MUSCLE CONTRACTION* M. L. GREASER University of Wisconsin

395 UPDATE: MUSCLE CONTRACTION* M. L. GREASER University of Wisconsin The mechanism by which muscles contract and e x e r t f o r c e has been t h e s u b j e c t of intense study f o r many years. Few problems i n biology have received as much a t t e n t i o n o r have been attacked by as many s c i e n t i f i c d i s c i p l i n e s. The concerted e f f o r t s of biochemists, physiologists, and anatomists have now led t o a f a i r l y d e t a i l e d understanding of t h e c o n t r a c t i l e process. I n t h i s r e p o r t I hope t o present a concise sumrnary of recent findings and i n t e g r a t e t h i s new information i n t o t h e evolving model of contraction. The number of s t u d i e s contributing t o t h i s model is overwhelming and I w i l l, therefore, not attempt t o reference each statement in t h i s r e p o r t. The reader should consult a number of e x c e l l e n t books and reviews f o r more d e t a i l e d information and s p e c i f i c literature references (Cold Spring Harbor Symposium on Q u a n t i t a t i v e Biology, 1972; The S t r u c t u r e and Function of Muscle, 1973; Weber and Murray, 1973; Fuchs, 1974; Offer, 1974; Ebashi, 1974; Squire, 1975; Pepe, 197.5; Macknnan and Holland, 1975; Szent-Gyorgyi, 1975; Goody e t a l., 1975; Ebashi, 1976; Gergely, 1976; Trentham e t a l., 1976) The understanding of muscle contraction has been g r e a t l y aided by t h e development of a c l e a r p i c t u r e of muscle micro-anatomy. Muscle is composed of a l a r g e rider of individual c e l l s or f i b e r s whose long axes are oriented i n t h e d i r e c t i o n of f o r c e development. The c o n t r a c t i l e f o r c e is produced by c y l i n d r i c a l organelles c a l l e d myofibrils which f i l l over 8 6 of t h e volume of t h e muscle f i b e r. A d i a g r a m t i c sketch of a f i b e r and a myofibril a r e shown i n f i g u r e 1. There are approximately one thousand myofibrils i n each f i b e r cross -sect ion. The a l t e r n a t i n g l i g h t and dark bands which a r e v i s i b l e using l i g h t and e l e c t r o n microscopy a r i s e from t h e arrangement of t h i c k and t h i n myofilaments which make up t h e major portion of t h e myofibril (figure 1). A more d e t a i l e d view of t h e t h i c k and t h i n filaments i s shown i n f i g u r e s 2 and 3. The t h i n filament contains t h r e e major p r o t e i n s. A two stranded polymer of globular-shaped a c t i n molecules makes up t h e backbone of t h e filaments Tropomyosin l i e s i n t h e grooves on both s i d e s of t h e polymer. It i s a rod shaped molecule composed of two polypeptide chains. Each chain is believed t o be completely a - h e l i c a l and t h e two chains are coiled about each other. Troponin i s bound t o t h e tropomyosin and is located a t 38.5 nm intervals along t h e t h i n filament. It is composed of t h r e e polypeptide chains: TN-T, t h e tropomyosin binding subunit; TN-I, t h e subunit which i n h i b i t s actomyosin ATPase a c t i v i t y ; and TN-C, t h e calcium binding subunit. The length of a tropomyosin molecule is such that it extends along 7 a c t i n s. Thus t h e molar stoichiometry of a c t i n : troporqyosin: troponin i n t h e t h i n f i l a m e n t i s 7 : l : l.. * Presented a t t h e 29th Annual Reciprocal Meat Conference of t h e American Meat Science Association, 1976.

396 Figure 1. Diagram of a portion of a muscle fiber and of a myofibril.

397 THIN FIlAMENT B A Figure 2. 4. Diagrams of t h i c k and t h i n f i l a m e n t s A. Longitudinal view (Redrawn from Murray and Weber, 1974, and Cohen, 1975). B. Cross s e c t i o n a l view. (Redrawn from Squire, 1974).

398 B TROPOMYOSIN TN-T 'C PROTEIN THICK FllAMENT THIN FILAMENT Figure 3. A. Thick filament cross section. B. Thin filament cross section. A second troponin complex has been omitted from the left side of the drawing. The tropomyosins are shown in the relaxed position. The arrows indicate the path that tropomyosin follows when calcium is bound to troponin. (Redrawn from Cohen, 1975).

399 The t h i c k filament s t r u c t u r e i s a l s o shown i n Figures 2 and 3. The major protein in t h e t h i c k filaments i s myosin, and t h e heads of t h e myosin molecules extend perpendicularly from t h e t h i c k filament s h a f t. These myosin heads c o n s t i t u t e t h e cross-bridges which reach out t o grab t h e a c t i n and cause a s l i d i n g of t h e two kinds of filaments past each other. S e t s of cross-bridges a r e located a t 14.3 nm i n t e r v a l s along t h e t h i c k filaments except f o r a small bare zone a t t h e very c e n t e r. The diagrams ( f i g u r e s 2 and 3 ) show t h r e e cross-bridges a t each i n t e r v a l, but models with two or four cross-bridges per s i t e cannot as y e t be r u l e d o u t. The t h i c k filament a l s o contains another p r o t e i n c a l l e d C p r o t e i n. T h i s protein binds t o t h e t h i c k f i l a m e n t shaft a t 42.9 nm i n t e r v a l s. Recent evidence suggests t h a t t h e r e a r e t h r e e C p r o t e i n molecules a t each binding s i t e and that they a r e wrapped perpendicular t o t h e t h i c k filament's long axis (figure 3 ). The function of t h i s protein and whether it r e l a t e s d i r e c t l y t o t h e c o n t r a c t i l e process has not been determined. Calcium ions a r e responsible f o r changing t h e myofibril from t h e r e s t i n g t o t h e contracting state. Calcium binds t o troponin and causes it t o change i t s shape. The troponin then moves tropomyosin t o a p o s i t i o n deeper i n t h e t h i n filament groove. The myosin binding s i t e on t h e a c t i n i s now exposed and t h e cross-bridges can a t t a c h and detach t o rachet t h e t h i c k and t h i n filaments past each other. When t h e calcium l e v e l is low, t h e troponin allows tropomyosin t o r o l l or s l i d e out of t h e t h i n filament grooves and i n t o a p o s i t i o n that s t e r i c a l l y prevents t h e myosin from binding t o a c t i n. The source of t h e calcium ions t o a c t i v a t e contraction i s an i n t r a c e l l u l a r membrane system c a l l e d t h e sarcoplasmic reticulum. Figure 4 d e p i c t s diagramatically how a portion of t h e sarcoplasmic reticulum i n mammalian s k e l e t a l muscle i s believed t o appear. This membrane system is composed of interconnected tubules and chambers which completely e n c i r c l e each myofibril. The sarcoplasmic reticulum m i n t a i n s a high i n t e r n a l calcium concentration during rest by means of an ATP-driven t r a n s p o r t system, thereby keeping t h e calcium ion concentration i n the sarcoplasm bathing t h e myofibrils a t 10-7 molar o r lower. When t h e sarcoplasmic reticulum receives a s i g n a l, it r e l e a s e s calcium i n t o t h e sarcoplasm which they r a p i d l y d i f f u s e s t o t h e troponin in t h e t h i n f i l a m e n t s. The sarcoplasmic reticulum pumps t h e calcium back i n s i d e i t s chambers after t h e c o n t r a c t i l e s i g n a l ceases. The message which causes t h e calcium r e l e a s e from t h e sarcoplasmic reticulum is conveyed by means of t h e transverse tubular system o r T system. The transverse tubules ( f i g u r e 4 ) are extensions of t h e e x t e r n a l c e l l membrane and function t o transmit t h e e x c i t a t o r y impulse r a p i d l y from t h e f i b e r surface t o t h e c e l l i n t e r i o r. The rapid passage of t h e s i g n a l throughout t h e muscle c e l l assures a n e a r l y synchronous a c t i v a t i o n of a l l t h e myofibrils. The nature of t h e s i g n a l from t h e T tubules t o t h e sarcoplasmic reticulum i s not p r e s e n t l y understood.

401 An e l e c t r o n micrograph of a portion of a muscle c e l l f u r t h e r demonstrates t h e arrangement of t h e myofibrils, t h e sarcoplasmic reticulum, and t h e T tubules ( f i g u r e 5 ). The combination of a T tubule plus t h e two c l o s e l y apposed e l e m n t s of t h e sarcoplasmic reticulum on e i t h e r side i s r e f e r r e d t o as a t r i a d. I n mammalian s k e l e t a l muscle t h e t r i a d s are located near t h e junctions between t h e A and I bands ( f i g u r e 5 ). The e n c k c l i n g of each myofibril by t h e sarcoplasmic reticulum mans t h a t t h e d i f f u s i o n distance f o r calcium is s h o r t and enables t h e c e l l t o change from r e l a x a t i o n t o contraction and back t o r e l a x a t i o n i n a r e l a t i v e l y s h o r t period of time. A c l e a r e r view of t h e events t h a t occur a t t h e cross-bridges has emerged a s a r e s u l t of recent enzyme k i n e t i c s studied on ATP hydrolysis by myosin and actomyosin. A four step scheme of t n e hydrolysis cycle is shown i n f i g u r e 6. In t h e absence of ATP, myosin and a c t i n bind t i g h t l y t o each other ( s t a t e 3 ), and t h i s bond is t h e same t h a t occurs during r i g o r mortis. ATP and Mg++ cause a c t i n and myosin t o d i s s o c i a t e ( s t a t e 4). The myosin then cleaves t h e ATP t o produce ADP and phosphate ( s t a t e 1) while it remains dissociated from the a c t i n. Following t h e hydrolysis s t e p t h e a c t i n and rqyosin combine (state 2 ) and the ADP and phosphate are released. Kinetic s t u d i e s i n d i c a t e that w i t h myosin alone t h e binding of ATP and its hydrolysis occw r a p i d l y but t h e r e l e a s e of ADP and phosphate is r e l a t i v e l y slow. Addition of a c t i n t o t h i s system markedly a c c e l e r a t e s t h e ATP s p l i t t i n g r a t e because it speeds t h e rate of ADP and phosphate release. Thus i n t h e absence of a c t i n the conversion from s t a t e 2 t o state 3 would be t h e rate l i m i t i n g s t e p.. This same cycle is Shawn i n figure 7 but displayed in terms of t h e During r e s t t h e crosscrossbridges and t h i c k and t h i n filaments bridges are not attached but contain bound D, phosphate, and Mg++ (state 1). Calcium a c t i v a t e d changes in the t h i n filament allow attachment of myosin cross-bridges t o a c t i n (state 2 ). The crossbridges then make t h e i r power strokes, s l i g h t l y displacing t h e two filaments r e l a t i v e t o each other. The cross-bridges are detached upon t h e binding of ATP ( s t a t e 4 ). Myosin then s p l i t s t h e ATP and the crossbridge r e t w n s t o i t s ''cocked" p o s i t i o n ( s t a t e 1). This cycle may be repeated many times during a s i n g l e contraction. The cross-bridges cycle independently during a contraction and as a r e s u l t only a s m l l percentage may be attached t o t h e t h i n filaments a t any one time. D u r i n g rest most of t h e cross-bridges w i l l be i n t h e s t a t e 1 position. Figures 6 and 7 show that Mg++ remains bound t o t h e myosin heads throughout t h e cross-bridge cycle. Whether t h i s is t h e case remains uncertain, but it has been shown t h a t Mg++ is required f o r ATP mediated d i s s o c i a t i o n of a c t i n and myosin and t h a t Mg++ can bind t o myosin i n t h e absence of ATP. L e t ' s now examine t h e whole sequence of events t h a t occur during a s i n g l e contraction cycle. A sumrnary of these events i s Shawn i n f i g u r e 8. The i n i t i a l a c t i v a t i o n s i g n a l comes from t h e nerve axon which r e l e a s e s a c e t y l choline a t t h e motor end p l a t e. The a c e t y l choline d i f f u s e s t o the muscle f i b e r surface and combines with s p e c i f i c receptors

402 Figure 5. An e l e c t r o n micrograph of a small portion of a porcine muscle c e l l. The regions between the myofibrils contain t h e sarcoplasmic reticulum (S.R.) and t h e T tubules ( T ). Triads (composed of two swollen elements of the sarcoplasmic reticulum plus t h e T tubule) are located near t h e.junctions between t h e A and I bands. (Photograph was provided by D r. W i n Stromer, Iawa S t a t e University),. X 30,000.

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1, NERVE RELEASE OF ACETYL CHOLINE 2, f"4m AM) T TUBULE DEFOlARIZATION 3, RULASE OF CALCIlM FRDM THE sc\fecopiasmic RETICUUM 4, CALCILM BINDING TO TRDPONIN 5, TFWININ MOVES TlXHWOSIN FROM BLOCKING POSITION 6, CROS BRIDGE AlTACH'IENT PND F I M SLIDING 7, s b M r n I C fieticulujl SEwSrERs CALCIUM 8, TRPUMYOSIN MxlEs TO BLOCKING POSITION 9, CRDSS BRIDGES AE ALL DETACHED d REMATION Figure 8. Muscle contraction cycle.

in the plasmalemma. The acetyl choline-receptor complex causes an alteration in the cell membrane '6 permeability, allowing sodium and potassium to move in and out of the cell respectively. The membrane's polarity in this region is lost, and the region of depolarization rapidly spreads out in all directions on the fiber's surface. The depolarization wave also passed down the T tubules to the cell's interior. The message then is transmitted to the sarcoplasmic reticulum which releases calcium into the sarcoplasm. Calcium diffuses into the myofibrils and binds to troponin on the thin filaments. Troponin changes its shape and moves tropomyosin from its blocking position to a place deeper in the actin helix groove. The myosin cross-bridges can then attach to the exposed sites on the actin, go through their power strokes, and cause the two sets of filaments to slide past each other. When the contraction signal ceases, the sarcoplasmic reticulum pumps calcium out of the sarcoplasm and back to its interior. Troponin changes its conformation and allows tropomyosin to mve back to the blocking position. The cross-bridges can no longer attach to the thin filaments and the muscle cell returns to the relaxed state, The whole process my then be repeated in response to another nerve s ignal. Literature Cited Cohen, C. 1975. The protein switch of muscle contraction. Sci. Am. 23336. Cold Spring Harbor Symposium on Quantitative Biology. 1972. Volume 37, The Mechanism of Muscle Contraction, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, Ekashi, S. 1974. Regulatory mechanism of muscle contraction with special reference to the Ca-troponin-tropomyosin system. Essays in Biochem. 1O:l. Ebashi, So 1976 38:293. Excitation-contraction coupling. Ann. Rev. Physiol. Fuchs, F, 1974. Striated muscle. Ann. Rev. Physiol. 36:461. Gergely, J. 1976. Excitation-contraction coupling-cardiac muscle events in the myofilament. Fed. Proc. 35:=83. Goody, R. S., K. C. Holmes, H. G. Mannherz, J. Barrington Leigh and G. Rosenbaum. 1975. Cross -bridge conf ormat ion as revealed by X-ray diffraction studies of insect flight muscles with ATP analogues Biophys. J. 15:687. 1975. Calcium transport in sarco- Macknnan, D. H. and P. C. Holland. plasmic reticulum. Ann. Rev. Biophys Bioeng. 4 : 377.

407. Murray, J M. and A. Weber. 1974. p r o t e i n s. Aci. Am. 230:58. The cooperative a c t i o n of muscle. Offer, G 1974. The molecular b a s i s of muscular b a s i s of muscular _. contraction. In Companion t o Biochemistry. Selected Topics f o r Further Studx. ed. A. T. Bull, J. R. Iagnado, J. 0. Thomas, K. F. pp. 623-671. Tipton, Longman Group Limited, London Peachey, L. D. 1965. The sarcoplasmic reticulum and transverse J. C e l l Biol. 25:209. tubules of the f r o g ' s s a r t o r i u s Pepe, F. A. 1975. S t r u c t u r e of muscle f i l a m e n t s from immunohistochemical and u l t r a s t r u c t u r a l s t u d i e s. J. Histochem. Cytochem. 2 3 5 4 3. Squire, J. M. 1974. Symmetry and three-dimensional arrangement of filaments i n v e r t e b r a t e s t r i a t e d muscle. J Mol. Biol. 90:153. Squire, J. M. 1975. Muscle filament s t r u c t u r e and muscle contraction. Ann. Rev. Biophys. Bioeng. 4:137. The S t r u c t u r e and Function of Muscle. Bourne, Academic Press, New York.. Szent-Gyorgyi, A. G. 19-77. Biophys J 15:707.. 1973. 2nd Edition, ed. G H. Calcium regulation of muscle contraction. Trentham, D. R., J. F. Eccleston, and C. R. Bagshaw. 1976. Kinetic analysis of ATPase mechanisms. Quart. Rev. Biophys. 9:217. Weber, A. and J, M. Murray, 1973. Molecular Control mechanisms i n muscle contraction. Physiol. Rev. 53:612. * * * M. L. Greaser: The present thinking on t h i s i s that t h e a c t i v a t e d stage r e a l l y is s t a t e 1 in Figure 7. The a c t i n combines with t h e crossbridge and it goes through i t s power stroke. The cross-bridge is more happy t o be over i n t h e t i l t e d position. That's why we f i n d it t h a t way in r i g o r. A 1 Pearson, Michigan S t a t e : position t o another one? Do they move from one attachment M. L. Greaser: Yes, there are many cycles of cross-bridge a t t a c h ment and detachment during a contraction cycle. Not a l l t h e crossbridges a r e attached a t t h e same t i m e. I n f a c t, only 10 or 20 percent may be attached a t one t i m e. So, we're going through a cycling process. Obviously, a cross-bridge has got t o f i n d an a c t i n t h a t ' s oriented i n t h e right d i r e c t i o n. We're only going t o f i n d one i n t h e right position every seventh a c t i n. The p e r i o d i c i t y of t h e cross-bridge is not exactly t h e same between t h e a c t i n and t h e myosin, So they don't j u s t release one and grab t h e seventh one darn t h e line.

408 A1 Pearson: The other question that 'I had deals with gap filaments. M. L. Greaser: Ron Locker from New Zealand has recently done some work in which he stretched bovine muscle way out beyond where there should be overlap between the thick and thin filaments. You would expect that 3.5 microns would be the maximum length to retain filament continuity in the myofibril. When he stretches these fibers to make sarcomere lengths of 9 or 10 microns he still finds that there's some material in the gap in between the thick and the thin filaments. It doesn't look like it's the same diameter as actin filaments. He thinks, I believe, there's some unknown protein and an additional set of filaments termed gap filaments in the myofibril. Now, you may have more information than I do about this. A1 Pearson: I think the way that he explains it, he says that the two thick filaments that have the gap filaments between adjacent ones, they go right through the 2 line and into the next sarcomere area and attach the two thick filaments together. Now, as far as I know, nothing has been related to muscle contraction in this area. He does feel that they have a very significant effect on tenderness. M. L. Greaser: This could well be. It could also make obsolete a number of the models that I have shown. I hope he's right. One thing I should point out is that the known myofibrillar proteins appear to account for more than go$ of the mass of the myofibril. If there are additional proteins that are involved in the filaments, the quantity of them must be fairly small or else they comigrate with some of the known muscle proteins. If we take a look at a polyacrylamide gel of a myofibril, we can account for almost all the bands as arising from myosin, actin, tropomyosin, troponin, C protein, a-actinin, and M protein. Uene Wierbicki: (Inaudible). Allen: I might add to Ebgene's comment. We have been working with some EM work on turkey red and white muscle during the last couple of years. One of the things that's very intersting is when you heat the white muscle to 82 degrees centigrade, which is the recommended internal temperature for turkey, all of the thin filaments in the I band disappear in the white muscle. In the red muscle, however, they are all still intact. The question I'd like to ask you is, one of the noticeable differences in both the raw state and the cooked state is there seems to be what we would call an N line in the red muscle but not in the white. What is the current status of thinking about this N line as a struc twe? M. L. Greaser: I haven't seen much about the N line in recent years. I can remember back when I started graduate school I did some electron microscopy on rat muscle. Im and behold, it had beautiful N lines. I have sort of kept an eye on the literature on the subject, but I don't have any recent information. Presumably, there is some kind of protein that's causing the N line's presence.

Allen: It appears t o become more pronounced a f t e r heating. The other t h i n g i s it appears that t h e A band goes s h o r t e r during heating than t h e I band does. Max Judge, Purdue: What's t h e nature of t h e bridge, t h e bridge i t s e l f? What's t h e chemical nature of t h e bridge? M. L. Greaser: The groups involved i n a t t a c h i n g t h e myosin t o t h e a c t i n a r e not known. The nature of t h e cross-bridge and t h e method of information transmission between t h e T tubules and t h e SR are t h e two major unanswered questions about t h e c o n t r a c t i l e process Dennis Campion, USDA: Do w e have any enzyme marker f o r t h e T tubules? When w e i s o l a t e it, dc we know that we have j u s t t h e T tubule? M. L. Greaser: The Na+-K+ ATPase could probably be used t o d i s t i n guish t h e T tubules from t h e sarcoplasmic reticulum fragments. However, a bigger problem would be that you wouldn't be a b l e t o d i f f e r e n t i a t e between T tubules and plasmalemma fragmnts. * * * Thank you very much. We have come a long way from Colonial America t o P r o t e i n C. I would l i k e t o a s k Gary Smith if Marion said anything t h a t you understood? B i l l StrinRer: Gary Smith: No. B i l l S t r i n g e r : I would l i k e t o t a k e t h i s moment t o thank a l l of t h e speakers f o r being on the program, f o r t h e i r very excellent present a t i o n s, and e s p e c i a l l y t h e i r good visuals. I don't t h i n k I have ever been t o a conference that has had as m n y good v i s u a l s, o r should I say l a c k of poor v i s u a l s, as we had a t t h i s one. I t h i n k you should be congratulated on that. I ' d like t o e s p e c i a l l y thank t h e program committee members, c e r t a i n l y t h e coordinators and chairmen f o r keeping us on t i m e, and f o r doing a very good job of organization. I know this i s going t o come up l a t e r, b u t I have t o personally thank Leon and Max and t h e i r staff here, f o r keeping us i n complete happiness. Everything we have needed they have provided it. They have t r i e d t o do everything, and have done everything, t o make t h i s meeting very successful. I have one last t h i n g f o r y m aembers, you people who have been i n attendance, you have a l s o done a great job of being i n here where t h e a c t i o n w a s going on. Many times I had nobody t o t a l k t o outside t h e meeting room. Now, on a more s e r i o u s note, w e a r e very happy t o have people from other countries with US. Some a r e members. Those that are not I have an a p p l i c a t i o n f o r you. For t h e other people that are v i s i t i n g us, we do have membership blanks a v a i l a b l e. The new form is a n attempt t o simplify and t o give something of t h e purpose of t h e organization and membership c l a s s i f i c a t i o n. We have with us, and I hope most of them

410 are still here, Edmond Prost from Poland. Edmond, would you stand please. The next gentleman from France, I may not do justice to your name, Renae Beautifunge. for this meeting Ed? W i l l you please stand. Are you here just Answer: Yes. Bill Stringer: Then, of course, Dr. Doug Rhodes, who we heard from in the Business Meeting. Is he still here? He is one of our members, but we're happy to have him here, Ben Heidelnan from Denmark, we're happy to have you here, too. Not from across the ocean, but from across the border, Dr. Howard Swatland, we're happy to have you here With US. * * *