Hindlimb Suspension Research Articles

Antioxidant mito-TEMPO prevents the increase in tropomyosin oxidation and mitochondrial calcium accumulation under 7-day rat hindlimb suspension

After the first day of muscle disuse (unloading) mitochondria-derived ROS accumulate in the postural-tonic soleus muscle. It is known that excess of ROS can lead to the accumulation of intramitochondrial calcium and overload of mitochondria with calcium, can negatively affect mitochondrial function and fatigue resistance of soleus muscle.We assumed that the use of mitochondrial ROS scavenger mito-TEMPO will be able to prevent the unloading-induced disruption of mitochondrial functions and will help maintain soleus muscle fatigue resistance.To test this hypothesis, male rats were divided into 3 groups (n = 16 in each): vivarium control with placebo (C), 7-day hindlimb suspension with placebo (7HS) and 7-day hindlimb suspension with intraperitoneal administration of the mimetic superoxide dismutase mito-TEMPO at a dose of 1 mg/kg (7HSM). In the 7HS group, increased fatigue of the soleus muscle was found in the ex vivo test, accompanied with increased activity of ETC complex I and “leak” respiration, as well as a twofold increased content of oxidized tropomyosin (a marker of ROS level in tissues) and increase in intramitochondrial calcium compared to C. In 7HSM, the activity of ETC complex I and “leak” respiration had no significant differences from the control group, and the increase in intramitochondrial calcium and the content of oxidized tropomyosin was partially prevented, however, muscle fatigue was also significantly higher than in the control group. Thus, mitochondrial ROS under 7-day muscle unloading contribute to the accumulation of intramitochondrial calcium and oxidation of tropomyosin, but do not have a significant effect on soleus muscle function.

Spirulina platensis components mitigate bone density loss induced by simulated microgravity: A mechanistic insight

In microgravity conditions, the consumption of Spirulina platensis (SP) as a renewable food source shows promise in mitigating osteoporosis due to its high nutritional content photosynthetic efficiency, environmental adaptability and positive effects on bone density, though the exact bioactive components and mechanisms remain unclear. Using a hindlimb suspension (HLS) model, this study investigated SP components: proteins (SPP), polysaccharides (SPS), lipids (SPL), and residue (SPR) on bone density and metabolism. Findings revealed that SPP and SPS significantly enhanced bone density and reduced oxidative stress. Activation of the FoxO3/Wnt/β-catenin pathway reduced FoxO3a expression and increased Wnt signaling molecules and β-catenin protein, boosting bone formation. Moreover, these components promoted beneficial gut bacteria like Turicibacter, reduced the Firmicutes-to-Bacteroidetes ratio, and enhanced SCFAs production, crucial for bone health. This study emphasized the potential of Spirulina nutrients in addressing space-induced osteoporosis and developing functional foods for long-term space missions.

Preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model

We previously demonstrated that transcutaneous CO2 application promotes muscle fiber-type switching, fracture healing, and osteogenesis by increasing blood flow and angiogenesis. Here, we aimed to investigate the preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model. Eleven-week-old male Sprague-Dawley rats were divided into hindlimb suspension (HS), HS with transcutaneous CO2 application (HSCO2), and control groups. HSCO2 rats were administered transcutaneous 100 % CO2 gas in their bilateral hindlimbs, five times a week for 20 min. After 3 weeks, we harvested the gastrocnemius, femur, and tibia for assessment. Histological analysis revealed a significant decrease in the gastrocnemius myofiber cross-sectional area in HS rats compared to the control rats, whereas HSCO2 rats exhibited a significant increase compared to HS rats. Micro-computed tomography showed significant bone atrophy in the trabecular and cortical bones of the femur in HS rats compared to those of the control rats, whereas significant improvement was noted in HSCO2 rats. Histological analysis of the proximal tibia revealed more marrow adipose tissue in the HS rats than in the control rats. However, in the HSCO2 rats, fewer marrow adipose tissue and osteoclasts were observed. Moreover, HSCO2 rats had more osteoblasts and higher expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) than the HS rats. The gastrocnemius and distal femur of HSCO2 rats also exhibited elevated PGC-1α and VEGF expression and upregulation of the myogenesis markers and osteogenesis markers compared to those of HS rats. This treatment effectively prevented disuse osteoporosis and muscle atrophy by promoting local angiogenesis and blood flow. PGC-1α is crucial for promoting this angiogenic pathway. Transcutaneous CO2 application may be a novel preventive procedure for disuse osteoporosis and muscle atrophy, complementing medication and rehabilitation.

Improvement of heat treated spirulina proteins and their enzymatic hydrolysates on osteoporosis under simulated microgravity

In the space microgravity environment, bone loss poses a formidable health challenge for astronauts. Dietary intake of renewable food components offers a feasible approach to mitigate bone loss associated with primary osteoporosis, aligning with the demands of long-term space missions. Recent research has highlighted the potential of Spirulina in ameliorating osteoporosis, with thermal processing enhancing the protein bioavailability. This study investigates the effects of different heat-treated Spirulina proteins (SPP) and their enzymatic hydrolysates on osteoporosis using a hindlimb suspension model (HLS) in mice to simulate microgravity conditions. The results demonstrate that heat-treated SPP and enzymatic hydrolysates significantly preserve skeletal morphology, suppress bone resorption, and enhance bone density, with the microwave-treated SPP hydrolysate (MWSPPH) showing the most pronounced improvement effects. Mechanistic insights reveal that MWSPPH enhances bone health by modulating the FoxO/Wnt/β-catenin signaling pathway. Specifically, MWSPPH reduces the expression of FoxO and facilitates the accumulation of β-catenin, while also indirectly modulating osteoclast-related signaling by altering RANKL and RANK expression. It also beneficially increases beneficial gut microbiota and the production of short-chain fatty acids, thereby improving bone density, suggesting its potential as a dietary strategy for managing osteoporosis in space environments.

Effects of Hindlimb Suspension on the Development of Hip Bone Morphologies in Growing Rats

Abnormal hip bone morphologies are associated with various diseases of the hip joint. Weight bearing, especially during growth, may be important to achieve normal acetabulum development. This study aimed to investigate whether hip bone morphologies were affected by hindlimb suspension (HS) in 4 week-old rats. In HS groups, tail suspension was applied for 0, 2, 4, and 8 weeks. Age-matched rats were used as controls. The complex of hip bones with lumbar and sacral vertebrae were assessed based on morphological indexes using three-dimensional reconstructed images from X-ray computed tomography. Acetabular widths (measured from cranial to caudal) unchanged and depths became larger in both groups with age. Acetabular lengths (from the ventral side to the dorsal side) became larger in control groups but unchanged in HS groups with age. In HS groups, acetabular width, length, and depths were smaller than the control groups at 4 and/or 8 weeks. Acetabular versions became enlarged (rotated inwards) with age in both groups, although this was particularly pronounced in HS groups. Histologically, triradiate cartilage layers in the acetabulum were thinner with age and almost disappeared at 8 weeks in both groups. However, HS decreased Safranin O staining and prolonged the presence of hypertrophic chondrocyte indicating alterations in the chondral ossification processes. Iliac wing angles remained unchanged and anterior superior iliac crest (ASIC) distances increased with age in controls. In contrast, HS groups showed narrowed iliac wing angles with small ASIC distances. These results suggest that reduced mechanical loading during growth can interfere with hip joint formation.

Proteomic Profiling of Hindlimb Skeletal Muscle Disuse in a Murine Model of Sepsis.

Sepsis leads to multiple organ dysfunction and negatively impacts patient outcomes. Skeletal muscle disuse is a significant comorbidity in septic patients during their ICU stay due to prolonged immobilization. Combination of sepsis and muscle disuse will promote a unique proteomic signature in skeletal muscle in comparison to disuse and sepsis separately. Following cecal ligation and puncture (CLP) or Sham surgeries, mice were subjected to hindlimb suspension (HLS) or maintained normal ambulation (NA). Tibialis anterior muscles from 24 C57BL6/J male mice were harvested for proteomic analysis. Proteomic profiles were assessed using nano-liquid chromatography with tandem mass spectrometry, followed by data analysis including Partial Least Squares Discriminant Analysis (PLS-DA), to compare the differential protein expression across groups. A total of 2876 differentially expressed proteins were identified, with marked differences between groups. In mice subjected to CLP and HLS combined, there was a distinctive proteomic signature characterized by a significant decrease in the expression of proteins involved in mitochondrial function and muscle metabolism, alongside a marked increase in proteins related to muscle degradation pathways. The PLS-DA demonstrated a clear separation among experimental groups, highlighting the unique profile of the CLP/HLS group. This suggests an important interaction between sepsis-induced inflammation and disuse atrophy mechanisms in sepsis-induced myopathy. Our findings reveal a complex proteomic landscape in skeletal muscle exposed to sepsis and disuse, consistent with an exacerbation of muscle protein degradation under these combined stressors. The identified proteins and their roles in cellular stress responses and muscle pathology provide potential targets for intervention to mitigate muscle dysfunction in septic conditions, highlighting the importance of addressing both sepsis and disuse concurrently in clinical and experimental settings.

Molecular Signaling Effects behind the Spontaneous Soleus Muscle Activity Induced by 7-Day Rat Hindlimb Suspension.

The elimination of ground reaction force (support withdrawal) vastly affects slow postural muscles in terms of their regulation and structure. One of the effects of support withdrawal in this study was an immediate postural muscle inactivation, followed by the daily gradual development of spontaneous activity of the slow postural soleus muscle in response to rat hindlimb suspension to mimic space flight. The origin of this activity is somewhat akin to muscle spasticity after spinal cord injuries and is the result of KCC2 content decline in the spinal cord's motor neurons. However, the physiological consequences of unloading-induced spontaneous activity remain unexplored. We have conducted an experiment with the administration of a highly specific KCC2 activator during 7-day unloading. For this experiment, 32 male Wistar rats were divided into 4 groups: C+placebo, C+CLP-290 (100 mg/kg b w), 7HS+placebo, and 7HS+CLP-hindlimb-suspended group with CLP-290 administration (100 mg/kg b w). The soleus muscles of the animals were dissected and analyzed for several proteostasis- and metabolism-related parameters. CLP-290 administration to the unloaded animals led to the upregulation of AMPK downstream (p-ACC) and mTOR targets (p-p70S6k and p-4E-BP) and an enhanced PGC1alpha decrease vs. the 7HS group, but neither prevented nor enhanced atrophy of the soleus muscle or myofiber CSA.

Achilles tendon enthesis behavior under cyclic compressive loading: Consequences of unloading and early remobilization

The Achilles tendon enthesis (ATE) anchors the Achilles tendon into the calcaneus through fibrocartilaginous tissue. The latter is enriched in type II collagen and proteoglycans (PGs), both of which give the enthesis its capacity to withstand compressive stress. Because unloading and reloading induce remodeling of the ATE fibrocartilage (Camy et al., 2022), chronic changes in the mechanical load could modify the mechanical response under compressive stress. Therefore, we investigated the ATE fatigue behavior in mice, under cyclic compressive loading, after 14 days of hindlimb suspension and 6 days of reloading. In addition, we performed a qualitative histological study of PGs in ATE fibrocartilage. The mechanical behavior of ATE was impaired in unloaded mice. A significant loss of 27 % in Δd (difference between the maximum and minimum displacements) was observed at the end of the test. In addition, the hysteresis area decreased by approximately 27 % and the stiffness increased by over 45 %. The increased stiffness and loss of viscosity were thrice and almost twice those of the control, respectively. In the reloaded entheses, where the loss of Δd was not significant, we found a significant 28 % decrease in the hysteresis area and a 26 % increase in stiffness, both of which were higher regarding the control condition. These load-dependent changes in the mechanical response seem partly related to changes in PGs in the uncalficied part of the ATE. These findings highlight the importance of managing compressive loading on ATE when performing prophylactic and rehabilitation exercises.

Spontaneous Tonic Activity Revealed in Rat Soleus Muscle by CLP290, a Novel Spinal Cord Potassium-Chloride Cotransporter Activator, during Hindlimb Suspension

Spontaneous Tonic Activity Revealed in Rat Soleus Muscle by CLP290, a Novel Spinal Cord Potassium-Chloride Cotransporter Activator, during Hindlimb Suspension

A Preclinical Model of Sepsis-Induced Myopathy with Disuse in Mice.

Sepsis is a major cause of in-hospital deaths. Improvements in treatment result in a greater number of sepsis survivors. Approximately 75% of the survivors develop muscle weakness and atrophy, increasing the incidence of hospital readmissions and mortality. However, the available preclinical models of sepsis do not address skeletal muscle disuse, a key component for the development of sepsis-induced myopathy. Our objective in this protocol is to provide a step-by-step guideline for a mouse model that reproduces the clinical setting experienced by a bedridden septic patient. Male C57Bl/6 mice were used to develop this model. Mice underwent cecal ligation and puncture (CLP) to induce sepsis. Four days post-CLP, mice were subjected to hindlimb suspension (HLS) for seven days. Results were compared with sham-matched surgeries and/or animals with normal ambulation (NA). Muscles were dissected for in vitro muscle mechanics and morphological assessments. The model results in marked muscle atrophy and weakness, a similar phenotype observed in septic patients. The model represents a platform for testing potential therapeutic strategies for the mitigation of sepsis-induced myopathy.

The relationship of sphingolipid mechanisms with oxidative stress and changes in mitochondria during functional unloading of postural muscles

Background. Prolonged inactivity of skeletal muscles is accompanied by the development of oxidative stress and changes in sphingolipid metabolism. The relationship of sphingolipid mechanisms with generation of reactive oxygen species (ROS) in muscles subjected to functional unloading has not been studied.The aim. To identify the relationship between changes in sphingomyelinase and ceramide abundance and ROS production in rat soleus muscle during functional unloading.Methods. Male Wistar rats were subjected to hindlimb suspension for 12 hours or 14 days with the acid sphingomyelinase (ASM) inhibitor amitriptyline (AMI). The levels of ASM, ceramide and ROS were determined by fluorescence microscopy on histological sections. Pro-oxidant enzymes (NADPH oxidases 2 and 4 (NOX2 and NOX4)), cytochrome c oxidase (COX IV), the regulator of mitochondrial biogenesis PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) in muscle homogenates were studied by Western blotting, which also was used for assessment of ceramide and ASM in the isolated mitochondrial fraction. The effects of sphingomyelinase and prooxidants on ceramide, ASM, ROS and NOX2 levels were studied in an ex vivo model by incubating the muscle with exogenous sphingomyelinase or H2O2.Results. 12-hour hindlimb suspension was accompanied by an increase in the level of ASM and ceramide in rat soleus muscle. Unloading for 14 days was characterized by an increase in ASM, ceramide, ROS, NOX2, NOX4 and a decrease in COX IV and PGC-1α levels. ASM and ceramide were also increased in the mitochondrial fraction of muscle. The ASM inhibitor amitriptyline partially or completely prevented the changes caused by the unloading. In the ex vivo model, the stimulating effect of exogenous sphingomyelinase on the ROS and NOX2 levels in rat soleus muscle was found, whereas H2O2 stimulated muscle ASM and ceramide production.Conclusion. A close relationship has been established between the sphingomyeli-nase pathway of ceramide formation and ROS production in skeletal muscle under conditions of functional unloading.

Deciphering the Therapeutic Role of Lactate in Combating Disuse-Induced Muscle Atrophy: An NMR-Based Metabolomic Study in Mice.

Disuse muscle atrophy (DMA) is a significant healthcare challenge characterized by progressive loss of muscle mass and function resulting from prolonged inactivity. The development of effective strategies for muscle recovery is essential. In this study, we established a DMA mouse model through hindlimb suspension to evaluate the therapeutic potential of lactate in alleviating the detrimental effects on the gastrocnemius muscle. Using NMR-based metabolomic analysis, we investigated the metabolic changes in DMA-injured gastrocnemius muscles compared to controls and evaluated the beneficial effects of lactate treatment. Our results show that lactate significantly reduced muscle mass loss and improved muscle function by downregulating Murf1 expression, decreasing protein ubiquitination and hydrolysis, and increasing myosin heavy chain levels. Crucially, lactate corrected perturbations in four key metabolic pathways in the DMA gastrocnemius: the biosynthesis of phenylalanine, tyrosine, and tryptophan; phenylalanine metabolism; histidine metabolism; and arginine and proline metabolism. In addition to phenylalanine-related pathways, lactate also plays a role in regulating branched-chain amino acid metabolism and energy metabolism. Notably, lactate treatment normalized the levels of eight essential metabolites in DMA mice, underscoring its potential as a therapeutic agent against the consequences of prolonged inactivity and muscle wasting. This study not only advances our understanding of the therapeutic benefits of lactate but also provides a foundation for novel treatment approaches aimed at metabolic restoration and muscle recovery in conditions of muscle wasting.

Impact of Hindlimb Disuse on Sepsis-Induced Myopathy in Older Adult Mice

BACKGROUND: Sepsis, a life-threatening condition due to an overactive immune response, adversely affects multiple organ systems. Despite its significance of accounting for 40% of total body mass, the skeletal muscle system is considered the forgotten organ system affected by sepsis. In addition to the inflammatory aspects of sepsis, patients in the intensive care unit (ICU) frequently experience prolonged periods of bed rest, which can lead to muscle disuse-induced myopathy characterized by muscle atrophy, weakness, and impaired regenerative capacity. Despite the higher incidence of sepsis in bedridden older adults, most research on sepsis-induced myopathy primarily focuses on younger adults under conditions that do not mimic bed rest. In this study, we simulated the clinical conditions of older adult ICU patients by combining sepsis with hindlimb disuse. Our hypotheses were that the combination of sepsis, disuse, and aging would exacerbate muscle atrophy and weakness, while also reducing the number of regenerative stem cells in muscle tissue. Methods: To investigate the functional and morphological responses of skeletal muscle to sepsis, we conducted experiments on 78-week-old male wild-type C57Bl6 mice. They underwent either cecal ligation and puncture (CLP, n = 17) or Sham (n = 14) surgeries. Four days post-surgery, the animals were allocated to hindlimb suspension (HLS — CLP, n=9; Sham, n=10) or normal ambulation (NA — CLP, n=8; Sham, n=4) for seven days. Terminal experiments were conducted after hindlimb suspension or normal ambulation treatments. We assessed peak force production ex vivo in solei muscles, examined the cross-sectional area (CSA) of relevant hindlimb muscles using immunohistochemistry, and quantified the abundance of stem cells through immunohistochemistry (e.g., PAX7+ cells) in muscle sections. Additionally, we evaluated markers of senescence by measuring the protein abundance of p16 and p53 in hindlimb muscle homogenates. Results: CLP surgery resulted in 50% mortality rate (p<0.05). Percentage body mass loss was as follows: 5.3 ± 4.2 for Sham/NA group, 13.3 ± 5.2 for Sham/HLS group, 7.3 ± 4.4 for CLP/NA group, and 17.7 ± 5.5 for CLP/HLS group. Peak absolute force was significantly lower in Sham/HLS and CLP/HLS compared to Sham/NA (Sham/NA = 277±2.4, Sham/HLS = 189±28.4, CLP/NA = 264±23.0, CLP/HLS = 180±48.9 mN, p<0.05). Mice exposed to CLP/HLS displayed a higher cumulative fiber area at smaller fiber sizes (800 to 1000 μm2) (p<0.05), indicating muscle atrophy. CLP/NA caused a 9.8% reduction in solei muscle mass (p < 0.05), while CLP/HLS led to an additional 30% loss in solei mass (p<0.05). Furthermore, CLP/HLS significantly decreased the number of PAX7+ stem cells by 75% (p<0.05). Notably, p16 was significantly increased in response to CLP/HLS (p<0.05), while no changes were observed in p53 abundance. CONCLUSION: This study demonstrates the role of muscle disuse in exacerbating skeletal muscle functional and morphological abnormalities in older adult septic mice, emphasizing the critical contribution of disuse to the pathophysiology of sepsis-induced myopathy. NIH R21AG072011 to OL. 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.

A possible physiological role of sclerostin in aging-associated skeletal muscle atrophy

Sarcopenia has a significant impact on quality of life by inhibiting the ability of daily living. Further, sarcopenia is a well-known risk factor for fall-associated fracture and frailty. Therefore, prevention of sarcopenia is an important issue for healthy life. Recently, it has been revealed that sclerostin (Sost) is a novel target for the treatment of aging-associated bone weakness, so-called osteoporosis, However, the physiological role of Sost in skeletal muscle is unknown. The purpose of this study was to investigate the effect of Sost for skeletal muscle mass. In order to investigate the effect of Sost on myogenic differentiation, we carried out cell culture experiment using mouse myoblast-derived cell line C2C12 cells. During differentiation, Sost (0, 10, 100, 1000 ng/ml) was administrated to the cell culture medium. Effects of anti-sclerostin antibody romosozumab (0, 2, 20, 200 μg/ml) on myotube formation of C2C12 was also investigated. Myotube diameter and the fusion index (FI) and evaluated. Furthermore, we also investigated the expression level of Sost in skeletal muscles of young (10-week old) and aged (100-week old) male mice (C57BL/6J). The effects of hindlimb unloading on the expression level of Sost in skeletal muscle was also investigated. All animal experimental procedures were carried out in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences by Japan Physiological Society, and approved by the Animal Use Committee of Toyohashi SOZO University. Soleus muscle (SOL) and plantaris muscle (PLA) were dissected before and after 2 weeks of hindlimb suspension. Expression level of Sost was evaluated by Western blotting. The relationship between Sost expression level in each muscle and muscle wet weight was analyzed using Pearson correlation coeffcient. The differences between groups were considered statistically significant at p < 0.05. Sost suppressed myogenic differentiation of C2C12 cells in a dose-dependent manner. On the other hand, romosozumab stimulated C2C12 differentiation in a dose-dependent manner. Expression level of Sost in SOL and PLA of aged mice was significantly higher than that in young animals. In SOL and PLA, a negative correlation was observed between the expression level of Sost and muscle weight was observed. Unloading-associated up-regulation of Sost was observed in PLA of aged mice. We demonstrated aging-associated up-regulation of Sost in mouse slow and fast skeletal muscles. Further, skeletal muscle weight was negatively correlated with the expression level of Sost in skeletal muscle. Therefore, it is suggested that skeletal muscle-derived Sost may play a role in aging-associated skeletal muscle atrophy. Sost may be a novel therapeutic target for sarcopenia. This work was supported by KAKENHI (18H03160, K.G.; 19K22825, K.G.; 19KK0254, K.G.;22H03474, K.G.; 22K19722, K.G.; 22H03319, K.G.; 22K18413, K.G.; 20K11221, T.K) from the Japan Society for the Promotion of Science, and a grant from DAIKO FOUNDATION (K.G.), Graduate School of Health Science, Toyohashi SOZO University (K.G.), and a research grant from Toyohashi SOZO university (K.G.). 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.

Combination of Parenteral Amino Acid Infusion and Intermittent Loading Exercise Ameliorates Progression of Postoperative Sarcopenia in Rat Model.

Postoperative sarcopenia is associated with poor outcomes in hospitalized patients. However, few studies have focused on short-term postoperative sarcopenia. Furthermore, the influence of nutritional management using amino acids (AAs) comprising a peripheral parenteral nutrition (PPN) solution and its combination with exercise (Exc) is unclear. Hence, we established a postoperative sarcopenic rat model to evaluate the effects of parenteral AA infusion combined with Exc on skeletal muscles and investigate the underlying mechanisms involved in the amelioration of muscle atrophy. Male F344 rats underwent surgery followed by hindlimb suspension (HS) for 5 days. The rats were divided into AA (-), AA (+), AA (-)-Exc, and AA (+)-Exc groups. They were continuously administered a PPN solution with or without AA at 98 kcal/kg/day. The Exc groups were subjected to intermittent loading for 1 h per day. Postoperative sarcopenic rats exhibited decreased muscle strength and mass and an upregulated ubiquitin-proteasome system, autophagy-lysosome system, and fast-twitch fiber-related genes, especially in the AA (-) group. The AA (+)-Exc group exhibited attenuated decreased muscle strength, increased gastrocnemius mass, and a suppressed upregulation of muscle atrophy- and fast-twitch fiber-related genes. Therefore, parenteral AA infusion combined with Exc may be effective in preventing postoperative sarcopenia in hospitalized patients.

Protective role of the ginsenoside Rg1 against methimazole-induced gestational hypothyroidism on reflexive behaviors, conditioned fear and cortical antioxidant levels in mice offspring

Ginsenoside Rg1(Rg1), a monomer of a tetracyclic triterpenoid derivative, possesses diverse medicinal properties attributed to its unique chemical structure and may have beneficial effects on fetal development. This study aimed to investigate the protective effects of prenatal exposure to Rg1 against Methimazole-induced gestational hypothyroidism on reflexive behaviors, conditioned fear, and cortical antioxidant levels in mouse offspring.40 female virgin mice and 12 male NMRI mice were assigned to four groups: group 1 served as the control, group 2 received Methimazole(MMI) at a concentration of 0.02% in their drinking water, group 3 received Rg1(150 mg/kg), and group 4 received both MMI and Rg1.Groups of 2–4 were administered the substances from days 1–9 of gestation. After delivery, pups were selected, and reflexive motor behaviors and conditioned fear were assessed. Additionally, levels of brain tissue catalase(CAT), malondialdehyde(MDA), superoxide dismutase(SOD), and glutathione peroxidase(GPx) levels were measured. Furthermore, postpartum immobility time in the forced swimming test (FST), tail suspension test (TST), and the number of squares crossed in the open field test (OFT)were determined. The results demonstrated that maternal exposure to Rg1 improved ambulation score, hind-limb suspension score, grip strength, front-limb suspension, hind-limb foot angle, negative geotaxis, surface righting, and conditioned fear in hypothyroidism-induced offspring(P<0.05). Rg1 decreased immobility time in the FST, and TST, and increased the number of squares crossed in the OFT in postpartum hypothyroidism-induced mice(P<0.05). Moreover, Rg1 reduced brain tissue MDA levels and increased brain tissue CAT, SOD, and GPx levels in mice and their offspring(P<0.05). These findings indicate that Rg1 mitigated postpartum depression in mice and improved reflexive motor behaviors in their pups.

Diet-induced hyperhomocysteinemia causes sex-dependent deficiencies in offspring musculature and brain function.

Hyperhomocysteinemia (HHcy), characterized by elevated homocysteine (Hcy) levels, is a known risk factor for cardiovascular, renal, and neurological diseases, as well as pregnancy complications. Our study aimed to investigate whether HHcy induced by a high-methionine (high-Met) diet exacerbates cognitive and behavioral deficits in offspring and leads to other breeding problems. Dietary HHcy was induced four weeks before mating and continued throughout gestation and post-delivery. A battery of behavioral tests was conducted on offspring between postnatal days (PNDs) 5 and 30 to assess motor function/activity and cognition. The results were correlated with brain morphometric measurements and quantitative analysis of mammalian target of rapamycin (mTOR)/autophagy markers. The high-Met diet significantly increased parental and offspring urinary tHcy levels and influenced offspring behavior in a sex-dependent manner. Female offspring exhibited impaired cognition, potentially related to morphometric changes observed exclusively in HHcy females. Male HHcy pups demonstrated muscle weakness, evidenced by slower surface righting, reduced hind limb suspension (HLS) hanging time, weaker grip strength, and decreased activity in the beaker test. Western blot analyses indicated the downregulation of autophagy and the upregulation of mTOR activity in HHcy cortexes. HHcy also led to breeding impairments, including reduced breeding rate, in-utero fetal death, lower pups' body weight, and increased mortality, likely attributed to placental dysfunction associated with HHcy. In conclusion, a high-Met diet impairs memory and cognition in female juveniles and weakens muscle strength in male pups. These effects may stem from abnormal placental function affecting early neurogenesis, the dysregulation of autophagy-related pathways in the cortex, or epigenetic mechanisms of gene regulation triggered by HHcy during embryonic development.

Pharmacological inhibition of endoplasmic reticulum stress mitigates osteoporosis in a mouse model of hindlimb suspension

Hindlimb suspension (HLS) mice exhibit osteoporosis of the hindlimb bones and may be an excellent model to test pharmacological interventions. We investigated the effects of inhibiting endoplasmic reticulum (ER) stress with 4-phenyl butyrate (4-PBA) on the morphology, physicochemical properties, and bone turnover markers of hindlimbs in HLS mice. We randomly divided 21 male C57BL/6J mice into three groups, ground-based controls, untreated HLS group and 4-PBA treated group (HLS+4PBA) (100mg/kg/day, intraperitoneal) for 21 days. We investigated histopathology, micro-CT imaging, Raman spectroscopic analysis, and gene expression. Untreated HLS mice exhibited reduced osteocyte density, multinucleated osteoclast-like cells, adipocyte infiltration, and reduced trabecular striations on micro-CT than the control group. Raman spectroscopy revealed higher levels of ER stress, hydroxyproline, non-collagenous proteins, phenylalanine, tyrosine, and CH2Wag as well as a reduction in proteoglycans and adenine. Furthermore, bone alkaline phosphatase and osteocalcin were downregulated, while Cathepsin K, TRAP, and sclerostin were upregulated. Treatment with 4-PBA partially restored normal bone histology, increased collagen crosslinking, and mineralization, promoted anti-inflammatory markers, and downregulated bone resorption markers. Our findings suggest that mitigating ER stress with 4-PBA could be a therapeutic intervention to offset osteoporosis in conditions mimicking hindlimb suspension.

Development of patellofemoral osteoarthritis with knee joint malalignment and lateral patellar dislocation after hindlimb suspension in growing rats.

Knee malalignment is a risk factor for patellar instability and patellofemoral osteoarthritis (PFOA), but etiologies remain unknown. We investigated the potential effects of decreased weight loading during growth on knee alignments and patellofemoral (PF) joint pathology. Hindlimb suspension (HS) was performed in 4-week-old female rats for 2, 4, and 8 weeks (HS groups). Age-matched rats were used as controls. Three-dimensional reconstructed images of the knee were obtained using X-ray computed tomography. Tibial tubercle-trochlear groove (TT-TG) distance, patellar tilt angle, and bisect offset were measured as indices of knee alignment. Histological analysis was also performed to evaluate the changes in cartilage and synovium in the PF joints. At Week 8, TT-TG distance, patella tilt angle, and bisect offset were significantly larger in the HS group than in the control group, respectively, indicating tibial external rotation, outward patellar tilt, and external displacement of the patella. Lateral patellar dislocation was frequently found in the HS group at Week 8 (five of eight knee joints, p < 0.05). Degenerative changes in the cartilage of the trochlear groove were observed at Week 8, and synovial changes such as hypertrophy and synovitis were observed at Weeks 4 and 8. Correlation analyses revealed significant relationships between the Mankin score and bisect offset, and between the OARSI synovitis score and all knee alignments indices. These results suggest that decreased weight loading on the lower extremities in growing rats resulted in knee malalignments characterized by external rotation of tibia and high incidence of lateral patellar dislocation with concomitant PFOA.

The mechanosensitive gene arrestin domain containing 2 regulates myotube diameter with direct implications for disuse atrophy with aging.

Arrestin domain containing 2 and 3 (Arrdc2/3) are genes whose mRNA contents are decreased in young skeletal muscle following mechanical overload. Arrdc3 is linked to the regulation of signaling pathways in nonmuscle cells that could influence skeletal muscle size. Despite a similar amino acid sequence, Arrdc2 function remains undefined. The purpose of this study was to further explore the relationship of Arrdc2/Arrdc3 expression with changes in mechanical load in young and aged muscle and define the effect of Arrdc2/3 expression on C2C12 myotube diameter. In young and aged mice, mechanical load was decreased using hindlimb suspension whereas mechanical load was increased by reloading previously unloaded muscle or inducing high-force contractions. Arrdc2 and Arrdc3 mRNAs were overexpressed in C2C12 myotubes using adenoviruses. Myotube diameter was determined 48-h posttransfection, and RNA sequencing was performed on those samples. Arrdc2 and Arrdc3 mRNA content was higher in the unloaded muscle within 1 day of disuse and remained higher up through 10 days. The induction of Arrdc2 mRNA was more pronounced in aged muscle than young muscle in response to unloading. Reloading previously unloaded muscle of young and aged mice restored Arrdc2 and Arrdc3 levels to ambulatory levels. Increasing mechanical load beyond normal ambulatory levels lowered Arrdc2 mRNA, but not Arrdc3 mRNA, in young and aged muscle. Arrdc2 overexpression only was sufficient to lower myotube diameter in C2C12 cells in part by altering the transcriptome favoring muscle atrophy. These data are consistent with Arrdc2 contributing to disuse atrophy, particularly in aged muscle.NEW & NOTEWORTHY We establish Arrdc2 as a novel mechanosensitive gene highly induced in response to mechanical unloading, particularly in aged muscle. Arrdc2 induction in C2C12 myotubes is sufficient to produce thinner myotubes and a transcriptional landscape consistent with muscle atrophy and disuse.