ARTICLE |
Correspondence to: Louis Lefaucheur, INRA, Unité Mixte de Recherches sur le Veau et le Porc, 35590 Saint-Gilles, France. E-mail: lefaucheur@st-gilles.rennes.inra.fr
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Summary |
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The accurate classification of skeletal muscle fiber types according to myosin heavy chain (MyHC) polymorphism remains a difficult task in the pig. Combined myofibrillar ATPase and metabolic enzyme histochemistry, in situ hybridization, and immunocytochemistry were performed on serial transverse sections of pig longissimus (L) and rhomboideus (R) muscles at 100 kg body weight to give a new insight into muscle fiber typing in the pig. Several monoclonal antibodies (MAbs) either specific for a single MyHC (I, IIa, or IIb) or of multiple MyHCs (IIa + IIx or I + IIx + IIb) were used. No monospecific IIx antibody was available for the pig. All three adult Type II isoforms were expressed in the white L muscle, whereas no IIb was observed in the red R muscle, which was confirmed using RNase protection analysis. In most fibers, the distribution of the transcripts closely matched that of the corresponding proteins. When observed, co-expression of MyHCs mostly occured for IIx and IIb in L muscle, and was more common at the protein (11.5%) than at the mRNA (2.2%) level. A minor proportion of myofibers showed a mismatch between MyHC mRNA and protein. According to the type grouping distribution of myofibers encountered in pig muscle, MyHC isoform expression followed the rank order of IIIa
IIx
IIb from the center to the periphery of the islets, concomitantly with a decrease in oxidative metabolism and an increase in fiber size. The developmental origin and functional significance of the type grouping distribution are discussed. (J Histochem Cytochem 50:719730, 2002)
Key Words: fiber types, pig skeletal muscle, myosin heavy chains, mATPase histochemistry, in situ hybridization, immunocytochemistry
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Introduction |
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STAINING for acid-labile myofibrillar ATPase (mATPase) histochemistry allows the identification of three main fiber types in adult pig skeletal muscles (
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Materials and Methods |
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Muscle Samples
A female Large White pig from the INRA (Institut National de la Recherche Agronomique) herd was slaughtered at the usual commercial body weight of 100 kg (170 days of age) by electrical stunning and exsanguination, in compliance with French national regulations applied in commercial slaughtering. Immediately after slaughter, two muscles with different locations, functions, and biochemical properties were sampled: the longissimus (L) at the last rib level, a fast-twitch glycolytic muscle involved in voluntary movements of the back, and the tubular portion of the rhomboideus (R), a postural mixed-oxydo-glycolytic muscle involved in supporting the head. Samples were mounted on flat sticks, frozen in isopentane cooled by liquid nitrogen, and stored at -80C until further analysis.
Histochemistry, In Situ Hybridization, and Immunocytochemistry
Histochemistry, ISH, and ICC were performed on 10-µm-thick serial transverse sections cut on a cryostat at -20C (Reichert-Jung, 2800 frigocut N; Heidelberg, Germany). A section was stained using conventional mATPase histochemistry after preincubation at pH 4.35 to identify types I, IIA and IIB fibers (
Riboprobes for In Situ Hybridization
Probes against the different MyHCs were subcloned in either pBluescript KS+ (Stratagene; La Jolla, CA) or pCR 2.1 (Invitrogen; Carlsbad, CA) vectors. Vectors were linearized with appropriate restriction enzymes. Thereafter, the cRNA probes were transcribed using either T3 or T7 RNA polymerases according to the manufacturer's instructions (Promega; Madison,WI) using [35S]-UTP as the labeled nucleotide (New England Nuclear; Boston, MA). The Type I MyHC probe was a 136-base pair (bp) fragment including the last 19 bp of the translated region (TR) and the entire 3'-untranslated region (3'-UTR) (Fig 1A). The IIa MyHC probe was a 134-bp fragment comprising the full-length 3'-UTR (Fig 1B). The IIx and IIb MyHC probes correspond to the 3' half portion of the 3'-UTR and are 43 and 53 bp fragments, respectively (Fig 1B).
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Monoclonal Antibodies
Different MyHC MAbs were tested: NLC-MHCs (Novocastra; Newcastle, UK), reactive with pig slow-twitch Type I MyHC (
RNA Isolation and RNase Protection Analysis
Total RNA was isolated from the L and R muscles using the guanidium thiocyanate method (32P]-CTP (New England Nuclear). The RPA protocol was as described by
32P]-CTP incorporated into each protected fragment. The relative amounts of Type IIa, IIx, and IIb mRNAs were expressed as a percentage of total Type II transcripts.
Statistical Analysis
Influence of fiber type and muscle on cross-sectional area was analyzed by a two-way ANOVA using the GLM procedure of
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Results |
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Histochemistry
The mATPase staining after acidic preincubation (pH 4.35) allowed a distinction between black Type I, unstained Type IIA, and gray Type IIB fibers in both L and R muscles (Fig 2). A type grouping distribution of fiber types was observed, consisting of islets of Type I fibers in the vicinity of which Type IIA and then Type IIB fibers are found. The percentages of Type I, IIA, and IIB fibers were 11%, 7%, and 82% in L muscle, and 68%, 12%, and 20% in R muscle. According to the SDH staining, fibers were classified as weakly, moderately, or highly oxidative (Fig 3B, fibers 3, 4 and 5, respectively). Whereas all Type I and IIA fibers were highly oxidative in both muscles, IIB fibers exhibited a heterogeneous SDH staining. Thus, IIB fibers were either weakly (fiber 3) or moderately (fiber 4) stained in L muscle (Fig 3A and Fig 3B) and were always moderately stained (fiber 3) in R muscle (Fig 4A and Fig 4B). About 20% and 80% of conventional IIB fibers were moderately and weakly oxidative in the L muscle, respectively.
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In Situ Hybridization
The presence of positive and negative fibers within the same sample allowed a clear identification of the specific signals. Serial sections processed for ISH with cRNA probes specific for each adult MyHC isoform showed that fibers 1, 2, 3, and 4 were exclusively stained with Type I, IIa, IIb, and IIx probes in L muscle (Fig 3C3F), showing that cRNA probes were highly specific for each MyHC isoform. An estimation carried out on 600 fibers in the L muscle showed that very few of them co-expressed two MyHC transcripts (I + IIa, 0.1%; IIa + IIx, 0.3%; IIx + IIb, 2.2%). In the L muscle, the percentages of fibers expressing Type I, IIa, IIx, and IIb transcripts were 10.9%, 6.7%, 17.5%, and 62.3%, respectively (Fig 5). In R muscle, no IIb transcript was observed (Fig 4F) and the proportions of fibers expressing Type I, IIa, and IIx mRNA were 68%, 12%, and 20%, respectively (Fig 5). Very few hybrid fibers were detected in this muscle. Based on the expression of MyHC mRNA, intensity of SDH staining was high for Types I and IIa fibers, moderate or high for Type IIx fibers, and low for Type IIb fibers (Fig 5). Influence of fiber type on myofiber CSA was different between the two muscles (Fig 6). CSA increased in the rank order of I = IIa < IIx < IIb in L muscle, and I < IIa = IIx in R muscle. A comparison between muscles showed that Types I and IIa were larger in R muscle (p<0.0001), whereas Type IIx fibers exhibited similar CSA in both muscles.
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RNase Protection Analysis
As expected, three protected fragments of 397, 338 and 265 bp, corresponding to Type IIx, IIa, and IIb mRNAs, were observed by RPA (Fig 7). All three Type II transcripts were present in the L muscle, whereas only the IIa and IIx transcripts were visible in the R muscle. A quantitative analysis of the Type II MyHCs showed that the proportions of IIa, IIx, and IIb mRNAs expressed as a percentage of total Type II transcripts were 7%, 41%, and 52% in the L muscle and 32%, 67%, and 0% in the R muscle, respectively.
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Immunocytochemistry
NLC-MHCs MAb.
In both muscles, Type I fibers, as evidenced by the mATPase staining (Fig 3A and Fig 4A), were reactive with NLC-MHCs, whereas remaining fibers were not (Fig 8A and Fig 9A). We conclude that NLC-MHCs is specific for Type I MyHC in the pig. At the mRNA level, all Type I fibers were labeled with the Type I cRNA probe (Fig 3C and Fig 4C).
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6B8 MAb.
All unstained fibers with the mATPase staining (Type IIA fibers) were reactive with 6B8 antibody in both muscles (Fig 8B and Fig 9B) and were labeled with the IIa cRNA probe (Fig 3D and Fig 4D). Most remaining fibers were unstained with 6B8 and contained either Type I, IIx, or IIb mRNA. Therefore, the present data identified 6B8 as an antibody specific for IIa MyHC in pig muscle. Interestingly, additional fibers that were stained either in black (Type I) or gray (Type IIB) after the mATPase staining were also reactive with the 6B8 antibody (Fig 8B, fibers 5 and 6; Fig 9B, fiber 5). Because these fibers were unreactive with the IIa cRNA probe (Fig 3D and Fig 4D), they contained the IIa protein without the corresponding mRNA. At the protein level, fiber 5 in L muscle is a hybrid fiber (I + IIa) which is converting to a Type I fiber (IIaI), as suggested by the presence of only the Type I mRNA (Fig 3C). Fibers 6 in L muscle (Fig 3A) and 5 in R muscle (Fig 4A) are hybrid fibers containing IIa + IIx proteins and are in the process of being converted to a Type IIx fiber (IIa
IIx), as suggested by the presence of only Type IIx mRNA (Fig 3E and Fig 4E).
SC-71 MAb. In the L muscle, SC-71 recognized Type IIA and a subpopulation of histochemically defined Type IIB fibers (Fig 8C, fiber 4) that were moderately oxidative (Fig 3B). These IIB fibers were positive with the IIx riboprobe (Fig 3E) and negative with the IIb riboprobe (Fig 3F). In the R muscle, this antibody recognized all Type IIA and IIB fibers (Fig 9C). All conventional Type IIB fibers (fiber 3) were reactive with the IIx probe (Fig 4E) and unreactive with the IIb probe (Fig 4F) in the R muscle. Therefore, our results show that SC-71 recognized both IIa and IIx MyHC in pig skeletal muscle, and that labeled conventional IIB fibers are in fact IIx fibers. It is noteworthy that Type IIA fibers (Fig 9C, fiber 2) were more intensely labeled with SC-71 than conventional Type IIB fibers (Fig 9C, fiber 3), suggesting that the affinity of SC-71 was higher for IIa than for IIx MyHC.
BF-F3 MAb. In the L muscle, BF-F3 recognized all histochemically defined Type IIB fibers that were unreactive with SC-71 (Fig 8E, fiber 3). These fibers were strongly reactive with the IIb riboprobe (Fig 3F), whereas they were unstained with the IIx probe (Fig 3E). In the R muscle, all conventional IIB fibers (fiber 3) were negative with the BF-F3 antibody (Fig 9E) and the IIb probe (Fig 4F), whereas they were positive with the IIx probe (Fig 4E). Therefore, the present data identified BF-F3 as being specific for IIb MyHC in pig skeletal muscle.
S5-7D4 MAb.
In the R muscle where no IIb MyHC was expressed, S5-7D4 (Fig 9D) recognized the same fibers as did SC-71 (Fig 9C). However, in contrast to SC-71, staining intensities were homogeneous between fibers with S5-7D4. Consequently, we conclude that S5-7D4 recognized IIa and IIx MyHC with the same affinity in pig muscle. In L muscle, S5-7D4 labeled the same fibers as did SC-71, as well as additional conventional IIB fibers with a lower intensity (Fig 8D, fiber 7). These fibers were also highly reactive with BF-F3, showing that they contained the IIb MyHC protein (Fig 8E). Interestingly, they were unreactive with the IIx probe (Fig 3E) and strongly labeled with the IIb probe (Fig 3F). Thus, at the protein level, our data identified fiber 7 as a hybrid (IIx + IIb) fiber that was converting to a IIb fiber (IIxIIb), as suggested by the presence of only the Type IIb mRNA.
S5-8H2 MAb.
In the L muscle, S5-8H2 uniformly labeled all except two fibers (Fig 8F) that were typed IIA by histochemistry (Fig 3A). Similar results were observed in R muscle (Fig 9F). Unstained fibers were reactive with the IIa probe and unreactive with either I, IIx, or IIb probe in both muscles. We conclude that S5-8H2 recognized all but Type IIa MyHC in pig muscle, i.e., I + IIx + IIb MyHCs. Fiber 5 in R muscle deserves special attention. This fiber was stained with the anti-IIa MyHC 6B8 antibody (Fig 9B), whereas no IIa mRNA was present (Fig 4D). In addition, it was moderately stained with S5-8H2 (Fig 9F), suggesting that an MyHC other than IIa was present. At the mRNA level, this fiber expressed only IIx mRNA (Fig 4E), suggesting that it was a hybrid IIa + IIx fiber at the protein level, converting to IIx (IIaIIx).
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Discussion |
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Pigs have been aggressively selected for rapid, large, and efficient accretion of muscle, which is believed to have deteriorated meat quality. Muscle fibers, quantitatively the most important compartment of skeletal muscle, have long been thought to be important factors influencing meat quality. However, identifying a superior fiber type for meat production remains a tough issue. Because a lack in the accuracy of myofiber typing can be a limiting factor in these studies, the present work was undertaken to bring new insights into muscle fiber types in the pig.
The specificity of each antibody is summarized in Table 1. They were either specific for a single MyHC (I, IIa, or IIb) or reacting with multiple MyHC isoforms (IIa + IIx or I + IIx + IIb). The main limiting factor in the pig remains the lack of a monospecific Type IIx antibody. A specific IIx MAb has been recently described in the mouse, guinea pig, rabbit, cat, and baboon (
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Until recently, the consensus among most researchers was that Type IIb MyHC, the isoform exhibiting the most rapid speed of contraction, was expressed only in small mammals, in relation to the relative higher speed of contraction of their muscles (
The four isoforms (I, IIa, IIx, IIb) were expressed at the mRNA and protein levels in the L muscle, a fast-twitch glycolytic muscle, whereas no IIb was observed in the R muscle, a mixed highly oxidative muscle. The absence of any IIb in R muscle was confirmed by RPA. This is in accordance with previous results showing no IIb expression in the deep red portion of pig semitendinosus muscle using ISH (
Within the typical islets, MyHC expression was spacially regulated from the center to the periphery with a gradual expression in the rank order of IIIa
IIx
IIb in the L muscle and I
IIa
IIx in the R muscle. In L muscle, a corresponding gradual decrease in oxidative metabolism (I = IIa > IIx > IIb), and increase in CSA (I = IIa < IIx < IIb) occurred, showing that metabolic and size properties of muscle fibers varied according to their MyHC content. The gradient in MyHC expression (I
IIa
IIx
IIb) corresponds to increasing mATPase activity, contractile velocity, tension cost, fatigability, and excitation threshold (
In most fibers, the distribution of Type I, IIa, IIx, and IIb transcripts closely matched that of the corresponding proteins, which confirms previous results showing that the expression of MyHC isoforms is mostly transcriptionally regulated (IIa, IIa
IIx or IIx
IIb, supporting the general idea of an obligatory pathway of MyHC gene expression in the rank order of I
IIa
IIx
IIb under normal physiological conditions (
In conclusion, the present integrated data confirm that fast-twitch Type IIB skeletal muscle fibers identified on the basis of their mATPase activities have been misclassified in the pig in most previous studies. The combination of ISH with ICC shows they should be subdivided into Types IIx and IIb, as well as hybrid IIx + IIb fibers. At the protein level, a monospecific Type IIx antibody is still not available to accurately type myofibers according to their MyHC composition in pig muscle. Interestingly, IIx fibers exhibited high or moderate SDH staining, whereas pure IIb fibers were weakly stained, showing that only weakly stained IIB fibers did correspond to real IIb fibers. However, the continuum between SDH-positive and -negative stainings makes the use of this classification not always very accurate. Recently,
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Acknowledgments |
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We are grateful to Dr David Gerrard for his generous gift of the monoclonal antibody 6B8, to Dr Gillian S. ButlerBrowne for critical reading of the manuscript, and to Véronique Corbière for her contribution in cloning the probes.
Received for publication August 31, 2001; accepted December 12, 2001.
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