Is Z-DiskDegradation Responsible for Postmortem Tenderization?'

"Richard G. Taylor, *Gee& H. Geesink,"Valery F. Thompson, TMohammad Koohmaraie,and "Darrel E. Go112

"Muscle Biology Group, University of Arizona, Tucson 85721 and ?US. Meat Animal ResearchCenter, Clay Center, NE 68933-0166

ABSTRACT: A number of studieshave suggested structures linking adjacent laterally at the that Z-disk degradation is a major factor contributing level of each Z-disk are also degraded during the first to postmortem tenderization. These conclusions seem 3 or 4 d of postmortem storage at 4"C, resulting in to have been based largely on experimental findings gapsbetween myofibrils in postmortem muscle. showing that the calpain system has a major role in Degradation of these structures would have important postmortem tenderization, and that when incubated effects on tenderness. The constituting these with myofibrils or muscle strips, purified calpain structures, andtitin (NZlines); , removes Z-disks. Approximately 65 to 80% of all ,and (three of the six toeight postmortem tenderization occurs during the first 3 or proteins constituting ); and desmin (fila- 4d postmortem, however, andthere is little or no mentslinking adjacent myofibrils) are all excellent ultrastructurally detectable Z-disk degradation during this period. Electron microscope studies described in substrates for the calpains, and nebulin, , vincu- thispaper show that,during the first 3 or 4 d of lin,and desmin are largelydegraded within 3 d postmortem storage at 4"C, both costameres and NZ postmortem in semimembranosus muscle. Electron lines are degraded. Costameres link myofibrils to the micrographs of myofibrils used inthe , and NZ lines have been reported to be fragmentation index assayshow that these myofibrils, areas where titin and nebulin filaments, which form a which have been assumedto be broken at their Z- cytoskeletal network linking thick and thin filaments, disks, in fact have intact Z-disks and are broken in respectively, to the Z-disk, coalesce. Filamentous their I-bands. KeyWords: Calpains,Costameres, Myofibril FragmentationIndex, NZLines, Postmortem Tenderization, Z-Disks

J. him. Sci. 1995. 73:1351-1367

Introduction mortem storage (Davey and Gilbert, 1969; Fukazawa and Yasui, 1967; Goll, 1968; Henderson et al., 1970). Light and electron microscope studies in the late It seemed likely that loss of Z-disk integrity would 1960s first showed that Z-disks occasionally were contribute to increasedtenderness, and indeed, my- disrupted and, in some instances in muscle stored at ofibrils prepared from muscle after 3 to 20 d of higher temperatures, were lost entirely during post- postmortem storage at 0 to 2°C were fragile and were broken intoshort segments of four to seven sarco- mereseach by homogenization (Daveyand Gilbert, This work was supported by grants from the USDA National 1969; Henderson etal., 1970). Immediately after ResearchInitiative Competitive GrantsProgram, 92-37206-7244; these structural observations, it was discovered that the Association; the National Live Stock and incubating muscle strips in a Ca2+-containing solution Meat Board; andthe Arizona AgncultureExperiment Station resulted in total degradation of Z-disks (Goll et al., Project 28, a contributing project to USDA Regional Project NC-131. 1970; Busch et al., 19721, and efforts were begun to We thank Janet Christner for her alacritous assistance with the manuscript, John Marchello for ensuring that we had a supply of identify the Ca2+-activated factor (called CAF at that animals available for thisstudy, Shoichi Ishiura,Institute of time) responsible for this degradation.Even before Molecular and Cellular Biosciences, University of Tokyo, for anti- CAF had been completely purified, it was shown that dystrophin antibodies, and ElizabethHuff Lonergan, Muscle Biology muscles having more CAF activityunderwent a Group, Iowa State Univ., Ames, for nebulinpurified from bovine greater degree of postmortem tenderizationthan . 2To whom correspondenceshould be addressed. muscles having less CAF activity (Goll et al., 1974). Received May 5, 1994. Moreover, incubation of myofibrils with purified CAF Accepted December 27, 1994. (now called m-calpain) caused the same or similar

1351 1352 TAYLOR ET AL. structural changes (Olson et al., 1976) and polypep- ments Are Not the Result of 2-Disk Degradation. It tide degradation (Olson et al., 1977) that postmortem has been suggested that postmortemdegradation of storagedid. Both CAF treatmentand postmortem T is simply an in situ estimate of calpain storage resulted in appearance of several polypeptide activity, but this argument would be more convincing fragments migrating in the 30,000-Da range in SDS- if degradation of a Z-disk could be shown to be PAGE. Earlier studies (Hay etal., 1973) hadreported related to tenderization. the appearanceof a 30-kDa polypeptide in postmortem There is Little or No Degradation of the Known 2- muscle, but the origin and cause of this fragment was Disk Proteins During the First 3 DaysPostmortem unknown at that time. It is now known thatthe WhenMost TenderizationOccurs. a- is the 30-kDa polypeptides result from degradation of tropo- major Z-disk protein, andthe filaments in Z-disks nin-T (Olson et al., 1977; Ho et al., 19941, and that containactin (Luther, 1991). Western blot analysis density of the 30-kDa band is related to ultimate finds no detectable degradation of a-actinin until after tenderness(McBride and Parrish, 1977). 2 wk of postmortemstorage at 2°C (Hwanand Bandman, 19891, and functionala-actinin can be CalpainActivity, 2-disks, and isolated from muscle after 13 d postmortem (Arakawa Postmortem Tenderization et al., 1970). Purified a-actininand are not degraded by purified calpain in invitro assays (Goll et Since theseearly observations, a greatdeal of evidence has accumulated indicating that the calpains al.,1991). It seems likely, therefore, that any have an important role in postmortem tenderization postmortem degradation of a-actinin or actin is due to (see Figure 1 for a summary). This evidence has led the cathepsins, which do degrade these two proteins to a prevailing view that the calpains are responsible (Okitaniet al., 1980; Matsukaraet al., 19811, and for 90% or more of thetenderization that occurs that this degradation occurs late, probably after 7 to during postmortem storage (Goll et al., 1992a), and 10d postmortem. furthermore,that this tenderization is the direct Two other proteins, titin and nebulin, are anchored result of calpain's ability to degrade Z-disks in skeletal at their N- and C-terminal ends,respectively, in the Z- muscle. Several observations, however, are not readily disk. The titin and nebulin polypeptide chains, how- accommodated by the "Z-disk dogma" of postmortem ever, both extend through the I-band and into the A- tenderization. band areas, and it seems unlikely that degradation of is Not Located in the 2-Disk, and Its these two proteins would resultin loss of Z-disks. Degradation and Appearance of the 30-kDa Frag- and desmin are both located at the periphery

A. Very little degradation of and actin occurs during postmortem storage at 2 to 4°C for 72 h (Bandman and Zdanis, 19881, even though most of the total postmortem tenderization occurs during this period. 1. Thecalpains are unique among the knownproteolytic in that they do notdegrade undenatured myosin andactin. a. "he known cathepsinsall degrade myosin andactin. B. Very little degradation of a-actinin, the major protein in skeletal muscle Z-disks, occurs during postmortem storage at 2 to 4°C for 72 h (Hwanand Bandman, 1989). 1. Several of the cathepsins and many other proteases (e.g., trypsin; Stromer et al., 1967) degrade the Z-disk structure, and this degradation is accompanied by degradation of a-actinin; the calpains,however, are unique in that they degrade Z-disk the structure, but they release a-actinin from this structure without degrading it to small fragments. 2. Degradation of myosin, actin, and a-actinin occurs during postmortem storage at 37"C, and this degradation may be due to the cathepsins. C. Thereis very little proteolysis of muscle proteinsduring postmortem storage at 2 to 4°C. 1. The structure remains largely intact, with theexception of loss of the N2 line and some variable but small loss of Z-disk integrity (Stromer et al., 1967). 2. Functional actomyosin (Robson et al., 1967) and a-actinin (Arakawa etal., 1970) can be isolated from skeletal muscle even after 13 d of postmortem storage at 2 to 4°C. 3. Many studies have failed to find significant increases in free amino acidseven after long periods of postmortem storage (Parrish et al., 1969). a. The cathepsins cause extensive degradation of proteins (Whipple andKoohmaraie, 1991), but the calpains causerelatively few cleavages and have little activity after muscle pH falls below 6.0. D. Severalstudies have shown thatincreasing Ca2+ concentration in muscle resultsin increased tenderness (Penny et al., 1974; Koohmaraie et al., 1988b, Koohmaraie and Shackelford, 1991). 1. Neitherthe cathepsins nor themulticatalytic protease is activated by Ca2+(Koohmaraie, 1992). E. A number of studies have shown that tenderness increases to a greater extent during postmortem storage in those muscles that have higher calpain (especially p-calpain) or lower calpastatin activities than in other muscles; low calpastatin activity especially seems highly associated withincreased postmortem tenderization(Koohmaraie, 1988, 1992; Wheeler et al., 1990).

Figure 1. Summary of some of the evidence that the calpain system has a major role in postmortem tenderization. This figure is adapted from a similar summary that appeared in Go11 et al. (1992a). Z-DISKTENDERIZATION DEGRADATION AND 1353 of the Z-disk and are not part of the Z-disk structure (Granger and Lazarides, 1978). Although these two proteins are readily degraded by the calpains (Davies et al., 1978; O'Shea et al., 1979), it seemsunlikely that their degradation would by itself cause loss of Z- disks. The Time During Which MostPostmortem Tenderi- zation Occurs Does Not Coincide with the Time That Changes in 2-Dish Structure or 2-Dish Proteins Are Observed. Anumber of studieshave reported that 31 2 """""~'"'~'~'"'"''" most postmortem tenderization occurs during the first336 0 1224 72 3 or 4d afterdeath (Go11 etal., 1964; Davey and Time Postmortem, h Gilbert, 1966; Parrish et al., 1973; Marsh et al., 1981; Dransfield, 1992; Wheeler and Koohmaraie, 1994). Figure 2. Changes in Warner-Bratzler shear force of The SDS-PAGE of myofibrils from postmortem mus- lamb longissimus thoracic et lumborum muscle during cle, however, indicates that the complex of polypep- postmortem storage. Insert shows the means and tides migrating at 30 kDa does not begin to appear standard deviations of the numbers plotted in the until after 3 d of postmortem storage at 2°C (Kooh- figure. Means without a common superscript differ maraie et al., 1984). Moreover, although Z-disks lose significantly at P < .05. This figureis taken from some structuralintegrity anddensity during Wheeler and Koohmaraie ( 1994). prolonged periods of postmortem storage at tempera- tures of 25°C or higher (Henderson et al., 1970), very few structural changescan be detected in Z-disks during the first 3 d postmortem at 2"C, and most Z- largechanges intenderness that occur duringthis disks show little ultrastructural signs of degradation period. A study comparing bos taurus and bos indicus even after 13 d or longer at 2 to 4°C (Stromer et al., found that the MFI for either of these breeds did not 1967). change between 1 and 3 dpostmortem andthen It hasbeen argued that only a few Z-disks, possibly increased significantly after 3 dof postmortem storage less than 5% of the total,would need to be degraded or (Whippleet al., 1990b). Conversely, two studies partly degraded to have a large effect on tenderness, monitoring postmortem changes in the MFI beginning and that the sampling problems inherent to electron at death and continuing until 7 or 14 d postmortem microscope analysis would make it difficult to detect found that61 to 64% of theincrease in MFI had occurred by d 3 postmortem compared withd7 this small number of disrupted Z-disks. Consequently, (Koohmaraie et al., 1988a) or d 14 (Koohmaraie et the myofibril fragmentationindex (MFI) has been al., 1987). Other studies thathave monitored changes widely adopted as a method of estimating Z-disk in MFI during the first 6 or 7 d postmortem indicate degradationin postmortem muscle. Myofibrils pre- that only 13 (Whipple et al.,1990b) to 30 to 38% pared from postmortem muscle are shorter than those (Olson et al., 1976; Whipple et al., 1990a;Shackelford prepared from at-death muscle (Stromer et al., 1974; et al., 1991) of the increase in MFI had occurred after Olson et al., 19761, suggesting that myofibrils are 3 d postmortem compared with 6 or 7 d postmortem. weakenedduring postmortem storageand therefore Hence, studies on changes in the MFI during the first are more easily broken intoshort fragments by 3 to 4dpostmortem have produced inconsistent homogenization. The MFI is a measure of the average results. More importantly,perhaps, no studieshave length of myofibrils andis significantlyrelated to been done to determine whether changes in the MFI tenderness (shorter myofibrils result in higher MFI, are indeed due to breakage at theZ-disk, as suggested which is related to greater tenderness; Maller et al., by the Z-disk theory, or whether these breaks occur 1973). Most studies on the MFI have related MFI at elsewhere inthe myofibril. some chosen time postmortem (usually between 3 and Estimates of percentage of total tenderization that 14 d) with tenderness at that same time, and only a occurs during the first 3d after death depend to a few studies have monitored changes in theMFI during large extent on the value obtained for tenderness at postmortemstorage (see Olson etal., 1976; Kooh- death. At-death muscle contains ATP and undergoes maraie et al., 1987, 1988a; Whipple et al. 1990a,b; severecontraction during cooking. It, therefore, has Shackelford et al., 1991). Although meat tenderness been difficult to obtain accurate estimates of at-death decreases markedly during the first 24 h postmortem tenderness. Modeling studies based on activity of p- andthen increases rapidly between 24 and 72 h calpain as a primary cause of postmortem tenderiza- postmortem (see Figure2and discussion inthe tion predict toughness at death as approximately 7.2 following paragraph),the MFI increases uniformly to 11.0 kg, toughness at 3 d postmortem as approxi- during the first 72 h postmortem (Olson et al., 1976; mately 5.5 to 7.2 kg,toughness at 5 d as approxi- Koohmaraie et al., 1987, 1988a; Whipple et al., 1990a, mately 5 to 7 kg, and ultimate toughness (25 d) as b; Shackelford et al., 1991) and does not reflect the approximately 3.5 to 4.0 kg(Dransfield, 1992). 1354 TAYLOR ET AL. Hence, this model suggests that approximately 65% of results of these three studies indicate that approxi- total postmortem tenderization occurs by d 3 postmor- mately 65 to 80% of allpostmortem tenderization tem. Warner-Bratzler shear measurements of bovine occurs during the first 72 h postmortem, before any semitendinosus muscle stored at 2 to 4°C have been largeultrastructural changes in Z-disk structure or reported to change from 7.3 kg at death to 4.1 kg after any significantdegradation of actin or a-actinin 3 postmortem,d andto 3.4 kgafter 13 d of occurs. postmortem storage (Go11 et al., 1964). Consequently, 82% of all postmortem tenderizationhad occurred CostamereStructures in SkeletalMuscle withinthe first 3 d postmortem inthis study. Recently, a concerted effort was made to obtain an In 1983, Craig and associates observed filamentous accurateestimate of changes inmeat toughness structures that linked myofibrils to the sarcolemma duringthe first 3 d postmortem (Wheelerand (Craig and Pardo,1983; Pardo et al., 1983; Figure 3). Koohmaraie, 1994). At-death muscle was clamped Thesestructures, which were namedcostameres, before excision from the carcass to prevent shortening contained y-actin, vinculin, P-, , and the and was then frozen until all ATP in the muscle had proteins, desmin and , been hydrolyzed. Shear force measurements indicated and occurred periodically along the myofibril at the that muscle was only moderatelytough at death, level of each I-band in skeletal muscle or Z-disk in increased in toughness during the first 24 h postmor- (Craig and Pardo, 1983; Pardo et al., tem, and then decreased rapidly in toughness during 1983). Subsequentstudies have shown that costa- the next 48 h postmortem (Figure 2). Based on the meres also contain 01 (the integrin that binds maximum toughness at 24 h, 77% of all postmortem to ), clathrin, , P-spectrin, and dystro- tenderization had occurred after 72 h postmortem. The phin,the proteinmissing in Duchenne and Becker

Costameres

Figure 3. Schematic diagram showing the structure and protein composition of costameres in striated muscle relative to Z-disks and the myofibrillar lattice. The probable position of titin filaments is shown in this diagram, but nebulin filaments have been omitted to simplify the diagram. The N-terminal end of the large titin molecule is anchored in the Z-disk. Desmin, vinculin, and ankyrin, three of the protein constituents of costameres, extend into the muscle where they encircle myofibrils at the Z-disk and run from myofibril to myofibril to link adjacent Z- disks laterally (Granger and Lazarides, 1978; Nelson and Lazarides, 1984; Terracio et al., 1990). DAPs = dystrophin associated proteins: DAG = dystrophin-associated glycans; S = skelemins. This diagram has been adopted from a similar diagram shown inPrice (1991). Z-DISK DEGRADATION AND TENDERIZATION 1355 musculardystrophy (Nelsonand Lazarides,1983, (Solon, OH); and the protease inhibitors used in the 1984; Shearand Block, 1985; Byers et al., 1991; preparation of myofibrils (Edmunds etal., 1991) were Danowski et al., 1992; Porter et al., 1992; Straub et from Sigma (St. Louis, MO). The reagents used for al., 19921, and that a second -like network the enhanced chemiluminescence ( ECL) reaction on extends from the M-line region of myofibrils to the Westernblots were purchased from DuPontNEN sarcolemma (Figure 3). Recent studies indicate that ResearchProduct (Boston, MA). The resinsand costameres are more robust structuresthan their reagents used for fixation in electron microscopy were ethereal appearance would indicate and that they are obtained from Ted Pella(Redding, CA). All other sites of force transduction to the substratum (Street, chemicals were analyticalreagent grade or better. 1983, Danowski et al., 1992). Consequently, degrada- tion of costameres may significantly weaken muscle MyofibrilPreparation and Myofibril structure. Two earlierstudies (Young et al., 1980; Fragmentation Index Robson et al., 1984) suggested that the cytoskeletal proteins,desmin, nebulin, andtitin, may have an Bovine semimembranosus and biceps femoris mus- important role in tenderness, but these studiesdid not cles from each of four animals were sampled within30 mentioncostameres. minafter exsanguination, and myofibrils were pre- With the possible exception of y-actin, whose pared according to Go11 et al. ( 1977). The muscle on susceptibility to the calpains has not been tested, all the right side of the animal was used for the at-death protein components of costameres,including dystro- sample, andsamples at 1, 3, 6 or 7, and14 d phin (Cottin etal., 1992), arerapidly degraded by the postmortem were taken from the left side to prevent calpains in in vitro assays. Also, the costameres are any effects associated with shortening of the muscle located at the surface of the where they that had been sampled within 30 min after exsangui- would be immediately exposed toany extracellular Ca2+ leaking into the muscle cell due to damage or nation. Myofibril fragmentationindex was done as weakening of the sarcolemma during postmortem described by Culler etal. (1978). storage (Jeacocke, 1993). Taylor et al. (1994) found that costamere structures are lost during the first 24 SodiumDodecyl Sulfate-Polyacrylamide Gel to 72 h postmortem and that thisloss parallels loss of Electrophoresis and Western Blotting NZ lines in postmortem muscle. Both these structural changes can be mimicked by calpaintreatment of Samples for SDS-PAGE were prepared at 0, 1, 3, muscle strips(Yamaguchi et al., 1983; Go11 et al., and 6 d postmortem by homogenizing a 5- g sample of 1991, 1992b; Taylor et al.,1994). Hence, loss of whole muscle tissuein 15 mL of 2% SDS, pH 7.0, costameres andthe NZline occur duringthe same usingthree 30-S bursts of a Brinkmann Polytron period that the major increase in tenderization occurs. homogenizer at 25,000 rpm. Five hundred-microliter Theavailable evidence, therefore,suggests that aliquots of the homogenate were dissolved by adding something besides orin addition to Z-disk degradation an equal volume of sample buffer (8 M urea, 2 M is involved in postmortem tenderization. Conse- thiourea, 3% SDS, .7% MCE, 50 mM Tris.HC1, pH quently, we have examined more closely the nature of 6.8,) and heating for 4 min at 70°C. This treatment the ultrastructural changes that occur during the first resulted in complete dissolution of the muscle sample. 7 d postmortem, whenthe major increase in tenderiza- Proteinconcentration inthe dissolved samplewas tion occurs, and have monitored the rate of degrada- measured by using Coomassie Brilliant Blue G-250 tion of nebulin, titin, and several costamere proteins (Coomassie Plus Protein Assay, Pierce, Rockford, IL), during postmortem storage to learn whether degrada- and the samples were diluted to a final concentration tion of these proteinsparallels major changes in of 4.0 mg/mL with a 5050 mixture of 2% SDS and the tenderness. Finally, we have examined ultrastructur- urea sample buffer. The diluted samples wereclarified ally the myofibrils produced during the MFI assay to by centrifuging2 min at 5,000 x g in atable-top determine whether these myofibrils are broken at the microcentrifuge at 22 to 25"C, and bromophenol blue Z-disk, as is commonly assumed, or whether they have tracking dye was added to a final concentration of .l%. beenbroken elsewhere along the myofibril. The Thissampling procedure ensured that all muscle results indicate that Z-disks remain fairly robust at proteins were retained in the sample being assayed least during the first 7 d of postmortem storage, and and that no fragments that might be produced during that myofibrils seem to be weakest in the I-band area. postmortem storage were lost by washing steps. The SDS-PAGE was done according to Wolfe et al. (1989), Experimental Procedures or for titin, nebulin, and dystrophin assays, according to Fritz et al. (1989). Both procedures used tall mini- Materials gels (.75 mm x 8.0 cm x 10.9 cm). After electrophore- sis, the gels were transferred electrophoretically to a Acrylamide (99.9%), bisacrylamide (99.99%), and nitrocellulose membrane (Hoefer Scientific Instru- sodium dodecyl sulfate (99%) were from Schwartd ments, San Francisco, CA) using a semi-dry blotting Mann;Tris (ultrapure, 99.8%) was from Amresco apparatus and the procedure recommended by Hoefer. 1356 TAYLOR ETAL. Transfer was for 90 min at 100 mA (or for 120 min at also examined ultrastructurallyusing this same 150 mA for dystrophin, nebulin, and titin) in 20 mM protocol. Tris, 150 mM glycine, .l% SDS, 10 mM MCE, 15% methanol. Membranes containing the transferred pro- teins were then incubatedovernight either with a Results monoclonal antibody (MAb) to vinculin (V284, Boehringer Mannheim); to nebulin (Nb2, Sigma), to UltrastructuralChanges During titin (T12, Sigma), or todesmin (Allen etal., 1991). A Postmortem Storage polyclonal antibody elicited against a whose sequencewas identical to residues 3495-3544 from Figure 4 is a panel of electron micrographs showing the dystrophin molecule (Ishiura et al., 1990) was bovine skeletal muscle sampled at death and after 1, used inthe dystrophinWestern blots, andalkaline 3, and 16 d of postmortem storage. The sarcolemma in phosphatase detection involving the reactionwith at-death muscle has periodic areas of invaginations at 5-bromo-4-chloro-3-indolyl phosphatase and nitroblue the level of the Z-disk and the M-line; the densely tetrazolium was used instead of ECL detection. These staining diffuse regions underlyingthese invagina- residues are from the C-terminal part of the dystro- tions are costameres. The sarcolemmal invaginations phin polypeptide and elicit antibodies that can cross- disappear during the first24 h of postmortem storage, react with dystrophin-related proteins (Ishiura et al., andin approximately 50% of the fibers, thesar- 1990; Khuranaet al., 1991). After washing, the colemma is pulled away from the underlying myofibril nitrocellulose membranes were incubated for 1 h with (Figure 4C). Because costameresevidently are a second antibody conjugated with horseradish peroxi- responsible for attachment of the sarcolemma to the dase,and the signal from the second antibodywas myofibril, these structural changes indicate that the detected by using the ECL procedure (exposure times costamereshave been extensivelydegraded already up to 50 min, depending on the experiment). Films within the first 24 h postmortem. After 72 h postmor- from the ECL assay were scanned densitometrically tem,the sarcolemma has moved away from the using a Molecular Dynamics Computing Densitometer myofibril inall the fibers (Figure 4D), and only witha heliudneonlaser light source. remnants of the densely stainingtransmembrane plaques that connected the sarcolemma to the my- ElectronMicroscopy ofibril in at-death muscle remain (Figure 4D). The nature of the proteinsconstituting these densely Biceps femoris muscle strips approximately 4 mm x staining patches of material is unclear. Most costa- 30 mm were isolated parallel with the long axis of the mere proteins (ankyrin, desmin, dystrophin, spectrin, muscle, were immobilized in muscle clamps (Fine talin,vinculin) are excellent substrates for the Science Tools, Foster City, CA), and were then calpains, as are many of the transmembrane proteins removed from the muscle and fixed immediately with ( 0-integrin, the DAF's and DAGS; see Matsumura and 2.5% glutaraldehyde in .lM cacodylate buffer, pH 7.3, Campbell, 1994). Consequently, it would be expected for 4 h at 4°C. Samples were rinsed with cacodylate that most of the proteins constituting the costamere/ buffer, en bloc stained with 1% tannic acid for 30 min, transmembrane complexwould be destroyed by the postfixed with 1% osmium for 1 h,rinsed with calpains duringthe first 72 h postmortem. The cacodylate buffer followedby three rinses with 25% densely staining patches may be complexes of mem- ethanol,and then en bEoc stainedwith 1% uranyl brane phospholipids, calpain-resistanttransmem- acetate in 25% ethanol for 30 min. The samples were brane proteins, andlarge polypeptides that remain dehydrated using a graded series of ethanol and were aftercalpain cleavage of proteinssuch as filamin, embedded in Spurr's resin. Thin sections were stained talin, and vinculin. The degree of separation between withlead citrateand uranyl acetate. Myofibrils the sarcolemma andthe myofibrils in postmortem prepared for the myofibril fragmentation index were muscle was variable and seemed to depend in part on

Table 1. Densitometric scans of Western blots of desmin,nebulin, and vinculin at different times postmortema

Desmin Vinculin Nebulin Time postmortem, d Biceps femoris Semimembranosus Biceps femoris Semimembranosus Biceps femoris Semimembranosus 100 100 0 100 100 100 100 100 1 107 94.8 92.2 61.5 75.4 71.0 3 88.0 44.2 75.4 39.1 26.0 16.8 6 90.5 25.2 53.7 14.6 22.4 3.5 aFigures are expressed as percentage of denistometric units obtained from the blot of at-death muscle. Z-DISK DEGRADATIONTENDERIZATION AND 1357 whether adjacent muscle cells were close together and ultrastructural change.“Tears” or breaksin the I- thereby hindered sarcolemmal separation or whether band begin to appearafter 3 dpostmortem and an open space allowed considerable separation (note become increasingprevalent with long periods of that the sarcolemma cannot be seen in Figure 4E). postmortemstorage (Ouali,1990). The important point, however, is not the extent of the Ultrastructure of Myofibrils Used in the Myofibril sarcolemmdmyofibrilseparation, butrather that it Fragmentation Index and Changes in Myofibril Frag- was a consistentstructural change observed inall mentationIndex During Postmortem Storage. The postmortem muscle examined andthat it indicates MFI is related to tenderness and has been usedwidely loss of the structure that attaches myofibrils to the as a method of estimatingmeat tenderness. It sarcolemma in living muscle. evidently has generally been assumed that postmor- Another characteristic ultrastructural change dur- tem storage weakensmyofibrils at theZ-disk and that ing postmortemaging of muscle is that adjacent this weakening results in rupture of the Z-disk when myofibrils within the muscle fiber become separated postmortem muscle is exposed to the shearingforces of (Figure 4A and4E). It is unclearwhether this blender blades. In agreement with earlier studies, the separation is due to loss of water as sarcolemmal MFI inthe present study increasedcontinuously integrity is lost; to the increasedosmolarity that during14 d of postmortem storage(Figure 5). occurs in postmortem muscle (Ouali, 1990), to degra- Absolute values of the MFI can vary significantlyfrom dation of the desminand M-filaments that connect animal to animal, but the relative increases in MFI adjacent myofibrils at the level of the Z-disk and M- shown in Figure 5 are similar to those that havebeen line, respectively (see Figure 3), or to a combination reportedearlier (Koohmaraie et al., 1987, 1988a; of theseand perhaps other factors. Although the Whipple et al.,1990a,b; Shackelford et al., 1991). latticespacing between actinand myosin filaments Consequently, ourMFI assays aremeasuring the decreases asthe pH decreases below6.0 (Matsuda same changes as those described in previous studies, and Podolsky 1986), the extent of this shrinkage is which showed thatthe MFI is highly related to very low in “rigor” fibers (i.e., fibers having no ATP, tenderness. If the change in MFI between 0 and 14 d which would be the case in postmortemmuscle). postmortem is taken as loo%, then 75% (semimem- Matsudaand Podolsky ( 1986) found that lattice branosus; a “fast-aging” muscle)to 14%(biceps, a spacing between filaments in rigor muscle decreases “slow-aging” muscle) of thetotal change in MFI by approximately 8 to 9%, not enough to account for occurred between 0 and 3 dpostmortem. These thelarge gaps seen between adjacent myofibrils in changes compare with those reportedin earlier studies postmortem muscle. Moreover, measurement of my- in which 61 to 64% of thetotal change in MFI of ofibril diameterindicates thatthe myofibrils in longissimus dorsi muscle after 7 (Koohmaraie et al., postmortem muscle haveapproximately thesame 1988a) or 14 d (Koohmaraie et al., 1987) of postmor- diameter as thosein at-death muscle (allowing for tem storage hadoccurred during the first3 d. Electron variationsdue to the plane at which they were micrographs of the myofibrils used in the MFI assays showed that Z-disks in these myofibrils were intact sectioned; see Figure 4). Consequently, it is unlikely and that ruptureof myofibrils in theMFI assay occurs thatthe separationbetween adjacent myofibrils in inthe I-bandand not at the Z-disk (Figure6). postmortem muscle is caused by a change in lattice Detailed ultrastructural analysis of myofibrils used in spacing of the filaments. a MFI assay is difficult because the myofibrils are Most Z-disks in postmortem muscle stored at 4°C randomly oriented. However, contrary to the assump- remain almost unchanged ultrastructurally, even after tion thatthe increasein MFI duringpostmortem 16 d of postmortem storage (Figure 4F). Except for storage is caused by weakening of Z-disks, no in- degradation of the costameres and loss of sarcolemmal stances of Z-disk breakage were observed inthe integrity, the ultrastructural changes most frequently myofibrils examined inthis study. observed in postmortem muscle are “tears” orgaps in the I-band area (Figure 4F). These tears or breaks WesternAnalysis of Postmortem Changes in often occur near intact Z-disks in the area previously Costamere Proteins and Nebulin and Titin occupied by the N2 line. Although not shown here, it is possible to find areas inpostmortem muscle where the Vinculin and desmin are two of theimportant Z-disks seem to havelost some structuralintegrity proteins found in costameres. Moreover, these two and appear diffuse with occasional breaks along their proteinsextend from the costamere structureto length (as has been reported previously; Fukazawa encircle Z-disks withinthe muscle cells (Figure 3, and Yasui, 1967; Davey and Gilbert, 1969; Goll, 1968; Granger and Lazarides, 1978; Richardson et al., 1981; Henderson et al., 1970).In muscle stored at 4”C, Robson et al., 1984; Terracio et al., 1990). Conse- however, suchareas of Z-disk disruptionare rare quently,Western blots were done to learnwhether during the first 4 to 7 d postmortem, and total loss of these proteins are degraded during postmortem stor- Z-disks is not observed. Conversely, loss of costamere age and whether thisdegradation temporally parallels structureswithin 72 h postmortem is a consistent the loss of costameres observed ultrastructurally. 1358 TAYLOR ET AL. Z-DISK DEGRADATION AND TENDERIZATION 1359 Western blots of whole skeletal showed tem, and substantial vinculin degradation occurs by 3 that little or no degradation of desmin occurs in either d postmortem even in the biceps femoris, a slow-aging the biceps femoris or the semimembranosus muscle muscle (Table 1, Figure 7). This loss of vinculin may during the first 24 h postmortem (Figure 7, Table 1). be associated with the changes in costamere structure Overhalf thetotal desminin the semitendinosus seen duringthe first 24 h postmortem (Figure 4). muscle, however, was degraded between 24 and 72 h Because vinculin seems very susceptible to postmor- after death (Figure7, Table l),a period during which tem degradation, we examined six lamb longissimus tenderness increases dramatically (Figure 2 1. Earlier muscles that had been classified as either tough or studies using Western analysis of bovine semitendino- tender on the basisof Warner-Bratzler shear force and sus muscle (Hwan and Bandman, 1989) also found that desminwas degraded in postmortem muscle, MFI measurements(Table 2). Westernanalysis of although desmin degradation was not detectable until these six samples showed that the ratio of degraded after 72 h postmortem in this muscle. The difference undegraded vinculin was 4- to 50-fold higher in the between the biceps femoris and the semimembranous tender muscles than in the tough muscles (Figure 7, muscles inrate of postmortem desmindegradation Table 2). A major polypeptide fragment that reacts (Figure 7, Table 1) is remarkable and underscores the with the anti-vinculinantibody is prominent in need to specify the muscle used in postmortem samples from thetender muscles (Figure7). This tenderization studies. Vinculin is very susceptible to polypeptide fragmentmigrates in SDS-PAGE at a degradationin postmortem muscle, and vinculin relative molecular weight of 90 kDa, which is the degradation begins during the first 24 h postmortem same size as the major proteolytic fragment produced (Table 1, Figure 7). Almost half the vinculin in by calpain digestion of purified vinculin (Go11 et al., semimembranosus was degraded after 24 h postmor- 1983b). Consequently, rate and extent of postmortem degradation of vinculin seemrelated to rateand extent of postmortemtenderization. Dystrophin is the protein encoded by the that

~ ~ ~ ~~~~ ~ ~~~~ ~~ ~~~~ ~~ is missing or disruptedin patients afflicted with Figure 4. Electron micrographs of sections of bovine Duchenne or Becker’s musculardystrophy (Mat- biceps femoris muscle sampled after different times of sumuraand Campbell, 1994). Extensiveim- postmortem storage at 4°C. (A) Electron micrograph of munolocalization studies have shown that dystrophin bovine skeletal muscle sampled within 45 min after exsanguination. Arrow and arrowhead indicate areas of density (costameres) where the sarcolemma is attached to the Z-disk and M-line, respectively. (B and C) Bovine biceps femoris muscle sampled 24 h after death. Both I these micrographs show structural disintegration and broadening of the sarcolemma compared withthe structure of at-death sarcolemma. The sarcolemma is clearly detached and has been displaced from the myofibril in C. This detachment is representative of approximately 50% of the structures observed after 24 h postmortem. (D and E) Bovine biceps femoris muscle after 3 d of postmortem storage. Arrowheads (D)point / biceps femoris to patches of densely staining material at the level of the Z-disk. These densely staining areas maybe remnants of the transmembrane patches containing , DAGS, and DAPs (see Figure 3). The arrow in D shows the detached membrane that has now been pulled away a considerable distance from the myofibril. E shows examples of widening of the distance between adjacent myofibrils in postmortem muscle and loss of 20 l I I I . material that seems to connect Z-disks from adjacent 0 3 6 9 12 15 myofibrils (arrow). This material probably contains Days post modem desmin and filamin (Richardson et al., 1981). (F)Bovine biceps femoris muscle after 16 d postmortem. The Z- disk structure is well-preserved even after 16 d Figure 5. Changes inMFI of two different muscles postmortem. Arrowheads point togaps in the I-band sampled after different times of postmortem storage. adjacent to the intact Z-disk. These gaps are observed in Points are means plus or minus standard errors (vertical muscle that has been stored for 4 d or longer at 4°C. lines) of measurements in six separate muscle samples Bar = 1 pm. each done in duplicate. 1360 TAYLOR ET AL.

Ir A

Figure 6. Electron micrographsshowing structure of biceps femoris myofibrils used in the MFI assay. (A) Myofibrils used in a MFI assay done after 24 h of postmortem storage. (B, C, and D) Myofibrils used in a MFI assay done after 7 d of postmortem storage. Myofibrils after both 1 and 7 d of postmortem storage at 4°C are sheared in the I-band or A-band areas of the myofibril and not at the Z-disk. These breaks seem to occur most frequently at an area closer to the Z-disk (Figure 6C and 6D) than the A-I junction. The break next to the Z-disk is especially evident athigher magnification [Figure 6D). As the MFI assay shows, the breaks are more frequent and the myofibril segments are shorterafter 7 d postmortem than they are after 1 d of postmortem storage. All Z-disks are intact in the myofibrils used in the MFI assay both after 1 and after 7 d postmortem. Bar = .5 pm for A, B, and C and .25 pm for D.

is locatedon theintracellular surface of thesar- degraded during postmortem storage (Figure8). Very colemma innormal skeletal muscle cells andis a little dystrophin degradationoccurs during thefirst 24 component of the costamere (Byers et al.,1991; Porter h after death, but inthe semitendinosus, dystrophin is et al., 1992; Straubet al., 1992). Dystrophin is degradedrapidly between 24 and 72 hpostmortem presentin at-death bovine skeletal muscle and is and is barely detectable after 6 d postmortem. Less

Table 2. Densitometricscans of Western blots of vinculinfrom tough andtender musclesa

Myofibril Vinculin Lane in Warner-Bratzler fragmentation area of degradation fragment Sample Figure 7 shear force index area of intact vinculin Tough 1 10.67 44.0 .l6 Tough 2 9.90 35.0 .l1 Tender 3 3.08 89.0 5.55 Tender 93.5 4 4.03 2.66 Tender 84.0 5 3.96 1.33 Tough 6 13.18 43.5 .37 Z-DISK DEGRADATION AND TENDERIZATION 1361

A 12345678

1 2 345 6 7 8 Figure 8. Western blots showing rate of degradation of dystrophin during postmortem storage. SDS-poly- acrylamide gels of samples from the biceps femoris and semitendinosus muscles were run, were transferred to nitrocellulose membranes as described in Experimental Procedures, and the nitrocellulose membraneswere probed with an anti-dystrophin antibody. Lanes 1-4 are biceps femoris muscle sampled at death (lane 1) and a after 1 d(lane 21, 3d (lane 3), and 6 d(lane 4) -- -- postmortem. Lanes 5-8 are semimembranosus muscle --- sampled at death (lane 5) and after 1 d (lane 6) 3 d [lane C 7), and 6 d (lane 8) postmortem. The Western analysis 123456 7 shown in this experiment used alkaline phosphatase detection rather than ECL detection, and the "grainy" Figure 7. Western blot analysis showing rates of appearance is from the reaction with 5-bromo- degradation of desmin and vinculin during postmortem 4-chloro-3-indolyl phosphase and nitroblue tetrazolium storage and the relative extent of vinculin degradation used in alkaline phosphatase detection. No prominent in tough andtender muscle. (A) Anti-desmin blots of reaction with dystrophin-related proteins was detected bovine biceps femoris muscle (lanes 1-4) and bovine in our studies. semimembranosus muscle [lanes 5-8). These muscle samples were taken from the same animal and, therefore, the ratio of postmortem desmin degradation can be compared directly in these two sets of blots. than 50% of the dystrophin inat-death muscle Lane 1, at-death biceps; lane 2, 1-d biceps; lane 3, remains after 6 d postmortem, even in the slow-aging 3-d biceps; lane 4, 6-d biceps; lane 5, at-death biceps femoris (Figure 8). Loss of dystrophin, there- semimembranous; lane 6, l-d semimembranosus; lane fore, may not be related to destruction of costameres 7, 3-d semimembranosus; lane 8, 6-d semimembrano- during the first 24 h postmortem but may contribute sus. Postmortem degradation of desmin obviously to the postmortem tenderization that occurs between occurs more rapidly in the semimembranosus, a "fast- 24 and 72 hpostmortem. aging" muscle,than in the biceps femoris. (B] Anti- Titin and nebulin have been reported to be consti- vinculin blots of bovine biceps femoris (lanes 1-4) and tuents of the NZ line (Funatsuet al., 1990),and bovine semimembranosus muscle (lanes 5-8). These ultrastructural studies have shown that the N2 line is muscle samples were also taken from the same animal, the one of the first structuralentities degraded by and the rate of postmortem vinculin degradation in calpain (Go11 et al., 1991, 1992b; Taylor et al., 1994). these two sets of blots can be compared directly. Lane The titin and nebulin molecules both extend through 1, at-death biceps; lane 2, 1-d biceps; lane 3, the entire I-band, where myofibrils seem to rupture 3-d biceps; lane 4, 6-d biceps; lane 5, at-death when being prepared for MFI assays (Figure 5), and semimembranosus; lane 6, l-d semimembranosus; lane anchor in the Z-disk (Furst et al., 1988; Wang and 7, 3-d semimembranosus; lane 8, 6-d semimembrano- Wright, 1988). Therefore, Western analysis was used sus. The rate of postmortem vinculin degradation also is to determine the rates at which nebulin and titin are much faster in the semimembranosus than in the biceps degradedduring postmortem storage andwhether femoris. (C] Anti-vinculin blots of tough [lanes 1, 2, and their degradation occurs during the first 3 d postmor- 6)and tender [lanes 3, 4, and 5) lamb longissimus tem atthe sametime as the largechanges in muscle [see Table 2 for details). Lane 7 contained tenderness occur. Approximately 25% of skeletal purified bovine cardiac muscle vinculin. The major muscle nebulin is degraded during the first 24 h after fragments in lanes 3, 4, and 5 migrate at an approximate death in both the biceps femoris and semitendinosus molecular weight of 90 kDa, corresponding to the major muscles (this degradation is not obvious from the polypeptide fragment produced by calpain digestion of Western blots shown in Figure 9 but is revealed when purified vinculin. the blots are scanned (Table 1). Degradation of 1362 TAYLOR ET AL. nebulinmay be related to loss of the N2 linein that it is difficult to avoid partial degradation of titin postmortem muscle. The Nb2 anti-nebulin MAb recog- tothe T2 form duringsampling. Little additional nizes an epitope near the N2 line in the large nebulin degradation of titin occurs duringthe first 24 h molecule (Furst et al., 1988). That this anti-nebulin postmortem, butthe remaining T1 form of titin is MAb also recognizes large fragments of the nebulin entirely degraded to the T2 form between 24 and 72 h molecule, only slightlysmaller thanthe 600- to of postmortem storage (Figure 9B). The T12 anti-titin 900-kDa intact nebulin molecule, indicates that post- MAb recognizes an epitope approximately .l pm from mortem degradation of nebulin occurs at one end of the Z-disk, and degradation of titin from the TIto the the nebulin polypeptide rather than in its center. If T2 form results from cleavage at the N-terminal end this degradation is at theZ-disk end (C-terminal end) (the Z-disk end) of the titin polypeptide (Furst etal., of thenebulin polypeptide, it would contribute to weakening of the Z-disk attachment at this point and may account for breaks in this area observed in the MFI myofibrils (Figure 6). Titinwasalready partly degraded thein 0-time (at-death) samples,because they contained Figure 9. Western blots showing rates of degradation approximately a 50-50 mixture of the T1 and T2 forms of nebulin and titin during postmortem storage. (A) of titin(Figure 9B). Earlier studies havereported Anti-nebulin blots of bovine biceps femoris and semi- membranosus muscles at death and after 1, 3, and 6 d of postmortem storage at 4°C. Lanes 1-4 are biceps femoris muscle at death (lane 1)and after 1 d (lane 2), 3 d (lane 3), and 6 d (lane 4) postmortem. Lanes 5-8 are semimembranosus muscle at death (lane 5)and after 1 d (lane 6), 3 d (lane 7),and 6 d (lane 8) postmortem. Lane 5, the at-death semimembranosus sample, inadvertantly had less protein loaded on it than the other lanes shown in this figure. (B) Anti-titin blots of bovine rectus abdominis muscle (lanes 1-4) and of myofibrils pre- 12345678 pared from the same rectus abdominis muscle (lanes 5-6). Lanes 1-4 are rectus abdominis muscle at death (lane l),and after 1 d (lane 2), 3 d (lane 3),and 6 d (lane 4) of postmortem storage at 4°C. Lanes 5-6 are myofibrils from rectus abdominis muscle at death (lane 5),and after 1 d (lane 6), 3 d (lane 71, and 6 d (lane 8) of postmortem storage at 4°C. The polypeptides migrating in the range of 150 to 180 kDa (6d) resemble polypeptides that are produced by digestion of my- ofibrils with purified calpain and that react with the TI, anti-titin MAb (Thompson et al.,1993). The polypep- tides above 200 kDa are not normally observed in calpain-treated muscle. The pattern of titin degradation 12345678 observed in myofibrils is identical to that observed in whole muscle strips, so preparing myofibrils did not wash titin fragments produced by postmortem storage from the myofibril. Even the smaller polypeptides migrating in the range of 150 to 350 kDa are identical to those seen in Western blots of intact muscle fibers. (C) Anti-titin blots of bovine biceps femoris and semiten- dinosus muscles at death and after 1,3, and 6 d of postmortem storage at 4°C. Large amounts of protein were loaded onto the gels in this experiment so the fragments produced by degradation of titin postmortem

. I- can be more easily detected. Lanes 1-4 are biceps rrlr -200kDa femoris muscle at death (lane 1)and after 1 d (lane2), 3 '-m d (lane 3),and 6 d (lane 4) postmortem. Lanes 5-8 are C semimembranosus muscle at death (lane 5) and after 1 d 12345678 (lane 6), 3 d (lane 7),and 6 d (lane 8) postmortem. The TI and T2 forms of titin are not distinguishable at these heavy loads and appear only asa broad band. Z-DISK DEGRADATION AND TENDERIZATION 1363 1988). Consequently, postmortem storage resultsin 1. An increased rigidity or strengthening followed by cleavage of the titinpolypeptide at a site closer than .l aweakening of the actidmyosin interaction. The pm from the Z-disk; this cleavage occurs sometime nature of thisstrengthening and weakening is not between 24 and 72 h postmortem at 4°C. This understood. Strengthening of the actidmyosin interac- degradation of titin together with nebulin degradation tion in postmortem muscle markedly increases tough- (Figure 9A) likely contributes to the increased I-band ness and occurs during the first 24 h postmortem (in fragility in myofibrils during postmortem storage. The bovine skeletal muscle stored at 4°C or earlier in other NZ line is approximately .22 pm from the Z-disk, so species such as poultry). This strengtheningis usually postmortem cleavage of both titin and nebulin occurs accompanied by sarcomere shortening such as occurs at a site that is closer to the Z-disk than the N2 line. to an extreme extent during cold shortening. Shorten- Some additional degradation of titin to polypeptides ingincreases the degree of toughening that occurs recognized by the Tl2 MAb occurs between 72 and 144 during strengthening of the actidmyosin interaction, h postmortem, but the major T2 polypeptide remains but it seemsunlikely that shorteningalone is (Figure 9B and 9C). Hence, the majorfraction of responsible for the large increase in toughness that postmortem titin degradation occurs between 24 and occurs during the first 24 hpostmortem. The large 72 h postmortem and temporally coincides with the decrease in toughness (increase in tenderness) that majorincrease in tenderness (Figure 3). occurs between 24 and 72 h postmortem (Figure 2) is We prepared myofibrils from muscle at different duepartly to a weakening of theactidmyosin times postmortem and then subjected these myofibrils interaction.Again, the nature of this weakening is to Western blot analysis to learn whether the washing unknown, but it resultsin gradual lengthening of steps used in preparing myofibrils removed any titin rigor-shortened (Stromer et al., 1967; Go11 fragments produced during postmortem storage. The et al., 1970), in a 20 to 80% increase in the Mg2+- results of this experiment showed that the major titin modified ATPase activity of myofibrils (Golland fragments that accumulateduring postmortem stor- Robson, 1967), andin an increase in the rate of age remain associated withthe myofibril andthat actomyosin superprecipitation(Goll et al., 1970). It similarresults concerning the postmortemdegrada- seems likely that the postmortem changes in ATPase tion of titin are obtained whether using whole muscle and superprecipitation involve the actidmyosin inter- or myofibrils (Figure 9B). action rather than changes in myosin itself because These results on rate of postmortem titin degrada- the EDTA-modified ATPase activity of myofibrils, which measures myosin ATPase activity alone at an tion differ from thosereported earlier by Fritz and ionic strength of .5M, remainsunchanged during Greaser (Fritz and Greaser, 1991; Fritz et al., 1993) postmortemstorage (Goll and Robson, 1967). but agree with Bandman and Zdanis( 1988), who also 2. Disruption and weakening of the connections used Western blotting to show that significant titin between thin filaments in the I-band and the Z-disk. degradation occurs in bovine semitendinosus muscle Thisdisruption may involve partialdegradation of between 24 and 72 h postmortem. Most investigators some as yetunidentified Z-disk proteins, butit (see Paxhia and Parrish,1988; Anderson and Parrish, probably is principally due to degradation of titin and 1989) find substantial degradation of titin to the T2 nebulin, which extend intothe Z-disk andare form during the first 3 dpostmortem, especially in anchored there by interaction with a Z-disk protein “light” (Paxhia and Parrish, 1988) or “tender” (Lon- (possibly a-actinin). Thetwo major Z-disk proteins, a- gergan,Parrish, and Robson, Iowa State Univ., actin and a-actinin, are not degraded during postmor- unpublished results) muscles. It is not clear whether tem storage, at least not to any appreciable extent, the TI and Tz forms of titin were resolved in Fritz and duringthe first 14 to 18 d postmortem at 4°C. Greaser (1991); the titin band in this study is broad 3. Degradation of the costameres and intermyofibril and is barelyseparated from nebulin inthe gels linkages. As shown in the present study, costameres shown. The Fritz et al. (1993) study used only two are partly degraded within 24 h postmortem and are time periods, 2 d and 16 d postmortem, and the titin almost completely destroyed after 72 hpostmortem. again may have already been in the T2 form in the Even partialdegradation of costameresmay cause firstsample. Hence, it is possible the Fritz and weakening of the sarcolemma and lead to increased Greaser studies measured degradation of the T2 form leakage of extracellular Ca2+ into muscle cells and to of titin, which is stable during postmortem storage, the increasedosmolarity observed in postmortem ratherthan the conversion of TIto T2. muscle (Ouali, 1990). Muscle osmolality increases by 60 to 70% during the first 24 h postmortem and then remains almost constant for 9 d postmortem (Ouali Discussion 1990). Consequently, osmolality per se does not seem to be causallyrelated to thelarge changes in The results of this study indicate that postmortem postmortem tenderness that occur during this period. tenderization involves a complex interplay among at Depending on the physiological state of the animal leastthree interacting events. at slaughterand on the conditions andtime of 1364 TAYLOR ET AL. postmortemstorage, any one or any combination of in that important roles are ascribed to degradation of thethree events described inthe preceding para- costameres, intermyofibril linkages, and disruption of graphs could have a predominant effect on postmor- the actin filament/Z-disk interaction, and less impor- tem tenderness. The very large increase in toughness tance is given to degradation of the Z-disk itself. The that occurs during the first 24 h postmortem (Figure model proposed also indicates that postmortemten- 2 is almost certainly due to event 1, S trengthening of derization is a complex process and involves a number the actidmyosin interaction. It is unclearhow much of of changes including degradation of certain cytoskele- the large decrease in toughness that occurs between tal proteins and changes in the actidmyosin interac- 24 and 72 h postmortem is due to weakening of the tion. actin myosin interactionand how much is due to Although the model for postmortem tenderization eventsand2 3. It seems likely, however, that proposed in this paper ascribes less importance to Z- weakening of the actidmyosin interactionaccounts for disk degradation than earlier models, it nevertheless only part of this large decrease, andthat events 2 and retains a central role for the calpain system in this 3 are responsible for part of this change (perhaps up process. Costamereproteins are all excellent sub- to 50%). Degradation of desmin,nebulin, titin, and stratesand are readilydegraded by the calpains. vinculin would contribute continuously toincreased Intermyofibrillinkages contain desmin and filamin, tenderness beginning shortly after death and continu- both of which are rapidly cleaved by the calpains. The ing for 4 to 6 dpostmortem, at which timethese calpains also rapidly degrade nebulin and convert titin proteins have been largely degraded (or for nebulin from the TI to T2 form, both in vitro and in situ in andtitin, clipped to smallerpolypeptides). The myofibrils (Thompson et al., 1993). Consequently, it contribution of this degradation to tenderness during seems likely that most proteolytic degradationas- the first 24 to 48 h postmortem is overshadowed by sociated with the postmortemtenderization that the large effect that strengthening of the actidmyosin occurs during the first 72 to 96 h postmortem (see interaction has on tenderness. As the actid myosin Figure 2) is due to the calpain system. The evidence interaction weakens between 24 and 72 h postmortem, thatthe calpainsystem hasan important role in the effects of desmin,nebulin, titin, and vinculin postmortemtenderization has been summarized degradation become evident. earlier (Goll et al., 1983a, 1992a; Koohmaraie, 1988, It also is difficult to estimate the extent to which 1992; Figure 1). Perhaps the most compelling part of disruption of thethin filament/Z-disk interaction this evidence is that neither myosin nor a-actinin is contributes to the major decrease in toughness be- degraded to any significant extent during postmortem tween 24 and 72 h postmortem. After 72 h of storage at 4°C (Bandmanand Zdanis,1988; Hwan postmortemstorage at 4"C, nebulin is almost com- and Bandman, 1989). The calpains are unique in that pletely degraded, and titin hasbeen almost completely they do not degrade a-actinin and myosin (Goll et al., converted to the T2 form. Because the T2 form of titin 1991, 1992b1, whereas both these proteins are rapidly lacks theN-terminal end (the Z-disk end) of the degraded by cathepsins B, D, and L (Okitani et al., intact titin molecule (the TI form of titin is approxi- 1980; Matsukaraet al., 1981). mately 2,600 to 2,800 kDa; the T2 form is approxi- Theability of calpainto remove N2 lines and to mately 1,600 to 2,100 kDa, so the T2 form is still a degradenebulin andtitin suggests that calpain is very large polypeptide), attachment of titin to the Z- responsible for postmortemweakening of thethin disk has been destroyed after 72 h postmortem at 4°C. filament/Z-disk interaction. It has been proposed that This degradation of titin and nebulin probably results the NZ lineresults from a coalescence of titinand in a significant weakening of the thin filament/Z-disk nebulinfilament in this region of the sarcomere interaction. (Funatsuet al.,1990). Degradation of N2 may be Costameres, also, are completely destroyed during responsible for the "tears" or breaksin the I-band thefirst 72 h of postmortem storage at 4"C, and region of postmortem muscle. These tears are themost Westernanalysis shows that two of theimportant consistently observed ultrastructural changes found in proteins constituting costameres, desmin and vinculin, postmortem muscle (Henderson et al., 1970; Penny, are almost completely degraded duringthis period. 1980; Ouali, 1990) and are seenmore frequently than Dystrophin, also a component of costameres, is sub- frank Z-disk degradation(Taylor et al., 1994). Our stantially degradedafter 72 hpostmortem. Conse- electron microscope observations of the myofibrils quently, to the extent that costameres contribute to used in MFI assays suggest that weakening of the thin muscle fiberstability, degradation of costameres filament/Z-disk interaction is a major contributor to would contribute significantly to postmortem tenderi- the postmortem increase in MFI. Ouali (1990) zation. It alsoseems likely that degradation of the originally suggested that degradation of the NZ line filamentouslinkages between adjacent myofibrils has an important role in postmortem tenderization. would contribute to postmortemtenderization. The Recently, Tatsumi and Takahashi (1992) found that modelfor postmortem tenderization proposed in the Ca2+induced fragmentation of nebulin filaments and present paper, therefore, differs from previous models suggested that such fragmentation would destabilize Z-DISK DEGRADATION AND TENDERIZATION 1365 thin filaments and would be a key factor in postmor- Anderson, T. J., and F. C. Parrish, Jr. 1989. Postmortem degrada- tem tenderization. It could also be suggested that tion of titin and nebulinof beef steaks varying in tenderness.J. Food Sci. 54:748. degradation of troponin T to 30-kDa fragments Arakawa, N., D. E. Goll, and J. Temple. 1970. Molecular properties weakens thinfilaments and that degradation of of postmortem muscle. 8. Effect of postmortem storage on a- troponin T, therefore, may be a direct contributor to actinin and the -troponin complex. J. Food sci. 35: postmortem tenderization ratherthan being simply an 703. insitu indicator of calpainactivity as previously Bandman, E., and D. Zdanis. 1988. An immunological method to assess protein degradation in post-mortem muscle. Meat Sci. suggested. Because the cathepsins arelocalized in the 22: 1. sarcoplasmicreticulum inthe I-band of skeletal Busch, W. A., M. H. Stromer, D. E. Goll, and A. Suzuki. 1972. (=a2+- muscle (Taylor et al., 1994), and because incubation specific removal of Z-lines from rabbit skeletal muscle. J. Cell of muscle with crude cathepsin mixtures results in I- Biol. 52:367. band degradation (Ouali, 19901, the cathepsins may Byers, T. J., L. M. Kunkel, and S. C. Watkins. 1991. The subcellular distribution of dystrophinin mouse skeletal,cardiac, and contribute to proteolysis in postmortem muscle after 6 . J. Cell Biol. 115:411. or 7 d postmortem when muscle pH is low and small Cottin, P., S. Poussard, D. Mornet, J. J. Brustis, M. Mohammad- amounts of myosin anda-actinin degradation are pour, J. Leger, and A. Ducastaing. 1992. In uitro digestion of observed (Bandmanand Zdanis, 1988;Hwan and dystrophin by calcium-dependent proteases, calpain I, and 11. Bandman,1989). Biochimie (Paris) 74:565. Craig, S. W., and J. V. Pardo. 1983. Gammaactin, spectrin, and It seems likely, therefore, that the calpains are a intermediate filament proteinscolocalize with vinculin at costa- major cause of proteolysis in skeletal muscle during meres, myofibril to sarcolemma attachment sites. Cell Motil. 3: the first 72 to 96hpostmortem, butthat this 449. proteolysis probably resultsin destruction of costa- Culler, R. D., F. E. Parrish, Jr., G. C. Smith, and H. R. Cross. 1978. Relationship of myofibril fragmentation index to certainchemi- meres and intermyofibrillar linkages and in weaken- cal, physical, and sensory characteristics of bovine longissimus ing of the thin filamentJZ-disk interaction rather than dorsi muscle. J. Food Sci. 43:1177. in direct Z-disk degradation, as previously proposed. Danowski, B.A., K. Imanaka-Yoshida, J. M. Sanger,and J. W. Hence, the answer to the question posed by the title of Sanger. 1992. Costameres are sitesof force transmission to the this paper is, probably not a major role. However, the substratum in adult ratcardiomyocytes. J. Cell Biol. 118:1411. Davey, C. L., and K. V.Gilbert. 1966. Studies in meat tenderness:11. questions as to how the calpains, with their high Ca2+ Proteolysis and the aging of beef. J. Food Sci. 31:135. requirement for proteolytic activity and in the pres- Davey, C. L., and K. V.Gilbert. 1969. Studies in meat tenderness. 7. ence of excess calpastatin, which can completely Changes in thefine structure of meat during aging.J. Food Sci. inhibit their activity, can everbe active in postmortem 34:69. muscle remain(Goll et al., 1983a, 1994). Davies, P.J.A., D. Wallach, M. C. Willinghan, I. Pastan, M. Yamaguchi, and R. M. Robson. 1978. Filamin-actin interaction. Dissociation of binding from gelation by Cazc-activated proteol- ysis. J. Biol. Chem. 253:4036. Implications Dransfield, E. 1992. Modeling post-mortem tenderization-111: Role of calpain I in conditioning.Meat Sci. 31:85. Edmunds, T., P. A. Nagainis, S. K. Sathe, V. F. Thompson, and D. E. Thefindings reported in this paperindicate that Goll. 1991. Comparison of the autolyzed and unautolyzed forms degradation of intermyofibrillar linkages and of costa- of p- and m-calpain from bovine skeletal muscle. Biochim. meres, structures that linkmyofibrils to the sarcolem- Biophys. Acta 1077:197. mal of muscle cells, and weakening of Fritz, J. D., and M. L. Greaser. 1991. Changes in titin and nebulin in the thin filament/Z-disk interaction have major roles postmortem bovine musclerevealed by , western blotting, and immunofluorescence microscopy. J. Food in postmortem tenderization of muscle. Z-disk degra- Sci. 56:607. dation, however, which was previously thought to Fritz, J. D., M. C. Mitchell, B. B. Marsh, and M. L. Greaser. 1993. have a major role in postmortem tenderization, does Titin content of beef in relation to tenderness. Meat Sci. 33:41. not occur to any significant extent during the first 3 or Fritz, J. D., D. B. Swartz, and M. L. Greaser. 1989. Factors affecting polyacrylamide gel electrophoresis and electroblotting of high- 4 d when most postmortem tenderization occurs. If molecular-weightmyofibrillar proteins. Anal. Biochem. 180: this conclusion is correct, future studies attempting to 205. enhancethe rate and uniformity of postmortem Fukazawa, T., and T. Yasui. 1967. The change in zigzag configura- tenderizationshould focuson determining how to tion of the Z-line of myofibrils. Biochim. Biophys. Acta 140:534. increase the rateof degradation of costamere proteins Funatsu, T., H. Higuchi, and S'i. Ishiwata. 1990. Elastic filaments in skeletal muscle revealed by selective removal of thin filaments such as desmin and vinculin, and of thin filament/Z- withplasma . J. Cell Biol. 110:53. diskproteins such as nebulin andtitin. Furst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. The organiza- tion of titinfilaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: A map of two nonrepetitive epitopes starting at the Z-line extends close LiteratureCited to the M-line. J. Cell Biol. 106:1563. Goll, D. E. 1968. The resolution of rigor mortis. Proc. 21st Annu. Allen, R. E., L. L. Rankin, E. A. Greene, L. K. Boxhorn, P. R. Pierce, Reciprocal Meat Conf. pp 16-46. National Live Stock and Meat and S. E. Johnson. 1991. Desmin is present in proliferating rat Board, Chicago, IL. muscle satellite cells but not in bovine muscle satellite cells. J. Goll, D. E., N. Arakawa, M. H. Stromer, W. A. Busch, and R. M. Cell. Physiol. 149525. Robson. 1970. Chemistry of muscle proteins as a food. In: E. J. 1366 TAYLOR ET AL.

Brisky, R. G. Cassens, and B. B. Marsh (Ed.) The Physiology zation and developmental expression of dystrophin related pro- and Biochemistry of Muscle as a Food 2. pp 755-800. The teinin skeletal muscle. Neuromuscl.Disorders 1:185. University of Wisconsin Press, Madison. Koohmaraie, M. 1988. The role of endogenous proteases in meat Goll, D. E., W. R. Dayton, I. Singh, and R. M. Robson. 1991. Studies tenderness. Proc. 41st. Annu. Recip. Meat Conf., pp 89-100. of the a-actinidactin interaction in theZ-disk by using calpain. National Live Stock and Meat Board, Chicago, IL. J. Biol. Chem. 266:8501. Koohmaraie, M. 1992. The role of Ca2+-dependent proteases (cal- Goll, D. E., D.W. Henderson, and E. A. Kline. 1964. Post-mortem pains) in post mortem proteolysis andmeat tenderness. Bi- changes in physical and chemical properties of bovine muscle. ochimie (Paris) 74:239. J. Food Sci. 29:590. Koohmaraie, M.,A. S. Babiker, R. A. Merkel, and T. R. Dutson. Goll, D. E., Y. Otsuka, P. A. Nagainis, J. D. Shannon, S. K. Sathe, 1988a. Role of Ca2+-dependentproteases and lysosomal en- and M. Mugurunla. 1983a. Role of muscle proteinases in main- zymes in postmortem changesin bovine skeletal muscle. J. tenance of muscle integrity and mass. J. Food Biochem. 7:137. Food Sci. 53:1253. Goll, D.E., and R. M. Robson. 1967. Molecular properties of post- Koohmaraie, M., A. S. Babiker, A. L. Schroeder, R. A. Merkel, and T. mortem muscle. 1. Myofibrillar nucleosidetriphosphatase ac- R. Dutson. 1988b. Acceleration of postmortem tenderization in tivity of bovine muscle. J. FoodSci. 32:323. ovine carcassesthrough activation of Ca2+-dependent pro- Goll, D. E., J. D. Shannon, T. Edmunds, S. K. Sathe, W. C. Kleese, teases. J. Food Sci. 53:1638. and P. A. Nagainis. 198313. Propertiesand regulation of the Koohmaraie, M., W. H. Kennick, E. A. Elgasin, and A. F. Anglemier. Ca2+- dependent proteinase. In: B. de Bernard, G. L. Sottocasa, 1984. Effects of postmortem storage on muscle protein degrada- G. Sandri, E. Carafoli, G. Sandri, A. N. Taylor, T. C. Vanaman, tion:Analysis by SDS-polyacrylamidegel electrophoresis. J. and R. J. P. Williams (Ed.) Calcium-Binding Proteinsin Food Sci. 49:292. Healthand Disease. pp 19-35. Elsevier Science Publishers, Koohmaraie, M., S. C. Seideman, J. E. Schollmeyer, T. R. Dutson, Amsterdam, The Netherlands. and J. D. Crouse. 1987. Effect of post-mortem storage on Ca2+- Goll, D. E., M. H. Stromer, D. G. Olson, W. R. Dayton, A. Suzuki, dependent proteases, their inhibitor and myofibril fragmenta- and R. M. Robson. 1974. The role of myofibrillar proteins in tion. Meat Sci. 19:187. meattenderness. Proc. MeatIndustry Res. Conf. pp 75-98. Koohmaraie, M., and S. D.Shackelford. 1991. Effect of calcium American Meat Institute Foundation, Arlington, VA. chloride infusion on the tenderness of lambs fed a &adrenergic Goll, D. E., M. H. Stromer, R. M. Robson, B. M. Luke, and K. S. agonist. J. Anim. Sci. 69:2463. Hammond. 1977. Extraction, purification, and localization of cr- Luther, P. K. 1991. Three-dimensional reconstruction of a simple Z- actinin from a synchronous insect flightmuscle. In: R. T. band in fish muscle. J. Cell Biol. 113:1043. Tregear, (Ed.) Insect Flight Muscle Pmc. Oxford Symp. pp Marsh, B. B., J. V. Lochner, G. Takahashi, and D. D. Kragness. 1540. North-Holland Publishing,Amsterdam, The Nether- 1981. Effects of early postmortem pH and temperature on beef lands. tenderness. Meat Sci. 5:479. Goll, D. E., R. G. Taylor, J. A. Christiansen, and V. F. Thompson. Matsukara, U., A. Okitani, T. Nishimuro, and H. Kato. 1981. Mode 1992a. Role of proteinasesand protein turnover in muscle of degradation of myofibrillar proteins by an endogenous pro- growth and meat quality. Proc. 44th Annu. Recip. Meat Conf. tease, cathepsin L. Biochim. Biophys. Acta 662:41. pp 25-36. National Live Stock and Meat Board, Chicago, IL. Matsumura, K., and K. P. Campbell. 1994. Dystrophin-glycoprotein Goll, D. E., V. F. Thompson, R. G. Taylor, and J. A. Christiansen. complex: Its role in the molecular pathogenesis of muscular 1992b. Role of the calpain system in muscle growth. Biochimie dystrophies.Muscle & Nerve 17:2. (Parisj 74:225. Matsuda, T., and R. J. Poldolsky. 1986. Ordering of the Goll, D. E., V. F. Thompson, R. G. Taylor, T. Edmunds, and J. Cong. lattice in musclefibers. J. MolBiol. 189:361. 1994. Properties and biological regulation of the calpain sys- McBride, M.A., andF. C. Parrish, Jr. 1977. The30,000- tem. In: A. Ouali, F. Smulders, and D. Demeyer (Ed.) Proc. component of tender bovine longissimus muscle. J. Food Sci. 42: 1627. Workshop on Expression, Regulation, and Role of Proteinases Moller, A. J., T. Vestergaard, and J. Wismer-Pedersen. 1973. My- in Muscle Development and Meat Quality. Audit TijdschriRen ofibril fragmentation in bovine longissimus dorsi as anindex of bv, Nijmegen,The Netherlands (In press). tenderness. J. Food Sci. 385324. Granger, B. L., and E.Lazarides. 1978. The existence of an insoluble Nelson, W. J., and E. Lazarides. 1983. Expression of the @-subunitof Zdisc scaffold in chicken skeletal muscle. Cell 15:1253. spectrin in nonerythroid cells. Proc. Nat. Acad. Sci. USA 80: Hay, J. D., R. W. Currie, and F. H. Wolfe. 1973. Polyacrylamide disc 363. gel electrophoresis of fresh andaged chicken muscleproteins in Nelson, W. J., and E. Lazarides. 1984. Goblin (ankyrin) in striated sodium dodecyl sulfate. J. Food Sci. 38:987. muscle: Identification of the potential membrane receptor for Henderson, D. W., D. E. Goll, and M. H. Stromer. 1970. A compari- erythroid spectrin inmuscle cells. Proc. Nat. Acad. Sci. USA 81: son of shortening and Z-line degradation in post-mortem bo- 3292. vine,porcine, and rabbit muscle. Am. J. Anst. 128:117. Okitani, A., U. Matsukara, H. Kato, and M. Fujimaki. 1980. Purifi- Ho, C-Y., M. H. Stromer, and R. M. Robson. 1994. Identification of cation and some properties of a myofibrillar protein-degrading the 30-kDapolypeptide in postmortem skeletal muscle as a protease, cathepsin L, from rabbit skeletal muscle. J. Biochem. degradation product of troponin-T. Biochimie (Paris)(In 87:1133. press). Olson, D. G., F. C. Parrish, Jr., W. R. Dayton, and D. E. Goll. 1977. Hwan, S-F., and E. Bandman. 1989. Studies of desmin and a-actinin Effect of postmortem storage and calcium activated factor on degradation in bovine semitendinosus muscle. J. Food Sci. 54: the myofibrillar proteins of bovine skeletal muscle. J. Food Sci. 1426. 42:117. Ishiura, B., K. Arahata, T. Tsukahara, R. Koga, H. Anraku, M. Olson, D. G., F. C. Parrish, Jr., and M. H, Stromer. 1976. Myofibril Yamaguchi, T. Kikuchi, I. Nonaka, and H. Sugita. 1990. Anti- fragmentation and shear resistance of three bovine muscles body against the C-terminal portion of dystrophin crossreacts during postmortemstorage. J. Food Sci. 41: 1036. with the 400 kDaprotein in the pia mater of dystrophin- O'Shea, J. M., R. M. Robson, T. W. Huiatt, M. K Hartzer, andM. H. deficientmdx mouse brain. J. Biochem. 107:510. Stromer. 1979. Purified desmin from adult mammalian skeletal Jeacocke, R. E. 1963. The concentrations of free magnesium and free muscle: a peptide mapping comparison withdesmins from calcium ions both increase in skeletal muscle fibers entering adult mammalian and aviansmooth muscle. Biochem. Biophys. rigor mortis. Meat Sci. 35:27. Res. Commun. 89:972. Khurana, T. S., S. C. Watkins, P. Chafey, J. Chelly, F.M.S. Tome, M. Ouali, A. 1990. Meattenderization: Possible causesand mechan- Fardeau, J-C. Kaplan, and L. M. Kunkel. 1991. Immunolocali- isms. A review. J. Muscle Foods 1:129. Z-DISKTENDERIZATION DEGRADATION AND 1367

Pardo, J. V., J.D.A. Siliciana, and S. W. Craig. 1983. Avinculin- Stromer, M. H., D. E. Goll, and L. E. Roth. 1967. Morphology of containing cortical lattice in skeletalmuscle: Transverse lattice rigor-shortened bovine muscle and the effect of trypsin on pre- elements(“costameres”) mark sites of attachment between and post-rigor myofibrils. J. Cell Biol. 34:431., . myofibrils and sarcolemma. Proc. Natl. Acad. Sci. USA 80:1008. Suzuki, A., K. Hoshino, E. Sasaki, N. Sano, M. Nakane, Y. Ikeuchi, Parrish, F.C., Jr., D. E. Goll, W. J. Newcomb, 111, B. V. delumen, H. and M. Saito. 1988. Postmortem changes of native connectin in M. Chaudhry, and E. A. Kline. 1969. Molecular properties of rabbit skeletal muscle. Agric. Bioi. Chem. 52:1439. postmortem muscle. 7. Changes in nonprotein nitrogen and free Tatsumi, R., and K. Takahashi. 1992. Calcium-induced fragmenta- amino acids of bovine muscle. J. Food Sci. 34:196. tion of skeletal muscle nebulin filaments. J. Biochem. 112:775. Parrish, F. C. Jr., R. G. Young, B. E. Miner, and L. D. Anderson. Taylor, R. G., A. Ouali, and D. E. Goll. 1994. localization 1973. Effect of postmortemconditions on certain chemical, during postmortem muscle tenderization. In: A. Quali, F. Srnul- morphological, and organoleptic properties of bovine muscle. J. ders, and D. Demeyer (Ed.) Proc. Workshopon Expression, Food Sci. 38:690. Regulation, and Role of Proteinases in MuscleDevelopment Paxhia, J. M., and F. C. Parrish, Jr. 1988. Effect of postmortem andMeat Quality. Audit Tijdschriften bv, Nijmegen, The storage on titin and nebulin inpork and poultry light and dark Netherlands (In press) muscles. J. Food Sci. 53:1599, 1670. Terracio, L., D. G. Simpson, L. Hilenski, W. Carver, R. S. Decker, N. Penny, I. F. 1980. The enzymology of conditioning. In: R. A. Lawrie Vinson, and T. K Borg. 1990. Distribution of vinculin in theZ- (Ed.) Development in Meat Science. Vol I. pp 115. Applied disk of striated muscle:Analysis by laser scanning confocal Science Publishers, Banking, U.K. microscopy. J. Cell. Physiol. 145:78. Penny, I. F., C. A. Voyle, and E. Dransfield. 1974. The tenderizeg effect of a muscle proteinase on beef. J. Sci. Food Agric. 25703. Thompson, V. F., R. G. Taylor, D. E. Goll, E. J. Huff, and R. M. Porter, G. A., G. M. Dmytrenko, J. C. Winkelman, and R. J. Block. Robson. 1993. qffect of h- and m-calpain on bovine skeletal 1992. Dystrophin colocalizes with 8-spectrin in distinct subsar- muscle myofibrils. Mol. Biol. Cell 4386a. colemmal domains in mammalian skeletalmuscle. J. Cell Biol. Wang, IC, and J. Wright. 1988. Architecture of the sarcomere matrix 117:997. of skeletal muscle: Immunoelectron microscopic evidence that Price, M. G. 1991. Striated muscle endosarcomeric and exosarco- suggests a set of parallel inextensible nebulin filaments an- merit lattices. In: S. K. Melhotra (Ed.) Advanced Structural chored at the Z-line. J. Cell Biol. 107:2199. Biology.Vol. 1. pp 175-207. JAIPress, Greenwich, CT. Wheeler, T. L., and M. Koohmaraie. 1994. Prerigor and postrigor Richardson, F. L., M. H. Stromer, T. W. Huiatt, and R. M. Robson. changes in tenderness of dvine longissimus muscle. J. Anim. 1981. Immunoelectron and immunofluorescence localization of Sci. 72:1232. desmin in mature avian muscles. Eur. J. Cell Biol. 26:91. Wheeler, T. L., J. W. Savell, H. R. Cross, D. K. Lunt,and S. B. Robson, R. M., D. E. Goll, and M. J. Main. 1967. Molecular proper- Smith. 1990. Mechanismsassociated withthe variation in ties of post-mortem muscle. 5. Nucleosidetriphosphatase ac- tenderness of meat from Brahmanand Hereford cattle. J. tivity of bovine myosin B. J. Food Sci. 32:544. Anim. Sci. 68:4206. Robson, R.M., J. M. OShea, and M. K. Hartzer. 1984. Role of new Whipple, G., and M. Koohmaraie. 1991. Degradation of myofibrillar cytoskeletal elements in maintenance of muscle integrity. J. proteins by extractable lysosomal enzymes and m-calpain, and Food Biochem. 8:l-24. the effects of zinc chloride. J. Anim. Sci. 69:4449. Shackelford, S. D., M. Koohmaraie, G. Whipple, T. L. Wheeler, M. F. Whipple, G., M. Koohmaraie, M. E. Dikeman,and J. D. Crouse. Miller, J. D. Crouse, and J.0. Reagan. 1991. Predictors of beef 1990a. Effects of high-temperature conditioning on enzymatic tenderness: Development and verification. J. Food Sci. 56:1130. activity and tenderness of Bos indicus longissimus muscle. J. Shear, C. R., and R. J. Block. 1985. Vinculin in subsarcolemmal Anim. Sci. 68:3654. densities in chicken skeletal muscle: localization and relation- Whipple, G., M. Koohmaraie, M. E. Dikeman, J. D. Crouse, M.C. ship to intracellular and extracellular structures. J. Cell Biol. Hunt, and R.D. Klemm. 1990b. Evaluation of attributes that 101:240. affect longissimusmuscle tendernessin bos taaurus and bos Straub, V., R. E.Bittner, J. J. Leger, andT. Voit. 1992. Direct visualization of the dystrophinnetwork in skeletalmuscle fiber indicus cattle. J. Anim. Sci. 68:2716. membrane. J. Cell Biol. 119:1183. Wolfe, F. H., S. K. Sathe, D. E. Goll, W. C. Kleese, T. Edmunds, and Street, S. F. 1983. Lateral transmission of tension in frog myofibers: S. M. Duperret. 1989. Chicken skeletal muscle has three Ca2+- a myofibrillar network and transverse cytoskeletal connections dependentproteinases. Biochim. Biophys. Acta 998:236. , are possible transmitters. J. Cell. Physiol. 114:346. Yamaguchi, M,, R. M. Robson, M. H. Stromer, N. R. Cholvin, and M. Stromer, M. H., D. E. Goll, W. J. Reville, D. G. Olson, W. R. Dayton, Izumoto. 1983. Properties of soleus muscle Z-lines and induce and R. M. Robson. 1974. Structural changes in muscle during Z-line analogs revealed by dissection with Ca2+-activated neu- postmortemstorage. Proc. 4thInt. Congr. of Food Sci. and tral protease. Anal. Rec. 206:345. Techno]., Vol. I. pp 401-418. Instituto Nacional de Ciencia y Young, 0.A., A. E. Graafhuis,and C. L. Davey. 1980-81. Post- Tecnologia de Alimentos Consejo Superior de Investigaciones mortem changes incytoskeletal proteins of muscle. Meat Sci. 5: Cientificas,Valencia, Spain. 41.