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With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: The present study aims to establish a method of whole mitochondrial DNA (mtDNA) sequencing for domestic dogs. Prior to proceeding with the experiment, the collection of relevant DNA samples was essential. Therefore, we decided to use oral mucosa DNA, which can be collected non-invasively. A polymerase chain reaction (PCR) was performed using the DNA collected from six dogs raised in Japan and four primer pairs as specific to the mtDNA, after which the amplified products were sequenced using next-generation sequencing. As a result, the whole mtDNA obtained from all dogs was correctly sequenced. Thus, we determined that the method we used in our study may be useful for future research on dog-related medical care and welfare. In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare. [ABSTRACT FROM AUTHOR]

The mechanistic/mammalian target of rapamycin (mTOR) is a well-known serine/threonine kinase that activates muscle protein synthesis in response to anabolic conditions after exercise or nutrition intake. Rapamycin, a canonical inhibitor of mTOR complex 1 (mTORC1), induces mitochondrial dysfunction in addition to attenuating the muscle protein synthesis response. These findings suggest that mTORC1 contributes to mitochondrial homeostasis; however, the involvement of mTOR complex 2 (mTORC2) and the relationship between mTORC1 and mTORC2 are unclear. Therefore, this study was conducted to determine the role of the mTOR complex in mitochondrial function in skeletal muscle cells using rapamycin and AZD8055, as an ATP-competitive mTOR kinase inhibitor that inhibits both mTORC1 and mTORC2. AZD8055 treatment more significantly reduced the expression of proteins associated with mitochondrial oxidative phosphorylation, fusion, and fission, and decreased the respiratory capacity than rapamycin treatment. In conclusion, mTORC1 and mTORC2 cooperatively regulate mitochondrial protein expressions and respiratory function in skeletal muscle cells. [ABSTRACT FROM AUTHOR]

Skeletal muscle is a highly pliable tissue and various adaptations such as muscle hypertrophy or atrophy are induced by overloading or disuse, respectively. However, the combined effect of overloading and disuse on the quantitative adaptation of skeletal muscle is unknown. Thus, the aim of this study was to investigate the effects of the combined stimuli of overloading and disuse on mouse skeletal muscle mass and the expression of regulatory factors for muscle protein anabolism or catabolism. Male mice from the Institute Cancer Research were subjected to denervation concomitant with unilateral functional overload or functional overload concomitant with unilateral denervation. Disuse and functional overload were induced by sciatic nerve transection (denervation) and synergist ablation, respectively, and the plantaris muscle was harvested 14 days after the operation. Our results showed that denervation attenuated functional overload‐induced muscle hypertrophy and functional overload partially ameliorated the denervation‐induced muscle atrophy. P70S6K phosphorylation, an indicator of mechanistic target of rapamycin complex 1 (mTORC1) activation, was not increased by unilateral functional overload in denervated muscles or by unilateral denervation in functional overloaded muscles. Denervation did not affect the increase of LC3‐II, a marker of autophagy activation, and ubiquitinated protein expression upon unilateral functional overload. Also, functional overload did not affect ubiquitinated protein expression during unilateral denervation. Thus, our findings suggest that functional overload‐induced muscle hypertrophy or denervation‐induced muscle atrophy was attenuated by the combined stimuli of overload and denervation. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin complex 1 (mTORC1) is a protein complex that regulates skeletal muscle protein synthesis and hypertrophy. mTORC1-mediated signaling activities are activated during denervation-induced skeletal muscle atrophy and suppressed during calorie restriction-induced atrophy. Mitochondria control the qualitative plasticity of skeletal muscles primarily through biogenesis, fusion, and fission. We recently showed that mTORC1 activation contributes toward mitochondrial homeostasis. In this study, we examined the role of mTORC1 in mitochondrial adaptation during denervation- or calorie restriction-induced skeletal muscle atrophy. Seven-week-old Institute of Cancer Research mice were subjected to 14 days of denervation or calorie restriction combined with the administration of the mTORC1 inhibitor—"rapamycin". Our results showed that although mTORC1 inhibition did not alter mitochondrial biogenesis, content and enzyme activity, it suppressed the activation of dynamin-related protein 1 (DRP1), a mitochondrial fission-related protein in denervated muscle, and reduced DRP1 expression in calorie-restricted muscle. Furthermore, calorie restriction-induced mitochondrial fragmentation was partially suppressed by mTORC1 inhibition. Taken together, our results indicate that mTORC1 activation upon denervation and inhibition upon calorie restriction contributes to qualitative changes in muscle plasticity by at least partially regulating the mitochondrial fission response. [ABSTRACT FROM AUTHOR]

Lactate is a metabolic product of glycolysis and has recently been shown to act as a signaling molecule that induces adaptations in oxidative metabolism. In this study, we investigated whether lactate administration enhanced muscle hypertrophy and protein synthesis responses during resistance exercise in animal models. We used male ICR mice (7–8 weeks old) were used for chronic (mechanical overload induced by synergist ablation: [OL]) and acute (high‐intensity muscle contraction by electrical stimulation: [ES]) resistance exercise models. The animals were intraperitoneally administrated a single dose of sodium lactate (1 g/kg of body weight) in the ES study, and once a day for 14 consecutive days in the OL study. Two weeks of mechanical overload increased plantaris muscle wet weight (main effect of OL: p < 0.05) and fiber cross‐sectional area (main effect of OL: p < 0.05), but those were not affected by lactate administration. Following the acute resistance exercise by ES, protein synthesis and phosphorylation of p70 S6 kinase and ribosomal protein S6, which are downstream molecules in the anabolic signaling cascade, were increased (main effect of ES: p < 0.05), but lactate administration had no effect. This study demonstrated that exogenous lactate administration has little effect on the muscle hypertrophic response during resistance exercise using acute ES and chronic OL models. Our results do not support the hypothesis that elevated blood lactate concentration induces protein synthesis responses in skeletal muscle. [ABSTRACT FROM AUTHOR]

Adipose tissue has been classified into white adipose tissue (WAT), brown adipose tissue (BAT), and beige adipose tissue the latter of which is produced as WAT changes into BAT due to exposure to cold temperature or exercise. In response to these stimulations, WAT produces heat by increasing mitochondrial contents and the expression of uncoupling protein 1 (UCP1), thus facilitating browning. Exercise is known to be one of the triggers for WAT browning, but the effects of exercise intensity on the browning of WAT remain to be unclear. Therefore, in this study, we aimed to examine the effects of high‐ or low‐intensity exercises on the browning of WAT. Mice performed high‐ or low‐intensity running on a treadmill running 3 days a week for four weeks. As per our findings, it was determined that four weeks of running did not significantly reduce inguinal WAT (iWAT) wet weight but did significantly reduce adipocytes size, regardless of exercise intensity. The protein expression level of UCP1 was significantly increased in iWAT by high‐intensity running. In addition, the expression of oxidative phosphorylation proteins (OXPHOS) in iWAT was significantly increased by high‐intensity running. These results demonstrated that high‐intensity exercise might be effective for increasing mitochondrial contents and heat production capacity in iWAT. Furthermore, we found that high‐intensity running increased the protein expression level of fibroblast growth factor 21 (FGF21) in skeletal muscle compared with that in low intensity running. We have also examined the relationship between browning of WAT and the expression of FGF21 in skeletal muscle and found a positive correlation between the protein expression of UCP1 in iWAT and the protein expression of FGF21 in gastrocnemius muscle. In conclusion, we suggest that high‐intensity exercise is effective for the browning of WAT and the increase of FGF21 in skeletal muscle. [ABSTRACT FROM AUTHOR]

Rheological techniques were utilized to determine the biological changes that occur in response to the codistribution of marginal actin fibers and subendothelial fibronectin in the endothelial cells of developing rat aorta. Analysis of fetal rat aortic endothelial cells indicated the role of increased tensile force and shear stress in inducing the codistribution of actin fibers and subendothelial fibronectin in developing rat aorta. Furthermore, the actin fibers, subendothelial fibronectin and stress fibers maintained the structural integrity of vascular endothelial cells.

Mechanistic/mammalian target of rapamycin (mTOR) is a central factor of protein synthesis signaling and plays an important role in the resistance training‐induced increase in skeletal muscle mass and subsequent skeletal muscle hypertrophy response. In particular, mTOR complex 1 (mTORC1) promotes protein synthesis in ribosomes by activating the downstream effectors, p70S6K and 4EBP1, in skeletal muscle and is highly sensitive to rapamycin, an mTOR inhibitor. Recently, resistance training has also been shown to affect mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria dynamically change their morphology through repeated fusion and fission, which may be key for controlling the quality of skeletal muscle. However, how the mechanisms of mitochondrial dynamics function during hypertrophy in skeletal muscle remains unclear. The aim of this study was to examine the impact of mTOR inhibition on mitochondrial dynamics during skeletal muscle hypertrophy. Consistent with previous studies, functional overload by synergist (gastrocnemius and soleus) ablation‐induced progressive hypertrophy (increase in protein synthesis and fiber cross‐sectional area) of the plantaris muscle was observed in mice. Moreover, these hypertrophic responses were significantly inhibited by rapamycin administration. Fourteen days of functional overload increased levels of MFN2 and OPA1, which regulate mitochondrial fusion, whereas this enhancement was inhibited by rapamycin administration. Additionally, overload decreased the levels of DRP1, which regulates mitochondrial fission and oxidative phosphorylation, regardless of rapamycin administration. These observations suggest that the relative reduction in mitochondrial function or content is complemented by enhancement of mitochondrial fusion and that this complementary response may be regulated by mTORC1. [ABSTRACT FROM AUTHOR]

Athletes train to improve strength and endurance to demonstrate maximum performance during competitions. Training methods vary but most focus on strength, endurance, or both. Concurrent training is a combination of two different modes of training. In this study, we combined resistance exercise (RE) and high‐intensity interval exercise (HIIE) to investigate the influence of the order of the concurrent training on signal molecules on hypertrophy and glycolysis in the skeletal muscle. The phosphorylation levels of mechanistic target of rapamycin (mTOR) signals, p70 S6 kinase (p70S6 K), ribosomal protein S6 (S6), and glycogen synthase kinase beta (GSK‐3β) were significantly increased in the HIIE first group compared with the control group. The combined training course did not affect the glycogen content and expression levels of proteins concerning glycolytic and metabolic capacity, suggesting that a combination of HIIE and RE on the same day, with HIIE prior to RE, improves hypertrophy response and glycolysis enhancement. [ABSTRACT FROM AUTHOR]

For many ball games, both resistance and endurance training are necessary to improve muscle strength and endurance capacity. Endurance training has been reported to inhibit muscle strength and hypertrophy, but some studies have reported that endurance exercise (EE) does not inhibit the effects of resistance exercise. Here, we examined the effect of short‐ or long‐duration EE on mouse skeletal muscle hypertrophy induced by functional overload (OL) at the molecular level. Plantaris muscle hypertrophy was induced by OL with synergist ablation in mice. Body mass was reduced with endurance training, but EE duration (30 or 90 min) had no effect. The ratio of plantaris muscle weight to body weight was higher in the OL and EE for 30 min (OL+EE30) and OL and EE for 90 min (OL+EE90) groups compared with the OL group. Expression of mechanistic target of rapamycin signaling proteins, which is related to protein synthesis and hypertrophy, was increased in the OL+EE30 group. Expression of Forkhead box‐containing protein O1, which is related to protein breakdown and atrophy, remained unchanged. However, microtubule‐associated protein 1 light chain 3, a known marker of autophagy, and MAFbx, which is related to protein breakdown, were significantly increased in the OL+EE90 group. Furthermore, markers of oxidative stress, ubiquitin and 4‐hydroxynonenal were also significantly increased in the OL+EE90 group compared with other groups. In conclusion, EE duration did not affect body mass and plantaris mass and did not interfere with mechanistic target of rapamycin signaling, but it did increase ubiquitinated proteins and oxidative stress. It is therefore necessary to consider training durations for EE when combining endurance and resistance training. [ABSTRACT FROM AUTHOR]

The regulation of cellular protein synthesis is a critical determinant of skeletal muscle growth and hypertrophy in response to an increased workload such as resistance exercise. The mechanistic target of rapamycin complex 1 (mTORC1) and its upstream protein kinase Akt1 have been implicated as a central signaling pathway that regulates protein synthesis in the skeletal muscle; however, the precise molecular regulation of mTORC1 activity is largely unknown. This study employed germline Akt1 knockout (KO) mice to examine whether upstream Akt1 regulation is necessary for the acute activation of mTORC1 signaling in the plantaris muscle following mechanical overload. The phosphorylation states of S6 kinase 1, ribosomal protein S6, and eukaryotic translation initiation factor 4E‐binding protein 1 which show the functional activity of mTORC1 signaling, were significantly increased in the skeletal muscle of both wildtype and Akt1 KO mice following an acute bout (3 and 12 hr) of mechanical overload. Akt1 deficiency did not affect load‐induced alteration of insulin‐like growth factor‐1 (IGF‐1)/IGF receptor mRNA expression. Also, no effect of Akt1 deficiency was observed on the overload‐induced increase in the gene expressions of pax7 and myogenic regulatory factor of myogenin. These observations show that the upstream IGF‐1/Akt1 regulation is dispensable for the acute activation of mTORC1 signaling and regulation of satellite cells in response to mechanical overload. [ABSTRACT FROM AUTHOR]

Denervation‐induced muscle atrophy increases signaling through both protein degradation and synthesis pathways. Renalase is a flavin adenine dinucleotide‐dependent amine oxidase that inhibits apoptosis and inflammation and promotes cell survival. This study aimed to elucidate the effect of renalase on denervation‐induced muscle atrophy. We used 7‐week‐old renalase knock‐out (KO) mice (a model of denervation‐induced muscle atrophy) and wild‐type (WT) mice (KO: n = 6, weight = 20–26 g; WT: n = 5, weight = 19–23 g). After their left legs were denervated, these mice were killed 1 week later. KO mice had lighter muscle weight than the WT mice. We observed an increase in molecular signaling through protein degradation pathway as well as oxidative stress in denervated muscles compared with that in sham‐operated muscles in both WT and KO mice. Additionally, we also observed the main effect of renalase in WT and KO mice. Mitochondrial oxidative phosphorylation protein content was lower in denervated muscles than in sham‐operated muscles in both WT and KO mice. However, a significant difference was noted in the reaction with Akt and p70S6K (components of the protein synthesis pathway) between WT and KO mice. In conclusion, mice with renalase deficiency demonstrated an attenuation of denervation‐induced muscle atrophy. This might be related to catecholamines because signaling through the protein synthesis pathway was increased following denervation in renalase KO mice compared with that in WT mice, despite showing no change in signaling through protein degradation pathways. [ABSTRACT FROM AUTHOR]

Concurrent training involves a combination of two different modes of training. In this study, we conducted an experiment by combining resistance and endurance training. The purpose of this study was to investigate the influence of the order of concurrent training on signal molecules in skeletal muscle. The phosphorylation levels of p70 S6 kinase, S6 ribosomal protein, and 4E‐binding protein 1, which are related to hypertrophy signaling, increased significantly in the resistance–endurance order group as compared with in control group not the endurance–resistance order group. The gene expressions related to metabolism were not changed by the order of concurrent training. The mitochondrial respiratory chain complex was evaluated by western blot. Although both groups of concurrent training showed a significant increase in MTCO1, UQCRC2, and ATP5A protein levels, we could not detect a difference based on the order of concurrent training. In conclusion, a concurrent training approach involving resistance training before endurance training on the same day is an effective way to activate both mTOR signaling and mitochondria biogenesis. [ABSTRACT FROM AUTHOR]

Background: Enzymatically modified isoquercitrin (EMIQ) is produced from rutin using enzymatic hydrolysis followed by treatment with glycosyltransferase in the presence of dextrin to add glucose residues. EMIQ is absorbed in the same way as quercetin, a powerful antioxidant reported to prevent disused muscle atrophy by targeting mitochondria and to have ergogenic effects. The present study investigated the effect of EMIQ on skeletal muscle hypertrophy induced by functional overload. Methods: In Study 1, 6-week-old ICR male mice were divided into 4 groups: sham-operated control, sham-operated EMIQ, overload-operated control, and overload-operated EMIQ groups. In Study 2, mice were divided into 3 groups: overload-operated whey control, overload-operated whey/EMIQ (low dose), and overload-operated whey/EMIQ (high dose) groups. The functional overload of the plantaris muscle was induced by ablation of the synergist (gastrocnemius and soleus) muscles. EMIQ and whey protein were administered with food. Three weeks after the operation, the cross-sectional area and minimal fiber diameter of the plantaris muscle fibers were measured. Results: In Study 1, functional overload increased the cross-sectional area and minimal fiber diameter of the plantaris muscle. EMIQ supplementation significantly increased the cross-sectional area and minimal fiber diameter of the plantaris muscle in both the sham-operated and overload-operated groups. In Study 2, EMIQ supplementation combined with whey protein administration significantly increased the cross-sectional area and minimal fiber diameter of the plantaris muscle. Conclusion: EMIQ, even when administered as an addition to whey protein supplementation, significantly intensified the fiber hypertrophy of the plantaris muscle in functionally overloaded mice. EMIQ supplementation also induced fiber hypertrophy of the plantaris in sham-operated mice. [ABSTRACT FROM AUTHOR]

Skeletal muscle represents a plentiful and accessible source of adult stem cells. Skeletal-muscle-derived stem cells, termed satellite cells, play essential roles in postnatal growth, maintenance, repair, and regeneration of skeletal muscle. Although it is well known that the number of satellite cells increases following physical exercise, functional alterations in satellite cells such as proliferative capacity and differentiation efficiency following exercise and their molecular mechanisms remain unclear. Here, we found that functional overload, which is widely used to model resistance exercise, causes skeletal muscle hypertrophy and converts satellite cells from quiescent state to activated state. Our analysis showed that functional overload induces the expression of MyoD in satellite cells and enhances the proliferative capacity and differentiation potential of these cells. The changes in satellite cell properties coincided with the inactivation of Notch signaling and the activation of Wnt signaling and likely involve modulation by transcription factors of the Sox family. These results indicate the effects of resistance exercise on the regulation of satellite cells and provide insight into the molecular mechanism of satellite cell activation following physical exercise. [ABSTRACT FROM AUTHOR]

The purposes of our study were to examine (1) Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) expressions in mouse skeletal muscle; and (2) the effect of 6-week training on Rhbg and Rhcg expressions. The levels of Rhbg and Rhcg expressions were much higher in the soleus (Sol) than in the plantaris (Pla) or gastrocnemius (Gas). Immunofluorescence microscopy demonstrated that Rhbg colocalizes with dystrophin, a plasma membrane protein marker, whereas Rhcg colocalizes with CD31, a vascular endothelial cell marker. In a 6-week swim training study, we set up two different training groups. Endurance ( END) group underwent swim training without load for 30 min and exercise time was increased by 30 min every 2 weeks. High-intensity interval training ( HIIT) group underwent 10-12 sets of swim training at 20 sec per set and intervals of 10 sec, with a load of 10% body weight. After 6 weeks of training, all mice performed exhaustive swimming performance test ( PT), with 9% of body weight to exhaustion. HIIT group could significantly swim more and showed significantly lower blood ammonia level compared with control ( CON) group at immediately after PT. Rhbg and Rhcg levels did not change after 6 weeks in both END and HIIT groups. Our results indicate that ammonia transporters are more abundant in slow fiber than fast fiber muscles and 6 weeks swim training suppresses blood ammonia elevation induced by high-intensity exercise with performance improvement, although the levels of ammonia transporter proteins does not change. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is one of the most common serious metabolic diseases that results in hyperglycemia due to defects of insulin secretion or insulin action or both. The present review focuses on the alterations to the diabetic neuronal tissues and skeletal muscle, including stem cells in both tissues, and the preventive effects of physical activity on diabetes. Diabetes is associated with various nervous disorders, such as cognitive deficits, depression, and Alzheimer’s disease, and that may be caused by neural stem cell dysfunction. Additionally, diabetes induces skeletal muscle atrophy, the impairment of energy metabolism, and muscle weakness. Similar to neural stem cells, the proliferation and differentiation are attenuated in skeletal muscle stem cells, termed satellite cells. However, physical activity is very useful for preventing the diabetic alteration to the neuronal tissues and skeletal muscle. Physical activity improves neurogenic capacity of neural stem cells and the proliferative and differentiative abilities of satellite cells. The present review proposes physical activity as a useful measure for the patients in diabetes to improve the physiological functions and to maintain their quality of life. It further discusses the use of stem cell-based approaches in the context of diabetes treatment. [ABSTRACT FROM AUTHOR]

Delay in the growth of skeletal muscle resulting from physical inactivity during the juvenile period may affect the subsequent quality of life. Therefore, it is important to clarify whether recovery of this muscle growth delay can be achieved by physical training thereafter. In the present study, mice were subjected to hindlimb suspension from 5 to 8 weeks of age to induce a delay of muscle growth and reloading from 8 weeks of age to recovery of muscle. Growth of the plantaris muscle, which is composed of fast-twitch fibers, was monitored at 5, 8, 11 and 14 weeks of age. This revealed that the growth of the plantaris had completely recovered from this delay by 11 weeks of age. Moreover, the recovery was accompanied by a significant increase of RNA content relative to control muscle. Since ribosomal RNA accounts for the majority of total RNA, it was thought that the machinery for protein synthesis had become activated in the recovering muscle. In addition, phosphorylated ribosomal protein S6, which is an important factor for efficient translation, was increased at the same time. These results suggested that protein synthesis was enhanced in recovering muscle. In addition, the recovering muscle showed activation of the Erk pathway, one of the pathways controlling the expression of ribosomal RNA. These results suggest that loss of skeletal muscle due to unloading during the juvenile period can be completely recovered by reloading, and that this recovery might require an increase of RNA content mediated by the Erk pathway. [ABSTRACT FROM AUTHOR]

Adult stem cells play an essential role in mammalian organ maintenance and repair throughout adulthood since they ensure that organs retain their ability to regenerate. The choice of cell fate by adult stem cells for cellular proliferation, self-renewal, and differentiationinto multiple lineages is critically important for the homeostasis and biological function of individual organs. Responses of stem cells to stress, injury, or environmental change are precisely regulated by intercellular and intracellular signaling networks, and these molecular events cooperatively define the ability of stem cell throughout life. Skeletal muscle tissue represents an abundant, accessible, and replenishable source of adult stem cells. Skeletal muscle contains myogenic satellite cells and muscle-derived stem cells that retain multipotent differentiation abilities. These stem cell populations have the capacity for long-term proliferation andhigh self-renewal. The molecularmechanisms associatedwithdeficits inskeletalmuscle and stemcell functionhave been extensively studied. Muscle-derived stem cells are an obvious, readily available cell resource that offers promise for cell-based therapy and various applications in the field of tissue engineering. This review describes the strategies commonly used to identify and functionally characterize adult stem cells, focusing especially on satellite cells, and discusses their potential applications. [ABSTRACT FROM AUTHOR]

To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatinknockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using realtime PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. [ABSTRACT FROM AUTHOR]

TAKEMASA, T., ABE, Y., KUMAGAI, C. and KIYOSAWA, H. Possible Involvement of MicroRNA-21 in Skeletal Muscle Hypertrophy. Adv. Exerc. Sports Physiol., Vol.18, No.1 pp.5-15, 2012. In order to elucidate the function of microRNAs (miRNAs) in the plasticity of skeletal muscle, we analyzed global changes in miRNA expression using the microarray technique during muscle hypertrophy induced by compensatory overload following bilateral Achilles tenotomy in mice. The most prominent change observed by microarray analysis was that of miR-21 expression. The expression of myogenic genes, myogenin and MEF2D was increased, and that of MyoD and MHCIIb was decreased in overloaded muscle. We also investigated the changes in expression of PDCD4, PTEN, and SPRY2, which are potential targets of miR- 21, and reduction in the expression of all three genes was observed in hypertrophic muscle We next investigated the expression of miR-21 and its prospective target genes using an in vitro differentiation system with C2C12 cultured myocytes through overexpressed or suppressed miR-21 by introducing the miR-21 precursor or inhibitor. When the miR-21 precursor was transfected, miR-21 expression was significantly increased, but the genes controlling muscle differentiation (MyoD, myogenin, MHCIIb, MEF2D) and possible miR-21 target genes (PDCD4, PTEN, and SPRY2) were not significantly changed in myoblasts. Similary, when we applied the miR-12 inhibitor to C2C12, the expression of myogenic genes was not significantly changed, although possible miR-21 target genes (PDCD4, PTEN, and SPRY2) tended to be up-regulated. These results raise the possibility that microRNA may regulate muscle plasticity during exercise and training [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) pathway positively regulates the cell growth through ribosome biogenesis in many cell type. In general, myostatin is understood to repress skeletal muscle hypertrophy through inhibition of mTOR pathway and myogenesis. However, these relationships have not been clarified in skeletal muscle undergoing atrophy. Here, we observed a significant decrease of skeletal muscle mass at 2 weeks after denervation. Unexpectedly, however, mTOR pathway and the expression of genes related to myogenesis were markedly increased, and that of myostatin was decreased. However, de novo ribosomal RNA synthesis and the levels of ribosomal RNAs were dramatically decreased in denervated muscle. These results indicate that ribosome biogenesis is strongly controlled by factors other than the mTOR pathway in denervated atrophic muscle. Finally, we assessed rRNA transcription factors expression and observed that TAFIa was the only factor decreased. TAFIa might be a one of the limiting factor for rRNA synthesis in denervated muscle. J. Cell. Physiol. 227: 1569-1576, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of training and detraining on the monocarboxylate transporter (MCT) 1 and MCT4 levels in the gluteus medius muscle of Thoroughbred horses. Twelve Thoroughbred horses were used for the analysis. For 18 weeks, all the horses underwent high-intensity training (HIT), with running at 90-110% maximal oxygen consumption () for 3 min, 5 days week. Thereafter, the horses either underwent detraining for 6 weeks by either 3 min of moderate-intensity training (MIT) at 70%, 5 days week (HIT-MIT group) or stall rest (HIT-SR group). The horses underwent an incremental exercise test, was measured and resting muscle samples were obtained from the middle gluteus muscle at 0, 18 and 24 weeks. The content of MCT1 and MCT4 proteins increased after 18 weeks of HIT. At the end of this period, an increase was noted in the citrate synthase activity, while phosphofructokinase activity remained unchanged. After 6 weeks of detraining, all these indexes returned to the pretraining levels in the HIT-SR group. However, in the HIT-MIT group, the increase in the MCT1 protein content and citrate synthase activity was maintained after 6 weeks of MIT, while the MCT4 protein content decreased to the pretraining value. These results suggest that the content of MCT1 and MCT4 proteins increases after HIT in Thoroughbred horses. In addition, the increase in the MCT1 protein content and oxidative capacity induced by HIT can be maintained by MIT of 70%, but the increase in the MCT4 protein content cannot be maintained by MIT. [ABSTRACT FROM AUTHOR]

Endurance exercise training pro- motes angiogenesis in skeletal muscle. In order to elucidate the mechanisms of exercise-induced angiogenesis in skeletal muscle, many researchers have focused on gene expression in skeletal muscle during and after an exercise, and it is well understood that acute endurance exercise induces temporarily increasing VEGF mRNA in skeletal muscle and secreting VEGF from active muscle. Because of these results, exercise-induced VEGF expression from skeletal muscle has been believed to be an early step of exercise-induced skeletal muscle angiogenesis. Recently it was revealed that inflammatory cells including macrophage secreted pro-angiogenesis cytokine including VEGF, which had an ability to facilitate angiogenesis in vivo. However the relationship between exercise-induced skeletal muscle angiogenesis and these inflammatory cells is not revealed. Therefore we had hypothesized that exercise-induced angiogenesis in skeletal muscle is facilitated by inflammatory cells secreting VEGF. To explore this hypothesis, we subjected ICR mice (male, 8 week-old) to acute endurance exercise (90 min, 20 m/min, 10° incline), sacrificed these mice by cervical dislocation at pre-exercise, 6 hour, 1 day, 2 day and 3 day after exercise, and harvested both gastrocnemius and plantaris muscle as hind-limb muscle. And, we used immunohistochemical method and observed significant increase of macrophage in hind-limb muscle at 1 day after exercise compared with pre exercise. Subsequently we observed that increment of percent- age of VEGF-secreting macrophage. These results suggest that migrating macrophages have a positive role in exercise-induced angiogenesis in skeletal muscle. [ABSTRACT FROM AUTHOR]

It has long been known that acute cold stress suppresses immune responses, and that swimming training increases tolerance to cold. The aim of the current study was to investigate whether the improved cold tolerance occasioned by swimming training prevented the suppression of immune responses as a result of acute cold stress, focusing in particular on the generation of suppressor macrophages and glucocorticoid hormones. This is because acute cold stress induces the generation of suppressor macrophages that express large numbers of receptors to the Fc portion of immunoglobulin G (MAC-1+Fc γ RII/IIIbright cells), leading to the suppression of splenocyte mitogenesis; moreover, the generation of MAC-1+Fc γγ RII/IIIbright cells is partly mediated by increased glucocorticoid levels during acute cold stress. The trained mice underwent a 6-week endurance swimming training (5 times/week) in water at 30γ for 90min. The swimming training remarkably increased brown adipose tissue mass, suggesting an improved cold tolerance. Indeed, when the swimming-trained mice were exposed to 5γ for 3h (acute cold stress), the rectal temperature was not substantially decreased, unlike the case for control mice. The proportion of MAC-1+Fc γ RII/IIIbright cells in peritoneal exudate cell population from the trained mice was unaffected by the acute cold stress. The swimming training significantly attenuated the increases in serum corticosterone levels in response to acute cold stress. Taken together, the results obtained suggegst that swimming training not only improves cold tolerance but also inhibits the generation of suppressor macrophages under acute cold stress. [ABSTRACT FROM AUTHOR]

Exercise dramatically increases oxygen consumption and causes oxidative stress. Superoxide dismutase (SOD) is important in the first-line defence mechanisms against oxidative stress. To investigate the effect of acute exercise on the expression of SOD, we examined the expression of mRNA for three SOD isozymes, in mice run on a treadmill to exhaustion. Six hours after exercise, the expression of extracellular SOD (EC-SOD) mRNA increased significantly in skeletal muscle and persisted for 24 h, whereas no change was observed for cytoplasmic and mitochondrial SOD mRNA. Moreover, acute exercise also induced EC-SOD mRNA in the aorta. These results suggest that a single bout of exercise is enough to augment the expression EC-SOD mRNA in skeletal muscle and the aorta, and may partly explain the beneficial effect of exercise. [ABSTRACT FROM AUTHOR]

Abstract: The effects of 3-week exercise training on the functions of peritoneal macrophages from BALB/c mice were investigated. Lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and proinflammatory cytokine production in macrophages from trained mice was markedly higher than those from control mice. Meanwhile, exercise training decreased the steady state level of β2-adrenergic receptor (β2AR) mRNA in macrophages. Overexpression of β2AR in the macrophage cell line RAW264 by transfecting with β2AR cDNA suppressed NO synthase (NOS) II expression but dose not influenced proinflammatory cytokine expression. When expression of transfected β2AR in RAWar cells was downregulated by a tetracycline repressor-regulated mammalian expression system, NOS II mRNA expression was significantly increased; this suggested that the changes in the β2AR expression level in macrophages associated with exercise training play a role in the regulation of NO production following LPS stimulation. These findings indicate that exercise training improves macrophage innate immune function in a β2AR-dependent and -independent manner. [Copyright &y& Elsevier]

Oligonol is a new lychee fruit-derived polyphenol converted into a low-molecular-form, which has improved absorption and strong antioxidative activity. The aim of the current study was to investigate the effects of Oligonol supplementation on perceived subjective mood states in addition to oxidative stress in 47 undergraduate athletes during 52 days of track-and-field training. This was a prospective single blind crossover study. The ratings of perceived exertion (RPE) responses were significantly (P<0.05) lower following Oligonol supplementation, suggesting that Oligonol intake caused the subjects to feel less fatigued during regular training. The results of our own questionnaire on fatigue and pain were as follows: Oligonol supplementation significantly (P<0.05)improved all the fatigue scores and tended to attenuate the feeling of three kinds of pains(muscular/anticular pain, lumbago, and menstrual pain), followed by a change fot the worse after the discontinuance of Oligonol intake. Unexpectedly, however, there were no definite changes in the Profile of Moods States (POMS) scores or oxidative stress markers (8-hydroxy-2'-deoxyguanosine and hexanoyllysine) in urine after Oligonol supplementation. The results obtained suggest that Oligonol supplementation in young athletes shows significant subjective positive effect particularly on the feeling of fatigue during regular track and-field training, possibly contributing to the maintenance of good conditioning. [ABSTRACT FROM AUTHOR]

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping. [ABSTRACT FROM AUTHOR]

Skeletal muscle is composed of several different types of myofiber: slow oxidative (SO), fast glycolytic oxidative and fast glycolytic. However, the classification is usually determined by myosin heavy chain typing rather than by metabolic index. In this study, the oxidative metabolic index was investigated as a possible method of myofiber typing. Myoglobin, which is involved in oxygen transport and storage in myofibers, and mitochondria, which are the central organelles for oxidative metabolism, were studied. High levels of myoglobin and mitochondria are believed to exist in SO fibers, but the current study showed that they are considerably richer in some fast type fibers. As myofiber typing using the oxidative metabolic index is important physiologically, an attempt was made to find a simple method for this purpose. Some mitochondrial proteins have been observed to auto-fluoresce but until now this effect was too faint to detect easily. Owing to the recent advances in cooling charge-coupled device technology, such auto-fluorescence can now be used for myofiber typing, and the simple and rapid method for doing so is reported here. [ABSTRACT FROM AUTHOR]

Previously, we reported that polyphenol-rich fraction (named E80) promotes skeletal muscle hypertrophy induced by functional overload in mice. This study indicates that E80 has potential for affecting skeletal muscle mass. Then, we evaluate the effect of E80 on atrophic and recovery conditions of skeletal muscle in mice. Hindlimb suspension (unloading) and relanding (reloading) are used extensively to observe disuse muscle atrophy and subsequent muscle mass recovery from atrophy. Eight-week old C57BL/6 mice were fed either a normal diet or a diet containing 0.5% E80 for two weeks under conditions of hindlimb suspension and a subsequent 5 or 10 days of reloading. We found that E80 administration did not prevent atrophy during hindlimb suspension, but promoted recovery of slow-twitch (soleus) muscle mass from atrophy induced by hindlimb suspension. After five days of reloading, we discovered that phosphorylation of the Akt/mammalian target of rapamycin (mTOR) pathway proteins, such as Akt and P70 ribosomal protein S6 kinase (S6K), was activated in the muscle. Therefore, E80 administration accelerated mTOR signal and increased protein synthesis in the reloaded soleus muscle. [ABSTRACT FROM AUTHOR]

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping. [ABSTRACT FROM AUTHOR]

Oxidative stress and mitochondrial dysfunction are associated with the aging process. However, the role of nuclear factor erythroid 2 ‐related factor 2 (Nrf2) in skeletal muscle during aging remains to be clarified. In the current study, we assessed whether the lack of Nrf2, which is known as a master regulator of redox homeostasis, promotes age‐related mitochondrial dysfunction and muscle atrophy in skeletal muscle. Here, we demonstrated that mitochondrial 4‐hydroxynonenal and protein carbonyls, markers of oxidative stress, were robustly elevated in aged Nrf2 knockout (KO) mice because of the decreased expression of Nrf2‐target antioxidant genes. Mitochondrial respiration declined with aging; however, there was no difference between Nrf2 KO and age‐matched WT mice. Similarly, cytochrome c oxidase activity was lower in aged WT and Nrf2 KO mice compared with young WT mice. The expression of Mfn1 and Mfn2 mRNA was lower in aged Nrf2 KO muscle. Mitochondrial reactive oxygen species production per oxygen consumed was elevated in aged Nrf2 KO mice. There was no effect of Nrf2 KO on muscle mass normalized to body weight. These results suggest that Nrf2 deficiency exacerbates age‐related mitochondrial oxidative stress but does not affect the decline of respiratory function in skeletal muscle. We demonstrated that mitochondrial 4‐hydroxynonenal and protein carbonyls, markers of oxidative stress, were robustly elevated in aged Nrf2 knockout mice, due to the decreased expression of Nrf2‐target antioxidant genes. However, Nrf2 deficiency did not affect the decline of mitochondrial respiratory function in aged skeletal muscle. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to elucidate the effect of early onset of exercise on muscle injury. Methods: Mice were divided into 3 groups, control group, injury group and exercise group. For injury group and exercise group, we forced micc to get muscle injury (eccentric-stimulation induced muscle injury). Then, for only exercise group, we let mice exercise (60 minutes swimming for 3 in every 4 days) after 7 days of muscle injury. All groups were sacrificed 21 days after muscle injury. Result and Discussion: we observed that the cross sectional area of the regenerating myofibers in exercise group was larger than that in injury group and the level of fibrosis in the exercise group was lower than that in injury group. Then, we measured mRNA level of collagenlα2 and collagen5α3, which are known to be fibrosis structural component. Collagen1α2 mRNA level are almost the same among 3 groups. On the other hand, in collagen5α3 mRNA level, exercise group showed less collagen5α3 mRNA level than injury group. Then, in order to reveal how exercise inhibits fibrosis, we measured mRNA level of TGF-β 1, which is a cytokine for promoting fibrosis. We observed that TGF-β 1 mRNA level of exercise group was less than that of injury group. Taken together, our data suggests that exercise improves healing process and exercise inhibit fibrosis via TGF-β 1 down regulation. [ABSTRACT FROM AUTHOR]

Purpose: Ammonia, one of the factors inducing fatigue, is transported by Rh b glycoprotein (Rhbg) and Rh c glycoprotein (Rhcg) in liver, kidney and other organs. The purpose of this study is examination of expression of Rhbg and Rhcg in skeletal muscle. Methods: Skeletal muscle samples (Soleus: Sol, Plantaris: Pia, Gastrocremius: Gas) were dissected from ICR mice (male, 8weeks old). Rhbg and Rhcg protein contents were analyzed by Western blot. Localization of Rhbg and Rhcg were analyzed by immunofluorescent microscopy. Myosin Heavy Chain (MHC) type composition of each muscles were measured by SDS-PAGE. Results: Rhbg and Rhcg protein much expressed in Sol than Pia and Gas. A positive correlation was found between MHC type I + II a/ II b and Rhbg/Rhcg expression. Immnofluorescent microscopy identified that Rhbg was colocalized with dystrophin, plasma membrane protein marker. On the other hand, staining for Rhcg showed colocalization with CD31 (vascular endothelial cell marker). Discussion: Ammonia transporters, both Rhbg and Rhcg, were rich in slow type fiber predominance muscle. Ammonia transporters may contribute in slow fiber than fast fiber. Rhbg and Rhcg localizations were different in types ofskeletal muscle. This result indicates that Rhbg and Rhbg have different role in skeletal muscle. [ABSTRACT FROM AUTHOR]

Purpose: We investigated the effects of exercise on adult satellite cell conversion focusing at Wnt signaling, which plays important roles in lineage control during embryonic myogenesis and postnatal development. Methods: Male C57BL/6J mice ages 8 weeks (adult) and 24 months (aged) were used and divided into control group and runner group. Runner group mice were housed individually in cages equipped with a running wheel and performed voluntary wheel running for 4 weeks. After 4 weeks of exercise, mice were sacrificed and the gastrocnemius muscles were dissected out quickly from each mouse. Results: We demonstrated that voluntary wheel running exercise, which was a low-stress exercise, converted satellite cells to activated state due to accelerated Wnt signaling. Our analyses showed that upregulated canonical Wnt/b-catenin signaling directly modulated chromatin structures of both MyoD and Myf5 genes, resulting in increases in the mRNA expression of Myf5 and MyoD and the number of proliferative Pax7(+)Myf5(+) and Pax7(+)MyoD(+) cells in skeletal muscle. The effect of Wnt signaling on the activation of satellite cells, rather than Wnt-mediated fibrosis, was observed in both adult and aged mice. Discussion: These results indicate that exercise-stimulated extracellular Wnts play a critical role in the regulation of satellite cells in adult and aged skeletal muscle. [ABSTRACT FROM AUTHOR]