Cold exposure increases slow‐type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats

The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism‐related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold‐exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow‐type MyHC1 content in the soleus muscle (a typical slow‐type fiber), while the intermediate‐type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast‐type fiber) and gastrocnemius (a mix of slow‐type and fast‐type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism‐related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus.     

Correlation between skeletal muscle fiber type and free amino acid levels in Japanese Black steers

Free amino acids are important components of tastants and flavor precursors in meat. To clarify the correlation between muscle fiber type and free amino acids, we determined the concentrations of various free amino acids and dipeptides in samples of different muscle tissues (n = 21), collected from 26‐month‐old Japanese Black steers (n = 3) at 2 days postmortem. The proportions of the myosin heavy chain (MyHC), slow (MyHC1) and fast (MyHC2) isoforms were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The contents of free amino acids and dipeptides were measured by high performance liquid chromatography (HPLC). The MyHC isoform composition varied among the tissue samples. The MyHC1 proportion ranged from 6.9% ± 3.9% to 83.3% ± 16.7%. We confirmed that there was a strong positive correlation between MyHC1 composition and total free amino acid concentrations, including those for two dipeptides. Among the 31 measured free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition. These results suggest that a high MyHC1 content induces high free amino acid contents in bovine muscles possibly because of greater oxidative metabolism. This high level of free amino acids could contribute to the intense flavor of meat that is rich in slow‐twitch fibers.     

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.     

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type‐specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin‐deficient double‐muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse‐transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC‐2x and ‐2a and less ‐slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster‐type muscles and on MyoD expression in slower‐type muscles, suggesting a possible muscle type‐specific effect of myostatin in skeletal muscle growth and maintenance.     

Effects of muscle type on beef taste‐traits assessed by an electric sensing system

To assess the role of muscle fiber type in beef taste‐traits, we analyzed cooked meats from bovine masseter, diaphragm, psoas major, longissimus thoracis, and semitendinosus muscles with an electric taste sensing system (INSENT SA402B). The system is composed of five taste sensors of polymer membranes fixing different lipids. The sensors, CT0, CA0, AAE, C00 and AE1 are designed to respond to the individual tastes of salty, sour, umami, bitter and astringent, respectively. The system found significant differences in the converted outputs of CA0 (cvCA0), C00 (cvC00) and AE1 (cvAE1) among the bovine muscles. The slow‐type muscles (masseter and diaphragm) showed lower cvCA0, higher cvC00, and higher cvAE1 than did the fast‐type muscles (psoas major, longissimus thoracis, and semitendinosus). Lactic acid content was different among muscle types and was highly related to the cvCA0 output and pH. carbonyl compounds and free fatty acids were higher in the slow‐type muscles. Free fatty acids were major components causing the difference in the C00 output among the muscle types. Iron content was also different among the muscle types and related to the cvC00 and cvAE1 outputs. These results suggested that the muscle fiber type affects the beef taste characteristics.     

Grazing‐induced changes in muscle microRNA‐206 and ‐208b expression in association with myogenic gene expression in cattle

To investigate the roles of microRNAs (miRNAs) in muscle type conversion, the effects of 4 months of grazing on the expression levels of miRNAs and mRNAs associated with skeletal muscle development were analyzed by quantitative RT‐PCR using the Biceps femoris muscle of Japanese Shorthorn cattle. After 4 months of grazing, the expression of muscle fiber type‐associated miR‐208b was higher in the grazed cattle than in the housed. In concordance with the pattern in miR‐208b expression, the expression of MyoD, a myogenic regulatory factor associated with the shifting of muscle property to the fast type, was lower in the grazed cattle after 4 months of grazing than in the housed cattle. In addition, the expression of MyHC‐2x (a fast type) was higher in the housed cattle than in the grazed, after 4 months of grazing. During the grazing period, miR‐206 expression decreased in the housed cattle, whereas expression in the grazed cattle did not change, but rather remained higher than that of the housed cattle even at 3 months after the grazing ended. These miRNAs including miR‐206 persisting with muscles of grazed cattle may be associated with regulation of muscle gene expression during skeletal muscle adaptation to grazing.     

Isolation and characterization of cytotoxic small peptides, α-casecidins, from bovine αs1-casein digested with bovine trypsin

Cytotoxic peptides were isolated from a trypsin digest of bovine αs1‐casein by a combination of ion‐exchange column chromatography and reversed‐phase high‐performance liquid chromatography as an indicator of cytotoxicity toward mouse spleen cells. Amino acids of the isolated peptides were sequenced as arginyl‐proly‐lysine, leucyl‐lysyl‐lysine and tyrosyl‐lysine, being compatible with sequences 1–3, 101–103 and 104–105 of bovine αs1‐casein, respectively. The isolated peptides displayed cytotoxicity toward healthy mouse T and B cells and human leukemic T and B cell lines in a commercially available serum‐free medium for lymphocytes, Celgrosser‐P, and were named α‐casecidins. Similar cytotoxicity was confirmed in chemically synthesized peptides corresponding to sequences 1–3, 101–103 and 100–105 of bovine αs1‐casein. The cytotoxicity induced by α‐casecidins was concluded to be because of necrosis, and was diminished in the presence of bovine serum albumin.     

Increased expression of NOR‐1 mRNA in the skeletal muscles of cold‐exposed neonatal chicks

Nuclear receptor subfamily 4, group A (NR4A) subgroup orphan receptors are rapidly induced by various physiological stimuli and have been suggested to regulate oxidative metabolism and muscle mass in mammalian skeletal muscle. The results showed that the NR4A subgroup orphan receptor, NOR‐1 (NR4A3), was acutely increased in skeletal muscles of neonatal chicks in response to short‐term cold exposure. The increased NOR‐1 gene expression was concomitant with cold‐induced changes in gene expression of both myostatin and proliferator‐activated receptor‐gamma coactivator‐1 (PGC‐1α), and the increase in skeletal muscle mass. These observations suggest that NOR‐1 might play a role in controlling skeletal muscle growth in cold‐exposed neonatal chicks.     

大白猪不同部位肌肉肌纤维表达特点

取3头本地大白猪4个不同部位的肌肉组织,运用免疫组化方法,测定快肌纤维和慢肌纤维的表达情况。免疫组化结果表明在猪的不同部位肌肉组织中,快肌纤维与慢肌纤维含量之间存在极显著差异(P<0.01)。快肌纤维的表达在股二头肌中最多,其次为背最长肌和内脊肌,头半棘肌最少。慢肌纤维的表达在头半棘肌中最多,其次为内脊肌和股二头肌,在背最长肌中表达最少。通过对猪4种骨骼肌中快肌纤维和慢肌纤维的免疫组化分析显示,二者的表达在猪不同部位的肌肉存在显著差异。     

Leucine regulates slow‐twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow‐twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow‐twitch muscle fibers and mitochondrial function‐related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho‐AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow‐twitch muscle fibers and mitochondrial function‐related gene expression. Finally, we showed that Sirt1 was required for leucine‐induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow‐twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.     

Increase in muscle endurance in mice by dietary Yamabushitake mushroom (Hericium erinaceus) possibly via activation of PPARδ

Skeletal muscle fiber is largely classified into two types: type 1 (slow‐twitch) and type 2 (fast‐twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator‐activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight‐week‐old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze‐dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake‐supplemented diet up‐regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake‐supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake‐supplemented diets.     

干扰DHX9对牛骨骼肌卫星细胞增殖与成肌分化的影响

为研究天冬氨酸-谷氨酸-丙氨酸-组氨酸盒解旋酶9 (DEAH (Asp-Glu-Ala-His)-box helicase 9,DHX9)对牛骨骼肌细胞增殖与分化的影响,利用已经建立的牛骨骼肌卫星细胞体外成肌分化模型,设计合成DHX9的si-RNA,采用荧光定量PCR和Western blot技术检测DHX9基因在成肌...     

Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (P<.05). There were no correlations between FOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important information about the composition of porcine skeletal muscle. The combination of the two methods may be essential to accurately characterize porcine skeletal muscles.     

过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响

旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。     

共轭亚油酸对体外培养的猪骨骼肌肌纤维类型组成的影响

旨在研究共轭亚油酸(conjugated linoleic acid,CLA)对体外培养的猪骨骼肌肌纤维类型组成的影响规律。以体外培养的原代猪骨骼肌卫星细胞为材料,在卫星细胞向肌纤维转化时添加不同水平CLA(0、50、100、150、200μg.mL-1),处理后第4、8和12天,分别采用相对定量RT-PCR测定肌纤维中MyHCⅠ、MyHC 2a、MyHC 2b和MyHC 2x 4种MyHC的基因表达。结果表明,肌纤维类型的组成随培养时间的延长发生显著变化,从第4到12天,MyHC2b型肌纤维比例显著上升,而其余3种类型的肌纤维比例均显著下降。添加50μg.mL-1CLA对肌纤维类型组成无显著影响。添加100μg.mL-1CLA主要影响第12天的肌纤维类型组成,而添加150~200μg.ml-1CLA则可显著改变第4~12天的肌纤维类型组成,即显著提高MyHC I和MyHC 2a型肌纤维比例,显著降低MyHC2x和MyHC2b型肌纤维比例。以上结果提示,添加CLA可使肌纤维类型组成发生变化,且该作用与添加水平和处理时间密切相关。CLA对肌纤维类型组成的影响主要表现为提高MyHCI和MyHC2a型肌纤维比例,降低MyHC 2b和MyHC 2x型肌纤维比例,这在一定程度上可解释CLA提高猪肉品质的原因。     

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.