Presence of an unidentified myosin isoform in certain bovine foetal muscles

Meat Science, Vol. 41, No. 3, 315-324, 1995 Elsevier Science Limited Printed in Great Britain 0309-1740/95 s9.50+ .oo 0309-1740(94)00082-4

ELSEVIER

Presence of an Unidentified Myosin Isoform in Certain Bovine Foetal Muscles Brigitte Picard, H&he Gagnihe, Jacques Francoise Pons & Yves Geay INRAZentre

Robelin,

de Clermont-Fd/Theix, Laboratoire Croissance et Metabolismes des Herbivores, Theix, St. Genes-Champanelle, 63122 France

(Received 30 September 1994; revised version received 24 November accepted 28 November 1994)

1994;

ABSTRACT Genetic variation in the establishment of bovine muscle jibre types was studied by comparing muscle dijherentiation at 210 days of foetal life in normal cattle and in ‘culard’ animals, which have muscular hypertrophy. The dtj-erent jibre types were determined by histochemical and immunohistochemical analyses with monoclonal antibodies spectjic to diflerent myosin heavy chain isoforms. The isoforms were separated by electrophoresis and quantified by the ELISA method. Four muscles with direrent contractile and metabolic characteristics were studied : Semitendinosus, Longissimus thoracis, Masseter (slow) and Cutaneus trunci (fast). Muscle jibres recognized by none of the antibodies used were observed in ‘culard’ foetuses in all the muscles studied and also in the Cutaneus trunci of normal animals. Electrophoretic analysis showed no particular myosin isoform in these muscles. It is possible therefore that the fibres contained a mysosin isoform until now unidentified in cattle, with a molecular weight the same as that of known isoforms. This newly observed isoform seems to be spectjic to muscles rich in IIBfibres such as Cutaneus trunci and to the muscles of adult ‘culard’ cattle.

INTRODUCTION Bovine skeletal muscle is of particular interest in metabolic studies since it contains fibre types (I, IIA and IIB) that affect the transformation of muscle into meat. It is the fibre types that govern meat maturation (Valin, 1988). They appear during foetal development and it has been shown in, several species (birds, rodents and mammals) that they arise from different myoblast populations (Miller $ 315

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et al.

Stockdale, 1987; Hoh et al., 1988; Vivarelli el al., 1988; Harris et al., 1989) in a process controlled by complex regulation mechanisms (for a review, see Buckingham, 1985). The different cell populations involved transitorily express different myosin heavy chain (MHC) isoforms. In mammals, 10 different isoforms have been evidenced (Emerson & Bernstein, 1987). The most common, MHCl, MHC2a and MHC2b, are found respectively in the three fibre types I, IIA and IIB, described by Brooke & Kaiser (1970). Two isoforms, referred to as embryonic MHC and neonatal MHC (Rushbrook & Stracher, 1979; Whalen et al., 1981), occur during foetal life. Others have been observed in particular muscles (for a review, see Staron & Pette, 1990), including superfast MHC in the Masseter of dog and cat (D’Albis et al., 1991) an MHC form specific to extraocular muscle (Wieczorek et al., 1985) and MHC 2 x or 2d, in muscles of rat and rabbit (Schiaffino et al., 1985; Schiaffino et al., 1986; Schiaffino et al., 1989; Parry & Zardini, 1990; Bar & Pette, 1988; Termin et al., 1989). In cattle, five myosin isoforms have been identified. They comprise two forms that occur solely during foetal development, foetal MHC and embryonic MHC (Robelin et al., 1993) and the three adult forms, MHCl, MHC2a and MHC2b. The development of these isoforms during foetal growth varies according to the type of muscle (Picard et al., 19946). The aim of this work was to determine to what extent variations in the temporal sequence of the establishment of fibre types are genetically determined. Muscle differentiation in normal Charolais foetuses was compared with that of Charolais foetuses possessing the ‘culard’ gene of muscular hypertrophy. At birth, the muscles of culard animals have more fibres than their normal counterparts (MacKeller, 1968; Dumont, 1982) and the fibres are bigger (Holmes & Ashmore, 1972). In addition, the muscles contain more of the larger type IIB fibres than those of the normal breed (Holmes & Ashmore, 1972). This study shows how the differences in muscle composition between culard and normal animals are expressed during the foetal stage. MATERIAL

AND METHODS

Tissue source

Thirty culard embryos of strain INRA 95 (INRA, Station de Genetique Quantitative et Appliquee, Jouy-en-Josas) were transplanted into Charolais/Salers crossbreed heifers. Sixteen foetuses were obtained from these transplantations and their development compared with that of 15 normal foetuses obtained by artificial insemination. Semitendinosus (ST), Longissimus thoracis (LT), Masseter (Ma, slow in the adult) and Cutaneus trunci (CT, fast in the adult) muscles were sampled in each of the aborted foetuses at 90, 130, 170 and 210 days of gestation. On average, three foetuses per stage were used. In this study, only results obtained at 210 days were considered. Histochemistry

and immunohistochemistry

The contractile type of fibres was determined according to Guth & Samaha (1970) on transverse serial sections 10 pm thick. ATPase activity was visualized after pre-incubation at pH 4.2. The metabolic characteristics of the fibres were

Presence of an unident$ed myosin isoform

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visualized by succinate dehydrogenase activity (SDH), an enzyme of oxidative metabolism, according to the method of Pearse (1968). The myosins present at this stage of foetal life were evidenced by immunofluorescence on serial frozen sections (Pons et al., 1986). The muscle sections were incubated with the monoclonal antibody for 30 min at 37°C. After washings with PBS, the second antibody (rabbit anti-mouse IgG labelled with dichlorotriazinylaminofluorescein, Interchim) diluted in PBS was applied for 30 min at 37°C. After washings with PBS (phosphate buffer), the sections were fixed with mowiol (Calbiochem, 475904). We used five monoclonal antibodies raised against different isoforms of myosin heavy chain. Antibody S (slow) was prepared from an adult human auricle specimen. It recognized isoform MHC 1 (slow). Antibody R (rapid) was specific for fast MHC (MHC 2a and MHC 2b). It was obtained from myosin of an adult rabbit Tibialis. Antibody F (foetal) was obtained from muscle of a 22 week-old bovine fetus. It was specific for a MHC present only during the foetal period, called MHC foetal. Antibody E (embryonic) was obtained from myosin of an adult humain atrium. It was specific for a MHC observed during the foetal period called MHC embryonic. The last antibody, A (alpha cardiac), was raised against human cardiac muscle. The specificity of these antibodies has been described previously (Pons et al., 1986 ; Marini et al., 1990). Their cross-reactions with myosin from foetal and adult cattle have been analyzed (Robelin et al., 1993). Protein preparation

Two hundred milligrams of frozen muscle were ground in 5 ml of buffer solution: 0.5 M NaCl, 20 mM sodium pyrophosphate, 50 mM Tris, 1 mM EDTA, 1mM dithiothreitol. After 10 min at 4°C the sample was centrifuged for 5 min at 2500 g. The supernatant was then mixed with glycerol at a final concentration of 50% (v/v) and stored at -20°C for several months. The protein concentration of the sample was determined according to the method of Bradford (1976), with a preparation of bovine serum albumin at 1 mg/ml (w/v) as standard. These protein preparations were used for electrophoretic analysis. Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on plates of 160*160*1.5 mm by the method of Laemmli (1970). The separation gel was a 5-10% polyacrylamide gradient with a cross-linking of 1.3, and the stacking gel was at 3.5%. To improve resolution, the separation gel also contained a 3&40% glycerol gradient, according to Sugiura & Murakami (1990). The samples stored at -20°C were diluted 50% (v/v) in a solution containing 34% (w/v) SDS, 20% (v/v) 1 M Tris pH 6.8, 17% (v/v) glycerol, 17% (w/v) Pyronin Y and 7.5% (v/v) mercaptoethanol. Diluted samples were incubated for 10 min at room temperature and then at 90°C for 10 min. The gel wells were loaded with 40 pg of protein. Migration was performed at a temperature of 4°C. Temperature control was essential to obtain a clear separation of the different MHC bands. For a uniform penetration of the proteins in the stacking gel, migration was begun at 50 V for 30 min and then continued for 110 V for 18 h. The gel was conventionally stained in a solution of R250 Coomassie Blue.

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Enzyme&ked

immunosorbent

assay (ELISA)

The method used was that of Winkelmann bovine MHC by Picard et al. (1994~).

& Lowey (1983), adapted to study

LT

ST normal

culard

normal

culard

S

R

I=

E

A

Fig. 1. Immunohistochemical Analysis of Transverse Serial Sections of the Muscles Semitendinosus (ST) and Longissimus thoracis (LT) of 210 Day-old Foetuses with Monoclonal Antibodies Directed against Slow MHC (S), Rapid MHC (R), Foetal MHC (F), Embryonic MHC (E) and Alpha Cardiac MHC (A). Cells Not Recognized by these Antibodies are Indicated by an Arrow (scale bars = 50 pm).

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Presence of an unidentiJied myosin isoform

RESULTS Immunohistochemical

analysis

The greatest difference between culard and normal foetuses was in their response to rapid (R) antibody. In ST, LT and Ma muscles in normal animals, the only cells not recognized by R antibody were large cells recognized by slow (S) antibody (Figs 1 and 2). In contrast, there were far more cells in culard animals that were not recognized by R antibody. Ma

CT nomwl

culard

normal

culard

F

Fig. 2.

Immunohistochemical Analysis of Transverse Serial Sections of the Muscles Cutaneus trunci (CT) and Masseter (Ma) of 210 Day-old Foetuses with Monoclonal Antibodies Directed against Slow MHC (S), Rapid MHC (R), Foetal MHC (F), Embryonic MHC (E) and Alpha Cardiac MHC (A). Cells Not Recognized by these Antibodies are Indicated by an Arrow (scale bar = 50 pm).

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Fig. 3.

Histochemical Analysis of the ST Muscle of a Culard Foetus (scale bar = 50 pm). 1 -Visualization of ATPase Activity; 2 -Visualization of SDH (oxidative) Activity; 3 Reaction with Antibody R (rapid); 4 - Reaction with Antibody S (Slow). The Arrows Indicate the Cells in Question.

Two categories of cells were observed: those which, as in normal animals, were recognized by S but not by R (Figs 1 and 2) and others recognized neither by R nor by S, F, E and A (Figs 1 and 2). In these figures, cells recognized by none of the antibodies are arrowed. In the CT muscle of both culard and normal animals, there were cells recognized by none of the antibodies S, R, F, E and X (Fig. 2). In contrast, CT, unlike the other three muscles, exhibited no difference between the two genetic types in the response to R antibody. These differences are summarized in Table 1.

TABL

1

Responses to Antibodies S (slow), R (rapid), ? (foetal), E (embryonic), X (alpha cardiac) of the Cells, in Normal and Culard Muscles. ( ) Indicates that Cells are Not Recognized by the Antibody. Normal

Large cells First generation Second generation

1 2 3

ST, Ma, LT

CT

Culard ST, Ma, LT, CT

(S-E F X)

(S _ E F X)

(S _ E F X)

(S R E F X) (_REFX)

(S R E F X) (_RFFX) (_____)

(S R E F X) (_REFX) (_____)

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Presence of an unidentified rnyosin isoform

ST

LT

1

2

CT

Ma

Fig. 4. Separation of the Different Isoforms of MHC from Normal (N) and Culard (C) Foetuses in the Muscles ST, LT, CT and MA by Electrophoresis (SDS-PAGE). (1) Corresponds to Adult Muscle CT with Rapid Isoforms MHC 2a and 2b; (2) Corresponds to Adult Muscle MA with Slow Isoform MHC 1.

Histochemical

analysis

The contractile and metabolic type of the fibres recognized by none of the antibodies was determined by analysis of serial cut sections made on ST muscle (Fig. 3). These fibres were fast-twitch because their ATPase activity was unstable at acid pH (Fig. 3). Their metabolism could not be clearly determined from SDH activity (Fig. 3), but these cells seemed to be less oxidative than those recognized by antibody R (Fig. 3). Electrophoresis Electrophoretic separation of the myosin isoforms of ST, LT, Ma and CT muscles according to molecular weight, showed no difference between the muscles of culard and normal animals. In all muscles and in both animal types, three bands were observed, although the lightest of these was present in very low concentrations (Fig. 4). ELISA The fast myosin heavy chain isoforms (MHCR, MHC2a and MHC2b) were assayed by the ELISA method with a monoclonal antibody. For ST, LT and Ma muscles MHCR concentrations were greater in normal than in culard foetuses (Fig. 5). In contrast, the MHCR concentrations in CT were comparable in the two genotypes (Fig. 5) but much lower than in the other three muscles.

DISCUSSION The development of foetal muscles in cattle involves two generations of cells (Robelin et al., 1993). The first generation consists of large cells giving rise to slow type I fibres, while the second generation is composed of smaller cells that give rise to fast type IIA and IIB fibres, and to type IIC fibres expressing both fast and slow myosin isoforms. At 210 days of foetal life the primary generation in the different muscles studied is completely differentiated into type I fibres (slow) (Picard et al., 19946). At the same stage, the secondary generation is made up of

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ST Fig. 5.

Quantitation

LT

Ma

CT

Muscle

of the Proportion of MHCR Binding in the Muscles ST, LT, MA, CT from Normal and Culard Foetuses.

two categories : type II fibres (fast), which still express embryonic and foetal MHC, and a few type IIC fibres (simultaneously fast and slow). Overall, this general pattern was observed in this study. However, one important difference emerged : the presence of cells that were recognized by none of the anbibodies used (S, R, F, E and A) in all four muscles (ST, LT, Ma and CT) in culard animals and in CT of normal animals. This finding was confirmed by an ELISA test which showed that CT behaved differently from the other three muscles. It had much lower MHCR concentrations than ST, LT and Ma. In addition, the concentrations were the same in both genotypes whereas in the other muscles concentrations were lower in culard animals. CT muscle, which is entirely fast in the adult, consists chiefly of type IIB fibres (Picard et al., 1994~). The muscles of adult culard animals also contain a high proportion of type IIB fibres (Gutman, 1967; Close, 1967; Ashmore 8~ Robinson, 1969; Holmes & Ashmore, 1972). It can therefore be deduced that the cells recognized by none of the antibodies correspond to fibres classified as IIB but containing a unique myosin isoform. These fibres might be the 11X or IID fibres found in rats and rabbits (Schiaffino et al., 1985, 1989; Bar & Pette, 1988). They contain an isoform, variously called 2 x (Schiaffino et al., 1985, 1986, 1989; Parry & Zardini, 1990) or 2d (Bar & Pette, 1988; Termin et al., 1989). These fibres were previously identified as type IIB but which have since been redesignated as 11X or IID. They have greater oxidative activity than type IIB fibres. They also have greater resistance to fatigue and are larger (Kelly et al., 1990). They are particularly numerous in the rat diaphragm (Pette & Staron, 1990). Published reports show that in adult culard animals type IIB fibres are larger than those in normal cattle (Ashmore & Robinson, 1969; Holmes & Ashmore, 1972). This observation is another argument in favour of the existence of these fibres in cattle, particularly in fast muscles. Electrophoretic results did not reveal any new myosin heavy chain isoform (Fig. 4). It is possible therefore that this particular isoform has the same molecular weight as that of one of the known isoforms. In several species, isoform MHC 2 x has been reported to have a molecular weight somewhere

Presence of an unidentified myosin isoform

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that of MHC 2a and MHC 2b (Schiaffino et al., 1989; Termin et al., 1990). It thus seems possible that these new fibres are of a similar type to IID or 11X and are present at about 210 days of foetal life in the muscles of culard animals and in muscles rich in fast fibres in normal cattle. It remains to be determined whether this type of fibre occurs in adult animals, but evidence suggests it does. Laframboise et al. (1990) showed that isoform MHC 2 x was present before birth in the Diaphragma of rats, whereas isofortn MHC 2b only appeared post-natally. Studies on culard animals have, so far, used only histochemical techniques and not antibodies specific to different myosin isoforms. This could explain why certain fibres classified as IIB in fact contain an isoform different from MHC2b. Termin et al. (1990) showed that the 11X and IID fibres histochemically were in all respects indistiguishable from those classified as IIB by Brooke 8z Kaiser (1970). An alternative hypothesis is that these fibres contain an isoform specific to the foetus. However, this hypothesis appears less plausible. Muscle differentiation takes place later in culard animals than in normal cattle (Quinn et al., 1990; Gerrard & Judge, 1993) and one would therefore expect to observe the isoform at an earlier stage of development in normal animals and in all muscles. between

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