Eicosapentaenoic acid increases proportion of type 1 muscle fibers through PPARδ and AMPK pathways in rats.

Muscle fiber type composition (% slow-twitch and % fast-twitch fibers) is associated with metabolism, with increased slow-twitch fibers alleviating metabolic disorders. Previously, we reported that dietary fish oil intake induced a muscle fiber-type transition in a slower direction in rats. The aim of this study was to determine the functionality of eicosapentaenoic acid (EPA), a unique fatty acid in fish oil, to skeletal muscle fiber type and metabolism in rats. Here, we showed that dietary EPA promotes whole-body oxidative metabolism and improves muscle function by increasing proportion of slow-twitch type 1 fibers in rats. Transcriptomic and metabolomic analyses revealed that EPA supplementation activated the peroxisome proliferator-activated receptor δ (PPARδ) and AMP-activated protein kinase (AMPK) pathways in L6 myotube cultures, which potentially increasing slow-twitch fiber share. This highlights the role of EPA as an exercise-mimetic dietary component that improves metabolism and muscle function, with potential benefits for health and athletic performance.

Up- and Downregulated Genes after Long-Term Muscle Atrophy Induced by Denervation in Mice Detected Using RNA-Seq.

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.

Beef extract supplementation promotes myoblast proliferation and myotube growth in C2C12 cells.

PURPOSE: We previously determined that the intake of beef extract for 4 weeks increases skeletal muscle mass in rats. Thus, this study aimed to clarify whether beef extract has a hypertrophic effect on muscle cells and to determine the signaling pathway underlying beef extract-induced myotube hypertrophy. METHODS: We assessed the effects of beef extract supplement on mouse C2C12 skeletal muscle cell proliferation and differentiation and myotube growth. In addition, the phosphorylation of Akt, ERK1/2, and mTOR following beef extract supplementation was examined by western blotting. Furthermore, the bioactive constituents of beef extract were examined using amino acid analysis and dialysis. RESULTS: In the proliferative stage, beef extract significantly increased myoblast proliferation. In the differentiation stage, beef extract supplementation did not promote myoblast differentiation. In mature myotubes, beef extract supplementation increased myotube diameter and promoted protein synthesis. Although Akt and ERK1/2 levels were not affected, beef extract supplementation increased mTOR phosphorylation, which indicated that the mTOR pathway mediates beef extract-induced myotube hypertrophy. The hypertrophic activity was observed in fractions of > 7000 Da. CONCLUSIONS: Beef extract promoted C2C12 myoblast proliferation and C2C12 myotube hypertrophy. Myotube hypertrophy was potentially induced by mTOR activation and active components in beef extract were estimated to be > 7000 Da.

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.

Fluorescence microscopy data on expression of Paired Box Transcription Factor 7 in skeletal muscle of APOBEC2 knockout mice.

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.

Data supporting possible implication of APOBEC2 in self-renewal functions of myogenic stem satellite cells: Toward understanding the negative regulation of myoblast differentiation.

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.

Slow-Myofiber Commitment by Semaphorin 3A Secreted from Myogenic Stem Cells.

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-neuropilin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fast-myosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreERT2 -Sema3Afl °x 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. Stem Cells 2017;35:1815-1834.

Mouse soleus (slow) muscle shows greater intramyocellular lipid droplet accumulation than EDL (fast) muscle: fiber type-specific analysis.

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.

APOBEC2 negatively regulates myoblast differentiation in muscle regeneration.

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.

Fast-to-slow shift of muscle fiber-type composition by dietary apple polyphenols in rats: Impact of the low-dose supplementation.

Our previous studies demonstrated that an 8-week intake of 5% (w/w) apple polyphenol (APP) in the diet improves muscle endurance of young-adult rats. In order to identify a lower limit of the dietary contribution of APP to the effect, the experiments were designed for lower-dose supplementation (8-week feeding of 0.5% APP in AIN-93G diet) to 12-week-old male Sprague-Dawley rats. Results clearly showed that the 0.5% APP diet significantly up-regulates slower myosin-heavy-chain (MyHC) isoform ratios (IIx and IIa relative to total MyHC) and myoglobin expression in lower hind-limb muscles examined (P < 0.05). There was a trend to increased fatigue resistance detected from measurements of relative isometric plantar-flexion force torque generated by a stimulus train delivered to the tibial nerve (F(98, 1372) = 1.246, P = 0.0574). Importantly, there was no significant difference in the animal body-phenotypes or locomotor activity shown as total moving distance in light and dark periods. Therefore, the present study encourages the notion that even low APP-intake may increase the proportions of fatigue-resistant myofibers, and has promise as a strategy for modifying performance in human sports and improving function in age-related muscle atrophy.

A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen.

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.

Semaphorin 3A promotes activation of Pax7, Myf5, and MyoD through inhibition of emerin expression in activated satellite cells.

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.

Transmembrane proteoglycans syndecan-2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early-differentiated myoblasts.

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed an 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) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan-2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan-2, 4 was abundant (two magnitudes higher than syndecan-1, 3) in early-differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2-induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non-knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan-2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2-syndecan-2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery.

Data in support of protocol for rat single muscle-fiber isolation and culture.

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.

Improvement of Endurance Based on Muscle Fiber-Type Composition by Treatment with Dietary Apple Polyphenols in Rats.

A recent study demonstrated a positive effect of apple polyphenol (APP) intake on muscle endurance of young-adult animals. While an enhancement of lipid metabolism may be responsible, in part, for the improvement, the contributing mechanisms still need clarification. Here we show that an 8-week intake of 5% (w/w) APP in the diet, up-regulates two features related to fiber type: the ratio of myosin heavy chain (MyHC) type IIx/IIb and myoglobin protein expression in plantaris muscle of 9-week-old male Fischer F344 rats compared to pair-fed controls (P < 0.05). Results were demonstrated by our SDS-PAGE system specialized for MyHC isoform separation and western blotting of whole muscles. Animal-growth profiles (food intake, body-weight gain, and internal-organ weights) did not differ between the control and 5% APP-fed animals (n = 9/group). Findings may account for the increase in fatigue resistance of lower hind limb muscles, as evidenced by a slower decline in the maximum isometric planter-flexion torque generated by a 100-s train of electrical stimulation of the tibial nerve. Additionally, the fatigue resistance was lower after 8 weeks of a 0.5% APP diet than after 5% APP, supporting an APP-dose dependency of the shift in fiber-type composition. Therefore, the present study highlights a promising contribution of dietary APP intake to increasing endurance based on fiber-type composition in rat muscle. Results may help in developing a novel strategy for application in animal sciences, and human sports and age-related health sciences.

Protocol for rat single muscle fiber isolation and culture.

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.

The growth-promoting activity of egg white proteins in the C2C12 myoblast cell line.

In this study, we examined the effects of several egg white proteins (ovalbumin, ovomucoid, ovotransferrin and lysozyme) on proliferation and myotube growth in C2C12 murine myoblast cells. Cell proliferation was measured using a water-soluble tetrazolium salt (WST-8)-based assay and then validated using Giemsa staining. Significant proliferative activities of C2C12 cells were observed in response to the addition of 10(-5) -10(-4) mol/L ovalbumin or ovomucoid. Ovotransferrin decreased C2C12 cell proliferation and lysozyme showed no significant effects on the proliferation of C2C12 cells. In contrast, the proliferative effects of ovalbumin and ovomucoid were not observed in 3T3-L1 murine preadipocyte cells. We also measured the effects of ovalbumin and ovomucoid on C2C12 myotube diameters by using histological analysis. In comparison to control cells, myotube diameters were significantly increased in cells cultured in 10(-6) -10(-4) mol/L ovalbumin or ovomucoid, suggesting that ovalbumin and ovomucoid stimulate the growth of myotubes. Thus, our results clearly demonstrated that ovalbumin or ovomucoid stimulated the proliferation of myoblasts and growth of myotubes.

Supplementary immunocytochemistry of hepatocyte growth factor production in activated macrophages early in muscle regeneration.

Regenerative intramuscular motor-innervation is thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies showed that resident myogenic stem cells, satellite cells, up-regulate a secreted neural-chemorepellent semaphorin 3A (Sema3A) during the early-differentiation period, in response to hepatocyte growth factor (HGF) elevated in injured muscle. However, a paracrine source of the HGF release is still unknown. Very recently, we proposed a possible contribution of anti-inflammatory macrophages (CD206-positive M2) by showing that M2 cells infiltrate predominantly at the early-differentiation phase (3-5 days post-injury) and produce/secrete large amounts of HGF. However, in understanding this concept there still remains a critical need to examine if phagocytotic pro-inflammatory macrophages (CD86-positive M1), another activated-phenotype still present at the early-differentiation phase concerned, produce HGF upon muscle injury. The current immunocytochemical study demonstrated that the HGF expression is negative for M1 prepared from cardiotoxin-injured Tibialis anterior muscle at day 5, in contrast to the intense fluorescent-signal of M2 served as a positive control. This supplementary result advances our understanding of a spatiotemporal burst of HGF secretion from M2 populations (not M1) to impact Sema3A expression, which ensures a coordinated delay in attachment of motoneuron terminals onto damaged and generating fibers during the early phase of muscle regeneration.

Implication of anti-inflammatory macrophages in regenerative moto-neuritogenesis: promotion of myoblast migration and neural chemorepellent semaphorin 3A expression in injured muscle.

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 immunocytochemistry 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.

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.