The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging

The objective was to study the potential role of calpain 3 in postmortem myofibril breakdown and meat tenderization. We determined the temporal changes in calpain 3 protein in the ovine m. longissimus thoracis et lumborum (LTL, n=4) during post-mortem storage. Concurrently, we also determined the kinetics of tenderization level, changes in MFI, degradation of nebulin and desmin, and autolysis of calpain 1. The autolysis of calpains 1 and 3 were strongly correlated with the kinetics of tenderization and changes in MFI. The best correlation was between the appearance of the autolyzed calpains 1 and 3 and nebulin degradation. Taken together, the results indicated that calpains 1 and/ or 3 might be playing a key role in post-mortem tenderization of LTL via the proteolysis of specific muscle structural proteins such as nebulin. This is the first report that relates calpain 3 to myofibrillar protein degradation in post-mortem skeletal muscle.     

Combination of low voltage electrical stimulation and early postmortem temperature conditioning on degradation of myofibrillar proteins in Korean native cattle (Hanwoo)

The combination effect of low voltage electrical stimulation (LVES) and early postmortem (PM) temperature conditioning (2, 16, and 30°C until 3 h PM) on degradation of myofibrillar proteins were determined from Korean native cattle (Hanwoo). Myofibrils were removed at 1, 2, 3, 7, and 14 days of PM storage (2°C) and analyzed for titin, nebulin, desmin, and troponin-T by SDS-PAGE and by Western blot analysis. Degradation rate of myofibrillar proteins was affected by the combination of LVES and temperature conditioning. LVES-30°C treatment resulted in faster degradation of titin, nebulin, desmin, and troponin-T during PM storage than the other treatments. Degradation of titin took place more slowly than nebulin, desmin or troponin-T.     

骨架蛋白降解对冰鲜草鱼质构的影响

利用免疫组化技术检测草鱼背肌白肉中骨架蛋白在冰藏条件下的变化,同时通过超微结构、剪切力、滴水损失率分析草鱼背肌组织精微结构和质构特性的变化,以期阐明草鱼骨架蛋白降解与鱼肉质构劣化之间的关系,从而为调控冰鲜淡水鱼品质提供理论依据。结果显示：冰藏21 d内伴肌球蛋白、伴肌动蛋白、抗肌营养不良蛋白分别降解了76.10%、78.21%、71.14%;冰藏14 d后草鱼肌纤维明暗带模糊,Z带、M带被破坏,肌纤维结构出现严重断裂松散现象。冰藏10 d内剪切力由81 N快速下降至32 N,滴水损失率由1.6%增加至7.8%;将骨架蛋白的灰度与剪切力及滴水损失率进行相关性分析发现,剪切力与伴肌球蛋白、抗肌营养不良蛋白的灰度呈极显著正相关（P＜0.01）;滴水损失率与伴肌球蛋白、抗肌营养不良蛋白的灰度值呈极显著负相关（P＜0.01）。综上所述,骨架蛋白降解可能是草鱼在冰藏期间肌纤维结构破坏以及质构劣化的重要原因。     

Postmortem Degradation of Titin and Nebulin of Beef Steaks Varying in Tenderness

Purified myofibrils were isolated from “tender” and “less-tender” bovine longissimus muscle at death and at 1, 3, 7, and 14 days of postmortem storage (4^oC). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect changes in the myofibrillar/cytoskeletal proteins, titin and nebulin. Titin and nebulin bands were observed to be less intense on gels from “tender” than from “less-tender” steaks. These results suggest that titin and nebulin were more rapidly degraded in “tender” than in “less-tender” steaks, and that the extent of beef loin steak tenderness may be dependent upon the postmortem degradation of titin and nebulin.     

Up- and down-regulation of longissimus tenderness parallels changes in the myofibril-bound calpain 3 protein

The objective of this study was to utilize Ca(2+) and Zn(2+) treatments of meat to critically explore the possible role of calpain 3 in meat tenderisation. Calpains 1 and 2 were also examined for comparative purpose. Control animals plus animals infused with CaCl(2), ZnCl(2) or H(2)O were used (six lambs per treatment) to determine the temporal changes in muscle calpain 3 protein in the Longissimus thoracis et lumborum (LTL) during post-mortem storage. Concurrently, the temporal changes of; (1) shear force, (2) sarcomere length, (3) proteolysis of titin and nebulin and (4) calpains 1 and 2 proteins were also determined. Infusing LTL with Ca(2+) or Zn(2+) caused significant up- and down-regulation of LTL tenderisation, respectively, compared to water infusion and the control animals. Furthermore, the rate of breakdown of calpain 3, the rate of proteolysis of titin and nebulin and the rate of meat tenderisation during post-mortem storage of LTL in the various treatments were highly correlated. These studies suggest that calpain 3, like calpain 1, may be involved in the tenderisation of meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin.     

极限pH对羊肉宰后成熟过程中肌原纤维蛋白特型的影响

为了研究羊肉宰后成熟过程中极限pH对肌原纤维蛋白特型即肌联蛋白、伴肌动蛋白、肌间线蛋白和肌钙蛋白-T降解及肌原纤维小片化指数的影响。本文选取50只羊的右侧背最长肌,贮存于4 ℃条件下,在宰后时间点分别为1 h、1、2、3、5 d和7 d时,测定其pH。按照宰后2 d的pH将肉样分成三组:高极限pH组(5.72±0.03),中极限pH组(5.54±0.01)和低极限pH组(5.40±0.02)。在每个宰后时间点,测定肌联蛋白、伴肌动蛋白、肌间线蛋白、肌钙蛋白-T降解程度和肌原纤维小片化指数(MFI)。结果表明:肌联蛋白在高极限pH组中宰后1 d开始降解;在宰后1 d时,高极限pH组肌间线蛋白相对灰度值显著低于中极限pH组和低极限pH组(p<0.05);肌钙蛋白-T在高极限pH组中,宰后1 d已出现降解条带。而伴肌动蛋白在中极限pH组中降解较快,在宰后1 d开始降解。另外在宰后1、2、3、5、7 d时,高极限pH组和中极限pH组的肌原纤维小片化指数显著高于低极限pH组的肌原纤维小片化指数(p<0.05)。极限pH通过影响这些肌原纤维蛋白降解来促进宰后肌肉成熟过程并且肌联蛋白、肌间线蛋白和肌钙蛋白-T的降解,加快了宰后前期嫩化过程。这为揭示宰后肉嫩度形成机理提供理论基础。     

Effect of Postmortem Storage on Titin and Nebulin in Pork and Poultry Light and Dark Muscles

Purified myofibrils were prepared from samples of chicken breast and thigh muscles and from light and dark portions of pork semitendinosus muscle at death and after storage at 4° and 22°C for 1, 3, and 7 days postmortem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the effect of postmortem storage of muscle on titin and nebulin. Results indicated that titin and nebulin were more rapidly degraded in light than in dark chicken muscle. In contrast, titin and nebulin were more rapidly degraded in dark than in light pork muscle.     

Desmin Degradation in Postmortem Fish Muscle

The degradation of desmin was studied in postmortem white dorsal muscle from sea bass, brown trout, turbot and sardine, stored at 0–4°C. Based on desmin specific antibody tests, results showed that the extent and rate of desmin degradation in postmortem fish muscle varied considerably among species. There was no degradation of desmin during the first 4 days postmortem storage of sea bass and brown trout, whereas 10 to 20% of muscle desmin was degraded during the first 4 days of postmortem storage of turbot. Sardine muscle presented a very complex pattern of desmin degradation products and aggregated desmin fragments detected within the first 24h. Most polypeptides were larger than the nondenatured desmin. Desmin degradation would not be a suitable marker for evaluation of postmortem changes of stored fish.     

THE DEGRADATION OF MYOFIBRILLAR PROTEINS IN BEEF AND LAMB USING DENATURING ELECTROPHORESIS - AN OVERVIEW

Degradation of specific myofibrillar proteins has been followed in many studies using SDS-PAGE (denaturing) electrophoresis and these have shown that proteins such as titin, nebulin, troponin-T, desmin, filamin and vinculin are degraded at different rates during postmortem storage of meat. Although informative, electrophoresis is usually restricted to qualitative interpretation of the gels and there have been few studies that have quantitatively linked the degradation of specific proteins to changes in toughness. Improved quantitative methods (including scanning densitometry, image analysis, and modeling approaches) will be necessary to determine the protein and/or protein alterations that cause postmortem tenderization. This approach is complementary to other measurements of proteolysis where the objective is to more adequately explain the variation in toughness.     

Studies of Desmin and α-Actinin Degradation in Bovine Semitendinosus Muscle

The degradation of desmin and α-actinin was studied in post-mortem bovine semitendinosus muscle. Using a desmin-specific monoclonal antibody, SDS-PAGE, and immunoblotting we show that desmin is easily degraded at 4°C during the aging process. Within 96 hr, fragments of degraded desmin are detected with our antibody probe. Further storage at 4°C results in an increase of proteolytic fragments and concomitant loss of intact desmin. By 3 wk post-mortem, little un-degraded desmin remains in the muscle. In contrast, α-actinin was degraded slowly at 4°C. Proteolytic fragments of α-actinin were not detectable with anti-α-actinin polyclonal antisera until the second week of incubation at 4°C. However, the degradation of α-actinin was accelerated when meat was incubated at 25°C or at 37°C.     

Changes in Titin and Nebulin in Postmortem Bovine Muscle Revealed by Gel Electrophoresis, Western Blotting and Immunofluorescence Microscopy

Postmortem storage of bovine psoas major muscle resulted in almost complete degradation of nebulin by 48 hr; however, some intact titin was still observed after 2 wk storage at 4°C. The percentage of myofibrils having four anti-titin bands per sarcomere (immunofluorescence microscopy) was less than 1% at 45 min but increased to 65% at 48 hr after death and remained stable thereafter. A similar four band pattern was seen in frozen sections of whole muscle at 48 hr or more postmortem. The time course of nebulin degradation appeared to correlate with the titin two to four band transition.     

Effect of heat shock protein 27 on the in vitro degradation of myofibrils by caspase‐3 and μ‐calpain

This study was designed to investigate the effect of heat shock protein 27 (HSP27) on the in vitro degradation of myofibrils induced by caspase‐3 or μ‐calpain. Myofibrillar proteins were prepared from at‐death beef muscles and incubated with caspase‐3 or μ‐calpain with and without HSP27, or with HSP27 alone, at 30 °C for 2 h, and protein degradation was assessed. Results showed that caspase‐3 promoted the degradation of titin, nebulin and troponin‐T, and μ‐calpain promoted the degradation of nebulin, desmin and troponin‐T, observed during normal PM ageing. Moreover, the addition of HSP27 restricted the degradation of troponin‐T in μ‐calpain‐ and caspase‐3‐treated myofilaments, and restricted the degradation of desmin in μ‐calpain‐treated myofilaments. Therefore, HSP27 may indirectly or directly interact with caspase‐3 and μ‐calpain, reducing their activity and mediating PM proteolysis of muscle proteins to affect meat tenderisation.     

CONTINUATION OF SYMPOSIUM: FUNDAMENTAL PROPERTIES OF MUSCLE PROTEINS IMPORTANT IN MEAT SCIENCE: ROLE OF NEW CYTOSKELETAL ELEMENTS IN MAINTENANCE OF MUSCLE INTEGRITY

Evidence suggests that desmin, titin and nebulin, three recently discovered proteins, have cytoskeletal roles in muscle cells. The three proteins have been purified from mature skeletal muscle and partially characterized. Properties of the three proteins are described, with special regard to their probable roles and importance in maintaining muscle cell integrity. Results will be shown that demonstrate ability of purified desmin to self-assemble into synthetic 10-nm (intermediate) diameter filaments. Taken together with immunoelectron microscope results (Richardson et al. 1981), it is evident that desmin is the major component of 10-nm filaments of mature skeletal muscle cells and that the desmin filaments link adjacent myofibrils at their Z-line levels and seemingly tie the myofibrils into the cell cyto-skeleton. Desmin is degraded at about the same rate as is the highly susceptible troponin-T in bovine semitendinosus muscle postmortem. Alterations in desmin and other recently discovered cytoskeletal proteins would be expected to disrupt muscle cell integrity and to have marked effects on properties of muscle important to its use as food.     

Location of and post‐mortem changes in some cytoskeletal proteins in pork and cod muscle

The cytoskeletal proteins actin, nebulin, spectrin, desmin, vinculin and talin were labelled immunohistochemically in sections of muscle from commercially available pigs and cod (Gadus morhua) taken pre‐rigor and from samples stored for several days. Actin, nebulin and spectrin gave similar labelling patterns in both pork and cod muscle which remained the same in stored samples. Desmin was intensely labelled at the cell boundaries and within the body of the cells in both pork and cod in the initial and the stored samples. Vinculin was readily labelled in pork muscle but showed only diffuse labelling in fish. Labelling for talin in pork muscle was intense at the sarcolemma but was not present in samples stored for 4 days. In contrast, the label for talin was concentrated at the myotendinous junction of the cod muscle throughout the storage period. These are the first reports of the detection and location of spectrin and vinculin in fish muscle and of the location of talin. The results are discussed in terms of muscle structure, function and post‐mortem tenderisation. © 2000 Society of Chemical Industry     

Effect of beef ultimate pH and large structural protein changes with aging on meat tenderness

This study investigated the effect of ultimate pH (pH_u) in beef on the degradation of large structural proteins during refrigerated storage using SDS-PAGE. M. longissimus dorsi from bull carcasses were selected and classified into three groups: low pH_u (≤ 5.79), intermediate pH_u (5.80–6.19) and high pH_u (≥ 6.2) muscles. Samples were then stored at − 1.5 °C for 1, 2, 7, 14, 21 and 28 days. Meat tenderness was measured at each aging time. Depending on meat pH_u, different protein patterns and degradation rates of structural proteins were found. Rapid changes of large structural proteins took place within 48 h post mortem. Besides titin and nebulin, degradation of filamin was clearly revealed. Two more large protein bands corresponding to myosin family members also exhibited fast decline with storage time. It suggested that the fast degradation of these proteins is a key factor in the improvement of meat tenderness.     

SDS-PAGE Conditions for Detection of Titin and Nebulin in Tender and Tough Bovine Muscles

Purified myofibrils were prepared from infraspinatus (tender) and rhomboideus (tough) muscles at 7 days postmortem and examined for myofibrillar/cytoskeleta1 protein degradation by using sodium dodecyl sulfate polyactylamide gel electrophoresis (SDS-PAGE). Four acrylamide/bisacrylamide ratios (37:1, 50:1, 75:l and 100:1) and two SDS-PAGE gel buffers (Tris-HCl, pH 8.0 and 8.9) were used to determine the optimum conditions for detection of titin and nebulin. Titin was degraded to a greater extent in myofibrils from the infraspinatus than in myofibrils from the rhomboideus. Very little nebulin was detected in either muscle. Use of acrylamide/bisacrylamide ratio of 37:1 and a gel buffer of pH 8.0 provided the most optimum conditions for detecting differences in the resolution of titin, nebulin and their apparent degradation products.     

Post mortem changes in porcine longissimus muscle incubated with 0.1 M calcium lactate

The effects of calcium lactate incubation at 5 °C on post mortem changes in porcine longissimus muscle were studied. Myofibrils were purified from control (CON) and calcium lactate‐incubated (CL) muscles. Samples were taken at 0, 1, 3, 7 and 14 days post mortem. SDS‐PAGE results showed that the disappearance of titin, nebulin, tropomyosin and troponin‐T and the appearance of a ～30 kDa component were more pronounced in CL samples than in CON samples. Western blots labelled with a monoclonal antibody to desmin also demonstrated that desmin degraded more quickly in CL samples. Our data suggested that calcium lactate incubation might accelerate the post mortem changes in porcine longissimus muscle via activation of calpains. © 2001 Society of Chemical Industry     

The effect of active caspase-3 on degradation of chicken myofibrillar proteins and structure of myofibrils

The objective of this study was to investigate the potential contribution of caspase-3 to meat postmortem tenderisation by examining the role of caspase-3 in the degradation of myofibrillar proteins and disruption of myofibril structure in vitro. Myofibrillar protein prepared from chicken muscle was incubated with EDTA or EDTA plus caspase-3 at 25 °C for 16 h and used for detecting muscle protein degradation and ultrastructure of myofibril. Results revealed that caspase-3 reproduced the degradation patterns of titin, nebulin and α-actinin during postmortem storage of meat, but caused little proteolysis of desmin and no appearance of 28–30 kDa peptides. Meanwhile, caspase-3 also induced the weakening in the I band adjacent to Z-lines, which occurred during meat postmortem ageing. Therefore, caspase-3 could account only for a part of the myofibrillar protein degradation observed in naturally aged meat and is likely involved in postmortem tenderisation of meat together with other endogenous proteases.     

In vitro study to evaluate the degradation of bovine muscle proteins post-mortem by proteasome and μ-calpain

The degradation of bovine muscle proteins by proteasome and ubiquitous calpains was explored via 2D gel proteome analysis by inhibition of the physiological level of the proteases by specific inhibitors. The inhibition of the proteasome chymotrypsin- and trypsin-like activity results in the lack of degradation of several fragments of structural proteins such as actin, troponin T, myosin light chain and nebulin. In addition the degradation of several sarcoplasmatic proteins was eliminated when proteasome was inhibited. The inhibition of the ubiquitous calpain only resulted in minor changes in the degradation pattern, which might indicate that p94, which is not inhibited by calpastatin, is involved in the degradation post-mortem. The results of the present study indicate a sequential degradation of the structural proteins post-mortem, where calpain initiates the disruption and destabilisation of the myofibrillar structure, and thereby allows the proteasome to act.