Regional neural activation defines a gateway for autoreactive T cells to cross the blood-brain barrier.

Although it is believed that neural activation can affect immune responses, very little is known about the neuroimmune interactions involved, especially the regulators of immune traffic across the blood-brain barrier which occurs in neuroimmune diseases such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T cells access the central nervous system via the fifth lumbar spinal cord. This location is defined by IL-6 amplifier-dependent upregulation of the chemokine CCL20 in associated dorsal blood vessels, which in turn depends on gravity-induced activation of sensory neurons by the soleus muscle in the leg. Impairing soleus muscle contraction by tail suspension is sufficient to reduce localized chemokine expression and block entry of pathogenic T cells at the fifth lumbar cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety of neurological diseases.

Exercise ameliorates high-fat diet-induced impairment of differentiation of adipose-derived stem cells into neuron-like cells in rats.

Adipose-derived stem cells (ADSCs) can differentiate into neurons under particular conditions. It remains largely unknown whether this differentiation potential is affected by physical conditions such as obesity, which modulates the functions of adipose tissue. In this study, we determined the impact of either a 9-week high-fat diet (60% fat; HFD) or 9-week exercise training on the differentiation potential of ADSCs into neuron-like cells in male Wistar rats. Rats were randomly assigned to a normal diet-fed (ND-SED) group, HFD-fed (HFD-SED) group, or exercise-trained HFD-fed group (HFD-EX). After a 9-week intervention, ADSCs from all groups differentiated into neuron-like cells. Expression of neuronal marker proteins (nestin, ßIII-tubulin, and microtubule-associated protein 2 [MAP2]) and the average length of cell neurites were lower in cells from HFD-SED rats than in other groups. Instead, protein expression of COX IV and Cyt-c, the Bax/Bcl-2 and LC3-II/I ratio, and the malondialdehyde level in culture medium were higher in cells from HFD-SED rats. No significant difference between ND-SED and HFD-EX rats was observed, except for the average length of cell neurites in MAP2. Thus, HFD impaired the differentiation potential of ADSCs into neuron-like cells, which was accompanied by increases in apoptotic activity and oxidative stress. Importantly, exercise training ameliorated the HFD-induced impairment of neurogenesis in ADSCs. The adipose tissue microenvironment could influence the differentiation potential of ADSCs, a source of autologous stem cell therapy.

Phosphorylated ERK1/2 protein levels are closely associated with the fast fiber phenotypes in rat hindlimb skeletal muscles.

The relationship between the extracellular signal-regulated kinase 1 and 2 (ERK1/2), one of the mitogen-activated protein kinases (MAPKs), and mammalian skeletal muscle fiber phenotype is unclear. We looked at this relationship in three in vivo conditions in male Wistar rats. First, the levels of phosphorylated (active) ERK1/2 protein were closely associated with the fiber type composition of sedentary rat hindlimb muscles: highest in the superficial portion of the gastrocnemius (100% fast fibers), lower in the plantaris (~ 80% fast fibers), and lowest in the soleus (~ 15% fast fibers). Second, during growth, there was a gradual decrease in the percentage of fast fibers from 40% at 3 weeks to 1.5% at 65 weeks and a concomitant gradual decrease in the levels of phosphorylated ERK1/2 in the soleus muscle. Third, sciatic nerve denervation induced a significant decrease in the weight of both the soleus and plantaris, but a slow-to-fast fiber type shift and increase in phosphorylated ERK1/2 protein were observed only in the soleus. Although only a few fast and fast + slow hybrid fibers of the denervated soleus muscle reacted positively to the anti-phosphorylated ERK1/2 antibody by immuno-histochemical analysis, our results suggest that the phosphorylated form of ERK1/2 seems to be closely related to the fast fiber phenotype program. Further evidence for this relationship was provided by the observation that several slow fiber phenotype-specific proteins, i.e., Hsp72, Hsp60, and PGC-1, changed in the opposite direction of the levels of phosphorylated ERK1/2 protein.

Temporary Loading Prevents Cancer Progression and Immune Organ Atrophy Induced by Hind-Limb Unloading in Mice.

Although the body's immune system is altered during spaceflight, the effects of microgravity (µG) on tumor growth and carcinogenesis are, as yet, unknown. To assess tumor proliferation and its effects on the immune system, we used a hind-limb unloading (HU) murine model to simulate µG during spaceflight. HU mice demonstrated significantly increased tumor growth, metastasis to the lung, and greater splenic and thymic atrophy compared with mice in constant orthostatic suspension and standard housing controls. In addition, mice undergoing temporary loading during HU (2 h per day) demonstrated no difference in cancer progression and immune organ atrophy compared with controls. Our findings suggest that temporary loading can prevent cancer progression and immune organ atrophy induced by HU. Further space experiment studies are warranted to elucidate the precise effects of µG on systemic immunity and cancer progression.

Effect of Circadian Rhythm on Clinical and Pathophysiological Conditions and Inflammation.

Circadian rhythms have long been known to regulate numerous physiological processes that vary across the diurnal cycle. The circadian clock system also controls various parameters of the immune system and its biological defense functions, allowing an organism to anticipate daily changes in activity and feeding and the associated risk of infection. Inflammation is an immune response triggered in living organisms in response to external stimuli. The risk of sepsis, an excessive inflammatory response, has been shown to have a diurnal variation. On the other hand, inflammatory responses are emerging to be induced by endogenous factors. Recent studies have suggested that chronic inflammation causes chronic diseases including rheumatoid arthritis, allergies, and aging-related diseases and that proteins encoded by clock genes affect the development of such chronic inflammatory diseases or increase the severity of their symptoms. Therefore, detailed understanding of circadian rhythm effects on inflammatory responses is expected to lead to new strategies for prevention or treatment of inflammatory diseases.

Suppression of Myostatin Stimulates Regenerative Potential of Injured Antigravitational Soleus Muscle in Mice under Unloading Condition.

Effects of myostatin (MSTN)-suppression on the regeneration of injured skeletal muscle under unloading condition were investigated by using transgenic mice expressing a dominant-negative form of MSTN (MSTN-DN). Both MSTN-DN and wild-type (WT) mice were subjected to continuous hindlimb suspension (HS) for 6 weeks. Cardiotoxin (CTX) was injected into left soleus muscle under anesthesia 2 weeks after the initiation of HS. Then, the soleus muscles were excised following 6-week HS (4 weeks after CTX-injection). CTX-injection caused to reduce the soleus fiber cross-sectional area (CSA) in WT mice under both unloading and weight-bearing conditions, but not in MSTN-DN mice. Under unloading condition, CTX-injected muscle weight and fiber CSA in MSTN-DN mice were significantly higher than those in WT mice. CTX-injected muscle had many damaged and regenerating fibers having central nuclei in both WT and MSTN-DN mice. Significant increase in the population of Pax7-positive nuclei in CTX-injected muscle was observed in MSTN-DN mice, but not in WT mice. Evidences indicate that the suppression of MSTN cause to increase the regenerative potential of injured soleus muscle via the increase in the population of muscle satellite cells regardless of unloading conditions.

Anti-interleukin-6 receptor antibody (MR16-1) promotes muscle regeneration via modulation of gene expressions in infiltrated macrophages.

BACKGROUND: Although rat anti-mouse IL-6 receptor (IL-6R) antibody (MR16-1) has been reported to effectively ameliorate various tissue damages, its effect on skeletal muscle regeneration has not been determined. Moreover, the localization, persistence and duration of action of this reagent in damaged tissues after systemic administration have not been assessed. METHODS: The MR16-1 was administered i.p. immediately after cardiotoxin (CTX)-induced muscle damage on mice. RESULTS: MR16-1 administered i.p. was observed only to the damaged muscle. This delivered MR16-1 was dramatically decreased from 3 to 7days post-injury concomitantly with a reduction of IL-6R expression. This reduction of the MR16-1 level in the damaged muscle was not rescued by additional administration of MR16-1, suggesting the short half-life of MR16-1 was not the factor for the remaining levels. In addition, a significant inhibitory effect of MR16-1 on phosphorylation of the signal transducer and activator of transcription 3 was observed in the macrophage-enriched area of damaged muscle 3days after injury. Finally, the acceleration of muscle regeneration observed at day 7 post-injury following MR16-1 treatment was associated with reduced expression of fibrosis-related genes, such as interleukin-10 and arginase, in the infiltrated macrophages. CONCLUSIONS: These results suggest that MR16-1 which was found primarily localized in infiltrated macrophages in the damaged muscle might facilitate muscle regeneration via immune modulation. GENERAL SIGNIFICANCE: These findings are deemed to provide further insight into the understanding not only of MR16-1 treatment on muscle regeneration, but also of the other anti-cytokine treatment on the cytokine-related disease.

Involvement of AMPK in regulating slow-twitch muscle atrophy during hindlimb unloading in mice.

AMPK is considered to have a role in regulating skeletal muscle mass. However, there are no studies investigating the function of AMPK in modulating skeletal muscle mass during atrophic conditions. In the present study, we investigated the difference in unloading-associated muscle atrophy and molecular functions in response to 2-wk hindlimb suspension between transgenic mice overexpressing the dominant-negative mutant of AMPK (AMPK-DN) and their wild-type (WT) littermates. Male WT (n = 24) and AMPK-DN (n = 24) mice were randomly divided into two groups: an untreated preexperimental control group (n = 12 in each group) and an unloading (n = 12 in each group) group. The relative soleus muscle weight and fiber cross-sectional area to body weight were decreased by ∼30% in WT mice by hindlimb unloading and by ∼20% in AMPK-DN mice. There were no changes in puromycin-labeled protein or Akt/70-kDa ribosomal S6 kinase signaling, the indicators of protein synthesis. The expressions of ubiquitinated proteins and muscle RING finger 1 mRNA and protein, markers of the ubiquitin-proteasome system, were increased by hindlimb unloading in WT mice but not in AMPK-DN mice. The expressions of molecules related to the protein degradation system, phosphorylated forkhead box class O3a, inhibitor of κBα, microRNA (miR)-1, and miR-23a, were decreased only in WT mice in response to hindlimb unloading, and 72-kDa heat shock protein expression was higher in AMPK-DN mice than in WT mice. These results imply that AMPK partially regulates unloading-induced atrophy of slow-twitch muscle possibly through modulation of the protein degradation system, especially the ubiquitin-proteasome system.

Habitual exercise training acts as a physiological stimulator for constant activation of lipolytic enzymes in rat primary white adipocytes.

It is widely accepted that lipolysis in adipocytes are regulated through the enzymatic activation of both hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) via their phosphorylation events. Accumulated evidence shows that habitual exercise training (HE) enhances the lipolytic response in primary white adipocytes with changes in the subcellular localization of lipolytic molecules. However, no study has focused on the effect that HE exerts on the phosphorylation of both HSL and ATGL in primary white adipocytes. It has been shown that the translocation of HSL from the cytosol to lipid droplet surfaces requires its phosphorylation at Ser-563. In primary white adipocytes obtained from HE rats, the level of HSL and ATGL proteins was higher than that in primary white adipocytes obtained from sedentary control (SC) rats. In HE rats, the level of phosphorylated ATGL and HSL was also significantly elevated compared with that in SC rats. These differences were confirmed by Phos-tag SDS-PAGE, a technique used to measure the amount of total phosphorylated proteins. Our results suggest that HE can consistently increase the activity of both lipases, thereby enhancing the lipolysis in white fat cells. Thus, HE helps in the prevention and treatment of obesity-related diseases by enhancing the lipolytic capacity.

Heat-Stress effects on the myosin heavy chain phenotype of rat soleus fibers during the early stages of regeneration.

INTRODUCTION: We investigated heat-stress effects on the adult myosin heavy chain (MyHC) profile of soleus muscle fibers at an early stage of regeneration. METHODS: Regenerating fibers in adult rats were analyzed 2, 4, or 6 days after bupivacaine injection. Rats were heat stressed by immersion in water (42 ± 1°C) for 30 minutes 24 hours after bupivacaine injection and every other day thereafter. RESULTS: No adult MyHC isoforms were observed after 2 days, whereas some fibers expressed only fast MyHC after 4 days. Heat stress increased fast and slow MyHC in regenerating fibers after 6 days. Regenerating fibers expressing only slow MyHC were observed only in heat-stressed muscles. Bupivacaine injection increased the number of Pax7(+) and MyoD(+) satellite cells in regenerating fibers, more so in heat-stressed rats. CONCLUSION: The results indicate that heat stress accelerates fast-to-slow MyHC phenotype conversion in regenerating fibers via activation of satellite cells.

AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12 skeletal muscle cells.

5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

Direct and indirect suppression of interleukin-6 gene expression in murine macrophages by nuclear orphan receptor REV-ERBα.

It is now evident that many nuclear hormone receptors can modulate target gene expression. REV-ERBα, one of the nuclear hormone receptors with the capacity to alter clock function, is critically involved in lipid metabolism, adipogenesis, and the inflammatory response. Recent studies suggest that REV-ERBα plays a key role in the mediation between clockwork and inflammation. The purpose of the current study was to investigate the role of REV-ERBα in the regulation of interleukin-6 (il6) gene expression in murine macrophages. REV-ERBα agonists, or overexpression of rev-erb α in the murine macrophage cell line RAW264 cells, suppressed the induction of il6 mRNA following a lipopolysaccharide (LPS) endotoxin challenge. Also, rev-erb α overexpression decreased LPS-stimulated nuclear factor κB (NFκB) activation in RAW264 cells. We showed that REV-ERBα represses il6 expression not only indirectly through an NFκB binding motif but also directly through a REV-ERBα binding motif in the murine il6 promoter region. Furthermore, peritoneal macrophages from mice lacking rev-erb α increased il6 mRNA expression. These data suggest that REV-ERBα regulates the inflammatory response of macrophages through the suppression of il6 expression. REV-ERBα may therefore be identified as a potent anti-inflammatory receptor and be a therapeutic target receptor of inflammatory diseases.

Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 µm(2)) gamma and medium-sized (300-700 µm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 µm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.

Microcurrent electrical nerve stimulation facilitates regrowth of mouse soleus muscle.

Microcurrent electrical nerve stimulation (MENS) has been used to facilitate recovery from skeletal muscle injury. However, the effects of MENS on unloading-associated atrophied skeletal muscle remain unclear. Effects of MENS on the regrowing process of unloading-associated atrophied skeletal muscle were investigated. Male C57BL/6J mice (10-week old) were randomly assigned to untreated normal recovery (C) and MENS-treated (M) groups. Mice of both groups are subjected to continuous hindlimb suspension (HS) for 2 weeks followed by 7 days of ambulation recovery. Mice in M group were treated with MENS for 60 min 1, 3, and 5 days following HS, respectively, under anesthesia. The intensity, the frequency, and the pulse width of MENS were set at 10 µA, 0.3 Hz, and 250 msec, respectively. Soleus muscles were dissected before and immediately after, 1, 3 and 7 days after HS. Soleus muscle wet weight and protein content were decreased by HS. The regrowth of atrophied soleus muscle in M group was faster than that in C group. Decrease in the reloading-induced necrosis of atrophied soleus was facilitated by MENS. Significant increases in phosphorylated levels of p70 S6 kinase and protein kinase B (Akt) in M group were observed, compared with C group. These observations are consistent with that MENS facilitated regrowth of atrophied soleus muscle. MENS may be a potential extracellular stimulus to activate the intracellular signals involved in protein synthesis.

Effects of heat stress on muscle mass and the expression levels of heat shock proteins and lysosomal cathepsin L in soleus muscle of young and aged mice.

Effects of heat stress on skeletal muscle mass in young and aged mice were investigated. Young (7-week) and aged (106-week) male C57BL/6J mice were randomly assigned to control and heat-stressed groups in each age. Mice in heat-stressed group were exposed to heat stress (41 °C for 60 min) in an incubator without anesthesia. Seven days after the exposure, soleus muscles were dissected from both hindlimbs. Protein content and the relative composition of Type II fibers in aged soleus were lower than those in young muscle. In aged soleus, higher baseline expression levels of HSP25, HSP72, and cathepsin L were observed compared with those in young muscle (p < 0.05). However, there were no significant differences in the expression levels of phosphorylated p70 S6 kinase (p-p70S6K), calpain 1, and calpain 2 of soleus between two age groups. A significant increase in muscle mass of both age groups was induced by heat stress (p < 0.05). Heat stress also upregulated the expressions of HSP25, HSP72, and p-p70S6K in both ages (p < 0.05). On the other hand, a significant decrease in cathepsin L expression by heating was observed in aged soleus, but not in young (p < 0.05). Both the percentage of Type I fibers and the expression of calpains in both age groups were unchanged following heat stress. Heat stress-associated downregulation of cathepsin L may be attributed to the upregulation of HSP72, which stabilizes lysosomal membranes (p < 0.05). Upregulations of HSP25, HSP72, and p-p70S6K and/or the downregulation of cathepsin L may play a role in heat stress-associated muscle hypertrophy in aged soleus muscle.

Responses of neuromuscular properties to unloading and potential countermeasures during space exploration missions.

We reviewed the responses of the neuromuscular properties of mainly the soleus and possible mechanisms. Sensory nervous activity in response to passive shortening and/or active contraction, associated with plantar-flexion or dorsi-flexion of the ankle joints, may play an essential role in the regulation of muscle properties. Passive shortening of the muscle fibers and sarcomeres inhibits the development of tension, electromyogram (EMG), and afferent neurogram. Remodeling of the sarcomeres, which decreases the total sarcomere number in a single muscle fiber causing recovery of the length in each sarcomere, is induced in the soleus following chronic unloading. Although EMG activity and tension development in each sarcomere are increased, the total tension produced by the whole muscle is still less owing to the lower sarcomere number. Therefore, muscle atrophy continues to progress. Moreover, walking or slow running by rear-foot strike landing with the application of greater ground reaction force, which stimulates soleus mobilization, could be an effective countermeasure. Periodic, but not chronic, passive stretching of the soleus may also be effective.

ATP spreads inflammation to other limbs through crosstalk between sensory neurons and interneurons.

Neural circuits between lesions are one mechanism through which local inflammation spreads to remote positions. Here, we show the inflammatory signal on one side of the joint is spread to the other side via sensory neuron-interneuron crosstalk, with ATP at the core. Surgical ablation or pharmacological inhibition of this neural pathway prevented inflammation development on the other side. Mechanistic analysis showed that ATP serves as both a neurotransmitter and an inflammation enhancer, thus acting as an intermediary between the local inflammation and neural pathway that induces inflammation on the other side. These results suggest blockade of this neural pathway, which is named the remote inflammation gateway reflex, may have therapeutic value for inflammatory diseases, particularly those, such as rheumatoid arthritis, in which inflammation spreads to remote positions.

Possible role of NF-ĸB signals in heat stress-associated increase in protein content of cultured C2C12 cells.

Heat stress is one of the hypertrophic stimuli on mammalian skeletal muscle. Nuclear factor-κB (NF-κB) signaling plays an important role in the regulation of skeletal muscle mass. However, the effects of heat stress on NF-κB signaling in skeletal muscle cells remain unclear. Effects of heat stress and/or administration of BAY11-7082, an inhibitor of NF-κB, on NF-κB signals and protein content of skeletal muscle were studied by using cell culture system. Differentiated mouse myoblasts (C2C12) were subjected to either (1) control (cultured at 37°C without BAY11-7082), (2) heat stress at 41°C for 60 min, (3) BAY11-7082 administration (1.25 µM) or (4) heat stress combined with BAY11-7082 administration. Heat shock protein 72 (HSP72) was upregulated by heat stress with or without administration of BAY11-7082. The increase in inhibitor of κBα (IκBα), which regulates the phosphorylation of NF-κB, and the decrease in phosphorylated NF-κB were also induced by administration of BAY11-7082 and/or heat stress. Protein content in C2C12 cells was increased by the administration of BAY11-7082 with a semi-logarithm fashion. Significant increases in the protein content of C2C12 cells were observed 48 h following heating with or without administration of BAY11-7082. These observations suggest that heat stress might increase muscle protein through the downregulation of NF-κB signaling. Inhibition of NF-κB induced by application of heat stress might be one of the hypertrophic stimuli on skeletal muscle cells.

Effects of creatine and its analog, ß-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts.

The effects of supplementation with creatine (Cr) and its analog, ß-guanidinopropionic acid (ß-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or ß-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and ß-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the ß-GPA group. Supplementation with ß-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas ß-GPA increased nucleolar sizes in the myotubes. These results suggest that ß-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.

Responses of muscle mass, strength and gene transcripts to long-term heat stress in healthy human subjects.

The present study was performed to investigate the effects of long-term heat stress on mass, strength and gene expression profile of human skeletal muscles without exercise training. Eight healthy men were subjected to 10-week application of heat stress, which was performed for the quadriceps muscles for 8 h/day and 4 days/week by using a heat- and steam-generating sheet. Maximum isometric force during knee extension of the heated leg significantly increased after heat stress (~5.8%, P < 0.05). Mean cross-sectional areas (CSAs) of vastus lateralis (VL, ~2.7%) and rectus femoris (~6.1%) muscles, as well as fiber CSA (8.3%) in VL, in the heated leg were also significantly increased (P < 0.05). Statistical analysis of microarrays (SAM) revealed that 10 weeks of heat stress increased the transcript level of 925 genes and decreased that of 1,300 genes, and gene function clustering analysis (Database for Annotation, Visualization and Integrated Discovery: DAVID) showed that these regulated transcripts stemmed from diverse functional categories. Transcript level of ubiquinol-cytochrome c reductase binding protein (UQCRB) was significantly increased by 10 weeks of heat stress (~3.0 folds). UQCRB is classified as one of the oxidative phosphorylation-associated genes, suggesting that heat stress can stimulate ATP synthesis. These results suggested that long-term application of heat stress could be effective in increasing the muscle strength associated with hypertrophy without exercise training.