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Sarcomere shortening and contraction nodes in stretched-restrained ovine myofibres during post mortem storage
Meat Science, Vol. 46, No. 4, 339-348, 1997 0 1997 Elsevier Science Ltd
All rights reserved. Printed in Great Britain PII:SO309-1740(97)00028-4
0309-1740/97 s17.oo+o.oo
ELSEVIER
Sarcomere Shortening and Contraction Nodes in StretchedRestrained Ovine Myofibres During Post Mortem Storage Atsushi Laboratory
Suzuki, Yuka Yamadera, of Functional
(Received
Kyoko
Kido & Kouichi
Morphology, Department of Animal Science, Faculty Tohoku University, Sendai 981, Japan
21 September
1996; revised 27 February
1997; accepted
Watanabe of Agriculture,
9 March
1997)
ABSTRACT Myojibres of muscles removed from the carcass shorten during rigor mortis. Whether myojibres of stretched and restrained muscle tissues shorten during post mortem storage was examined. Muscle strips removed from the semimembranosus muscle of sheep before rigor onset were stretched and restrained. The muscle strips werefixed separately in formalin solution from 1.5 hr to 12 days post mortem, and embedded in parafin. Sections were stained with Mallory-azan. Some myojibres in the pre-rigor-excised and stretche& restrained muscIe strips had contraction nodes at 1 day post mortem. The contraction node did not increase in frequency hereafter. Two to several successive sarcomeres shortened regularly to form the nodes parallel to the Z-line. In addition, several successive sarcomeres shortened partly and successively from one side to the other side to form the nodes oblique to the Z-line. Oblique and irregular contraction nodes appear to cause kinks or bends of myojibres. Sarcomere lengths were not always uniform in individual myojibres. Formation of contraction nodes and shortening of partial sarcomeres in the stretche6 restrained myojibres indicate that the myojibres shorten individually during rigor mortis and vary in shortening states as in muscIes left on the carcass. 0 1997 Elsevier Science Ltd
INTRODUCTION
Isolated skeletal muscles shorten in rigor mortis (Smith, 1950; Locker and Hagyard, 1963) and have short sarcomere lengths (Takahashi et al., 1967, 1995; Henderson et al., 1970). Some myofibres form contacture nodes or kink or crinkle in post mortem storage (Paul er al., 1944, 1952). Active contraction of myofibres in rigor mortis is believed to produce the characteristic nodes, whereas the crinkles or kinks occur in the passively retracted myofibres by outside stress generated by the actively contracted myofibres (Paul et al., 1944, 1952). Myofibres change in sarcomere length during rigor mortis. Transversely broken myofibres are observed in post mortem ageing (Paul et al., 1944, 1952; Gann and Merkel, 1978; Suzuki, 1986). Myofibres of the longissimus dorsi and biceps femoris muscles, from the horizontally placed carcass with limbs perpendicular to the vertebrae, are longer in sarcomere length 339
A. Suzuki et al.
340
than those of the carcass suspended vertically from the Acilles tendon in post-rigor in cattle (Herring et al., 1965a; Hostetler et al., 1972). These facts indicate that the method of carcass suspension before rigor onset influences sarcomere lengths in post mortem muscles. Pre-rigor excised and free muscles are shorter in sarcomere length, whereas pre-rigor excised and stretched-restrained muscles are longer (Herring et al., 19656). The longer sarcomeres of myofibres in the muscles of the horizontally placed carcass or in the stretched-restrained muscles raise a question as to whether their myofibres are unshortened during rigor mortis. The purpose of this study was to examine whether myofibres of stretched and restrained muscle tissues excised from the carcass before rigor onset change in sarcomere length and form contraction nodes in post mortem storage as well as myofibres of muscles attached to the skeleton. MATERIALS
AND METHODS
The semimembranosus muscle from female sheep (Suffolk x Corriedale) aged 2 years were used in this study. At 1.5 hr after slaughter, six longitudinal strips of muscles (4 mmx6 mm x 2 cm) were excised from the carcasses. These samples were stretched and tied to sticks at both ends of the strips to prevent them from contracting. One of these stretchedrestrained strips was immediately fixed with Baker’s formol-calcium (Pearse, 1968). The others were put on a sheet of moist filter paper in petri dishes. The petri dishes were covered, wrapped in plastic wrap, and kept at room temperature (18-20°C) for 6 hr after slaughter. At 6 hr post mortem, one of them was taken out and immersed in the fixative. After 6 hr post mortem, the stretched-restrained strips in the petri dishes were stored in a refrigerator at 45°C. These were taken out separately from the petri dish at 1, 3, 6 and 12 days post mortem, and then immersed in the fixative without taking the muscle strips off sticks. For comparison, two muscle strips were excised from the semimembranosus muscle, from the side placed horizontally on a table at 1.5 hr, 6 hr, 1 day, 3 days, 6 days and 12 days post mortem. One was tied to a stick without stretching (excised-restrained strip) and another was untied (excised-unrestrained strip). These were immersed in the fixative. The tied samples were cut off the sticks after fixation and before dehydration. All the samples were dehydrated and embedded in paraffin. Longitudinal sections, 3 km thick, were stained with Mallory-Heidenhain’s azan stain (Lillie, 1965). The lengths of a 30 sarcomere segment in 10 myofibres in muscle sections were measured from images enlarged at 3400x magnification. Differences between the sarcomere lengths were compared using the Student’s t-test.
RESULTS Stretched-restrained
muscle tissue
Myofibres were straight from 1.5 hr to 12 days post mortem [Fig. l(a) and (b)]. Crossstriations composed of the A-band and I-band with the Z-line were seen clearly in post mortem ageing. At 1 day post mortem, two or several successive sarcomeres shortened in places in some myofibers, and consequently contraction nodes were formed (Figs l(b) and 2). The I-bands disappeared in the nodes owing to shortening. The contraction nodes formed by the relatively regular shortening of two or several sarcomeres looked like a bamboo joint (Fig. 2). No such phenomena were observed at 1.5 and 6 hr post mortem.
341
ovine muscle tissue at 1.5 hr post mortem. Sarcomere lengths are almost uniform and 2.65 pm in average. Mallory-Heidenhain’s azan stain. White bar length = 15 1pm. (b) Myofibres of the stretched-restrained ovine muscle tissue at 1 day post mortem. Three myofibres have contraction nodes (arrows) (like bamboo joints). Sarcomeres vary in length: 2.73 pm in A, 2.75 ,um in B, 2.20 pm in C, 2.49 pm in D, 2.26 pm in E, 2.19 pm in F, 2.26 pm in G, 2.30pm in H and 2.51 pm in I, which are mean values determined from a 5 sarcomere segFig. 1. (a) Myofibres of the stretched-restrained
ment. Mallory-Heidenhain’s
azan stain. White bar length = 15.1 pm.
In a number of sarcomeres, the I-band was discerned at one side of a myofibre, but was invisible by shortening at the other side [Fig. 2(b) and (c)l. The structural arrangements of the sarcomere were disorganised at the contraction nodes [Fig. 2(c)]. The I-bands of several successive sarcomeres shortened partly and successively from one side to the other in a myofibre to form an oblique contraction node non-parallel to the Z-line [Fig. 3(a)]. Such irregular and active shortening of sarcomeres appeared to produce kinks or bends in the myofibre. The number of nodes varied from myofibre to myofibre. Not all myofibres had the contraction node. The characteristic rigor nodes corresponding to the structural features shown by Paul et al. (1944) were observed infrequently from 1.5 or 6 hr post mortem onward, regardless of the type of muscle strip [Fig. 3(b)]. Sarcomere shortening of the contraction nodes, which looked like bamboo joints, occurred much more frequently than did the rigor nodes. The sarcomere lengths were not always uniform in the regions that did not form the contraction nodes, although the sarcomeres remained stretched between the nodes (Fig. l(b)). The sarcomere lengths were observed to vary from region to region in a myofibre, in which no contraction nodes was observed.
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The myofibres tended to have more nodes at 1 and 3 day post mortem than did those at 6 and 12 day post mortem. After 1 day post mortem, the arrangement of the sarcomere was disturbed in places [Fig. 1(b)]. The disarrangement appeared to occur more in 6 and 12 day post mortem. No myofibres were broken transversely from 1.5 hr to 12 day post mortem storage. Muscle tissue excised from carcass
In the excised and restrained muscle tissue, the contraction nodes and disarrangements of the sarcomeres occurred at 1 day post mortem as in the stretched-restrained muscle tissue.
Fig. 2. Contraction nodes in the stretched-restrained ovine myofibres at 1 day post mortem. These figures are enlarged from part of Fig. l(b). Contraction nodes are formed by the shortening of two or three (a) to several [(b), (c )] sarcomeres. Dark bands are the A-bands, light bands the I-bands and fine lines the Z-lines. The Z-lines that arrows indicate [(b),( c )] are invisible on one side by uneven shortening of the I-band. Part of the sarcomere structures (asterisk) of the contraction node (c) are disorganised. Mallory-Heidenhain’s azan stain. White bar length = 4.7 pm.
Contraction node of rigor ovine myojbres
343
The irregularly shortening sarcomeres were observed at this stage, indicating that myofibres kinked or bent actively [Fig. 3(c)]. Almost all myofibres showed clear cross-striations. Myofibres bent as a wave in places at 1 day post mortem. Bent myofibres decreased after 3 days post mortem.
Fig. 3. (a) Contraction node of the stretched-restrained ovine myofibre at 1 day post mortetn. Sarcomeres between two arrows in both sides of the myofibre shorten irregularly to form a contraction node oblique to the Z-line, which appears to cause kink in myofibres. Mallory-Heidenhain’s azan stain. White bar length = 3.8 Wm. (b) Contraction node in a myofibre of the excised-restrained ovine muscle tissue at 6 hr post mortem. This node corresponds to the characteristic rigor node (R) shown by Paul et al. (1944). Many sarcomeres shorten extremely. Myofibres (C) adjacent the myofibre forming the rigor node are crinkled passively at its side. Mallory-Heidenhain’s azan stain. White bar length = 8.7 km. (c) Contraction node in a kinked myofibre of the excised-restrained ovine muscle strip at 1 day post mortem. Sarcomere structures (asterisk) are disorganised partly in contraction nodes oblique to the Z-line. Large arrows indicate the H-band, which means the stretched sarcomeres, and small arrows the Z-line. The contraction node of the kinked (k) myofibre does not affect the sarcomeres of an adjacent (A) myofibre. Mallory-Heidenhain’s azan stain, White bar length = 4 grn.
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ovine muscle tissue at 1 day post mortem. K: kinked myofibre. Mallory-Heidenhain’s azan stain. White bar length= 30.1 pm. (b) Transversely cracked myofibres of the excised-restrained ovine muscle tissue at 3 days post mortem. Arrows indicate cracks. Mallory-Heidenhain’s azan stain. White bar length = 18.5 pm. (c) Transversely cracked and broken myofibres of the excised-unrestrained ovine muscle tissue at 6 days post mortem. Arrows indicate cracks and an asterisk a broken myofibre. Mallory-Heidenhain’s azan stain. White bar length = 37.4 pm. (d) Transversely broken myofibres of the exciseddunrestrained ovine muscle tissue at 12 days post mortem. Asterisks indicate broken myofibres. Mallory-Heidenhain’s azan stain. White bar length = 37.4 pm. Fig. 4. (a) Kinked myofibres in the excised-unrestrained
The excised and unrestrained muscle tissues had both straight myofibres and bent myofibres as waves at 1.5 and 6 hr post mortem. At 1 day post mortem, bent or kinked myofibres occurred more in the unrestrained muscle tissue than in the restrained muscle tissues; some myofibres had several contraction nodes [Fig. 4(a)]. In many contraction nodes, the sarcomeres were shortened too extremely to discern the structures [Fig. 4(a)]. Occurrence of bent myofibres was higher in frequency at 1 day post mortem and lower after 3 days post mortem; many myofibres were straight [Fig. 4(a),(c) and (d)]. At 3 days post mortem, some myofibres in the excised-restrained and excised-unrestrained muscle tissues were transversely broken in places [Fig. 4(b)]. At 1 day post mortem,
Contraction node of rigor ovine myojibres
345
TABLE 1 Sarcomere Lengths in Post Mortem Muscle Tissue from the Semimembranousus Muscle of Sheep Post mortem age
Excise&unrestrained(~)
1 hr 6hr 1 day 3 days 6 days 12 days
1.94kO.O6’@ 2.01 f 0.096 2.12AO.04’ 2.04 f 0. 180bcd 2.05 AI0.09” 2.09 * 0.07cd
Excised-restrained(m) 242 f 0.05” 249 i 0.09” 2.15*0.16b 2.24 f 0. lob” 2.34ztO.13” 2.24k0.11bc
Stretchekrestrained
(p)
2.65*0.13& 2.75*0.10b 2.55*0.15a 2.96ztO.19’ 2.61 f 0.09” 2.6OztO.11”
‘Mean f SEM determined from 10 myofibres (30 sarcomeres per myofibre). dCdSacromere lengths with different superscripts in columns are significantly different (P < 0.05).
transverse cracks which showed breaks of myofibrils were found in a few myofibres. The breaks of myofibres in the muscle tissue excised from the carcass increased with storage time [Fig. 4(b),(c) and (d)]. Length of ssrcomeres
Table 1 shows the sarcomere lengths, except for the extremely shortened sarcomeres of the contraction nodes. No regular changes common to the three different muscle strips were observed in sarcomere length during post mortem ageing. Sarcomere lengths of the unrestrained muscle tissues were shorter at 1.5 hr after slaughter than at 1 day post mortem, and unchanged from 3 days post mortem onward. In the excised-restrained muscle strips, sarcomere lengths were shorter at 1 day post mortem than at 1.5 and 6 hr post mortem and changed little hereafter. The myofibres of stretched-restrained muscle strips appeared to shorten at 1 day post mortem, but varied in sarcomere length, regardless of post mortem ageing. The unrestrained muscle strips had the shortest sarcomere lengths and the stretched-restrained muscle strips had the largest sarcomere lengths at all the stages (P < O-01). DISCUSSION
Extreme shortening in two adjoining sarcomeres or several successive sarcomeres resulted in the formation of contraction nodes. The occurrence of the contraction nodes in myofibres of the stretched-restrained muscle tissues at 1 day post mortem, as found in the
restrained and unrestrained muscle tissues excised from the carcass, shows that the myofibres contract actively in rigor mortis. The finding is consistent with the results of previous studies showing that muscles or sarcomeres shortened during post mortem storage (Smith, 1950; Locker and Hagyard, 1963; Takahashi et al., 1967, 1995; Henderson et al., 1970). The scattered formation of contraction nodes, and non-uniform shortening of the sarcomeres within a myofibre, may be related to the structure of the connective tissue network of the endomysium. The contraction node (like a bamboo joint) in the stretched-restrained myofibres is a type of rigor node (Paul et al., 1944), but differs in structural features from characteristic rigor nodes, as shown by Paul et al. (1944). Characteristic rigor nodes seem to occur in pre- and post-rigor because they were observed from 1.5 hr post mortem onward. Various shortening modes of the sarcomeres in contraction nodes could be observed clearly in the
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stretched-restrained myofibres, because the contraction nodes of the stretched-restrained myofibres shortened less than the ones of the unrestrained myofibres. The kinked or crinkled myofibres in the muscles excised from carcasses stored post mortem are similar to those shown by Paul et al. (1944, 1952). The kinks of myofibres are considered to occur through passive retraction (Paul et al., 1944). Observation of the sarcomeres in obliquely formed contraction nodes indicates that some kinked myofibres are formed actively by irregular and partial shortening of the sarcomeres. The excised muscle develops tension in an isometric state immediately after death and subsequently loses this tension (Jungk et al., 1967). The occurrence of contraction nodes in myofibres coincides with tension development. Tension developed by formation of the contraction nodes may stretch the sarcomeres to some extent among the nodes. Myofibres are transversely broken at 1 day post mortem in the bovine muscle (Paul, 1944) or 3 days post mortem in the ovine muscle (Suzuki, 1986). The breaks of myofibres were observed in the muscle tissues excised from the carcass during post mortem ageing. Breaks of myofibres appeared to be associated with decreases in bent myofibres after 3 days post mortem. The breaks of myofibres appear to release tension developed in them. The tension release may relax myofibres bent in waves and consequently the myofibres straighten to some extent. Fragmentation of myofibrils at the I-band and Z-disc junction occurs at 48 hr post mortem, and the quantity of transverse breaks increased hereafter in the bovine muscles (Gann and Merkel, 1978). Similar increases in the extent of myofibre breakage were observed in this study. The breaks of myofibres are believed to make postrigor meat tender (Paul et al., 1952; Gann and Merkel, 1978; Pearson, 1986). The myofibrils have been shown to become fragile during post mortem ageing (Taylor et al., 1995). Proteolysis by calpains in skeletal muscle during post mortem storage results in destruction of costameres and intermyofibrillar linkages and weakening of the filament/ Z-disc interaction, which are considered to have an important role in meat tenderization (Taylor et al., 1995). On the contrary, translocated paratropomyosin induced by increases in the calcium levels in sarcoplasmic reticulum has been proposed as a key factor in meat tenderization (Takahashi et al., 1995). Longer sarcomere lengths of the stretched-restrained myofibres than those of the unrestrained myofibres in ovine muscle tissues agree with the results from bovine muscles shown by Herring et al. (1965b). The sarcomere length of post mortem stored muscles depends on the conditions to which the muscles are exposed or in which muscle tissues are fixed (Herring et al., 1965~; Hostetler ef al., 1972). The sarcomeres were observed to shorten at 1 day post mortem in the excised-restrained muscle tissues, but not in the unrestrained muscle tissue. At this stage, the sarcomeres of the stretched-restrained muscle tissues showed a tendency to shorten. The sarcomere lengths in the three different muscle tissues did not increase or decrease with post mortem ageing. Post mortem ageing has been reported to have no influence on sarcomere lengths in the muscles of sheep (Bouton er al., 1973) or of turkeys (Johnson and Bowers, 1976). The unrestrained muscle tissues from the stored carcass seem to be influenced by formalin fixation because they had the shortest sarcomere lengths. Pre-rigor unrestrained muscle tissues had more bent myofibres than did post-rigor unrestrained muscles, indicating that the myofibres of the pre-rigor muscles are influenced more by fixation than those of the post-rigor muscles. Pre-rigor myofibres, sensitive to the fixative, presumably bent during fixation. Bending and shortening of myofibres by formalin fixation is inhibited in the restrained muscle tissues, because the restrained muscle tissues had no bent myofibres or shortest sarcomere lengths at 1.5 hr post mortem. We assumed that the stretched-restrained myofibres contracted actively during rigor mortis and consequently myofibres should be broken by the tension developed within them. However, the myofibres were unbroken during post mortem storage. No physical
Contraction node of rigor ovine myojibres
347
force was applied to the muscle strips from storage through fixation. Maximum tension in living muscles is generated at sarcomere lengths in the range of 2.05 and 2.25 pm (Gordon et al., 1966). The tension in the stretched-restrained muscle tissue may be too weak to make myofibres fragile post-rigor because the sarcomere lengths were beyond 2.5 ,um. In unstretched muscle samples at 90 hr post mortem, myofibres have been reported to be unbroken, whereas myofibres are broken in stretched muscle samples (Davey and Dickson, 1970). Marked extension of myofibres leads to fracture (Mutungi et al., 1995). The myofibres which were stretched and restrained before rigor onset shortened individually but not greatly during rigor mortis and varied in their shortening states. Although muscles attached to the skeleton in the carcass cannot shorten freely if the joints are fixed by antagonistic function of the other muscles or gravitation in the suspended carcass during rigor mortis, the myofibres appear to shorten individually and vary in shortening states; causing uniform, partial or irregular shortening along a myofibre. In muscles attached to the skeleton, shortening of individual myofibres in rigor mortis may not result in shortening of the muscle as a whole because the connective tissues within muscles, which bind the myofibres and fascicles together, permit a certain degree of free motion between them.
REFERENCES Bouton, P. E., Harris, P. V., Shorthose, W. R. and Baxter, R. I. (1973) A comparison of the effects of aging, conditioning and skeletal restraint on the tenderness of mutton. Journal of Food Science 38, 932-937. Davey, C. L. and Dickson, M. R. (1970) Studies in meat tenderness. 8. Ultra-structural changes in meat during aging. Journal of Food Science 35, N-60. Gann, G. L. and Merkel, R. A. (1978) Ultrastructural changes in bovine Iongissimus muscle during postmortem ageing. Meat Science 2, 129-144. Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966) The variation in isometric tension with sarcomere length in vertebrate muscle fibres. The Journal of Physiology 184, 17&192. Henderson, D. W., Goll, D. E. and Stromer, M. H. (1970) A comparison of shortening and Z line degradation in post-morten bovine, porcine, and rabbit muscle. The American JournaI of Anatomy 128, 117-136. Herring, H. K., Cassens, R. G. and Briskey, F. J. (1965a) Further studies on bovine muscle tenderness as influenced by carcass position, sarcomere length, and fiber diameter. JournaI of Food Science 30, 1049-1054. Herring, H. K., Cassens, R. G. and Briskey, E. J. (19656) Sarcomere length of free and restrained bovine muscles at low temperature as related to tenderness. Journal of the Science of Food and Agriculture 16, 379-384. Hostetler, R. L., Link, B. A., Landmann, W. A. and Fitzhugh, H. A., Jr. (1972) Effect of carcass suspension on sarcomere length and shear force of some major bovine muscles. Journal of Food Science 37, 132-l 35. Johnson, P. G. and Bowers, J. A. (1976) Influence of aging on the electrophoretic and structural characteristics of turkey breast muscle. Journal of Food Science 41, 255-261. Jungk, R. A., Snyder, H. E., Goll, D. E. and McConnell, K. G. (1967) Isometric tension changes and shortening in muscle strips during post-mortem aging. Journal of Food Science 32, 158% 161. Lillie, R. D. (1965) Histopathologic Technic and Practical Histochemistry, 3rd edn, p. 545. McGrawHill, New York. Locker, R. H. and Hagyard, C. J. (1963) A cold shortening effect in beef muscles. Journal of the Science of Food and Agriculture 14, 787-793. Mutungi, G., Purslow, P. and Warkup, C. (1995) Structural and mechanical changes in raw and cooked single porcine muscle fibres extended to fracture. Meat Science 40, 217-234.
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Paul, P., Lowe, B. and McClurg, B. R. (1944) Changes in histological structure and palatability of beef during storage. Food Research 9, 221-233. Paul, P., Bratzler, L. J., Farwell, E. D. and Knight, K. (1952) Studies on tenderness of beef. I. Rate of heat penetration. Food Research 17, 504-5 10. Pearse, A. G. E. (1968) Histochemistry. Theoretical and Applied, 3rd edn, Vol. 1, p. 601. J. & A. Churchill, London. Pearson, A. M. (1986) Physical and biochemical changes occurring in muscle during storage and preservation. In Muscle as Food, ed. P. J. Bechtel, pp. 103-134. Academic Press, New York. Smith, R. D. (1950) Studies on rigor morns. Part II. Qualitative observations on the post mortem shortening of muscles. The Anatomical Record 108,207-216. Suzuki, A. (1986) A histochemical change in myofibers of sheep muscle in refrigeration. Tohoku Journal of Agricultural Research 36, 135-143. Takahashi, K., Fukazawa, T. and Yasui, T. (1967) Formation of myofibrillar fragments and reversible contraction of sarcomeres in chicken pectoral muscle. Journal of Food Science 32,409-413. Takahashi, K., Hattori, A. and Kuroyanagi, H. (1995) Relationship between the translocation of paratropomyosin and the restoration of rigor-shortened sarcomeres during post-mortem ageing of meat - A molecular mechanism of meat tenderization. Meat Science 40,413-423. Taylor, R. G., Geesink, G. H., Thompson, V. F., Koohmaraie, M. and Goll, D. E. (1995) Is Z-disk degradation responsible for postmortem tenderization? Journal of Animal Science 73,1351-l 367.
All rights reserved. Printed in Great Britain PII:SO309-1740(97)00028-4
0309-1740/97 s17.oo+o.oo
ELSEVIER
Sarcomere Shortening and Contraction Nodes in StretchedRestrained Ovine Myofibres During Post Mortem Storage Atsushi Laboratory
Suzuki, Yuka Yamadera, of Functional
(Received
Kyoko
Kido & Kouichi
Morphology, Department of Animal Science, Faculty Tohoku University, Sendai 981, Japan
21 September
1996; revised 27 February
1997; accepted
Watanabe of Agriculture,
9 March
1997)
ABSTRACT Myojibres of muscles removed from the carcass shorten during rigor mortis. Whether myojibres of stretched and restrained muscle tissues shorten during post mortem storage was examined. Muscle strips removed from the semimembranosus muscle of sheep before rigor onset were stretched and restrained. The muscle strips werefixed separately in formalin solution from 1.5 hr to 12 days post mortem, and embedded in parafin. Sections were stained with Mallory-azan. Some myojibres in the pre-rigor-excised and stretche& restrained muscIe strips had contraction nodes at 1 day post mortem. The contraction node did not increase in frequency hereafter. Two to several successive sarcomeres shortened regularly to form the nodes parallel to the Z-line. In addition, several successive sarcomeres shortened partly and successively from one side to the other side to form the nodes oblique to the Z-line. Oblique and irregular contraction nodes appear to cause kinks or bends of myojibres. Sarcomere lengths were not always uniform in individual myojibres. Formation of contraction nodes and shortening of partial sarcomeres in the stretche6 restrained myojibres indicate that the myojibres shorten individually during rigor mortis and vary in shortening states as in muscIes left on the carcass. 0 1997 Elsevier Science Ltd
INTRODUCTION
Isolated skeletal muscles shorten in rigor mortis (Smith, 1950; Locker and Hagyard, 1963) and have short sarcomere lengths (Takahashi et al., 1967, 1995; Henderson et al., 1970). Some myofibres form contacture nodes or kink or crinkle in post mortem storage (Paul er al., 1944, 1952). Active contraction of myofibres in rigor mortis is believed to produce the characteristic nodes, whereas the crinkles or kinks occur in the passively retracted myofibres by outside stress generated by the actively contracted myofibres (Paul et al., 1944, 1952). Myofibres change in sarcomere length during rigor mortis. Transversely broken myofibres are observed in post mortem ageing (Paul et al., 1944, 1952; Gann and Merkel, 1978; Suzuki, 1986). Myofibres of the longissimus dorsi and biceps femoris muscles, from the horizontally placed carcass with limbs perpendicular to the vertebrae, are longer in sarcomere length 339
A. Suzuki et al.
340
than those of the carcass suspended vertically from the Acilles tendon in post-rigor in cattle (Herring et al., 1965a; Hostetler et al., 1972). These facts indicate that the method of carcass suspension before rigor onset influences sarcomere lengths in post mortem muscles. Pre-rigor excised and free muscles are shorter in sarcomere length, whereas pre-rigor excised and stretched-restrained muscles are longer (Herring et al., 19656). The longer sarcomeres of myofibres in the muscles of the horizontally placed carcass or in the stretched-restrained muscles raise a question as to whether their myofibres are unshortened during rigor mortis. The purpose of this study was to examine whether myofibres of stretched and restrained muscle tissues excised from the carcass before rigor onset change in sarcomere length and form contraction nodes in post mortem storage as well as myofibres of muscles attached to the skeleton. MATERIALS
AND METHODS
The semimembranosus muscle from female sheep (Suffolk x Corriedale) aged 2 years were used in this study. At 1.5 hr after slaughter, six longitudinal strips of muscles (4 mmx6 mm x 2 cm) were excised from the carcasses. These samples were stretched and tied to sticks at both ends of the strips to prevent them from contracting. One of these stretchedrestrained strips was immediately fixed with Baker’s formol-calcium (Pearse, 1968). The others were put on a sheet of moist filter paper in petri dishes. The petri dishes were covered, wrapped in plastic wrap, and kept at room temperature (18-20°C) for 6 hr after slaughter. At 6 hr post mortem, one of them was taken out and immersed in the fixative. After 6 hr post mortem, the stretched-restrained strips in the petri dishes were stored in a refrigerator at 45°C. These were taken out separately from the petri dish at 1, 3, 6 and 12 days post mortem, and then immersed in the fixative without taking the muscle strips off sticks. For comparison, two muscle strips were excised from the semimembranosus muscle, from the side placed horizontally on a table at 1.5 hr, 6 hr, 1 day, 3 days, 6 days and 12 days post mortem. One was tied to a stick without stretching (excised-restrained strip) and another was untied (excised-unrestrained strip). These were immersed in the fixative. The tied samples were cut off the sticks after fixation and before dehydration. All the samples were dehydrated and embedded in paraffin. Longitudinal sections, 3 km thick, were stained with Mallory-Heidenhain’s azan stain (Lillie, 1965). The lengths of a 30 sarcomere segment in 10 myofibres in muscle sections were measured from images enlarged at 3400x magnification. Differences between the sarcomere lengths were compared using the Student’s t-test.
RESULTS Stretched-restrained
muscle tissue
Myofibres were straight from 1.5 hr to 12 days post mortem [Fig. l(a) and (b)]. Crossstriations composed of the A-band and I-band with the Z-line were seen clearly in post mortem ageing. At 1 day post mortem, two or several successive sarcomeres shortened in places in some myofibers, and consequently contraction nodes were formed (Figs l(b) and 2). The I-bands disappeared in the nodes owing to shortening. The contraction nodes formed by the relatively regular shortening of two or several sarcomeres looked like a bamboo joint (Fig. 2). No such phenomena were observed at 1.5 and 6 hr post mortem.
341
ovine muscle tissue at 1.5 hr post mortem. Sarcomere lengths are almost uniform and 2.65 pm in average. Mallory-Heidenhain’s azan stain. White bar length = 15 1pm. (b) Myofibres of the stretched-restrained ovine muscle tissue at 1 day post mortem. Three myofibres have contraction nodes (arrows) (like bamboo joints). Sarcomeres vary in length: 2.73 pm in A, 2.75 ,um in B, 2.20 pm in C, 2.49 pm in D, 2.26 pm in E, 2.19 pm in F, 2.26 pm in G, 2.30pm in H and 2.51 pm in I, which are mean values determined from a 5 sarcomere segFig. 1. (a) Myofibres of the stretched-restrained
ment. Mallory-Heidenhain’s
azan stain. White bar length = 15.1 pm.
In a number of sarcomeres, the I-band was discerned at one side of a myofibre, but was invisible by shortening at the other side [Fig. 2(b) and (c)l. The structural arrangements of the sarcomere were disorganised at the contraction nodes [Fig. 2(c)]. The I-bands of several successive sarcomeres shortened partly and successively from one side to the other in a myofibre to form an oblique contraction node non-parallel to the Z-line [Fig. 3(a)]. Such irregular and active shortening of sarcomeres appeared to produce kinks or bends in the myofibre. The number of nodes varied from myofibre to myofibre. Not all myofibres had the contraction node. The characteristic rigor nodes corresponding to the structural features shown by Paul et al. (1944) were observed infrequently from 1.5 or 6 hr post mortem onward, regardless of the type of muscle strip [Fig. 3(b)]. Sarcomere shortening of the contraction nodes, which looked like bamboo joints, occurred much more frequently than did the rigor nodes. The sarcomere lengths were not always uniform in the regions that did not form the contraction nodes, although the sarcomeres remained stretched between the nodes (Fig. l(b)). The sarcomere lengths were observed to vary from region to region in a myofibre, in which no contraction nodes was observed.
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The myofibres tended to have more nodes at 1 and 3 day post mortem than did those at 6 and 12 day post mortem. After 1 day post mortem, the arrangement of the sarcomere was disturbed in places [Fig. 1(b)]. The disarrangement appeared to occur more in 6 and 12 day post mortem. No myofibres were broken transversely from 1.5 hr to 12 day post mortem storage. Muscle tissue excised from carcass
In the excised and restrained muscle tissue, the contraction nodes and disarrangements of the sarcomeres occurred at 1 day post mortem as in the stretched-restrained muscle tissue.
Fig. 2. Contraction nodes in the stretched-restrained ovine myofibres at 1 day post mortem. These figures are enlarged from part of Fig. l(b). Contraction nodes are formed by the shortening of two or three (a) to several [(b), (c )] sarcomeres. Dark bands are the A-bands, light bands the I-bands and fine lines the Z-lines. The Z-lines that arrows indicate [(b),( c )] are invisible on one side by uneven shortening of the I-band. Part of the sarcomere structures (asterisk) of the contraction node (c) are disorganised. Mallory-Heidenhain’s azan stain. White bar length = 4.7 pm.
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The irregularly shortening sarcomeres were observed at this stage, indicating that myofibres kinked or bent actively [Fig. 3(c)]. Almost all myofibres showed clear cross-striations. Myofibres bent as a wave in places at 1 day post mortem. Bent myofibres decreased after 3 days post mortem.
Fig. 3. (a) Contraction node of the stretched-restrained ovine myofibre at 1 day post mortetn. Sarcomeres between two arrows in both sides of the myofibre shorten irregularly to form a contraction node oblique to the Z-line, which appears to cause kink in myofibres. Mallory-Heidenhain’s azan stain. White bar length = 3.8 Wm. (b) Contraction node in a myofibre of the excised-restrained ovine muscle tissue at 6 hr post mortem. This node corresponds to the characteristic rigor node (R) shown by Paul et al. (1944). Many sarcomeres shorten extremely. Myofibres (C) adjacent the myofibre forming the rigor node are crinkled passively at its side. Mallory-Heidenhain’s azan stain. White bar length = 8.7 km. (c) Contraction node in a kinked myofibre of the excised-restrained ovine muscle strip at 1 day post mortem. Sarcomere structures (asterisk) are disorganised partly in contraction nodes oblique to the Z-line. Large arrows indicate the H-band, which means the stretched sarcomeres, and small arrows the Z-line. The contraction node of the kinked (k) myofibre does not affect the sarcomeres of an adjacent (A) myofibre. Mallory-Heidenhain’s azan stain, White bar length = 4 grn.
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ovine muscle tissue at 1 day post mortem. K: kinked myofibre. Mallory-Heidenhain’s azan stain. White bar length= 30.1 pm. (b) Transversely cracked myofibres of the excised-restrained ovine muscle tissue at 3 days post mortem. Arrows indicate cracks. Mallory-Heidenhain’s azan stain. White bar length = 18.5 pm. (c) Transversely cracked and broken myofibres of the excised-unrestrained ovine muscle tissue at 6 days post mortem. Arrows indicate cracks and an asterisk a broken myofibre. Mallory-Heidenhain’s azan stain. White bar length = 37.4 pm. (d) Transversely broken myofibres of the exciseddunrestrained ovine muscle tissue at 12 days post mortem. Asterisks indicate broken myofibres. Mallory-Heidenhain’s azan stain. White bar length = 37.4 pm. Fig. 4. (a) Kinked myofibres in the excised-unrestrained
The excised and unrestrained muscle tissues had both straight myofibres and bent myofibres as waves at 1.5 and 6 hr post mortem. At 1 day post mortem, bent or kinked myofibres occurred more in the unrestrained muscle tissue than in the restrained muscle tissues; some myofibres had several contraction nodes [Fig. 4(a)]. In many contraction nodes, the sarcomeres were shortened too extremely to discern the structures [Fig. 4(a)]. Occurrence of bent myofibres was higher in frequency at 1 day post mortem and lower after 3 days post mortem; many myofibres were straight [Fig. 4(a),(c) and (d)]. At 3 days post mortem, some myofibres in the excised-restrained and excised-unrestrained muscle tissues were transversely broken in places [Fig. 4(b)]. At 1 day post mortem,
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TABLE 1 Sarcomere Lengths in Post Mortem Muscle Tissue from the Semimembranousus Muscle of Sheep Post mortem age
Excise&unrestrained(~)
1 hr 6hr 1 day 3 days 6 days 12 days
1.94kO.O6’@ 2.01 f 0.096 2.12AO.04’ 2.04 f 0. 180bcd 2.05 AI0.09” 2.09 * 0.07cd
Excised-restrained(m) 242 f 0.05” 249 i 0.09” 2.15*0.16b 2.24 f 0. lob” 2.34ztO.13” 2.24k0.11bc
Stretchekrestrained
(p)
2.65*0.13& 2.75*0.10b 2.55*0.15a 2.96ztO.19’ 2.61 f 0.09” 2.6OztO.11”
‘Mean f SEM determined from 10 myofibres (30 sarcomeres per myofibre). dCdSacromere lengths with different superscripts in columns are significantly different (P < 0.05).
transverse cracks which showed breaks of myofibrils were found in a few myofibres. The breaks of myofibres in the muscle tissue excised from the carcass increased with storage time [Fig. 4(b),(c) and (d)]. Length of ssrcomeres
Table 1 shows the sarcomere lengths, except for the extremely shortened sarcomeres of the contraction nodes. No regular changes common to the three different muscle strips were observed in sarcomere length during post mortem ageing. Sarcomere lengths of the unrestrained muscle tissues were shorter at 1.5 hr after slaughter than at 1 day post mortem, and unchanged from 3 days post mortem onward. In the excised-restrained muscle strips, sarcomere lengths were shorter at 1 day post mortem than at 1.5 and 6 hr post mortem and changed little hereafter. The myofibres of stretched-restrained muscle strips appeared to shorten at 1 day post mortem, but varied in sarcomere length, regardless of post mortem ageing. The unrestrained muscle strips had the shortest sarcomere lengths and the stretched-restrained muscle strips had the largest sarcomere lengths at all the stages (P < O-01). DISCUSSION
Extreme shortening in two adjoining sarcomeres or several successive sarcomeres resulted in the formation of contraction nodes. The occurrence of the contraction nodes in myofibres of the stretched-restrained muscle tissues at 1 day post mortem, as found in the
restrained and unrestrained muscle tissues excised from the carcass, shows that the myofibres contract actively in rigor mortis. The finding is consistent with the results of previous studies showing that muscles or sarcomeres shortened during post mortem storage (Smith, 1950; Locker and Hagyard, 1963; Takahashi et al., 1967, 1995; Henderson et al., 1970). The scattered formation of contraction nodes, and non-uniform shortening of the sarcomeres within a myofibre, may be related to the structure of the connective tissue network of the endomysium. The contraction node (like a bamboo joint) in the stretched-restrained myofibres is a type of rigor node (Paul et al., 1944), but differs in structural features from characteristic rigor nodes, as shown by Paul et al. (1944). Characteristic rigor nodes seem to occur in pre- and post-rigor because they were observed from 1.5 hr post mortem onward. Various shortening modes of the sarcomeres in contraction nodes could be observed clearly in the
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stretched-restrained myofibres, because the contraction nodes of the stretched-restrained myofibres shortened less than the ones of the unrestrained myofibres. The kinked or crinkled myofibres in the muscles excised from carcasses stored post mortem are similar to those shown by Paul et al. (1944, 1952). The kinks of myofibres are considered to occur through passive retraction (Paul et al., 1944). Observation of the sarcomeres in obliquely formed contraction nodes indicates that some kinked myofibres are formed actively by irregular and partial shortening of the sarcomeres. The excised muscle develops tension in an isometric state immediately after death and subsequently loses this tension (Jungk et al., 1967). The occurrence of contraction nodes in myofibres coincides with tension development. Tension developed by formation of the contraction nodes may stretch the sarcomeres to some extent among the nodes. Myofibres are transversely broken at 1 day post mortem in the bovine muscle (Paul, 1944) or 3 days post mortem in the ovine muscle (Suzuki, 1986). The breaks of myofibres were observed in the muscle tissues excised from the carcass during post mortem ageing. Breaks of myofibres appeared to be associated with decreases in bent myofibres after 3 days post mortem. The breaks of myofibres appear to release tension developed in them. The tension release may relax myofibres bent in waves and consequently the myofibres straighten to some extent. Fragmentation of myofibrils at the I-band and Z-disc junction occurs at 48 hr post mortem, and the quantity of transverse breaks increased hereafter in the bovine muscles (Gann and Merkel, 1978). Similar increases in the extent of myofibre breakage were observed in this study. The breaks of myofibres are believed to make postrigor meat tender (Paul et al., 1952; Gann and Merkel, 1978; Pearson, 1986). The myofibrils have been shown to become fragile during post mortem ageing (Taylor et al., 1995). Proteolysis by calpains in skeletal muscle during post mortem storage results in destruction of costameres and intermyofibrillar linkages and weakening of the filament/ Z-disc interaction, which are considered to have an important role in meat tenderization (Taylor et al., 1995). On the contrary, translocated paratropomyosin induced by increases in the calcium levels in sarcoplasmic reticulum has been proposed as a key factor in meat tenderization (Takahashi et al., 1995). Longer sarcomere lengths of the stretched-restrained myofibres than those of the unrestrained myofibres in ovine muscle tissues agree with the results from bovine muscles shown by Herring et al. (1965b). The sarcomere length of post mortem stored muscles depends on the conditions to which the muscles are exposed or in which muscle tissues are fixed (Herring et al., 1965~; Hostetler ef al., 1972). The sarcomeres were observed to shorten at 1 day post mortem in the excised-restrained muscle tissues, but not in the unrestrained muscle tissue. At this stage, the sarcomeres of the stretched-restrained muscle tissues showed a tendency to shorten. The sarcomere lengths in the three different muscle tissues did not increase or decrease with post mortem ageing. Post mortem ageing has been reported to have no influence on sarcomere lengths in the muscles of sheep (Bouton er al., 1973) or of turkeys (Johnson and Bowers, 1976). The unrestrained muscle tissues from the stored carcass seem to be influenced by formalin fixation because they had the shortest sarcomere lengths. Pre-rigor unrestrained muscle tissues had more bent myofibres than did post-rigor unrestrained muscles, indicating that the myofibres of the pre-rigor muscles are influenced more by fixation than those of the post-rigor muscles. Pre-rigor myofibres, sensitive to the fixative, presumably bent during fixation. Bending and shortening of myofibres by formalin fixation is inhibited in the restrained muscle tissues, because the restrained muscle tissues had no bent myofibres or shortest sarcomere lengths at 1.5 hr post mortem. We assumed that the stretched-restrained myofibres contracted actively during rigor mortis and consequently myofibres should be broken by the tension developed within them. However, the myofibres were unbroken during post mortem storage. No physical
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force was applied to the muscle strips from storage through fixation. Maximum tension in living muscles is generated at sarcomere lengths in the range of 2.05 and 2.25 pm (Gordon et al., 1966). The tension in the stretched-restrained muscle tissue may be too weak to make myofibres fragile post-rigor because the sarcomere lengths were beyond 2.5 ,um. In unstretched muscle samples at 90 hr post mortem, myofibres have been reported to be unbroken, whereas myofibres are broken in stretched muscle samples (Davey and Dickson, 1970). Marked extension of myofibres leads to fracture (Mutungi et al., 1995). The myofibres which were stretched and restrained before rigor onset shortened individually but not greatly during rigor mortis and varied in their shortening states. Although muscles attached to the skeleton in the carcass cannot shorten freely if the joints are fixed by antagonistic function of the other muscles or gravitation in the suspended carcass during rigor mortis, the myofibres appear to shorten individually and vary in shortening states; causing uniform, partial or irregular shortening along a myofibre. In muscles attached to the skeleton, shortening of individual myofibres in rigor mortis may not result in shortening of the muscle as a whole because the connective tissues within muscles, which bind the myofibres and fascicles together, permit a certain degree of free motion between them.
REFERENCES Bouton, P. E., Harris, P. V., Shorthose, W. R. and Baxter, R. I. (1973) A comparison of the effects of aging, conditioning and skeletal restraint on the tenderness of mutton. Journal of Food Science 38, 932-937. Davey, C. L. and Dickson, M. R. (1970) Studies in meat tenderness. 8. Ultra-structural changes in meat during aging. Journal of Food Science 35, N-60. Gann, G. L. and Merkel, R. A. (1978) Ultrastructural changes in bovine Iongissimus muscle during postmortem ageing. Meat Science 2, 129-144. Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966) The variation in isometric tension with sarcomere length in vertebrate muscle fibres. The Journal of Physiology 184, 17&192. Henderson, D. W., Goll, D. E. and Stromer, M. H. (1970) A comparison of shortening and Z line degradation in post-morten bovine, porcine, and rabbit muscle. The American JournaI of Anatomy 128, 117-136. Herring, H. K., Cassens, R. G. and Briskey, F. J. (1965a) Further studies on bovine muscle tenderness as influenced by carcass position, sarcomere length, and fiber diameter. JournaI of Food Science 30, 1049-1054. Herring, H. K., Cassens, R. G. and Briskey, E. J. (19656) Sarcomere length of free and restrained bovine muscles at low temperature as related to tenderness. Journal of the Science of Food and Agriculture 16, 379-384. Hostetler, R. L., Link, B. A., Landmann, W. A. and Fitzhugh, H. A., Jr. (1972) Effect of carcass suspension on sarcomere length and shear force of some major bovine muscles. Journal of Food Science 37, 132-l 35. Johnson, P. G. and Bowers, J. A. (1976) Influence of aging on the electrophoretic and structural characteristics of turkey breast muscle. Journal of Food Science 41, 255-261. Jungk, R. A., Snyder, H. E., Goll, D. E. and McConnell, K. G. (1967) Isometric tension changes and shortening in muscle strips during post-mortem aging. Journal of Food Science 32, 158% 161. Lillie, R. D. (1965) Histopathologic Technic and Practical Histochemistry, 3rd edn, p. 545. McGrawHill, New York. Locker, R. H. and Hagyard, C. J. (1963) A cold shortening effect in beef muscles. Journal of the Science of Food and Agriculture 14, 787-793. Mutungi, G., Purslow, P. and Warkup, C. (1995) Structural and mechanical changes in raw and cooked single porcine muscle fibres extended to fracture. Meat Science 40, 217-234.
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Paul, P., Lowe, B. and McClurg, B. R. (1944) Changes in histological structure and palatability of beef during storage. Food Research 9, 221-233. Paul, P., Bratzler, L. J., Farwell, E. D. and Knight, K. (1952) Studies on tenderness of beef. I. Rate of heat penetration. Food Research 17, 504-5 10. Pearse, A. G. E. (1968) Histochemistry. Theoretical and Applied, 3rd edn, Vol. 1, p. 601. J. & A. Churchill, London. Pearson, A. M. (1986) Physical and biochemical changes occurring in muscle during storage and preservation. In Muscle as Food, ed. P. J. Bechtel, pp. 103-134. Academic Press, New York. Smith, R. D. (1950) Studies on rigor morns. Part II. Qualitative observations on the post mortem shortening of muscles. The Anatomical Record 108,207-216. Suzuki, A. (1986) A histochemical change in myofibers of sheep muscle in refrigeration. Tohoku Journal of Agricultural Research 36, 135-143. Takahashi, K., Fukazawa, T. and Yasui, T. (1967) Formation of myofibrillar fragments and reversible contraction of sarcomeres in chicken pectoral muscle. Journal of Food Science 32,409-413. Takahashi, K., Hattori, A. and Kuroyanagi, H. (1995) Relationship between the translocation of paratropomyosin and the restoration of rigor-shortened sarcomeres during post-mortem ageing of meat - A molecular mechanism of meat tenderization. Meat Science 40,413-423. Taylor, R. G., Geesink, G. H., Thompson, V. F., Koohmaraie, M. and Goll, D. E. (1995) Is Z-disk degradation responsible for postmortem tenderization? Journal of Animal Science 73,1351-l 367.