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Electrophoretic separation of bovine muscle myosin heavy chain isoforms
Meat Science 53 (1999) 1±7
www.elsevier.com/locate/meatsci
Electrophoretic separation of bovine muscle myosin heavy chain isoforms B. Picard*, C. Barboiron, M.P. Duris, H. GagnieÂre, C. Jurie, Y. Geay Laboratoire Croissance et MeÂtabolismes des Herbivores, INRA, Theix, 63 122 Saint-GeneÁs Champanelle, France Received 29 September 1998; received in revised form 25 January 1999; accepted 29 January 1999
Abstract This study concerns the de®nition of the optimum conditions for separation of adult and developmental myosin heavy chain (MHC) isoforms in bovine muscle. The various techniques published do not result in good separation of the MHC in this species. The trials carried out concerned the concentration of acrylamide and N, N0 -methylene-bis-acrylamide, and more particularly the concentration of Tris in the separating gel. The ®nding was that analysis of adult isoforms and developmental isoforms require dierent conditions. A acrylamide gradient of 3.5±10% with 200 mM Tris pH 8.8 gives good resolution for adult isoforms. Under these conditions 3 fast adult isoforms are revealed. However, study of MHC isoforms throughout foetal life in bovines is complex, and requires the combined use of more than one gel (gradient 3.5±10% at 200 mM Tris and gradient 3.5±10% at 250 mM Tris). # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Muscle; Myosin; Electrophoresis; Bovine
1. Introduction The study of bovine muscle ®bres is of primordial interest because they play a fundamental role in modeling meat quality and tenderness. Post-mortem meat tenderization processes transforming muscle into meat involve enzymatic and physicochemical mechanisms, which, in intensity and amplitude, are largely governed by the proportion of the distinct ®bre types (Ouali, 1990). The variable contraction velocity of muscles depends, at least in part, on the polymorphism of myosin heavy chain (MHC). To date, a total of 10 distinct MHC isoforms encoded by a multigene family, have been elucidated in mammalian skeletal muscle (for review: Staron & Pette, 1990). In adult the most abundant are MHC I, MHC IIa, MHC IIb and MHC IIx expressed respectively in ®bres identi®ed as types I, IIA, IIB and IIX. This last type has not been evidenced to date in bovine muscle (Picard, Duris, & Jurie, 1998b). Other isoforms are present during foetal life (MHC embryonic, neonatal or foetal) or in special muscles (MHC Itonic, MHC alpha cardiac, MHC eom, MHC IIM). * Corresponding author. Tel.: +33-04-73-62-4056; fax: +33-04-7362-4622. E-mail address: [email protected] (B. Picard)
The technique of Laemmli (1970) on polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) has been used for the determination of muscle MHC phenotype since 1975. Burridge and Bray (1975) were the ®rst to report a dierence in the electrophoretic mobilities of MHC from skeletal and smooth muscles. Since then the technique has constantly been improved over the years. Fast and slow MHC isoforms were ®rst separated from avian and mammalian muscle (Carraro & Catani, 1983; Rushbrook & Stracher, 1979). Then, Danieli-Betto, Zerbato, and Betto (1986) were able to resolve the IIa and IIb MHC in rat skeletal muscle. Several laboratories separated another mammalian MHC isoform called IId or IIx (BaÈr & Pette, 1988; Sugiua & Murakami, 1990). Dierent authors in several species found that the addition of glycerol has a sharpening eect on the electrophoretic bands (Carraro & Catani; Danieli-Betto et al.; Laframboise et al., 1990; Rushbrook & Stracher). Also, Sugiura and Murakami showed that the temperature of migration was of great importance for the quality of MHC resolution. The addition of 2-mercaptoethanol has been shown to increase the resolution of MHC (Agbulut, Zhenlin, Mouly, & Butler-Browne, 1996; Blough, Rennie, Zhang, & Peter, 1996; Fritz, Swartz, & Greaser, 1989). The separation of MHC IIa-MHC IIb or MHC IIa-MHC
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B. Picard et al. / Meat Science 53 (1999) 1±7
IIx has been improved by the development of gradient gels (BaÈr & Pette, 1988; Caizzo, Herrich, & Baldwin, 1991; Danieli-Betto et al., 1986; Perrie & Brumford, 1984). More recently a new protocol was developed by Talmadge and Roy (1993) to separate the skeletal muscle MHC of adult rats. It was adapted for use with different species (rabbit: Janmot & D'Albis, 1994; horse: Rivero, Talmadge, & Edgerton, 1997). The dierent conditions of electrophoretic separation vary a lot between species. To date no technique has been published for the resolution of bovine MHC. Only Young and Davey (1981) have studied bovine myosin but with peptide mapping. The dierent methods quoted above do not allow the distinction between MHC IIa and MHC IIb. So, the aim of this work is to determine the best conditions of the separation of both developmental and adult MHC isoforms in bovine skeletal muscle. 2. Materials and methods 2.1. Preparation of muscle samples Adult muscles with dierent composition of ®bre types: Semitendinousus (ST), Longissimus thoracis (LC), Biceps femoris (BF), Triceps Brachii (TB), Masseter (M), Cutaneus trunci (CT) were taken after slaughter of Charolais bulls, 15 months old. Semitendinosus muscle of Charolais and double-muscled strain INRA 95 foetuses were taken after the slaughter of the mother at gestational ages of 110, 230, 260 days. Samples of muscles were prepared and frozen in liquid nitrogen as described by Robelin et al. (1991). The muscles were extracted as previously described (Picard, GagnieÁre, Robelin, Pons, & Geay, 1995). The protein concentrations of the samples were determined according to the method of Bradford (1976). 2.2. Conditions of electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on plates of 1601601.5 mm in Hoer system, by the method of Laemmli (1970) (Table 1). The stacking gel was at 3.5% of polyacrylamide with a cross-link of 1.3%. The separating gel was a 3.5±10% polyacrylamide gradient with a cross-link of 1.3% and a concentration of 200 mM of Tris pH 8.8 for adult muscles or 250 mM for foetal muscles. To improve resolution, the separating gel also contained a 30±40% glycerol gradient according to Suguira and Murakami (1990). The samples were diluted 50% in a solution containing 1.4% (w/v) SDS, 10% (v/v) 1 mM Tris pH 6.8, 17% (v/v) glycerol, 0.6% (w/v) Pyronin Y and 7.5% mercaptoethanol. They were incubated for 10 min at room temperature and then at 90 C for 10 min. Wells were
Table 1 Preparation of the polyacrylamide gels
Acrylamide 40% N-N0 -methylene bisacrylamide 2% Tris 200 mM pH 8.8 H2O Glycerol SDS 10% Temed Ammonium persulfate 1%
3.5%
10%
1.10 ml 0.29 ml 1.3 ml 5.5 ml 3.8 ml 200 ml 10 ml 500 ml
3.10 ml 0.83 ml 1.3 ml 1.7 ml 5.1 ml 200 ml 10 ml 500 ml
probed with 3 mg of protein. A standard Laemmli (1970) running buer was used. The electrophoretic run was carried out at a constant voltage of 50 V for 30 min, followed by 110 V for 18 h at a temperature of 4 C. After migration the gels were stained in a solution of R 250 Coomassie Blue. The proteins were ®xed in trichloracetic acid 15% (w/v) for 30 min at room temperature. Then, the gels were incubated in the colouration solution containing: propanol 2 25% (v/v), acetic acid 90% (vlv), Coomassic Blue R250 0.2% (w/v), for 45 min at 68 C. Gels were rinsed and decolourated for 1 h 30 min in a solution of: ethanol 95 35% (v/v), acetic acid 90% (v/v) at room temperature, under agitation. The bands were identi®ed as MHC IIb, IIa, I (Fig. 2) on the basis of previously determined migration pattern (Picard, Robelin, Pons, & Geay, 1994; Young & Davey, 1981). 3. Results The preliminary trials on adult muscles, in 7% polacrylamide gels (data not shown) or according to the technique of Talmadge and Roy (1993) did not permit the resolution of fast MHC (IIa and IIb). Talmadge and Roy conditions gave a good separation of rat MHC, taken as control, but MHC of dierent bovine muscles were not resolved (Fig. 1). So the use of gradient gel seems to be necessary for the study of bovine MHC isoforms. Dierent concentrations of polyacrylamide gradients were tested: 5±8%, 3.5±8%, 3.5±9%, 3.5±10%. The gradient 3.5±10% (with 300 mM Tris pH 8.8) gave the best separation of the three MHC (I, IIa and IIb). Then dierent cross-link values were analysed to improve the resolution. Fig. 2 shows that a cross-link of 1.3% gives the best results. Among the various factors governing correct resolution of MHC, the Tris concentration in the separating gel is extremely important. The Tris enables the pH to be maintained constant in the gel, it provides ions which aect the mobility of the proteins and thus, the
B. Picard et al. / Meat Science 53 (1999) 1±7
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Fig. 1. Separation of bovine MHC according to Talmadge and Roy (1993). 1. rat gastrocnemius (5 mg of proteins); 2±8 are bovine muscles; 2. Sternomendibularis (10 and 5 mg), 3. Splenius (10 and 5 mg), 4. Soleus (5 mg), 5. Diaphragma (5 mg), 6. Semitendinosus (10 and 5 mg), 7. Longissimus thoracis (10 and 5 mg), 8. Cutaneus trunci (10 and 5 mg).
Fig. 2. In¯uence of the cross-link (C) on the separation of bovine MHC. a. C=0.9%, b. C=1.3%, c. C=3%, d. C=4%. 1. Semitendinosus, 2. Cutaneus trunci, 3. Masseter, 4. Longissimus thoracis.
clarity of the bands obtained. So to improve separation of the MHC of bovine muscle even further, the in¯uence of various concentrations of Tris (375, 300, 250, 200, 150, 125, 100 mM) was tested on separation of MHC present during foetal life on the one hand, and on the other in adult muscle. In the various adult muscles, only two bands could be observed at the concentration of 375 mM (Fig. 3), MHC I and a very concentrated band of higher molecular weight. When the Tris concentration was reduced, the distance separating the two bands increased (300 and 250 mM). By reducing it still further (200, 165, 150 mM) the fast isoforms were separated. However, if the concentration is too low (125, 100 mM) separation is mediocre (data not shown). So all these trials show that the optimum separation of MHC isoforms in adult bovine muscle is obtained with an acrylamide concentration gradient of 3.5±10% with a cross-link of 1.3% and a concentration of 200 mM Tris pH 8.8 in the separating gel. Under these conditions 3 bands can be distinguished in the fast MHC (Figs. 3 and 4). These three bands are present in the four mixed muscles analysed: ST, LT, BF and TB in varying proportions. In ST, the higher molecular weight band is the most concentrated, with the two others having a similar concentration. In LT, the lower molecular weight fast band is the most concentrated, the middle band being only faintly present.
In BF the two higher bands are the most concentrated. Finally in TB the middle band has the lowest concentration (Fig. 4). The distance between these three bands was low, and more particularly between the heaviest two, so densitometric analysis is not always feasible. Fig. 3 shows that the best separation of all MHC present in foetal muscle was obtained at a concentration of 250 mM Tris in the separating gel. For foetus of 110 days (Fig. 3) with 200 mM Tris only two bands were observed, one with the same mobility as MHC I, the other migrating to the level of MHC IIa identi®ed as MHC emb according to Picard et al. (1994). With 250 mM Tris (Fig. 3) three bands were observed: MHC I and two MHC of higher PM. In double-muscled foetuses yet a fourth band was revealed (Fig. 3). By increasing the concentration of Tris, a better separation of higher molecular weight isoforms was obtained. When the concentration was increased yet again to 300 mM, separation of these bands was improved even further. On the other hand, at 375 mM there was less distance between MHC I and the higher bands so the resolution was less good. In older foetuses aged 230 and 260 days, three bands were observed with the 200 mM concentration (Fig. 3). When the concentration was increased, the separation between the two high molecular weight bands was reduced (Fig. 3). At 375 mM they were no longer separated at all.
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Fig. 3. Electrophoresis of muscle bovine MHC from foetal and adult stages: in¯uence of Tris concentration. 1. adult Masseter; 2±8 correspond to Semitendinosus of foetuses: 2. 110 day-old, 3. 230 day-old, 4. 260 day-old; samples 5±8 are double-muscled foetuses: 5 and 6. 110 day-old, 7. 230 dayold, 8. 260 day-old; a±d are adult muscles: a. Semitendinosus, b. Triceps brachii, c. Longissimus thoracis, d. Biceps femoris.
4. Discussion
4.1. Adult MHC
These various trials clearly show that the concentration of Tris in the separating gel is a fundamental limiting criteria for good resolution of myosin isoforms in bovines. It would appear that the optimum concentration for separation of adult isoforms is not the same as that used to separate developmental isoforms.
The 200 mM concentration gives a good resolution for the adult isoforms (MHC I, IIa and IIb). Furthermore a third band is even revealed in the fast isoforms in the various muscles studied, which is a very original result. However, the distance between the three bands is not yet satisfactory enough to allow densitometric analysis.
B. Picard et al. / Meat Science 53 (1999) 1±7
Fig. 4. Fast MHC of adult bovine muscles. 1. Masseter, 2. Semitendinosus, 3. Triceps brachii, 4. Longissimus thoracis, 5. Biceps femoris.
Addition of 2-mercaptoethanol in the upper buer as suggested by Blough et al. (1996), could perhaps further improve the separation obtained. However, it is obvious that the relative proportions of these three isoforms, for a given animal, is very dierent from one muscle to another. This is the ®rst work which has made it possible to show three fast isoforms in bovine muscle. Until now only isoforms MHC I, IIa and IIb had been described in adult bovine muscle (Totland & Kryvi, 1991; Jurie, Robelin, Picard, & Geay, 1995). The third fast MHC revealed must obviously correspond to MHC IIx. This isoform has now been identi®ed in a large number of species (rat, mouse, rabbit, pig, human) (Gorza, 1990; Schiano & Reggiani, 1994), so it seems logical that it would be present in all mammals, in greater or lesser proportions. According to HamalaõÈnen and Pette (1993) the muscles of large mammals, whose fast movements are much smaller than in small species such as the rat and the mouse, would contain little if any MHC IIb. So it can be assumed that the standard histochemistry techniques used in bovines classify ®bres IIX and IIB both as IIB ®bres, without making any distinction between the two. Immunohistochemistry trials have been carried out with various antibodies (Picard, Duris, & Jurie, 1998a) to check this hypothesis. The various results obtained plead in its favour. Antibody BF F3 (Schiano et al., 1989) which recognises MHC IIb in the rat and the mouse recognises no isoform at all in bovine ST muscle. Similarly antibody BF 35 which recognises all MHC except MHC IIx in the rat and mouse, recognises all MHC except IIb in bovines (Duris, Picard, & Geay, 1998). Furthermore antibody S5 8H2 which recognises both MHC I and IIb in bovine muscle (Picard et al., 1998a) identi®es MHC I, IIb and IIx in pig muscle. Similarly a antibody speci®c to MHC IIa and IIx in the pig (Lefaucheur & Ecolan, 1998) recognises all the fast MHC in bovines. All these results would seem to indicate that the MHC IIb and IIx in bovine muscle have very similar structures and are coidenti®ed by the anti IIb or IIx antibodies used. This would explain why they are very dicult to separate
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according to their molecular weight. The revelation of a third band calls the nomenclature used into question, considering that the highest molecular weight band corresponds to MHC IIb and the other to MHC IIa. It now remains to identify these three fast MHC more precisely in bovine muscle. This identi®cation could have some implications in the study of meat quality, particularly, tenderness. It is well established that the composition of muscle ®bres condition the speed of ageing of meat (Ouali, 1990). However, muscles with a similar proportion of the dierent types of ®bres present dierent speed of ageing. So, these dierences could be explained in part, by the proportions of the three fast MHC. 4.2. Developmental MHC Electrophoretic study of MHC isoforms present during foetal life is even more complex. The Tris concentration of 250 mM alone made it possible to observe all the bands present at the various ages. At 110 days, the 200 mM concentration revealed only one band at the high molecular weight level, whereas two bands are visible with 250 mM. If the Tris concentration is increased still further (375 mM) the separation between the MHC is reduced. It might be thought that these two high molecular weight isoforms correspond to MHC IIa and IIb. But under these conditions there is no separation between these two isoforms. In addition the results we obtained previously with Western-blot and ELISA assays (Picard et al, 1994) showed that the MHC IIa and IIb fast adult isoforms only appear from 210 days foetal life on. These two isoforms have been identi®ed as embryonic MHC (higher molecular weight) and foetal MHC (medium molecular weight) (Picard et al., 1993; Robelin et al., 1993). Study of 230 and 260 day foetuses with 200 mM Tris revealed two high molecular weight bands, migrating to the same level as the adult isoforms MHC IIa and MHC IIb. Nevertheless analysis of the gel at 250 mM Tris always revealed two high molecular weight bands at 230 and 260 days, whereas under these conditions the fast adult isoforms were not separated. So the combined observation of these two gels (200 and 250 mM Tris) prompt us to conclude that at least three high molecular weight bands are present in 230 and 260 days foetuses. This is con®rmed by our earlier results with Western-blot and ELISA which show that at this stage the slow, fast adult (IIa and IIb) and foetal isoforms are present (Picard et al., 1994). The proportion of foetal isoform drops considerably during this period in parallel with a strong increase in the proportion of fast adult isoforms. Complete analysis of the MHC isoforms present during the ®nal third of foetal life in bovines therefore requires the combined use of two complementary gels, one at 200 mM and the other at 250 mM Tris. But at the beginning of life, for as long as the
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fast adult isoforms are not present, the 250 mM concentration is sucient to reveal the various isoforms present. 5. Conclusion The whole of this study shows how important it is to have complete mastery over the electrophoresis conditions and that interpretation of the results obtained must be very prudent. It is necessary to use other techniques (Western blot, immunohistology or ELISA) to con®rm identi®cation of the bands observed. Nevertheless, it would appear that separation of all adult isoforms in bovine muscles is obtained with an acrylamide gradient of 3.5±10% containing 200 mM Tris pH 8.8. These conditions make separation of three fast isoforms possible, which until now had never been obtained in bovine muscle. It now remains to improve the electrophoresis conditions still further to be capable of quantifying these three bands by densitometry. Each of them also needs to be identi®ed speci®cally. However, separation of developmental isoforms proves to be far more complex. At the beginning of foetal life, a 3.5± 10% acrylamide gel containing 250 mM Tris pH 8.8 makes it possible to reveal all the isoforms present. However, from the time when fast adult isoforms appear onwards, it becomes necessary to use two types of gel to reveal all the isoforms present. Given that these conditions are very complex, we need to ®nd a solution to reveal all these isoforms with one and the same gel. References Agbulut, O., Zhenlin, L., Mouly, V., & Butler-Browne, G. S. (1996). Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms. Biol. Cell., 88, 131±135. BaÈr, A., & Pette, D. (1988). Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett., 235, 153±155. Blough, E. R., Rennie, E. R., Zhang, F., & Peter, J. R. (1996). Enhanced electrophoretic separation and resolution of myosin heavy chains in mammalian and avian skeletal muscles. Anal. Biochem., 233, 31±35. Bradford, M. M. (1976). A fast and sensitive method for the quanti®cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72, 248±254. Burridge, K., & Bray, D. (1975). Puri®cation and structural analysis of myosins from brain and other non-muscle tissues. J. Mol. Biol., 99, 1±14. Caizzo, V. L., Herrick, R. E., & Baldwin, K. (1991). In¯uence of hyperthyroõÈdism on maximal shortening velocity and myosin isoform distribution in skeletal muscles. Am. J. Physiol., 261, 285±295. Carraro, U., & Catani, C. (1983). A sensitive SDS-PAGE method separating myosin heavy chain isoforms of rat skeletal muscles reveals the heterogeneous nature of embryonic myosin. Biochem. Biophys. Res. Commun., 116, 793±802. Danieli-Betto, D., Zerbato, E., & Betto, R. (1986). Type 1, 2A and 2B myosin heavy chain electrophoretic analysis of rat muscle ®bers. Biochem. Biophys. Res. Commun., 116, 981±987.
Duris, M. P., Picard, B., & Geay, Y. (1998). Characterisation of the speci®city of dierent anti myosin heavy chain antibodies in bovine muscle. Meat Science, submitted. Fritz, J. D., Swartz, D. R., & Greaser, M. L. (1989). Factors aecting polyacrylamide gel electrophoresis and electroblotting of high molecular weight myo®brillar proteins. Anal. Biochem., 108, 205± 210. Gorza, L. (1990). Identi®cation of a novel type 2 ®ber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J. Histochem. Cytochem., 38, 257±265. HamalaõÈ nen, N., & Pette, D. (1993). The histochemical pro®les of fast ®ber types IIB, IID and IIA in skeletal muscle of mouse, rat and rabbit. J. Histochem. Cytochem., 41, 733±743. Janmot, C., & D'Albis, A. (1994). Electrophoretic separation of developmental and adult rabbit skeletal muscle myosin heavy chain isoforms: example of application to muscle denervation study. FEBS Lett., 353, 13±15. Jurie, C., Robelin, L., Picard, B., & Geay, Y. (1995). Postnatal changes in the biological characteristics of semitendinosus muscle in male limousin cattle. Meat Science, 41, 125±135. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227, 680± 685. Laframboise, W. A., Daood, M. J., Guthrie, R. D., Moretti, P., Schiano, S., & Ontell, M. (1990). Electrophoretic separation and immunological identi®cation of type 2X myosin heavy chain in rat skeletal muscle. Biochem. Biophys. Acta, 1035, 109±112. Lefaucheur, L., & Ecolan, P. (1998). Composition en chaõÃnes lourdes de myosine des ®bres musculaires de type II chez le porc. INRA Prod. Anim., 11, 152±154. Ouali, A. (1990). Meat tenderization: possible causes and mechanisms. A review. J. Muscle Foods, 1, 129±165. Perrie, W. T., & Brumford, S. J. (1984). Correlation of myosin heavy chain with ATPase staining of skeletal and cardiac muscle ®bers. Biochem. Soc. Trans., 12, 825±826. Picard, B., GagnieÁre, H., Robelin, J., Pons, F., & Geay, Y. (1995). Presence of an unidenti®ed myosin isoform in certain bovine foetal muscles. Meal Science, 41, 315±324. Picard, B., Robelin, J., Pons, F., & Geay, Y. (1994). Comparison of the foetal development of ®bre types in four bovine muscles. J. Muscle Res. Cell Motil., 15, 473±486. Picard, B., Duris, M. P., & Jurie, C. (1998a). CaracteÂrisation des chaõÃnes lourdes de myosine dans le muscle bovin. INRA Prod. Anim., 11, 150±152. Picard, B., Duris, M. P., & Jurie, C. (1998b). Classi®cation of bovine muscle ®bres by dierent histochemical techniques. Histochem. J., 30, 473±479. Rivero, J. L. L., Talmadge, R. J., & Edgerton, V. R. (1997). A sensitive electrophoretic method for the quanti®cation of myosin heavy chain isoforms in horse skeletal muscle: histochemical and immunocytochemical veri®cation. Electrophoresis, 18, 1967±1972. Robelin, J., Lacourt, A., BeÂchet, D., Ferrara, M., Briand, Y., & Geay, Y. (1991). Muscle dierentiation in the bovine fetus: a histological and histochemical approach. Growth Dev. Aging, 55, 151±160. Robelin, J., Picard, B., Listrat, A., Jurie, C., Barboiron, C., Pons, F., & Geay, Y. (1993). Myosin transitions in semitendinosus muscle during fetal development of cattle: immunocytochemical and electrophoretic analyses. Repro. Nutr. Dev., 33, 25±41. Rushbrook, J. I., & Stracher, A. (1979). Comparison of adult, embryonic, and dystrophic myosin heavy chain from chicken muscle by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and peptide mapping. Proc. Natl. Acad. Sci. USA, 76, 981±987. Schiano, S., Gorza, L., Satore, S., Saggin, L., Ausoni, S., Vianello, M., Gundersen, K., & Lomo, T. (1989). Three myosin heavy chain isoforms in type 2 skeletal muscle ®bres. J. Muscle Res. Cell Motil, 10, 197±205.
B. Picard et al. / Meat Science 53 (1999) 1±7 Schiano, S., & Reggiani, C. (1994). Myosin isoforms in mammalian skeletal muscle. J. Appl. Physiol., 77, 493±501. Staron, R. S., & Pette, D. (1990). Correlation between myo®brillar ATPase activity and myosin heavy chain composition in rabbit muscle ®bers. Histochem., 86, 19±23. Sugiura, T., & Murakami, N. (1990). Separation of myosin heavy chain isoforms in rat skeletal muscles by gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis. Biomed. Res., 11, 87±91.
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Talmadge, R., & Roy, R. R. (1993). Electrophoretic separation of rat skeletal muscle myosin heavy chain isoforms. J. Appl. Physiol. 75, 2337±2341. Totland, G. K., & Kryvi, H. (1991). Distribution patterns of muscle ®bre type in major muscles of the bulls (Bos taurus). Anat. Embryol., 184, 441±450. Young, O. A., & Davey, L. (1981). Electrophoretic analysis of proteins from single bovine muscle ®bres. Biochem. J., 195, 317±327 .
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Electrophoretic separation of bovine muscle myosin heavy chain isoforms B. Picard*, C. Barboiron, M.P. Duris, H. GagnieÂre, C. Jurie, Y. Geay Laboratoire Croissance et MeÂtabolismes des Herbivores, INRA, Theix, 63 122 Saint-GeneÁs Champanelle, France Received 29 September 1998; received in revised form 25 January 1999; accepted 29 January 1999
Abstract This study concerns the de®nition of the optimum conditions for separation of adult and developmental myosin heavy chain (MHC) isoforms in bovine muscle. The various techniques published do not result in good separation of the MHC in this species. The trials carried out concerned the concentration of acrylamide and N, N0 -methylene-bis-acrylamide, and more particularly the concentration of Tris in the separating gel. The ®nding was that analysis of adult isoforms and developmental isoforms require dierent conditions. A acrylamide gradient of 3.5±10% with 200 mM Tris pH 8.8 gives good resolution for adult isoforms. Under these conditions 3 fast adult isoforms are revealed. However, study of MHC isoforms throughout foetal life in bovines is complex, and requires the combined use of more than one gel (gradient 3.5±10% at 200 mM Tris and gradient 3.5±10% at 250 mM Tris). # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Muscle; Myosin; Electrophoresis; Bovine
1. Introduction The study of bovine muscle ®bres is of primordial interest because they play a fundamental role in modeling meat quality and tenderness. Post-mortem meat tenderization processes transforming muscle into meat involve enzymatic and physicochemical mechanisms, which, in intensity and amplitude, are largely governed by the proportion of the distinct ®bre types (Ouali, 1990). The variable contraction velocity of muscles depends, at least in part, on the polymorphism of myosin heavy chain (MHC). To date, a total of 10 distinct MHC isoforms encoded by a multigene family, have been elucidated in mammalian skeletal muscle (for review: Staron & Pette, 1990). In adult the most abundant are MHC I, MHC IIa, MHC IIb and MHC IIx expressed respectively in ®bres identi®ed as types I, IIA, IIB and IIX. This last type has not been evidenced to date in bovine muscle (Picard, Duris, & Jurie, 1998b). Other isoforms are present during foetal life (MHC embryonic, neonatal or foetal) or in special muscles (MHC Itonic, MHC alpha cardiac, MHC eom, MHC IIM). * Corresponding author. Tel.: +33-04-73-62-4056; fax: +33-04-7362-4622. E-mail address: [email protected] (B. Picard)
The technique of Laemmli (1970) on polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) has been used for the determination of muscle MHC phenotype since 1975. Burridge and Bray (1975) were the ®rst to report a dierence in the electrophoretic mobilities of MHC from skeletal and smooth muscles. Since then the technique has constantly been improved over the years. Fast and slow MHC isoforms were ®rst separated from avian and mammalian muscle (Carraro & Catani, 1983; Rushbrook & Stracher, 1979). Then, Danieli-Betto, Zerbato, and Betto (1986) were able to resolve the IIa and IIb MHC in rat skeletal muscle. Several laboratories separated another mammalian MHC isoform called IId or IIx (BaÈr & Pette, 1988; Sugiua & Murakami, 1990). Dierent authors in several species found that the addition of glycerol has a sharpening eect on the electrophoretic bands (Carraro & Catani; Danieli-Betto et al.; Laframboise et al., 1990; Rushbrook & Stracher). Also, Sugiura and Murakami showed that the temperature of migration was of great importance for the quality of MHC resolution. The addition of 2-mercaptoethanol has been shown to increase the resolution of MHC (Agbulut, Zhenlin, Mouly, & Butler-Browne, 1996; Blough, Rennie, Zhang, & Peter, 1996; Fritz, Swartz, & Greaser, 1989). The separation of MHC IIa-MHC IIb or MHC IIa-MHC
0309-1740/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(99)00021-2
2
B. Picard et al. / Meat Science 53 (1999) 1±7
IIx has been improved by the development of gradient gels (BaÈr & Pette, 1988; Caizzo, Herrich, & Baldwin, 1991; Danieli-Betto et al., 1986; Perrie & Brumford, 1984). More recently a new protocol was developed by Talmadge and Roy (1993) to separate the skeletal muscle MHC of adult rats. It was adapted for use with different species (rabbit: Janmot & D'Albis, 1994; horse: Rivero, Talmadge, & Edgerton, 1997). The dierent conditions of electrophoretic separation vary a lot between species. To date no technique has been published for the resolution of bovine MHC. Only Young and Davey (1981) have studied bovine myosin but with peptide mapping. The dierent methods quoted above do not allow the distinction between MHC IIa and MHC IIb. So, the aim of this work is to determine the best conditions of the separation of both developmental and adult MHC isoforms in bovine skeletal muscle. 2. Materials and methods 2.1. Preparation of muscle samples Adult muscles with dierent composition of ®bre types: Semitendinousus (ST), Longissimus thoracis (LC), Biceps femoris (BF), Triceps Brachii (TB), Masseter (M), Cutaneus trunci (CT) were taken after slaughter of Charolais bulls, 15 months old. Semitendinosus muscle of Charolais and double-muscled strain INRA 95 foetuses were taken after the slaughter of the mother at gestational ages of 110, 230, 260 days. Samples of muscles were prepared and frozen in liquid nitrogen as described by Robelin et al. (1991). The muscles were extracted as previously described (Picard, GagnieÁre, Robelin, Pons, & Geay, 1995). The protein concentrations of the samples were determined according to the method of Bradford (1976). 2.2. Conditions of electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on plates of 1601601.5 mm in Hoer system, by the method of Laemmli (1970) (Table 1). The stacking gel was at 3.5% of polyacrylamide with a cross-link of 1.3%. The separating gel was a 3.5±10% polyacrylamide gradient with a cross-link of 1.3% and a concentration of 200 mM of Tris pH 8.8 for adult muscles or 250 mM for foetal muscles. To improve resolution, the separating gel also contained a 30±40% glycerol gradient according to Suguira and Murakami (1990). The samples were diluted 50% in a solution containing 1.4% (w/v) SDS, 10% (v/v) 1 mM Tris pH 6.8, 17% (v/v) glycerol, 0.6% (w/v) Pyronin Y and 7.5% mercaptoethanol. They were incubated for 10 min at room temperature and then at 90 C for 10 min. Wells were
Table 1 Preparation of the polyacrylamide gels
Acrylamide 40% N-N0 -methylene bisacrylamide 2% Tris 200 mM pH 8.8 H2O Glycerol SDS 10% Temed Ammonium persulfate 1%
3.5%
10%
1.10 ml 0.29 ml 1.3 ml 5.5 ml 3.8 ml 200 ml 10 ml 500 ml
3.10 ml 0.83 ml 1.3 ml 1.7 ml 5.1 ml 200 ml 10 ml 500 ml
probed with 3 mg of protein. A standard Laemmli (1970) running buer was used. The electrophoretic run was carried out at a constant voltage of 50 V for 30 min, followed by 110 V for 18 h at a temperature of 4 C. After migration the gels were stained in a solution of R 250 Coomassie Blue. The proteins were ®xed in trichloracetic acid 15% (w/v) for 30 min at room temperature. Then, the gels were incubated in the colouration solution containing: propanol 2 25% (v/v), acetic acid 90% (vlv), Coomassic Blue R250 0.2% (w/v), for 45 min at 68 C. Gels were rinsed and decolourated for 1 h 30 min in a solution of: ethanol 95 35% (v/v), acetic acid 90% (v/v) at room temperature, under agitation. The bands were identi®ed as MHC IIb, IIa, I (Fig. 2) on the basis of previously determined migration pattern (Picard, Robelin, Pons, & Geay, 1994; Young & Davey, 1981). 3. Results The preliminary trials on adult muscles, in 7% polacrylamide gels (data not shown) or according to the technique of Talmadge and Roy (1993) did not permit the resolution of fast MHC (IIa and IIb). Talmadge and Roy conditions gave a good separation of rat MHC, taken as control, but MHC of dierent bovine muscles were not resolved (Fig. 1). So the use of gradient gel seems to be necessary for the study of bovine MHC isoforms. Dierent concentrations of polyacrylamide gradients were tested: 5±8%, 3.5±8%, 3.5±9%, 3.5±10%. The gradient 3.5±10% (with 300 mM Tris pH 8.8) gave the best separation of the three MHC (I, IIa and IIb). Then dierent cross-link values were analysed to improve the resolution. Fig. 2 shows that a cross-link of 1.3% gives the best results. Among the various factors governing correct resolution of MHC, the Tris concentration in the separating gel is extremely important. The Tris enables the pH to be maintained constant in the gel, it provides ions which aect the mobility of the proteins and thus, the
B. Picard et al. / Meat Science 53 (1999) 1±7
3
Fig. 1. Separation of bovine MHC according to Talmadge and Roy (1993). 1. rat gastrocnemius (5 mg of proteins); 2±8 are bovine muscles; 2. Sternomendibularis (10 and 5 mg), 3. Splenius (10 and 5 mg), 4. Soleus (5 mg), 5. Diaphragma (5 mg), 6. Semitendinosus (10 and 5 mg), 7. Longissimus thoracis (10 and 5 mg), 8. Cutaneus trunci (10 and 5 mg).
Fig. 2. In¯uence of the cross-link (C) on the separation of bovine MHC. a. C=0.9%, b. C=1.3%, c. C=3%, d. C=4%. 1. Semitendinosus, 2. Cutaneus trunci, 3. Masseter, 4. Longissimus thoracis.
clarity of the bands obtained. So to improve separation of the MHC of bovine muscle even further, the in¯uence of various concentrations of Tris (375, 300, 250, 200, 150, 125, 100 mM) was tested on separation of MHC present during foetal life on the one hand, and on the other in adult muscle. In the various adult muscles, only two bands could be observed at the concentration of 375 mM (Fig. 3), MHC I and a very concentrated band of higher molecular weight. When the Tris concentration was reduced, the distance separating the two bands increased (300 and 250 mM). By reducing it still further (200, 165, 150 mM) the fast isoforms were separated. However, if the concentration is too low (125, 100 mM) separation is mediocre (data not shown). So all these trials show that the optimum separation of MHC isoforms in adult bovine muscle is obtained with an acrylamide concentration gradient of 3.5±10% with a cross-link of 1.3% and a concentration of 200 mM Tris pH 8.8 in the separating gel. Under these conditions 3 bands can be distinguished in the fast MHC (Figs. 3 and 4). These three bands are present in the four mixed muscles analysed: ST, LT, BF and TB in varying proportions. In ST, the higher molecular weight band is the most concentrated, with the two others having a similar concentration. In LT, the lower molecular weight fast band is the most concentrated, the middle band being only faintly present.
In BF the two higher bands are the most concentrated. Finally in TB the middle band has the lowest concentration (Fig. 4). The distance between these three bands was low, and more particularly between the heaviest two, so densitometric analysis is not always feasible. Fig. 3 shows that the best separation of all MHC present in foetal muscle was obtained at a concentration of 250 mM Tris in the separating gel. For foetus of 110 days (Fig. 3) with 200 mM Tris only two bands were observed, one with the same mobility as MHC I, the other migrating to the level of MHC IIa identi®ed as MHC emb according to Picard et al. (1994). With 250 mM Tris (Fig. 3) three bands were observed: MHC I and two MHC of higher PM. In double-muscled foetuses yet a fourth band was revealed (Fig. 3). By increasing the concentration of Tris, a better separation of higher molecular weight isoforms was obtained. When the concentration was increased yet again to 300 mM, separation of these bands was improved even further. On the other hand, at 375 mM there was less distance between MHC I and the higher bands so the resolution was less good. In older foetuses aged 230 and 260 days, three bands were observed with the 200 mM concentration (Fig. 3). When the concentration was increased, the separation between the two high molecular weight bands was reduced (Fig. 3). At 375 mM they were no longer separated at all.
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Fig. 3. Electrophoresis of muscle bovine MHC from foetal and adult stages: in¯uence of Tris concentration. 1. adult Masseter; 2±8 correspond to Semitendinosus of foetuses: 2. 110 day-old, 3. 230 day-old, 4. 260 day-old; samples 5±8 are double-muscled foetuses: 5 and 6. 110 day-old, 7. 230 dayold, 8. 260 day-old; a±d are adult muscles: a. Semitendinosus, b. Triceps brachii, c. Longissimus thoracis, d. Biceps femoris.
4. Discussion
4.1. Adult MHC
These various trials clearly show that the concentration of Tris in the separating gel is a fundamental limiting criteria for good resolution of myosin isoforms in bovines. It would appear that the optimum concentration for separation of adult isoforms is not the same as that used to separate developmental isoforms.
The 200 mM concentration gives a good resolution for the adult isoforms (MHC I, IIa and IIb). Furthermore a third band is even revealed in the fast isoforms in the various muscles studied, which is a very original result. However, the distance between the three bands is not yet satisfactory enough to allow densitometric analysis.
B. Picard et al. / Meat Science 53 (1999) 1±7
Fig. 4. Fast MHC of adult bovine muscles. 1. Masseter, 2. Semitendinosus, 3. Triceps brachii, 4. Longissimus thoracis, 5. Biceps femoris.
Addition of 2-mercaptoethanol in the upper buer as suggested by Blough et al. (1996), could perhaps further improve the separation obtained. However, it is obvious that the relative proportions of these three isoforms, for a given animal, is very dierent from one muscle to another. This is the ®rst work which has made it possible to show three fast isoforms in bovine muscle. Until now only isoforms MHC I, IIa and IIb had been described in adult bovine muscle (Totland & Kryvi, 1991; Jurie, Robelin, Picard, & Geay, 1995). The third fast MHC revealed must obviously correspond to MHC IIx. This isoform has now been identi®ed in a large number of species (rat, mouse, rabbit, pig, human) (Gorza, 1990; Schiano & Reggiani, 1994), so it seems logical that it would be present in all mammals, in greater or lesser proportions. According to HamalaõÈnen and Pette (1993) the muscles of large mammals, whose fast movements are much smaller than in small species such as the rat and the mouse, would contain little if any MHC IIb. So it can be assumed that the standard histochemistry techniques used in bovines classify ®bres IIX and IIB both as IIB ®bres, without making any distinction between the two. Immunohistochemistry trials have been carried out with various antibodies (Picard, Duris, & Jurie, 1998a) to check this hypothesis. The various results obtained plead in its favour. Antibody BF F3 (Schiano et al., 1989) which recognises MHC IIb in the rat and the mouse recognises no isoform at all in bovine ST muscle. Similarly antibody BF 35 which recognises all MHC except MHC IIx in the rat and mouse, recognises all MHC except IIb in bovines (Duris, Picard, & Geay, 1998). Furthermore antibody S5 8H2 which recognises both MHC I and IIb in bovine muscle (Picard et al., 1998a) identi®es MHC I, IIb and IIx in pig muscle. Similarly a antibody speci®c to MHC IIa and IIx in the pig (Lefaucheur & Ecolan, 1998) recognises all the fast MHC in bovines. All these results would seem to indicate that the MHC IIb and IIx in bovine muscle have very similar structures and are coidenti®ed by the anti IIb or IIx antibodies used. This would explain why they are very dicult to separate
5
according to their molecular weight. The revelation of a third band calls the nomenclature used into question, considering that the highest molecular weight band corresponds to MHC IIb and the other to MHC IIa. It now remains to identify these three fast MHC more precisely in bovine muscle. This identi®cation could have some implications in the study of meat quality, particularly, tenderness. It is well established that the composition of muscle ®bres condition the speed of ageing of meat (Ouali, 1990). However, muscles with a similar proportion of the dierent types of ®bres present dierent speed of ageing. So, these dierences could be explained in part, by the proportions of the three fast MHC. 4.2. Developmental MHC Electrophoretic study of MHC isoforms present during foetal life is even more complex. The Tris concentration of 250 mM alone made it possible to observe all the bands present at the various ages. At 110 days, the 200 mM concentration revealed only one band at the high molecular weight level, whereas two bands are visible with 250 mM. If the Tris concentration is increased still further (375 mM) the separation between the MHC is reduced. It might be thought that these two high molecular weight isoforms correspond to MHC IIa and IIb. But under these conditions there is no separation between these two isoforms. In addition the results we obtained previously with Western-blot and ELISA assays (Picard et al, 1994) showed that the MHC IIa and IIb fast adult isoforms only appear from 210 days foetal life on. These two isoforms have been identi®ed as embryonic MHC (higher molecular weight) and foetal MHC (medium molecular weight) (Picard et al., 1993; Robelin et al., 1993). Study of 230 and 260 day foetuses with 200 mM Tris revealed two high molecular weight bands, migrating to the same level as the adult isoforms MHC IIa and MHC IIb. Nevertheless analysis of the gel at 250 mM Tris always revealed two high molecular weight bands at 230 and 260 days, whereas under these conditions the fast adult isoforms were not separated. So the combined observation of these two gels (200 and 250 mM Tris) prompt us to conclude that at least three high molecular weight bands are present in 230 and 260 days foetuses. This is con®rmed by our earlier results with Western-blot and ELISA which show that at this stage the slow, fast adult (IIa and IIb) and foetal isoforms are present (Picard et al., 1994). The proportion of foetal isoform drops considerably during this period in parallel with a strong increase in the proportion of fast adult isoforms. Complete analysis of the MHC isoforms present during the ®nal third of foetal life in bovines therefore requires the combined use of two complementary gels, one at 200 mM and the other at 250 mM Tris. But at the beginning of life, for as long as the
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fast adult isoforms are not present, the 250 mM concentration is sucient to reveal the various isoforms present. 5. Conclusion The whole of this study shows how important it is to have complete mastery over the electrophoresis conditions and that interpretation of the results obtained must be very prudent. It is necessary to use other techniques (Western blot, immunohistology or ELISA) to con®rm identi®cation of the bands observed. Nevertheless, it would appear that separation of all adult isoforms in bovine muscles is obtained with an acrylamide gradient of 3.5±10% containing 200 mM Tris pH 8.8. These conditions make separation of three fast isoforms possible, which until now had never been obtained in bovine muscle. It now remains to improve the electrophoresis conditions still further to be capable of quantifying these three bands by densitometry. Each of them also needs to be identi®ed speci®cally. However, separation of developmental isoforms proves to be far more complex. At the beginning of foetal life, a 3.5± 10% acrylamide gel containing 250 mM Tris pH 8.8 makes it possible to reveal all the isoforms present. However, from the time when fast adult isoforms appear onwards, it becomes necessary to use two types of gel to reveal all the isoforms present. Given that these conditions are very complex, we need to ®nd a solution to reveal all these isoforms with one and the same gel. References Agbulut, O., Zhenlin, L., Mouly, V., & Butler-Browne, G. S. (1996). Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms. Biol. Cell., 88, 131±135. BaÈr, A., & Pette, D. (1988). Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett., 235, 153±155. Blough, E. R., Rennie, E. R., Zhang, F., & Peter, J. R. (1996). Enhanced electrophoretic separation and resolution of myosin heavy chains in mammalian and avian skeletal muscles. Anal. Biochem., 233, 31±35. Bradford, M. M. (1976). A fast and sensitive method for the quanti®cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72, 248±254. Burridge, K., & Bray, D. (1975). Puri®cation and structural analysis of myosins from brain and other non-muscle tissues. J. Mol. Biol., 99, 1±14. Caizzo, V. L., Herrick, R. E., & Baldwin, K. (1991). In¯uence of hyperthyroõÈdism on maximal shortening velocity and myosin isoform distribution in skeletal muscles. Am. J. Physiol., 261, 285±295. Carraro, U., & Catani, C. (1983). A sensitive SDS-PAGE method separating myosin heavy chain isoforms of rat skeletal muscles reveals the heterogeneous nature of embryonic myosin. Biochem. Biophys. Res. Commun., 116, 793±802. Danieli-Betto, D., Zerbato, E., & Betto, R. (1986). Type 1, 2A and 2B myosin heavy chain electrophoretic analysis of rat muscle ®bers. Biochem. Biophys. Res. Commun., 116, 981±987.
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Talmadge, R., & Roy, R. R. (1993). Electrophoretic separation of rat skeletal muscle myosin heavy chain isoforms. J. Appl. Physiol. 75, 2337±2341. Totland, G. K., & Kryvi, H. (1991). Distribution patterns of muscle ®bre type in major muscles of the bulls (Bos taurus). Anat. Embryol., 184, 441±450. Young, O. A., & Davey, L. (1981). Electrophoretic analysis of proteins from single bovine muscle ®bres. Biochem. J., 195, 317±327 .