sign in sign up
<<
Home , Costamere, Nebulette, Nebulin, Protein filament, Synemin, Titin

BIOCHEMISTRY - IMPACT ON MEAT TENDERNESS

Postmortem Changes in the Myofibrillar and Other C'oskeletal in Muscle

RICHARD M. ROBSON*, ELISABETH HUFF-LONERGAN', FREDERICK C. PARRISH, JR., CHIUNG-YING HO, MARVIN H. STROMER, TED W. HUIATT, ROBERT M. BELLIN and SUZANNE W. SERNETT

introduction filaments (), and integral Z-line region (a-, Cap Z), as well as proteins of the intermediate filaments (, The of "typical" vertebrate cells contains paranemin, and ), Z-line periphery () and three filament systems, namely the -7-nm diameter underlying the membrane (filamin, -containing , the -1 0-nm diameter in- , , and ) are listed along with an esti- termediate filaments (IFs), and the -23-nm diameter tubu- mate of their abundance, approximate molecular weights, lin-containing (Robson, 1989, 1995; Robson and number of subunits per molecule. Because the et al., 1991 ).The contractile myofibrils, which are by far the are the overwhelming components of the skeletal major components of developed cells and cytoskeleton, the approximate percentages of the cytoskel- are responsible for most of the desirable qualities of muscle eton listed for the myofibrillar proteins (e.g., , actin, foods (Robson et al., 1981,1984, 1991 1, can be considered , a-actinin, etc.) also would represent their ap- the highly expanded corollary of the system proximate percentages of total myofibrillar protein. of non-muscle cells. The myofibrils, IFs, cell membrane skel- eton (complex protein-lattice subjacent to the ), Some Important Characteristics, Possible and attachment sites connecting these elements will be con- Roles, and Postmortem Changes of Key sidered as comprising the muscle cell cytoskeleton in this Cytoskeletal Proteins review. A subset of the proteins listed in Table 1 are known to Selected Myofibrillar and Other Cytoskeletal undergo postmortem proteolysis and/or to be highly sensi- Proteins in Developed Mammalian Skeletal tive to proteolysis. These include titin, , desmin, Muscle Cells paranemin, synemin, filamin, and the -T (TN-T) sub- unit of troponin. Nearly all, if not all, of these proteins are The entire cytoskeleton of a muscle cell probably consists likely to have important roles in organizing and maintaining of hundreds of different kinds of proteins, including many the integrity and strength of the contractile myofibrils and of present in very small amounts, and a large number yet-to-be the overall cytoskeleton of the skeletal muscle cell. A sum- discovered. A list of some selected cytoskeletal proteins is mary of important characteristics, possible roles, and post- shown in Table 1. Proteins of the myofibrillar thick filaments mortem changes of these seven proteins are described in a (myosin, C-protein, and H-protein), M-line region (myomesin, "table format," which hopefully will enable readers of this M-protein, creatine , and skelemin), thin filaments (ac- review to get a concise overview of the respective proteins. tin, tropomyosin, troponin, , and nebulin), titin Although space limitations unfortunately do not permit in- clusion of many of the original citations for specific charac- teristics and postmortem changes of proteins described *Richard M. Robson, Muscle Biology Group, 3 7 IO Mo- herein, most can be found within the citations that are in- lecular Biology Building, Iowa State University, Ames, IA cluded. Many of the earlier studies reported on titin, nebulin, 500 7 1-3260. desmin, and synemin also can be found in the reviews by 'Elisabeth Huff-Lonergan, Department of Animal and Robson and Huiatt (1983), Robson et al. (1984, 19911, and Diary Sciences, Auburn University, Auburn, AL 36849. Robson (1995). Reciprocal Meat Conference Proceedings, Volume SO, Titin (Tables 2 and 3) and nebulin (Tables 4 and 5) are 1997. two very large, long proteins that run in parallel with the

50th Annual Reciprocal Meat Conference 43 -TABLE 1, Some Selected Myofibrillar and Other Cytoskeletal Proteins in Developed Mammalian Skeletal Muscle Cells. -Protein Primary Myofibrillar/Cytoskeletal Location Approximate o/o of Cytoskeleton Approx. MW (# of Subunits) Myosin Thick filament 45 520,000 C-Protein (M~BP-C) Thick filament 2 130,000 H-Protein (My6P-H) Thick filament 1 74,000 Myomesin M-line 2 185,000 M-Protein M-line 1 165,000 Creatine Kinase M-line <1 80,000 Skelemin M-line (peripheral) <1 195,000 Actin Thin filament 20 42,000 Tropomyosin Thin filament 5 66,000 Troponin2 Thin filament 5 69,000 Tropomodul in Thin filament-free end <1 41,000 Titin2 Longitudinal sarcomeric 10 3,700,000 filaments (Z- to M-line) Nebulin2 Parallels (part of) thin 3 773,000 filaments to the Z-line a- Acti ni n Z-line (integral) 2 204,000 Cap Z Z-line (integral) <1 66,000 Desmin' Intermediate filaments <1 212,000 at Z-line (peripheral) Paranemin2 Intermediate filaments <1 356,000 at Z-line (peripheral) Synemin' Intermediate filaments <1 372,000 at Z-line (peripheral) Filamin2 Z-line (peripheral) <1 560,000 Costamere-Mem brane Dystrophin -Mem brane <1 854,000 Talin Costamere-Membrane <1 5 36,000 Vinculin Costamere-Membrane <1 11 6,000

long axis of the , myofibrils, and muscle cell. heretofore were considered to be IF-associated proteins (for These haproteins, therefore, should contribute significantly review, see Bellin et al., 19971, have recently been shown in to maintaining the longitudinal continuity and integrity of our lab to actually be IF proteins (Table 9). Whereas both muscle cells. Both proteins undergo proteolysis early post- proteins are present in all types (skeletal, cardiac, and smooth! mortem and are excellent substrates for the calpain pro- of developing and mature muscle cells, paranemin is down- teolytic system. regulated and present in only very small amounts in mature Filamin is generally considered as playing an important skeletal muscle cells, and synemin is down-regulated and cytoskeletal cross-linking role in cells, but much more infor- present in only very small amounts in mature mation is needed to understand its role in postmortem muscle cells. It is likely that these two proteins form heteropolymeric (Table 6). Two recent reports indicate that filarnin undergoes IFs with desmin in mature skeletal muscle cells, with Postmortem proteolysis (Uytterhaegen et al., 1994; Huff- paranemin present in a very low molar ratio, and synemin Lonergan et al., 1996a), but not as early postmortem as do present in a low molar ratio, to desmin. Both paranemin nebdin and titin (Huff-Lonergan et al., 1996a). and synemin may play an important cytoskeletal role in link- The major protein composing the IFs of adult skeletal ing desmin IFs to other structures in muscle cells (Table 10). muscle cells is desmin (Table 7). The IFs in skeletal muscle Because they are both extremely sensitive to proteolysis, it cells Primarily run perpendicular to the long axis of the is likely that paranemin and synemin are degraded early myofibrils and skeletal muscle cell (Robson et al., 1981; postmortem and, in turn, decrease ability of the IFs to main- Robson, 1995). As a result, the IFs are believed to play a tain muscle cell integrity (Table 10). major role in maintaining overall muscle cell integrity (Table One of the proteins that has been studied extensively, 8). In contrast to the titin and nebulin components, post- both with regard to its role in regulation of contractionhe- mortem degradation of the IFs would be expected to result laxation of muscle (for review of its characteristics and role in loss Of integrity perpendicular to the long axis of muscle in muscle, see Farah and Reinach, 1995) and in postmor- cells (Table 8).Two proteins, paranemin and synemin, which tem proteolysis (Ho et al., 1994; Huff-Lonergan et al., 1995,

American Meat Science Association TABLE 2. Some Important Characteristics of Muscle Titin.

1. Titin is a fairly insoluble, huge myofibrillar protein present in skeletal (Mr= 3.7 x 10‘) and cardiac (Mr = 3 x 106) muscle cells of vertebrates and invertebrates (many contain much smaller ) (for recent titin reviews, see Labeit et ai., 1997; Maruyama, 1997). It was discovered by Dr. Kuan Wang, University of Texas at Austin, in 1979 (Wang et a]., 1979). Titin is sometimes called connectin, and comprises about 8-10y0 of total myofibrillar protein in vertebrate skeletal muscle. Titin is high in b-sheet (-60%) and b-turn (-3O’h) structures, and very low in a-helix (Kimura et al., 1992). 2. A titin molecule spans -one half the width of a (Trinick, 19921, Le., from the middle ofthe Z-line (titin’s N-terminus; Gautel etal., 1996) to just past the middle of the M-line (titin’s C-terminus; Obermann et al., 1996) and, thus, forms a third filament system within the myofibrillar sarcomere. 3. Intact skeletal muscle titin (T1 or a-connectin) has usually been isolated and purified in the presence of denaturing solvents. A degraded (M~= 2.2 x 106) ”native” form of skeletal muscle titin (T2 or b-connectin), missing a large piece Of the Z-line end of titin, has often been isolated without the use of denaturing solvents and observed by electron microscopy. Titin is a very long mm) thin molecule, resembling a long string of -4 nm diameter pearls. 4. That part of titin located within the sarcomeric A-band is evidently bound firmly to the outside of the thick filament, and is relatively inelastic. Much of the part of titin located between the end of a thick filament and the Z-line is “elastic” (Maruyama et al., 1989; Wang et a/., 19911, The part of titin near the Z-line is associated with the thin filaments (Trombitas et ai., 19971, but, overall, titin is not as firmly attached to thin filaments as it is to the thick filaments. 5. The cDNAsequence of titin is known (Labeit and Kolmerer, 1995a). Much of the titin molecuk consists of two domains (similar to fibronectin type 111 and immunoglobulin C2), each about 100 amino acids long. Both are present in the A-band part of titin. The c2 domains, but not type 111 domains, are present in the I-band part of titin nearer to the Z-line. Multiple copies of a Pro-Glu-Val-Lys (PEVK) repeat are present in one region (near Nr-line) of the I-band part of titin, and may account for much of titin’s elasticity and resting (passive) tension of muscle at longer Sarcomere lengths (Gautel and Goulding, 1996; Linke et al., 1996). The elasticity and foldindunfolding properties of individual titin molecules are now under intense scrutiny (e.g., Kellermayer et al., 1997; Rief et al., 1997; Tskhovrebova et al., 1997). 6. Purified titin is degraded to T2 (b-connectin) by calpain (Kimura et al., 1992).Titin in purified bovine myofibrils also is degraded by calpain (Zeece et al., 1986; Huff-Lonergan et al., 1996a; Suzuki et al., 1996). Interestingly, muscle-specific calpain (~941binds to at least two sites (Nz-line region and near C-terminus) on the titin molecule (Kinbara et al., 1997). 7. Several titin isoforms varying in size have been identified (Wang et al., 1991; Labeit and Kolmerer, 1995a), and have been correlated to muscle stiffness and elasticity. It has been hypothesized that isoform and species differences in the PEVK region of titin may be related to meat tenderness (Tanabe et al., 1997). 8. Titin interacts in vitro with myosin (rod part, not the S1 heads), C-protein, AMP deaminase, M-line proteins (myomesin and M-protein), actin, and the integral Z-line protein, a-actinin (Trinick, 1994; Keller, 1995; Labeit et al., 1997; Maruyama, 1997; Ohtsuka et al., 1997). Adapted from Robson et a/. (1991)and Robson (19951.

TABLE 3. Possible Roles and Postmortem Changes of Muscle Titin. Possible Roles of Titin 1. In developing muscle, titin is one of the first sarcomeric proteins detected during , and may play a role as part of a morphogenetic scaffolding during sarcomeric organization (Handel et al., 1989; Fulton and Isaacs, 1991; van der LOOP et al., 1996; Maruyama, 1997). 2. In mature muscle, titin forms a third filament system within the myofibrils that provides sarcomeric alignment (e.g., keeps myosin thick filaments in register, regulates the length of the myosin thick filaments; Bennett and Gautel, 1996), and helps maintain overall structural integrity of the sarcomeres, myofibrils and muscle cells (for reviews, see Labeit et al., 1997; Maruyama, 1997). Titin appears to act as a molecular blueprint or ruler for the layout of much of the sarcomere, even including width and structure of the Z-lines (Gautel et al., 1996). 3. Apart of the titin molecule/filament in the I-band and, thus, not associated with the A-band (thickfilaments), appears to provide an elastic element of the sarcomeres, accounts for the ”gap filaments” observed in over-stretched bovine skeletal muscle many years ago by Dr. Ron Locker and associates (1 975), and appears to be involved in the generation of resting (passive)tension (Wang et al., 1993; Labeit and Kolmerer, 1995a; Labeit et al., 1997; Maruyama, 1997). Postmortem Changes in Titin 1. It is reasonable to expect that titin plays a very significant role in determining degree of overall integrity, or strength, of myofibrils, muscle cells and . Titin comprises longitudinal structures in the . It is the only protein that is Present throughout the entire length of the sarcomere, and, for that matter, throughout the entire length of the very long myofibrils. 2. Titin is one of the most proteolytically-susceptible myofibrillar proteins, and undergoes significant degradation during postmortemaging of bovine muscle (Lusby et al., 1983; Bandman and Zdanis, 1988; Fritz and Greaser, 1991 ; Huff-Lonergan et ai., 1995, 1996a,b; Taylor et al., 1995). The rate of degradation of T1 (intact titin) is related to tenderness measurements, with significant degradation occurring in tender beef samples by one day postmortem, and nearly complete degradation occurring by three days postmortem. In tougher beef samples, rate of postmortem degradation of T1 is slower, but significant degradation occurs between one and three days postmortem (Huff-Lonergan et ai., 1996a). 3. The rate of postmortem degradation of titin (Tl) in muscle from Bo5 indicus crossbred cattle (less tender meat) is slower (Ho et a[., 1997) than the rate in muscle (relatively tender meat) from Bos taurus cattle (Ho et al., 1996). 4. The rate of postmortem degradation of Tl in muscle from Callipyge lambs, which produce tougher meat, is slower than occurs in muscle samples from normal lambs (Koohmaraie et al., 1995). 5. The titin in purified bovine myofibrils is an excellent substrate for the calpains using postmortem-like conditions (Huff-Lonergan et ai., 1996a). The resulting major proteolytic fragments of titin appear similar to those observed during postmortem aging of bovine muscle.

50th Annual Reciprocal Meat Conference 45 TABLE 3. (Continued) 6. Electron microscope studiesof postmortem bovine muscle (e.g., Taylor et al., 1995; Ho et al., 1996, 1997) suggest that one ofthe more Pronounced ultrastructural changes are breaks near the myofibrillar Z-lines, a region occupied by titin. 7. Overall, the degree of titin degradation appears to parallel measurements of meat tenderness.

TABLE 4. Some Important Characteristics of Skeletal Muscle Nebulin.

1. Nebulin is an insoluble, very high molecular weight myofibrillar protein (M, = 6 to 9 x 105) present in skeletal (not cardiac or smooth) muscle cells of vertebrates. It accounts for about 3 to 4% of total myofibrillar protein. Nebulin was discovered by Dr. Kuan Wang, University of Texas at Austin, in 1980. A mini-nebulin-like protein (; Mr = 1 x 105) has been identified in cardiac muscle (Moncman and Wang, 1995). 2. Nebulin is a very elongated (-1 mm), very thin (-1 nm) molecule running in parallel with the thin actin filaments (i.e., it is part of the sarcomeric thin filaments). Nebulin's N-terminus is near the distal (free) end of the thin filament and its C-terminus is located in the 2-line (Chen et a]., 1993; Wax et al.. 1996). 3. There is a positive correlation between the size of nebulin isoforms and the length of thin filaments from different species and from different skeletal muscles within a species (Kruger et al., 1991; Labeit et at., 1991). 4. Complete primary structure studies (Labeit and Kolmerer. 1995b) of human nebulin revealed 185 repeating copies of -35-residue modules, which comprise 97O/b of nebulin's mass, with each module representing the structural domain that binds to one actin monomer. Most of these modules are very basic, pl = 10, and are predicted to be a-helical (Chen and Wang, 1994; Pfuhl et al., 19961, but have not always been found to be SO by actual measurements (Chen and Wang, 1994). There also are 22 seven-module super repeats corresponding to the 38.5 nm thin filament repeats along the 3-D contour of actin in the thin filament. 5. vitro studies indicate that some of nebulin's many quasi-repeatingdomains (modules) can bind to F-actin, tropomyosin, troponin, (see Wang et al., 1996), and, perhaps via its C-terminal domains, to a-actinin in the Z-line (Nave et at., 1990). Expressed fragments representing the part of nebulin (N-terminal half) in association with the distal (free) end of thin filaments also bind in vitro to myosin and myosin heads (Jinand &'a%, 1991; Root and Wang, 1994; Wang et al., 1996). Cross-linking studies indicate that nebulin modules bind to actin's N-terminus in subdomain 1 (Shih et al., 1997). 'Adapted from Robson et a!. (1 997) and Robson (l995J.

TABLE 5. Possible Roles and Postmortem Changes of Skeletal Muscle Nebulin. Possible Roles of Nebulin 1. In developing muscle cells, nebulin becomes associated with nascent myofibrils after myosin and titin. Likewise, the mature striated patterns of myosin and titin precede development of the nebulin striations (Kurpakus, 1988; Moncman and Wang, 1996). The actin filaments then form thin filaments of uniform length. Thus, nebulin appears to play an important role in organization of the thin filaments during myofibrillogenesis. 2. In the mature sarcomere, nebulin presumably continues to act as a template for assembly (i.e., acts as a protein ruler regulating thin filament length) and/or scaffold for stability of the thin filaments. Rather than constitute a separate set of longitudinal filaments in the sarcomere as was Once believed, nebulin evidently comprises of the thin filament structure and runs along each of the two long F-actin grooves (Jinand Wang, 1991; Labeit et al., 1991 ;Wang et al., 1996). Because nebulin interacts with tropomyosin and troponin in vitro, it likely runs in association with them in the two actin grooves ("ang et al., 1996). Nebulin appears to wrap around the outer edges of the actin filament at or near where troPomYosin, myosin S-1 and some other actin binding proteins interact with actin (Shih et al., 1997). 3. Nebdin may help linkjanchor the thin filament firmly to the Z-line structure. 4. Nebdin may participate in active contraction of skeletal muscle, and help regulate actin and myosin interaction (Jinand \Wan% 1991; Root and WaW, 1994; Wang et ai., 1996). Postmortem Changes in Nebulin 1. It is reasonable to assume that nebulin, which runs the entire -1 mm length of the thin filaments, and may help anchor the thin filaments to the Z- lines, is responsible for a significant degree of the overall cytoskeletal integrity of myofibrils and muscle cells, and, in turn, the integrity of muscle tissue. 2. Nebdin is one of the most proteolytically-susceptible myofibrillar proteins, and undergoes significant degradation during postmortem aging of bovine muscle (Lusby et al., 1983; Fritz and Greaser, 1991; Huff-Lonergan et al., 1995, 1996a,b; Taylor et al., 19951. Intact nebulin is degraded even slightly faster than is intact titin (Huff-Lonergan et al., 1996a,b). The rate of postmortem degradation of intact nebulin is related to tenderness measurements, with some degradation occurring in myofibrils from more tender beef samples by one day postmortem, and complete degradation of intact nebulin occurring by three days postmortem. In myofibrils from tougher beef samples, rate of postmortem degradation of intact nebulin is slower, but very significant degradation occurs between one and three days postmortem (Huff-Lonergan et al., 1996a). 3. The rate of postmortem degradation of nebulin in muscle from 50s indicus crossbred cattle, which produce less tender meat, is slower (Ho et al., 1997) than the rate in muscle from 50s taurus cattle, which are genetically predisposedto produce relatively tender meat (Ho et al., 1996). 4. The nebdin in purified bovine myofibrils is an excellent substrate for the calpains using postmortem-like conditions (Huff-Lonergan et al., 1996a). 5. Results of electron microscope studies of postmortem bovine muscle (e.g., Taylor et al., 1995; Ho et al., 1996, 1997) suggest that one of the more Pronounced ultrastructural changes is the occurrence of breaks near the myofibrillar Z-lines, a region occupied by nebulin. 6. Overall, the degree of nebulin degradation appears to parallel measurements of meat tenderness.

46 American Meat Science Association TABLE 6. Some Important Characteristics, Possible Roles, and Postmortem Changes of Muscle Filamin. Important Characteristics of Filamin Filamin (also called ABP = actin-binding protein) is an -500,000 molecular weight homodimer (Wang et al., 1975; Hartwig and Stossel, 1975). Several filamin isoforms exist, but adult skeletal and cardiac muscle are extremely similar, if not identical (Price et al., 1994). The C-terminal ends of the two polypeptides are linked to each other to form -V-shaped hornodimers, whereas the two N-termini form the tips of the V-shaped molecule, and contain actin-binding domains (Gorlin et al., 1990). Calpain cleaves the -250 kDa filamin subunits into -240 and -1 0 kDa fragments (Davies et al., 1978). The small cleavage fragment is from the C-termini of each of the two subunits in the homodimer. As a result, the 240 kDa fragment can bind to, but no longer crosslink, actin filaments. Filamin is located at the periphery of myofibrillar Z-lines (see Price et al., 1994, and refs. therein), and probably at the costameres located along the sarcolemma that have a periodicity that matches the Z-lines of the nearby peripheral layer of myofibrils. The costameric location of filarnin has not been clearly documented. Possible Role(s) of Filamin 1. Filamin, with its homodimeric structure, can crosslink actin filaments in parallel and in orthogonal arrays (see Price et al., 1994). There is suggestive (circumstantial) evidence that filamin also may associate with intermediate filaments (see discussion in Price et al., 1994). Taken in toto, filamin appears to be a cytoskeletal cross-linking protein. 2. It seems we don’t know much about the precise role(s) of filamin in skeletal muscle cells! Postmortem Changes in Filamin 1. Filamin in skeletal muscle samples, which have been injected with CaC12 to stimulate proteolysis and tenderization, exhibits increased degradation (Uytterhaegen et al., 1994). 2. Filamin is degraded at different rates in myofibrils isolated from naturally-aged beef muscles having different shear force values, with some filamin degraded by three days postmortem in tender beef samples, but not until about seven to fourteen days in tougher beef samples. Thus, filarnin is degraded more slowly postmortem than are nebulin and titin (Huff-Lonergan et al., 1996a). Filamin degradation may be involved in tenderization, via disruption of key cytoskeletal linkages, and/or is at least an indicator of postmortem proteolysis. 3. Filarnin in purified bovine myofibrils is a substrate for the calpains using postmortem-like conditions (Huff-Lonergan et al., 1996a). The resulting major proteolytic fragment of filamin is similar to that observed during postmortem aging of bovine muscle. 4. Much more must be learned regarding filamin’s properties (e.g., location, interacting proteins, etc.) and function in skeletal muscle in order for us to address in detail the significance of postmortem changes in filamin.

TABLE 7. Some Important Characteristics of Muscle Desmin.

1. Desmin is one of the major types/isomers of protein comprising 10-nm diameter intermediate filaments (IFs) that, in turn, are part ofthe cytoskeleton of nearly all animal cells (Robson, 1989, 1995). 2. Desmin is a fairly insoluble cytoskeletal protein (Mrof subunit = 53,000) present in skeletal, cardiac and most cells of vertebrates. A tetramer (Mr= 21 2,000) of desmin comprises the protofilament building block of the IFs (Ip et al., 1985). Desmin is easily degraded by several proteinases, including calpain (O’Shea et al., 1979). 3. Desmin was successfully purified from mammalian skeletal muscle in our lab (O‘Shea et al., 1979, 19811. The purified protein has the ability, via several structural intermediates, starting with the tetrameric protofilament building block, to self-assemble into synthetic, 1 0-nm diameter, very long (>1-2 mm) filaments (Ip et al., 1985). IFs are much more dynamic structures than was originally believed (e.g., their assernbly/disassembly is mediated by covalent modifications; Robson, 1989; Huang et al., 1993; Zhou et al., 1996, Graves et al., 1997; lnagaki et al., 1997), but our understanding of the control of their assembly/disassembly properties is still very incomplete. 4. lmmunoelectron microscope localization studies in our lab indicated that desmin IFs encircle the Z-line and radiate out perpendicular to the myofibril axis to ensnare and connect adjacent myofibrils (Richardson et al., 1981). The desmin IFs also link myofibrillar Z-lines to subcellular organelles, such as nuclei and mitochondria, and the 2-lines of the peripheral layer of cellular myofibrils to the cell membrane skeleton (Robson, 1995).

’Adapted from Robson et al. (1991) and Robson (1995).

50th Annual Reciprocal Meat Conference 47 TABLE 8. Possible Roles and Postmortem Changes of Muscle Desmin. Possible Roles of Desmin 1. Desmin IFs may help align and tie together adjacent myofibrils in developing muscle cells, but this remains unclear. Studies involving isometric force measurements of single myotubes, with truncated desmin expression, indicate a critical role for desmin IFs in providing mechanical stability for force development (Feng et al., 1994). 2. Desmin IFs contribute to resting tension in adult muscle cells, but only at unusually long sarcomere lengths b4.5mm) (Wang et al., 1993). 3. In the developed muscle cell, desmin IFs are believed to play an important cytoskeletal role in connecting the myofibrils and, in turn, tie or anchor the myofibrils to subcellular organelles and the cell membrane, i.e., desmin IFs may play a significant role in maintaining overall integrity and organization of the skeletal muscle cell (Robson, 1995). In some muscles (especially those heavily used such as the diaphragm), desmin ”knockout” mice exhibit severe disruption of muscle cellular organization (Milner et al., 1996), which also indicates a major role for desmin in maintaining integrity of the muscle cell cytoskeleton. Postmortem Changes in Desmin 1. It is reasonable to assume that desmin IFs, which primarily run perpendicular to the long axis of the myofibrils and muscle cell, and connect all adjacent myofibrils and the peripheral layer of cellular myofibrils to the cell membrane skeleton, are important in helping to maintain overall muscle cell integrity. 2. Desmin is degraded in postmortem muscle (Robson et al., 1980, 1981 ; Koohmaraie et al., 1984a,b; Hwan and Bandman, 1989; Taylor et a/., 1995; Ho et al., 1996, 1997; Huff-Lonergan et al., 1996a). The rate of degradation of desmin, however, is slower than that of nebulin and titin (Huff-LOnergan et al., 1996a; Ho et ai., 1996). 3. The rate of postmortem degradation of desmin in bovine muscle is related to tenderness measurements, with some degradation occurring in tender samples by three days postmortem, but not until about seven to fourteen days in tougher beef samples (Huff-Lonergan et al., 1996a). 4. 80th purified porcine skeletal muscle desmin (OShea et al., 1979) and the desrnin in purified bovine myofibrils (Huff-Lonergan et al., 1996a) are good substrates for the calpains. 5. Results of electron microscope studies of postmortem bovine muscle (e.g., Taylor et al., 19951 suggest that filamentous structures, presumably desmin IFs (a) linking adjacent myofibrils laterally at their Z-lines are degraded, resulting in gaps between myofibrils, and (b) linking the peripheral layer of myofibrils to costameres, are degraded. 6. Overall, postmortem desmin degradation is consistent with increased meat tenderness.

TABLE 9. Some Important Characteristics of Muscle Paranemin and Synemin. Important Characteristics of Paranemin 1. Until recently, paranemin has generally been considered as one of the types of -associated proteins (IFAPs). It is generally found together with desmin and/or , a desmin homologue (Price and Lazarides, 1983). 2. Paranemin has recently been cloned and completely sequenced in our lab (Hemken et al., 1996, 1997). Although the Mr of the paranemin Polypeptide subunit= 280,000 by SDS-PAGE, the predicted M, of the paranemin polypeptide subunit = 178,000 from the sequence. The sequencing studies also have shown that paranemin is a novel IF protein rather than an IFAP. Paranemin likely forms heteropolymeric IFs with desmin in mature skeletal muscle cells, with paranemin present in a very low molar ratio to desmin. 3. Localization studies show that paranemin is localized with desmin/vimentin IFs in developing muscle cells, and with the desmin IFs at the Z-line (PeriPheY) of mature striated muscle myofibrils (Hemken et al., 1997). Important Characteristics of Synemin 1. Until recently, synemin has generally been considered as one of the types of intermediate filament-associated proteins (IFAPs). It is generally found together with desmin and/or the desmin homologue, vimentin (Price and Lazarides, 1983). 2. SYnemin is a rather insoluble cytoskeletal protein (Mr of polypeptide subunit by SDS-PAGE = 230,000; forms a dimer of MI = 460,000) present in skeletal, cardiac and smooth muscle cells ofvertebrates. Partial primary structure studies in our lab (Becker et al., 1995) first revealed that synemin is a novel IF protein rather than an IFAp. we (Bellin et a\., 1996, 1997) have now obtained synemin’s complete sequence !Mr Of polypeptide SUbunit = 186,000 from sequence). Synemin, by itself, will not form IFs in vitro. It more likely forms heteropolymeric IFs with desmin in which SYnemin is present in a low molar ratio to desmin. 3. immunofluorescence localization studies show that synemin is co-localized with desmin at the Z-line (periphery) of striated muscle myofibrils. lmrnunoelectron microscope studies in our lab indicate that synemin is attached to, more likely is of, the desmin IFs that link myofibrils together, and myofibrils to other cellular structures. 4. SYnemin interacts in vitro with desmin, altering IF assembly and dynamics, and, under some conditions, also with the integral Z-line protein a- actinin and with the costarneric protein vinculin (Bellin et al., 1996; Sernett et al., 1996, 1997a,b).

American Meat Science Association TABLE 10. Some Possible Roles and Postmortem Changes of Muscle Paranemin and Synemin.

Possible Roles of Paranemin and Synemin 1. In developing muscle cells, paranemin and synemin may Play an important cytoskeletal role in linking desrnin and/or vimentin IFs to other structures such as the Z-lines of assembling myofibrils. 2. In mature striated muscle cells, paranemin and synemin may Play a significant cytoskeletal role in linking desmin IFs to other structures such as the myofibrillar Z-lines and discrete sites, called costameres, along the cell membrane skeleton (i.e., contribute to overall muscle cell integrity and organization) (Bellin et al., 1997; Hemken et al., 1997; Sernett et al., 1997a,b).

Postmortem Changes in Paranemin and Synemin 1. Our results to date suggest that paranemin and synemin will form heteropolymeric IFs with desmin and/or vimentin. 2. Both paranemin and synemin are proteolytically labile, and require use of Potent Protease inhibitor cocktails during their purification (Sernett et ai., 1996; Bellin et al., 1997; Hemken et al., 1997). We also have found that purified synemin is an excellent substrate for the calpains. Because both paranemin and synemin are very proteolytically sensitive, it is reasonable to suggest that postmortem degradation of these newly identified IF proteins would hinder the ability of desrnin IFs to link adjacent myofibrils, and the peripheral layer of cellular myofibrils to the costameric regions at the sarcolemma. Interestingly, synemin binds in VitrOtO vinculin (Sernettet ai., 1997a,b) and dystrophin (S.W. Sernett and R.M. Robson, unpublished observations), two costameric proteins other investigators (Taylor et al., 1995) have shown are degraded during postmortem aging of bovine muscle. 3. The precise role(s) of paranemin and synemin in postmortem muscle will require further studies.

TABLE 11. Postmortem Changes in Muscle Troponin-T.

1. Troponin-T (TN-T), first shown in the early 1970’s by Greaser and Gergely (1 971) to be one of the three TN subunits, is a well characterized component of the tropomyosin-troponin regulatory complexes located along the length of the thin filaments (for review, see Farah and Reinach, 1995). 2. TN-T (-37 kDa by SDS-PAGE) is extensively degraded postmortem, coincident with appearance of polypeptides at -30 kDa (JMacBrideand Parrish, 1977; Olson and Parrish, 1977; Penny and Dransfield, 1979). Ho et at. (1994) have recently shown unambiguously, by use of Western blotting, that the -30 kDa polypeptides in aged bovine muscle are products of TN-T degradation. 3. The rate of degradation of TN-T is related to tenderness measurements, with significant degradation occurring in more tender beef samples by three days postmortem. In tougher beef samples, rate of TN-T degradation is slower, but significant degradation occurs by seven days postmortem (Huff-Lonergan et al., 1996a). 4. The rate of postmortem degradation of TN-T in muscle from Callipyge lambs, which produce tougher meat, is slower than occurs in muscle samples from normal lambs (Koohmaraie et al., 1995). 5. The TN-T in purified bovine myofibrils is a substrate for the calpains using postmortem-like conditions (Huff-Lonergan etal., 1996a). The resulting major proteolytic fragments of TN-T appear similar to those observed during postmortem aging of bovine muscle. 6. Whether degradation of TN-T may just be an indicator of postmortem proteolysis, and/or specifically contribute to the increase in tenderness postmortem, remains unclear (see discussions in Ho et al., 1994; Huff-Lonergan et al., 1995,’1996a).

TABLE 12. Summary of Postmortem Changes in the Myofibrillar and Other Cytoskeletal Proteins in Muscle.

1. The major myofibrillar/structural proteins, myosin and actin, are not proteolytically degraded in conventionally-aged meat. 2. Postmortem proteolysis of key myofibrillar (titin, nebdin, TN-T) and other cytoskeletal (dystrophin, filamin, vinculin, desmin) proteins (and probably ones not yet examined) is a significant factor contributing to meat tenderness. 3. We must have a reasonable understandingof properties and role(s) of a protein in muscle before we can easily address the importance/impact of postmortem changes in that protein to meat tenderness. 4. Exactly which protein(s) being degraded postmortem is most important in tenderness is not known, and it may not be important that we know. It is more likely that it is the overall proteolysis of several cytoskeletal proteins that is important. 5. It is unlikely that all of, or even much of, a given protein undergoing postmortem proteolysis must be degraded to impact tenderness (i.e., a small number of proteolytic cleavageshreakswithin the cytoskeleton may result in a large, disproportionate increase in tenderness). 6. The proteins undergoing proteolysis are directly attached to kg., titin, nebulin), closely associated with (filamin, desmin, paranemin, synemin, some TN-Ts), or near (dystrophin, vinculin) the myofibrillar Z-lines. 7. One of the more likely and useful results of knowing which proteins are degraded, as well as the identity of their degraded products, is that someone will use this information as an indicator of proteolysis and devise a simple, practical testiassay for predicting meat tenderness. 8. Postmortem changes, other than proteolysis, have not been addressed in this presentation (e.g., protein denaturation effects due to changes in pH and ionic strength). 9. Rich Robson’s major professor, Darrel E. Golf, was correct when he told him as a young graduate student in 1964 that the biochemical changes in postmortem muscle are complex!

50th Annual Reciprocal Meat Conference 49 l9964, is the TN-T subunit of troponin (Table 11). How- Chen, M.-J.C.:Shih, C.-L.; Wang, K. 1993. Nebulin as an actin zipper. A two-module nehulin fragment promotes actin nucleation and stabilizes ever, it remains unclear as to whether degradation of TN-T actin filaments. J. Biol. Chem. 268, 20327-20334. Simply an indicator of postmortem proteolysis, and/or may Davies, P.J.A.; Wallach, D.; Willingham, M.C.; Pastan, I.; Yamaguchi, M.; Play a direct role in the increase in tenderness during post- Robson, R.M. 1978. Filamin-actin interaction. Dissociation of binding from gelation by Ca*+-activatedproteolysis. J. Biol. Chem. 253, 4036- mortem aging. Two costameric proteins listed in Table 1, 4042. namely dystrophin and vinculin, also have been shown to Farah. C.S.; Reinach, F.C. 1995. The troponin complex and regulation of undergo postmortem proteolysis (see Taylor et al., 1995, and . FASEB 1. 9, 755-767. Feng, H.S.; Yang, Z.H.; Yu, K.R.; Hijikata, T.; Holtzer, H.; Sweeney, H.L. references therein), but have not been described in detail in 1994. Truncated desmin expression and isometric force measurement this review. The other costameric protein listed in Table 1, in isolated quail skeletal myotubes. Mol. Biol. Cell 5, 27a. talin, has not yet been examined in postmortem muscle, but Fritz, J.D.; Creaser, M.L. 1991. Changes in titin and nebulin in postmortem bovine muscle reveaied by , Western bloaing and IS Proteolytically labile (Zhang et al., 1996). immunofluorescence microscopy. I. Food Sci. 56, 607-61 0, 61 5. Fulton, A.B.; Isaacs, W.B. 1991. Titin, a huge, elastic sarcomeric protein with a probable role in morphogenesis. BioEssays 13, 157-161. Conclusions Cautel, M.; Coulding, D. 1996. A molecular map of titinkonnectin elastic- A Summary of the postmortem changes in the myofibril- ity reveals two different mechanisms acting in series. FEBS Lett. 385, 11-14. larand other cytoskeletal proteins in muscle that have been Gautel, M.; Goulding, D.; Bullard, 6.; Weber, K.; Furst, D.O. 1996. The revieWd in this paper is presented in Table 12. Although a central Z-disk region of titin is assembled from a novel repeat in vari- great deal of progress has been made in understanding the able copy numbers. J. Cell Sci. 109, 2747-2754. Goll, D.E.; Boehm,M.L.;Geesink, G.H.;Thompson,V.F. 1997. What causes Properties and roles of proteins and structures that undergo postmortemtenderization? Proc. Recip. Meat Conf. 50. [paper in press). Postmortem proteolysis, we obviously have a lot of work Gorlin, J.B.; Yamin, R.; Egan, S.; Stewart, M.; Stossel, T.P.; Kwiatkowski, yet to do. Although there is a growing consensus among D.J.;Hartwig, J.H. 1990. Human endothelial actin-binding protein (ABP- 280, nonmuscle filamin):A molecular leafspring.]. Cell Biol. 111,1089- many scientists that postmortem proteolysis, and especially 11 05. that resulting from the calpain system, is the major mecha- Craves, D.J,; Huiatt, T.W.; Zhou, H.; Huang, H.-Y.; Sernett, S.W.; Robson. nlSm underlying postmortem tenderization (Koohmaraie, R.M.; McMahon, K.K. 1997. Regulatoryrole of arginine-specific mono ADP-ribosyltransferasein muscle cells. In Biological Significance of 1992, 1994, 1996; Uytterhaegen et al., 1994), other pro- Mono-ADP-Ribosyiation in Animal Tissues (F. Haag and F. Nolte, eds.), vocative views do exist (Goll et al., 1997). Plenum Press. (review in press). Greaser, M.L.; Gergely, J. 1971. Reconstitution of troponin activity from three protein components, J. Biol. Chem. 246, 4226-4233. Acknowledgments Handel, S.E.;Wang, S.-M.;Creaser, M.L.;Schultz, E.; Bulinski, J.C.; Lessard, J.L. 1989. Skeletal muscle myofibriliogenesis as revealed with a mono- we are very grateful to Lynn Newbold for typing the clonal antibody to titin in combination with detection of the a- and g- manuscript. R. M. Robson also thanks Rob Bellin, Suzy isoforms of actin. Dev. Biol. 132, 35-44. Sernettand Elisabeth Lonergan for lots of assistance in prepa- Hartwig, J.H.;Stossel, T.P. 1975. Isolation and properties of actin, myosin, and a new actin-binding protein in rabbit alveolar macrophages.J. Biol. ration of the oral presentation of this paper. The work de- Chem. 250, 5696-5705.. scribed in this review and that came from our lab was SUP- Hemkm..~ ,, P.M.: Becker. ., B.; Bellin, R.M.: Huiatt, T.W.; Robson, R.M. 1996. Ported in part by grants from the USDA-NRICGP (Award Nucleotide sequence of para'nemin reveals it is a novel intermediate filament protein. Mol. Biol. Cell 7, 557.3. 96-35206-37441, American Association, Iowa Affili- Hemken, P.M.; Bellin, R.M.; Sernett, S.W.; Becker, 6.;Huiatt, T.W.; Robson, ate, Muscular Dystrophy Association and Beef Industry Coun- R.M. 1997. Molecular characteristics of the novel intermediate fila- cil Of the National Live Stock and Meat Board. journal Paper ment protein paranemin. (paper submitted). Ho, C.-Y.; Stromer, M.H.; Robson R.M. 1994. Identification of the 30 kDa Number 1-1 7491 of the Iowa Agriculture and Home ECO- polypeptide in postmortem skeletal muscle as a degradation product of nomics Experiment Station, Ames, IA 5001 1, Projects 3349, troponin-T. Biochimie 76, 369-375. 3444 and 2127, and supported by Hatch Act and State of Ho, C.-Y.; Stromer, M.H.; Robson, R.M. 1996. Effect of electrical stimula- Iowa Funds. tion on postmortem titin, nebulin, desmin, and troponin-T degradation and ultrastructural changes in bovine longissimus muscle. J. Anim. Sci. 74, 1563-1575.

Ho.~, C.-Y.:- I Stromer. M.H.: Rouse, G.; Robson. R.M. 1997. Effects of electri- References cal stimulation and postmortem storage on changes in titin, nebulin, Bandman, E.; Zdanis, D. 1988. An immunological method to assess pro- desmin, troponin-T, and muscle ultrastructure in 60s indicus crossbred tein degradation in post-mortem muscle. Meat Sci. 22, 1-19. cattle. J. Anim. Sci. 75, 366-376. Beckert B.; Bellin, R.M.; Sernett, S.W.; Huiatt, T.W.; Robson, R.M. 1995. Huang, H.-Y.; Graves, D.J.; Robson, R.M.; Huiatt, T.W. 1993. ADP- SYnemincontains the rod domain of intermediate filaments. Biochem. Ribosylation of the intermediate filament protein desmin and inhibi- BiophYs. Res. Commun. 21 3, 796-802. tion of desmin assembly in vitro by muscle ADP-ribosyltransferase. R.M.; Sernett, S.W.; Becker, B.; Huiatt, T.W.; Robson, R.M. 1996. Biochem. Biophys. Res. Commun. 197, 570-577. sequence and molecular interaction studies of the interme- Huff-Lonergan, E.; Parrish, jr,, F.C.; Robson R.M. 1995. Effects of postmor- diate filament protein synemin. Mol. Biol. Cell 7, 555a. tem aging time, animal age, and sex on degradationof titin and nebulin Bel1in,R-M.; Sernett, S.W.; Robson, R.M. 1997. Synemin. In Guidebook to in bovine longissimus muscle. 1. Anim. Sci. 73, 1064-1073. the CYtoskeletal and Motor Proteins, Second Edition (T. Kreis and R. Huff-Lonergan, E.; Mitsuhashi, T.; Beekman, D.D.; Parrish, Jr., F.C.; Olson, Vale,edS.), Oxford Univ. Press. (review in press). D.G.; Robson, R.M. 1996a. Proteolysis of specific muscle structural Bennett, p.M.; Gautel, M. 1996. Titin domain patterns correlate with the proteins by m-calpain at low pH and temperature is similar to degrada- disposition of myosin at the end of the thick filament. J. Mol. Biol. tion in postmortem bovine muscle. J. Anim. Sci. 74, 993-1008. 259, 896-9133. Huff-Lonergan, E.; Mitsuhashi, T.; Parrish, Jr., F.C.; Robson R.M. 1996b. Chens M.-]G.; Wang, K. 1994. Conformational studies of a two-module SDS-PAGE and Western blotting comparisons of purified myofibrils and fragment of nebulin and implications for actin association. Arch. whole muscle preparations for evaluating titin and nebulin in postmor- Biochem. Biophys. 310, 310-317. tem bovine muscle. J. Anim. Sci. 74, 779-785.

American Meat Science Association Hwan, S.F.; Bandman, E. 1989. Studies of desmin and a-actinin degrada- Maruyama, K.; Matsuno, A.; Higuchi, H.; Shimaoka, S.; Kimura, S.; Shimizu, tion in bovine semitendinosus muscle. J. Food Sci. 54, 1426-1430. T. 1989. Behaviour of connectin (titin) and nebulin in skinned muscle Inagaki, M.; Inagaki, N.; Takahashi, T.; Takai, Y. 1997. Phosphorylation- fibres released after extreme stretch as revealed by immunoelectron dependent control of structures of intermediate filaments: A novel ap- microscopy. J. Muscle Res. Cell Motil. 10, 350-359. proach using site- and phosphorylation state-specific antibodies. J. Milner. D.J.; Weitzer, G.; Tran, K.; Bradley, A,; Capetanaki, Y. 1996. Dis- Biochem. 121, 407-41 4. ruption of muscle architecture and myocardial degeneration in mice Ip, W.; Heuser, J.E.; Pang, Y.-Y.S.; Hartzer, M.K.; Robson, R.M. 1985. Sub- lacking desmin. J. Cell Biol. 134, 1255-1270. unit structure of desmin and vimentin protofilaments and how they as- Moncman, C.L.; Wang, K. 1995. Nebulette: A 107 kD nebulin-like protein semble into intermediate filaments. Ann. New York Acad. Sci. 455, in cardiac muscle. Cell Motil. Cytoskeleton 32, 205-225. 185-199. Moncman, C.L.; Wang, K. 1996. Assembly of nebulin into the sarcomeres Jin, J.-P.; Wang, K. 1991. Nebulin as a giant actin-binding template protein of avian skeletal muscle. Cell Motil. Cytoskeleton 34, 167-184. in skeletal muscle sarcomere. Interaction of actin and cloned human Nave, R.; Furst, D.O.; Weber, K. 1990. Interaction of a-actinin and nebulin nebulin fragments. FEBS Lett. 281, 93-96. in vitro. FEBS Lett. 269, 163-166. Keller 111, T.C.S. 1995. Structure and function of titin and nebulin. Curr. Oberman, W.M.J.; Gautel, M.; Steiner, F.; van der Ven, P.F.M.; Weber, K.; Opin. Cell Biol. 7, 32-38. Furst, D.O. 1996. The structure of the sarcomeric M band: Localization Kellermayer, M.S.Z.; Smith, S.B.; Cranzier, H.L.; Bustamante, C. 1997. Fold- of defined domains of rnyomesin, M-protein, and the 250-kD carboxy- ing-unfolding transitions in single titin molecules characterized with terminal region of titin by immunoelectron microscopy. J. Cell Biol. laser tweezers. Science 276, 11 1 2-1 11 6. 134, 1441-1453. Kimura, S.; Matsuura, T.; Ohtsuka, S.; Nakauchi, Y.; Matsuno, A.; Maruyama, Ohtsuka, H.;Yajima, H.;Maruyama, K.; Kimura, S. 1997. Binding of the N- K. 1992. Characterization and localization of a-connectin (titin 1): An terminal 63 kDa portion of connectin/titin to a-actinin as revealed by elastic protein isolated from rabbit skeletal muscle. J. h.luscle Res. Cell the yeast two-hybrid system. FEBS Lett. 401, 65-67. Motil. 13, 39-47. Olson, D.G.; Parrish, Jr., F.C. 1977. Relationship of myofibril fragmenta- Kinbara, K.; Sorimachi, H.; Ishiura. S.; Suzuki, K. 1997. Muscle-specific tion index to measures of beefsteak tenderness. J. Food Sci. 42, 506- calpain, p94, interacts with the extreme C-terminal region of connectin, 509. a unique region flanked by two immunoglobulin C2 motifs. Arch. O’Shea, J.M.; Robson, R.M.; Huiatt, T.W.; Hartzer, M.K.; Stromer, M.H. Biochem. Biophys. 342, 99-107. 1979. Purified desmin from adult mammalian skeletal muscle: A pep- Koohmaraie, M. 1992. The role of Ca2+-dependentproteases (calpains) in tide mapping comparison with desmins from adult mammalian and post-mortem proteolysis and meat tenderness. Biochimie 74, 239-245. avian smooth muscle. Biochem. Biophys. Res. Commun. 89,972-980. Koohmaraie, M. 1994. Muscle proteinases and meat aging. Meat Sci. 36, O’Shea, J.M.; Robson, R.M.; Hartzer, M.K.; Huiatt, T.W.; Rathbun, W.E.; 93-1 04. Stromer, M.H. 1981. Purification of desmin from adult mammalian skel- Koohmaraie, M. 1996. Biochemical factors regulating the toughening and etal muscle. Biochem. J. 195, 345-356. tenderization processes of meat. Meat Sci. 43, S193-S201. Penny, I.F.; Dransfield, E. 1979. Relationshipbetween toughness and tropo- Koohmaraie, M.; Kennick, W.H.; Anglemier, A.F.; Elgasim, E.A.; Jones, T.K. nin-T in conditioned beef. Meat Sci. 3, 135-141. 1984a. Effect of postmortem storage on cold-shortened bovine muscle: Pfuhl, M.;Winder, S.J.; Morelli, M.A.C.; Labeit, S.; Pastore, A. 1996. Corre- Analysis by SDS-polyacrylamide gel electrophoresis. 1. Food Sci. 49, lation between conformational and binding properties of nebulin re- 290-291. peats. J. Mol. Biol. 257, 367-384. Koohmaraie, M.;Kennick, W.H.; Elgasim. E.A.; Anglemier, A.F. 1984b. Ef- Price, M.C.; Lazarides, E. 1983. Expression of intermediate filament-asso- fects of postmortem storage on muscle protein degradation: Analysis ciated proteins paranemin and synemin in chicken development. J. Cell by SDS-polyacrylamide gel electrophoresis.J. Food Sci. 49, 292-293. Biol. 97, 1860-1 874. Koohmaraie, M.; Shackelford, S.D.; Wheeler, T.L.; Lonergan, S.M.; Doumit, Price, M.G.; Caprette, D.R.: Comer, R.H. 1994. Different temporal patterns M.E. 1995. A muscle hypertrophy condition in lamb (callipyge): Char- of expression result in the same type, amount and distribution of filamin acterization of effects on muscle growth and meat quality traits. j. Anim. (ABP) in cardiac and skeletal myofibrils. Cell Motil. Cytoskeleton 27, Sci. 73, 3596-3607. 248.261. Kruger, M.; Wright, 1.; Wang, K. 1991. Nebulin as a length regulator of thin Richardson, F.L.; Stromer, M.H.; Huiatt, T.W.; Robson, R.M. 1981. filaments of vertebrate skeletal muscles: Correlation of thin filament lmmunoelectron and fluorescence n~icroscopelocalization of desmin length, nebulin size, and epitope profile. J. Cell Biol. 115, 97-107. in mature avian muscles. Eur. J. Cell Biol. 26, 91-101. Kurpakus, M.A. 1988. The role of titin and nebulin in myofibril assembly in Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J.M.; Gaub, H.E. 1997. cultured embryonic chick muscle cells. Ph.D. Dissertation, Iowa State Reversible unfolding of individual titin immunoglobulin domains by University, Ames, IA. AFM. Science 276, 1109-1112. Labeit, S.; Kolmerer, B. 1995a. Titins: Giant proteins in charge of muscle Robson, R.1‘4. 1989. Intermediate filaments. Curr. Opin. Cell Biol. 1, 36- ultrastructure and elasticity. Science 270, 293-296. 43. Labeit, S.; Kolmerer, B. 1995b. The complete primary structure of human Robson, R.M. 1995. Myofibrillar and cytoskeletal structures and proteins nebulin and its correlation to muscle structure. J. Mol. Biol. 248, 308- in mature skeletal muscle cells. In Expression of Tissue Proteinases and 31 5. Regulationof Protein Degradation as Related to Meat Quality (A. Ouali, Labeit, S.; Gibson, T,; Lakey, A.; Leonard, K.; Zevani, M.; Knight, P.; Wardale, 0.1. Demeyer and F.J.M. Smulders, eds.), ECCEAMST, Utrecht, The 1.; Trinick, J. 1991. Evidence that nebulin is a protein-ruler in muscle Netherlands, pp.267-288. thin filaments. FEBS Lett. 282, 31 3-331. Robson, R.M.; Huiatt, T.W. 1983. Roles of the cytoskeletal proteins desmin, Labeit, S.; Kolmerer, B.; Linke, W.A. 1997. The giant protein titin. Emerging titin and nebulin in muscle. Proc. Recip. Meat Conf. 36, 116-124. roles in physiology and pathophysiology. Circ. Res. 80, 290-294. Robson, R.M.; Stromer, M.H.; Huiatt, T.W.; O’Shea, J.M.; Hartzer, M.K.; Linke, W.A.; Ivemeyer, M.;Olivieri, N.;Kolmerer, B.; Ruegg, J.C.; Labeit, S. Richardson, F.L.; Rathbun, W.E. 1980. Biochemistry and structure of 1996. Towards a molecular understanding of the elasticity of titin. J. desmin and the recently discovered muscle cell cytoskeleton. Proc. Mol. Biol. 261, 62-71. 26th Eur. Meeting Meat Res. Workers, Vol. 1, pp. 22-25. Locker, R.H.; Leet, N.G. 1975. of highly-stretched beef muscle. Robson, R.M.; Yamaguchi, M.; Huiatt, T.W.; Richardson, F.L.; O’Shea, J.M.; I. The fine structure of grossly stretched single fibers. J. Ultrastruct. Res. Hartzer, M.K.; Rathbun, W.E.; Schreiner, P.J.; Stromer, M.H.; Pang, S.: 52. 64-75. Evans, R.R.; Ridpath, J.F. 1981. Biochemistry and molecular architec- Lushy, M.L.; Ridpath, J.F.; Parrish, Jr., F.C.; Robson, R.M. 1983. Effect of ture of muscle cell 10-nm filaments and Z-line. Roles of desmin and a- postmortem storage on degradation of the recently discovered myo- actinin. Proc. Recip. Meat Conf. 34, 5-11. fibrillar protein titin in bovine longissimus muscle. J. Food Sci. 48. 1789- Robson, R.M.; O’Shea, J.M.; Hartzer, M.K.; Rathbun, W.E.; LaSalle, F.; 1790, 1795. Schreiner, P.J.; Kasang, L.E.; Stromer, M.H.; Lusby, M.L.; Ridpath, J.F.; MacBride, M.A.; Parrish, Jr., F.C. 1977. The 30,000- component of Pang, Y.-Y.; Evans, R.R.; Zeece, M.G.; Parrish, F.C.; Huiatt, T.W. 1984. tender bovine longissimus muscle. J. Food Sci. 42, 1627-1629. Role of new cytoskeletal elements in maintenance of muscle integrity. Maruyama, K. 1997. Connectidtitin, giant elastic protein of muscle. FASEB J. Food Biochem. 1, 1-24. J. 11, 341-345. Robson, RM.; Huiatt, T.W.; Parrish, Jr., F.C. 1991, Biochemical and struc- tural propertiesof titin, nebulin and intermediate filaments. Proc. Recip. Meat Conf. 44, 7-20.

50th Annual Reciprocal Meat Conference 51 Root, D.D.; Wang, K. 1994. Calmodulin-sensitive interaction of human van der Loop, F.T.L.; van der Ven, P.F.M.; Furst, D.E.; Gautel, M.;van Eys, nebulin fragments with actin and myosin. Biochemistry 33, 12581- C.J.J.M.;Ramaekers, F.C.S. 1996. Integration of titin into the sarcom- 12591. eres of cultured differentiating human skeletal muscle cells. Eur. J. Cell Sernett, S.W.; Robson, R.M.; Bellin, R.M.; Bilak, S.R.; Stromer, M.H.;Huiatt, Biol. 69, 301-307. T.W. 1996. Interactions of muscle synemin with both desmin and with Wang, K.;Ash, J.F.; Singer, S.J. 1975. Filarnin, a new high-molecular-weight Z-line and costameric proteins. J. Anim. Sci. 74 (Suppl. l), 50. protein found in smooth muscle and non-muscle cells. Proc. Natl. Acad. Sernett, S.W.; Bellin, R.M.; Huiatt, T.W.; Robson, R.M. 1997a. Molecular Sci. USA 72, 4483-4486. characterization and interaction studies of synemin, an intermediate Wang, K.; McClure, I.; Tu, A. 1979. Titin: Major myofibrillar components filament protein. I.him. Sci. 75 (Suppl. 11, 55. of striated muscle. Proc. Natl. Acad. Sci. USA 76, 3698-3702. Sernett, S.W.; Bellin, R.M.; Huiatt, T.W.; Robson, R.M. 1997b. Molecular Wang, K.; McCarter, R.; Wright, 1.; Beverly, 1.; Ramirez-Mitchell, R. 1991. properties of synemin and its interactions with specific proteins in the Regulationof skeletal muscle stiffness and elasticity by titin isoforms:A muscle cell cytoskeleton. Proc. Recip. Meat Conf. 50. (abstractin press). test of the segmental extension model of resting tension. Proc. Natl. Shih, C.-L.; Chen, M.-J.C.; Linse, K.; Wang, K. 1997. Molecular contacts Acad. Sci. USA 88, 7101-7105. between nebulin and actin: Cross-linking of nebulin modules to the N- Wang, K.; McCarter, R.; Wright, J,; Beverly, J.; Ramirez-Mitchell, R. 1993. terminus of actin. 1997. Biochemistry 36, 1814-1825. Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin- Suzuki, A.; Kim, K.; Ikeuchi, Y. 1996. Proteolytic cleavage of connectin/ myosin composite filament is a dual-stagemolecular spring. Biophys. 1. titin. Adv. Biophys. 33, 53-64, 64, 11 61 -1 1 77. Tanabe, R.; Muroya, S.; Nakajima, I.; Chikuni, K.; Nakai, H. 1997. J. Food Wang, K.; Knipfer, M.; Huang, Q.-Q.; van Heerden, A.; Hsu, L. C.-L.; Sci. 62, 451 -453, 461, Gutierrez, G.;Quian, X.-L.; Stedman, H. 1996. Human skeletal muscle Taylor, R.C.; Ceesink, G.H.; Thompson, V.F.; Koohmaraie, M.; Coll, D.E. nebulin sequence encodes a blueprint for thin filament architecture. 1995. 1s Z-disk degradation responsiblefor postmortem tenderization? Sequence motifs and affinity profiles of tandem repeats and terminal J. Anim. Sci. 73, 1351-1367. SH3. J, Biol. Chem. 271, 4304-4314. Trinick, 1. 1992. Molecular rulers in muscle?Curr. Biol. 2, 75-77. Zeece, M.C.; Robson, R.M.; Lusby, M.L.; Parrish, Jr., F.C. 1986. Effect of Trinick, 1. 1994. Titin and nebulin: protein rulers in muscle. Trends Biochem. calcium activated protease (CAF) on bovine myofibrils under different Sci. 19, 405-409. conditions of pH and temperature.J. Food Sci. 51, 797-803. TrombitAs, K.; Creaser, M.L.; Pollack, C.H. 1997. interaction between titin Zhang, 1.; Robson, R.M.; Schmidt, J.M.; Stromer, M.H. 1996. Talin can and thin filaments in intact cardiac muscle. 1. Muscle Res. Cell Motil. crosslink actin filaments into both networks and bundles. Biochem. 18, 345-351. Biophys. Res. Commun. 21 8, 530-537. Tskhovrebova, L.; Trinick, 1.; Sleep, J.A.; Simmons, R.M. 1997. Elasticity Zhou, H.; Huiatt, T.W.; Robson, R.M.; Sernett. S.W.: Craves, D.J. 1996. and unfoldingofsingle molecules of the giant muscle protein titin. Nature Characterization of ADP-ribosylation sites on desmin and restoration 387, 308-31 2. of desmin intermediatefilament assembly by de-ADP-ribosylation.Arch. Uytterhaegen, L; Ctaeys, E.; Demeyer, D. 1994. Effects of exogenous pro- Biochem. Biophys. 334, 214-222. tease effectors on beef tenderness development and myofibrillar degra- dation and solubility. 1. Anim. Sci. 72, 1209-1223.

American Meat Science Association