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.

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.

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.

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.

Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch.

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 µM (GsMTx-4), 10 µM (nifedipine), or 100 µM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 µM) 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.

Growth factor regulation of neural chemorepellent Sema3A expression in satellite cell cultures.

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)-ßs 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-ß2 and -ß3 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-ß 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.

Deficiency in APOBEC2 leads to a shift in muscle fiber type, diminished body mass, and myopathy.

The apoB RNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of proteins includes APOBEC1, APOBEC3, and activation-induced deaminase, all of which are zinc-dependent cytidine deaminases active on polynucleotides and involved in RNA editing or DNA mutation. In contrast, the biochemical and physiological functions of APOBEC2, a muscle-specific member of the family, are unknown, although it has been speculated, like APOBEC1, to be an RNA-editing enzyme. Here, we show that, although expressed widely in striated muscle (with levels peaking late during myoblast differentiation), APOBEC2 is preferentially associated with slow-twitch muscle, with its abundance being considerably greater in soleus compared with gastrocnemius muscle and, within soleus muscle, in slow as opposed to fast muscle fibers. Its abundance also decreases following muscle denervation. We further show that APOBEC2-deficient mice harbor a markedly increased ratio of slow to fast fibers in soleus muscle and exhibit an approximately 15-20% reduction in body mass from birth onwards, with elderly mutant animals revealing clear histological evidence of a mild myopathy. Thus, APOBEC2 is essential for normal muscle development and maintenance of fiber-type ratios; although its molecular function remains to be identified, biochemical analyses do not especially argue for any role in RNA editing.

High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo.

Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases.

Possible implication of satellite cells in regenerative motoneuritogenesis: HGF upregulates neural chemorepellent Sema3A during myogenic differentiation.

Regenerative coordination and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory mechanisms of such coordination remain unclear, although both attractive and repulsive axon guidance molecules may be involved in the signaling pathway. Here we show that expression of a neural secreted chemorepellent semaphorin 3A (Sema3A) is remarkably upregulated in satellite cells of resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers, when treated with hepatocyte growth factor (HGF), established as an essential cue in muscle fiber growth and regeneration. When satellite cells were treated with HGF in primary cultures of cells or muscle fibers, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Other growth factors had no inductive effect except for a slight effect of epidermal growth factor treatment. Sema3A upregulation was HGF dose dependent with a maximum (about 7- to 8-fold units relative to the control) at 10-25 ng/ml and occurred exclusively at the early-differentiation stage, as characterized by the level of myogenin expression and proliferation (bromodeoxyuridine incorporation) of the cells. Neutralizing antibody to the HGF-specific receptor, c-met, did not abolish the HGF response, indicating that c-met may not mediate the Sema3A expression signaling. Finally, in vivo Sema3A was upregulated in the differentiation phase of satellite cells isolated from muscle regenerating following crush injury. Overall, the data highlight a heretofore unexplored and active role for satellite cells as a key source of Sema3A expression triggered by HGF, hence suggesting that regenerative activity toward motor innervation may importantly reside in satellite cells and could be a crucial contributor during postnatal myogenesis.

Differential expression of sarcoplasmic and myofibrillar proteins of rat soleus muscle during denervation atrophy.

Denervation is known to induce skeletal muscle atrophy and fiber-type transitions, the molecular mechanisms of which are poorly understood. To investigate the effect of denervation on skeletal muscle, proteomic analysis was performed to compare denervated soleus muscle with normal soleus muscle. The muscles were fractionated to myofibrillar and sarcoplasmic fractions, which were analysed using two-dimensional gel electrophoresis (2-DE), followed by MALDI-TOF-MS. At least 30 differentially regulated proteins were identified in the sarcoplasmic fractions of normal and denervated soleus muscles. This group included metabolic enzymes, signaling molecules, chaperones, and contractile proteins. We also found two proteins, APOBEC-2 (RNA-editing enzyme) and Gamma-synuclein (breast cancer related protein), which have not been recognized as denervation-induced proteins to date. Our results might prove to be beneficial in elucidating the molecular mechanisms of denervation-induced muscle atrophy.

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.

Matrix metalloproteinase-2 mediates stretch-induced activation of skeletal muscle satellite cells in a nitric oxide-dependent manner.

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, mediate HGF release from the matrix and this step in the pathway is downstream from NO synthesis [Yamada, M., Tatsumi, R., Kikuiri, T., Okamoto, S., Nonoshita, S., Mizunoya, W., et al. (2006). Matrix metalloproteinases are involved in mechanical stretch-induced activation of skeletal muscle satellite cells. Muscle Nerve, 34, 313-319]. Experiments reported herein provide evidence that MMP2 may be involved in the NO-dependent release of HGF in vitro. Whole lysate analyses of satellite cells demonstrated the presence of MMP2 mRNA and the protein. When rat satellite cells were treated with 30 microM sodium nitroprusside a NO donor or mechanical cyclic stretch for 2h period, inactive proMMP2 (72 kDa) was converted into 52-kDa form and this processing was abolished by adding a NO synthase inhibitor l-NAME (10 microM) to the stretch culture. The 52-kDa species was also generated by treatment of the recombinant MMP2 protein with 1 microM NOC-7 that can spontaneously release NO under physiological conditions without any cofactor, and its activating activity was demonstrated by applying the NOC-7-treated MMP2 to satellite cell culture. HGF release was detected in NOC-7-MMP2-conditioned media by western blotting; very little HGF was found in media that were generated from cultures receiving NOC-7-treated MMP2 (10 ng/ml) plus 250 ng/ml tissue inhibitor-1 of metalloproteinases. Therefore, results from these experiments provide evidence that NO-activated MMP2 may cause release of HGF from the extracellular matrix of satellite cells and contribute to satellite cell activation.

Protocol for high-resolution separation of rodent myosin heavy chain isoforms in a mini-gel electrophoresis system.

Skeletal muscle comprises several fiber types classified based on their contractile and metabolic properties. Skeletal muscle fiber types are classified according to their myosin heavy chain isoforms (MyHC I, IIa, IIx, and IIb). We attained good separation of MyHC isoforms in a mini-gel system by modifying a previously developed electrophoresis protocol. Increased glycerol and decreased cross-linking agent concentrations improved the separation of MyHC isoforms. Sample preparation with dithiothreitol and protease inhibitors produced clear MyHC band boundaries. This protocol included silver staining, with a linear range. The protocol provided high resolution and a highly accurate assay of rodent MyHC isoforms.

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.

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide.

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.

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.

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.