Calcium-Induced Fragmentation of Skeletal Muscle Nebulin Filaments'

J. Biochem. 112, 775-779 (1992) Calcium-Induced Fragmentation of Skeletal Muscle Nebulin Filaments' Ryuichi Tatsumi and Koui Takahashi Meat Science Laboratory, Department of Animal Science, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, August 6, 1992 When chicken breast muscle myofibrils were treated with a solution containing 0.1 mm CaCl2 and 30 fig of leupeptin/ml, nebulin filaments were fragmented into 200-, 180-, 40-, 33-, and 23-kDa subfragments. All the subfragments except the 180-kDa one were released from the myofibrils. The fragmentation of nebulin filaments seems to be induced by the binding of large amounts of calcium ions. Similar changes took place in nebulin filaments in post-mortem skeletal muscle. It has been proposed that nebulin co-exists with thin (actin) filaments and participates in stabilizing their organization [Wang, K. & Wright, J. (1988) J. Cell Biol. 107, 2199-2212]. Thus, the above result suggests that Ca-induced fragmentation of nebulin filaments destabilizes the organization of thin filaments and is a key factor in meat tenderization during post-rigor aging. Nebulin is a giant structural protein, found by Wang and Williamson (1), existing in the sarcomeres of a wide range of skeletal muscle myofibrils (2, 3). In most vertebrates, nebulin accounts for 3-4% of the total myofibrillar protein (1), and its molecular mass varies from 600 to 900 kda in various muscle tissues (2, 3). Wang and Wright (4) have shown that nebulin constitutes a set of long inextensible longitudinal filaments that span the space between the Z-disk and the distal region of a thin (actin) filament, and proposed that nebulin might interact with and adhere to thin filaments, thereby stabilizing their organization. Wang described the decrease in nebulin during postmortem storage of rabbit skeletal muscle (5). On reexamination of post-mortem changes in nebulin, we confirmed that nebulin decreases during storage of skeletal muscle of various animals. We sought an essential factor which would induce post-mortem fragmentation of nebulin filaments using isolated myofibrils. This study shows that nebulin filaments are non-enzymatically fragmented by calcium ions. When chicken breast muscle myofibrils were treated with a solution containing 0.1 mm CaCl2 and 30ug of leupeptin/ml, 200-, 180-, 40-, 33-, and 23-kDa subfragments were produced; all the subfragments except the 180-kDa one were released from the myofibrils. There is a possibility that the fragmentation of nebulin filaments is induced by the binding of large amounts of calcium ions to specific regions of nebulin filaments. MATERIALS AND METHODS Preparation of Myofibrils-Breast muscle (Musculus pectoralis superficialis) of chicken, and back muscle (Musculus longissimus thoracis) of rabbit, pig, horse, and cow were used. If necessary, freshly dissected muscles were treated antiseptically by wrapping them with gauze containing 1 mm NaN3, and stored at 5'C. Myofibrils were ' This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. prepared from fresh and stored muscles by the method of Perry and Grey (6), leupeptin (Peptide Institute, Osaka) being present at 30ug/ml throughout the preparation to inhibit proteases. Ca-Treatment of Myofibrils-As described in our previous paper (7), freshly prepared myofibrils (15 mg/ml) were treated with a solution containing 0.1 M KCI, 0.1 mm CaC12, 30,u g of leupeptin/ml, 1 mm dithiothreitol, 1 mm NaN,, and 10 mm Tris-maleate buffer, ph 7.0, at 5'C with gentle stirring. At appropriate times, the myofibril suspensions were centrifuged at 10,000 rpm for 30 min, and both the precipitates and supernatant solutions were analyzed by SDS-PAGE and immunoblotting. SDS-PAGE-According to the method of Fairbanks et al. (8), linear 2-12% polyacrylamide gradient gels were prepared, and 4-10,u l of the samples was applied. The gels were stained and destained by the method of Laemmli (9). Preparation of the samples for gel electrophoresis and densitometry of the stained gels were performed as described previously (7). Immunoblotting-According to the method of Matsuura et al. (10), the protein bands obtained on SDS-PAGE were transferred electrophoretically onto nitrocellulose membranes (Bio-Rad Laboratories, California), and the membranes were cut into strips and treated with antinebulin antiserum, which was kindly provided by Prof. K. Maruyama of the University of Chiba. RESULTS Figure 1 shows the post-mortem changes in nebulin of chicken breast muscle myofibrils. The band of nebulin decreased with post-mortem time and completely disappeared within 1 d of storage, in parallel with the disappearance of a-connectin. The disappearance of nebulin during post-mortem aging was a common phenomenon in all skeletal muscles of various animals, but the initial lag time and the rate of decrease in the amount of nebulin differed in the muscles of different species (Fig. 2). More detailed determination of the changes in nebulin was carried out by Vol_ 112. No. 6. 1992 775

776 R. Tatsumi and K. Takahashi means of the immunoblotting technique using antinebulin antiserum (Fig. 3). In myofibrils prepared from stored muscle, immunologically stained polypeptides appeared under the band of nebulin, with a decrease in the amount of nebulin. However, these polypeptides disappeared, and a 180-kDa polypeptide was produced on prolonged storage. Because the molecular mass of the 180-kDa fragment accounted for only about 26% of the mass of the original nebulin (700 kda), the remaining portion of nebulin must have been released from the myofibrils during postmortem aging. Similar changes in nebulin could be induced by Catreatment of freshly prepared myofibrils (Fig. 4). After centrifugation of the Ca-treated myofibrils, both the precipitated myofibrils and supernatant solutions were sub- Fig. 1. Post-mortem changes in nebulin of chicken breast muscle. Myofibrils were prepared from chicken breast muscle stored at 5 C, dissolved in a SDS sample buffer solution containing 1% SDS, 5 mm EDTA, 5 mm Tris-HCl buffer, ph 8.0, 1% /3-mercaptoethanol, and 10% glycerol, and then boiled for 2 min. Samples were subjected to SDS-PAGE on linear 2-12% polyacrylamide gradient gels. Only the top portions of the stained gels are shown. Lane a, freshly prepared myofibrils; lanes b-f, myofibrils prepared from stored muscle: lane b, for 0.25 d; lane c, for 0.5 d; lane d, for 1 d; lane e, for 3 d; lane f, for 7 d. a, a-connectin;,q,q-connectin; N, nebulin; MHC, myosin heavy chain. Fig. 2. Post-mortem changes in the amount of nebulin in various skeletal muscles. Skeletal muscle specimens of various species were stored at 5 C. Myofibrils were prepared at appropriate times, treated with the SDS sample buffer solution, and then subjected to SDS-PAGE as shown in Fig. 1. The ratio of the nebulin band to the myosin heavy chain band was determined by densitometry of the stained gels, and expressed as a percentage of the ratio in fresh myofibrils. chicken; :, pig; -, horse; A, cow;, rabbit. Fig. 3. Detection of nebulin subfragments bound to myofibrils during post-mortem storage of muscle. Myofibrils were prepared from chicken breast muscle stored at 5 C, treated with the SDS Fig. 1. The separated proteins were transferred electrophoretically onto nitrocellulose membranes. Individual strips were blocked with a Tris buffered salt solution (0.5 M NaCl, 20 mm Tris-HC1 buffer, ph 7.5) containing 3% gelatin, and then incubated with antinebulin antiserum (diluted 500 times) in the Tris buffered salt solution containing 1% gelatin (lanes b-h). The binding of antibodies was detected by means of the horseradish peroxidase reaction using goat anti-rabbit IgG diluted 2,000 times with the Tris buffered salt solution containing 1% gelatin. Lane a, myofibrillar proteins stained with Coomassie Brilliant Blue R-250; lane b, fresh myofibrils; lanes c-h, myofibrils prepared from stored muscle: lane c, for 0.25 d; lane d, for 0.5 d; lane e, for 1 d; lane f, for 3 d; lane g, for 7 d; lane h, for 14 d. a, a-connectin; Q, 8-connectin; N, nebulin; MHC, myosin heavy chain; A, actin. Fig. 4. Changes in nebulin during Ca-treatment of myofibrils. Myofibrils (15 mg/ml) prepared from chicken breast muscle were treated with a solution containing 0.1 M KCI, 0.1 mm CaC12,30,Ug of leupeptin/ml, 1 mm dithiothreitol, 1 mm NaN,, and 10 mm Trismaleate buffer, ph 7.0, at 5 C. After the Ca-treatment, the myofibril suspensions were centrifuged. The precipitated myofibrils were treated with the SDS sample buffer solution and then subjected to SDS-PAGE as shown in Fig. 1. Lane a, fresh myofibrils; lanes b-f, myofibrils treated with 0.1 mm CaC12:lane b, for 1 d; lane c, for 3 d; lane d, for 4 d; lane e, for 5 d; lane f, for 7 d. a, a-connectin; Q, /3-connectin; N, nebulin; MHC, myosin heavy chain. J. Biochem.

Ca-Induced Fragmentation of Nebulin Filaments 777 Fig. 5. Detection of the 180-kDa subfragment produced during Ca-treatment of myofibrils. Myofibrils (15 mg/ml) prepared from chicken breast muscle were treated with 0.1 mm CaC12 as shown in Fig. 4. After the Ca-treatment, the myofibril suspensions were centrifuged. The precipitated myofibrils were treated with the SDS Fig. 1. The separated proteins were transferred electrophoretically onto nitrocellulose membranes, and then individual strips were treated with antinebulin antiserum and with goat anti-rabbit IgG, as shown in Fig. 3 (lanes b-h). Lane a, myofibrils stained with Coomassie Brilliant Blue R-250; lane b, fresh myofibrils; lanes c-h, myofibrils treated with 0.1 mm CaC12: lane c, for 1 d; lane d, for 3 d; lane e, for 5 d; lane f, for 7 d; lane g, for 9 d; lane h, for 11 d. C, a- and fl-connectins; N, nebulin; MHC, myosin heavy chain; A, actin. Fig. 6. Detection of nebulin subfragments released from myofibrils during Ca-treatment of myofibrils. Myofibrils (15 mg/ ml) prepared from chicken breast muscle were treated with 0.1 mm CaCl2 as shown in Fig. 4. After the Ca-treatment, the myofibril suspensions were centrifuged. The supernatant solutions were treated with the SDS sample buffer solution and then subjected to SDS-PAGE as shown in Fig. 1. The separated proteins were transferred electrophoretically onto nitrocellulose membranes, and then individual strips were treated with antinebulin antiserum and with goat antirabbit IgG, as shown in Fig. 3. Lane a, myofibrils stained with Coomassie Brilliant Blue R-250. Lanes b-h, supernatant solutions of Ca-treated myofibrils: lane b, for 1 d; lane c, for 3 d; lane d, for 7 d; lane e, for 11 d; lane f, for 13 d; lane g, for 15 d; lane h, for 17 d. C, a- and Q-connectins; N, nebulin; MHC, myosin heavy chain; A, actin; TM, tropomyosin; Tn-C, troponin C. jected to immunoblotting. In this case, the changes in nebulin occurred more slowly and clearly than those in post-mortem muscle. While the 180-kDa fragment bound to myofibrils (Fig. 5), the 200-, 90-, 40-, 33-, and 23-kDa fragments were found in the supernatant solution (Fig. 6, lanes e and f), first the 90-kDa fragment and eventually the other four fragments (Fig. 6, lanes g and h). These results show that nebulin filaments are fragmented into 200-, 180-, 40-, 33-, and 23-kDa subfragments on Ca-treatment of myofibrils, regardless of the animal species or muscle type, and all the subfragments other than the 180-kDa one are released from the myofibrils. The fragmentation of nebulin filaments occurred uniquely in the presence of calcium ions (Fig. 7). When myofibrils were treated with a solution containing 0.1 mm CaCl2 in the absence of leupeptin, nebulin disappeared completely within 3 h, indicating that myofibrils are contaminated by some protease when they are prepared by the usual method. The changes in the amount of nebulin were completely inhibited in the presence of both 5 mm EGTA and 30 k g of leupeptin/ml. Leupeptin (11) was the most effective of the various protease inhibitors tested, i.e., antipain, E-64, pepstatin A, phenylmethanesulfonyl fluoride, and diisopropylfluorophosphate. Calcium-specific fragmentation of nebulin filaments was induced by the treatment of myofibrils with a solution containing 0.1 mm CaCl2 and 30 u g of leupeptin/ml. The addition of 15 k g each of calpain-inhibitors I and II (Nacalai Tesque, Kyoto)/ ml did not affect the rate of the Ca-specific fragmentation of nebulin filaments. Figure 8 shows the dependence of the calcium-specific fragmentation of nebulin filaments on the calcium ion concentration. The fragmentation occurred above 10 k M and was maximum at 0.1 mm. The addition of 2 mm MgC12 did not affect the Ca-dependence of the fragmentation of nebulin filaments. The calcium-specific fragmentation of nebulin filaments into five kinds of subfragments was affected by ph and temperature (Fig. 9). When myofibrils were treated at 5 C with a solution containing 0.1 mm CaC12, at various phs, and then analyzed by SDS-PAGE, the calcium-specific fragmentation of nebulin was found to be maximum around ph 7, and was suppressed at acidic phs. When myofibrils were treated with 0.1 mm CaCl2 at ph 7.0, the rate of fragmentation of nebulin increased almost linearly with increasing temperature until 25'C, and slowed down thereafter. DISCUSSION Fragmentation of nebulin filaments is induced by 0.1 mm Ca'. There is a possibility that nebulin filaments are Vol. 112, No. 6, 1992

778 R. Tatsumi and K. Takahashi Fig. 7. Calcium-specific fragmentation of nebulin filaments. Myofibrils (15 mg/ml) prepared from chicken breast muscle were treated witi a solution containing 0.1 M KCI, 30,ug of leupeptin/ml, 1 mm dithiothreitol, 1 mm NaN3, 10 mm Tris-maleate buffer, ph 7.0, and 0.1 mm CaC12 ( ) or 5 mm EGTA (0), at 5 C. At appropriate times during the treatment, the myofibril suspensions were centrifuged, and the precipitated myofibrils were treated with the SDS Fig. 1. The ratio of the nebulin band to the myosin heavy chain band was determined by densitometry of the stained gels and expressed as a percentage of the ratio in intact myofibrils. o, 0.1 mm CaC12 in the absence of leupeptin. Fig. 9. Temperature and ph dependence of the calcium-specific fragmentation of nebulin filaments. Myofibrils (15 mg/ml) prepared from rabbit back muscle were treated with a solution containing 0.1 M KCI, 0.1 mm CaC1,, 30 pg of leupeptin/ml, 1 mm dithiothreitol, 1 mm NaN,, and 10 mm Tris-maleate buffer, ph 7.0, at various temperatures for 3 h (!), or with a solution containing 0.1 M KCI, 0.1 mm CaCl,, 30y g of leupeptin/ml, 1 mm dithiothreitol, 1 mm NaN,, and 10 mm Tris-maleate buffer at various phs, at 5 C for 48 h (o). After the Ca-treatment, the myofibrils were precipitated, treated with the SDS sample buffer solution and then subjected to SDS-PAGE as shown in Fig. 1. The ratio of the nebulin band to the myosin heavy chain band was determined by densitometry of the stained gels and expressed as a percentage of the ratio in intact myofibrils. Fig. 8. Dependence of the calcium-specific fragmentation of nebulin filaments on the calcium ion concentration. Myofibrils (15 mg/ml) prepared from rabbit back muscle were treated with a solution containing 0.1 M KCI, various concentrations of CaCl2r 30,ug of leupeptin/ml, 1 mm dithiothreitol, 1 mm NaN,, and 10 mm Tris-maleate buffer, ph 7.0, for 48 hat 5 C. After centrifugation, the precipitated myofibrils were treated with the SDS sample buffer solution and then subjected to SDS-PAGE as shown in Fig. 1. The ratio of the nebulin band to the myosin heavy chain band was determined by densitometry of the stained gels and expressed as a percentage of the ratio in intact myofibrils. non-enzymatically fragmented on the binding of calcium ions, though we cannot completely exclude the possibility of proteolysis by an unknown protease which is active under the present experimental conditions. We will report in a following paper that nebulin is a calcium-binding protein, and that calcium ions bind to the 200-, 40-, and 23-kDa subfragments (12). The Ca-specific fragmentation of nebulin filaments is similar to the Ca-specific splitting of connectin filaments; connectin, which exists as an elastic filament connecting a thick filament with a Z-disk in a sarcomere (13, 14), is non-enzymatically split into f8- connectin and a 1,200-kDa subfragment on the binding of large amounts of calcium ions (0.1 mm) to the l-connectin portion of a-connectin (7). These high-molecular-weight proteins, nebulin and connectin, seem to show common characteristics in the presence of large amounts of calcium ions. Actually, the ph dependence and temperature dependence of the Ca-specific fragmentation of nebulin filaments shown in Fig. 9 are entirely the same as those of the Ca-specific splitting of connectin filaments (Fig. 8 of Ref. 7). At the physiological concentration of calcium ions, it is possible that the properties of nebulin filaments change during the contraction-relaxation cycle of skeletal muscle. Somerville and Wang reported (15, 16) that nebulin is phosphorylated in vivo. They also pointed out the possibility that phosphorylated nebulin is involved in regulatory functions. The phosphorylation of and calcium-binding on nebulin filaments might play roles in muscle contraction. In post-mortem muscles, the ultimate sarcoplasmic J. Biochem.

Ca-Induced Fragmentation of Nebulin Filaments 779 calcium ion concentration is increased to about 0.2 mm (Ji, J.R. & Takahashi, K., manuscript in preparation), due to the disappearance of the Call -accumulating ability of sarcoplasmic reticular and mitochondrial membranes (17, 18). This concentration of calcium ions is approximately 2,000 times higher than that in resting muscle. The Ca-specific fragmentation of nebulin filaments takes place under these conditions. The Ca-specific fragmentation of nebulin filaments must destabilize the organization of thin filaments, and thus contribute to the tenderization of meat during post-rigor aging. In addition to the weakening of Z-disks (19), the weakening of rigor linkages formed between actin and myosin (20, 21), and the splitting of connectin filaments (7), all of which we have already reported to be important factors in the tenderization of meat during post-rigor aging, the fragmentation of nebulin filaments appears to be a fourth factor. All of these forms of structural weakening of myofibrils occur non-enzymatically at 0.1 mm Ca". The results presented in this paper support the "calcium theory of meat tenderization" proposed by us (7, 22). We wish to express our gratitude to Prof. K. Maruyama, Dr. S. Kimura, and Mr. T. Matsuura of the Faculty of Science, the University of Chiba, for providing the antinebulin antiserum, and for the technical guidance concerning the SDS-PAGE and immunoblotting techniques. REFERENCES 1. Wang, K. & Williamson, C.L. (1980) Proc. Natl. Acad. Sci. USA 77,3254-3258 2. Hu, D.H., Kimura, S., & Maruyama, K. (1986) J. Biochem. 99, 1485-1492 3. Locker, R.H. & Wild, D.J.C. (1986) J. Biochem. 99, 1473-1484 4. Wang, K. & Wright, J. (1988) J. Cell Biol. 107, 2199-2212 5. Wang, K. (1985) in Cell and Muscle Motility (Shay, J.W., ed.) Vol. 6, pp. 315-369, Plenum Press, New York and London 6. Perry, S.V. & Grey, T.C. (1956) Biochem. J. 64, 184-192 7. Takahashi, K., Hattori, A., Tatsumi, R., & Takai, K. (1992) J. Biochem. 111, 778-782 8. Fairbanks, G., Steck, T.L., & Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617 9. Laemmli, U.K. (1970) Nature 227, 680-685 10. Matsuura, T., Kimura, S., Ohtsuka, S., & Maruyama, K. (1991) J. Biochern. 110, 474-478 11. Aoyagi, T., Miyata, S., Nanbo, M., Kojima, F., Matsuzaki, M., Ishizuka, M., Takeuchi, T., & Umezawa, H. (1969) J. Antibiot. 22,558-568 12. Tatsumi, R., Hattori, A., & Takahashi, K. (1992) J. Biochem. 112,780-785 13. Maruyama, K., Yoshioka, T., Higuchi, H., Ohashi, K., Kimura, S., & Natori, R. (1985) J. Cell Biol. 101, 2167-2172 14. FUrst, D.O., Osborn, M., Nave, R., & Weber, K. (1988) J. Cell Biol. 106,1563-1572 15. Somerville, L.L. & Wang, K. (1987) Biochem. Biophys. Res. Commun. 147, 986-992 16. Somerville, L.L. & Wang, K. (1988) Arch. Biochem. Biophys. 262,118-129 17. Greaser, M.L., Cassens, R.G., Hoekstra, W.G., & Briskey, E.J. (1969) J. Food Sci. 34, 633-637 18. Buege, D.R. & Marsh, B.B. (1975) Biochem. Biophys. Res. Commun. 65, 478-482 19. Takahashi, K., Kim, O.H., & Yano, K. (1987) J. Biochem. 101, 767-773 20. Takahashi, K., Yamanoue, M., Murakami, T., Nishimura, T., & Yoshikawa, R. (1987) J. Biochem. 102,1187-1192 21. Yamanoue, M. & Takahashi, K. (1988) J. Biochem. 103, 843-847 22. Takahashi, K. (1992) Biochimie 74, 247-250 Vol. 112, No. 6, 1992