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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.19 n.2 Temuco ago. 2001

http://dx.doi.org/10.4067/S0716-98682001000200008 

FIBER MUSCLE TYPES IN ADULT FEMALE PIGS AS DETERMINED BY
COMBINATING HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL METHODS

TIPOS DE FIBRAS MUSCULARES EN CERDAS DETERMINADOS POR COMBINACIÓN DE TÉCNICAS HISTOQUÍMICAS E INMUNOHISTOQUÍMICAS

*Guillermo H. Graziotti; *Gustavo Delhon; *Clara M. Ríos & **José-Luis L. Rivero.

* Department of Physiology and Basic Sciences, Faculty of Veterinary Sciences, University of Buenos Aires, Buenos Aires, Argentina.
** Department of Comparative Anatomy and Pathological Anatomy (Muscle Biology Laboratory), University of Cordoba, Cordoba, Spain.

SUMMARY: Swine skeletal muscle fibers have traditionally been classified as types I, IIA, IIB and their hybrid forms by determining the activity of myosin ATPase (mATPase). More recently, molecular, electrophoretic and immunohistochemical approaches have allowed the identification of IIx isoform-containing fibers.

In this study, myosin isoforms present in different types of muscle fibers were identified by combining histochemical and immunohistochemical methods. Samples from the M. longissimus dorsi were collected from primiparus pigs (mean body weight, 150 Kg) 45 minutes after slaughtering and subsequently frozen in dry ice-cooled acetone. Serial cross sections were processed for mATPase and NADH-TR activities and for immunohistochemistry. Five histochemical fiber groups were identified: type I (dark), type IIA (light) and three intermediate types. A panel of monoclonal antibodies identified the slow ßI, IIa, IIx and IIb isoforms present in types I, IIA, IIX and IIB fibers and the hybrid group IIAX.

KEY WORDS: 1. Pig; 2. Muscular fibers; 3. Myosin; 4. Isoforms; 5. Histochemical; 6. Immunohistochemical.

INTRODUCTION

Four fiber types (I, IIA, IIX/IID, and IIB) have been identified in adult small mammal limb and diaphragm muscles by using histochemical detection of myosin adenosin triphosphatase (mATPase), (Termin et al., 1989; Gorza, 1990; Lind & Kernell, 1991; Hämäläinen & Pette, 1993; for techniques see Brooke & Kaiser, 1970, and Guth & Samaha 1970). These fiber types contain  the I or ß slow, IIa, IIx / IId, or IIb myosin heavy chain (MHC) isoforms, respectively, as determined by combined immunohisto-chemistry using specific monoclonal antibodies (Mabs) against MHC isoforms, and/or electrophoresis and mATPase histochemistry (Schiaffino et al., 1989; GorzaTalmadge and Roy, 1993; Sant'Ana Pereira et al., 1995). Termin et al. had suggested that MHC isoforms IIx and IId are identical.

            The presence of MHC isoforms I, IIa, and IIx in the diaphragm and limb muscles has been  documented in humans (Smerdù et al., 1994), horses (Rivero et al. 1996), cats (Talmadge et al., 1996), and bovines (Tanabe et al., 1998). The isoforms I, IIa, and IIb were described for sheep skeletal muscle (Maier et al., 1992). Electrophoretic analysis of swine skeletal muscle identified three fast MHC isoforms: II, IIx, and IIb (Lefaucheur et al., 1998; Bee et al., 1999).

When fiber metabolic properties and mATPase activity are combined, muscle fibers are classified as slow oxidative type I (SO), fast oxidative glycolytic type IIA ( FOG) and fast glycolytic type IIB (FG) (Delp & Duan, 1996). Ruusunen and Poulanne (1997), classified pig skeletal muscle as light or dark according to its fiber composition; in light muscle FG fibers are dominant whereas in dark muscle, FOG and SO fibers predominate.

Given the relationship between muscle fiber composition and meat quality, we examined the MHC isoform profile in swine myofibrils. To avoid any variability due to differences in the production system, animals from an experimental unit composed of primiparus pigs were used. We identified four MHC isoforms expressed as pure fiber types I, IIA, IIX, and IIB, and the hybrid type IIAX.

MATERIAL AND METHOD

Animals: Fifteen Duroc X Landrace 330 day old primiparus pigs were used for these studies. The animals were fed ad libitum with standard commercial meals, during lactation but restricted during pregnancy. The animals were slaughtered 24 days post-partum at an average body weight of 150 Kg.

Samples: Forty five minutes after slaughtering, samples from the M. longissimus dorsi at the level of the last rib were collected. Muscle samples were immediately frozen in dry ice-cooled acetone and stored at _80ºC. Ten µm thick serial sections were obtained in a Reichert-Jung 1800 cryostate and mounted on 1% poly-L-lysine-coated slides.

Histochemistry: Cross sections were preincubated in acid solution (pH 4.6) for two minutes at 21-23 ºC following a modification of the method originally described by Brooke and Kaiser, (Nwoye et al., 1982). To asses the metabolic profile of fibers, additional sections were stained for nicotinamide adenine tetrazolium reductase (NADH-TR) reaction (Dubowitz, 1985). As controls for the mATPase reaction, samples from rat vastous lateralis muscle were simultaneously processed.

Immunocytochemistry: Frozen sections were processed for indirect immunohistochemistry as described by Rivero et al. (1996). Monoclonal antibodies (Mabs) directed to the various myosin heavy chain isoforms (see Table I for specificities) were used as primary antibodies. Common fields from several sections treated for the different techniques were microphotographed with a M35W camera mounted on a Zeiss 9901 microscope.

Table. I. Specificity of monoclonal antibodies (Mabs) against adult rat skeletal myosin heavy chain isoforms used in the study.

Mabs
I
IIa
IIx
IIb
Reference
A4.1519 1/10
-
+
-
+-
Hughes & Blau,1992
A.474 1/10
-
+
+-
-
Hughes & Blau, 1992
BF- 35 1/1000
+
+
-
+
Schiaffino et al., 1989
S5-8H2 1/100
+
-
+
+
Barrey et al., 1998

RESULTS

The fiber types identified in this study in rat and pig skeletal muscle are detailed in Table II

Tabla II: Histochemical and immunohistochemical characterization of skeletal muscle fiber types in rats and pigs according to the myosin heavy chain isoforms they express.

DISCUSSION

Five fiber types were identified in pig skeletal muscle based on histochemical and immunohistochemical methods. Since we used rat muscle as a control, the immunohisto-chemical phenotype was determined assuming the high structural homology between MHC isoforms from different mammals and the fact that most divergences between isoforms are found within the same species (Schiaffino and Reggiani, 1996).

In Fig. 2, fiber 3 was classified as containing the IIx MHC isoform alone. Its low to moderate staining for mATPase after acid preincubation is in agreement with a pattern of increasing stability (IIa>IIx>IIb>I) of MHC isoforms as described by Gorza for mouse muscle, although it is known that the mATPase phenotype is not exclusively dependent on MHC content (Hämäläinen and Pette, Linnane et al., 1999, Rivero et al., 1999). In our assays, rat IIX muscle fibers were on the same stability level at mATPase after acid preincubation (Fiber 3, Fig 1). As in rats (Table I ), pig MHC IIx isoform was labelled by MAb S5-8H2, but not by MAb BF-35 which do not recognize the IIx isoform. Note the  reaction with MAb S5-8H2 in rat fiber muscle (Plate F, Fig 1). This reactivity was not described in previous works. The IIX immunohisto-chemical phenotype in rat and pig muscle fibers can be distinguished by their reactivities against Mabs A4.74 and A4.1519 (Table II). These MAbs are specific for the rat MHC IIa isoform  (Hughes & Blau, 1992); it is likely that common epitopes exist in rat IIa and pig IIx MHC isoforms, although Lefaucheur et al. have documented low homology at the nucleotide level in the 3' untranslated region, and the fifth aminoacid from the stop codon, where pig IIx changes valine by isoleucine, where   rat IIa MHC isoform is present.

We found similar moderate metabolic activities in rat (Gorza) and pig IIX fibers. In pig muscle, Fernandez et al., (1995) divided the IIB type group according to metabolic reaction.

The immunohistochemical  phenotype of pig IIB fibers (Fiber 4, fig 2) differed slightly from rat fibers despite of the high molecular homology of IIb isoforms between these species (Lefaucheur et al.). In this work pig fibers remained unreactive when tested against Mab BF-35. Nevertheless, mATPase and NADH-TR reactivities were similar for rat and pig muscle fibers when Yorkshire pigs about 100 Kg in weight were tested (Lefaucheur et al.,).

These authors described the IIBX hybrid fiber type instead of the IIAX type found in this work (Fiber 5, Fig. 2). We based our conclusions on the moderate immunohisto-chemical staining with MAb BF-35, and the moderate mATPase and NADH-TR reactivities which are dependent on MHC content (Rivero et al., 1999).

In conclusion, five fiber types, I, IIA, IIX, IIB and IIAX were identified in the pig population examined in this work. These results differ from those of Lefaucheur et al., in regard to the MHC isoform distribution in hybrid fibers. This difference could be related to the animal population examined, which is different in age, weight and herd control. Interchange between rat MHC isoforms following the pattern I´IIa´IIx´IIb, has been described, (Gorza). In pig muscle, Ruusunen (1994), found a change in fiber composition due to fibrillar interconversion which varied according to the age and weight of the animal. Similarly, Harrison et al. (1996) showed that MHC differentiation was influenced by the energy content of the diet and the temperature.

Fig. 1A and B. Rat vastous lateralis stained for mATPase activity after preincubation in acid  (pH 4.6, A) or alkaline (pH: 10.5, B) solution.
C-F. Rat muscle fiber immunohistochemistry with Mab´s against specific MHC isoforms (see Table I for Mab specificities).
C: Mab A4.1519 1/10;
D: Mab A4.74 1/10;
E: Mab BF-35 1/1000;
F: Mab S5-8H2 1/100.
G: NADH-TR activity in rat muscle. Fiber 1: Type I; Fiber 2: Type IIA; Fiber 3: Type IIX; Fiber 4: Type IIB. Magnification: X100.

Fig. 2. A-   Pig M. longissimus dorsi stained for mATPase activity after preincubation in acid  (pH 4.6) solution
B-E: Pig muscle fiber immunohistochemistry with Mab´s against specific MHC isoforms (see Table I for Mab specificities).
B: Mab A4.1519 1/10;
C: Mab A4.74 1/10;
D: Mab BF-35 1/1000;
E: Mab S5-8H2 1/100.
F: NADH-TR activity in pig muscle.
Fiber 1: Type I; Fiber 2: Type IIA; Fiber 3: Type IIX; Fiber 4: Type IIB; Fiber 5: type IIAX. Magnification: X 100.

ACKNOWLEDGMENTS

The Mabs A 4.74 and A 4.1519 were obtained from the Developmental Studies Hybridoma Bank (University of Iowa). Mabs BF-35 and S5-8H2 were generous gifts from Dr S. Schiaffino (University of Padova, Italy) and Dr E. Barrey (INRA, France), respectively.

RESUMEN: Las fibras musculares esqueléticas del cerdo han sido tradicionalmente  tipificadas como I, IIA y IIB, más sus formas híbridas, determinando la actividad de la enzima miosina ATPasa miofibrilar. Recientemente, la utilización de anticuerpos monoclonales contra isoformas de cadena pesada de miosina, electroforesis y estudios moleculares, han documentado la existencia, además, de la isoforma IIx. El objetivo de este estudio fue determinar la existencia de las isoformas de miosina, presentes en los distintos tipos fibrilares, utilizando técnicas histoquímicas e inmunohistoquímicas  combinadas, dentro de una unidad experimental compuesta por muestras musculares del M. longíssimo del dorso, en cerdas de 150 kg promedio. Las muestras  fueron obtenidas 45 minutos después de la faena, mediante escisión y congeladas en acetona enfriada con hielo seco. Cortes seriados de 10µm de espesor fueron tratados por la técnicas de mATPasa modificada por Nwoye (1982), a pH 4.6 , NADH-TR  e inmunohistoquímica.

Histoquímicamente fueron identificados cinco grupos de fibras: tipo I oscuras, IIA claras, y tres tipos intermedios. Los ensayos inmunohistoquímicos permitieron identificar las isoformas ß lenta I, IIa, IIx y IIb presentes en fibras de tipo I, IIA, IIX, IIB y un grupo híbrido IIAX.

PALABRAS CLAVE: 1. Cerdo; 2. Fibras musculares; 3. Miosina; 4. Isoformas; 5. Histoquímica; 6. Inmunohistoquímica.

Supported by Grant U.B.A.C.Y.T  IG 001

Correspondence to:
Guillermo H. Graziotti
Area of Anatomy
Physiology and Basic Sciences Department
Faculty of Veterinary Sciences
Universidad de Buenos Aires
Chorroarín 280
Capital 1427
Buenos Aires
ARGENTINA

Email: ggrazio@fvet.uba.ar

Recibido : 23-04-2001
Aceptado: 30-05-2001

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