Low-dose simulated galactic cosmic radiation exposure after hindlimb unloading has long-term impact on skeletal muscle metabolic proteins in female mice.

Prolonged skeletal muscle inactivity causes muscle fibre atrophy. Redox imbalance has been considered one of the major triggers of skeletal muscle disuse atrophy, but whether redox imbalance is actually the major cause or simply a consequence of muscle disuse remains of debate. Here we hypothesized that a metabolic stress mediated by PGC-1α down-regulation plays a major role in disuse atrophy. First we studied the adaptations of soleus to mice hindlimb unloading (HU) in the early phase of disuse (3 and 7 days of HU) with and without antioxidant treatment (trolox). HU caused a reduction in cross-sectional area, redox status alteration (NRF2, SOD1 and catalase up-regulation), and induction of the ubiquitin proteasome system (MuRF-1 and atrogin-1 mRNA up-regulation) and autophagy (Beclin1 and p62 mRNA up-regulation). Trolox completely prevented the induction of NRF2, SOD1 and catalase mRNAs, but not atrophy or induction of catabolic systems in unloaded muscles, suggesting that oxidative stress is not a major cause of disuse atrophy. HU mice showed a marked alteration of oxidative metabolism. PGC-1α and mitochondrial complexes were down-regulated and DRP1 was up-regulated. To define the link between mitochondrial dysfunction and disuse muscle atrophy we unloaded mice overexpressing PGC-1α. Transgenic PGC-1α animals did not show metabolic alteration during unloading, preserving muscle size through the reduction of autophagy and proteasome degradation. Our results indicate that mitochondrial dysfunction plays a major role in disuse atrophy and that compounds inducing PGC-1α expression could be useful to treat/prevent muscle atrophy.

The effects of prolonged hind-limb unloading on titin antibody localization and expression of titin isozymes of single fibers from the synergistic slow-twitch soleus (SOL) and fast-twitch plantaris (PLN) of adult rats were studied after 14 and 28 days of hind-limb unloading (HU). Titin antibody localization and expression was not altered at 14 days of HU. However, there was a 4% loss in antibody to Z-band distance (Ab-Z) in the SOL and an increase of 8% in PLN Ab-Z after 28 days of HU. The titin and myosin heavy chain composition of single fibers and small bundles of fibers from control and unloaded muscles were examined using 2% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked loss of relative amounts of titin in both SOL and PLN following 28 days of HU. As the protein loads for these measures were identical, the authors conclude that these findings represent an actual loss of titin density rather than a decreased value due to a loss of total muscle mass. Laser scanning densitometry of the titin bands show a marked decrease in density and molecular weight in unloaded SOL. In the PLN, marked losses of titin density were accompanied by decreased electrophoretic motility. The results demonstrate that the titin isoform composition and titin antibody localization of skeletal muscle is altered during hind-limb unloading. Furthermore, as titin is responsible for positional stability of the sarcomere and the fiber during contraction, change in isoforms during HU may predispose atrophied muscle to injury during reuse and recovery.

Background:Disuse osteoporosis is a prevalent complication among patients with rheumatoid arthritis (RA). The treatment of disuse osteoporosis has been reported to diminish the efficacy of conventional osteoporosis drugs. Therefore, alternative...

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.

BackgroundPrevious data in humans suggest that extreme physical inactivity (EPI) affects iron metabolism differently between sexes. Our objective was to deepen the underlying mechanisms by studying rats of both sexes exposed to hindlimb unloading (HU), the reference experimental model mimicking EPI.MethodsEight‐week‐old male and female Wistar rats were assigned to control (CTL) or hindlimb unloading (HU) conditions (n = 12/group). After 7 days of HU, serum, liver, spleen, and soleus muscle were removed. Iron parameters were measured in serum samples, and ICP‐MS was used to quantify iron in tissues. Iron metabolism genes and proteins were analysed by RT‐qPCR and Western blot.ResultsCompared with control males, control females exhibited higher iron concentrations in serum (+43.3%, p < 0.001), liver (LIC; +198%, P < 0.001), spleen (SIC; +76.1%, P < 0.001), and transferrin saturation (TS) in serum (+53.3%, P < 0.001), contrasting with previous observations in humans. HU rat males, but not females, exhibited an increase of LIC (+54% P < 0.001) and SIC (+30.1%, P = 0.023), along with a rise of H‐ferritin protein levels (+60.9% and +134%, respectively, in liver and spleen; P < 0.05) and a decrease of TFRC protein levels (−36%; −50%, respectively, P < 0.05). HU males also exhibited an increase of splenic HO‐1 and NRF2 mRNA levels, (p < 0.001), as well as HU females (P < 0.001). Concomitantly to muscle atrophy observed in HU animals, the iron concentration increased in soleus in females (+26.7, P = 0.004) while only a trend is observed in males (+17.5%, P = 0.088). In addition, the H‐ferritin and myoglobin protein levels in soleus were increased in males (+748%, P < 0.001, +22%, P = 0.011, respectively) and in females (+369%, P < 0.001, +21.9%, P = 0.007, respectively), whereas TFRC and ferroportin (FPN) protein levels were reduced in males (−68.9%, P < 0.001, −76.8%, P < 0.001, respectively) and females (−75.9%, P < 0.001, −62.9%, P < 0.001, respectively). Interestingly, in both sexes, heme exporter FLVCR1 mRNA increased in soleus, while protein levels decreased (−39.9% for males P = 0.010 and −49.1% for females P < 0.001).ConclusionsTaken together, these data support that, in rats (1) extreme physical inactivity differently impacts the distribution of iron in both sexes, (2) splenic erythrophagocytosis could play a role in this iron misdistribution. The higher iron concentrations in atrophied soleus from both sexes are associated with a decoupling between the increase in iron storage proteins (i.e., ferritin and myoglobin) and the decrease in levels of iron export proteins (i.e., FPN and FLVCR1), thus supporting an iron sequestration in skeletal muscle under extreme physical inactivity.

Rapamycin relieves anxious emotion and synaptic plasticity deficits induced by hindlimb unloading in mice

Background: Hindlimb unloading (HU) mice is a ground-based model that simulates the effects of microgravity. Since microgravity significantly affects the immune system, understanding immune cell function under these conditions is crucial for developing strategies to protect astronauts from infections and malignancies during long space missions.Objective: To evaluate how microgravity affects neutrophils and T cells as the key components of innate and adaptive immunity, the activity of these cells in HU mice was compared with untreated control mice.Material and Methods: In this experimental study, 10 HU male BALB/c mice and 10 untreated control mice were included. Neutrophil-to-lymphocyte ratio (NLR) was evaluated and neutrophil function was assessed using the DHR assay. T cell proliferation was evaluated using the CFSE-dilution assay. IL-4 and IFN-ɣ production by T cell subsets was determined by intracellular cytokine staining with flow cytometry.Results: The capacity for reactive oxygen species (ROS) production in neutrophils did not differ between HU mice and control mice however, NLR was higher in HU mice. The proliferation of both CD4+ and CD8+ T cells was slightly reduced in HU mice. More notably, IL-4 production by CD4+ T cells and IFN-ɣ production by both CD4+ and CD8+ T cells were significantly decreased in HU mice. Conclusion: Hindlimb unloading, simulating microgravity, impairs immune cell functions by reducing cytokine production and T cell proliferation. The increased NLR in HU mice could indicate a heightened inflammatory response. These insights are essential for advancing space biology and medicine, ensuring astronaut health during prolonged space travel.

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

Simulated microgravity accurately models long-duration spaceflight effects on bone and skeletal muscle in skeletally immature mice☆

Previous data indicate that 14 days of continuous HU in rats produces cardiac autonomic imbalance. We hypothesized that a daily period of normal posture may resolve these effects. Male, Sprague‐Dawley rats were randomly assigned to control (CC; n=3), continuous HU (HUC; n=4) or intermittent HU (HUI; n= 5) groups. Control rats were maintained in the normal posture. HUC rats underwent continuous elevation of the hindlimbs for 14‐days while HUI rats were returned to the normal posture for 30 minutes each day during the HU procedure. On the 14th day, mean arterial pressure (MAP) and heart rate (HR) were measured followed by selective cardiac autonomic blockade administered via i.v. injection of methylatropine and propranolol. Both HU groups exhibited significant resting tachycardia compared to CC rats, but HR was lower in HUI vs. the HUC group (CC: 366±3.9; HUC 474±7.5; HUI: 434±1.6 bpm). However, both HU groups had significantly less parasympathetic (CC: 60±7.7; HUC: 25±10.9; HUI: 15±5.2 Δbpm) and greater sympathetic cardiac tone (CC: 16±7.9; HUC: 91±10.9; HUI: 77±9.2 Δbpm) than CC rats, with no difference between HU groups with respect to either autonomic parameter. Baseline MAP was not different among any of the groups. Data indicate that while daily recovery from HU may partially restore resting HR, it is not a sufficient stimulus to reverse the autonomic imbalance produced by HU‐induced deconditioning. (Supp. by: NIH HL14388 &amp; DK 066086).

Background: Hindlimb-unloaded (HU) mice are animal models of simulated microgravity that exhibit pathological changes in several organs; however, relevant findings regarding the kidneys remain elusive. We investigated the possible contribution of protein dysregulation through endoplasmic reticulum (ER) stress to renal pathology in HU mice. Methods: We categorized male C57BL/6j mice into ground-based control and HU groups and treated them daily with a placebo or 4-phenylbutyrate (4PBA, an ER stress inhibitor), respectively, for 3 weeks. HU mice showed reduced body weight, whereas kidney weight remained unchanged. Results: Treatment with 4PBA increased the kidney weight of HU mice. The histopathological changes in HU mouse kidneys, including the widening of the Bowman’s capsule and increased glomerular area, were reversed through 4PBA treatment, accompanied by a 4PBA-induced reduction in the expression of several ER stress markers. Particularly, we found a reduction in the splicing of the X-box binding protein, indicating reduced ER stress in 4PBA-treated HU mice. Thus, 4PBA may use additional mechanisms to suppress ER stress and prevent renal pathology in HU mice. Conclusion: Our findings are the first to suggest that HU causes disruption of renal microarchitecture and that 4PBA may be a potent drug for kidney restoration under HU. Our preliminary findings have translational potential for conditions that mimic HU, such as prolonged bed rest.

Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S. -J. Lee. Nature 387: 83-90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation (r = 0.725, P < 0. 0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

Skeletal muscle atrophy is often debilitating. In women the decline in lean muscle mass accelerates at the onset of menopause due in part to lower estrogen (E2) levels. We evaluated the role of E2 to regulate skeletal muscle mass and maintain contractile function over 14 days of hindlimb unloading (HU) and 3 days of reloading (REC) in E2 replete (estrogen receptor‐α knock‐out, ERαKO) and E2 deficient (aromatase knockout, ARKO) mice, compared to wild type (WT) animals. Mice were separated into 3 groups: 1) CON: ambulatory controls, 2) HU: 14days of HU, 3) REC: 14 days of HU + 3 days REC. We observed differential effects on body weight (BW) after HU and REC. BW significantly decreased in ARKO mice (−10.9%) and ERαKO (−10.5%) after HU, while BW of WT mice was not different. Neither ARKO nor ERαKO recovered to pre‐HU BW values following REC. Expected declines in muscle mass of soleus (SOL), plantaris (PLAN), gastrocnemius (GAS), and tibialis anterior (TA) in WT mice after HU (Table 1) were observed. ARKO mice trended towards losing more muscle mass during HU and recovered slower than WT. Percent atrophy of muscle mass loss in HU &amp; REC compared to CON. WT SOL PLAN GAS TA HU −38 −20 −15 −11 REC −18 −6 −6 −2 ARKO SOL PLAN GAS TA HU −43 −30 −23 −12 REC −34 −17 −19 −15 ERαKO SOL PLAN GAS TA HU −46 −9 −15 −4 REC −25 −7 −7 −8 The ERαKO muscle mass profile resembled that of WT mice, suggesting that the presence of E2 rather than the absence of ERα mediates estrogen effects during HU and REC. These data suggest that estrogen deficiency slows skeletal muscle mass recovery. Support: HD‐058834 &amp; University of MO Life Sciences Fellowship.

The altered gut microbes of astronauts during space travel may contribute to health issues after their return to Earth. Previously, an association between the elevated endoplasmic reticulum (ER) stress and gut microbial dysbiosis has been described. Herein, we induced gut microbial changes in mice under a simulated microgravity environment in an established model of hindlimb unloaded (HU) mice. The intestinal metabolomic profiles under microgravity conditions using the HU model were examined, along with the potential role of 4-phenylbutyric acid (4-PBA), a potent ER stress inhibitor. For a microgravity environment, the mice were suspended in special cages individually for three weeks. Mice were sacrificed, and gut dissections were performed, followed by amplicon sequencing analysis of bacterial species via DNA extraction and 16S rRNA analysis. The results indicate that the gut bacterial communities of mice differed under gravity and microgravity conditions. Principal component analyses revealed differences in the bacterial community structure in all groups. Around 434 operational taxonomic units (OTUs) were specific to mice seen in controls, while 620 OTUs were specific to HU mice. Additionally, 321 bacterial OTUs were specific to HU mice treated with 4-PBA. When the relative abundance of taxa was analyzed, Bacteroidetes dominated the gut of control and HU mice treated with 4-PBA.. In contrast, the untreated HU mice were dominated by Firmicutes. At the genus level, a reduction in beneficial species of Akkermansia and Lactobacillus was observed in HU but not the unloaded–treated and control mice. Furthermore, an increase in the relative abundance of Lachnospiraceae and Enterorhabdus, associated with inflammation, was observed in HUmice but not in controls and unloaded-treated mice. Following treatment with 4-PBA, the ratio of Firmicutes to Bacteroidetes was restored in unloaded–treated mice, comparable to controls. Of note, beneficial microbes such as Akkermansia and Lactobacillus were observed in unloaded–treated mice but not or in lesser relative abundance in HU mice. Nonetheless, microbial diversity was reduced in unloaded–treated mice compared to controls, and future studies are needed to mitigate this finding. These may comprise the addition of pre-/pro- and postbiotic species in the diet to increase microbial diversity. Overall, the findings suggest that 4-PBA, a potent ER stress inhibitor, may have therapeutic value in treating patients on prolonged bed rest or astronauts during spaceflight.

A large set of FoxOs-dependent genes play a primary role in controlling muscle mass during hindlimb unloading. Mitochondrial dysfunction can modulate such a process. We hypothesized that endurance exercise before disuse can protect against disuse-induced muscle atrophy by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) expression and preventing mitochondrial dysfunction and energy-sensing AMP-activated protein kinase (AMPK) activation. We studied cross sectional area (CSA) of muscle fibers of gastrocnemius muscle by histochemistry following 1, 3, 7, and 14 days of hindlimb unloading (HU). We used Western blotting and qRT-PCR to study mitochondrial dynamics and FoxOs-dependent atrogenes’ expression at 1 and 3 days after HU. Preconditioned animals were submitted to moderate treadmill exercise for 7 days before disuse. Exercise preconditioning protected the gastrocnemius from disuse atrophy until 7 days of HU. It blunted alterations in mitochondrial dynamics up to 3 days after HU and the expression of most atrogenes at 1 day after disuse. In preconditioned mice, the activation of atrogenes resumed 3 days after HU when mitochondrial dynamics, assessed by profusion and pro-fission markers (mitofusin 1, MFN1, mitofusin 2, MFN2, optic atrophy 1, OPA1, dynamin related protein 1, DRP1 and fission 1, FIS1), PGC1α levels, and AMPK activation were at a basal level. Therefore, the normalization of mitochondrial dynamics and function was not sufficient to prevent atrogenes activation just a few days after HU. The time course of sirtuin 1 (SIRT1) expression and content paralleled the time course of atrogenes’ expression. In conclusion, seven days of endurance exercise counteracted alterations of mitochondrial dynamics and the activation of atrogenes early into disuse. Despite the normalization of mitochondrial dynamics, the effect on atrogenes’ suppression died away within 3 days of HU. Interestingly, muscle protection lasted until 7 days of HU. A longer or more intense exercise preconditioning may prolong atrogenes suppression and muscle protection.