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M-protein in chicken cardiac muscle
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Experimental
M-PROTEIN
Cell Research
IN CHICKEN
124 (1979) 39-45
CARDIAC
MUSCLE
E. E. STREHLER, G. PELLONI, C. W. HEIZMANN’ and H. M. EPPENBERGER Institut
fir
Zellbiologie,
ETH HGnggerberg,
CH-8093
Ziirich,
Switzerland
SUMMARY Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M= 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confumed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.
The M-line, a characteristic electron-dense region in vertebrate skeletal muscle runs transversely through the middle of the Hregion, the region where thick filaments are free of myosin cross-bridges. Chicken cardiac muscle differs from chicken skeletal muscle and from cardiac and skeletal muscle of a number of vertebrate species in that the H-zone of its myofibrils lacks electrondense material in the M-line region [ 1, 21. Much work has been reported on the elucidation of the structure [3-S] and protein composition [9-161 of the M-line of skeletal muscle. Among the proteins identified as possible M-line components only the homodimeric isoenzyme of creatine kinase consisting of two M subunits (MM-CK, subunit M=40000) [lo, 131 and an M=165000 protein, called M-protein [15], are still thought to represent true constituents of the M-band [13, 15, 17-211. The M-line structure could be responsible for maintaining the thick filaments in proper register. However, thick filaments of chicken cardiac muscle form a regular array [22, 231, al-
though the myofibrils of this type of muscle lack a visible M-line. Moreover, in chicken cardiac muscle the homodimeric form of creatine kinase consisting of two B subunits (BB-CK) is the predominant creatine kinase form [24] and this enzyme has been shown to be more strongly bound to the Z-line region than to the H-zone in isolated chicken heart myofibrils [2]. It was therefore of interest to investigate whether the other component attributed to skeletal muscle M-line structure, the Mprotein with M,. = 165 000, could be detected in chicken cardiac muscle and, if so, at which myofibrillar region it was localized. MATERIALS
AND
METHODS
Purification of M-protein from chicken breast muscle Chicken muscle was obtained from Kneuss, Magenwil, Switzerland. The isolation of M-protein was done as 1 Present address: Institut fur Pharmakologie und Biochemie der vet. med. Fakulmt, Winterthurerstrasse 260, CH-8057 Ziirich, Switzerland. Exp Cd
Res 124 (1979)
40
Strehler et al. serum were prepared as described [25]. Anti-M-protein IgG fraction was further purified by antigen-affinity chromatography [26].
Immunojluorescence
localization
M-protein was localized in chicken heart myolibrils by the indirect immunofluorescence technique [18]. Preimmune serum and specific antiserum were diluted 1 : 50 in solution A containing 0.1 M KCI, 1 mM EGTA, 5 mM EDTA, 1 mM DTT, pH 7. The fluorescein-conjugated goat-anti-rabbit IgG (Grand Island Biological Co.) was diluted 1 : 100 in solution A.
Preparation and incubation with antibody offiber bundles from chicken heart muscle
Fig. I. Double immunodiffusion test of afftnity purified anti-chicken breast muscle M-protein-IgG (1 mg/ml, center well) against high salt extract from a, chicken breast muscle; h, chicken heart muscle (about 10 mglml).
described [15]. In the final step of purification phosphorylase b was separated from M-protein by affinity chromatography on adenosine-5’ monophosphate (5’AMP)-Sepharose 4B. About 25 mg of protein, dissolved in 50 ml of 50 mM Tris-HCl, 1 mM EDTA, 0.3 mM dithiothreitol (DTT), 0.1 mM phenylmethane sulfonyl fluoride (PMSF), 10m6M Pepstatin, pH 7.9, was loaded on a 0.8~6 cm 5’-AMP Senharose 4B (Pharmacia) column equilibrated in the same buffer. Homoaeneous M-urotein (as iudged from SDS-nolyacryla&de gel electrophoresis)-was eluted in the breakthrough fraction whereas phosphorylase b was retained due to its affinity for AMP.
Preparation of antibody
and purification
Antiserum against chicken breast muscle M-protein was elicited in rabbits. A solution containing 0.3 mg of purified M-protein in 1.5 ml of 50 mM Tris-HCl, 1 mM EDTA, 0.3 mM DTT, 0.1 mM PMSF, 10eB M Pepstatin, nH 7.9 was emulsified with 1.5 ml complete Freunds adjuvant. One-half of this emulsion was injected subcutaneously at different sites of the back and the other half intramuscularly into the thigh muscle of a rabbit. Additional injections were given 1, 2, and 5 weeks later. They were identical with the first except that the protein solution was emulsified with incomplete instead of complete Freund’s adjuvant. One week after the last injection the rabbits were bled from the carotis artery. The centrifuged serum was stored at -20°C. For purification of the antibody the IgG fractions of both preimmune serum (control) and of anti-M-protein Exp Cell
Res 124 11979)
Hearts from freshly killed chicken were immediately removed and placed in cold solution A containing 50% (vol/vol) glycerol. Rapid rupturing of membranes was achieved by alternating solution A plus glycerol with solution A alone every 3 h for 24 h. Before use, hearts were washed in solution A with several changes over a 3 h period to remove the glycerol. Small fiber bundles were obtained by tearing the hearts into pieces with an injection needle. The fiber bundles were incubated for 24 h with either specific or control antiserum (or IgG) at the concentrations given in the text. Then the fiber bundles were washed for 1 h in solution A to remove unbound antibody. Incubations with specific and control antibodies were done on different fragments of the same fiber bundle.
Electron microscotv_ . The fiber bundle preparations were prefixed in 0.2 M cacodylate buffer, pH 7.3, containing 1.5% glutaraldehyde and 1.5 % acrolein at 4°C for 12 h. Fixation in 0.2 M cacodylate buffer, pH 7.3 containing 1% osmium tetroxide for 2 h at 4°C was followed by dehvdration in ethanol and embeddina in Eoon 812. Longitudinal sections (about 50 nm) &re l&t-stained [33] with 2 % uranyl acetate in HZ0 for 45 min followed by lead citrate for 15 min. A Siemens Elmiskop 102 was used at 100 kV.
Other methods Protein determination. Protein was determined by the Biuret method 1271. Electrophoresis and immunoreplica technique. SDS-polyacrylamide slab gel electrophoresis was carried out as described [28]. Unstained gels were used in the immunoreplica technique [ 19, 291;in which a 0.6 % agarose gel containing anti-M-protein serum (diluted 1: 1) was noured onto the unfixed slab eel. After incub&ion at 37°C for about 12 h the ov&lay gel was washed for about 3 days in 0.15 M NaCl, 0.01 M nhosphate buffer, pH 7 and then stained with Coomassie brilliant blue. Ouchterlony double immunodiffusion. Ouchterlony
M-Protein
in chicken
cardiac
muscle
41
Myofibrillar extracts. 0.5 ml of freshly prepared suspensions of breast and heart myotibrils (about 20 vol/wt) were centrifuged for 1 min in a microcapillary centrifuge model MB (International Equipment Co., Boston, Mass.) and the pellets incubated at 4°C in 0.5 ml of either low salt (5 mM Tris-HCl, pH 7.7,l mM DTT) or hiah salt extraction buffer (0.1 M uhosuhate buffer, pH &, 0.6 M KCl, 1 mM EDTA, 1 mM MgCl*, 10 mM Na.,P,O,. . - 0.3 mM DTT). After extraction (24 72 h) the myofibril suspensions were centrifuged for 15 min in the microcapillary centrifuge and the supernatants used for double immunodiffusion and gel electrophoresis.
RESULTS Characterization
5
6
7
Detection of a cross-reacting protein species in chicken heart muscle
1234 Fig. 2. SDS-polyacrylamide (5 %) gel electrophoresis of proteins from low and high salt extracts from chicken heart muscles and identification of a heart M-protein by immunoreplication. For preparation of low and high salt extracts see Materials and Methods. I, Low salt extract (72 h, 3 pp); 2, high salt extract (24 h, 5 pg); 3, high salt extract (72 h, 3 pg); 4, M, marker proteins (approximate values: chicken debranching enzyme, 165000; rabbit muscle phosphorylase b, 94000; bovine serum albumin, 68 000; rabbit muscle pyruvate kinase, 57000); 5-7, immunoreplica of a part of the same gel duplicating lanes 1-3 not fixed and stained directly but instead overlaid with an agarose gel containing anti-chicken breast muscle M-protein serum. Single precipitin lines were found in lanes l-3 at the positions designed by the arrows.
double immunodiffttsion
1301.
of the antibody
The antiserum obtained against chicken breast muscle M-protein gave a single precipitin line when tested against the homologous antigen in the Ouchterlony double immunodiffusion test. The titer of the antiserum was 1: 64. There was no cross-reaction of the antibody against phosphorylase b and glycogen debranching enzyme, two possible contaminants of the M-protein preparation (not shown).
was performed as described
Preparation of myofibrils. Myotibrils from chicken heart muscle were prepared according to Kundrat & Pepe [31] with the precautions given by Heizmann et al. [32].
When affinity-column purified anti-chicken breast muscle M-protein-IgG was reacted against high salt extracts of heart myofibrils a precipitin line formed that fused with the precipitin line obtained against breast muscle extract (fig. 1). However, the crossreacting protein species from cardiac muscle extract seems not to be quite identical with the breast muscle protein. There must be at least one antigenic site not common to both proteins, as can be seen in fig. 1; the precipitin line formed between the antibody and the heart antigen fuses with the line formed between the antibody and the homologous antigen extracted from breast muscle but a spur can be seen in the latter reaction. The immunoreplica technique was used to identify the cardiac muscle protein Exp
Cell RPS 124 (1979)
42
Strehler
et al.
Fig. 3. Localization of M-protein in isolated chicken heart muscle myofibrils by the indirect immunofluorescence technique. (a), (c) Phase contrast; (b),
(d) immunofluorescent staining of the same myofibrils as in (a) and (c); (b) incubation with control serum; (d) incubation with anti-M-protein serum.
on 5 % polyacrylamide slab gels in the presence of SDS of low and high salt extracts from chicken heart muscle. Faint but distinct precipitin lines were formed at the sites of antibody-antigen reaction showing the heart protein to have a (subunit) molecular weight similar to that of breast muscle M,.= 165 000 M-protein (fig. 2). For simplicity we shall refer to the two closely related proteins as ‘heart M-protein’ and ‘skeletal M-protein’, or simply as ‘M-protein’ where the experimental procedure does not distinguish between them.
isolated chicken heart myofibrils, strong fluorescence at the H-zone and only weak fluorescence at the Z-line was observed
Localization of heart M-protein within heart myofibrils
When the indirect immunofluorescence technique was used to localize M-protein in EXJI Cell Res 124 (1979)
(fig. 3).
More information on the localization of M-protein within the structure of heart myofibrils could be obtained by electron microscopy of longitudinal sections of antibody-incubated cardiac muscle fiber bundles. Heavy staining of the region in the middle of the H-zone (corresponding to the M-line region of chicken breast muscle myofibrils) was observed when fibers previously incubated with anti-M-protein serum (1.5 mglml) were used (fig. 4 b). In contrast, longitudinal sections of fiber bundles incubated with preimmune serum (or IgG, 1 mglml) lacked an electron dense M-line
M-Protein
in chicken cardiac muscle
43
Fig. 4. Thin sections of heart fiber bundles. (a) Incubation with preimmune (control) IgG (1 mglml). The Hzone lacks an electron-dense M-line. (b) Staining of
the region in the middle of the H-zone by anti-chicken breast muscle M-protein-serum (1.5 mglml). H, Hzone; Z, Z-line.
(fig. 4a). The absence of electron-dense Mlines in chicken heart muscle has been reported by many workers [ 1,2,21].
ondary M-bridges are thought to link each M-filament to four adjacent M-filaments [ 81. Much less is known on the structure of the H-zone of myofibrils from muscles lacking a visible M-line, such as chicken cardiac muscle. However, the absence of an electron-dense M-line suggests that primary Mbridges arranged in the same way as in skeletal muscle are not present in chicken cardiac muscle. Whether structural elements comparable to the secondary Mbridges and to the M-filaments exist in this type of muscle is not known. Among the proteins thought to represent true M-line components in skeletal muscle, MM-CK has been tentatively associated with primary M-bridges [lo, 17-191. After selective removal, by monovalent anti-
DISCUSSION In vertebrate skeletal muscle myofibrils, transverse arrays of primary M-bridges are believed to make the principal contribution to the electron density of the M-line, as seen in electron micrographs [3-6]. These primary M-bridges link each myosin filament to six neighbouring longitudinally oriented M-filaments [4, 71. Thus, in these muscles myosin filaments seem to be interconnetted by the following structural arrangement: primary M-bridge - M-filament - primary M-bridge. In addition, Y-shaped sec-
Exp Cell
Res 124 (1979)
44
Strehler
et al.
bodies against MM-CK, of that MM-CK tightly bound to the myofibrillar structure an electron-dense M-line was no longer observed in electron micrographs of longitudinal sections of skeletal muscle fibers [19]. MM-CK would thus appear to be essential to the structure of primary Mbridges, the M-line elements responsible for the prominent, electron-dense, transverse striations within the M-line. The absence of MM-CK from chicken heart myofibrils [2, 24, 341, together with the lack of a visible M-line in chicken cardiac muscle [ 1,2], supports this idea. M-protein, the other protein thought to be an M-line component that has been isolated so far, has been proposed as a possible component of the M-filaments of skeletal muscle [20, 211. The results presented in this study show that a similar protein is found in chicken cardiac muscle, where it is localized predominantly within a region of heart myofibrils corresponding to the Mline region in skeletal muscle. Unlike MMCK, M-protein thus seems to be a structural component not restricted to those myofibrils that possess a visible M-line. The function of the M-line structure is not yet clear. This structure might be responsible for maintaining the proper register of thick filaments during contraction when interfilamentary distances increase [20, 21, 341. In skeletal muscle myolibrils primary M-bridges interconnecting myosin filaments via M-filaments might fulfill this function. A mechanism preventing the ‘rupture’ of these bridges during contraction should then be expected to exist [34]. However, M-filaments and secondary M-bridges or other structural elements not detected until now might as well be mainly responsible for the regular arrangement of thick filaments. Chicken cardiac myofibrils, for example, lack a visible M-line and thus posExp Cell
Res 124 (IY79)
sibly lack primary M-bridges; yet, their thick filaments form a regular array [22, 231. Moreover, skeletal muscle myofibrils from which MM-CK (and concomitantly the visible M-line) has been removed still show an unchanged arrangement of thick filaments [19]. The presence of primary Mbridges in certain types of muscles possibly reflects a structural adaptation of these muscles. Perhaps the degree of order ‘required’ for fast twitch fibers is higher than for tonic or cardiac fibers and the primary M-bridges might represent an additional feature that provides a greater degree of order for the specific needs of fast twitch muscles. Evidence has been reported recently showing the M-protein to interact with myosin subfragment 2, the flexible hinge region of myosin, but not with light meromyosin and subfragment 1 [20]. This interaction seems to occur mainly in the overlap region of thick filaments as can be concluded from the localization of M-protein within isolated myotibrils [15]. These results, together with the finding that M-protein is localized within the M-region not only of chicken skeletal but also of chicken cardiac muscle myofibrils, make this component a likely candidate for a structural element which helps to keep the proper register of thick filaments during contraction. It remains to be elucidated, however, whether M-protein is associated with myofibrils in all types of striated muscle, or, indeed, with other nonmyotibrillar, actomyosin systems. Much work will also have to be done to assess the exact function and structural localization of this M-protein within the Hzone of skeletal and cardiac muscle myotibrils . The authors wish to thank Dr D. C. Turner for help in preparing this manuscript. This work was supported by Grant 3.187-0.77 from
M-Protein the Swiss National Science Foundation and by a grant to H.M.E. from the Muscular Dystrophy Association, IIIC.
References 1. Sommer, J R & Johnson, A, Z Zellforsch mikrosk Anat 98 (1%9) 437. 2. Wallimann, T, Kuhn, H J, Pelloni, G, Turner, D C & Eppenberger, H M, J cell bio175 (1977) 318. 3. Sjiistriim, M & Squire, J M, J mol biol 109 (1977) 49. 4. Knappeis, G G & Carlsen, F, J cell biol 38 (1968) 202. 5. Pepe, F A, Progr biophys mol bio122 (1971) 77. 6. Huxley, H E, The structure and function of muscle (ed G H Boume) vol. 1, p. 301. Academic Press, New York (1972). 7. Thomell, L E & Sjiistrom, M, J microscopy 104 ( 1975) 263. Luther, P & Squire, J, J mol biol 125 (1978) 313. Z: Masaki. T. Takaiti. 0 & Ebashi, S. J biochem (Tokyoj 64(1968) 909. 10. Morimoto, K & Harrington, W F, J biol them 247 (1972) 3052. 11. Eaton, B L & Pepe, F A, J cell biol55 (1972) 681. 12. Ma&i, T & Takaiti, 0, J biochem (Tokyo) 71 (1972) 355. 13. Turner, D C, Wallimann, T & Eppenberger, H M, Proc natl acad sci US 70 (1973) 702. 14. Ma&i, T & Takaiti, 0, J biochem (Tokyo) 75 (1974) 367. 15. Trinick, J & Lowey, S, J mol biol 113 (1977) 343. 16. Heizmann, C W & Eppenberger, H M, J biol them 253 (1978) 270.
in chicken cardiac
muscle
45
17. Wallimann, T, Turner, D C & Eppenberger, H M, FEBS proc meet 31 (1975) 119. 18. - J cell bio175 (1977) 297. 19. Wallimann, T, Pelloni, Cl, Turner, D C & Eppenberger, H M, Proc natl acad sci US 75 (1978) 42%. 20. Mani, R S & Kay, C M, Biochim biophys acta 536 (1978) 134. 21. Herasymowych, 0 S, Mani, R S & Kay, C M, Biochim biophys acta 534 (1978) 38. 22. Peachy, L D & Huxley, A F, J cell biol 13 (1%2) 177. 23. Mayr, R, Tissue and cell 3 (1971) 433. 24. Eppenberger, H M, Dawson, D M & Kaplan, N 0, J biol them 242 (1967) 204. 25. Sober, H A & Peterson, E A, Fed proc fed Am sot exp biol 17 (1958) 1116. 26. Perriard, J-C, Caravatti, M, Perriard, E R & Eppenberger, H M, Arch biochem biophys 191(1978) 90. 27. Layne, E, Methods enzymo13 (1957) 447. 28. Laemmli, U K, Nature 227 (1970) 680. 29. Showe, M K, Isobe, E & Onorato, L, J mol biol 107 (1976) 55. 30. Ouchterlony, 0, Handbook of experimental immunology (ed D M Weir) p. 655. Blackwell Scientific Publications, Oxford (1967). 31. Kundrat, E & Pepe, F A, J cell bio148 (1971) 340. 32. Heizmann, C W, Blauenstein, I E 8~ Eppenberger, H M, Experientia 34 (1978) 38. 33. Reynolds, E S, J cell biol 17 (1963) 208. 34. Eppenberger, H M, Wallimann, T, Kuhn, H J & Turner, D C, Isozymes (ed C Market?) vol. 2, p. 409. Academic Press, New York (1975). Received April 24, 1979 Accepted May 31, 1979
Exp Cell
Res 124 (1979)
Experimental
M-PROTEIN
Cell Research
IN CHICKEN
124 (1979) 39-45
CARDIAC
MUSCLE
E. E. STREHLER, G. PELLONI, C. W. HEIZMANN’ and H. M. EPPENBERGER Institut
fir
Zellbiologie,
ETH HGnggerberg,
CH-8093
Ziirich,
Switzerland
SUMMARY Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M= 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confumed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.
The M-line, a characteristic electron-dense region in vertebrate skeletal muscle runs transversely through the middle of the Hregion, the region where thick filaments are free of myosin cross-bridges. Chicken cardiac muscle differs from chicken skeletal muscle and from cardiac and skeletal muscle of a number of vertebrate species in that the H-zone of its myofibrils lacks electrondense material in the M-line region [ 1, 21. Much work has been reported on the elucidation of the structure [3-S] and protein composition [9-161 of the M-line of skeletal muscle. Among the proteins identified as possible M-line components only the homodimeric isoenzyme of creatine kinase consisting of two M subunits (MM-CK, subunit M=40000) [lo, 131 and an M=165000 protein, called M-protein [15], are still thought to represent true constituents of the M-band [13, 15, 17-211. The M-line structure could be responsible for maintaining the thick filaments in proper register. However, thick filaments of chicken cardiac muscle form a regular array [22, 231, al-
though the myofibrils of this type of muscle lack a visible M-line. Moreover, in chicken cardiac muscle the homodimeric form of creatine kinase consisting of two B subunits (BB-CK) is the predominant creatine kinase form [24] and this enzyme has been shown to be more strongly bound to the Z-line region than to the H-zone in isolated chicken heart myofibrils [2]. It was therefore of interest to investigate whether the other component attributed to skeletal muscle M-line structure, the Mprotein with M,. = 165 000, could be detected in chicken cardiac muscle and, if so, at which myofibrillar region it was localized. MATERIALS
AND
METHODS
Purification of M-protein from chicken breast muscle Chicken muscle was obtained from Kneuss, Magenwil, Switzerland. The isolation of M-protein was done as 1 Present address: Institut fur Pharmakologie und Biochemie der vet. med. Fakulmt, Winterthurerstrasse 260, CH-8057 Ziirich, Switzerland. Exp Cd
Res 124 (1979)
40
Strehler et al. serum were prepared as described [25]. Anti-M-protein IgG fraction was further purified by antigen-affinity chromatography [26].
Immunojluorescence
localization
M-protein was localized in chicken heart myolibrils by the indirect immunofluorescence technique [18]. Preimmune serum and specific antiserum were diluted 1 : 50 in solution A containing 0.1 M KCI, 1 mM EGTA, 5 mM EDTA, 1 mM DTT, pH 7. The fluorescein-conjugated goat-anti-rabbit IgG (Grand Island Biological Co.) was diluted 1 : 100 in solution A.
Preparation and incubation with antibody offiber bundles from chicken heart muscle
Fig. I. Double immunodiffusion test of afftnity purified anti-chicken breast muscle M-protein-IgG (1 mg/ml, center well) against high salt extract from a, chicken breast muscle; h, chicken heart muscle (about 10 mglml).
described [15]. In the final step of purification phosphorylase b was separated from M-protein by affinity chromatography on adenosine-5’ monophosphate (5’AMP)-Sepharose 4B. About 25 mg of protein, dissolved in 50 ml of 50 mM Tris-HCl, 1 mM EDTA, 0.3 mM dithiothreitol (DTT), 0.1 mM phenylmethane sulfonyl fluoride (PMSF), 10m6M Pepstatin, pH 7.9, was loaded on a 0.8~6 cm 5’-AMP Senharose 4B (Pharmacia) column equilibrated in the same buffer. Homoaeneous M-urotein (as iudged from SDS-nolyacryla&de gel electrophoresis)-was eluted in the breakthrough fraction whereas phosphorylase b was retained due to its affinity for AMP.
Preparation of antibody
and purification
Antiserum against chicken breast muscle M-protein was elicited in rabbits. A solution containing 0.3 mg of purified M-protein in 1.5 ml of 50 mM Tris-HCl, 1 mM EDTA, 0.3 mM DTT, 0.1 mM PMSF, 10eB M Pepstatin, nH 7.9 was emulsified with 1.5 ml complete Freunds adjuvant. One-half of this emulsion was injected subcutaneously at different sites of the back and the other half intramuscularly into the thigh muscle of a rabbit. Additional injections were given 1, 2, and 5 weeks later. They were identical with the first except that the protein solution was emulsified with incomplete instead of complete Freund’s adjuvant. One week after the last injection the rabbits were bled from the carotis artery. The centrifuged serum was stored at -20°C. For purification of the antibody the IgG fractions of both preimmune serum (control) and of anti-M-protein Exp Cell
Res 124 11979)
Hearts from freshly killed chicken were immediately removed and placed in cold solution A containing 50% (vol/vol) glycerol. Rapid rupturing of membranes was achieved by alternating solution A plus glycerol with solution A alone every 3 h for 24 h. Before use, hearts were washed in solution A with several changes over a 3 h period to remove the glycerol. Small fiber bundles were obtained by tearing the hearts into pieces with an injection needle. The fiber bundles were incubated for 24 h with either specific or control antiserum (or IgG) at the concentrations given in the text. Then the fiber bundles were washed for 1 h in solution A to remove unbound antibody. Incubations with specific and control antibodies were done on different fragments of the same fiber bundle.
Electron microscotv_ . The fiber bundle preparations were prefixed in 0.2 M cacodylate buffer, pH 7.3, containing 1.5% glutaraldehyde and 1.5 % acrolein at 4°C for 12 h. Fixation in 0.2 M cacodylate buffer, pH 7.3 containing 1% osmium tetroxide for 2 h at 4°C was followed by dehvdration in ethanol and embeddina in Eoon 812. Longitudinal sections (about 50 nm) &re l&t-stained [33] with 2 % uranyl acetate in HZ0 for 45 min followed by lead citrate for 15 min. A Siemens Elmiskop 102 was used at 100 kV.
Other methods Protein determination. Protein was determined by the Biuret method 1271. Electrophoresis and immunoreplica technique. SDS-polyacrylamide slab gel electrophoresis was carried out as described [28]. Unstained gels were used in the immunoreplica technique [ 19, 291;in which a 0.6 % agarose gel containing anti-M-protein serum (diluted 1: 1) was noured onto the unfixed slab eel. After incub&ion at 37°C for about 12 h the ov&lay gel was washed for about 3 days in 0.15 M NaCl, 0.01 M nhosphate buffer, pH 7 and then stained with Coomassie brilliant blue. Ouchterlony double immunodiffusion. Ouchterlony
M-Protein
in chicken
cardiac
muscle
41
Myofibrillar extracts. 0.5 ml of freshly prepared suspensions of breast and heart myotibrils (about 20 vol/wt) were centrifuged for 1 min in a microcapillary centrifuge model MB (International Equipment Co., Boston, Mass.) and the pellets incubated at 4°C in 0.5 ml of either low salt (5 mM Tris-HCl, pH 7.7,l mM DTT) or hiah salt extraction buffer (0.1 M uhosuhate buffer, pH &, 0.6 M KCl, 1 mM EDTA, 1 mM MgCl*, 10 mM Na.,P,O,. . - 0.3 mM DTT). After extraction (24 72 h) the myofibril suspensions were centrifuged for 15 min in the microcapillary centrifuge and the supernatants used for double immunodiffusion and gel electrophoresis.
RESULTS Characterization
5
6
7
Detection of a cross-reacting protein species in chicken heart muscle
1234 Fig. 2. SDS-polyacrylamide (5 %) gel electrophoresis of proteins from low and high salt extracts from chicken heart muscles and identification of a heart M-protein by immunoreplication. For preparation of low and high salt extracts see Materials and Methods. I, Low salt extract (72 h, 3 pp); 2, high salt extract (24 h, 5 pg); 3, high salt extract (72 h, 3 pg); 4, M, marker proteins (approximate values: chicken debranching enzyme, 165000; rabbit muscle phosphorylase b, 94000; bovine serum albumin, 68 000; rabbit muscle pyruvate kinase, 57000); 5-7, immunoreplica of a part of the same gel duplicating lanes 1-3 not fixed and stained directly but instead overlaid with an agarose gel containing anti-chicken breast muscle M-protein serum. Single precipitin lines were found in lanes l-3 at the positions designed by the arrows.
double immunodiffttsion
1301.
of the antibody
The antiserum obtained against chicken breast muscle M-protein gave a single precipitin line when tested against the homologous antigen in the Ouchterlony double immunodiffusion test. The titer of the antiserum was 1: 64. There was no cross-reaction of the antibody against phosphorylase b and glycogen debranching enzyme, two possible contaminants of the M-protein preparation (not shown).
was performed as described
Preparation of myofibrils. Myotibrils from chicken heart muscle were prepared according to Kundrat & Pepe [31] with the precautions given by Heizmann et al. [32].
When affinity-column purified anti-chicken breast muscle M-protein-IgG was reacted against high salt extracts of heart myofibrils a precipitin line formed that fused with the precipitin line obtained against breast muscle extract (fig. 1). However, the crossreacting protein species from cardiac muscle extract seems not to be quite identical with the breast muscle protein. There must be at least one antigenic site not common to both proteins, as can be seen in fig. 1; the precipitin line formed between the antibody and the heart antigen fuses with the line formed between the antibody and the homologous antigen extracted from breast muscle but a spur can be seen in the latter reaction. The immunoreplica technique was used to identify the cardiac muscle protein Exp
Cell RPS 124 (1979)
42
Strehler
et al.
Fig. 3. Localization of M-protein in isolated chicken heart muscle myofibrils by the indirect immunofluorescence technique. (a), (c) Phase contrast; (b),
(d) immunofluorescent staining of the same myofibrils as in (a) and (c); (b) incubation with control serum; (d) incubation with anti-M-protein serum.
on 5 % polyacrylamide slab gels in the presence of SDS of low and high salt extracts from chicken heart muscle. Faint but distinct precipitin lines were formed at the sites of antibody-antigen reaction showing the heart protein to have a (subunit) molecular weight similar to that of breast muscle M,.= 165 000 M-protein (fig. 2). For simplicity we shall refer to the two closely related proteins as ‘heart M-protein’ and ‘skeletal M-protein’, or simply as ‘M-protein’ where the experimental procedure does not distinguish between them.
isolated chicken heart myofibrils, strong fluorescence at the H-zone and only weak fluorescence at the Z-line was observed
Localization of heart M-protein within heart myofibrils
When the indirect immunofluorescence technique was used to localize M-protein in EXJI Cell Res 124 (1979)
(fig. 3).
More information on the localization of M-protein within the structure of heart myofibrils could be obtained by electron microscopy of longitudinal sections of antibody-incubated cardiac muscle fiber bundles. Heavy staining of the region in the middle of the H-zone (corresponding to the M-line region of chicken breast muscle myofibrils) was observed when fibers previously incubated with anti-M-protein serum (1.5 mglml) were used (fig. 4 b). In contrast, longitudinal sections of fiber bundles incubated with preimmune serum (or IgG, 1 mglml) lacked an electron dense M-line
M-Protein
in chicken cardiac muscle
43
Fig. 4. Thin sections of heart fiber bundles. (a) Incubation with preimmune (control) IgG (1 mglml). The Hzone lacks an electron-dense M-line. (b) Staining of
the region in the middle of the H-zone by anti-chicken breast muscle M-protein-serum (1.5 mglml). H, Hzone; Z, Z-line.
(fig. 4a). The absence of electron-dense Mlines in chicken heart muscle has been reported by many workers [ 1,2,21].
ondary M-bridges are thought to link each M-filament to four adjacent M-filaments [ 81. Much less is known on the structure of the H-zone of myofibrils from muscles lacking a visible M-line, such as chicken cardiac muscle. However, the absence of an electron-dense M-line suggests that primary Mbridges arranged in the same way as in skeletal muscle are not present in chicken cardiac muscle. Whether structural elements comparable to the secondary Mbridges and to the M-filaments exist in this type of muscle is not known. Among the proteins thought to represent true M-line components in skeletal muscle, MM-CK has been tentatively associated with primary M-bridges [lo, 17-191. After selective removal, by monovalent anti-
DISCUSSION In vertebrate skeletal muscle myofibrils, transverse arrays of primary M-bridges are believed to make the principal contribution to the electron density of the M-line, as seen in electron micrographs [3-6]. These primary M-bridges link each myosin filament to six neighbouring longitudinally oriented M-filaments [4, 71. Thus, in these muscles myosin filaments seem to be interconnetted by the following structural arrangement: primary M-bridge - M-filament - primary M-bridge. In addition, Y-shaped sec-
Exp Cell
Res 124 (1979)
44
Strehler
et al.
bodies against MM-CK, of that MM-CK tightly bound to the myofibrillar structure an electron-dense M-line was no longer observed in electron micrographs of longitudinal sections of skeletal muscle fibers [19]. MM-CK would thus appear to be essential to the structure of primary Mbridges, the M-line elements responsible for the prominent, electron-dense, transverse striations within the M-line. The absence of MM-CK from chicken heart myofibrils [2, 24, 341, together with the lack of a visible M-line in chicken cardiac muscle [ 1,2], supports this idea. M-protein, the other protein thought to be an M-line component that has been isolated so far, has been proposed as a possible component of the M-filaments of skeletal muscle [20, 211. The results presented in this study show that a similar protein is found in chicken cardiac muscle, where it is localized predominantly within a region of heart myofibrils corresponding to the Mline region in skeletal muscle. Unlike MMCK, M-protein thus seems to be a structural component not restricted to those myofibrils that possess a visible M-line. The function of the M-line structure is not yet clear. This structure might be responsible for maintaining the proper register of thick filaments during contraction when interfilamentary distances increase [20, 21, 341. In skeletal muscle myolibrils primary M-bridges interconnecting myosin filaments via M-filaments might fulfill this function. A mechanism preventing the ‘rupture’ of these bridges during contraction should then be expected to exist [34]. However, M-filaments and secondary M-bridges or other structural elements not detected until now might as well be mainly responsible for the regular arrangement of thick filaments. Chicken cardiac myofibrils, for example, lack a visible M-line and thus posExp Cell
Res 124 (IY79)
sibly lack primary M-bridges; yet, their thick filaments form a regular array [22, 231. Moreover, skeletal muscle myofibrils from which MM-CK (and concomitantly the visible M-line) has been removed still show an unchanged arrangement of thick filaments [19]. The presence of primary Mbridges in certain types of muscles possibly reflects a structural adaptation of these muscles. Perhaps the degree of order ‘required’ for fast twitch fibers is higher than for tonic or cardiac fibers and the primary M-bridges might represent an additional feature that provides a greater degree of order for the specific needs of fast twitch muscles. Evidence has been reported recently showing the M-protein to interact with myosin subfragment 2, the flexible hinge region of myosin, but not with light meromyosin and subfragment 1 [20]. This interaction seems to occur mainly in the overlap region of thick filaments as can be concluded from the localization of M-protein within isolated myotibrils [15]. These results, together with the finding that M-protein is localized within the M-region not only of chicken skeletal but also of chicken cardiac muscle myofibrils, make this component a likely candidate for a structural element which helps to keep the proper register of thick filaments during contraction. It remains to be elucidated, however, whether M-protein is associated with myofibrils in all types of striated muscle, or, indeed, with other nonmyotibrillar, actomyosin systems. Much work will also have to be done to assess the exact function and structural localization of this M-protein within the Hzone of skeletal and cardiac muscle myotibrils . The authors wish to thank Dr D. C. Turner for help in preparing this manuscript. This work was supported by Grant 3.187-0.77 from
M-Protein the Swiss National Science Foundation and by a grant to H.M.E. from the Muscular Dystrophy Association, IIIC.
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17. Wallimann, T, Turner, D C & Eppenberger, H M, FEBS proc meet 31 (1975) 119. 18. - J cell bio175 (1977) 297. 19. Wallimann, T, Pelloni, Cl, Turner, D C & Eppenberger, H M, Proc natl acad sci US 75 (1978) 42%. 20. Mani, R S & Kay, C M, Biochim biophys acta 536 (1978) 134. 21. Herasymowych, 0 S, Mani, R S & Kay, C M, Biochim biophys acta 534 (1978) 38. 22. Peachy, L D & Huxley, A F, J cell biol 13 (1%2) 177. 23. Mayr, R, Tissue and cell 3 (1971) 433. 24. Eppenberger, H M, Dawson, D M & Kaplan, N 0, J biol them 242 (1967) 204. 25. Sober, H A & Peterson, E A, Fed proc fed Am sot exp biol 17 (1958) 1116. 26. Perriard, J-C, Caravatti, M, Perriard, E R & Eppenberger, H M, Arch biochem biophys 191(1978) 90. 27. Layne, E, Methods enzymo13 (1957) 447. 28. Laemmli, U K, Nature 227 (1970) 680. 29. Showe, M K, Isobe, E & Onorato, L, J mol biol 107 (1976) 55. 30. Ouchterlony, 0, Handbook of experimental immunology (ed D M Weir) p. 655. Blackwell Scientific Publications, Oxford (1967). 31. Kundrat, E & Pepe, F A, J cell bio148 (1971) 340. 32. Heizmann, C W, Blauenstein, I E 8~ Eppenberger, H M, Experientia 34 (1978) 38. 33. Reynolds, E S, J cell biol 17 (1963) 208. 34. Eppenberger, H M, Wallimann, T, Kuhn, H J & Turner, D C, Isozymes (ed C Market?) vol. 2, p. 409. Academic Press, New York (1975). Received April 24, 1979 Accepted May 31, 1979
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