Hindlimb Unloading Group Research Articles

Effects of hindlimb unloading on the mevalonate and mechanistic target of rapamycin complex 1 signaling pathways in a fast-twitch muscle in rats.

Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.

Effects of exercise prehabilitation on muscle atrophy and contractile properties in hindlimb-unloaded rats.

Effective strategies for rapid recovery after surgery are needed. Therefore, we investigated the effects of exercise prehabilitation (EP) and hindlimb unloading (HU) on muscle loss and contractility. Twenty-two Sprague-Dawley rats (12 wk old) were divided into normal control (NCON, n = 5), hindlimb unloading control (HCON, n = 10), and exercise prehabilitation followed by hindlimb unloading (Ex-preH, n = 7) groups. Ex-PreH performed exercise training for 14 days before hindlimb unloading for 14 days. Body composition was evaluated, along with muscle strength and function. The soleus (SOL) and extensor digitorum longus (EDL) muscle contractile properties were analyzed at the whole-muscle level. The titin concentration and myosin heavy chain (MHC) type composition were analyzed. There were no effects of Ex-preH on total mass, lean mass, or muscle weight. Physical function was significantly higher in the Ex-preH group than in the HCON group (39.5° vs. 35.7°). The SOL twitch force (19.6 vs. 7.1 mN/m2 ) and specific force (107.3 vs. 61.2 mN/m2 ) were greater in Ex-preH group than in HCON group. EDL shortening velocity was higher in Ex-preH group than in HCON group (13.2 vs. 5.0 FL/s). The SOL full-length titin level was higher in Ex-preH group than in HCON group. Exercise prehabilitation did not prevent muscle mass loss followed by muscle wasting, although it minimized the reduction of physical function. Therefore, exercise prehabilitation should be considered for rapid functional recovery after disuse due to surgery and injuries.

Acute effect of electrical stimulation on muscle protein synthesis and break-down in the soleus muscle of hindlimb unloaded rats.

Electrical stimulation (ES) is effective for disuse-induced muscle atrophy. However, the acute effect of ES on muscle protein synthesis (MPS) and muscle protein breakdown (MPB) remains unclear. We investigated the effect of a single-session ES treatment on mTORC1 signaling, MPS, and MPB in the soleus muscle of 2-week hindlimb unloaded rats. Sprague Dawley rats (n = 12 male) were randomly divided into control (CON) and hindlimb unloaded (HU) groups. After 2 weeks, the right soleus muscle was percutaneously stimulated and underwent supramaximal isometric contractions. The left soleus muscle served as an internal control. We collected soleus muscle samples 6 h after ES. Two weeks of HU decreased p70S6K and S6rp activation, downstream factors for mTORC1 signaling, and SUnSET method-assessed MPS, but increased the LC3-II/I ratio, an indicator of autophagy. ES on disused muscle successfully activated mTORC1 signaling but did not affect MPS. Contrary, ES decreased ubiquitinated proteins expression and LC3B-II/I ratio. HU might affect mTORC1 activation and MPS differently in response to acute ES possibly due to excessive ROS production caused by ES. Our findings suggest that ES applied to disused skeletal muscles may suppress MPB, but its effect on MPS appears to be attenuated.

Transcranial photobiomodulation mitigates learning and memory impairments induced by hindlimb unloading in a mouse model of microgravity exposure by suppression of oxidative stress and neuroinflammation signaling pathways

Prolonged microgravity exposure causes cognitive impairment. Evidence shows that oxidative stress and neuroinflammation are involved in the causation. Here, we explore the effectiveness of transcranial near-infrared photobiomodulation (PBM) on cognitive deficits in a mouse model of simulated microgravity.24 adult male C57BL/6 mice were assigned into three groups (8 in each); control, hindlimb unloading (HU), and HU + PBM groups. After surgery to fit the suspension fixing, the animals were housed either in HU cages or in their normal cage for 14 days. The mice in the HU + PBM group received PBM (810 nm laser, 10 Hz, 8 J/cm2) once per day for 14 days. Spatial learning and memory were assessed in the Lashley III maze and hippocampus tissue samples were collected to assess oxidative stress markers and protein expression of brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-related factor 2 (Nrf2), Sirtuin 1 (Sirt1), and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).Behavioral testing showed that the PBM-treated animals had a shorter latency time to find the target and fewer errors than the HU group. PBM decreased hippocampal lipid peroxidation while increasing antioxidant defense systems (glutathione peroxidase, superoxide dismutase, and total antioxidant capacity) compared to HU mice. PBM increased protein expression of Sirt1, Nrf2, and BDNF while decreasing NF-κB compared to HU mice.Our findings suggested that the protective effect of PBM against HU-induced cognitive impairment involved the activation of the Sirt1/Nrf2 signaling pathway, up-regulation of BDNF, and reduction of neuroinflammation and oxidative stress in the hippocampus.

Enhancing microbial diversity as well as multi-organ health in hind-limb unloaded mice

During space travel, the gut microbiota is changed which can lead to health-related issues. Previously, we utilized the hind-limb unloaded (HU) mouse, which is an established ground-based in-vivo model of microgravity and observed altered gut microbiota. In this study, we evaluated the beneficial effects of novel bacterial conditioned media in HU mice to understand if they can offset the effects of unloading in the HU mouse model. We aimed to explore the influence of bacterial conditioned media on diversity and quantity of intestinal microbes in HU mice, and investigated the microarchitecture of mice retinas and kidneys to evaluate the potential systemic effects of bacterial conditioned media in HU mice. Four-month-old, male C57/Bl6 mice were separated into groups: including the ground-based control group, the HU group mice fed with vehicle as placebo (HU-placebo mice), and the HU group fed with bacterial conditioned media (HU-CP mice) and kept under controlled environmental conditions for three weeks. Next, mice were sacrificed; gut dissections were conducted, and metagenomic analysis of bacterial species was performed via DNA extraction and 16S rRNA analysis. The results revealed an HU-induced reduction in intestinal microbial diversity, and an increase in pathogenic bacteria dominated by Firmicutes (45%). In contrast, supplementation with bacterial conditioned media for three weeks led to a significant increase in gut microbial diversity with noticeable changes in the OTUs abundance in the HU mice. Additionally, HU-induced muscle weakness and structural abnormalities in the retina and kidney were partially prevented with bacterial conditioned media. Moreover, a greater diversity of several bacteria in the HU-CP was observed including, Bacteriodota, Firmicutes, Proteobacteria, Actionobacteriota, Verrucomicorbiota, Cyanobacteria, Gemmatimonadota, Acidobacteriota, Chloroflexi, Myxococcota, and others. Prospective research involving molecular mechanistic studies are needed to comprehend the systemic effects of bacterial metabolites conditioned media on experimental animal models under chronic stress.

In situ fluorescence-photoacoustic measurement of the changes of brown adipose tissue in mice under hindlimb unloading.

Space travel causes rapid weight loss of astronauts, but the underlying reasons are still obscure. Brown adipose tissue (BAT) is a well-known thermogenesis tissue that is innervated by sympathetic nerves, and norepinephrine stimulation can promote the thermogenesis and angiogenesis of BAT. Herein, the structural and physiological changes of BAT as well as serological indicators were investigated in mice under hindlimb unloading (HU) to simulate a weightless environment in space. The results showed that long-term HU could induced the thermogenic activation of BAT by upregulating the mitochondrial uncoupling protein. Further, peptide-conjugated indocyanine green was developed to target the vascular endothelial cells of BAT. Noninvasive fluorescence-photoacoustic imaging presented the neovascularization of BAT on the micron scale in the HU group, accompanying by the increase of vessel density. Downward trend of serum triglyceride and glucose level of mice treated with HU proved the more heat production and energy consumption in BAT compared with the control group. This study suggested that HU may be an effective strategy to curb the occurrence of obesity, whereas fluorescence-photoacoustic dual-modal imaging showed capability of assessing BAT activity.NEW & NOTEWORTHY We found that the mechanism of weight loss of astronauts in space flight may be that hindlimb unloading (HU) promotes the activation of brown adipose tissue (BAT) and the increase of uncoupling protein (UCP1) expression, which accelerates the body's heat production. Meanwhile, the activation of BAT is accompanied by the proliferation of blood vessels. With the help of peptide CPATAERPC conjugated indocyanine green targeting to vascular endothelial cells, fluorescence-photoacoustic imaging has selectively tracked the vascular structure of BAT on the micron scale, which provided noninvasive imaging tools to in situ measure the changes of BAT.

Differential bone remodeling mechanism in hindlimb unloaded and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse within the same species.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/β-catenin signaling pathway (GSK-3β and phospho-β-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3β was increased, and the protein expression of phospho-β-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

Effects of repetitive transcranial magnetic stimulation on neuronal excitability and ion channels in hindlimb unloading mice

Weightlessness in the space environment affects astronauts' learning memory and cognitive function. Repetitive transcranial magnetic stimulation has been shown to be effective in improving cognitive dysfunction. In this study, we investigated the effects of repetitive transcranial magnetic stimulation on neural excitability and ion channels in simulated weightlessness mice from a neurophysiological perspective. Young C57 mice were divided into control, hindlimb unloading and magnetic stimulation groups. The mice in the hindlimb unloading and magnetic stimulation groups were treated with hindlimb unloading for 14 days to establish a simulated weightlessness model, while the mice in the magnetic stimulation group were subjected to 14 days of repetitive transcranial magnetic stimulation. Using isolated brain slice patch clamp experiments, the relevant indexes of action potential and the kinetic property changes of voltage-gated sodium and potassium channels were detected to analyze the excitability of neurons and their ion channel mechanisms. The results showed that the behavioral cognitive ability and neuronal excitability of the mice decreased significantly with hindlimb unloading. Repetitive transcranial magnetic stimulation could significantly improve the cognitive impairment and neuroelectrophysiological indexes of the hindlimb unloading mice. Repetitive transcranial magnetic stimulation may change the activation, inactivation and reactivation process of sodium and potassium ion channels by promoting sodium ion outflow and inhibiting potassium ion, and affect the dynamic characteristics of ion channels, so as to enhance the excitability of single neurons and improve the cognitive damage and spatial memory ability of hindlimb unloading mice.

Effect of Microgravity on the Gut Microbiota Bacterial Composition in a Hindlimb Unloading Model.

We utilised a ground-based microgravity hindlimb unloading (HU) mouse model to elucidate the gut microbiota bacterial changes in mice under a simulated microgravity environment. Four-month-old, male C57/Bl6 mice were randomly divided into ground-based controls and the HU groups and kept under controlled environmental conditions. For the microgravity environment, the mice were suspended in special cages individually for 20 days. At the end of the suspension, the mice were sacrificed; gut dissections were performed, followed by a metagenomic analysis of bacterial species, which was carried out by extracting DNA and 16S rRNA analysis. The results revealed that the gut bacterial communities of mice under gravity and microgravity were different. Notably, our findings revealed differences in the bacterial community structure. Around 449 bacterial OTUs were specific to mice kept under normal gravity versus 443 bacterial OTUs under microgravity conditions. In contrast, 694 bacterial OTUs were common to both groups. When the relative abundance of taxa was analyzed, Bacteroidetes dominated the gut (64.7%) of normal mice. Conversely, mice in the microgravity environment were dominated by Firmicutes (42.7%), and the relative abundance of Bacteroidetes differed significantly between the two groups (p < 0.05). The distribution of Muribaculaceae between normal mice versus microgravity mice was significantly different, at 62% and 36.4%, respectively (p < 0.05). Furthermore, a significant decrease in 11 bacteria was observed in mice under simulated microgravity, including Akkermansia muciniphila, Eubacterium coprostanoligenes, Bacteroides acidifaciens, Clostridium leptum, Methylorubrum extorquens, Comamonas testosterone, Desulfovibrio fairfieldensis, Bacteroides coprocola, Aerococcus urinaeequi, Helicobacter hepaticus, and Burkholderiales. Further studies are needed to elucidate gut bacterial metabolites of these identified bacterial species in microgravity conditions and normal environment. Notably, the influence of these metabolites on obesity, neuroprotection, musculoskeletal and cardiovascular dysfunction, longevity, inflammation, health, and disease in astronauts ought to be investigated and will be important in developing procedures against adverse effects in astronauts following space travel.

Muscle Atrophy Evaluation via Radiomics Analysis Using Ultrasound Images: A Cohort Data Study.

Existing methods for muscle atrophy evaluation based on muscle size measures from ultrasound images are inadequate in precision. Radiomics has been widely used in various medical studies, but its validity for the evaluation of muscle atrophy has not been fully explored. This study presents a radiomics analysis for muscle atrophy evaluation using ultrasound images. The hindlimb unloading rat model was developed to simulate weightlessness muscle atrophy and ultrasound images of the hind limbs were acquired for both the hindlimb unloaded (HU) and control groups during a 21-day HU period. A total of 368 radiomics features were extracted and the stable and informative features were selected through a two-stage feature selection procedure. The feature change trajectory of the stable features was analyzed using the hierarchical clustering method. Finally, an adaptive longitudinal feature selection and grading network, ALNet, was developed to evaluate muscle atrophy. The clustering trajectories of ultrasound image features showed similar trends to the changes in muscle atrophy at the molecular level. The best grading accuracy achieved by the ALNet was 79.5% for the Soleus (Sol) muscle and 82.6% for the Gastrocnemius (Gas) muscle. The test-retest is essential in performing radiomics analysis on ultrasound images. The longitudinal feature selection is important for muscle atrophy grading. The ultrasound image features of the Gas muscle have better discrimination ability than that of the Sol muscle. This study proves for the first time the capability of ultrasound image features for muscle atrophy evaluation.

Effects Of Hindlimb Unloading And Long Acting Reversible Contraception On Markers Of Mitophagy In Muscle

Spaceflight has been shown to cause severe muscle atrophy. While loss of contractile proteins has been well documented, there is little research on the effects on the mitochondria and mitophagy. Long-acting reversible contraceptives (LARC; progestin-only) will be likely be recommended for female astronauts on deep space mission, however, the impact on muscle is unknown. PURPOSE: The purpose of this study was to examine the effects of hindlimb unloading (HU) and LARC on muscle mitophagy. METHODS: Female Sprague-Dawley rats (n = 52; 4-mo-old) were singly housed and randomly assigned to placebo (PL) and LARC groups, via an implanted slow-release etonogestrel pellet (0.00ug/d vs. 0.30ug/d). A week later, animals were further randomized to weight bearing (WB) and HU groups for 6 weeks. At the end of the 6-week HU period animals were euthanized. Tissues collected and snap frozen for later analysis. Western blot analysis was run to quantify markers of mitophagy (Parkin, NIX) as well as glycolytic enzymes (GAPDH). Signal values were normalized against the loading control for each gel. Statistics were conducted using RStudio. RESULTS: Despite increasing their food intake during HU (p < 0.01), HU animals lost weight and weighed less than WB animals starting on HU week 2 (p < 0.01). Weight wet of the gastrocnemius was significantly different after HU (WB: 2.07 g +/- 0.08 vs HU: 1.58 g +/- 0.11). A significant difference was observed between the WB and HU groups for both Parkin (1.01 +/- 0.33 vs 0.74 +/- .25, p = .006) and NIX (1.10 +/- 0.38 vs 0.82 +/- 0.43, p = .040), respectively. No significant difference was found in GAPDH. LARC had no effect on mitophagy or glycolytic enzyme content. There was no significant interaction between HU and LARC on mitophagy or glycolytic markers. CONCLUSION: After 6-weeks of unloading, markers of mitophagy were significantly decreased. This could be due to either impairments in the mitophagy process or reduced enzyme content due to reduced muscle fiber size as indicated by reduced muscle weight after HU. LARC implantation did not alter the impact of HU on mitophagy markers. Further analysis into mitochondrial protein content as well as respiratory capacity will provide insight into mitochondrial health and function.

Improvement of Osteoporosis in Rats With Hind-Limb Unloading Treated With Pulsed Electromagnetic Field and Whole-Body Vibration.

Physical factors have been used to address disuse osteoporosis, but their effects and mechanism remain unclear. The purpose of this study was to determine the effects of pulsed electromagnetic field (PEMF) and whole-body vibration (WBV) on disuse osteoporosis to increase knowledge about treating osteoporosis. A disuse osteoporosis rat model was developed by hind-limb unloading (HU) for 6weeks. Forty 4-month-old female Sprague-Dawley rats were divided into 5 groups and given the following interventions: HU, HU treated with PEMF (HUP), HU treated with WBV (HUW), HU treated with both PEMF and WBV (HUPW), and no intervention (controls). After 8weeks of intervention, measurements were taken. HU induced a decrease in bone mineral density (BMD), whereas HUP, HUW, and HUPW increased it. Moreover, the bone resorption markers tartrate-resistant acid phosphatase (TRAP) and C-terminal peptide of type 1 collagen in the HU group significantly increased, whereas the osteogenesis markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly decreased. The markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly increased, but TRAP and C-terminal peptide of type 1 collagen significantly decreased in the HUPW, HUP, and HUW groups compared with the HU group. In particular, HUPW effectively increased osteocalcin and decreased TRAP compared with HUP and WBV. Microcomputed tomography analysis of the femur indicated that HUPW improved trabecular number, bone volume over total volume, bone surface over bone volume, trabecular separation, and the structure model index compared with HUP and that it improved bone surface over bone volume, trabecular separation, and structure model index compared with HUW. The HUPW group showed a significant increase in maximum load compared with the HUW group and a significant increase in elastic modulus compared with the HUP group. PEMF, WBV, and their combination all attenuated bone resorption and enhanced osteogenesis. WBV and the combination of treatments have great potential to improve osteogenesis compared with PEMF. In addition, HUPW significantly attenuated bone resorption compared with HUW and HUP. The results of this study indicated that HUPW could effectively improve disuse osteoporosis compared with HUP, given that trabecular number and bone volume over total volume are associated with disuse osteoporosis. Moreover, BMD recovered well with HUP, HUW, and HUPW but the bone structure-especially mechanical performance-did not, indicating that osteoporosis should be evaluated with BMD and mechanical performance, not with BMD in isolation.

Mitigating sarcoplasmic reticulum stress limits disuse-induced muscle loss in hindlimb unloaded mice

Muscle disuse in the hindlimb unloaded (HU) mice causes significant atrophy and weakness. However, the cellular and molecular mechanisms driving disuse-muscle atrophy remain elusive. We investigated the potential contribution of proteins dysregulation by sarcoplasmic reticulum (SR), a condition called SR stress, to muscle loss during HU. Male, c57BL/6j mice were assigned to ground-based controls or HU groups treated with vehicle or 4-phenylbutyrate (4-PBA), a potent inhibitor of SR stress, once a day for three weeks. We report that the 4-PBA reduced the SR stress and partly reversed the muscle atrophy and weakness in the HU mice. Transcriptome analysis revealed that several genes were switched on (n = 3688) or differentially expressed (n = 1184) due to HU. GO, and KEGG term analysis revealed alterations in pathways associated with the assembly of cilia and microtubules, extracellular matrix proteins regulation, calcium homeostasis, and immune modulation during HU. The muscle restoration with 4-PBA partly reversed these changes along with differential and unique expression of several genes. The analysis of genes among the two comparisons (HU-v vs. control and HU-t vs. HU-v.) shows 841 genes were overlapped between the two comparisons and they may be regulated by 4-PBA. Altogether, our findings suggest that the pharmacological suppression of SR stress may be an effective strategy to prevent disuse-induced muscle weakness and atrophy.

Calycosin prevents bone loss induced by hindlimb unloading

Bone loss induced by microgravity exposure seriously endangers the astronauts’ health, but its countermeasures still have certain limitations. The study aims to find potential protective drugs for the prevention of the microgravity-induced bone loss. Here, we utilized the network pharmacology approach to discover a natural compound calycosin by constructing the compound-target interaction network and analyzing the topological characteristics of the network. Furthermore, the hind limb unloading (HLU) rats’ model was conducted to investigate the potential effects of calycosin in the prevention of bone loss induced by microgravity. The results indicated that calycosin treatment group significantly increased the bone mineral density (BMD), ameliorated the microstructure of femoral trabecular bone, the thickness of cortical bone and the biomechanical properties of the bone in rats, compared that in the HLU group. The analysis of bone turnover markers in serum showed that both the bone formation markers and bone resorption markers decreased after calycosin treatment. Moreover, we found that bone remodeling-related cytokines in serum including IFN-γ, IL-6, IL-8, IL-12, IL-4, IL-10 and TNF-α were partly recovered after calycosin treatment compared with HLU group. In conclusion, calycosin partly recovered hind limb unloading-induced bone loss through the regulation of bone remodeling. These results provided the evidence that calycosin might play an important role in maintaining bone mass in HLU rats, indicating its promising application in the treatment of bone loss induced by microgravity.

Molecular and Metabolic Mechanism of Low-Intensity Pulsed Ultrasound Improving Muscle Atrophy in Hindlimb Unloading Rats.

Low-intensity pulsed ultrasound (LIPUS) has been proved to promote the proliferation of myoblast C2C12. However, whether LIPUS can effectively prevent muscle atrophy has not been clarified, and if so, what is the possible mechanism. The aim of this study is to evaluate the effects of LIPUS on muscle atrophy in hindlimb unloading rats, and explore the mechanisms. The rats were randomly divided into four groups: normal control group (NC), hindlimb unloading group (UL), hindlimb unloading plus 30 mW/cm2 LIPUS irradiation group (UL + 30 mW/cm2), hindlimb unloading plus 80 mW/cm2 LIPUS irradiation group (UL + 80 mW/cm2). The tails of rats in hindlimb unloading group were suspended for 28 days. The rats in the LIPUS treated group were simultaneously irradiated with LIPUS on gastrocnemius muscle in both lower legs at the sound intensity of 30 mW/cm2 or 80 mW/cm2 for 20 min/d for 28 days. C2C12 cells were exposed to LIPUS at 30 or 80 mW/cm2 for 5 days. The results showed that LIPUS significantly promoted the proliferation and differentiation of myoblast C2C12, and prevented the decrease of cross-sectional area of muscle fiber and gastrocnemius mass in hindlimb unloading rats. LIPUS also significantly down regulated the expression of MSTN and its receptors ActRIIB, and up-regulated the expression of Akt and mTOR in gastrocnemius muscle of hindlimb unloading rats. In addition, three metabolic pathways (phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism) were selected as important metabolic pathways for hindlimb unloading effect. However, LIPUS promoted the stability of alanine, aspartate and glutamate metabolism pathway. These results suggest that the key mechanism of LIPUS in preventing muscle atrophy induced by hindlimb unloading may be related to promoting protein synthesis through MSTN/Akt/mTOR signaling pathway and stabilizing alanine, aspartate and glutamate metabolism.

Evaluation of the cargo contents and potential role of extracellular vesicles in osteoporosis.

Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). Extracellular vesicles (EVs) remain unexplored in the context of osteoporosis. Towards this, EVs were isolated from plasma of a discovery cohort with 8 non-osteoporotic and 8 osteoporotic individuals, and nanoparticle tracking analysis (NTA) revealed a significantly higher EV concentration in osteoporotic individuals (P = 0.003). Moreover, EVs concentration showed a linear correlation with bone mineral density (BMD) values (linear correlation coefficient r = 0.9542, deviation from zero, p < 0.001). Results using a mouse model of osteoporosis confirmed that the number of EVs in mice from hindlimb unloading group was significantly higher than that from the age-matched control group (p = 0.015). TaqMan Real-Time PCR demonstrated that miR-335-5p, -320a, -483-5p, and miR-21-5p, were significantly higher expressed in osteoporotic patients compared with non-osteoporotic individuals. Quantitative real-time PCR shown that Wnt1, Wnt5a, Wnt7a, and Wnt9a mRNAs were lower expressed in osteoporosis derived EVs. In vitro functional assay indicated that osteoporosis derived EVs resulted in reduced mineralization in SaOS-2 cells. In conclusion, these results suggest that osteoporosis increased the secretion of EVs which carry higher expression of miRNAs and decreased expression of Wnt signals, further decreased the mineralization capacity in human osteoblasts.

Peptidyl Inhibition of Nox2 Enhances Stress Response and Mitigates Muscle Fiber Atrophy with Simulated Microgravity

Skeletal muscle is a dynamic and pliable tissue, which contains up to 75% of all proteins in the body. The regulation of proteins synthesis and degradation determines the muscle responsive adaptations to alterations in mechanical loading, nutrition availability, exercise intensity, etc. The pathway by which skeletal muscle cells are equipped with the ability of converting changes in mechanical force to biochemical signals is entitled “mechanotransduction”. The translocation of mu-splice variant of neuronal Nitric Oxide Synthase (nNOSµ) from sarcolemma to sarcoplasm is ascertained a causal player in mechanotransduction in unloaded skeletal muscle. In addition, Reactive Oxygen Species (ROS) are demonstrated to be causal in the translocation of nNOSμ, suppression of anabolic signaling, and muscle fiber atrophy. Mitochondria is known to be one of the sources of ROS production during unloading. Moreover, the sarcolemmal NADPH oxidase-2 (Nox2), a mechano-sensitive source of ROS, is also revealed to be increased in unloaded skeletal muscles. The upregulated Nox2 would further contribute directly to nNOSμ translocation, perturbation in heat shock proteins (e.g., HSP70) and antioxidant enzyme (e.g., MnSOD), and muscle atrophy during unloading. Upstream mechanisms and downstream effects involved in the regulation of Nox2 activity during exposure to weightlessness are partially understood due to their complex nature. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor reported to protect cells in response to stressors. We postulate that peptidyl inhibition of Nox2 would alleviate the disruption of HSP70, MnSOD, and Nrf2, and the translocation of sarcolemmal nNOS during unloading and, therefore, muscle fiber atrophy. 4-month-old F344 rats were partitioned into 3 groups: ambulatory control (CON), hindlimb unloaded (HU), and hindlimb unloaded + 5 mg/kg/day peptidyl Nox2 inhibitor gp91dstat (HUG). At the end of the study (7 days), soleus muscles were removed and frozen in isopentane chilled in liquid N2. Samples were then fractionized for further analysis by western blot, immunofluorescence, etc. Our data revealed that exposure to mechanical unloading significantly decreases the protein level of HSP70, MnSOD, and NrF2. Although, no significant protective effect of gp91dstat treatment was observed in MnSOD protein abundance, Nrf2 and HSP70 levels were notably protected during unloading when Nox2 was inhibited, suggestive of the preservation of antioxidant protection. In addition, the nNOS sarcolemmal activity, which was reduced in the hindlimb unloading study group, was elevated with the use of peptidyl Nox2 inhibitor. In conclusion, this study provides direct evidence that upregulated Nox2 can act as a causal role in skeletal muscle mechanotransduction and cellular remodeling during mechanical unloading.

Hindlimb unloading-induced reproductive suppression via Downregulation of hypothalamic Kiss-1 expression in adult male rats

BackgroundSpaceflights-induced microgravity can alter various physiological processes in human’s body including the functional status of the reproductive system. Rodent model of tail-suspension hindlimb unloading is extensively used to stimulate the organs responses to the microgravity condition. This study explores the potential effects of hindlimb unloading on testicular functions and spermatogenesis in adult male rats and the underlying mechanism/s.MethodsTwenty Sprague-Dawley rats were allotted into two groups: normally loaded group (control; all arms were in touch with the grid floor) and hindlimb unloaded group (HU; only the forearms were in contact with the grid floor).ResultsFollowing 30 days of exposure, the HU group saw a decline in body weight, testicular and epidydimal weights, and all semen parameters. The circulating concentrations of gonadotropin-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone significantly decreased, while levels of kisspeptin, corticosterone, inhibin, prolactin and estradiol (E2) increased in the HU group. Intratesticular levels of 5α-reductase enzyme and dihydrotestosterone (DHT) were suppressed, while the levels of aromatase and kisspeptin were significantly elevated in the HU group. Hypothalamic kisspeptin (Kiss1) mRNA expression levels were downregulated while its receptors (Kiss1R) were upregulated in the HU group. On the contrary, the mRNA expression levels of testicular Kiss1 were upregulated while Kiss1R were downregulated. The pituitary mRNA expression levels of FSHβ and LHβ decreased in the HU group. The levels of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and nitric oxide (NO) concentrations, and total antioxidant capacity (TAC) were elevated while malondialdehyde (MDA) concentrations declined in the testes of HU group. The testes of the HU rats showed positive immunostaining of caspase-3, heat shock protein 70 (HSP70) and Bcl2.ConclusionsAltogether, these results revealed an inhibitory effect of hindlimb unloading on kisspeptin signaling in the hypothalamic-pituitary-testicular axis with impaired spermatogenesis and steroidogenesis.

Hindlimb unloading causes regional loading-dependent changes in osteocyte inflammatory cytokines that are modulated by exogenous irisin treatment

Disuse-induced bone loss is characterized by alterations in bone turnover. Accruing evidence suggests that osteocytes respond to inflammation and express and/or release pro-inflammatory cytokines; however, it remains largely unknown whether osteocyte inflammatory proteins are influenced by disuse. The goals of this project were (1) to assess osteocyte pro-inflammatory cytokines in the unloaded hindlimb and loaded forelimb of hindlimb unloaded rats, (2) to examine the impact of exogenous irisin during hindlimb unloading (HU). Male Sprague Dawley rats (8 weeks old, n = 6/group) were divided into ambulatory control, HU, and HU with irisin (HU + Ir, 3×/week). Lower cancellous bone volume, higher osteoclast surfaces (OcS), and lower bone formation rate (BFR) were present at the hindlimb and 4th lumbar vertebrae in the HU group while the proximal humerus of HU rats exhibited no differences in bone volume, but higher BFR and lower OcS vs. Con. Osteocyte tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), RANKL, and sclerostin were elevated in the cancellous bone of the distal femur of HU rats vs. Con, but lower at the proximal humerus in HU rats vs. Con. Exogenous irisin treatment increased BFR, and lowered OcS and osteocyte TNF-α, IL-17, RANKL, and sclerostin in the unloaded hindlimb of HU + Ir rats while having minimal changes in the humerus. In conclusion, there are site-specific and loading-specific alterations in osteocyte pro-inflammatory cytokines and bone turnover with the HU model of disuse bone loss, indicating a potential mechanosensory impact of osteocyte TNF-α and IL-17. Additionally, exogenous irisin significantly reduced the pro-inflammatory status of the unloaded hindlimb.

Protein Supplementation Enhances the Effects of Intermittent Loading on Skeletal Muscles by Activating the mTORC1 Signaling Pathway in a Rat Model of Disuse Atrophy.

Inactivity leads to skeletal muscle atrophy, whereas intermittent loading (IL) during hind limb unloading (HU) attenuates muscle atrophy. However, the combined effects of IL and protein supplementation on disuse muscle atrophy are unclear. Therefore, we investigated the effects of IL and a high-protein oral nutritional supplement (HP) during HU on skeletal muscle mass and protein synthesis/breakdown. Male F344 rats were assigned to the control (CON), 14-day HU (HU), IL during HU (HU + IL), and IL during HU followed by HP administration (2.6 g protein/kg/day; HU + IL + HP) groups. Soleus and gastrocnemius muscles were sampled 30 min after the last IL and HP supplementation. HU decreased relative soleus and gastrocnemius muscle masses. Relative muscle masses and p70 ribosomal protein S6 kinase/ribosomal protein S6 phosphorylation in soleus and gastrocnemius muscles were higher in the HU + IL group than the HU group and further higher in the HU + IL + HP group than the HU + IL group in gastrocnemius muscle. Therefore, protein administration plus IL effectively prevented skeletal muscle atrophy induced by disuse, potentially via enhanced activation of targets downstream of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway.