Myosin: Formation and maintenance of thick filaments

Skeletal muscle consists of bundles of myofibers containing millions of myofibrils, each of which is formed of longitudinally aligned sarcomere structures. Sarcomeres are the minimum contractile unit, which mainly consists of four components: Z‐bands, thin filaments, thick filaments, and connectin/titin. The size and shape of the sarcomere component is strictly controlled. Surprisingly, skeletal muscle cells not only synthesize a series of myofibrillar proteins but also regulate the assembly of those proteins into the sarcomere structures. However, authentic sarcomere structures cannot be reconstituted by combining purified myofibrillar proteins in vitro, therefore there must be an elaborate mechanism ensuring the correct formation of myofibril structure in skeletal muscle cells. This review discusses the role of myosin, a main component of the thick filament, in thick filament formation and the dynamics of myosin in skeletal muscle cells. Changes in the number of myofibrils in myofibers can cause muscle hypertrophy or atrophy. Therefore, it is important to understand the fundamental mechanisms by which myofibers control myofibril formation at the molecular level to develop approaches that effectively enhance muscle growth in animals.     

Post mortem development of meat quality as related to changes in cytoskeletal proteins of chicken muscles

1. A procedure was developed to separate high and medium molecular weight myofibrillar proteins from chicken muscular tissue with a high resolution by flat bed sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent detection by either a general protein stain or Western blotting. These procedures were used to analyse the degradation process of cytoskeletal proteins in chicken breast and leg muscles during meat ageing. 2. This study demonstrates the degradation of all the examined cytoskeletal proteins: titin, nebulin and desmin as well as vinculin, a protein component of the costamere structure. All the examined proteins were found to be degraded during ageing of chicken breast and leg muscles. 3. Degradation of titin, nebulin and desmin started at 3 h post mortem in breast muscle. Intact titin and nebulin disappeared within 1 d. Intact desmin and vinculin were not detectable after 3 d post mortem. In leg muscle, the degradation process of all the examined proteins evolved much more slowly than in breast chicken muscles. 4. The changes observed in shear force, myofibrillar fragmentation and cooking loss were related to changes in cytoskeletal proteins and used to identify marker proteins or degradation products for the purpose of monitoring the development of meat ageing. The ageing process was faster in breast muscle than in leg muscle. 5. Significant correlations were found between degradation processes of titin, nebulin, and desmin and shear force, as well as myofibril fragmentation index of breast and leg muscles.     

Effect of porcine stress syndrome on the solubility and degradation of myofibrillar/cytoskeletal proteins.

This study examined the effect of stress classification (stress-positive, stress-carrier, stress-negative) of pigs on selected properties of postmortem muscle, including protein solubility and degradation of proteins such as titin. Longissimus muscle samples were removed 45 min postslaughter, divided into samples, and stored at 0 to 2 degrees C for analysis at 0, 1, 3, 5, and 7 d postmortem. Whole-muscle samples (homogenates) and purified myofibrils were prepared from each sample for analysis by SDS-PAGE. A portion of each muscle sample also was extracted 1) with a low-ionic-strength solution to obtain a sarcoplasmic protein fraction and 2) with two different high-ionic-strength solutions to obtain a myofibrillar/cytoskeletal protein fraction for measurement of protein solubility and for analysis of extracts by SDS-PAGE. No significant differences were observed between muscle from stress-negative and stress-carrier animals in this study. Sarcoplasmic (P less than .05) and myofibrillar/cytoskeletal (P less than .01) protein solubility was lower in muscle samples from stress-positive animals than in muscle samples from stress-carrier and stress-negative animals at all postmortem times studied. The high molecular weight protein titin was degraded more slowly postmortem in muscle from stress-positive than in muscle from stress-negative animals, as observed by SDS-PAGE analysis of whole-muscle samples (homogenates) an myofibrils. The combination of lowered protein solubility and reduced rate of postmortem degradation of structural proteins such as titin may explain, at least in part, the reduced quality and protein functionality of muscle from stress-positive pigs.     

The effect of post‐mortem storage on the water‐soluble proteins of breast muscles of uneviscerated pheasant and chicken carcasses

Electropherograms, obtained by polyacrylamide gel electrophoresis, of water‐soluble proteins extracted from the breast muscles of uneviscerated pheasant and chicken carcasses which had been stored at 10 °C were compared. The results indicated that chicken muscle proteins remained largely unchanged, while marked changes occurred in pheasant muscle proteins especially after 10 d post‐mortem.     

Physicochemical properties of water-soluble myofibrillar proteins prepared from chicken breast muscle

The solubility of skeletal muscle myofibrillar proteins in water was examined. The solubility of the proteins was found to be sensitive to ionic strength and pH of the solution. At the ionic strength of less than 12 mM and neutral pH, more than 80% of myofibrillar proteins were solubilized. Heating at a temperature of more than 70°C was required for the proteins to retain their solubility. The solubility of freeze‐dried protein powder prepared from water‐soluble myofibrillar proteins was also examined, and it was found that addition of trehalose and heating were essential for re‐solubilization in water. Amino acid composition of water‐soluble myofibrillar proteins was found to be almost the same as that of myofibrillar proteins.     

Proteome analysis of whole and water‐soluble proteins in masseter and semitendinosus muscles of Holstein cows

To assess both quantitative and qualitative differences between the slow‐ and fast‐type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two‐dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water‐soluble protein fraction. Two slow‐type myofibrillar proteins (myosin light chain‐1 slow‐b and myosin light chain‐2 slow), and aconitase‐2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast‐type myofibrillar proteins (myosin light chain‐1 fast, myosin light chain‐2 fast, myosin light chain‐3 fast and tropomyosin‐1), and three enzymes of glycolytic pathway (enolase‐3, aldolase‐A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow‐ and fast‐type muscles.     

Involvement of μ/m‐calpain in the proteolysis and meat quality changes during postmortem storage of chicken breast muscle

The objective of this study was to investigate the role of calpain isotypes, especially poultry‐specific μ/m‐calpain in the proteolysis and meat quality changes of chicken breast muscle during postmortem storage. Calpain activity was detected by casein zymography, while the degradation of titin, desmin and Troponin‐T was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and western blot. Meat quality indicators such as water holding capacity and tenderness were also studied. The correlation analysis between calpain activity, proteolysis and the changes in meat quality indicators indicated that there were strong correlations for μ‐calpain during the first 12 h of storage, while such strong correlations for μ/m‐calpain were only found in samples stored from 12 h to 7 days. Our study suggested that μ‐calpain played a major role in meat quality changes while μ/m‐calpain could also be involved but played a limited role in the proteolysis and meat quality changes during 12 h to 7 days postmortem storage of chicken breast muscle.     

Acceleration of post‐mortem changes in Tsaiya duck (Anas platyrhynchos) breast muscle by lactic acid marination

1. The effect of lactic acid marination at 5°C on post mortem changes in breast muscle pectoralis major of spent layer Tsaiya duck was studied.

2. Myofibrils were prepared from 0.1 M and 0.2 M lactic acid marinated muscle and control (non‐marinated samples) sampled at 0, 1, 3, 7 and 14 d post mortem.

3. Changes in myofibril fragmentation index (MFI), myofibrillar proteins and Z‐line structure were examined.

4. Marination of duck breast muscle in lactic acid at 5°C enhanced fragmentation of myofibrils and degradation of myofibrillar proteins and Z‐line structure as compared to control samples.

5. In summary, lactic acid marination at 5°C can accelerate the post mortem degradation of myofibrils in Tsaiya duck breast muscle.     

Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.     

Inosine‐5'‐monophosphate is a candidate agent to resolve rigor mortis of skeletal muscle

The object of the present study was to reveal the action of inosine‐5'‐monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19‐0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0–3.3 m mol/L adenosine‐5'‐diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP‐induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments.     

In vitro degradation of bovine myofibrils is caused by &#956;-calpain, not caspase-3

Tenderness is a key palatability trait influencing perception of consumers of meat quality and is influenced by a multitude of factors, including postmortem proteolysis. A fundamental understanding of this biological mechanism regulating tenderness is necessary to decrease variability and increase consumer satisfaction. However, reports regarding the enzyme systems involved in postmortem tenderization are conflicting. Therefore, the objective of this study was to determine if caspase-3 is responsible for the degradation of myofibrillar proteins during aging. Bovine semitendinosus muscles were removed from 2 carcasses. Muscle from the left side of each carcass was excised 20 min postmortem and utilized for in vitro analysis of protein degradation. Muscle strips were dissected from the semitendinosus, restrained to maintain length, and placed in a neutral buffer containing protease inhibitors. Upon rigor completion, myofibrils were isolated from each strip and sarcomere length was determined. Samples with similar sarcomere lengths were selected to minimize the effect of sarcomere length on proteolysis. Myofibrils were then incubated at 22°C with μ-calpain, caspase-3, or μ-calpain + caspase-3 for 0.25, 1, 3, 24, 48, or 72 h at optimum pH for enzyme activity. The semitendinosus from the right side of each carcass was excised 1 d postmortem, cut into 2.54-cm steaks, vacuum-packaged, and allowed to age for 2, 4, 7, or 10 d to evaluate normal protein degradation during beef aging. Proteolysis of troponin T, α-actinin, and desmin was monitored using SDS-PAGE and Western blotting techniques, whereas proteolysis of titin and nebulin was monitored using SDS-vertical agarose gel electrophoresis and Western blotting. Analysis of Western blots revealed no change in abundance of intact troponin T, desmin, titin, or nebulin over time in myofibrils incubated with caspase-3. However, abundance of these proteins subjected to digestion with μ-calpain and μ-calpain + caspase-3 revealed degradation patterns similar to in situ samples. No degradation of α-actinin was observed in in vitro or in situ samples. Results of this study indicate μ-calpain, not caspase-3, is responsible for the degradation of key myofibrillar proteins during beef aging.     

Isolation of chicken taste buds for real‐time Ca2+ imaging

We isolated chicken taste buds and used a real‐time Ca²⁺ imaging technique to investigate the functions of the taste cells. With RT‐PCR, we found that isolated chicken taste bud‐like cell subsets express chicken gustducin messenger RNA. Immunocytochemical techniques revealed that the cell subsets were also immunopositive for chicken gustducin. These results provided strong evidence that the isolated cell subsets contain chicken taste buds. The isolated cell subsets were spindle‐shaped and approximately 61–75 μm wide and 88–98 μm long, and these characteristics are similar to those of sectional chicken taste buds. Using Ca²⁺ imaging, we observed the buds' response to 2 mmol/L quinine hydrochloride (a bitter substance) and their response to a mixture of 25 mmol/L L‐glutamic acid monopotassium salt monohydrate and 1 mmol/L inosine 5′‐monophosphate disodium salt, umami substances. The present study is the first morphological demonstration of isolated chicken taste buds, and our results indicate that the isolated taste buds were intact and functional approaches for examining the taste senses of the chicken using Ca²⁺ imaging can be informative.     

AA鸡胸肌和腿肌中水分含量的差异研究

为了比较AA鸡胸肌和腿肌中水分含量的差异,选取来自江苏省东台市和宿迁市的49日龄AA鸡进行屠宰,测定其胸肌和腿肌中的水分含量。结果表明,2种来源的AA鸡胸肌中的含水量分别为73.70%和73.98%,腿肌中的含水量分别为75.44%和74.56%;2种来源的AA鸡腿肌中的水分含量都极显著地高于胸肌（P〈0.01）。研究结果为AA鸡肉品质标准的制定以及肉品质的改善提供了基础数据。     

云南武定鸡肉品质分析

本试验对230日龄武定鸡不同性别和部位的肉质物理特性指标进行了测定分析。结果显示:①性别对230日龄武定鸡的肉质物理特性影响不大,仅在嫩度上母鸡优于公鸡,且差异显著(P<0.05);②部位对230日龄武定鸡肉质的物理特性有很明显的影响(P<0.05),胸肌的嫩度、贮藏损失、L值、b值均显著高于腿肌,而a值、pH值显著低于腿肌,只有系水率在胸部和腿部间没有显著差异(P>0.05);③阉割可以改善武定鸡肉质。     

云南武定鸡肉品质分析

本试验对230日龄武定鸡不同性别和部位的肉质物理特性指标进行了测定分析。结果显示：①性别对230日龄武定鸡的肉质物理特性影响不大,仅在嫩度上母鸡优于公鸡,且差异显著（P〈0.05）;②部位对230日龄武定鸡肉质的物理特性有很明显的影响（P〈0.05）,胸肌的嫩度、贮藏损失、L值、b值均显著高于腿肌,而a值、pH值显著低于腿肌,只有系水率在胸部和腿部间没有显著差异（P〉0.05）;③阉割可以改善武定鸡肉质。     

Effect of stresses before slaughter on changes to the physiological, biochemical and physical characteristics of duck muscle.

1. This experiment was designed to study the effects of fasting and enforced exercise on the physiological, biochemical and physical characteristics of duck muscle. 2. Sixty 75-d-old male ducks weighing 3.0 +/- 0.2 kg were assigned to three treatments: a control, and an 8 and 24 h fast plus enforced exercise for 10 min. The ducks were then sacrificed and the carcass stored at 4 degrees C for 24 h. 3. Although the pH and serum lactate contents gradually increased with fasting time the responses were not significant. The ultimate pH was elevated and the lactate of breast and thigh muscles was lower in stressed birds. 4. The activity of lactic dehydrogenase was significantly increased by the stress, and the activities of creatine phosphokinase and alkaline phosphatase were also increased slightly. However, no effect was found on the ATPase activity of the myofibrillar protein of either breast or thigh muscle as a result of the stress. The ATPase activity of myofibrillar protein of breast muscle significantly increased with storage time. 5. The extractability of myofibrillar protein increased with storage time for all treatments. The SDS-PAGE patterns of myofibrillar proteins were also studied. 6. Consequently DFD-like muscle was observed in the breast and thigh muscles of ducks which had been stressed.     

Breed differences and genetic parameters of myoglobin concentration in porcine longissimus muscle

An evaluation of porcine longissimus myoglobin concentration was conducted to determine breed and gender differences for myoglobin content, estimate genetic parameters for myoglobin concentration, and determine the relationship between myoglobin content and objective measures of muscle color. Data from centrally tested (n = 255), purebred Yorkshire (42), Duroc (61), Hampshire (17), Chester White (28), Berkshire (67), Poland China (28), and Landrace (12) barrows and gilts from the 1999 National Barrow Show Sire Progeny Test were used. Ultimate pH and Hunter L were measured on the 10th-rib face 24 h postmortem. A section of bone-in loin containing the 10th rib was taken to the Iowa State University Meats Laboratory. At 48 h postmortem, Hunter L, CIE L*, a*, and b*, Japanese color score, and water-holding capacity were measured on the face of the 10th-rib loin chop. A slice from the 10th-rib loin section was evaluated for percentage of i.m. fat. The resulting loin chop was used for the determination of soluble myoglobin concentration (mg/g, wet basis). Chester White, Hampshire, and Duroc pigs had the highest (P < 0.05) myoglobin concentration (0.92, 0.95, and 0.85 mg/g, respectively), whereas Landrace had the lowest (0.62 mg/g; P < 0.05). No gender differences were detected for myoglobin concentration. The heritability estimate for soluble myoglobin concentration was 0.27. Residual correlations between soluble myoglobin and CIE L*, a*, b*, Hunter L (24 h), Hunter L (48 h), and Japanese color score were -0.17, 0.23, -0.15, -0.16, -0.13, and 0.13, respectively. These correlations are low but in the desired direction. The residual correlation between soluble myoglobin and intramuscular fat percent was 0.18. Results show that myoglobin concentration has a moderate heritability and could be used in a selection program to make pork loins darker in color.     

兔腰大肌和鸡胸大肌三聚磷酸酶特性的比较研究

分离纯化兔腰大肌和鸡胸大肌中的三聚磷酸盐酶(TPPase),比较不同物种TTPase的酶学特性和作用条件。试验结果,显示兔腰大肌和鸡胸大肌的TPPase的最适温度分别为35℃和30℃;最适pH分别是6和5。Mg2+对兔腰大肌和鸡胸大肌的TPPase都有激活作用,在0~20 mmol/L范围内,鸡胸大肌TPPase活性随Mg2+浓度增加而缓慢上升,3 mmol/L的Mg2+对兔腰大肌TPPase激活作用最明显。Ca2+对兔腰大肌TPPase有激活作用,而对鸡胸大肌TPPase有抑制作用。高浓度的EDTA-Na4和KIO3对两种肉TPPase都有明显的抑制效果。EDTA-Na2对兔腰大肌TPPase的活性没有显著影响,但在高于0.5 mmol/L时,对鸡胸大肌TPPase的活性抑制效果明显。     

Characterization of myofibrillar adenosine triphosphatase activity and liberation of actin from myofibrils upon heating chicken breast meat

Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg²⁺‐ATPase (adenosine triphosphatase) and Ca²⁺‐ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine‐5′‐monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding (‘Bond A’) between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond (‘Bond B’, more heat‐stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.