BACKGROUND: Animal models of spontaneous osteoarthritis (OA) are sparse and not well characterized. The purpose of the present study is to examine OA-related changes and mechanisms in senescence-accelerated mouse prone 8 (SAMP8) that displays a phenotype of accelerated aging.  METHODS: Knees of male SAMP8 and SAM-resistant 1 (SAMR1) mice as control from 6 to 33 weeks of age were evaluated by histological grading systems for joint tissues (cartilage, meniscus, synovium, and subchondral bone), and µCT analysis. Gene expression patterns in articular cartilage were analyzed by real-time PCR. Immunohistochemistry was performed for OA-related factors, senescence markers, and apoptosis. RESULTS: Starting at 14 weeks of age, SAMP8 exhibited mild OA-like changes such as proteoglycan loss and cartilage fibrillation. From 18 to 33 weeks of age, SAMP8 progressed to partial or full-thickness defects with exposure of subchondral bone on the medial tibia and exhibited synovitis. Histological scoring indicated significantly more severe OA in SAMP8 compared with SAMR1 from 14 weeks [median (interquartile range): SAMR1: 0.89 (0.56-1.81) vs SAMP8: 1.78 (1.35-4.62)] to 33 weeks of age [SAMR1: 1.67 (1.61-1.04) vs SAMP8: 13.03 (12.26-13.57)]. Subchondral bone sclerosis in the medial tibia, bone mineral density (BMD) loss of femoral metaphysis, and meniscus degeneration occurred much earlier than the onset of cartilage degeneration in SAMP8 at 14 weeks of age. CONCLUSIONS: SAMP8 are a spontaneous OA model that is useful for investigating the pathogenesis of primary OA and evaluating therapeutic interventions.

Tendon is a fibrous connective tissue, that is, transmitting the forces that permit body movement. However, tendon/ligament biology is still not fully understood and especially, the role of miRNAs in tendon/ligament is sparse and uncharacterized in in vivo models. The objectives of this study were to address the function of DICER using mice with tendon/ligament-specific deletion of Dicer (Dicer conditional knockout; cKO), and to identify key miRNAs in tendon/ligament. Dicer cKO mice exhibited hypoplastic tendons through structurally abnormal collagen fibrils with downregulation of tendon-related genes. The fragility of tendon did not significantly affect the tensile strength of tendon in Dicer cKO mice, but they showed larger dorsiflexion angle in gait compared with Control mice. We identified two miRNAs, miR-135a and miR-1247, which were highly expressed in the Achilles tendon of Control mice and were downregulated in the Achilles tendon of Dicer cKO mice compared with Control mice. miR-135a mimic increased the expression of tendon-related genes in injured Achilles tendon-derived fibroblasts. In this study, Dicer cKO mice exhibited immature tendons in which collagen fibrils have small diameter with the downregulation of tendon-related genes such as transcriptional factor, extracellular matrix, and miRNAs. Thus, DICER plays an important role in tendon maturation, and miR-135a may have the potential to become key miRNA for tendon maturation and healing.

<title>Abstract</title>Muscle atrophy occurs in a variety of physiological and pathological conditions. Specific molecular networks that govern protein synthesis and degradation play important roles in controlling muscle mass under diverse catabolic states. MicroRNAs (miRNAs) were previously found to be regulators of protein synthesis and degradation, and their expressions in skeletal muscle were altered in muscle wasting conditions. However, functional roles of miRNAs in muscle atrophy are poorly understood. In this study, we generated tamoxifen-inducible <italic>Dicer</italic> knockout (iDicer KO) mice and subjected them to 2 weeks of single hindlimb denervation. The expression of <italic>Dicer</italic> mRNA was significantly reduced in muscle of the iDicer KO mice compared to that of WT mice. The loss of <italic>Dicer</italic> moderately reduced levels of muscle-enriched miRNAs, miR-1, miR-133a and miR-206 in both innervated and denervated muscles of the iDicer KO mice. We also found that the extent of denervation-induced muscle atrophy as well as changes of signaling molecules related to protein synthesis/degradation pathways in the iDicer KO mice were comparable to these in WT mice. Taken together, <italic>Dicer</italic> knockout in adult skeletal muscle did not affect denervation-induced muscle atrophy.

Discovery of blood biomarkers to evaluate exercise-induced muscle damage have attracted many researchers and coaches. This study aimed to determine changes in circulating myomesin 3 fragments as a novel biomarker for exercise-induced muscle damage. Nine healthy males performed 10 sets of 40 repetitions of one-leg calf-raise exercise by the load corresponding to the half of their body weight. Muscle symptoms were evaluated by a visual analog scale (VAS). Blood samples were collected before and 2, 4, 24, 48, 72, and 96 h post-exercise. Plasma myomesin 3 fragments levels were significantly increased at 96 h after the eccentric exercise. The myomesin 3 fragments levels were correlated with other biomarkers of muscle damage and the muscle symptoms. These results suggest that the circulating myomesin 3 fragments levels are potential biomarkers reflecting eccentric exercise-induced muscle damage.

Skeletal muscle hypertrophy can be induced by hormones and growth factors acting directly as positive regulators of muscle growth or indirectly by neutralizing negative regulators, and by mechanical signals mediating the effect of resistance exercise. Muscle growth during hypertrophy is controlled at the translational level, through the stimulation of protein synthesis, and at the transcriptional level, through the activation of ribosomal RNAs and muscle-specific genes. mTORC1 has a central role in the regulation of both protein synthesis and ribosomal biogenesis. Several transcription factors and co-activators, including MEF2, SRF, PGC-1α4, and YAP promote the growth of the myofibers. Satellite cell proliferation and fusion is involved in some but not all muscle hypertrophy models.

<title>Abstract</title>An evolutionarily conserved mechanism, use of non-optimal codons, slows ribosomes during translation to allow proper folding of nascent polypeptides. However, until now, it was unknown whether any eukaryote-specific mechanisms exist for this purpose. Here, we propose that miRNAs slow translating ribosomes to prevent protein misfolding, with little negative effect on protein abundance. To prove this, we bioinformatically analyze ribosome profiling and miRNA binding sites and biochemically confirm that miRNA deficiency causes severe misfolding, which is rescued by slowing translating ribosomes. We demonstrate that non-cleaving shRNAs, targeting regions where elongation rates become faster in miRNA-deficient cells, improve protein folding with minimal effects on protein abundance. These results reveal broader functionality of miRNAs and a previously unknown mechanism to prevent protein misfolding.

<sec><title>One Sentence Summary</title>Eukaryote use of miRNAs prevents protein misfolding in a target-specific manner.

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The urinary level of the titin fragment has been considered a non-invasive and sensitive biomarker for muscle damage in clinical cases. However, there is little evidence regarding changes in the urinary titin fragment in response to exercise-induced muscle damage. In this study, we aimed to investigate whether the urinary titin fragment reflects the magnitude of muscle damage induced by two lower-limb eccentric exercises. In this study, healthy young male subjects performed drop jump (n=9) and eccentric ergometer exercise (n=9). Blood and urine samples were collected at various time points before and after the exercises. Although perceived muscle soreness assessed by sit-to-stand tasks was increased at 24h and 48h after both drop jump and the eccentric ergometer exercise groups, the pressure pain threshold was not changed. Changes of the urinary titin fragment, plasma myomesin 3 fragments, creatine kinase (CK), and myoglobin (Mb) after the eccentric exercises were increased but not statistically significant. Meanwhile, we found that the changes in the urinary titin fragment levels in response to both drop jump and the eccentric ergometer exercise were correlated with those of plasma CK and Mb levels. These results provide evidence that the urinary titin fragment level is a non-invasive biomarker reflecting the magnitude of eccentric exercise-induced muscle damage.

Skeletal muscle is a dynamic tissue with two unique abilities; one is its excellent regenerative ability, due to the activity of skeletal muscle-resident stem cells named muscle satellite cells (MuSCs); and the other is the adaptation of myofiber size in response to external stimulation, intrinsic factors, or physical activity, which is known as plasticity. Low physical activity and some disease conditions lead to the reduction of myofiber size, called atrophy, whereas hypertrophy refers to the increase in myofiber size induced by high physical activity or anabolic hormones/drugs. MuSCs are essential for generating new myofibers during regeneration and the increase in new myonuclei during hypertrophy; however, there has been little investigation of the molecular mechanisms underlying MuSC activation, proliferation, and differentiation during hypertrophy compared to those of regeneration. One reason is that `degenerative damage' to myofibers during muscle injury or upon hypertrophy (especially overloaded muscle) is believed to trigger similar activation/proliferation of MuSCs. However, evidence suggests that degenerative damage of myofibers is not necessary for MuSC activation/proliferation during hypertrophy. When considering MuSC-based therapy for atrophy, including sarcopenia, it will be indispensable to elucidate MuSC behaviors in muscles that exhibit non-degenerative damage, because degenerated myofibers are not present in the atrophied muscles. In this review, we summarize recent findings concerning the relationship between MuSCs and hypertrophy, and discuss what remains to be discovered to inform the development and application of relevant treatments for muscle atrophy.

Objectives A number of previous studies reported physiological responses and adaptations after eccentric muscle contraction of limb muscles. In contrast, no study has determined physiological response after eccentric contraction of trunk muscles. The purpose of the present study was to compare the functional and metabolic changes after eccentric or concentric exercises of trunk extensor muscles.Methods In this randomised, crossover study, 10 men performed a single bout of 50 maximal voluntary concentric and eccentric contractions of the trunk extensor with an interval of 2 weeks between bouts. The activities of the paraspinal muscles were recorded during concentric and eccentric contractions. Muscle soreness, muscle function, blood lipid profiles and glycaemic responses were measured before, immediately after and at 24, 48, 72 and 96 hours after each bout.Results The lumbar multifidus and iliocostalis lumborum activities during eccentric contractions were significantly higher than those during concentric contractions (p<0.05). The maximal strength and muscle endurance of the trunk extensor were not decreased even after the eccentric contractions. Compared with concentric contractions, muscle soreness was significantly increased at 24, 48, 72 and 96 hours after eccentric contractions (p<0.05). The TG, TC and LDL-C were significantly lower at 48, 72 and 96 hours after eccentric contractions (p<0.05), while blood glucose levels and HOMA-IR were significantly greater at 48 and 72 hours after eccentric contractions (p<0.05).Conclusion This study indicated that eccentric contractions of the trunk extensor had positive effects on the lipid profile and the glycaemic response.

Skeletal muscle has a remarkable regenerative capacity, which is orchestrated by multiple processes, including the proliferation, fusion, and differentiation of the resident stem cells in muscle. MicroRNAs (miRNAs) are small noncoding RNAs that mediate the translational repression or degradation of mRNA to regulate diverse biological functions. Previous studies have suggested that several miRNAs play important roles in myoblast proliferation and differentiation in vitro. However, their potential roles in skeletal muscle regeneration in vivo have not been fully established. In this study, we generated a mouse in which the Dicer gene, which encodes an enzyme essential in miRNA processing, was knocked out in a tamoxifen-inducible way (iDicer KO mouse) and determined its regenerative potential after cardiotoxin-induced acute muscle injury. Dicer mRNA expression was significantly reduced in the tibialis anterior muscle of the iDicer KO mice, whereas the expression of muscle-enriched miRNAs was only slightly reduced in the Dicer-deficient muscles. After cardiotoxin injection, the iDicer KO mice showed impaired muscle regeneration. We also demonstrated that the number of PAX7(+) cells, cell proliferation, and the myogenic differentiation capacity of the primary myoblasts did not differ between the wild-type and the iDicer KO mice. Taken together, these data demonstrate that Dicer is a critical factor for muscle regeneration in vivo.

Exercise training is considered an effective way to prevent age-related skeletal muscle loss. However, the molecular mechanism has not been clarified. Growth and differentiation factor 11 (GDF11) has been controversially considered a regulator of skeletal muscle aging. In this study, we examined whether GDF11 is associated with skeletal muscle aging and the effects of exercise training on age-related skeletal muscle loss. First, we observed that Gdf11 mRNA and protein expression levels in young (5-month-old, n = 6) and aged (22-to 26-month-old, n = 5) mice were not significantly different. Aged mice were then divided into sedentary (n = 5) and exercise (n = 6) groups. The exercise group performed moderate-intensity treadmill running for 6 weeks. Treadmill exercise training increased Gdf11 mRNA expression in the soleus muscle, but its protein expression was not altered. In contrast, the GDF11 level in the plantaris muscle was not changed at either the mRNA or protein level. Collectively, our data demonstrate that GDF11 levels do not change during aging, and that treadmill exercise training increased Gdf11 mRNA expression in a predominantly slow-twitch muscle.

The contractile and metabolic properties of adult skeletal muscle change in response to endurance exercise. The mechanisms of transcriptional regulation in exercise-induced skeletal muscle adaptation, including fiber-type switching and mitochondrial biogenesis, have been investigated intensively, whereas the role of microRNA (miRNA)-mediated posttranscriptional gene regulation is less well understood. We used tamoxifen-inducible Dicer1 knockout (iDicer KO) mice to reduce the global expression of miRNAs in adult skeletal muscle and subjected these mice to 2 wk of voluntary wheel running. Dicer mRNA expression was completely depleted in fast-twitch plantaris muscle after tamoxifen injection. However, several muscle-enriched miRNAs, including miR-1 and miR-133a, were reduced by only 30-50% in both the slow and fast muscles. The endurance exercise-induced changes that occurred for many parameters (i.e., fast-to-slow fiber-type switch and increases in succinate dehydrogenase, respiratory chain complex II. and citrate synthase activity) in wild type (WT) also occurred in the iDicer KO mice. Protein expression of myosin heavy chain Ha, peroxisome proliferator-activated receptor-gamma coactivator- 1 alpha, and cytochrome c complex IV was also increased in the iDicer KO mice by the voluntary running. Furthermore, there was no significant difference in oxygen consumption rate in the isolated mitochondria between the WT and iDicer KO mice. These data indicate that muscle-enriched miRNAs were detectable even after 4 wk of tamoxifen treatment and there was no apparent specific endurance-exercise-induced muscle phenotype in the iDicer KO mice.

MicroRNAs are small regulatory noncoding RNAs that repress gene expression at the posttranscriptional level. Previous studies have reported that the expression of miR-23, miR-27, and miR-24, driven from two miR-23-27-24 clusters, is altered by various pathophysiological conditions. However, their functions in skeletal muscle have not been clarified. To define the roles of the miR-23-27-24 clusters in skeletal muscle, we generated double-knockout (dKO) mice muscle-specifically lacking the miR-23-27-24 clusters. The dKO mice were viable and showed normal growth. The contractile and metabolic features of the muscles, represented by the expression of the myosin heavy chain and the oxidative markers PGC1-alpha and COX IV, were not altered in the dKO mice compared with wild-type mice. The dKO mice showed increased cross-sectional areas of the oxidative fibers. However, this dKO did not induce functional changes in the muscles. The dKO mice also showed normal adaptation to voluntary wheel running for 4 weeks, including the glycolytic-to-oxidative fiber type switch, and increases in mitochondrial markers, succinate dehydrogenase activity, and angiogenesis. In conclusion, our data demonstrate that the miR-23-27-24 clusters have subtle effects on skeletal muscle development and endurance-exercise-induced muscle adaptation.

The capillary network is distributed throughout the body. and its reconstruction is induced under various pathophysiological conditions. MicroRNAs are small noncoding RNAs that regulate gene expression via posttranscriptional mechanisms and are involved in many biological functions, including angiogenesis. Previous studies have shown that each microRNA of miR-23 clusters, composed of the miR-23a cluster (miR-23a-27a-24-2) and miR-23b cluster (miR-23b-27b-24-1), regulates angiogenesis in vitro. However, the role of miR-23 clusters. located within a single transcription unit, in angiogenesis in vivo has not been elucidated. In the present study. we generated vascular endothelial cell (EC)-specific miR-23 cluster double-knockout (DKO) mice and demonstrated sprouting angiogenesis under various conditions, including voluntary running exercise. hindlimb ischemia, skin wound healing, and EC sprouting from aorta explants. Here, we demonstrated that EC-specific miR-23 DKO mice are viable and fertile, with no gross abnormalities observed in pups or adults. The capillary number was normally increased in the muscles of these DKO mice in response to 2 wk of voluntary running and hindlimb ischemia. Furthermore, we did not observe any abnormalities in skin wound closure or EC sprouting from aortic ring explains in EC-specific miR-23 cluster DKO mice. Our results suggest that endothelial miR-23 clusters are dispensable for embryonic development and postnatal angiogenesis in vivo.NEW & NOTEWORTHY We generated vascular endothelial cell (EC)-specific miR-23a/b cluster double-knockout mice and determined sprouting angiogenesis under various conditions, including voluntary running exercise. hindlimb ischemia, skin wound healing, and EC sprouting from aorta explants. We demonstrated that the double-knockout mice were viable and fertile, with no gross abnormalities in exercise- and ischemia-induced angiogenesis and skin wound closure or EC sprouting from aortic ring explants.

骨格筋は、多様な外的刺激に適応し、自身の収縮・代謝特性を変化させる高い可塑性を有している。これまでの多くの研究から、転写コアクチベーターであるPeroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α)が、骨格筋の可塑性を制御する中心的な分子の1つであることが理解されている。PGC-1αは、筋ミトコンドリア新生などの収縮・代謝特性の制御に加え、種々の因子を介して骨格筋を栄養する毛細血管ネットワークの再構築にも関与するなど、その作用は広範にわたる。(著者抄録)

血管内皮機能は血圧の恒常性維持に重要な因子である。近年、血管内皮細胞におけるマイクロRNAが血管内皮機能を調節することが明らかにされている。しかしながら、血圧の恒常性維持におけるマイクロRNAの役割は不明である。そこで我々は、血管内皮細胞特異的Dicer欠損マウスを用いて、安静時および8週間の食塩水摂取後に血圧を測定することで、血圧調節における血管内皮細胞のマイクロRNAの役割を検討した。血管内皮細胞特異的Dicerホモ欠損マウスは胎生致死であったため、ヘテロ欠損マウス(Het)を用いて血行動態の解析を行った。Hetマウスの肺におけるDicer mRNA発現は、WTマウスと比較して有意に減少した。しかしながら、8週間の食塩水摂取の前後において、心拍数、収縮期血圧および拡張期血圧のいずれにも顕著な差は認められなかった。本研究において、血管内皮細胞特異的Dicer欠損マウスが胎生致死であることから、血管内皮細胞のDicerおよびマイクロRNAはマウス胚発生に必須であることが示された。加えて、血管内皮細胞特異的Dicerヘテロ欠損マウスでは、血行動態に顕著な表現型は観察されなかった。(著者抄録)

Many studies have attempted to determine the associations between blood biomarkers and exercise-induced muscle damage. However, poor correlations between the changes in biomarker levels and the magnitude of muscle symptoms have been reported. Recent advances in proteomic tools offer a strategy for the comprehensive analysis of protein expression, which can be used to identify biomarkers. Here, we used a proteomic analysis to identify urinary proteins that appear in response to a calf-raise exercise, including repetitive eccentric muscle contractions, and found that a titin (also known as connectin) N-terminal fragment molecule appears in the urine after eccentric exercise. We measured the titin fragment in urine samples from nine individuals before and after eccentric exercise using a newly-established enzyme-linked immunosorbent assay and found that the titin fragment excretion rate increased 96 h after the exercise (5.1 to 77.6 pg/min, p < 0.01). The changes in the titin fragment excretion rate were correlated strongly with blood markers of muscle damage and with muscle symptoms. These findings suggest that the urinary titin fragment is potentially a noninvasive biomarker of muscle damage.

It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid-induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid-induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15). Heat stress recovered the dexamethasone-induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress-induced protection against glucocorticoid-induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650-664, 2017. (c) 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

Introduction: Disuse-induced skeletal muscle atrophy is a serious concern; however, there is not an effective mouse model to elucidate the molecular mechanisms. We developed a noninvasive atrophy model in mice. Methods: After the ankle joints of mice were bandaged into a bilateral plantar flexed position, either bilateral or unilateral hindlimbs were immobilized by wrapping in bonsai steel wire. Results: After 3, 5, or 10 days of immobilization of the hip, knee, and ankle, the weight of the soleus and plantaris muscles decreased significantly in both bilateral and unilateral immobilization. MAFbx/atrogin-1 and MuRF1 mRNA was found to have significantly increased in both muscles, consistent with disuse-induced atrophy. Notably, the procedure did not result in either edema or necrosis in the fixed hindlimbs. Conclusions: This method allows repeated, direct access to the immobilized muscle, making it a useful procedure for concurrent application and assessment of various therapeutic interventions.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.

Large variability exists in muscle adaptive response to resistance exercise (RE) training between individuals. Recent studies have revealed a significant role for microRNAs (miRNAs) in skeletal muscle plasticity. In this study, we investigated how RE affects miRNA expression and whether the variability of muscle hypertrophy to RE training may be attributed to differential miRNA regulation in the skeletal muscle. To screen high and low responders to RE, we had 18 young men perform arm curl exercise training. After screening, all the men performed 12 wk of lower body RE training, but only the high or low responders participated in the acute RE test before training. Muscle biopsies were obtained from the vastus lateralis muscle at baseline, 3 h after acute RE, and after the training period. Total RNA was extracted from the skeletal muscle, and miRNA expression (800 miRNAs) was analyzed. RE training increased the cross-sectional area of the biceps brachii (-1.7-26.1%), quadriceps (2.2-16.8%), and hamstrings (1.6-18.4%). Eighty-five and 102 miRNAs were differentially expressed after acute and chronic RE, respectively (P < 0.05). Seventeen miRNAs, especially 23b-3p, 26a-5p, 32-5p, 148b-3p, and 376a-3p, were differentially expressed at baseline, and 23 miRNAs, especially let-7a-5p, 95, 148a-3p, and 376a-3p, and 26 miRNAs, especially 30d-5p and 376a-3p, were differentially regulated after acute and chronic RE, respectively, in the skeletal muscle between high and low responders, indicating that the expression patterns of several miRNAs are altered by acute or chronic RE, and that miRNAs are involved in skeletal muscle adaptation to RE training.

Reduced estrogen secretion and low calcium (Ca) intake are risk factors for bone loss and arterial calcification in female rodents. To evaluate the effects of Ca intake at different amounts on bone mass changes and arterial calcification, 8-wk-old female Wistar rats were randomly placed in ovariectomized (OVX) control and OVX with vitamin D-3 plus nicotine (VDN) treatment groups. The OVX with VDN rats were then divided into six groups to receive different amounts of Ca in their diets: 0.01%, 0.1%, 0.3%, 0.6%, 1.2%, or 2.4% Ca. After 8 wk of administration, low Ca intake groups with 0.01% and 0.1% Ca diets had significantly reduced bone mineral density (BMD) and bone mechanical properties as compared with those of the other groups, whereas high Ca intake groups with 1.2% and 2.4% Ca diets showed no differences as compared with the 0.6% Ca intake group. For both the 0.01% and 2.4% Ca intake groups, Ca levels in their thoracic arteries were significantly higher as compared with those of the 0.6% Ca diet group, and that was highly correlated with serum PTH levels. An increase in relative BMP-2 mRNA expression in the arterial tissues of the 0.01% and 2.4% Ca diet groups was also observed. These results suggested that extremely low Ca intake during periods of estrogen deficiency may be a possible risk for the complications of reduced BMD and arterial calcification and that extremely high Ca intake may promote arterial calcification with no changes in BMD.

Background: It has been reported that strenuous exercise increases the number of bone marrow-derived progenitor cells such as CD34(+) cells in the blood, but no previous studies have investigated the changes in circulating CD34(+) cells following resistance exercise. This study tested the hypothesis that the number of CD34(+) cells in the blood would increase after eccentric exercise of the elbow flexors, but decrease in recovery, and the magnitude of the changes would be dependent on the magnitude of muscle damage.Methods: Nine men (28.0 +/- 6.6 years) performed exercises consisting of 10 sets of six maximal voluntary eccentric contractions of the elbow flexors with their non-dominant arm. Six of them performed the same exercise with the same arm 4 weeks later. Changes in indirect markers of muscle damage were measured before, within 10 min after, and at 24, 48, 72, and 96 h after eccentric exercise. Differential leukocyte counts (total leukocytes, neutrophils, lymphocytes, monocytes) and CD34(+) cells in the blood were measured before, immediately after, and at 2, 24, 48, 72, and 96 h following the exercises.Results: After eccentric exercise, significant (p < 0.05) decreases in maximal voluntary isometric contraction torque and increases in delayed onset muscle soreness and plasma creatine kinase activity were observed. However, no significant changes in leukocytes and CD34(+) cells were evident. The changes in muscle damage markers were significantly (p < 0.05) smaller following the second exercise session as compared with the first exercise session, but the changes in leukocytes and CD34(+) cells were not significantly different between sessions.Conclusion: These results did not support the hypothesis, and showed that eccentric exercise-induced muscle damage to the elbow flexors did not influence the number of circulating CD34(+) cells. Copyright (C) 2014, Shanghai University of Sport. Production and hosting by Elsevier B.V. All rights reserved.

Mesenchymal stem cell (MSC) transplantation is used for treatment of many diseases. The paracrine role of MSCs in tissue regeneration is attracting particular attention. We investigate the role of MSC exosomes in skeletal muscle regeneration. MSC exosomes promote myogenesis and angiogenesis in vitro, and muscle regeneration in an in vivo model of muscle injury. Although MSC exosomes had low concentrations of muscle-repair-related cytokines, a number of repair-related miRNAs were identified. This study suggests that the MSC-derived exosomes promote muscle regeneration by enhancing myogenesis and angiogenesis, which is at least in part mediated by miRNAs such as miR-494. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Skeletal muscles contain several subtypes of myofibers that differ in contractile and metabolic properties. Transcriptional control of fiber-type specification and adaptation has been intensively investigated over the past several decades. Recently, microRNA (miRNA)-mediated posttranscriptional gene regulation has attracted increasing attention. MiR-23a targets key molecules regulating contractile and metabolic properties of skeletal muscle, such as myosin heavy-chains and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1 alpha). In the present study, we analyzed the skeletal muscle phenotype of miR-23a transgenic (miR-23a Tg) mice to explore whether forced expression of miR-23a affects markers of mitochondrial content, muscle fiber composition, and muscle adaptations induced by 4 weeks of voluntary wheel running. When compared with wild-type mice, protein markers of mitochondrial content, including PGC-1 alpha, and cytochrome c oxidase complex IV (COX IV), were significantly decreased in the slow soleus muscle, but not the fast plantaris muscle of miR-23a Tg mice. There was a decrease in type IId/x fibers only in the soleus muscle of the Tg mice. Following 4 weeks of voluntary wheel running, there was no difference in the endurance exercise capacity as well as in several muscle adaptive responses including an increase in muscle mass, capillary density, or the protein content of myosin heavy-chain IIa, PGC-1 alpha, COX IV, and cytochrome c. These results show that miR-23a targets PGC-1 alpha and regulates basal metabolic properties of slow but not fast twitch muscles. Elevated levels of miR-23a did not impact on whole body endurance capacity or exercise-induced muscle adaptations in the fast plantaris muscle.

The pathogenesis of bone disorders in young male athletes has not been well understood. We hypothesized that bone fragility is caused by low energy availability, due to insufficient food intake and excessive exercise energy expenditure in young male athletes. To examine this hypothesis, we investigated the influence of food restriction on bone strength and bone morphology in exercised growing male rats, using three-point bending test, dual-energy X-ray absormetry, and micro-computed tomography. Four-week-old male Sprague-Dawley rats were divided randomly into the following groups: the control (Con) group, exercise (Ex) group, food restriction (R) group, and food restriction plus exercise (REx) group after a 1-wk acclimatization period. Thirty-percent food restriction in the R and REx groups was carried out in comparison with that in the Con group. Voluntary running exercise was performed in the Ex and REx groups. The experimental period lasted 13 wk. At the endpoint of this experiment, the bone strength of the femurs and tibial BMD in the REx group were significantly lower than those in the Con group. Moreover, trabecular bone volume and cortical bone volume in the REx group were also significantly lower than those in the Con group. These findings indicate that food restriction causes low bone strength and microarchitectural deterioration in exercised growing male rats.

Hypoxia is an important modulator of endurance exercise-induced oxidative adaptations in skeletal muscle. However, whether hypoxia affects resistance exercise-induced muscle adaptations remains unknown. Here, we determined the effect of resistance exercise training under systemic hypoxia on muscular adaptations known to occur following both resistance and endurance exercise training, including muscle cross-sectional area (CSA), one-repetition maximum (1RM), muscular endurance, and makers of mitochondrial biogenesis and angiogenesis, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), citrate synthase (CS) activity, nitric oxide synthase (NOS), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF-1), and capillary-to-fiber ratio. Sixteen healthy male subjects were randomly assigned to either a normoxic resistance training group (NRT, n = 7) or a hypoxic (14.4% oxygen) resistance training group (HRT, n = 9) and performed 8 weeks of resistance training. Blood and muscle biopsy samples were obtained before and after training. After training muscle CSA of the femoral region, 1RM for bench-press and leg-press, muscular endurance, and skeletal muscle VEGF protein levels significantly increased in both groups. The increase in muscular endurance was significantly higher in the HRT group. Plasma VEGF concentration and skeletal muscle capillary-to-fiber ratio were significantly higher in the HRT group than the NRT group following training. Our results suggest that, in addition to increases in muscle size and strength, HRT may also lead to increased muscular endurance and the promotion of angiogenesis in skeletal muscle.

Athletes, in particular endurance athletes and dancers, are chronically exposed to a state of low energy availability due to insufficient dietary energy intake and massive exercise energy expenditure. Low energy availability sometimes causes bone fragility, thereby increasing the risk of bone disorders. Although the decrease in energy availability shows no sexual dimorphism, epidemiological studies have reported that bone disorders are less frequent in male athletes than in female athletes. We hypothesized that bone tissue was not affected by low energy availability in males. The purpose of this study was to examine the influence of food restriction combined with voluntary running training on bone morphology and strength in adult male rats. Fourteen-week-old male Sprague-Dawley rats were divided randomly into four groups: control (C) group, food restriction (R) group, exercise (Ex) group, and food restriction plus exercise (REx) group. For the R and REx groups, 30 % food restriction was carried out in comparison with the C group. Bone strength, bone mineral density (BMD), bone architecture, and bone turnover rate were measured after a 13-week experimental period. Bone strength was not significantly lower in the REx group compared with the C group. BMD and trabecular bone volume showed no difference among groups. These findings indicate that bone morphology and strength were little affected by food restriction combined with exercise training in adult male rats.

center dot The discovery of microRNAs (miRNAs) has established new mechanisms that control health, but little is known about the regulation of skeletal muscle miRNAs in response to exercise.center dot This study investigated components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and 206), and several miRNAs dysregulated in muscle myopathies, and showed that 3 h following an acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and miR-181a were all increased, while miR-9, -23a, -23b and -31 were decreased.center dot Short-term training increased miR-1 and miR-29b, while miR-31 remained decreased.center dot Negative correlations were observed between miR-9 and HDAC4 protein, miR-31 and HDAC4 protein and between miR-31 and NRF1 protein, 3 h after exercise.center dot miR-31 binding to the HDAC4 and NRF1 3 untranslated region (UTR) reduced luciferase reporter activity.center dot Exercise rapidly and transiently regulates several miRNA species potentially involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis.Abstract The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and -206), and several miRNAs dysregulated in muscle myopathies (miR-9, -23, -29, -31 and -181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate-intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and -181a were all increased. In contrast miR-9, -23a, -23b and -31 were decreased. Short-term training increased miR-1 and -29b, while miR-31 remained decreased. Negative correlations were observed between miR-9 and HDAC4 protein (r=-0.71; P= 0.04), miR-31 and HDAC4 protein (r =-0.87; P= 0.026) and miR-31 and NRF1 protein (r =-0.77; P= 0.01) 3 h following exercise. miR-31 binding to the HDAC4 and NRF1 3 untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise-mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health.

Recent studies have revealed a new aspect of physiological regulation in which microRNAs (miRNAs) play fundamental roles in diverse biological and pathological processes. Furthermore, it was recently discovered that miRNAs are stably secreted into blood and that circulating miRNAs may play important roles in cell-cell communication. Here, we examined whether the circulating miRNA profile is affected by acute resistance exercise. Twelve males performed a resistance exercise session (bench press and leg press), consisting of five sets of 10 repetitions at 70% of maximum strength, with a 1 min rest between sets. Blood samples were taken before exercise, and at 0 and 60 min, 1 day, and 3 days after exercise. The circulating miRNA profile was determined by microarray analysis. Quantitative real-time PCR confirmed that the miR-149* level increased three days after resistance exercise. In contrast, the miR-146a and miR-221 levels decreased three days after resistance exercise. Our findings suggest that circulating miRNA levels change in response to acute resistance exercise, and miRNAs may play important roles in resistance-exercise-induced adaptation.

Akimoto T, Okuhira K, Aizawa K, Wada S, Honda H, Fukubayashi T, Ushida T. Skeletal muscle adaptation in response to mechanical stress in p130cas(-/-) mice. Am J Physiol Cell Physiol 304: C541-C547, 2013. First published January 16, 2013; doi: 10.1152/ajpcell.00243.2012.-Mammalian skeletal muscles undergo adaptation in response to changes in the functional demands upon them, involving mechanical-stress-induced cellular signaling called "mechanotransduction." We hypothesized that p130Cas, which is reported to act as a mechanosensor that transduces mechanical extension into cellular signaling, plays an important role in maintaining and promoting skeletal muscle adaptation in response to mechanical stress via the p38 MAPK signaling pathway. We demonstrate that muscle-specific p130Cas(-/-) mice express the contractile proteins normally in skeletal muscle. Furthermore, muscle-specific p130Cas(-/-) mice show normal mechanical-stress-induced muscle adaptation, including exercise-induced IIb-to-IIa muscle fiber type transformation and hypertrophy. Finally, we provide evidence that exercise-induced p38 MAPK signaling is not impaired by the muscle-specific deletion of p130Cas. We conclude that p130Cas plays a limited role in mechanical-stress-induced skeletal muscle adaptation.

Low calcium (Ca) intake is the one of risk factors for both bone loss and medial elastocalcinosis in an estrogen deficiency state. To examine the effect of different amounts of Ca intake on the relationship between bone mass alteration and medial elastocalcinosis, 6-wk-old female SD rats were randomized into ovariectomized (OVX) control or OVX treated with vitamin D-3 plus nicotine injection (VDN) groups. The OVX treated with VDN group was then divided into 5 groups depending on the different Ca content in their diet, 0.01%, 0.1%, 0.6%, 1.2%, and 2.4% Ca intakes. After 8 wk of experimentation, the low Ca intake groups of 0.01% and 0.1% showed a low bone mineral density (BMD) and bone properties significantly different from those of the other groups, whereas the high Ca intake groups of 1.2% and 2.4% showed no difference compared with the OVX control. Only in the 0.01% Ca intake group, a significantly higher Ca content in the thoracic artery was found compared with that of the OVX control. Arterial tissues of the 0.01% Ca intake group showed an increase of bone-specific alkaline phosphatase (BAP) activity, a marker of bone mineralization, associated with arterial Ca content. However, the high Ca intake did not affect arterial Ca content nor arterial BAP activity. These results suggested that a low Ca intake during periods of rapid bone loss caused by estrogen deficiency might be one possible cause for the complication of both bone loss and medial elastocalcinosis.

Skeletal muscle mitochondrial dysfunction is believed to play a role in the progression and severity of amyotrophic lateral sclerosis (ALS). The regulation of transcriptional co-activators involved in mitochondrial biogenesis and function in ALS is not well known. When compared with healthy control subjects, patients with ALS, but not neurogenic disease (ND), had lower levels of skeletal muscle peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) mRNA and protein and estrogen-related receptor-alpha (ERR alpha) and mitofusin-2 (Mfn2) mRNA PGC-1 beta, nuclear respiratory factor-1 (NRF-1) and Mfn1 mRNA as well as cytochrome C oxidase subunit IV (COXIV) mRNA and protein were lower in patients with ALS and ND. Both patient groups had reductions in citrate synthase and cytochrome c oxidase activity. Similar observations were made in skeletal muscle from transgenic ALS G93A transgenic mice. In vitro, PGC-1 alpha and PGC-1 beta regulated Mfn1 and Mfn2 in an ERR alpha-dependent manner. Compared to healthy controls, miRNA 23a, 29b, 206 and 455 were increased in skeletal muscle of ALS patients. miR-23a repressed PGC-1 alpha translation in a 3' UTR dependent manner. Transgenic mice over expressing miR-23a had a reduction in PGC-1 alpha, cytochome-b and COXIV protein levels. These results show that skeletal muscle mitochondrial dysfunction in ALS patients is associated with a reduction in PGC-1 alpha signalling networks involved in mitochondrial biogenesis and function, as well as increases in several miRNAs potentially implicated in skeletal muscle and neuromuscular junction regeneration. As miR-23a negatively regulates PGC-1 alpha signalling, therapeutic inhibition of miR-23a may be a strategy to rescue PGC-1 alpha activity and ameliorate skeletal muscle mitochondrial function in ALS. (C) 2012 Elsevier Inc. All rights reserved.

Nakamura, D, Suzuki, T, Yasumatsu, M, and Akimoto, T. Moderate running and plyometric training during off-season did not show a significant difference on soccer-related high-intensity performances compared with no training controls. J Strength Cond Res 26(12): 3392-3397, 2012-Several investigators have reported the effects of reduced training and interrupted training on athletic performance, but few reports are available for soccer players. The purpose of this study was to examine, using the Yo-Yo intermittent recovery level 2 (YoYoIR2) test and sprint performance, the effects on soccer players of a reduced training program consisting of either moderate running training, plyometric training. After the completion of a competitive season, 29 male soccer players were divided into 3 groups: the running group (n = 13), the plyometric group (n = 11), and the control group (n = 5). Both training groups completed either running or plyometric training sessions 2 d.wk(-1) for 3 weeks, whereas the control group was not allowed to perform any training. The subjects performed YoYoIR2 and 20-m sprint tests before (pre) and after (post) the experimental period. Neither training group showed any significant training effects on the YoYoIR2 performance or 20-m sprint times compared with the control group. This study suggests that neither endurance running nor plyometric training 2 d.wk(-1) for 3 weeks has a significant effect on high-intensity performance compared with a nontraining regimen. However, our results do not support complete inactivity. These results may have important implications for the management of training cessation for a few weeks.

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. (C) 2012 Elsevier Inc. All rights reserved.

AIZAWA, K., M. IEMITSU, S. MAEDA, N. MESAKI, T. USHIDA, and T. AKIMOTO. Endurance Exercise Training Enhances Local Sex Steroidogenesis in Skeletal Muscle. Med. Sci. Sports Exerc., Vol. 43, No. 11, pp. 2072-2080, 2011. Purpose: Endurance training improves skeletal muscular function including energy metabolism and structure. Sex steroid hormones partly contribute to the exercise-induced muscular adaptations. Recently, we demonstrated that skeletal muscle contains steroidogenic converting enzymes to synthesize sex steroid hormones and an acute endurance exercise activates local steroidogenesis in skeletal muscle. However, whether chronic endurance training leads to enhanced steroidogenesis in skeletal muscle is unknown. Here, we examined changes in steroidogenic enzymes and sex steroid hormones in the skeletal muscle after chronic endurance exercise training. Methods: Eleven male rats were divided into two groups: sedentary (n = 6) and trained (n = 5). Endurance training was performed on a treadmill (30 m.min(-1), 30 min) for 5 d.wk(-1) for 12 wk. The posttraining harvesting was performed 48 h after the last exercise training. Results: The mRNA expressions of 3 beta-HSD, aromatase cytochrome P450, and 5 alpha-reductase in the skeletal muscle of trained rats were significantly higher than those of sedentary rats (P < 0.05). The protein expressions of aromatase cytochrome P450 and 5 alpha-reductase in the skeletal muscle of trained rats were also significantly higher than those of sedentary rats (P < 0.05). The muscular dihydrotestosterone (DHT) concentrations in the skeletal muscle of trained rats were significantly higher than those of sedentary rats (P < 0.01), but there was no change in dehydroepiandrosterone, total testosterone, free testosterone, and estradiol. Furthermore, muscle weight corrected for body weight of trained rats was moderately correlated with the level of muscular DHT concentration in trained rats (r = 0.41, P < 0.05). Conclusions: Endurance exercise training enhances the muscular DHT concentration through 5 alpha-reductase in the skeletal muscle of rats, suggesting that local bioactive androgen metabolism may participate in exercise training-induced skeletal muscular adaptation.

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.

It is not known whether local androgen metabolism is involved in the mechanisms underlying the dehydroepiandrosterone (DHEA) administration-induced improvement of bone mineral density (BMD) in an estrogen-deficiency state. The aim of the present study was to clarify whether DHEA administration would improve local androgen metabolism and BMD in cancellous site of tibia of ovariectomized (OVX) rats. Twenty-two female rats, 6 weeks old, were randomized into three groups: sham-operated rats, OVX control rats, and OVX rats that received DHEA treatment. DHEA was administered intraperitoneally at 20 mg/kg body weight for 8 weeks. The concentrations of free testosterone and dihydrotestosterone (DHT) in cancellous site of tibia did not change as a result of ovariectomy, while the DHT concentration increased following DHEA administration. We revealed that DHEA administration improved the reduction of 17 beta- and 3 beta-hydroxysteroid dehydrogenases and clearly reversed the reduction of 5 alpha-reductase types 1 and 2 and androgen receptor in the cancellous site of tibia of OVX rats. DHEA administration suppressed estrogen deficiency relative to the decrease in the cancellous BMD, which was positively associated with local DHT concentration. These findings indicate that DHEA administration enhances local bioactive androgen metabolism in the cancellous tibia of young OVX rats, suggesting that local DHT may play a part in the DHEA administration-induced improvement of cancellous BMD.

Preventing skeletal muscle atrophy is critical for maintaining quality of life, but it is often a challenging goal for the elderly and patients with severe conditions. We hypothesized that acupuncture in place of exercise training is an alternative non-pharmacological intervention that can help to prevent muscle atrophy. To elucidate the effects of acupuncture on skeletal muscle atrophy caused by hindlimb suspension (HS), we performed acupuncture on mice according to two different methods: acupuncture with electrical stimulation (EA: electroacupuncture) and without electrical stimulation (MA: manual acupuncture). A needle was retained in the gastrocnemius muscle for 30 min every day for 2 weeks in the EA and MA groups. In the EA group, 30 min of repetitive electrical stimulation (1 Hz, 1 ms pulse width, 6.5 m]A intensity) was also applied. HS significantly reduced muscle mass and the cross-sectional area of the soleus muscles. This HS-induced reduction was significantly improved in the EA group, although the level of improvement remained insufficient when compared with the control group. We found that the mRNA expression levels of atrogin-1 and MuRF1, which play a principal role in muscle-specific degradation as E3 ubiquitin ligases, were significantly increased in the HS group compared to the control group. EA and MA reduced the HS-induced upregulation of atrogin-1 (p < 0.01 in EA and MA) and MuRF1 (p < 0.01 in EA) mRNAs. We also found that the expression levels of PI3K, Akt1, TRPV4, adenosine A1 receptor, myostatin, and SIRT1 mRNAs tended to be increased by HS. EA and MA further increased the HS-induced upregulation of Akt1 (p < 0.05 in MA) and TRPV4 (p < 0.05 in MA) mRNAs. We concluded that acupuncture partially prevented skeletal muscle atrophy. This effect might be due to an increase in protein synthesis and a decrease in protein degradation. (C) 2011 Elsevier Inc. All rights reserved.

Introduction of membrane-impermeant substances into living cells is the key method to understand contemporary cellular processes by investigating cellular responses and phenotypes. Here, we performed gold ion beam exposure into live cells by using the focused ion beam implantation method, which was originally developed to precisely control semiconductor device performances. We evaluated the viability of the gold-irradiated cells by measuring the concentration of adenosine triphosphate (ATP), which is an intracellular energy source produced in the mitochondrial membrane. The viability of the irradiated cells was found to be 20% higher than that of the unirradiated control cells. The atoms might promote the energy generating processes within the mitochondrion. Our results suggest that the viability of living cells can be modulated by accurately controlling the dopant atom numbers. Our technique may be considered as a potential tool in life and medical sciences to quantitatively elucidate the dose-dependent effects of dopants. Biotechnol. Bioeng. 2011;108: 222-225. (C) 2010 Wiley Periodicals, Inc.

The purpose of this study was to investigate the relation between alterations of salivary secretory immunoglobulin A (SIgA) and the occurrence of upper respiratory tract infections (URTI) in rugby football players.We examined the relationship between the onset of URTI and the daily alterations of SIgA. levels in 32 male collegiate rugby football players (20.5 +/- 1.3 years) during Summer training camp for 36 days.Total of 6 in 32 subjects had the appearance of URTI symptoms (18%). SIgA secretion rate decreased significantly in the middle of training camp compared to the baseline (P < 0.05). Furthermore, SIgA secretion rate during the appearance of URTI (13.7 +/- 1.1 mu g/min) were significantly lower than that without symptoms (19.2 +/- 1.4 mu g/min, P < 0.01). These results suggest that serial monitoring of SIgA may be useful to assess the risk status of URTI affection in athletes.

Endurance exercise promotes mitochondrial biogenesis in skeletal muscle possibly by inducing peroxisome proliferator-activated receptor gamma coactivator la gene transcription through activation of the p38 mitogen-activated protein kinase pathway. A novel imaging analysis revealed the importance of activating transcription factor 2 and histone deacetylase 5/myocyte enhancer factor 2 in the regulation of the Pgc-1 alpha gene in skeletal muscle in vivo.

The influence of age and gender on salivary secretory immunoglobulin A (SIgA) in response to moderate exercise training was studied in 158 elderly subjects, Subjects were assigned to an exercise training group (EXC: 51 males, 74 females) or a non-exercise control group (CON: 11 males, 22 females). The subjects in each group were separated into four age-gender subgroups (60-69-yr-old males, over 70-yr-old males, 60-69-yr-old females, over 70-yr-old females) and compared by age and gender Subjects in EXC participated in exercise sessions 5-days a week for 6 months. Saliva samples were collected both before and after the study period. The SIgA secretion rates were significantly increased after training (p < 0.05) in all the age-gender subgroups of EXC (60-69 males: 41%, over 70 males: 55%, 60-69 females: 40%, over 70 females: 38%); no age- or gender-related differences were observed. On the other hand, none of the age-gender subgroups of CON showed significant changes in the SIgA secretion rate; also, there were no age- or gender-related differences. In conclusion, enhancement of mucosal immune function following regular moderate exercise training occurs in elderlies in their 60s and over 70 years, and in both, males and females.

Skeletal muscle undergoes active remodeling in response to endurance exercise training, and the underlying mechanisms of this remodeling remain to be defined fully. We have recently obtained evidence that voluntary running induces cell cycle gene expression and cell proliferation in mouse plantaris muscles that undergo fast-to-slow fiber-type switching and angiogenesis after long-term exercise. To ascertain the functional role of cell proliferation in skeletal muscle adaptation, we performed in vivo 5-bromo-2'-deoxyuridine (BrdU) pulse labeling (a single intraperitoneal injection), which demonstrated a phasic increase (5- to 10-fold) in BrdU-positive cells in plantaris muscle between days 3 and 14 during 4 wk of voluntary running. Daily intraperitoneal injection of BrdU for 4 wk labeled 2.0% and 15.4% of the nuclei in plantaris muscle in sedentary and trained mice, respectively, and revealed the myogenic and angiogenic fates of the majority of proliferative cells. Ablation of resident stem cell activity by X-ray irradiation did not prevent voluntary running-induced increases of type IIa myofibers and CD31-positive endothelial cells but completely blocked the increase in muscle mass. These findings suggest that resident stem cell proliferation is not required for exercise-induced type IIb-to-IIa fiber-type switching and angiogenesis but is required for activity-dependent muscle growth. The origin of the angiogenic cells in this physiological exercise model remains to be determined.

Mammalian skeletal muscles undergo adaptation in response to alteration in functional demands by means of a variety of cellular signaling events. Previous experiments in transgenic mice showed that an active form of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is capable of stimulating peroxisome proliferator-activated receptor gamma-coactivator 1alpha (PGC-1alpha) gene expression, promoting fast-to-slow fiber type switching and augmenting mitochondrial biogenesis in skeletal muscle. However, a role for endogenous CaMKIV in skeletal muscle has not been investigated rigorously. We report that genetically modified mice devoid of CaMKIV have normal fiber type composition and mitochondrial enzyme expression in fast-twitch skeletal muscles and responded to long-term ( 4 wk) voluntary running with increased expression of myosin heavy chain type IIa, myoglobin, PGC-1alpha, and cytochrome c oxidase IV proteins in plantaris muscle in a manner similar to that of wild-type mice. Short-term motor nerve stimulation ( 2 h at 10 Hz) likewise increased PGC-1alpha mRNA expression in tibialis anterior muscles in both Camk4(-/-) and wild-type mice. In addition, we have confirmed that no detectable CaMKIV protein is expressed in murine skeletal muscle. Thus CaMKIV is not required for the maintenance of slow-twitch muscle phenotype and endurance training-induced mitochondrial biogenesis and IIb-to-IIa fiber type switching in murine skeletal muscle. Other protein kinases sharing substrates with constitutively active CaMKIV may function as endogenous mediators of activity-dependent changes in myofiber phenotype.

In response to sustained increase in contractile activity, mammalian skeletal muscle undergoes adaptation with enhanced mitochondrial biogenesis and fiber type switching. The peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was recently identified as a key regulator for these adaptive processes. To investigate the sequence elements in the PGC-1alpha gene that are responsible for activity-dependent transcriptional activation, we have established a unique system to analyze promoter activity in skeletal muscle of living mice. Expression of PGC-1alpha-firefly luciferase reporter gene in mouse tibialis anterior muscle transfected by electric pulse-mediated gene transfer was assessed repeatedly in the same muscle by using optical bioluminescence imaging analysis before and after low-frequency ( 10 Hz) motor nerve stimulation. Nerve stimulation ( 2 h) resulted in a transient 3-fold increase ( P &lt; 0.05) in PGC-1&alpha; promoter activity along with a 1.6-fold increase (P &lt; 0.05) in endogenous PGC-1alpha mRNA. Mutation of two consensus myocyte enhancer factor 2 (MEF2) binding sites ( - 2901 and - 1539) or a cAMP response element (CRE) ( - 222) completely abolished nerve stimulation-induced increase in PGC-1alpha promoter activity. These findings provide direct evidence that contractile activity-induced PGC-1alpha promoter activity in skeletal muscle is dependent on the MEF2 and the CRE sequence elements. The experimental methods used in the present study have general applicability to studies of gene regulation in muscle.

The purpose of this study was to investigate differences between underwater and land-based exercise in leg muscle activity. Nine healthy males (mean age : 21.7 +/- 0.5 years, mean height : 173.4 +/- 2.2 cm) had electrodes placed on their left leg muscles (Tibialis Anterior; TA, Medial Gastrocnemius; MG, Soleus; SOL, Rectus Femoris; RF, and Biceps Femoris; BF), and their muscle activity was measured during various exercises. The subjects performed six types of exercise such as the forward walk, backward walk, squat, calf raise, leg range, and one leg wave, both in the water (waist level) and on land. These exercises were categorized as 3 types of leg movement according to direction; horizontal, vertical, and mixed movement. In the forward walk and backward walk, categorized as horizontal movements, the integrated electromyogram (IEMG) significantly increased during underwater exercise compared with on land. In the squat, as a vertical movement, the IEMG showed a similar change under both conditions. In the calf raise, as a vertical movement, and leg range and one leg wave, as mixed movements, the IEMG significantly decreased during underwater exercise compared with on land. These results suggest that leg muscle activity during underwater exercise is different based on the movement direction of the legs. In a word, it was apparent that movement in a horizontal direction underwater provides greater activity for leg muscles than on land; but movement in a vertical and/or mixed direction underwater provides less activity for leg muscles than on land. In prescribing an exercise program, it may be helpful to understand the differences between underwater and land-based exercise in leg muscle activity.

[Objective] The aim of this investigation was to evaluate salivary dehydroepiandrosterone (DHEA) responses during a competitive period among female football players.
[Methods] Subjects were college female football players (n=9). Saliva and blood samples were collected at 18 : 00 (6 : 00 pm) and the profile of mood state (POMS) was recorded during a period of normal training (Pre), 3 days of competition (Competition), and a recovery period (Post). Levels of salivary DHEA, cortisol, and serum creatin kinase (CK), urea nitorogen (UN), were determined.
[Results] The levels of salivary DHEA significantly increased during competition (2 nd days) compared-with Pre (p&lt;0.05), and significantly decreased after competition compared with Pre (p&lt; 0.05). The levels of salivary cortisol significantly increased during the competition (2 nd and 3 rd days) compared with Pre (p&lt;0.05). Whereas it decreased after competition compared with Pre. The levels of serum CK significantly increased during the competition (2 nd days) compared with Pre (P&lt;0.05). The levels of serum UN did not change during the study. The fatigue score-of POMS significantly increased during competition (2 nd days) compared with Pre (P&lt;0.05).
[Conclusion] These data suggest that DHEA could be a useful endocrinological indicator for evaluating training status in female athletes.

This study examined changes among young females of resting serum dehydroepiandrosterone sulfate (DHEAS) concentration after an 8-week period of resistance training. Nineteen healthy untrained young females [training group: age 18.9 (0.3) years, n=10, control group: age 19.3 (1.0) years, n=9; mean (SD)] were recruited in this study. The training group participated in an 8-week resistance training program (2 days per week on nonconsecutive days). The control group did not involve in any resistance training or regular exercise during the study period. Muscular strength, anthropometry, and resting hormonal levels were measured before and after training in both groups. Serum concentrations of DHEAS, dehydroepiandrosterone (DHEA), testosterone and cortisol were measured by radioimmunoassay. Body mass (2.4%) and lean body mass (2.4%) were significantly increased in the training group (P&lt;0.05), but not in the control group. The training also significantly increased one-repetition maximum (1-RM) values (P&lt;0.05). In the training group, resting concentration of serum DHEAS significantly increased after training (P&lt;0.05). Percent change of DHEAS in the training group was greater than that of the control group (P&lt;0.05). In the training group, the change of DHEAS level was positively correlated with the change of lean body mass during the training (r=0.61; P&lt;0.05). Serum DHEA, testosterone and cortisol concentrations did not change in either group during the training. The dramatic increase of resting serum DHEAS concentration after training indicates that DHEAS might be an anabolic hormone marker of adaptation to resistance training among young females. Results are presented as mean (SD).

筋力の向上に効果的な運動としてレジスタンストレーニングが知られている。近年では、加齢に伴う筋機能の低下を予防する目的でレジスタンス運動が広く用いられている。それゆえ、レジスタンス運動による筋機能向上のメカニズムを明らかにすることは、生涯スポーツの観点からもとくに重要と考えられる。レジスタンス運動は血中アンドロゲン（男性ホルモン）を増大させ、筋肥大に働くことは知られている。しかし、男性と比べて血中アンドロゲンレベルが絶対的に少ない女性や高齢者では、運動による応答性はみられない多く報告がある。本研究では、女性の筋機能の向上に関与するアンドロゲンとして、デヒドロエピアンドロステロン（ＤＨＥＡ）に着目した。若年女性を対象に8週間のレジスタンストレーニングを施行したところ、トレーニング後に血中ＤＨＥＡ濃度が明らかに増大し、筋肥大率と正の相関関係を示した。以上の結果により、トレーニングにより血中ＤＨＥ

[Objective] The purpose of this study was to determine whether icing treatment has an effect on muscle reaction time and functional performance of a sprained ankle. [Methods] Subjects were six persons (healthy group) and six persons who had an acute ankle sprain (patient group). Anterior talar translation and talar tilt of the six patient group were evaluated by the stress X-ray measure. Ice treatment was applied for 20 minutes. [Results] The following points were clarified : 1) Reaction times of peroneus longus (PL) and peroneus brevis (PB) for the sprained ankle group (SA) were significantly longer than those for the non-sprained ankle group (NA) (p&lt;0.05). Length of time of standing on one leg with closed eyes for SA was significantly shorter than for NA (p&lt;0.05). 2) After icing treatment, reaction time of PL for SA tended to be shorter. Furthermore, PB significantly decreased after icing treatment (p&lt;0.05). Side-steps for SA were significantly increased after icing treatment (p&lt;0.05). 3) Reaction times of PL and PB for NA significantly increased after icing treatment (p&lt;0.05). Standing on one leg with closed eyes for NA significantly decreased after icing treatment (p&lt;0.05). [Conclusion] It was shown that icing treatment of a sprained ankle leads to a shorter muscle reaction time and an increase in the number of side-steps. Therefore, it was concluded that icing treatment of a sprained ankle might be able to improve incapacitated neuromuscular function and functional performance by acute trauma.

PURPOSE: We examined the levels of autoantibodies against prethrombin-1 and fragment-1 in pregnant women to determine the type of autoantibodies that were associated with severe, preeclampsia or spontaneous abortion.
SUBJECTS AND METHODS: We measured autoantibodies bound to prethrombin-1, prethromhin-1, and fragment-1 by using an enzyme-linked immunosorbent assay (ELISA) in 12 healthy nonpregnant women, 36 women with normal pregnancies, 28 pregnant women with severe preeclampsia, and 19 pregnant women who subsequently had spontaneous abortion.
RESULTS: Plasma samples in 10 (36%) of the 28 women with severe preeclampsia and 11 (58%) of the 19 women with spontaneous abortion were positive for antiprothrombin antibodies as compared with 3 (9%) of the 36 women with normal pregnancies. AU II of the positive samples from women who had spontaneous abortions were positive for antiprethrombin-1 antibody, but only 1 was positive for antifragment-1 antibody. The mean ( +/- SD) titer of antiprethrombin-1 1 antibodies in patients with spontaneous abortion (36 +/- 9 U) was higher than that in women with normal pregnancies (10 +/- 4 U; P &lt; 0.01). Antiprethrombin-1 antibody was detected in only 28 women with severe preeclampsia, whereas all 10 women with antiprethrombin antibodies were positive for antifragment-1 antibody. The antifragment-1 antibody titer in patients with severe pre eclampsia (49 +/- 15 U) was higher than in women with normal pregnancies(l3 +/- 6 U, P &lt;0.01).
CONCLUSIONS: There is a strong and specific association between various types of antiprothrombin antibodies with severe preeclampsia and spontaneous abortion. (C)2001 by Excerpta Medica, Inc.

Interleukin-12 (IL-12) is a cytokine that was originally identified as natural killer cell stimulatory factor. It induces the activity of T-helper 1 (Th1) cells and exhibits strong anti-tumor activity. In this study, we studied the effects of brief anaerobic maximal exercise on circulating levels of IL-12. Six healthy males [mean (SD) 25.2 (2.6) years] performed a modified Wingate test exercise (resistance 0.075 kg/kg of body mass). The exercise consisted of five bouts of maximal cycling for 10 s, with rest intervals of 50 s between them. Blood samples were taken before, immediately after, 30 min after, 60 min after and 120 min after the exercise. Plasma concentrations of IL-12 were measured using an enzyme-linked immunosorbent assay. Data were corrected for hemoglobin and hematocrit measurements. Plasma concentrations of IL-12 averaged [mean (SD)] 234.2 (40.9) pg/ml before, 305.2 (62.1) pg/ml immediately after, 202.8 (24.2) pg/ml 30 min after, 239.7 (35.1) pg/ml 60 min after, and 199.6 (49.2) pg/ml 120 min after the exercise. We showed that plasma concentrations of IL-12 increased significantly immediately after brief anaerobic maximal cycle ergometer exercise (P &lt; 0.01).

Objective. Some investigators have reported that anti-prothrombin autoantibodies (aPT) in lupus anticoagulant positive sera were detectable by ELISA. Discrepancies in aPT ELISA were observed by some investigators. To clarify this situation, we tested the binding of aPT positive sera to purified prothrombin under various conditions. Methods. We performed aPT ELISA under different conditions. The variables we tested were: ELISA plate (untreated or gamma irradiated polystyrene plates), buffer (phosphate buffered saline or Tris buffered saline), detergent (presence or absence of Tween-20), and antigen condition (intact or fragmented prothrombin). Results. Anti-PT from patients with lupus or antiphospholipid syndrome were similarly bound to prothrombin with both buffers. Addition of Tween-20 to the buffer increased reactivity in the irradiated plate assay, but decreased reactivity in the untreated plate assay. Reactivities in 90% of lupus sera were decreased by the use of fragmented prothrombin. In contrast, the reactivity of serum from a healthy subject was remarkably increased by antigen fragmentation. Conclusion. The discrepant ELISA results in measurement of aPT in the various reports may have been due to the use of detergent in the buffer and condition of the prothrombin used as antigen. In our experiments the best ELISA condition for measurement of aPT was achieved using buffer with Tween-20 detergent, with prothrombin directly coated onto irradiated polystyrene plates.

Autoantibodies against prothrombin, including lupus anticoagulant antibodies (LAC), have been identified in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To identify the epitopes of LAC in patients with SLE and APS, we analyzed B cell epitopes of anti-prothrombin Abs. Prothrombin was purified from fresh plasma samples from healthy subjects, and fragmented by thrombin. Two fragments (prethrombin-1, 50 kDa, and fragment-1, 22 kDa) were separated and used for further experiments. The two fragments were coated on irradiated plate and the binding activities of sera from 13 patients with anti-prothrombin Abs (SLE, 7; APS, 4; SLE + APS, 2) were determined by using ELISA. The assay was conducted under the following conditions: use of irradiated plates, and TBS containing Tween-20. We detected two types of anti-prothrombin Abs. The first was anti-prethrombin-1 (n = 5) while the other was Ab against fragment-1 (n = 8). There were no patients with Abs that showed binding activities to both fragments. A higher proportion of patients with thrombosis were positive for anti-prethrombin-1 Abs (80%) than for anti-fragment-1 Abs (25%). Two patients with anti-prethrombin-1 Ab were positive for LAC and negative for anti-cardiolipin-beta(2) glycoprotein I antibody (aCL-beta(2)GPI). Our results strongly support the notion that both prethrombin-1 and fragment-1 on prothrombin molecule are B cell epitopes.

It is generally accepted that secretory immunoglobulin A (sIgA) is a major effector of local immunity in the oral cavity. The salivary sIgA level is known to be temporarily decreased after an acute single bout of intense exercise. On the other hand, the effect of continuous exercise training on salivary sIgA has been controversial. In the present study, we collected timed saliva samples from collegiate kendoists using the reliable saliva collection method that has already bren reported. The collection was performed before, during and after a traditional high-intensity 10-day training camp during the coldest part of the winter. We investigated the effect of repetitious intense exercise training on resting sIgA levels in saliva.
The subjects were 19 males and 8 females (age:19.9+/-0.5 years) who took part in the camp. We obtained saliva samples before the camp, on the first and the 10 th days of the camp, and 4 and 10 days after the camp at 5 p. m. The concentration of sIgA was measured by ELISA, and the sIgA secretion rate was calculated.
The resting sIgA secretion rate decreased significantly during the camp. It remained at a lower level 4 and 10 days after the camp compared to the initial level, a;though it tended to recover gradually. Prolonged suppression of the resting sIgA secretion rate during a traditional winter kendo training camp might be induced by repetition of high-intensity training.

To investigate the effect of intense exercise on immunological factors in saliva, we measured secretory immunoglobulin A (sIgA), lactoferrin and fibronectin. We used a reliable saliva collection method that has already been reported. Timed saliva samples were obtained from 16 healthy young males. Samples were collected before, immediately after, 1 day after and 5 days after endurance running (42.195 km). The concentrations mu g/ml) of sIgA, lactoferrin and fibronectin were measured hy ELISA, and the secretion rates (mu g/min) of each were calculated. Immediately alter the exercise, the concentration of total protein in saliva increased significantly, but the sIgA secretion rate decreased to 53%. The secretion rates of both lactoferrin and fibronectin did not change significantly. The local immune system plays an important role in mucosal surface defense against upper respiratory tract infection. The sIgA level in the oral cavity was temporarily decreased after intense exercise. A decreased sIgA secretion rate might partly explain the increased susceptibility to upper respiratory tract infection after endurance exercise.

Objective.-Considering the adverse effects of exercise-induced cortisol secretion on health in athletes, it is important to determine the environmental and individual factors that contribute to the variations in exercise-induced cortisol secretion. In this study, the effects of cold environment exposure and cold acclimatization on exercise-induced salivary cortisol responses were investigated.Methods.-Short track skaters (n = 11), who usually practice under cold conditions, and inline skaters (n = 11), who usually practice under room temperature conditions, participated in a randomized crossover study. All participants cycled for 60 minutes at 65% (V) over doto(2) max under cold (ambient temperature: 5 +/- 1 degrees C, relative humidity 41% +/- 9%) and room temperature (ambient temperature: 21 +/- 1 degrees C, relative humidity 35% +/- 5%) conditions. The participants had a 120-minute bed rest recovery phase at room temperature after both exercise bouts. Cortisol levels were measured in saliva samples collected pre-exercise and postexercise at 1 minute, 30 minutes, 60 minutes, and 120 minutes.Results.-Both short track and inline skaters exhibited clear cortisol responses to exercise under cold and room temperature conditions. The magnitude of the cortisol response, however, was different between skaters and conditions. The inline skaters exhibited significantly higher cortisol values under cold conditions than under room temperature conditions (7.6 nmol/L and 4.2 nmol/L, respectively). However, the short track skaters exhibited significantly higher cortisol values under cold conditions compared to room temperature conditions (8.7 nmol/L and 5.4 nmol/L, respectively).Conclusions.-The effects of cold environment exposure on exercise-induced cortisol response were different between skaters who usually practice under cold or room temperature conditions. These results can be interpreted as acclimatization to either cold or room temperature conditions attenuating the cortisol response, suggesting that acclimatization may be beneficial in reducing the exercise-induced cortisol response.