Language：English   Publishing type：Research paper (scientific journal)  

Skeletal muscle atrophy occurs rapidly as a result of inactivity. Although there are many reports on changes in gene expression during the early phase of muscle atrophy, the patterns of up-and downregulated gene expression after long-term and equilibrated muscle atrophy are poorly understood. In this study, we comprehensively examined the changes in gene expression in long-term denervated mouse muscles using RNA-Seq. The murine right sciatic nerve was denervated, and the mice were housed for five weeks. The cross-sectional areas of the hind limb muscles were measured using an X-ray CT system 35 days after denervation. After 28 d of denervation, the cross-sectional area of the muscle decreased to approximately 65% of that of the intact left muscle and reached a plateau. Gene expression in the soleus and extensor digitorum longus (EDL) muscles on the 36th day was analyzed using RNA-Seq and validated using RT-qPCR. RNA-Seq analysis revealed that three genes-Adora1, E230016M11Rik, and Gm10718-were upregulated and one gene-Gm20515-was downregulated in the soleus muscle; additionally, four genes-Adora1, E230016M11Rik, Pigh, and Gm15557-were upregulated and one gene-Fzd7-was downregulated in the EDL muscle (FDR < 0.05). Among these genes, E230016M11Rik, one of the long non-coding RNAs, was significantly upregulated in both the muscles. These findings indicate that E230016M11Rik could be a candidate gene for the maintenance of atrophied skeletal muscle size and an atrophic state.

DOI： 10.3390/life13051111

Language：Others  

Language：English   Publishing type：Research paper (scientific journal)  

The Nudt family has been identified as enzymes performing Coenzyme A to 3'5'-ADP + 4'-phospho pantetheine catalysis. The members of this family have been shown to be particularly involved in lipid metabolism, while their involvement in gene regulation through regulating transcription or mRNA metabolism has also been suggested. Here, we focused on peroxisomal NUDT7, possessing enzymatic activity similar to that of its paralog, peroxisomal NUDT19, which is involved in mRNA degradation. No reports have been published about the Nudt family in zebrafish. Our transcriptomic data showed that the Nudt family members are highly expressed around zygotic gene activation (ZGA) in developing zebrafish embryos. Therefore, we confirmed the computational prediction that the products of the nudt7 gene in zebrafish were localized in the peroxisome and highly expressed in early embryogenesis. The depletion of nudt7 genes by the CRISPR/Cas9 system did not affect development; however, it decreased the rate of transcription in ZGA. In addition, H3K27ac ChIP-seq analysis demonstrated that this decrease in transcription was correlated with the genome-wide decrease of H3K27ac level. This study suggests that peroxisomal Nudt7 functions in regulating transcription in ZGA via formation of the H3K27ac domain in active chromatin.

DOI： 10.1093/jb/mvac086

Language：English   Publishing type：Research paper (scientific journal)  

Protein tyrosine residue (Y) nitration, a post-translational chemical-modification mode, has been associated with changes in protein activity and function; hence the accumulation of specific nitrated proteins in tissues may be used to monitor the onset and progression of pathological disorders. To verify the possible impact of nitration on postnatal muscle growth and regeneration, a pilot study was designed to examine the nitration/dysfunction of hepatocyte growth factor (HGF), a key ligand that is released from the extracellular tethering and activates myogenic stem satellite cells to enter the cell cycle upon muscle stretch and injury. Exposure of recombinant HGF (a hetero-dimer of α- and β-chains) to peroxynitrite induces Y nitration in HGF α-chain under physiological conditions. Physiological significance of this finding was emphasized by Western blotting that showed the NK1 segment of HGF (including a K1 domain critical for signaling-receptor c-met binding) undergoes nitration with a primary target of Y198. Peroxynitrite treatment abolished HGF-agonistic activity of the NK1 segment, as revealed by in vitro c-met binding and bromodeoxyuridine-incorporation assays. Importantly, direct-immunofluorescence microscopy of rat lower hind-limb muscles from two aged-groups (2-month-old "young" and 12-month-old "retired/adult") provided in vivo evidence for age-related nitration of extracellular HGF (Y198). Overall, findings provide the insight that HGF/NK1 nitration/dysfunction perturbs myogenic stem cell dynamics and homeostasis; hence NK1 nitration may stimulate progression of muscular disorders and diseases including sarcopenia.

DOI： 10.1016/j.bbrep.2022.101295

Language：English  

Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.

DOI： 10.1093/jb/mvab030

Language：English   Publishing type：Research paper (scientific journal)  

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.

DOI： 10.3390/ijms22094499

Language：English   Publishing type：Research paper (scientific journal)  

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.

DOI： 10.1093/jb/mvab030

Language：Japanese  

Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

To clarify the relationship between the fiber type composition and meat quality, we performed metabolomic analysis using porcine longissimus dorsi (LD) muscles. In the LD of pigs raised outdoors, the expression of myosin heavy chain (MyHC)1 (slow-twitch fiber marker protein) was significantly increased compared with that of MyHC1 in pigs raised in an indoor pen, suggesting that rearing outdoors could be considered as an exercise treatment. These LD samples were subjected to metabolomic analysis for examining the profile of most primary and secondary metabolites. We found that the sex of the animal and exercise stimulation had a strong influence on the metabolomic profile in the porcine skeletal muscles, and this difference in the metabolomic profile is likely in part due to the changes in the muscle fiber type. We also examined the effects of cooking (70 °C for 1 h). The effect of exercise on the metabolomic profile was also maintained in the cooked muscle tissues. Cooking treatment resulted in an increase in some of the metabolite levels while decreasing in some other metabolite levels. Thus, our study could indicate the effect of the sex of the animal, exercise stimulus, and cooking on the metabolomic profile of pork meat.

DOI： 10.3390/metabo10010010

Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1111/asj.13199

Language：Others   Publishing type：Research paper (scientific journal)  

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

DOI： 10.1111/asj.13199

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1111/asj.13185

Language：English  

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.

DOI： 10.1111/asj.13185

Language：English  

Satellite cells (SCs) are myogenic stem cells that play an important role in skeletal muscle regeneration and hypertrophy. Primary cultures of SCs are useful to analyze cell functions; however, it is difficult to obtain highly pure SCs from young rats with the conventional procedures. The purpose of this study is to establish a purification method for SC isolation from young rats and quantitatively evaluate the purification procedure employing Percoll, a common research tool to purify cells. We elucidated the purity of SCs collected by Percoll density gradient centrifugation using real-time RT-qPCR and immunocytochemistry for desmin. Percoll treatment increased the purity of SCs isolated from young rats to nearly 90%, which was comparable to that achieved with the conventional method using middle-aged rats.

DOI： 10.1007/978-1-4939-8897-6_6

Language：English   Publishing type：Research paper (scientific journal)  

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3-L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets.

DOI： 10.1111/asj.13050

Language：Others  

Paired box 7 inhibits differentiation in 3T3-L1 preadipocytes.

DOI： 10.1111/asj.13050

Language：English   Publishing type：Research paper (scientific journal)  

The data presented in this article are related to the research articles entitled “APOBEC2 negatively regulates myoblast differentiation in muscle regeneration” and “Data supporting possible implication of APOBEC2 in self-renewal functions of myogenic stem satellite cells: toward understanding the negative regulation of myoblast differentiation” (Ohtsubo et al., 2017a, 2017b) [1,2]. This article provides in vivo phenotypical data to show that Paired Box Transcription Factor 7 (Pax7)-positive cell number (per myofiber) is significantly lower in APOBEC2 (a member of apoB mRNA editing enzyme, catalytic polypeptide-like family)-knockout muscle than the control wild-type tissue at the same age of 8-wk-old in mice. The emerging results support an essential role for APOBEC2 in the self-renewal functions of myogenic stem satellite cells, namely the re-establishment of quiescent status after activation and proliferation of myoblasts.

DOI： 10.1016/j.dib.2018.02.063

Language：Japanese  

Study of dietary fat and skeletal muscle fiber type.

Language：English   Publishing type：Research paper (scientific journal)  

The data presented in this article are related to the research articles entitled “APOBEC2 negatively regulates myoblast differentiation in muscle regeneration” and “Data supporting possible implication of APOBEC2 in self-renewal functions of myogenic stem satellite cells: toward understanding the negative regulation of myoblast differentiation” (Ohtsubo et al., 2017a, 2017b) [1,2]. This article provides in vivo phenotypical data to show that Paired Box Transcription Factor 7 (Pax7)-positive cell number (per myofiber) is significantly lower in APOBEC2 (a member of apoB mRNA editing enzyme, catalytic polypeptide-like family)-knockout muscle than the control wild-type tissue at the same age of 8-wk-old in mice. The emerging results support an essential role for APOBEC2 in the self-renewal functions of myogenic stem satellite cells, namely the re-establishment of quiescent status after activation and proliferation of myoblasts.

DOI： 10.1016/j.dib.2018.02.063

Language：English   Publishing type：Research paper (scientific journal)  

Additional data supporting possible implication of APOBEC2 in self-renewal functions of myogenic stem satellite cells.

DOI： 10.1016/j.dib.2018.02.063

Language：English   Publishing type：Research paper (scientific journal)  

Apobec2 is a member of the activation-induced deaminase/apolipoprotein B mRNA editing enzyme catalytic polypeptide cytidine deaminase family expressed in differentiated skeletal and cardiac muscle. We previously reported that Apobec2 deficiency in mice leads to a shift in muscle fiber type, myopathy, and diminished muscle mass. However, the mechanisms ofmyopathy caused by Apobec2 deficiency and its physiologic functions are unclear. Here we show that, although Apobec2 localizes to the sarcomeric Z-lines in mouse tissue and cultured myotubes, the sarcomeric structure is not affected in Apobec2-deficient muscle. In contrast, electron microscopy reveals enlarged mitochondria and mitochondria engulfed by autophagic vacuoles, suggesting that Apobec2 deficiency causes mitochondrial defects leading to increased mitophagy in skeletal muscle. Indeed, Apobec2 deficiency results in increased reactive oxygen species generation and depolarized mitochondria, leading to mitophagy as a defensive response. Furthermore, the exercise capacity of Apobec2^-/- mice is impaired, implying Apobec2 deficiency results in ongoing muscle dysfunction. The presence of rimmed vacuoles in myofibers from 10-mo-old mice suggests that the chronic muscle damage impairs normal autophagy. We conclude that Apobec2 deficiency causes mitochondrial defects that increase muscle mitophagy, leading to myopathy and atrophy. Our findings demonstrate that Apobec2 is required for mitochondrial homeostasis to maintain normal skeletal muscle function.

DOI： 10.1096/fj.201700493R

Language：English   Publishing type：Research paper (scientific journal)  

Apobec2 is a member of the activation-induced deaminase/apolipoprotein B mRNA editing enzyme catalytic polypeptide cytidine deaminase family expressed in differentiated skeletal and cardiac muscle. We previously reported that Apobec2 deficiency in mice leads to a shift in muscle fiber type, myopathy, and diminished muscle mass. However, the mechanisms ofmyopathy caused by Apobec2 deficiency and its physiologic functions are unclear. Here we show that, although Apobec2 localizes to the sarcomeric Z-lines in mouse tissue and cultured myotubes, the sarcomeric structure is not affected in Apobec2-deficient muscle. In contrast, electron microscopy reveals enlarged mitochondria and mitochondria engulfed by autophagic vacuoles, suggesting that Apobec2 deficiency causes mitochondrial defects leading to increased mitophagy in skeletal muscle. Indeed, Apobec2 deficiency results in increased reactive oxygen species generation and depolarized mitochondria, leading to mitophagy as a defensive response. Furthermore, the exercise capacity of Apobec2-/- mice is impaired, implying Apobec2 deficiency results in ongoing muscle dysfunction. The presence of rimmed vacuoles in myofibers from 10-mo-old mice suggests that the chronic muscle damage impairs normal autophagy. We conclude that Apobec2 deficiency causes mitochondrial defects that increase muscle mitophagy, leading to myopathy and atrophy. Our findings demonstrate that Apobec2 is required for mitochondrial homeostasis to maintain normal skeletal muscle function.

DOI： 10.1096/fj.201700493R

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/stem.2639

Language：English   Publishing type：Research paper (scientific journal)  

Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cell membrane receptor (neuropilin2-plexinA3) > myogenin-myocyte enhancer factor 2D slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropilinl/plexinAl, A2 may enhance slow-fiber formation by activating signals that inhibit fast myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2-Sema3Af" activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease.

DOI： 10.1002/stem.2639

Language：English   Publishing type：Research paper (scientific journal)  

This paper provides in vitro phenotypical data to show that APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, may implicate in self-renewal functions of myogenic stem satellite cells, namely in the re-establishment of quiescent status after activation and proliferation of myoblasts in single-myofiber culture.

DOI： 10.1016/j.dib.2017.03.051

Language：English   Publishing type：Research paper (scientific journal)  

This paper provides in vitro phenotypical data to show that APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, may implicate in self-renewal functions of myogenic stem satellite cells, namely in the re-establishment of quiescent status after activation and proliferation of myoblasts in single-myofiber culture.

DOI： 10.1016/j.dib.2017.03.051

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016

Language：English   Publishing type：Research paper (scientific journal)  

Skeletal muscle is the main tissue of lipid metabolism and accordingly is critical for homeostasis and energy production; however, the determinants of lipid accumulation in skeletal muscle are unknown. Here, we examined whether the soleus muscle (predominantly slow-twitch fibers) has a higher lipid accumulation capacity than that of the extensor digitorum longus (EDL, predominantly fast-twitch fibers) muscle in mice. Soleus and EDL muscles were harvested from male C57BL/6J mice. The mRNA levels of genes involved in fatty acid import and triglyceride synthesis and accumulation were examined in soleus and EDL muscles. The intramyocellular lipid (IMCL) droplets of muscle cross sections and isolated single fibers were visualized by staining with BODIPY493/503, and fiber types were determined by immunofluorescent detection of myosin heavy chain (MyHC) isoforms. We detected higher mRNA expression of genes related to lipid accumulation in the soleus than the EDL. We also observed a marked increase of IMCL in single fibers from the soleus, but not the EDL, after treatment with a high-fat diet plus denervation. Interestingly, greater accumulation of IMCL droplets was observed in type 2A and 2X fibers (MyHC2A- and MyHC2X-positive fibers) than type 1 fibers (MyHC1-positive fibers) in soleus muscles. These results suggest that the soleus contains more IMCL owing to the higher population of type 2A fibers, and the difference in lipid accumulation between the soleus and EDL could depend on fiber type composition.

DOI： 10.1007/s10974-017-9468-6

Language：English   Publishing type：Research paper (scientific journal)  

Recently we found that the deficiency of APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, leads to a diminished muscle mass and increased myofiber with centrally-located nuclei known as dystrophic phenotypes. APOBEC2 expression is predominant in skeletal and cardiac muscles and elevated exclusively at the early-differentiation phase of wild-type (WT) myoblast cultures; however the physiological significance is still un-known. Here we show that APOBEC2 is a key negative regulator of myoblast differentiation in muscle regeneration. APOBEC2-knockout (A2KO) mice myoblast cultures displayed a normal morphology of primary myotubes along with earlier increase in fusion index and higher expression levels of myosin heavy chain (MyHC), myogenin and its cooperating factor MEF2C than WT myoblasts. Similar response was observable in APOBEC2-knockdown cultures of WT myoblasts that were transfected with the specific siRNA at the differentiation phase (not proliferation phase). Importantly, cardiotoxin-injured A2KO gastrocnemius muscle provided in vivo evidence by showing larger up-regulation of neonatal MyHC and myogenin and hence earlier regeneration of myofiber structures with diminished cross-sectional areas and minimal Feret diameters. Therefore, the findings highlight a promising role for APOBEC2 in normal progression of regenerative myogenesis at the early-differentiation phase upon muscle injury. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bioce1.2017.02.005

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s10974-017-9468-6

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biocel.2017.02.005

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0166080

Language：English   Publishing type：Research paper (scientific journal)  

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.

DOI： 10.1371/journal.pone.0166080

Language：English  

Experiments reveal a novel mechanism to regulate myofiber types and its activation by functional food ingredients.

Language：English   Publishing type：Research paper (scientific journal)  

We previously showed that Semaphorin 3A (Sema3A) expression was induced when quiescent muscle satellite cells were stimulated by hepatocyte growth factor and became activated satellite cells (ASCs). However, how Sema3A regulates genes in the early phase of ASCs remains unclear. In this study, we investigated whether Sema3A signaling can regulate the early phase of ASCs, an important satellite cell stage for postnatal growth, repair, and maintenance of skeletal muscle. We showed that expression of the myogenic proliferation regulatory factors Pax7 and Myf5 was decreased in myoblasts transfected with Sema3A siRNA. These cells failed to activate expression MyoD, another myogenic proliferation regulatory factor, during differentiation. Interestingly, some of the Sema3A-depleted cells did not express Pax7 and MyoD and had enlarged nuclei and very large cytoplasmic areas. We also observed that Pax7 and Myf5 expression was increased in Myc-Sema3A overexpressing myoblasts. BrdU analysis indicated that Sema3A regulated proliferation of ASCs. These findings suggest that Sema3A signaling can modulate expression of Pax7, Myf5, and MyoD. Moreover, we found that expression of emerin, an inner nuclear membrane protein, was regulated by Sema3A signaling. Emerin was identified by positional cloning as the gene responsible for the X-linked form of Emery-Dreifuss muscular dystrophy (X-EDMD). In conclusion, our results support a role for Sema3A in maintaining ASCs through regulation, via emerin, of Pax7, Myf5, and MyoD expression.

DOI： 10.1002/2211-5463.12050

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/2211-5463.12050

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.14814/phy2.12553

Language：English   Publishing type：Research paper (scientific journal)  

This data article contains data related to the research article entitled, "Protocol for rat single muscle-fiber isolation and culture" by Komiya et al. [1]. It has yet to be shown whether adult myosin heavy chain (MyHC) isoforms are expressed at a readily detectable level in cultured myotubes. In this study, we examined whether adult MyHC isoforms could be detected in myotubes differentiated from rat satellite cells using a Western blotting assay and specific antibodies against slow MyHC, fast MyHC and pan-MyHC. Results showed that slow adult MyHC isoforms were faintly detected in myotubes, suggesting that rat myotubes express adult MyHC isoforms although that amount is very low.

DOI： 10.1016/j.dib.2015.04.016

Language：English   Publishing type：Research paper (scientific journal)  

To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2015.03.034

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.dib.2015.04.016

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ab.2015.03.034

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biocel.2014.05.032

Language：English   Publishing type：Research paper (scientific journal)  

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) triggered its expression exclusively at the early-differentiation phase. In order to verify this concept, the present study was designed to clarify a paracrine source of HGF release. In vitro experiments demonstrated that activated anti-inflammatory macrophages (CD206-positive M2) produce HGF and thereby promote myoblast chemoattraction and Sema3A expression. Media from pro-inflammatory macrophage cultures (M1) did not show any significant effect. M2 also enhanced the expression of myoblast-differentiation markers in culture, and infiltrated predominantly at the early-differentiation phase (3-5 days post-injury); M2 were confirmed to produce HGF as monitored by in vivo/ex vivo immunocytochemistiy of CD11b/CD206/HGF-positive cells and by HGF in situ hybridization of cardiotoxin- or crush-injured tibialis anterior muscle, respectively. These studies advance our understanding of the stage-specific activation of Sema3A expression signaling. Findings, therefore, encourage the idea that M2 contribute to spatiotemporal up-regulation of extracellular Sema3A concentrations by producing HGF that, in turn, stimulates a burst of Sema3A secretion by myoblasts that are recruited to site of injury. This model may ensure a coordinated delay in re-attachment of motoneuron terminals onto damaged fibers early in muscle regeneration, and thus synchronize the recovery of muscle-fiber integrity and the early resolution of inflammation after injury. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biocel.2014.05.032

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0080152

Language：English   Publishing type：Research paper (scientific journal)  

Muscle damage induces massive macrophage infiltration of the injury site, in which activated pro-inflammatory and anti-inflammatory phenotypes (currently classified as M1 and M2, respectively) have been documented as distinct functional populations predominant at different times after the conventional acute injury by intramuscular injection of snake venoms (cardiotoxin, notexin) or chemicals (bupivacaine hydrochloride, barium chloride). The present study employed a muscle-crush injury model that may better reflect the physiologic damage and repair processes initiated by contusing a gastrocnemius muscle in the lower hind-limb of adult mice with hemostat forceps, and examined the time-course invasion of M1 and M2 macrophages during muscle regeneration by immunocytochemistry of CD197 and CD206 marker proteins. CD197-positive M1 macrophages were observed exclusively at 1-4 days after crush followed by the alternative prevalence of CD206-positive M2 at 7 days of myogenic differentiation, characterized by increasing levels of myogenin messenger RNA expression. Preliminary PCR analysis showed that M2 may produce hepatocyte growth factor (HGF) in culture, providing additional benefit to understanding that M2 populations actively promote regenerative myogenesis (muscle fiber repair) and moto-neuritogenesis (re-attachment of motoneuron terminals onto damaged fibers) through their time-specific infiltration and release of growth factor at the injury site early in muscle regeneration. © 2013 Japanese Society of Animal Science.

DOI： 10.1111/asj.12105

Language：English   Publishing type：Research paper (scientific journal)  

Dietary fat plays a major role in obesity, lipid metabolism, and cardiovascular diseases. To determine whether the intake of different types of dietary fats affect the muscle fiber types that govern the metabolic and contractile properties of the skeletal muscle, we fed male Wistar rats with a 15% fat diet derived from different fat sources. Diets composed of soybean oil (n-6 polyunsaturated fatty acids (PUFA)-rich), fish oil (n-3 PUFA-rich), or lard (low in PUFAs) were administered to the rats for 4 weeks. Myosin heavy chain (MyHC) isoforms were used as biomarkers to delineate the skeletal muscle fiber types. Compared with soybean oil intake, fish oil intake showed significantly lower levels of the fast-type MyHC2B and higher levels of the intermediate-type MyHC2X composition in the extensor digitorum longus (EDL) muscle, which is a fast-type dominant muscle. Concomitantly, MyHC2X mRNA levels in fish oil-fed rats were significantly higher than those observed in the soybean oil-fed rats. The MyHC isoform composition in the lard-fed rats was an intermediate between that of the fish oil and soybean oil-fed rats. Mitochondrial uncoupling protein 3, pyruvate dehydrogenase kinase 4, and porin mRNA showed significantly upregulated levels in the EDL of fish oil-fed rats compared to those observed in soybean oil-fed and lard-fed rats, implying an activation of oxidative metabolism. In contrast, no changes in the composition of MyHC isoforms was observed in the soleus muscle, which is a slow-type dominant muscle. Fatty acid composition in the serum and the muscle was significantly influenced by the type of dietary fat consumed. In conclusion, dietary fat affects the expression of genes related to the contractile and metabolic properties in the fast-type dominant skeletal muscle, where the activation of oxidative metabolism is more pronounced after fish oil intake than that after soybean oil intake. © 2013 Mizunoya et al.

DOI： 10.1371/journal.pone.0080152

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/asj.12014

Language：English   Publishing type：Research paper (scientific journal)  

Resident myogenic stem cells, satellite cells, up-regulate a secreted multi-functional modulator, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle-crush injury and treatment with hepatocyte growth factor (HGF) or basic fibroblast growth factor (FGF2). Here, we add evidence that the Sema3A expression and secretion induced by the growth factors is significantly higher in primary cultures from adult rat soleus than from the fast-twitch extensor digitorum longus (EDL) muscle. The higher Sema3A response, revealed by quantitative PCR and Western blotting of cell lysates and conditioned media, may account for the higher myogenin expression of soleus muscle satellite cells early in differentiation since addition of recombinant Sema3A stimulates myogenin expression in cultures. These experiments also showed that mRNA expression of plexin A2, which together with neuropilins, constitutes Sema3A composite-receptors, was higher in satellite cells from soleus than EDL with no difference in plexin A1 and A3 and neuropilin-1 and 2 levels. These comparative studies, therefore, highlight a possible Sema3A-plexin A2-myogenin signaling axis that may ensure promoting early differentiation by soleus muscle satellite cells. Crown Copyright (c) 2012 Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biocel.2012.10.003

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biocel.2012.10.003

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1740-0929.2012.01057.x

Language：English   Publishing type：Research paper (scientific journal)  

Time-coordinated prevalence of extracellular HGF, FGF2 and TGF-ss 3 in crush-injured skeletal muscle
Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3A upon in vivo muscle-crush injury. The study also revealed that Sema3A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)-beta 2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-beta 3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescenceWestern blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 28?days post-crush, whereas active TGF-beta 3 increases after 12?days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-beta 3 that drive regulation of Sema3A expression during regenerative intramuscular moto-neuritogenesis.

DOI： 10.1111/j.1740-0929.2012.01057.x

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Hara M, Tabata K, Suzuki T, Do MQ, Mizunoya W, Nakamura M, Nishimura S, Tabata S, Ikeuchi Y, Sunagawa K, Anderson JE, Allen RE, Tatsumi R. Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch. Am J Physiol Cell Physiol 302: C1741-C1750, 2012. First published March 28, 2012; doi:10.1152/ajpcell.00068.2012.-When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 mu M (GsMTx-4), 10 mu M (nifedipine), or 100 mu M (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 mu M) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade.

DOI： 10.1152/ajpcell.00068.2012

Language：English   Publishing type：Research paper (scientific journal)  

Background: The zebrafish is well established as a model organism for the study of vertebrate embryogenesis, but transgenic lines enabling restricted gene expression are still lacking for many tissues. Results: We first generated the hoxb1a(beta-globin):eGFPum8 line that expresses eGFP in hindbrain rhombomere 4 (r4), as well as in facial motor neurons migrating caudally from r4. Second, we generated the hoxb1a(beta-globin) Gal4VP16um60 line to express the exogenous Gal4VP16 transcription factor in r4. Lastly, we prepared the UAS(beta-actin):hoxa3aum61 line where the hoxa3a gene, which is normally expressed in r5 and r6, is under control of Gal4-regulated UAS elements. Crossing the hoxb1a(beta-globin):Gal4VP16um60 line to the UAS(beta-actin):hoxa3aum61 line drives robust hoxa3a expression in r4. We find that transgenic expression of hoxa3a in r4 does not affect hoxb1a expression, but has variable effects on migration of facial motorneurons and formation of Mauthner neurons. While cases of somatic transgene silencing have been reported in zebrafish, we have not observed such silencing to date, possibly because of our efforts to minimize repetitive sequences in the transgenic constructs. Conclusion: We have generated three transgenic lines that will be useful for future studies by permitting the labeling of r4-derived cells, as well as by enabling r4-specific expression of various transgenes. Developmental Dynamics 241:11251132, 2012. (c) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.23794

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Do MKQ, Sato Y, Shimizu N, Suzuki T, Shono J, Mizunoya W, Nakamura M, Ikeuchi Y, Anderson JE, Tatsumi R. Growth factor regulation of neural chemorepellent Sema3A expression in satellite cell cultures. Am J Physiol Cell Physiol 301: C1270-C1279, 2011. First published August 24, 2011; doi: 10.1152/ajpcell.00257.2011.-Successful regeneration and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory signals of such coordination remain unclear, although axon-guidance molecules may be involved. Recently, satellite cells, resident myogenic stem cells positioned beneath the basal lamina and at high density at the myoneural junction regions of mature fibers, were shown to upregulate a secreted neural chemorepellent semaphorin 3A (Sema3A) in response to in vivo muscle-crush injury. The initial report on that expression centered on the observation that hepatocyte growth factor (HGF), an essential cue in muscle fiber growth and regeneration, remarkably upregulates Sema3A expression in early differentiated satellite cells in vitro [Tatsumi et al., Am J Physiol Cell Physiol 297: C238-C252, 2009]. Here, we address regulatory effects of basic fibroblast growth factor (FGF2) and transforming growth factor (TGF)-beta s on Sema3A expression in satellite cell cultures. When treated with FGF2, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Sema3A upregulation by FGF2 was dose dependent with a maximum (8- to 1-fold relative to the control) at 2.5 ng/ml (150 pM) and occurred exclusively at the early differentiation stage. The response was highly comparable in dose response and timing to effects of HGF treatment, without any additive or synergistic effect from treatment with a combination of both potent upregulators. In contrast, TGF-beta 2 and -beta 3 potently decreased basal Sema3A expression; the maximum effect was at very low concentrations (40 and 8 pM, respectively) and completely cancelled the activities of FGF2 and HGF to upregulate Sema3A. These results therefore encourage the prospect that a time-coordinated increase in HGF, FGF2, and TGF-beta ligands and their receptors promotes a programmed strategy for Sema3A expression that guarantees successful intramuscular motor reinnervation by delaying sprouting and reattachment of motoneuron terminals onto damaged muscle fibers early in regeneration pending restoration of muscle fiber contractile integrity.

DOI： 10.1152/ajpcell.00257.2011

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation.

DOI： 10.1089/hyb.2009.0117

Language：English   Publishing type：Research paper (scientific journal)  

CHD1 is a subfamily member of the CHD family, which possesses a chromodomain, a helicase domain, and a DNA-binding domain. The CHD family regulates gene expression by contributing to ATP-dependent chromatin remodeling. CHD1 exists in the transcriptionally active region and alters the chromatin structure. Little is known about the function of endogenous CHD1, however, and studies have been hindered by the lack of an antibody specific for CHD1 in mammals. In the present study, we established a monoclonal antibody specifically against CHD1 using the rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody showed specific binding to CHD1, allowing us to identify the deduced full-length CHD1. In addition, cell immunostaining clearly revealed the nuclear localization of CHD1. This monoclonal antibody will be useful for further analysis of CHD1 function in mammals.

DOI： 10.1089/hyb.2009.0106

Language：English   Publishing type：Research paper (scientific journal)  

Dhx9/NDHII/RHA is a member of the DEAH family of proteins, which possess a double-stranded RNA-binding domain (dsRBD) and a helicase domain. The DEAH protein family plays a critical role in RNA metabolism. DEAH family members function as ATP-dependent RNA helicases and regulation of transcription. In the present study, we report the establishment of a monoclonal antibody specific for Dhx9 using the rat medial iliac lymph node method. Immunoblot analysis using our antibody against Dhx9 detected full-length Dhx9. In addition, immunocytochemical staining using our antibody against Dhx9 revealed the nuclear localization of Dhx9. This monoclonal antibody against Dhx9 will allow for further detailed studies of Dhx9 expression.

DOI： 10.1089/hyb.2009.0107

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 29

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

CHD2 is a member of the CHD family that contains chromodomain, helicase domain as well as DNA-binding domain. The CHD family is involved in gene expression and transcription by ATP-dependent chromatin remodeling. Analysis of mutant mouse revealed that CHD2 is involved in development as well as hematopoiesis, which suggests the involvement of CHD2 in gene expression. However, CHD2 has not yet been analyzed biochemically as there is no specific antibody against it. Here, we report on the establishment of specific monoclonal antibody (MAb) against CHD2 utilizing a rat medial iliac lymph node method. Through cell immunostaining utilizing established MAb to CHD2, we confirmed that CHD2 was localized in euchromatin. Additionally, IP-Western revealed that the expression level of full-length CHD2 did not change during the differentiation stage. Additionally, a specific signal was confirmed around 95 kDa at the undifferentiated stage. This clearly indicated that CHD2 was involved in specific gene expression at this stage. Thus, this antibody can contribute to elucidating the function of CHD2 in cell expression.

DOI： 10.1089/hyb.2009.0090

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function.

DOI： 10.1089/hyb.2009.0066

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Brm-related gene-1 (Brg1) is a catalytic subunit of the SWI/SNF chromatin remodeling enzyme complex that has ATPase activity. This complex facilitates chromatin remodeling for gene expression by utilizing energy for ATP hydrolysis. It is well known that the SWI/SNF chromatin remodeling enzyme complex is essential for cell differentiation, cell cycle regulation, and embryogenesis. Here we report the establishment of a hybridoma cell line for producing an antibody against Brg1 subunit by the rat medial iliac lymph node method. Immunoblot analysis showed that our antibody can specifically recognize Brg1. It was revealed by immunocytochemistry that Brg1 is located in euchromatin of C2C12 myoblast nuclei. These data suggested this antibody is useful for analyzing molecular function of Brg1 protein in cells.

DOI： 10.1089/hyb.2009.0041

Language：English   Publishing type：Research paper (scientific journal)  

Brm is a subunit of the SWI/SNF complex that has a ATPase activity. It is well known that the complex plays a major role in cell processes, such as proliferation, differentiation, and DNA repair of cells. Here we report the production of monoclonal antibody (1H7A10) against Brm by rat medial iliac lymph node method. Immunoblot analysis with the antibody revealed the specific recognition of Brm and increase of Brm protein level in skeletal muscle differentiation. Immunocytochemistry analysis shows nuclear localization in myoblast C2C12 and involvement of transcription in the late stages of differentiation.

DOI： 10.1089/hyb.2009.0044

Language：English   Publishing type：Research paper (scientific journal)  

Pax7 is a nuclear localization protein, well known as a member of the paired box family. It is expressed at a very early stage of muscle differentiation and is also found in muscle satellite cells that are recognized as muscle stem cells. Pax7 is also recognized as a tumor cell marker since it is greatly expressed in various types of tumor cells. Pax7 has homology among other paired family members and is not easy to distinguish one from the others. In this study, we report on the establishment of monoclonal antibodies (MAb) against Pax7 using a rat medial iliac lymph node method. The quality of the antibody was examined by immunoblotting analysis. It was confirmed that the antibody can specifically recognize the Pax7 protein. It was also revealed that the MAb antibody successfully recognizes the nuclear localized Pax7 protein in Ewing's sarcoma cells by immunocytochemistry. The antibody can clearly show the regions of euchromatin and heterochromatin where hoechst is positive.

DOI： 10.1089/hyb.2009.0039

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Hox proteins form complexes with Pbx and Meis cofactors to control gene expression, but the role of Meis is unclear. We demonstrate that Hoxb1-regulated promoters are highly acetylated on histone H4 (AcH4) and occupied by Hoxb1, Pbx, and Meis in zebrafish tissues where these promoters are active. Inhibition of Meis blocks gene expression and reduces AcH4 levels at these promoters, suggesting a role for Meis in maintaining AcH4. Within Hox transcription complexes, Meis binds directly to Pbx and we find that this binding displaces histone deacetylases (HDACs) from Hoxb1-regulated promoters in zebrafish embryos. Accordingly, Pbx mutants that cannot bind Meis act as repressors by recruiting HDACs and reducing AcH4 levels, while Pbx mutants that bind neither HDAC nor Meis are constitutively active and recruit CBP to increase AcH4 levels. We conclude that Meis acts, at least in part, by controlling access of HDAC and CBP to Hox-regulated promoters.

DOI： 10.1016/j.devcel.2009.08.007

Language：English   Publishing type：Research paper (scientific journal)  

Background: Members of the NET subfamily of zinc-finger proteins are related to the Sp-family of transcription factors and are required during embryogenesis. In particular, Nlz1/Znf703 and Nlz2/Znf503 are required for formation of rhombomere 4 of the vertebrate hindbrain. While NET family proteins have been hypothesized to regulate transcription, it remains unclear if they function as activators or repressors of transcription.
Results: Here we demonstrate that Nlz proteins repress transcription both in cell lines and in developing zebrafish embryos. We first use standard cell culture-based reporter assays to demonstrate that Nlz1/Znf703 represses transcription of a luciferase reporter in four different cell lines. Structure-function analyses and pharmacological inhibition further reveal that Nlz1-mediated repression requires histone deacetylase activity. We next generate a stable transgenic zebrafish reporter line to demonstrate that Nlz1 promotes histone deacetylation at the transgenic promoter and repression of transgene expression during embryogenesis. Lastly, taking a genetic approach we find that endogenous Nlz proteins are required for formation of hindbrain rhombomere 4 during zebrafish embryogenesis by repressing expression of non-rhombomere 4 genes.
Conclusion: We conclude that Nlz1/Znf703 acts as a repressor of transcription and hypothesize that other NET family members function in a similar manner.

DOI： 10.1186/1471-213X-8-108

Language：English   Publishing type：Research paper (scientific journal)  

C2H2 zinc finger proteins make up one of the largest protein families in eukaryotic organisms. Recent study in several different systems has identified a set of novel zinc finger proteins that appear to form a distinct subfamily that we have named the NET family. Members of the NET family (Noc, Nlz, Elbow, and Tlp-1) share two protein motifs-a buttonhead box and an Sp motif-with zinc finger proteins from the Sp family. However, the NET family is uniquely characterized by a single atypical C2H2 zinc finger, in contrast to the Sp family that contains three tandem C2H2 fingers. Here, we review current information about the biochemical function and in vivo role for members of this subfamily. In general, NET family proteins are required during embryonic development. They appear to act by regulating transcription, most likely as repressors, although they are unlikely to bind DNA directly. In the future, it will be important to directly test if NET family proteins control transcription of specific target genes, perhaps via interactions with DNA-binding transcription factors, as well as to further explore their function in vivo. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcb.20255

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Serum response factor and the (CC(A/T)(6)GG) (CArG) box interact to promote the transcription of c-fos and muscle genes; this tissue-specific activity may require co-regulators for serum response factor. The a, integrin promoter contains two cis-elements besides the CArG box: a TAAT sequence, a consensus binding site for homeoproteins, and a GATA-binding box. As a candidate TAAT-binding factor, we cloned an NK family homeobox gene, Nkx-3.2, which is expressed mainly in smooth muscle tissues and skeletal structures. Nkx-3.2, serum response factor, and GATA-6 were co-expressed only in the medial smooth muscle layer of arteries. These three transcription factors formed a complex with their corresponding cis-elements and cooperatively transactivated smooth muscle genes, including a, integrin, SM22alpha, and caldesmon. Cardiac muscle-specific members of the NK and GATA families exist, and the triad of Nkx-2.5, serum response factor, and GATA-4 also transactivated the cardiac atrial natriuretic factor gene, which contains a CArG-like box, a GATA-binding box, and an NK-binding element. Our findings demonstrate that smooth and cardiac muscle have a shared transcriptional machinery and that the GATA and NK families confer muscle specificity on the serum response factor/CArG interaction.

DOI： 10.1074/jbc.M111824200

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Tropomyosin (TM) is a regulatory protein of actomyosin system. Muscle type-specific expression of TM isoforms is generated from different genes and by alternative splicing. beta -TM isoforms in chicken skeletal and smooth muscles are encoded by a single gene and transcribed from the same promoter. We previously reported a smooth muscle cell (SMC) phenotype-dependent change in beta -TM expression (Kashiwada, K., Nishida, W,, Hayashi, K,, Ozawa, K., Yamanaka, Y,, Saga, H,, Yamashita, T,, Tohyama, M,, Shimada, S,, Sate, K,, and Sobue, K, (1997) J. Biol. Chem. 272, 15396-15404), and identified beta -TM as an SMC-differentiation marker. Here, we characterized the transcriptional machinery of the beta -TM gene in SMCs. Promoter and gel mobility shift analyses revealed an obligatory role for serum response factor and its interaction with the CArG box sequence in the SMC-specific transcription of the beta -TM gene in differentiated SMCs, We further isolated a novel homologue of the Barx homeoprotein family, Barx1b, from chicken gizzard, Barx1b was exclusively localized to SMCs of the upper digestive organs and their attached arteries and to craniofacial structures. Serum response factor and Barx1b bound each other directly, coordinately transactivated the beta -TM gene in differentiated SMCs and heterologous cells, and formed a ternary complex with a CArG probe. Taken together, these results suggest that SRF and Barx1b are coordinately involved in the SMC-specific transcription of the beta -TM gene in the upper digestive organs and their attached arteries.

DOI： 10.1074/jbc.m101127200

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Using monoclonal antibody against the 45 kDa postsynaptic density protein, we isolated a novel isoform of Homer/vesl, The NH2-terninal region containing a PDZ domain of this protein is identical to that of Homer/vesl, and the COOH-terminal region containing unique leucine zippers shows self-multimerization. We named this protein PSD-Zip45, In addition to specific binding of PSD-Zip45 mediated by a PDZ domain to the metabotropic glutamate receptors 1 alpha or 5, the distribution of PSD-Zip45 transcripts is highly consistent with that of metabotropic glutamate receptor transcripts. The PSD-Zip45 is, therefore, the first candidate as receptor anchoring proteins containing leucine zipper motifs in the central nervous system. (C) 1998 Federation of European Biochemical Societies.

DOI： 10.1016/s0014-5793(98)01256-3

Event date： 2019.11 - 2019.12

Language：English   Presentation type：Oral presentation (general)  

Venue：Yonago International Cenference Center   Country：Japan  

Event date： 2019.11 - 2019.12

Language：English   Presentation type：Oral presentation (general)  

Venue：Yonago International Cenference Center   Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岩手大学   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：麻布大学   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：麻布大学   Country：Japan  

Event date： 2018.6

Language：English  

Country：France  

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in non-myogenic lineage cells. We performed experiments using stably expressed Pax7 in 3T3-L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. These cells showed decreased expression of PDGFR, PPAR and Fabp4 and inhibited forming lipid droplets. However, they could not differentiate into myogenic cells. We think the Pax7 functions to pause cell differentiation and needs co-factors to induce into a specific cell type. To see the function of Pax7 in a cell type where possible myogenic co-factors may be available, we decided to use smooth muscle cells (SMCs). The SMCs culture model with E15 chick gizzard has been developed. The cells are cultured in the serum free medium supplemented with BSA and IGF-1. The cells are currently analyzed for the marker gene expression to find out if they keep the differentiation state.

Event date： 2010.7

Venue：Carefree, AZ   Country：United States  

Venue：Madison, WI   Country：United States  

Event date： 2024.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2024.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2024.6

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.12

Language：Others   Presentation type：Oral presentation (general)  

Venue：九州大学   Country：Other  

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.9

Language：English   Presentation type：Oral presentation (general)  

Country：United Kingdom  

Event date： 2023.8

Language：English  

Venue：千里ライフサイエンスセンター   Country：Japan  

Event date： 2023.8

Language：Japanese  

Venue：千里ライフサイエンスセンター   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2019.11 - 2019.12

Language：English   Presentation type：Oral presentation (general)  

Venue：Yonago International Cenference Center   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：麻布大学   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎ウエスレヤン大学 ウエスレー館   Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2018.3 - 2013.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8 - 2017.11

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：English  

Country：United States  

Event date： 2017.8

Language：English  

Country：United States  

Event date： 2017.8

Language：English  

Country：United States  

Event date： 2017.8

Language：English  

Country：Korea, Republic of  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：English  

Country：Japan  

Event date： 2017.8

Language：English  

Country：Japan  

Event date： 2017.8

Language：English  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese  

Country：Japan  

Event date： 2017.8

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8

Language：English  

Country：Japan  

Event date： 2017.8

Language：English  

Country：Japan  

Event date： 2017.8

Language：English   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8

Language：English   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2017.8

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2016.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2013.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：広島   Country：Japan  

Event date： 2012.12

Language：English  

Venue：福岡   Country：Japan  

Event date： 2012.8

Venue：Lucca Italy   Country：Italy  

Event date： 2012.8

Venue：Lucca Italy   Country：Italy  

Event date： 2012.8

Venue：Lucca Italy   Country：Italy  

Event date： 2012.3 - 2017.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：名古屋大学   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：名古屋大学   Country：Japan  

Language：Others  

Country：Other  

Language：Japanese  

Country：Other  

Development of Differentiated Smooth Muscle Cell Culture with chick embryo gizzard

Language：Others  

Country：Other  

Language：Others  

Country：Other  

Language：Others  

Country：Other  

Language：Others  

Country：Other  

Language：Others  

Country：Other  

Language：Others  

Country：Other  

Language：Others  

Language：Others  

Language：Others  

Language：Others  

Language：Japanese  

Sema3A依存的な遅筋型筋線維形成機構の食品機能学的制御:リンゴポリフェノールによる促進と活性成分の同定

Language：Japanese  

筋幹細胞分泌因子Sema3Aによる遅筋型筋線維形成誘導:ヒラメ筋の損傷・再生実験による実証

Language：Japanese  

筋成長・再生における筋幹細胞分泌因子Semaphorin 3Aの機能

Language：Japanese  

マウス骨格筋の再生に伴う筋線維タイプ組成の経時的解析

Language：Japanese  

長期筋萎縮で発現変動する遺伝子の網羅的探索

Language：Japanese  

4種類の新規ミオシン重鎖抗体を用いた筋線維タイプ同定法

Language：English  

マウス遅筋及び速筋を用いた代謝物プロファイリング

Language：Japanese  

筋肥大・再生における異種細胞間コミュニケーションダイナミクス―筋幹細胞による運動神経支配の再構築制御(仮説)を話題にして―

Language：Japanese  

Language：Japanese  

PPARδアゴニスト投与によってマウス骨格筋で発現変動する遺伝子の網羅的解析

Language：Japanese  

遅筋は速筋に比べ脂肪酸取込および脂肪合成能に優れる

Language：English  

Functional interaction between Sema3A and Pax7 in proliferation and differentiation of myoblasts
Poster presentation

Language：English  

APOBEC2 deficiency up-regulates myoblast differentiation
Poster Presentation

Language：English  

Sema3A secreted from satellite cells promotes slow-twitch fiber generation
Poster Presentation

Language：English  

M2 macrophages may implicate in regenerative moto-neuritogenesis, by promoting myoblast migration and Sema3A expression
Poster Presentation

Language：Japanese  

Language：English  

Semaphorin 3A secreted from myogenic stem cells promotes slow-twitch muscle fiber generation
Poster Presentation

Language：Japanese  

頭脳循環を加速する若手研究者戦略的海外派遣プログラムー(3)ドイツライプニッツ家畜生物学研究所およびミュンヘン工科大学を訪問して

Language：English  

Eicosapentaenoic acid affects skeletal muscle fiber types relating factors via PPARδ pathway in isolated fibers of rats
Poster presentation

Language：Japanese  

マウス骨格筋線維を用いた筋内脂肪量評価系の構築

Language：English  

APOBEC2 deficiency causes increased autophagy and abnormal mitochondria in skeletal muscle
Poster presentation

Language：English  

Change of skeletal muscle fiber types by n-3 poly unsaturated fatty acid
Poster presentation

Language：English  

Heparan/chondroitin sulfate chains may mediate HGF/FGF2-induced up-regulation of neural chemorepellent Sema3A in satellite cell cultures
Poster presentation

Language：English  

Semaphorin 3A regulates the early differentiation of satellite cells
Poster presentation

Language：English  

Mechano-Sensing Calcium-Influx Machinery that Instigates Skeletal Muscle Satellite Cell Activation
Selected for Oral presentation

Language：Japanese  

頭脳循環を加速する若手研究者戦略的海外派遣プログラムー(1)ドイツライプニッツ家畜生物学研究所を訪問して

Language：Japanese  

A homeodomain protein, Barx and serum response factor (SRF) regulate transcription of β-tropomyosin gene in the smooth muscle cells.

Language：Japanese  

Transcriptional regulation of α1 integrin gene in the smooth muscle cells

Language：Japanese  

Cloning and claracterization of Zip 45, a novel PSD protein containing leucine zipper motifs

Type：Competition, symposium, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：1

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

Type：Competition, symposium, etc. 

ファシリテーター

P＆P萌芽的若手研究採択課題「代謝刷り込みウシモデルの分子生物学的アプローチによる食肉品質研究　—国際若手女性研究者チームの研究教育波及プロジェクト—」の一環として、九州大学女性研究者キャリア開発センター、生物産業創成学コースと共催で女子学生を対象としたワークショップ「女性研究者と一緒につくるオーダーメイドのキャリアプラン」を開催した。

企画、司会、進行

Audience：Infants,　Schoolchildren,　Junior students,　High school students

Audience：General,　Scientific,　Company,　Civic organization,　Governmental agency

Type：Seminar, workshop