The importance of subfragment 2 and C‐terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C‐terminus on thick filament assembly. C‐terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C‐terminus of LMM.     

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.     

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.     

Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts

Cell‐cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB‐GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB‐DsRed. mMB‐GFP and chMB‐DsRed were cocultured and induced to differentiate. After 24 h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP⁺/DsRed⁺ hybrid myotubes were able to survive and grew to hyper‐multinucleated mature form. We also found that undifferentiated mMB‐GFP efficiently fuse to the chMB‐DsRed‐derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast‐based fusogenic technique will open up an alternative direction to create novel hybrid products.     

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.     

A rhabdomyosarcoma arising in the larynx of a dog

A neoplastic nodular lesion, 2 × 3 cm in diameter, was found in the larynx of a 6-year-old spayed female dog. The tumor was ill-circumscribed, consisting histologically of large round cells with abundant cytoplasm interspersed with small round cells with less cytoplasm and occasional multinucleated cells (myotubes). Immunohistochemically, tumor cells were positive for myoglobin, desmin and vimentin in varying degrees, but negative for S-100 protein, GFAP or cytokeratin. Cytoplasmic myofilaments/myofibrils with a dense Z-line-like structure were seen, the fine structures of which were complemented by PTAH stain. Based on these findings, the tumor was diagnosed as a rhabdomyosarcoma, a very rare tumor in the larynx of dogs.     

Electron microscopic observations on canine mixed mammary tumors, with special reference to cytoplasmic filamentous components

Three chief cell types were studied in 8 canine mixed mammary tumors--luminal epithelial cells, myoepithelial cells, and filament cells, the last being the "stellate" cells recognized by light microscopists. Most cytoplasmic filaments in the myoepithelial cells were 5 to 8 nm thick, whereas another type of filaments were scattered, 8 to 10 nm thick. Almost all cytoplasmic filaments in the filament cells were 8 to 10 nm thick. In the adenomatous parts of the tumors, myoepithelial cells were chiefly seen, along with luminal epithelial cells. Filament cells were rarely observed without intercellular accumulation of mucoid. The filament cells were largely confined to the myxomatous and chondromatous parts of the tumors. The presence of cells of transitional forms in adenomatous areas suggested that the filament cells were derived from the myoepithelial cells.     

Protein metabolism in chicken muscle cell cultures treated with cimaterol

Primary muscle cell cultures were prepared from the leg muscle of 12-d broiler chicken embryos. The partitioning agent cimaterol (10(-6) to 10(-10) M) was added on d 1 and each day thereafter, and cells were studied after 7 d in culture. Cimaterol had no effect at any level either on the percentage of nuclei within multinucleated myotubes or on the total number of nuclei within myotubes. At 10(-7) M cimaterol, the quantity of the myofibrillar protein fraction was increased by 25.1 +/- 8.0% (P less than .05) and the quantity of myosin heavy chain was increased by 30.9 +/- 4.5% (P less than .05). To understand the basis for the increase in myofibrillar protein, the incorporation rate of [3H]Leu was measured in pulse labeling experiments. The apparent synthesis rate of the soluble protein fraction and the crude myofibrillar fraction was not significantly increased by cimaterol; however, cimaterol levels greater than 10(-8) M caused a 10 to 12% increase (P less than .05) in the incorporation rate of [3H]Leu into myosin heavy chain. The effect of cimaterol on release of [3H]Leu from prelabeled protein also was assessed in pulse-chase experiments; the apparent rate of protein degradation was inhibited by 10 to 15% (P less than .05) at the higher levels of cimaterol. Dot blot analysis indicated that the quantity of myosin heavy chain mRNA was elevated in cimaterol-treated cultures. Thus, the increased quantity of myofibrillar proteins in embryonic broiler muscle cell cultures is the combined result of a stimulation in the rate of protein synthesis and an inhibition in the rate of protein degradation.     

Light microscopy and image analysis of thin filament lengths utilizing dual probes on beef, chicken, and rabbit myofibrils

Image analysis procedures for immunofluorescence microscopy were developed to measure muscle thin filament lengths of beef, rabbit, and chicken myofibrils. Strips of beef cutaneous trunci, rectus abdominis, psoas, and masseter; chicken pectoralis; and rabbit psoas muscles were excised 5 to 30 min postmortem. Fluorescein phalloidin and rhodamine myosin subfragment-1 (S1) were used to probe the myofibril structure. Digital images were recorded with a cooled charge-coupled device controlled with IPLab Spectrum software (Signal Analytics Corp.) on a Macintosh operating system. The camera was attached to an inverted microscope, using both the phase-contrast and fluorescence illumination modes. Unfixed myofibrils incubated with fluorescein phalloidin showed fluorescence primarily at the Z-line and the tips of the thin filaments in the overlap region. Images were processed using IPLab and the National Institutes of Health's Image software. A region of interest was selected and scaled by a factor of 18.18, which enlarged the image from 11 pixels/microm to approximately 200 pixels/microm. An X-Y plot was exported to Spectrum 1.1 (Academic Software Development Group), where the signal was processed with a second derivative routine, so a cursor function could be used to measure length. Fixation before phalloidin incubation resulted in greatest intensity at the Z lines but a more-uniform staining over the remainder of the thin filament zone. High-resolution image capture and processing showed that thin filament lengths were significantly different (P < 0.01) among beef, rabbit, and chicken, with lengths of 1.28 to 1.32 microm, 1.16 microm, and 1.05 microm, respectively. Measurements using the S1 signal confirmed the phalloidin results. Fluorescent probes may be useful to study sarcomere structure and help explain species and muscle differences in meat texture.     

牛骨骼肌卫星细胞的分离鉴定和诱导分化

本研究对牛骨骼肌卫星细胞进行体外分离培养、诱导分化和鉴定,采用胶原酶和胰酶联用的酶消化法分离肌卫星细胞,应用差速贴壁法进行纯化,观察卫星细胞及诱导分化后肌管的形态结构,并利用标志基因的反转录PCR（RT-PCR）和免疫荧光染色方法对分化前后细胞进行鉴定。结果显示,分离出的肌卫星细胞呈梭形生长,生长状态良好,RT-PCR和免疫荧光染色显示肌卫星细胞Pax7和MyoD呈阳性表达,纯化后的肌卫星细胞纯度大于93%;诱导分化后,卫星细胞融合生长,形成的肌管状态良好,分化标志基因MyoG和MHC呈阳性表达。本研究建立了一套从牛肌肉组织中分离和鉴定肌卫星细胞的方法,可以为肌肉的发育分化和肉牛肉质改良研究提供良好的细胞模型。     

Decorin activates Akt downstream of IGF‐IR and promotes myoblast differentiation

Decorin, a small leucine‐rich proteoglycan, plays an important role in cellular activities through modification of growth factors. It also acts as a signaling molecule to non‐muscle cells through epidermal growth factor receptor or insulin‐like growth factor I receptor (IGF‐IR). However, it is unclear if decorin acts as a signaling molecule to myogenic cells. In this study, we investigated the effect of decorin on the differentiation of myoblasts and the signaling via IGF‐IR to myogenic cells. C2C12 myoblasts cultured in media containing decorin for 72 h showed more extensive formation of multinucleated myotubes than control cells cultured in the same media without decorin. The protein expressions of myogenin and myosin heavy chian were higher in decorn‐treated cells than in control cells. These results suggest that decorin enhances the differentiation of myoblasts. Western blot analysis and immunocytochemistry showed that IGF‐IR was expressed in myoblasts and myotubes. Furthermore, Akt, which is downstream of IGF‐IR, was more phosphorylated in myoblasts cultured in media containing decorin than those in media without decorin. These results suggest that decorin activates Akt downstream of IGF‐IR and enhances the differentiation of myogenic cells.     

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.     

Pax‐3 and Pax‐7 Label Muscle Progenitor Cells During Myotomal Myogenesis in Coregonus lavaretus (Teleostei: Coregonidae)

In Coregonus lavaretus, prior the mesoderm segmentation, in cells adjacent to the notochord called adaxial cells MyoD and slow myosin heavy chain (MyHC‐slow) proteins were observed. After somite formation, adaxial cells migrate towards the lateral part of the myotome and form a layer of red muscles. Deeper cells differentiate into white muscle fibres. In situ hybridization using Pax‐3 molecular probe revealed, that after somitogenesis, Pax‐3 is expressed in a layer of cells superficial to the myotome resembling the “external cells” (found in many teleosts species) or dermomyotome described in Amniota. During later developmental stages Pax‐3 gene is expressed in cells in intermyotomal space and then in myoblasts between myotubes. In these cells Pax‐7 protein was also observed. Pax‐3/7 positive cells which have migrated into the myotomes differentiate into satellite cells/secondary myoblasts and participate in hypertrophic and hyperplastic growth of muscles.     

Isolation of mycoplasmas from pneumonic sheep lungs in the Sudan

A cell type with properties similar to those of the brush cell was recognised in the bronchial epithelium of calves. It occurred in small numbers and was not found in airways distal to the small bronchi. The ultrastructural features are described, emphasising the two constant characteristics (i) a dense population of thick apical microvilli and (ii) cytoplasmic filament bundles which extent into the microvilli.     

Accumulation of newly synthesized myosin heavy chain during thyroxine-induced myofiber type transition

Slow-tonic myofiber to fast-twitch myofiber conversion was induced in chickens by feeding thyroxine. Incorporation of newly synthesized myosin heavy chain (MHC) into myofibers and myofibrils was followed by immunofluorescence with antibodies specific for fast-twitch MHC and slow-tonic MHC. Presence of more than one type of myosin heavy chain was detected in thyroxine-induced transitional myofibers of chicken pectoralis. Myofibers undergoing a transition were histochemically identical to immunologically cross-reacting, transitional myofibers of normal anterior latissimus dorsi. Newly synthesized MHC appeared to be incorporated uniformly across the cross sectional area of transitional myofibers and incorporated homogeneously into each sarcomere of transitional myofibrils. These observations are consistent with a theory of continuous protein exchange between myofibrillar protein and a non-myofibrillar protein pool, such that every sarcomere of a myofibril, and every myofibril of a myofiber, would be turned over simultaneously.     

Canine laryngeal rhabdomyoma

Three canine laryngeal tumors were diagnosed as oncocytomas by light microscopy, but were determined to be rhabdomyomas following ultrastructural and immunocytochemical examination. Tumors consisted of large eosinophilic cells interspersed with smaller dark cells. Large tumor cells had a granular, intensely eosinophilic cytoplasm. Scattered through the tumors were a few elongated cells with cytoplasmic cross striations and multiple nuclei. Tumor cells from all three dogs contained numerous mitochondria and bundles of myofibrils with electron-dense Z-lines typical of striated muscle cells. Intracellular myoglobin and desmin were detected in the tumors by immunocytochemistry. Comparisons are made with a previous report of canine laryngeal oncocytomas.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Regulation of bovine satellite cell proliferation and differentiation by insulin and triiodothyronine

Satellite cells activity contributes to postnatal muscle growth. Herein, we have studied the respective influence of insulin and triiodothyronine (T3) on the proliferation and differentiation of primary bovine satellite cells isolated from Semitendinosus muscle of Montbéliard steers. Under basal conditions, satellite cells proliferated until the fifth day of culture, began to fuse into myotubes and expressed differentiation markers such as connectin, myogenin, and myosin heavy chain (MHC) isoforms. Insulin behaved as an effective mitogen. Moreover, it promoted extensive myotube formation and enhanced differentiation as shown by an increase in the accumulation of differentiation markers. Maximal differentiation occurred with insulin physiological range concentrations. A delay in the stimulation of differentiation was registered with a high dose that promoted maximal proliferation. Conversely, T3 decreased cell proliferation in a dose-dependent manner. In addition, fusion and biochemical differentiation (accumulation of connectin, MyoD1, myogenin, and myosin heavy chain isoforms) were also enhanced. Bovine satellite cells seemed to respond differentially to insulin and T3 for proliferation. Interestingly, both hormones displayed a myogenic influence. Our observations suggest that both hormones could influence bovine satellite cells in vivo and contribute to the regulation of postnatal muscle growth.     

Tertiary myotubes in postnatal growing pig muscle detected by their myosin isoform composition.

The postnatal development of skeletal muscles was studied in growing pigs from 8 to 210 d of age. Indirect immunoperoxidase staining of frozen sections of porcine semimembranosus muscle and longissimus muscle revealed a distinct population of small fibers (tertiary myotubes) that were stained specifically by an antibody (anti-NE) selective for the developmental (embryonic and neonatal) isoforms of muscle myosin. At 8 d of age the other larger fibers were already anti-NE negative and differentiated into Types I and II. A gradual decrease in the number of anti-NE positive fibers together with a gradual increase in area of the remaining positive fibers was observed throughout the pigs' growth. These results may indicate that hyperplastic growth does not cease at birth. Possible mechanisms to explain the origin of these tertiary myotubes containing developmental isoforms of myosin are suggested.     

Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-I, and sera on protein turnover in porcine myotube cultures

Porcine myogenic cells isolated from 50 to 55-d porcine fetuses were frozen and stored in liquid nitrogen until they were needed to establish cultures. Approximately 75.8 +/- .59% of the clonal cultures established from these frozen stocks produced myotubes and 60.8 +/- 2.3% of the nuclei in differentiated mass cultures were in myotubes. Differentiated cultures contained higher levels of creatine phosphokinase activity than undifferentiated cultures. Additionally, differentiated cultures incorporated [35S]methionine into putative myosin heavy chain, alpha-actinin, and actin more rapidly than did undifferentiated cultures. Insulin, insulin-like growth factor I, and sera stimulated total protein synthesis rate and decreased total protein degradation rate in myotube cultures. Based on our initial characterization, we believe that we have developed an effective and practical procedure for isolating and culturing fetal porcine myogenic cells.