Characteristics of myofibres in the masseter muscle of mice during postnatal growth period

Functional maturation of muscles is related to the constitutional proportion of muscle protein isoforms during development and growth. Although the mouse masseter muscle (MS) is classified as a fast limb muscle, its functions are different from those of a limb muscle. This study investigated the differentiation of myosin heavy chain (MHC) isoforms during the postnatal development periods in mouse MS and mouse tibialis anterior (TA), which is a fast limb muscle. Many anti-MHC slow-type-positive fibres were observed in neonatal MS and TA; these fibres decreased during development. Adult MS was composed of anti-MHC fast-type-positive fibres. MHC isoforms in MS were composed of MHC-2a and MHC-2d soon after birth. MHC-2b was expressed, but MHC-2a was not seen after 21 days. Expression of MHC-2b agreed with the weaning period, that is 2-3 weeks after birth. This fact suggested that the transformation from suckling to mastication changed the MHC isoforms during this period. In this study, the expressions of MHC-2b agree with the weaning period.     

Myosin Heavy Chain Transitions in Dog Skeletal Muscle Fibres during Post-natal Development

Myosin heavy chain (MHC) isoform expression was established in the masseter, axial and limb skeletal muscles in 18 young dogs from late prenatal to 6 months of age. Results obtained by immunohistochemical procedure using 11 monoclonal antibodies, SDS-PAGE electrophoresis, Western-blotting and enzyme-histochemical reactions were compared to those found in skeletal muscles of adult dogs. In the skeletal muscles of both fetuses in the last stages of development and neonatal dogs, the developmental isoforms MHC-emb and MHC-neo were prevalent. During the first 10 days after birth, the level of embryonic isoform expression declined, as did that of the neonatal isoform during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased. By 6 weeks of age the presence of MHC-IIx became obvious. This transitional pattern was similar in all muscles except in the rhomboideus, where secondary slow fibres were present before birth. In the other muscles, only one primary, centrally located, slow fibre was present in a fascicle and the secondary slow fibres evolved from developmentally fast fibres between the second and third weeks of age. Our results indicate that timing of muscle maturation depends on its activity right after birth. The fastest developing muscles were the masseter muscle and the diaphragm. They were followed by others, postural and locomotory muscles. A pronounced swap from developmental to adult isoforms was noted between the 4^th and 6^th week of age. At this age puppies are weaned and begin playing intensively and investigating their surroundings. The results of our study indicate that dog skeletal muscles exhibit an immature morphology at birth and develop relatively slowly compared to those of other domestic animals.     

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre‐ and postnatal development

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre‐ and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.     

Relationships between tropomyosin and myosin heavy chain isoforms in bovine skeletal muscle

The composition of tropomyosin (TPM) and myosin heavy chain (MyHC) isoforms was analyzed in 10 physiologically different bovine muscles ( masseter , diaphragm, tongue, semispinalis, pectoralis profundus , biceps femoris, psoas major , semimembranosus, longissimus thoracis and semitendinosus ) to clarify the relationships between TPM and MyHC isoforms in different muscle fiber types. The content of TPM1 and TPM3 was different in muscles according to their function in muscle contraction, although the content of TPM2 was constantly about 50% of the total TPM in all muscles. The content of TPM1 was higher in semimembranosus , longissimus thoracis and semitendinosus, while that of TPM3 was higher in masseter and diaphragm. The high positive correlation between MyHC-slow content and TPM3 content ( r  = 0.92) suggested a coexpression of TPM3 and MyHC-slow isoforms in a muscle fiber. MyHC-slow and TPM3 were expressed at the same level in masseter and diaphragm, whereas there was more TPM3 than MyHC-slow in tongue and semispinalis , so it appears that the excess TPM3 in tongue and semispinalis is expressed with other MyHC isoforms. MyHC-2a was the only fast type isoform expressed in tongue and semispinalis . Therefore, the excess TPM3 was composed of myofibrils with MyHC-2a. The results suggested that a fiber expressing MyHC-2a would be regulated delicately by changing the TPM isoform types.     

The Structure of the Masseter Muscle in the Giraffe (Giraffa camelopardalis)

In the giraffe (Giraffa camelopardalis), the masseter muscle was divided into several layers. The superficial and more medial (second) tendinous sheets of the masseter muscle fused with each other at the dorso-caudal part and a fleshy portion was located between these tendinous sheets. In the rostral part, the most superficial tendinous sheet turned around as a compact tendon and attached to the facial crest (Crista facialis). The turned tendinous sheet, however, never fused with the second tendinous sheet and this layer of the masseter muscle, that originated from the facial crest with the turned sheet, was inserted into the mandible with its fleshy portion. In the cattle, goat, sheep and Sika deer, the rostral layer of the masseter muscle arises from the facial crest with its fleshy portion and is inserted into the tubercle on the mandible through the strong tendinous sheet. In this study, the takin also showed the same structure of the masseter muscle. In the giraffe, however, the rostral layer inserted into the mandible through the strong tendinous sheet could not be distinguished, thus, there was no conspicuous tubercle on the mandible. Moreover in the masseteric region of the skull.,the giraffe was similar to the Sika deer in several ways. However, it is suggested that the giraffe exerts smaller forces on the cheek teeth than does the Sika deer because of its longer Margo interalveolaris.     

Age-related changes in metabolic properties of equine skeletal muscle associated with muscle plasticity

The purpose of the present study was to determine the age-related changes in myosin heavy chain (MHC) composition and muscle oxidative and glycolytic capacity in 18 horses ranging in age from two to 30 years. Muscle samples were collected by excisional biopsy of the semimebranosus muscle. MHC expression and the key enzymatic activities were measured. There was no significant correlation between horse age and the proportions of type-IIA and type-IIX MHC isoforms. The percentage of type-I MHC isoforms decreased with advancing age. Muscle citrate synthase activity decreased, whereas lactate dehydrogenase activity increased with increasing age. Muscle 3-OH acyl CoA dehydrogenase activity did not change with ageing. The results suggest that, similar to humans, the oxidative capacity of equine skeletal muscle decreases with age. The age-related changes in muscle metabolic properties appear to be consistent with an age-related transition in MHC isoforms of equine skeletal muscle that shifts toward more glycolytic isoforms with age.     

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.     

microRNA-21和TGFBI基因在猪骨骼肌生长发育中的表达分析

试验旨在研究microRNA-21(miR-21)和转化生长因子β诱导(TGFBI)基因在长白猪骨骼肌中的表达相关性。采用实时荧光定量PCR(qPCR)方法分析了miR-21和TGFBI基因在长白成年猪中的组织表达谱,同时比较分析了其在长白猪不同发育阶段(出生前和出生后共28个发育点)背最长肌中的表达相关性。结果表明,miR-21在长白成年猪的各个组织中均表达,且分布相对平衡,在不同发育阶段的背最长肌中呈现波浪式的表达趋势;TGFBI基因在长白猪胚胎期的背最长肌中高表达,在整个背最长肌发育过程中呈现出不同的表达丰度。相关性分析表明,在胚胎期骨骼肌发育过程中,miR-21和TGFBI表达之间为显著的负相关,TGFBI可能是miR-21的调控靶标基因。     

Immunohistochemical identification and fiber type specific localization of protein kinase C isoforms in equine skeletal muscle

OBJECTIVE: To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy. ANIMALS: 5 healthy adult Dutch Warmblood horses. PROCEDURE: In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (alpha, beta1, beta2, delta, epsilon, or zeta)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis. RESULTS: The PKC alpha, beta1, beta2, delta, epsilon, and zeta isoforms were localized within the fibers of equine skeletal muscle. In addition, PKC alpha and beta2 were detected near or in the plasma membrane of muscle cells. For some PKC isoforms, distribution was specific for fiber type. Staining of cell membranes for PKC alpha was observed predominantly in fibers that reacted positively with myosin heavy chain (MHC)-IIa; PKC delta and epsilon staining were more pronounced in MHC-I-positive fibers. In contrast, MHC-I negative fibers contained more PKC zeta than MHC-I-positive fibers. Distribution of PKC beta1 was equal among the different fiber types. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that PKC isoforms are expressed in equine skeletal muscle in a fiber type-specific manner. Therefore, the involvement of PKC isoforms in signal transduction in equine skeletal muscle might be dependent on fiber type.     

Unique expression patterns of myosin heavy chain genes in the ductus arteriosus and uterus of rabbits.

In smooth muscle tissue, two smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2) and two non-muscle MHC isoforms (NMA, NMB) have been identified. The purpose of our study was to clarify whether smooth muscle MHC mRNA expression reflects the physiological and functional state of the muscle. We studied the expression pattern of MHC mRNAs, using the S1-nuclease mapping procedure, in functionally and morphologically changeable organs; the ductus arteriosus (DA) during development (25 and 29 days of gestation, and from 3-day-old neonates) and uteri from virgin, day-10 pregnant (P10) and day-29 pregnant (P29) rabbits. The results demonstrated that SM2 expression was greater in the fetal DA than in the fetal aortic and pulmonary arteries, but that it decreased significantly following closure of DA. In the gravid uterus, SM1 expression was significantly (P<0.05) strong compared to other MHC mRNAs from virgin to P10 rabbits. During pregnancy, NMB expression showed a tendency to increase until P10, and after P10, SM2 expression increased dramatically and NMB expression decreased to give almost a mirror image of the SM2 expression. Smooth muscle type (SM1, SM2) was significantly (P<0.05) strong compared to non-muscle type expression (NMA, NMB) at P29. These data suggest that smooth muscle MHC mRNA, especially SM2 expression reflects the physiological and functional state of the smooth muscle.     

Amino acid sequences of multiple fast and slow troponin T isoforms expressed in adult bovine skeletal muscles

Multiple nucleotide sequences of complementary DNA (cDNA) of bovine troponin T (TnT) isoforms expressed in the adult skeletal muscles were determined to facilitate the elucidation of the TnT degradation progress during postmortem aging of muscles. Fresh muscle samples were excised from the lingual, masseter, pectoralis, diaphragm, psoas major, longissimus thoracis, spinnalis, semitendinosus, semimembranosus, and biceps femoris muscles of three Holstein cows within 1 h of slaughter. Complementary DNA fragments of fast and slow TnT isoforms expressed in each muscle were amplified by reverse-transcribed PCR. Consequently, four major fragments of fast TnT and two fragments of slow TnT, all of which contained the complete coding region, were obtained. The sequence determination of these fragments revealed that at least eight and two isoforms were generated by the alternative splicing from bovine fast and slow TnT messenger RNA, respectively. In the fast TnT isoforms, five small variable exons were observed; three of these five exons were in the amino (N)-terminal region. The calculated molecular weight of fast and slow TnT isoforms ranged from 29,816 to 32,125 and from 30,166 to 31,284, respectively. The deduced amino acid sequences revealed that the N-terminal region of all the TnT isoforms was extremely glutamic acid-rich. Reverse-transcribed PCR analysis revealed that expression of each of these isoforms was distributed in a fast or slow muscle-specific manner. Given that TnT degradation has been reported to accompany a decrease in glutamic acid content in the conventional 30-kDa degradation product, the sequence data suggested that the 30-kDa fragment seem to be generated by the proteolytic removal of the glutamic acid-rich N-terminal ends. The multiplicity of TnT isoforms may result in a complicated pattern of TnT degradation on SDS-PAGE gel during beef aging.     

Comparison of sensitivity of sodium currents to tetrodotoxin in equine muscle specimens with that in murine and human muscle specimens

OBJECTIVE: To determine sensitivity of equine skeletal muscle to tetrodotoxin and compare that with sensitivity of murine and human skeletal muscles. SAMPLE POPULATION: Semimembranosus, vastus lateralis, triceps brachii, and masseter muscle specimens from 22 euthanatized horses, vastus lateralis muscle biopsy specimens from 25 clinically normal humans, and diaphragmatic muscle specimens from 6 mice. PROCEDURE: Electrically elicited twitch responses were measured in muscle specimens incubated in medium alone and with tetrodotoxin (100 nM, 400 nM, 1.6 microM for equine specimens and 100 nM, 200 nM, 400 nM, 800 nM, 1.6 microM for murine and human specimens). Percentages of tetrodotoxin-sensitive and -resistant sodium channels were determined and compared among muscles and species. RESULTS: 2 sodium channels with different sensitivities to tetrodotoxin were identified in equine muscle. One was blocked with 100 nM tetrodotoxin and the other was unaffected by tetrodotoxin at concentrations up to 1.6 microM. The only difference detected among the 4 equine muscles was that masseter muscle specimens had a higher percentage of tetrodotoxin-sensitive channels than triceps brachii muscle specimens. Tetrodotoxin-resistant sodium channels constituted 31 to 66% of the equine muscle twitch response, which was greater than that determined for normal human and murine muscle (< 5%). CONCLUSION AND CLINICAL RELEVANCE: Equine skeletal muscle contains a high percentage of tetrodotoxin-resistant sodium channels. The 4 equine muscles evaluated were more similar to each other than to murine and human muscles. Shifts in expression of sodium channel subtypes may play a role in the manifestation of certain myopathies.     

Fiber type composition of rostral and caudal portions of the digastric muscle in the dog

The distribution of muscle fiber types in rostral and caudal portions of the musculus digastricus (digastric muscle) was studied in 6 dogs. Staining procedures which stain specifically for type IIM fibers, a fiber type found in other muscles supplied by the trigeminal nerve, were used. Rostral and caudal portions of the muscle were compared because the rostral portion is innervated by the trigeminal nerve, and the caudal portion is innervated by the facial nerve. The musculus triceps brachii (triceps muscle), which contains fiber types I and IIA, and the musculus masseter (masseter muscle), which contains type IIM, were used as controls. Mean fiber diameters were calculated for each of the muscles. Both portions of the digastric muscle exhibited the same histochemical behavior, possessing types I and IIA myofibers. Neither portion contained type IIM fibers. Type I fibers in the masseter muscle were histochemically different from type I fibers in the other muscles studied. Type II fibers predominated in all 3 muscles, but there were significantly (P less than 0.001) more type I fibers in the triceps muscle than in either portion of the digastric muscle or in the masseter muscle. Type II fibers were significantly larger than type I fibers in the caudal digastric (P less than 0.01) and masseter (P less than 0.05) muscles. There was no difference in the size of type I or type II fibers between any of the muscles studied (P greater than 0.20).     

CPT1A基因在广西麻鸡的表达研究

该研究旨在阐明CPT1A基因在广西麻鸡组织表达和时序表达谱,为研究鸡体内脂肪代谢提供一定的基础资料.选取胚胎期到性成熟时的广西麻鸡,在不同发育时间点采集胸大肌、腓肠肌、心脏和肝脏组织样品,利用实时荧光定量方法,分析CPT1A基因在不同组织不同发育阶段表达规律.组织表达结果显示,CPT1A在广西麻鸡不同发育阶段4种组织中...     

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.     

吐鲁番黑羊MSTN/Smad信号通路基因表达的发育性变化

为探讨MSTN/Smad信号通路基因对吐鲁番黑羊肌肉生长发育的影响,采用实时定量PCR法,分别对1～6月龄吐鲁番黑羊的腿肌和尾脂MSTN/Smad信号通路基因进行检测.结果表明：MSTN/Smad信号通路基因在腿肌和尾脂组织中均有表达,MSTN/Smad信号通路基因在吐鲁番黑羊不同生长阶段的腿肌和尾脂中的表达没有出现随月龄的增加而一直增加或下降的趋势.     

MYH3基因在动物肌肉发育、能量代谢及生产性能中的研究进展

肌球蛋白重链3（myosin heavy chain 3,MYH3）基因编码胚胎型肌球蛋白重链蛋白,控制肌肉的牵引滑动。MYH3基因是肌肉分化的重要标志基因,能够调控肌肉发育及能量代谢,在动物整个肌肉发育过程中均发挥重要作用。MYH3基因在不同物种间高度保守,且在动物体内多组织中均有表达,在胚胎期肌肉组织和肌肉再生过程中表达量较高。它受转录因子、microRNA、lncRNA及环境营养因子等多种因素影响,也可调控其他基因的功能。MYH3基因突变可以改变TGF-β信号通路和MAPK信号通路相关蛋白的磷酸化水平;影响ATP酶活性,使ATP水解时间延长,延长横桥周期;影响肌肉的能量代谢,最终引发肌肉能量代谢疾病。MYH3基因拷贝数变化、突变或表达量变化与动物的体尺、胴体重、屠宰重、生长性能具有显著的相关性。MYH3基因在大理石花纹高、肌内脂肪高的肌肉组织中表达量高,被认为是影响动物肌肉嫩度、剪切力和肉色红度的重要候选基因。MYH3基因的高表达与骨骼肌中氧化Ⅰ型肌纤维的含量、肌纤维直径和慢肌纤维含量有关。作者介绍了MYH3基因的基本结构特点,指出了其与肌肉组织发育及相关影响因子之间的调控作用,阐述了MYH3基因与动物肌肉能量代谢、生长性能和肉品质之间的关系,为进一步研究MYH3基因与肌肉发育调控和肉质性能改良提供参考。     

microRNA与胎盘发育关系的研究进展

microRNA (miRNA)是一类大小约22 nt的内源性非编码RNA,它们通过剪切靶基因的转录产物或抑制靶基因转录产物的翻译,在转录后起到调控靶基因表达的作用。大量研究结果表明,动物体内的miRNA参与了胚胎的早期发育、脑和神经发育、心脏发育、肌肉及骨骼发育等动物发育的各个方面。已有证据表明,一些miRNA在胎盘中特异表达,在胎盘的正常发育过程中起到了重要作用。作者对参与胎盘发育的miRNAs进行了综述。     

Myosin heavy chain composition in normal and atrophic equine laryngeal muscle

The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. Left and right thyroarytenoideus (TA) and cricoarytenoideus dorsalis (CAD) were obtained from 7 horses affected with left-sided intrinsic laryngeal muscle atrophy and from 2 normal horses. Frozen sections were evaluated histologically for degree of atrophy and fiber type composition. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of muscle protein. Histologic atrophy was seen in all atrophic muscles and some right-sided muscles of 3 affected horses, as well as the left TA of 1 normal horse. Fiber type grouping or loss of type I muscle fibers was observed in the left-sided laryngeal muscles in all but 1 affected horse, as well as in the right muscles of 2 affected horses, and the left TA of 1 normal horse. SDS-PAGE showed 2 bands corresponding to the type I and type IIB myosin isoforms in the CAD and TA of the 2 normal horses. Affected horses demonstrated a trend toward increased expression of the type IIB isoform and decreased expression of the type I isoform in atrophic muscles. This study confirmed the presence of histologic abnormalities in grossly normal equine laryngeal muscle, and it demonstrated an increased expression of type IIB MHC with a concurrent decreased expression of type I MHC in affected muscles. Evaluation of muscle fiber changes at the cellular level under denervated and reinnervated conditions may aid in assessing future strategies for reinnervation or regeneration of atrophic laryngeal muscle.