodies and riboprobes usable by histology and the development of a TaqMan real-time polymerase chain reaction assay allow for significant advances in the characterization of myosin heavy-chain polymorphism in pig muscle, and show that previous classification of fibers in I, IIA, and IIB by histoenzymology is inaccurate in pig skeletal muscle. The dramatic increase of the IIx:IIb ratio in longissimus muscle of Meishan pigs at both the messenger ribonucleic acid and protein levels suggests a transcriptional regulation, and raises several questions about its significance for muscle growth and meat quality, as well as about the mechanisms involved in the regulation of the IIx and IIb genes between muscles and breeds. Further work is needed at the promoter level to better understand these differences.


   Footnotes
 
1 We acknowledge D. Gerrard, Dept. of Anim. Sci., Purdue Univ., West Lafayette, IN, for donation of the monoclonal antibody 6B8. The authors wish to thank J. C. Caritez for his technical assistance in breeding animals, and J. Chevalier for computational assistance in preparing the paper. This work was financially supported by INRA. Back

2 Correspondence—phone: +33-2-23-48-56-43; fax: +33-2-23-48-50-80; e-mail: lefaucheur{at}st-gilles.rennes.inra.fr.

Received for publication December 4, 2003. Accepted for publication March 22, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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