Hindlimb Unloading Model Research Articles

Mesenchymal stem cell transplant as an intervention to ameliorate disuse-induced muscle atrophy in a mouse model of simulated microgravity

BackgroundThe hindlimb unloaded (HU) mouse model exhibits disuse-induced muscle atrophy. However, effective interventions remain elusive. We investigated the therapeutic potential of mesenchymal stem cells (MSC) transplant on muscle decline in HU mice. MethodsWe divided 4-month-old male c57BL/6j mice into controls and HU mice treated with PBS as placebo (HU-PBS) or MSCs (HU-MSC; one million cells/100 μl PBS into gastrocnemius muscles once a week) for three weeks. We measured muscle mass, grip strength, and an unbiased transcriptome analysis of gastrocnemius muscles. ResultsMSC treatment prevented muscle atrophy and improved grip strength in HU mice. Transcriptome analysis revealed MSC-induced unique (557 genes) and differential (1,214 genes) expressions of several genes compared to the HU-PBS group. GO and KEGG term analysis revealed an HU-induced downregulation of pathways associated with the regulation of contractile apparatus, neuromuscular junction, and satellite cell function, which were partly reversed with MSC treatment. Lastly, MSC treatment also upregulated the pathways controlling muscle differentiation and growth in the HU mice. ConclusionAltogether, we report the therapeutic potential of MSCs in treating disuse-induced muscle atrophy and weakness. Our study may help unravel novel molecular mechanisms involved in MSCs-induced muscle restoration.

SDSSD peptide modified polyvinylamine − A novel bone-targeting RNA delivery system

Bone-related disorders have become a prevailing concern in older men and postmenopausal women. Traditional methods of treatment, though have benefits, their inability to target specific cells or tissues increases their ineffectiveness and side effects. RNA therapy has emerged as an alternative to conventional therapy owing to its specific targeting ability. However, the lack of appropriate RNA vectors remains a major problem for successful RNA therapy. Here, we designed a delivery system, (SDSSD-PVAm (PS)) based on a cationic polymer, polyvinylamine (PVAm), and a bone-targeting peptide, SDSSD (Serine-Aspartate-Serine-Serine-Aspartate), to deliver a therapeutic RNA, anti-miR-138-5p, to the bone tissue. The results showed that the SDSSD modification not only had no negative effect on the RNA-carrying or protection abilities of PVAm but also improved targeting and RNA delivery to osteoblasts and improve osteogenic potential of the cells by improving the expression of osteogenic genes both at RNA and protein level. Recombinant anti-miR-138-5p delivered through PS was highly enriched in the bone tissue, and the resultant nanoparticles could circulate for a longer time in the blood with negligible toxicity. The results showed that improved RNA delivery through PS significantly increased the rate of bone formation and improved trabecular and cortical bone microarchitecture, resulting in enhanced bone mass in ovariectomy (OVX) and hind limb unloading (HLU) model mice. These results demonstrate that SDSSD-PVAm is a novel RNA delivery system for bone-related disorders, such as osteoporosis.

Melatonin Regulates Osteoblast Differentiation through the m6A Reader hnRNPA2B1 under Simulated Microgravity.

Recent studies have confirmed that melatonin and N6-methyladenosine (m6A) modification can influence bone cell differentiation and bone formation. Melatonin can also regulate a variety of biological processes through m6A modification. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) serves as a reader of m6A modification. In this study, we used the hindlimb unloading model as an animal model of bone loss induced by simulated microgravity and used 2D clinorotation to simulate a microgravity environment for cells on the ground. We found that hnRNPA2B1 was downregulated both in vitro and in vivo during simulated microgravity. Further investigations showed that hnRNPA2B1 could promote osteoblast differentiation and that overexpression of hnRNPA2B1 attenuated the suppression of osteoblast differentiation induced by simulated microgravity. We also discovered that melatonin could promote the expression of hnRNPA2B1 under simulated microgravity. Moreover, we found that promotion of osteoblast differentiation by melatonin was partially dependent on hnRNPA2B1. Therefore, this research revealed, for the first time, the role of the melatonin/hnRNPA2B1 axis in osteoblast differentiation under simulated microgravity. Targeting this axis may be a potential protective strategy against microgravity-induced bone loss and osteoporosis.

Effects of Ginsenoside Rb1 on the Crosstalk between Intestinal Stem Cells and Microbiota in a Simulated Weightlessness Mouse Model.

Exposure to the space microenvironment has been found to disrupt the homeostasis of intestinal epithelial cells and alter the composition of the microbiota. To investigate this in more detail and to examine the impact of ginsenoside Rb1, we utilized a mouse model of hindlimb unloading (HU) for four weeks to simulate the effects of microgravity. Our findings revealed that HU mice had ileum epithelial injury with a decrease in the number of intestinal stem cells (ISCs) and the level of cell proliferation. The niche functions for ISCs were also impaired in HU mice, including a reduction in Paneth cells and Wnt signaling, along with an increase in oxidative stress. The administration of Rb1 during the entire duration of HU alleviated the observed intestinal defects, suggesting its beneficial influence on epithelial cell homeostasis. Hindlimb unloading also resulted in gut dysbiosis. The supplementation of Rb1 in the HU mice or the addition of Rb1 derivative compound K in bacterial culture in vitro promoted the growth of beneficial probiotic species such as Akkermansia. The co-housing experiment further showed that Rb1 treatment in ground control mice alone could alleviate the defects in HU mice that were co-housed with Rb1-treated ground mice. Together, these results underscore a close relationship between dysbiosis and impaired ISC functions in the HU mouse model. It also highlights the beneficial effects of Rb1 in mitigating HU-induced epithelial injury by promoting the expansion of intestinal probiotics. These animal-based insights provide valuable knowledge for the development of improved approaches to maintaining ISC homeostasis in astronauts.

Simulated Microgravity Alters Gene Regulation Linked to Immunity and Cardiovascular Disease.

Microgravity exposure induces a cephalad fluid shift and an overall reduction in physical activity levels which can lead to cardiovascular deconditioning in the absence of countermeasures. Future spaceflight missions will expose crew to extended periods of microgravity among other stressors, the effects of which on cardiovascular health are not fully known. In this study, we determined cardiac responses to extended microgravity exposure using the rat hindlimb unloading (HU) model. We hypothesized that exposure to prolonged simulated microgravity and subsequent recovery would lead to increased oxidative damage and altered expression of genes involved in the oxidative response. To test this hypothesis, we examined hearts of male (three and nine months of age) and female (3 months of age) Long-Evans rats that underwent HU for various durations up to 90 days and reambulated up to 90 days post-HU. Results indicate sex-dependent changes in oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) and antioxidant gene expression in left ventricular tissue. Three-month-old females displayed elevated 8-OHdG levels after 14 days of HU while age-matched males did not. In nine-month-old males, there were no differences in 8-OHdG levels between HU and normally loaded control males at any of the timepoints tested following HU. RNAseq analysis of left ventricular tissue from nine-month-old males after 14 days of HU revealed upregulation of pathways involved in pro-inflammatory signaling, immune cell activation and differential expression of genes associated with cardiovascular disease progression. Taken together, these findings provide a rationale for targeting antioxidant and immune pathways and that sex differences should be taken into account in the development of countermeasures to maintain cardiovascular health in space.

Physical exercise restores adult neurogenesis deficits induced by simulated microgravity

Cognitive impairments have been reported in astronauts during spaceflights and documented in ground-based models of simulated microgravity (SMG) in animals. However, the neuronal causes of these behavioral effects remain largely unknown. We explored whether adult neurogenesis, known to be a crucial plasticity mechanism supporting memory processes, is altered by SMG. Adult male Long-Evans rats were submitted to the hindlimb unloading model of SMG. We studied the proliferation, survival and maturation of newborn cells in the following neurogenic niches: the subventricular zone (SVZ)/olfactory bulb (OB) and the dentate gyrus (DG) of the hippocampus, at different delays following various periods of SMG. SMG exposure for 7 days, but not shorter periods of 6 or 24 h, resulted in a decrease of newborn cell proliferation restricted to the DG. SMG also induced a decrease in short-term (7 days), but not long-term (21 days), survival of newborn cells in the SVZ/OB and DG. Physical exercise, used as a countermeasure, was able to reverse the decrease in newborn cell survival observed in the SVZ and DG. In addition, depending on the duration of SMG periods, transcriptomic analysis revealed modifications in gene expression involved in neurogenesis. These findings highlight the sensitivity of adult neurogenesis to gravitational environmental factors during a transient period, suggesting that there is a period of adaptation of physiological systems to this new environment.

Effects of N-Acetyl-Carnosine Treatment on Aging and Carbonyl Stress

By 2050 the number of individuals over the age of 80y will triple to reach 426 million and the rate of age-related comorbidities has also increased precipitously as our global population has continued to age. Currently, there is no clinically approved treatment for age-related functional decline creating demand for the development of such treatments. Our lab previously demonstrated that carbonyl stress is exacerbated in muscles during a hindlimb unloading (HU) model of inactivity. Importantly, the accumulation of proteins modified by the reactive lipid carbonyl 4HNE, as well as muscle dysfunction and atrophy, were attenuated in HU mice treated with the carbonyl-scavenging dipeptide N-acetyl-carnosine (N-Ac-Carn). Therefore, we hypothesized that N-Ac-Carn could prevent carbonyl stress in aging muscles and other tissues. 17-month-old male and female C57Bl6J mice from the NIA colony were treated for 6-months with 80 mM N-Ac-Carn or normal (CON) water. Before and after the 6-month intervention we assessed several markers of health including glucose tolerance, kidney, muscle, bone, and cognitive function. N-Ac-Carn preserved ex vivo muscle force production in soleus ( p=0.0015) and EDL ( p=0.0008) muscles from female but not male mice. N-Ac-Carn treated mice also possessed superior bone volume compared to CON ( p=0.0046). Following terminal tissue collection, we also assessed muscles for carbonyl stress by probing for proteins modified by total carbonyls, 4HNE, MDA, and methylglyoxal. N-Ac-Carn did not attenuate 4HNE, MDA, or total carbonyls in gastrocnemius. However, N-Ac-Carn showed strong trends for attenuating 4HNE burden in both soleus and EDL muscles. N-Ac-Carn is an endogenously produced dipeptide that is safe for human consumption and affordable making it a promising therapeutic strategy for the mitigation of age-related functional decline. AG074535-02S2. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition

ObjectiveSkeletal muscle plasticity and remodeling are critical for adapting tissue function to use, disuse, and regeneration. The aim of this study was to identify genes and molecular pathways that regulate the transition from atrophy to compensatory hypertrophy or recovery from injury. Here, we have used a mouse model of hindlimb unloading and reloading, which causes skeletal muscle atrophy, and compensatory regeneration and hypertrophy, respectively. MethodsWe analyzed mouse skeletal muscle at the transition from hindlimb unloading to reloading for changes in transcriptome and extracellular fluid proteome. We then used qRT-PCR, immunohistochemistry, and bulk and single-cell RNA sequencing data to determine Mustn1 gene and protein expression, including changes in gene expression in mouse and human skeletal muscle with different challenges such as exercise and muscle injury. We generated Mustn1-deficient genetic mouse models and characterized them in vivo and ex vivo with regard to muscle function and whole-body metabolism. We isolated smooth muscle cells and functionally characterized them, and performed transcriptomics and proteomics analysis of skeletal muscle and aorta of Mustn1-deficient mice. ResultsWe show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. We show that Mustn1 is secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle extracellular fluid, particularly during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates. ConclusionsWe show that, in addition to its previously reported intracellular localization, Mustn1 is a microprotein secreted from smooth muscle cells into the muscle extracellular space. We explore its role in muscle ECM deposition and remodeling in homeostasis and upon muscle injury. The role of Mustn1 in fibrosis and immune infiltration upon muscle injury and dystrophies remains to be investigated, as does its potential for therapeutic interventions.

Characterization of the mechanical properties of the mouse Achilles tendon enthesis by microindentation. Effects of unloading and subsequent reloading

The fibrocartilaginous tendon enthesis, i.e. the site where a tendon is attached to bone through a fibrocartilaginous tissue, is considered as a functionally graded interface. However, at local scale, a very limited number of studies have characterized micromechanical properties of this transitional tissue. The first goal of this work was to characterize the micromechanical properties of the mineralized part of the healthy Achilles tendon enthesis (ATE) through microindentation testing and to assess the degree of mineralization and of carbonation of mineral crystals by Raman spectroscopy. Since little is known about enthesis biological plasticity, our second objective was to examine the effects of unloading and reloading, using a mouse hindlimb-unloading model, on both the micromechanical properties and the mineral phase of the ATE. Elastic modulus, hardness, degree of mineralization, and degree of carbonation were assessed after 14 days of hindlimb suspension and again after a subsequent 6 days of reloading. The elastic modulus gradually increased along the mineralized part of the ATE from the tidemark to the subchondral bone, with the same trend being found for hardness. Whereas the degree of carbonation did not differ according to zone of measurement, the degree of mineralization increased by >70 % from tidemark to subchondral bone. Thus, the gradient in micromechanical properties is in part explained by a mineralization gradient. A 14-day unloading period did not appear to affect the gradient of micromechanical properties of the ATE, nor the degree of mineralization or carbonation. However, contrary to a short period of unloading, early return to normal mechanical load reduced the micromechanical properties gradient, regardless of carbonate-to-phosphate ratios, likely due to the more homogeneous degree of mineralization. These findings provide valuable data not only for tissue bioengineering, but also for musculoskeletal clinical studies and microgravity studies focusing on long-term space travel by astronauts.

Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Mechanical Unloading Promotes Osteoclastic Differentiation and Bone Resorption by Modulating the MSC Secretome to Favor Inflammation.

Aging, space flight, and prolonged bed rest have all been linked to bone loss, and no effective treatments are clinically available at present. Here, with the rodent hindlimb unloading (HU) model, we report that the bone marrow (BM) microenvironment was significantly altered, with an increased number of myeloid cells and elevated inflammatory cytokines. In such inflammatory BM, the osteoclast-mediated bone resorption was greatly enhanced, leading to a shifted bone remodeling balance that ultimately ends up with disuse-induced osteoporosis. Using Piezo1 conditional knockout (KO) mice (Piezo1fl/fl;LepRCre), we proved that lack of mechanical stimuli on LepR+ mesenchymal stem cells (MSCs) is the main reason for the pathological BM inflammation. Mechanically, the secretome of MSCs was regulated by mechanical stimuli. Inadequate mechanical load leads to increased production of inflammatory cytokines, such as interleukin (IL)-1α, IL-6, macrophage colony-stimulating factor 1 (M-CSF-1), and so on, which promotes monocyte proliferation and osteoclastic differentiation. Interestingly, transplantation of 10% cyclic mechanical stretch (CMS)-treated MSCs into HU animals significantly alleviated the BM microenvironment and rebalanced bone remodeling. In summary, our research revealed a new mechanism underlying mechanical unloading-induced bone loss and suggested a novel stem cell-based therapy to potentially prevent disuse-induced osteoporosis.

Serum multi-omics analysis in hindlimb unloading mice model: Insights into systemic molecular changes and potential diagnostic and therapeutic biomarkers

Microgravity, in space travel and prolonged bed rest conditions, induces cardiovascular deconditioning along with skeletal muscle mass loss and weakness. The findings of microgravity research may also aid in the understanding and treatment of human health conditions on Earth such as muscle atrophy, and cardiovascular diseases. Due to the paucity of biomarkers and the unknown underlying mechanisms of cardiovascular and skeletal muscle deconditioning in these environments, there are insufficient diagnostic and preventative measures. In this study, we employed hindlimb unloading (HU) mouse model, which mimics astronauts in space and bedridden patients, to first evaluate cardiovascular and skeletal muscle function, followed by proteomics and metabolomics LC-MS/MS-based analysis using serum samples. Three weeks of unloading caused changes in the function of the cardiovascular system in c57/Bl6 mice, as seen by a decrease in mean arterial pressure and heart weight. Unloading for three weeks also changed skeletal muscle function, causing a loss in grip strength in HU mice and atrophy of skeletal muscle indicated by a reduction in muscle mass. These modifications were partially reversed by a two-week recovery period of reloading condition, emphasizing the significance of the recovery process. Proteomics analysis revealed 12 dysregulated proteins among the groups, such as phospholipid transfer protein, Carbonic anhydrase 3, Parvalbumin alpha, Major urinary protein 20 (Mup20), Thrombospondin-1, and Apolipoprotein C-IV. On the other hand, metabolomics analysis showed altered metabolites among the groups such as inosine, hypoxanthine, xanthosine, sphinganine, l-valine, 3,4-Dihydroxyphenylglycol, and l-Glutamic acid. The joint data analysis revealed that HU conditions mainly impacted pathways such as ABC transporters, complement and coagulation cascades, nitrogen metabolism, and purine metabolism. Overall, our results indicate that microgravity environment induces significant alterations in the function, proteins, and metabolites of these mice. These observations suggest the potential utilization of these proteins and metabolites as novel biomarkers for assessing and mitigating cardiovascular and skeletal muscle deconditioning associated with such conditions.

The Critical Role of The Piezo1/β-catenin/ATF4 Axis on The Stemness of Gli1+ BMSCs During Simulated Microgravity-Induced Bone Loss.

Disuse osteoporosis is characterized by decreased bone mass caused by abnormal mechanical stimulation of bone. Piezo1 is a major mechanosensitive ion channel in bone homeostasis. However, whether intervening in the action of Piezo1 can rescue disuse osteoporosis remains unresolved. In this study, a commonly-used hindlimb-unloading model is employed to simulate microgravity. By single-cell RNA sequencing, bone marrow-derived mesenchymal stem cells (BMSCs) are the most downregulated cell cluster, and coincidentally, Piezo1 expression is mostly enriched in those cells, and is substantially downregulated by unloading. Importantly, activation of Piezo1 by systemically-introducing yoda1 mimics the effects of mechanical stimulation and thus ameliorates bone loss under simulated microgravity. Mechanistically, Piezo1 activation promotes the proliferation and osteogenic differentiation of Gli1+ BMSCs by activating the β-catenin and its target gene activating transcription factor 4 (ATF4). Inhibiting β-catenin expression substantially attenuates the effect of yoda1 on bone loss, possibly due to inhibited proliferation and osteogenic differentiation capability of Gli1+ BMSCs mediated by ATF4. Lastly, Piezo1 activation also slightly alleviates the osteoporosis of OVX and aged mice. In conclusion, impaired function of Piezo1 in BMSCs leads to insufficient bone formation especially caused by abnormal mechanical stimuli, and is thus a potential therapeutic target for osteoporosis.

Stromal Lineage Precursors from Rodent Femur and Tibia Bone Marrows after Hindlimb Unloading: Functional Ex Vivo Analysis.

Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb-tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions.

Role of myofascia in the recovery of bone loss after reloading in tail-suspended rats

Astronauts experience severe musculoskeletal degradation in space. After returning to earth, bone loss recovery did not parallel muscle recovery. Fascia may play an important role as a considerable part of muscle force is transmitted to bone through fascia. However, little is known about how fascia changes in microgravity and its effect on the development of musculoskeletal degeneration. In this study, a rat hindlimb unloading model was used to simulate microgravity (21 days of tail suspension) and then reloading for 7 days, 14 days and 21 days. After different periods, bone mineral density (BMD) and microstructure of the tibia were analyzed by μCT, the mechanical properties of the tibia were determined by the three-point bending test and the mechanical properties of fascia were explored by tensile test. The results show that, after tail suspension, collagen Ⅰ in myofascia was significantly increased (p < 0.05), the myofascial elastic modulus was significantly decreased (p < 0.05), BMD of the tibia was significantly decreased (p < 0.05) and the microstructure of the tibia had deteriorated. After 21 days of reloading, there was no significant improvement in the myofascial elastic modulus as well as in the tibial maximum load. Correlation analysis results showed that the myofascial elastic modulus was significantly correlated with the tibial maximum load. In summary, the deterioration of mechanical property of myofascia induced by tail suspension did not recover when the time of reloading was the same as the time of unloading. This indicates that the delayed bone recovery after being exposed to microgravity was associated with deterioration of myofascia.

Cold stress during room temperature housing alters skeletal response to simulated microgravity (hindlimb unloading) in growing female C57BL6 mice

Laboratory mice are typically housed at temperatures below the thermoneutral zone for the species, resulting in cold stress and premature cancellous bone loss. Furthermore, mice are more dependent upon non-shivering thermogenesis to maintain body temperature during spaceflight, suggesting that microgravity-induced bone loss may be due, in part, to altered thermogenesis. Consequently, we assessed whether housing mice at room temperature modifies the skeletal response to simulated microgravity. This possibility was tested using the hindlimb unloading (HLU) model to mechanically unload femora. Humeri were also assessed as they remain weight bearing during HLU. Six-week-old female C57BL6 (B6) mice were housed at room temperature (22 °C) or near thermoneutral (32 °C) and HLU for 2 weeks. Compared to baseline, HLU resulted in cortical bone loss in femur, but the magnitude of reduction was greater in mice housed at 22 °C. Cancellous osteopenia in distal femur (metaphysis and epiphysis) was noted in HLU mice housed at both temperatures. However, bone loss occurred at 22 °C, whereas the bone deficit at 32 °C was due to failure to accrue bone. HLU resulted in cortical and cancellous bone deficits (compared to baseline) in humeri of mice housed at 22 °C. In contrast, fewer osteopenic changes were detected in mice housed at 32 °C. These findings support the hypothesis that environmental temperature alters the skeletal response to HLU in growing female mice in a bone compartment-specific manner. Taken together, species differences in thermoregulation should be taken into consideration when interpreting the skeletal response to simulated microgravity.

Percutaneous electrical stimulation-induced muscle contraction prevents the decrease in ribosome RNA and ribosome protein during pelvic hindlimb suspension.

Skeletal muscle unloading leads to muscle atrophy. Ribosome synthesis has been implicated as an important skeletal muscle mass regulator owing to its translational capacity. Muscle unloading induces a reduction in ribosome synthesis and content, with muscle atrophy. Percutaneous electrical muscle stimulation (pEMS)-induced muscle contraction is widely used in clinics to improve muscle mass. However, its efficacy in rescuing the reduction in ribosomal synthesis has not been addressed thus far. We examined the effects of daily pEMS treatment on ribosome synthesis and content during mouse hindlimb unloading. Male C57BL/6J mice were randomly assigned to sedentary (SED) and hindlimb unloading by pelvic suspension (HU) groups. Muscle contraction was triggered by pEMS treatment of the right gastrocnemius muscle of a subset of the HU group (HU + pEMS). Hindlimb unloading for 6 days significantly lowered 28S rRNA, rpL10, and rpS3 expression, which was rescued by daily pEMS treatment. The protein expression of phospho-p70S6K and UBF was significantly higher in the HU + pEMS than in the HU group. The mRNA expression of ribophagy receptor Nufip1 increased in both the HU and HU + pEMS groups. Protein light chain 3 (LC3)-II expression and the LC3-II/LC3-I ratio were increased by HU, but pEMS attenuated this increase. Our findings indicate that during HU, daily pEMS treatment prevents the reduction in the levels of some proteins associated with ribosome synthesis. In addition, the HU-induced activation of ribosome degradation may be attenuated. These data provide insights into ribosome content regulation and the mechanism of attenuation of muscle atrophy by pEMS treatment during muscle disuse.NEW & NOTEWORTHY Muscle inactivity reduces ribosome synthesis and content during atrophy. Whether percutaneous electrical muscle stimulation (pEMS)-induced muscle contraction rescues the ribosome synthesis and content during muscle unloading is unclear. Using a mouse hindlimb-unloading model with pelvic suspension, we provide evidence that daily pEMS-induced muscle contraction during hindlimb unloading rescues the reduction in the expression of some ribosome synthesis-related proteins and ribosome content in the gastrocnemius muscle.

Activation of Focal Adhesion Kinase Restores Simulated Microgravity-Induced Inhibition of Osteoblast Differentiation via Wnt/Β-Catenin Pathway.

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/β-catenin pathway. However, the mechanism by which SMG alters the Wnt/β-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/β-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/β-catenin and Wnt/β-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/β-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.

Kinetics of bone loss in the murine hindlimb unloading model at two temperatures: standard 22°C vs. thermoneutral 28°C

Kinetics of bone loss in the murine hindlimb unloading model at two temperatures: standard 22°C vs. thermoneutral 28°C

Hind-limb unloading in rodents: Current evidence and perspectives

Due to scarcity of available biological samples from returning space explorers, alternative models, which improve the understanding of spaceflight-related pathophysiology are needed. This review presents an update of available literature on the hind-limb unloading model (HLU). HLU has been used as a model to simulating microgravity in rodents since the early 1980s. This model, by removing the gravitational load from hind limbs of the animals, is widely used for studying the effects of the hindlimb unloading on the cardiovascular and musculoskeletal physiology. Recent updates in the literature with regards to HLU, its advantages as well as how this model can be used to understand the effects of bedrest confinement and/or spaceflight are provided.