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.

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.

Effects of microgravity exposure and fructo-oligosaccharide ingestion on the proteome of soleus and extensor digitorum longus muscles in developing mice.

Short-chain fatty acids produced by the gut bacterial fermentation of non-digestible carbohydrates, e.g., fructo-oligosaccharide (FOS), contribute to the maintenance of skeletal muscle mass and oxidative metabolic capacity. We evaluated the effect of FOS ingestion on protein expression of soleus (Sol) and extensor digitorum longus muscles in mice exposed to microgravity (µ-g). Twelve 9-week-old male C57BL/6J mice were raised individually on the International Space Station under µ-g or artificial 1-g and fed a diet with or without FOS (n = 3/group). Regardless of FOS ingestion, the absolute wet weights of both muscles tended to decrease, and the fiber phenotype in Sol muscles shifted toward fast-twitch type following µ-g exposure. However, FOS ingestion tended to mitigate the µ-g-exposure-related decrease in oxidative metabolism and enhance glutathione redox detoxification in Sol muscles. These results indicate that FOS ingestion mildly suppresses metabolic changes and oxidative stress in antigravity Sol muscles during spaceflight.

Depression of Bone Density at the Weight-Bearing Joints in Wistar Hannover Rats by a Simulated Mechanical Stress Associated With Partial Gravity Environment.

The partial gravity environment in space can negatively affect bone health. This survey aimed to study the reaction of different parts of the lower limb bones of rats to partial gravity and the effects of different degrees of gravity on these bony parts. We used 15 8-week-old male Wistar Hannover rats were used at the beginning of the experiment. The degree of mechanical stress was modified, but the ankle joint was maintained at ∼30°, ∼120°, or ∼160° with or without plaster fixation during 10-day hindlimb suspension. Computed tomography was performed to measure the bone parameters [bone mineral density (BMD), trabecular BMD, cortical BMD, and cortical thickness] of each studied group of the whole, proximal, middle, and distal femur and distal tibia. BMD, trabecular BMD, and cortical thickness of the distal femur and proximal tibia of the simulated mechanical stress associated with partial gravity groups were significantly lower than those of the control group; the effect of different degrees of gravity on the same area of hindlimb bone had no significant difference. The simulated mechanical stress associated with partial gravity had the most significant effect on the bone close to the knee joint, with the largest weight-bearing response.

Role of 72-kDa Heat Shock Protein in Heat-stimulated Regeneration of Injured Muscle in Rat.

The regeneration of injured muscles is facilitated by intermittent heat stress. The 72-kDa heat shock protein (HSP72), the level of which is increased by heat stress, is likely involved in this effect, but the precise mechanism remains unclear. This study was conducted to investigate the localization and role(s) of HSP72 in the regenerating muscles in heat-stressed rats using immunohistochemistry. Heat stress was applied by immersion of the rat lower body into hot water (42C, 30 min, every other day) following injection of bupivacaine into the soleus muscles. After 1 week, we found that HSP72 was expressed at high levels not only in the surviving myofibers but also in the blood vessels of the regenerating muscles in heated rats. In addition, leukocytes, possibly granulocytes, expressing cluster of differentiation 43 within the blood capillaries surrounding the regenerating myofibers also highly expressed HSP72. In contrast, marked expression of HSP72 was not observed in the intact or regenerating muscles without heat stress. These results suggest that heat-stress-induced HSP72 within the myofibers, blood vessels, and circulating leukocytes may play important roles in enhancing regeneration of injured muscles by heat stress. Our findings would be useful to investigate cell-specific role(s) of HSP72 during skeletal muscle regeneration.

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.

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.

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 a 9-week exercise training regimen on expression of developmental genes related to growth-dependent fat expansion in juvenile rats.

This study examined the association between changes in mRNA expression of development-related genes including those of the homeobox (Hox) family and growth-dependent increases in inguinal, mesenteric, and epididymal white adipose tissue (WAT) at 4, 6, 10, and 14 weeks of age in rats. We also examined the effects of a 9-week exercise training regimen starting at 5 weeks of age on the mRNA levels of the genes of interest. HoxC8, HoxC9, Gpc4, Bmpr1a, Pparγ, Pgc1α, Adrb3, Hsl, leptin, and adiponectin in each type of WAT - except HoxA5, Gpc4, and Pgc1α in epididymal - showed a positive association between WAT weights and WAT mRNA levels; however, the slope of the regression lines exhibited fat depot-specific differences. HoxA5 showed no significant association, and Gpc4 and Pgc1α showed a negative association in epididymal WAT. After exercise training, the mean HoxA5, HoxC8, HoxC9, HoxC10, Gpc4, Pparγ, and Pgc1α mRNA levels in inguinal WAT were outliers on the regression line between mean mRNA level and WAT weight in control rats - that is, mean HoxA5 and Pgc1α mRNA level was higher, whereas HoxC8, HoxC9, HoxC10, Gpc4, and Ppar levels were lower in exercise-trained rats than in same-age controls. Pparγγ and adiponectin levels were upregulated in epididymal WAT, while HoxA5 was downregulated, but HoxC9, Gpc4, Pparγ, and adiponectin levels were upregulated in mesenteric WAT. These results suggest that some of the developmental genes tested may have fat depot-specific roles in the growth-dependent expansion of WAT, and that Hox genes that are activated in response to exercise training also vary among different WAT types.

Differential response of adipose tissue gene and protein expressions to 4- and 8-week administration of ß-guanidinopropionic acid in mice.

ß-Guanidinopropionic acid (ß-GPA) feeding inhibits growth-associated gain of body mass. It remains unknown, however, whether and how ß-GPA feeding affects growth-associated increase in white adipose tissue (WAT) mass. We examined the effects of 4- and 8-week ß-GPA feeding on serum myostatin levels and expression of genes and proteins related to adipogenesis, lipolysis, and liposynthesis in epididymal WAT (eWAT) and brown adipose tissue (BAT) in 3-week-old, juvenile male mice. Body, eWAT, and muscle weights were significantly lower in ß-GPA-fed mice than in controls after feeding. Four- but not 8-week-ß-GPA feeding increased the serum myostatin level. Incubation of C2C12 myotubes with ß-GPA (1 mM) significantly promoted myostatin mRNA expression. The protein expression of peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC-1α) and peroxisome proliferator-activated receptor α (PPARα) was up-regulated in GPAF eWAT at week 4, but down-regulated at week 8. There was no significant difference in the protein expression of adipocyte triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) between groups in eWAT. In BAT, no significant difference was found in the protein expression of PGC-1α, PPARα, ATGL, and HSL between ß-GPA-fed and control mice, whereas that of FAS and ACC was significantly lower in ß-GPA-fed mice at week 8. Uncoupling protein 1 was expressed higher in ß-GPA-fed mice both at weeks 4 and 8 than that in controls. Thus, the mechanism by which ß-GPA feeding in early juvenile mice inhibits growth-associated increase in eWAT mass may differ between early and later periods of growth.

The effects of heat stress on morphological properties and intracellular signaling of denervated and intact soleus muscles in rats.

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (Atrogin-1), and muscle RING-finger protein-1 (MuRF-1), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of Atrogin-1, but not MuRF-1 transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which are negative regulators of Atrogin-1 and MuRF-1 transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect Atrogin-1 and MuRF-1 transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1α Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.

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.

Effects of icing or heat stress on the induction of fibrosis and/or regeneration of injured rat soleus muscle.

The effects of icing or heat stress on the regeneration of injured soleus muscle were investigated in male Wistar rats. Bupivacaine was injected into soleus muscles bilaterally to induce muscle injury. Icing (0 °C, 20 min) was carried out immediately after the injury. Heat stress (42 °C, 30 min) was applied every other day during 2-14 days after the bupivacaine injection. Injury-related increase in collagen deposition was promoted by icing. However, the level of collagen deposition in heat-stressed animals was maintained at control levels throughout the experimental period and was significantly lower than that in icing-treated animals at 15 and 28 days after bupivacaine injection. Furthermore, the recovery of muscle mass, protein content, and muscle fiber size of injured soleus toward control levels was partially facilitated by heat stress. These results suggest that, compared with icing, heat stress may be a beneficial treatment for successful muscle regeneration at least by reducing fibrosis.

Differences in histone modifications between slow- and fast-twitch muscle of adult rats and following overload, denervation, or valproic acid administration.

Numerous studies have reported alterations in skeletal muscle properties and phenotypes in response to various stimuli such as exercise, unloading, and gene mutation. However, a shift in muscle fiber phenotype from fast twitch to slow twitch is not completely induced by stimuli. This limitation is hypothesized to result from the epigenetic differences between muscle types. The main purpose of the present study was to identify the differences in histone modification for the plantaris (fast) and soleus (slow) muscles of adult rats. Genome-wide analysis by chromatin immunoprecipitation followed by DNA sequencing revealed that trimethylation at lysine 4 and acetylation of histone 3, which occurs at transcriptionally active gene loci, was less prevalent in the genes specific to the slow-twitch soleus muscle. Conversely, gene loci specific to the fast-twitch plantaris muscle were associated with the aforementioned histone modifications. We also found that upregulation of slow genes in the plantaris muscle, which are related to enhanced muscular activity, is not associated with activating histone modifications. Furthermore, silencing of muscle activity by denervation caused the displacement of acetylated histone and RNA polymerase II (Pol II) in 5' ends of genes in plantaris, but minor effects were observed in soleus. Increased recruitment of Pol II induced by forced acetylation of histone was also suppressed in valproic acid-treated soleus. Our present data indicate that the slow-twitch soleus muscle has a unique set of histone modifications, which may relate to the preservation of the genetic backbone against physiological stimuli.

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.

Effects of long time exposure to simulated micro- and hypergravity on skeletal architecture.

This manuscript reports the structural alterations occurring in mice skeleton as a consequence of the longest-term exposition (90 days) to simulated microgravity (hindlimb unloading) and hypergravity (2g) ever tested. Bone microstructural features were investigated by means of standard Cone Beam X-ray micro-CT, Synchrotron Radiation micro-CT and histology. Morphometric analysis confirmed deleterious bone architectural changes in lack of mechanical loading with a decrease of bone volume and density, while bone structure alterations caused by hypergravity were less evident. In the femurs from hypergravity-exposed mice, the head/neck cortical thickness increment was the main finding. In addition, in these mice the rate of larger trabeculae (60-75 µm) was significantly increased. Interestingly, the metaphyseal plate presented a significant adaptation to gravity changes. Mineralization of cartilage and bone deposition was increased in the 2g mice, whereas an enlargement of the growth plate cartilage was observed in the hindlimb unloaded group. Indeed, the presented data confirm and reinforce the detrimental effects on bone observed in real space microgravity and reveal region-specific effects on long bones. Finally these data could represent the starting point for further long-term experimentations that can deeply investigate the bone adaptation mechanisms to different mechanical force environments.

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.

Responses of skeletal muscles to gravitational unloading and/or reloading.

Adaptation of morphological, metabolic, and contractile properties of skeletal muscles to inhibition of antigravity activities by exposure to a microgravity environment or by simulation models, such as chronic bedrest in humans or hindlimb suspension in rodents, has been well reported. Such physiological adaptations are generally detrimental in daily life on earth. Since the development of suitable countermeasure(s) is essential to prevent or inhibit these adaptations, effects of neural, mechanical, and metabolic factors on these properties in both humans and animals were reviewed. Special attention was paid to the roles of the motoneurons (both efferent and afferent neurograms) and electromyogram activities as the neural factors, force development, and/or length of sarcomeres as the mechanical factors and mitochondrial bioenergetics as the metabolic factors.