ARTICLE |
Correspondence to: José-Luis L. Rivero, Laboratorio de Biopatología Muscular, Departamento de Anatomía, Universidad de Córdoba, Campus de Rabanales, Edificio de Sanidad Animal, Crtra Madrid a Cadiz, km. 396, 14014 Cordoba, Spain. E-mail: an1lorij@uco.es
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Summary |
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Skeletal muscle fiber types classified on the basis of their content of different myosin heavy chain (MHC) isoforms were analyzed in samples from hindlimb muscles of adult sedentary llamas (Lama glama) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry, and quantitative histochemistry of fiber metabolic and size properties. The immunohistochemical technique allowed the separation of four pure (i.e., expressing a unique MHC isoform) muscle fiber types: one slow-twitch (Type I) and three fast-twitch (Type II) phenotypes. The same four major fiber types could be objectively discriminated with two serial sections stained for mATPase after acid (pH 4.5) and alkaline (pH 10.5) preincubations. The three fast-twitch fiber types were tentatively designated as IIA, IIX, and IIB on the basis of the homologies of their immunoreactivities, acid denaturation of their mATPase activity, size, and metabolic properties expressed at the cellular level with the corresponding isoforms of rat and horse muscles. Acid stability of their mATPase activity increased in the rank order IIA>IIX>IIB. The same was true for size and glycolytic capacity, whereas oxidative capacity decreased in the same rank order IIA>IIX>IIB. In addition to these four pure fibers (I, IIA, IIX, and IIB), four other fiber types with hybrid phenotypes containing two (I+IIA, IIAX, and IIXB) or three (IIAXB) MHCs were immunohistochemically delineated. These frequent phenotypes (40% of the semitendinosus muscle fiber composition) had overlapped mATPase staining intensities with their corresponding pure fiber types, so they could not be delineated by mATPase histochemistry. Expression of the three fast adult MHC isoforms was spatially regulated around islets of Type I fibers, with concentric circles of fibers expressing MHC-IIA, then MHC-IIX, and peripherally MHC-IIB. This study demonstrates that three adult fast Type II MHC isoproteins are expressed in skeletal muscle fibers of the llama. The general assumption that the very fast MHC-IIB isoform is expressed only in small mammals can be rejected. (J Histochem Cytochem 49:10331044, 2001)
Key Words: skeletal muscle, muscle fiber, immunohistochemistry, ATPase, fiber size
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Introduction |
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Skeletal muscle cells fall into several specialized classes, termed fiber types, which show differences in morphological, contractile, and metabolic properties. Myosin is the most abundant protein in muscle and makes up the primary component of the thick filaments. Its molecule is composed of two heavy chains (MHCs) and four light chains. Both kinds of chains occur as several distinct isoforms coded by different genes (
From the literature available, it has been suggested that the molecular diversity of adult skeletal muscle fibers is species-specific, and important physiological differences between species related to body size have already been reported (
The llama (Lama glama) is a South American cud-chewing mammal related to the camel, but smaller and without a hump. It was domesticated in the Andes and is now used as a beast of burden and a source of food. The adult body weight of this mammal is 101 ± 18 kg (mean ± SD) for females and 116 ± 22 kg for males (
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Materials and Methods |
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Six elderly (810-year-old) and clinically healthy llamas (all males) were used. Semitendinosus muscle, biceps femoris muscle, and vastus lateralis muscle were dissected and cleaned of connective tissues. Muscle samples (1 x 1 x 0.5 cm) were removed from the midbelly region of these muscles, frozen by immersion in isopentane kept in liquid nitrogen, and stored at -80C until analyzed.
Samples were transferred to a cryostat at -20C, serially sectioned at 10-µm, and mounted on poly-L-lysine-coated glass slides for immunohistochemistry and histochemistry. Samples from the rat extensor digitalis longus muscle and biopsies from the equine gluteus medius muscle were also removed and used as standard references of skeletal muscle of two well-known species expressing three (rat) and two (horse) fast MHC isoforms. These samples were frozen and processed in the same way as the llama samples.
Serial sections were reacted with a panel of monoclonal antibodies (MAbs) specific for MHC isoforms (Table 1). The specificity of the majority of these MAbs for rat (
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Additional sections were stained for qualitative myofibrillar ATPase activity and acid (pH 4.24.6, 2 min) and alkaline (range of pH 10.310.6, 10 min) preincubations by using a modification (-glycerophosphate dehydrogenase (GPD;
To characterize fiber types according to their MHC content expressed at the protein level and to determine the relative proportion and mean fiber size, a region of the cross-sections containing 150175 fibers was selected for further analyses. The sections stained for immunohistochemistry, myofibrillar ATPase histochemistry, and metabolic properties (SDH and GPD) were surveyed to find regions free of artifact. Serial sections were visualized and analyzed using a Leica DMLS microscope (Leica Microsistemas; Barcelona, Spain), a Leica high-resolution color charge-coupled device camera (Leica Microsistemas), an eight-bit Matrox meteor frame-grabber (Matrox Electronic Systems; Barcelona, Spain), combined with image-analyzing software (Visiolog 5, Noemi; Microptic, Barcelona, Spain). With the use of the mATPase staining after acid preincubation, a fiber mask was drawn along the cell borders of the desired number of fibers. Images of the remaining immunohistochemical and histochemical sections were then fitted into the fiber mask. Single fibers were subsequently identified and qualitatively classified as positive or negative for a given immunohistochemical reaction. The fiber type distribution of each muscle biopsy was established by immunohistochemistry.
For each fiber analyzed, a mean optical density (OD) was determined for qualitative mATPase after acid and alkaline preincubation, as well as for the SDH and GPD reactions. In addition, the cross-sectional areas of the same individual muscle fibers were determined in the SDH histochemical reaction. Measurements for each fiber were made in duplicate in two consecutive serial sections, and the mean of both was used to quantify the intensity of the reaction and size of an individual fiber.
Quantitative data were averaged according to fiber type and differences between mean values analyzed by a one-way ANOVA. In the presence of a significant F ratio, post hoc comparisons of means were provided by a Fisher's least significant difference test. Statistical significance was accepted at p<0.05.
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Results |
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Rat
Seven muscle fiber types were delineated using IHC MAbs against specific MHC isoforms in the control sample of rat muscle (Fig 1AD; Table 2). Four pure muscle fiber types were identified according to their MHC content: I, IIA, IIX, and IIB (e.g., fibers 1, 3, 5, and 7, respectively, in Fig 1). A few fibers (e.g., fiber 2 in Fig 1) demonstrated coexpression of MHC I and MHC IIA and corresponded to classical IIC fibers (
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The four pure fiber types (I, IIA, IIX, and IIB) were also delineated by myofibrillar ATPase (mATPase) histochemistry using combined staining after acid (pH 4.35) and alkaline (pH 10.5) preincubations (Fig 1E and Fig 1F). Type I fibers stained dark after acid preincubation and very light after alkaline preincubation (e.g., fiber 1 in Fig 1E and Fig 1F). The reverse was true for IIA fibers (e.g., fiber 3 in Fig 1E and Fig 1F). Type IIX and IIB fibers stained medium after preincubation at pH 4.35 (e.g., fibers 5 and 7, respectively, in Fig 1E), but IIX fibers stained darker than IIB fibers after alkaline pretreatment (Fig 1F). Quantitative differences in the intensity of staining for mATPase after both acid and alkaline preincubations of these four main fiber types are presented in Table 3. Consequently, these four fiber types could be objectively discriminated with only two sections stained for mATPase histochemistry (Fig 2A). In addition, intermediate hybrid fibers containing MHC I plus MHC IIA were also delineated with these two mATPase histochemical reactions (Fig 2A), because they stained medium to dark after both acid and alkaline preincubations (e.g., fiber 2 in Fig 1E and Fig 1F). Conversely, fast hybrid fibers (IIAX and IIXB) had mean ATPase activities intermediate between those of their respective pure MHC fiber types (Table 3). As a consequence, all these fibers had overlapping ATPase activities and could not be objectively divided into discrete categories (Fig 2A).
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Muscle fiber types expressing different MHC isoforms also showed important differences in both their intensity of staining for SDH and GPD and their mean cross-sectional areas (Fig 1G and Fig 1H and Table 3).
Horse
Five different fiber populations were demonstrated immunohistochemically according to the MHC isoform they express in the horse gluteus medius muscle (Fig 3A3E; Table 2). Three were pure fibers containing a unique MHC isoform and were identified as Types I, IIA, and IIX (e.g., fibers labeled 1, 3, and 5 in Fig 3). A few fibers were demonstrated to be hybrid fibers coexpressing MHC I and MHC IIA (e.g., fiber 2 in Fig 3). Many other fibers were hybrid fibers containing the two fast MHC isoforms, i.e., IIA and IIX (e.g., fiber 4 in Fig 3).
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On the basis of mATPase reactions after acid (pH 4.45) and alkaline (pH 10.35) preincubations, equine muscle fibers could be objectively divided into three categories (Fig 3F and Fig 3G). Type I fibers were acid-stable and alkaline-labile (e.g., fiber 1 in Fig 3F and Fig 3G). Type IIA fibers were acid-labile and partially alkaline-stable (e.g., fiber 3 in Fig 3F and Fig 3G). Type IIX fibers were partially acid-stable and alkaline-stable (e.g., fiber 5 in Fig 3F and Fig 3G). Quantitative differences observed in the staining intensity of the three major fiber types for both mATPase methods were statistically significant (Table 3), and they could be discriminated into discrete clusters of fibers (Fig 2B). Type C fibers were also histochemically delineated (e.g., fiber 2 in Fig 3F and Fig 3G). A continuum in the staining intensity for mATPase was observed, however, between pure IIA and IIX fibers (Fig 3F and Fig 3G). In fact, hybrid IIAX fibers (e.g., fiber 4 in Fig 3F and Fig 3G) had lower mATPase activity after acid preincubation than pure IIX fibers, but this activity was not different between the two fiber types after alkaline pretreatment (Table 3). As a consequence, hybrid Type IIAX fibers were graphically scattered across their respective pure MHC fiber types (Fig 2B).
The MHC content also had a significant impact on SDH and GPD activities, as well as on areas, of equine skeletal muscle fiber types (Fig 3H and Fig 3I; Table 3).
Llama
Eight different fiber populations were demonstrated immunohistochemically in hindlimb muscles of adult llamas with the panel of MAbs specific for MHC isoforms used in the present study (Fig 4A4F; Table 2). These phenotypes were classified as four pure fibers containing a single MHC isoform (named I, IIA, IIX, and IIB) and four hybrid fibers with two (i.e., I+IIA, IIAX, and IIXB) or three (i.e., IIAXB) different MHC isoforms. Despite this, the immunoreactivity patterns of these MAbs to MHC isoforms in llama skeletal muscle fibers were not identical to those recorded in rat and horse skeletal muscle fibers (Table 2), and the results of the present study are not conclusive regarding the identity of the MHC isoforms expressed in llama skeletal muscle. However, we have adopted a conventional terminology to describe the various muscle fiber phenotypes found in this species to maintain a consistent nomenclature across different species.
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On the basis of mATPase reactions after acid (pH 4.5) and alkaline (pH 10.5) preincubations, the muscle fibers could be divided into four basic categories (Fig 4G, Fig 4H, Fig 5A, and Fig 5B). Type I fibers stained dark after acid and light after alkaline preincubations (e.g., fiber 1 in Fig 4G and Fig 4H). Type IIA stained light after acid preincubation and medium after alkaline pretreatment (e.g., fiber 3 in Fig 4G and Fig 4H). Type IIX fibers had moderate mATPase activity after acid and high activity after alkaline preincubations (e.g., fiber 5 in Fig 4G and Fig 4H). Type IIB fibers also stained medium by mATPase after acid and alkaline preincubations, but they stained darker than IIX fibers after acid pretreatment (e.g., fiber 7 in Fig 4G and Fig 4H). To sum up, after acid preincubation, Type I fibers had the highest mATPase activity, followed in the rank order by IIB>IIX>IIA (Fig 5A; Table 3). The mATPase activity after alkaline preincubation increased in rank order I<IIA<IIB<IIX (Fig 5B; Table 3). Once again, hybrid fibers had intermediate staining intensities for mATPase histochemistry (Table 3). When the relationship between acid and alkaline ATPase activities of the same individual fibers was plotted, a clear grouping of muscle fiber types with homogeneous MHC content was possible in the llama (Fig 2C). Hybrid C fibers also were delineated, but fast hybrid fibers (i.e., IIAX, IIXB, and IIAXB) were admixed with their respective MHC pure phenotypes (Fig 2C).
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The staining intensities of SDH (Fig 4I and Fig 5C) and GPD (not shown) activities also formed a continuum, but differences could be elicited between fiber types of presumed homogeneous MHC content (Table 3). Type I fibers had the highest SDH activity, followed in rank order by IIA>IIX>IIB. The staining intensities of GPD reaction was consistently ranked according to fiber type such that IIB>IIX>IIA>I. Hybrid fibers generally showed intermediate SDH and GPD activities in between their respective pure MHC fiber types. The cross-sectional areas of llama muscle fibers also varied significantly according to the MHC isoform they express (Table 3). Type I and IIA fibers were the smallest, type IIB the largest, and type IIX of intermediate fiber size. There was an inverse relationship between fiber area and fiber SDH activity (r=0.81, p<0.001) in the llama muscle, but this relationship did not allow a certain grouping of myofibers (Fig 2D).
After a population of 972 fibers in the semitendinosus muscle of the llama were examined, 58.7% of fibers were pure phenotypes (i.e., expressing a single MHC isoprotein), whereas the remaining 41.3% were hybrid fibers with two or even three MHCs. Within the pure phenotypes, IIX fiber was the most common (37.9%), followed in rank order by IIB (14.4%), IIA (3.6%), and I (2.8%). The most common hybrid phenotype was IIXB (23.5%), followed by IIAXB (11.3%), IIAX (6.1%), and I+IIA (0.4%).
Distribution of the three fast fiber types in skeletal muscle fiber types was spatially regulated around typical islets of Type I fibers (Fig 6A). Fibers expressing MHC IIA were contiguous to those expressing MHC I (Fig 6B and Fig 6C). Pure IIX fibers were in the direct vicinity of Type I and IIA fibers, and hybrid IIXB and IIAXB were located mostly within primary fascicles between the islets of Type I fibers (Fig 6D). However, pure IIB fibers were located mainly at the periphery of the rosettes near the edges of primary fascicles (Fig 6E).
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Discussion |
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By using different MAbs, it was possible to identify four MHC isoforms in adult llama skeletal muscle: the MHC-ß/slow or MHC I and three fast (Table 4). Whereas the identity of one of these three fast MHC isoforms seems to be clearly an MHC IIA isoform, the present results are not conclusive regarding the final characterization and identification of the other two fast MHCs. We have provisionally referred to these isoforms as IIX and IIB on the basis of their immunolabeling, enzyme histochemistry, and size homologies at the cellular level with the corresponding isoforms of rat and horse muscles (Table 4). This adoption is based on the assumption that the structure of each MHC isotype is generally conserved between species, whereas greater sequence divergence may be found between different isotypes within the same species (
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Fiber typing in mammalian skeletal muscle has extensively been identified by myofibrillar ATPase histochemical methods based on acid (
The limitation of histochemical ATPase procedures is clearly illustrated in the present study by the fact that a large number (30% of those examined) of histochemically designated IIB fibers in the llama actually are hybrid IIXB (27%) and IIAXB (13%) fibers. Similarly, a large number (6% of the fiber population) of hybrid Type IIAX fibers were admixed with their corresponding pure phenotypes. These data emphasize the advantages of an immunohistochemical analysis to detect fibers with mixed MHC composition.
The phenotypic differences in fiber size, oxidative capacity and glycolytic capacity seen between llama fiber types were, in general, linked to the MHC content and very similar to those observed in rat muscle (
Results from the present study have demonstrated the presence of MHC IIB isoprotein at a very high percentage (49.2% in semitendinosus muscle) of the fibers in several hindlimb muscles of adult llamas. Interestingly, the percentage of Type II fibers was greater than 90% in all these muscles. In agreement with our data,
The differential distribution of the four MHC isoforms identified in llama skeletal muscle defines four major fiber types containing a single MHC isoform (i.e., I, IIA, IIX, and IIB) and a number of intermediate hybrid fiber populations containing (a) both MHC-I and MHC-IIA (i.e., Type I+IIA MHC fibers or Type "C" fibers), (b) two of the three fast MHCs (i.e., Type IIAX and IIXB fibers), or (c) even the three fast MHC isoforms (i.e., Type IIAXB fibers). The large number of fibers expressing IIB and/or IIX MHC isoforms (93.2% of those examined in semitendinosus muscle) may well be related to the very low level of fitness of animals included in the study, because inactivity induces a transition of MHC isoform in the order IIIA
IIX
IIB (
Some previous studies have revealed fibers that simultaneously coexpress three different MHC isoforms in muscles of elderly subjects (
Finally, another interesting observation of the present study is the spatial distribution of muscle fiber types in llama skeletal muscle (Fig 6). Whereas in most mammalian species muscle fibers belonging to the same motor unit are randomly distributed and are mixed with other muscle units, exhibiting a classical "mosaic" pattern, llama muscle exhibits unique rosette patterns consisting of islets of a single slow fiber, surrounded by concentric circles of fast fibers expressing successively MHC IIA, then IIX, and finally IIB. Accordingly, the vast majority of the fibers expressing MHC IIB are located at the periphery of primary fascicles. Similar spatial distribution patterns have already been described in pig muscles (
This study demonstrates that at least four adult MHC isoforms, one slow-twitch and three fast-twitch, are expressed at the protein level in llama limb muscles. These isoforms have provisionally been referred to as Types I, IIA, IIX, and IIB, based on the homologies of their immunoreactivies with a panel of MAbs, acid and alkaline stabilities of mATPase histochemistry, and their metabolic and size properties with the corresponding isoforms of rat and horse muscles. Nevertheless, further studies are needed to fully characterize these MHC isoforms. The present study confirms that the very fast MHC-IIB isoform is not exclusively expressed in skeletal muscles of small species of mammals but is also present in predominantly fast-contracting muscles of larger mammals such as the llama. It is noteworthy that conventional IIB fibers, as defined by traditional mATPase histochemistry, constitute a heterogeneous population and should be characterized either as pure (IIB) or as hybrid (IIXB and IIAXB) phenotypes. This improvement in accuracy of muscle fiber typing is of practical importance to better understand the involvement of fiber types in locomotor, growth, and meat quality traits of the llama.
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Acknowledgments |
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Supported by the grant U.B.A.C.Y. TV039 and the Spanish D.G.E.S.I.C. PB98-1016.
We thank Dr Stefano Schiaffino (University of Padova, Italy) for his generous gift of monoclonal antibodies. The antibody S5-8H2 is a generous gift from Dr Eric Barrey (INRA, France). We also thank Dr Antonio Serrano (University of Padova, Italy) for critically reading the manuscript, and Dr Linda Linnane for checking the English.
Received for publication January 1, 2001; accepted March 17, 2001.
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