Purpose: This study aimed to examine the exercise-mediated musculoskeletal recovery following hindlimb suspension (HS) in order to identify whether bone modeling and muscle hypertrophy would eventuate in a synchronized manner during recovery stage. Methods: To identify whether 2-week HS would be sufficient to induce a significant reduction of physiological indices in both tibia and adjacent hindlimb muscles, a total of 20 rats was randomized into 2-week HS (n = 10) and age-matched control group (n = 10, CON). Another batch of rats were randomly assigned to three different groups to identify recovery intervention effects following suspension: (1) 2-week HS followed by 4-week spontaneous reloading recovery (HRE, n = 7). (2) 2-week HS followed by 4-week progressive resistance ladder climbing exercise (HEX, n = 7). (3) Age-matched control (CON, n = 7). DXA, micro-CT, and 18F-sodium fluoride (NaF) imaging, and EIA analysis were utilized to measure tibia bone indices. Hindlimb muscle wet weight and grip strength were measured to evaluate muscle mass and strength, respectively. Results: In study 1, bone quality values [bone volume/total volume (BV/TV): -27%, areal bone mineral density (aBMD): -23%, mineral contents: -7.9%, mineral density: -4.1%, and bone density: -38.9%] and skeletal muscle weight (soleus: -46.8%, gastrocnemius: -19.6%, plantaris: -20.8%, TA: -22.8%, and EDL: -9.9%) were significantly lower in HS group compared to CON group. In study 2, micro-CT and DXA-based bone morphology (bone density, BT/TV, and aBMD) were fully recovered in HRE or HEX group. However, suspension-induced dysregulation of bone mineral metabolism was returned to age-matched control group in only HEX group, but not in HRE group. A greater level of biomarkers of bone formation (P1NF) and resorption (CTX-1) was observed in only HRE group compared to CON. The hindlimb skeletal muscle mass was significantly lower in both HRE and HEX groups compared to CON group. Hindlimb grip strength was the greatest in HEX group, followed by CON and HRE groups. Conclusion: Following HS, progressive resistance exercise promotes recovery rates of bone and skeletal muscle strength without a significant increase in muscular mass, suggesting that exercise-induced reacquisition of bone and muscle strength is independent of muscle hypertrophy during early recovery stage.

Nitric oxide (NO), produced by NO-synthases via L-arginine oxidation, is an essential trigger for signaling processes involved in structural and metabolic changes in muscle fibers. Recently, it was shown that L-arginine administration prevented the decrease in levels of the muscle cytoskeletal proteins, desmin and dystrophin, in rat soleus muscle after 14 days of hindlimb unloading. Therefore, in this study, we investigated the effect of L-arginine administration on the degree of atrophy changes in the rat soleus muscles under unloading conditions, and on the content, gene expression, and phosphorylation level of titin, the giant protein of striated muscles, able to form a third type of myofilaments-elastic filaments. A 7-day gravitational unloading [hindlimb suspension (HS) group] resulted in a decrease in the soleus weight:body weight ratio (by 31.8%, p < 0.05), indicating muscle atrophy development. The content of intact titin (T1) decreased (by 22.4%, p < 0.05) and the content of proteolytic fragments of titin (T2) increased (by 66.7%, p < 0.05) in the soleus muscle of HS rats, compared to control rats. The titin gene expression and phosphorylation level of titin between these two groups were not significantly different. L-Arginine administration under 7-day gravitational unloading decreased the degree of atrophy changes and also prevented the decrease in levels of T1 in the soleus muscle as compared to HS group. Furthermore, L-arginine administration under unloading resulted in increased titin mRNA level (by 76%, p < 0.05) and decreased phosphorylation level of T2 (by 28%, p < 0.05), compared to those in the HS group. These results suggest that administration of L-arginine, the NO precursor, under unloading decreased the degree of atrophy changes, increased gene expression of titin and prevented the decrease in levels of T1 in the rat soleus muscle. The results can be used to search for approaches to reduce the development of negative changes caused by gravitational unloading in the muscle.

It is known that nitric oxide (NO) may affect myosin heavy chain (MyHC) isoform mRNA transcription in skeletal muscles. The content of NO in soleus muscles decreases during rat hindlimb unloading as well as slow MyHC mRNA transcription. We aimed to detect which signaling pathways are involved in NO-dependent prevention of hindlimb-suspension (HS)-induced changes in MyHCs' expression pattern. Male Wistar rats were divided into four groups: cage control group (C), hindlimb suspended for 7 days (7HS), hindlimb suspended for 7 days with L-arginine administration (7HS+A) (500 mg/kg body mass), and hindlimb suspended for 7 days with both L-arginine (500 mg/kg) and NO-synthase inhibitor L-NAME administration (50 mg/kg) (7HS+A+N). L-arginine treatment during 7 days of rat HS prevented HS-induced NO content decrease and slow MyHC mRNA transcription decrease and attenuated fast MyHC IIb mRNA transcription increase; it also prevented NFATc1 nuclear content decrease, calsarcin-2 expression increase, and GSK-3β Ser 9 phosphorylation decrease. Moreover, L-arginine administration prevented the HS-induced myh7b and PGC1α mRNAs content decreases and slow-type genes repressor SOX6 mRNA transcription increase. All these slow fiber-type protective effects of L-arginine were blocked in HS+A+N group, indicating that these effects were NO-dependent. Thus, NO decrease prevention during HS restores calcineurin/NFATc1 and myh7b/SOX6 signaling.

As astronauts prepare to undertake new extra-terrestrial missions, innovative diagnostic tools are needed to better assess muscle deconditioning during periods of weightlessness and partial gravity. Electrical impedance myography (EIM) has been used to detect muscle deconditioning in rodents exposed to microgravity during spaceflight or using the standard ground-based model of hindlimb unloading via tail suspension (HU). Here, we used EIM to assess muscle changes in animals exposed to two new models: hindlimb suspension using a pelvic harness (HLS) and a partial weight-bearing (PWB) model that mimics partial gravity (including Lunar and Martian gravities). We also used a simple needle array electrode in lieu of surface or ex vivo EIM approaches previously employed. Our HLS results confirmed earlier findings obtained after spaceflight and tail suspension. Indeed, one EIM measure (i.e., phase-slope) that was previously reported as highly sensitive, was significantly decreased after HLS (day 0: 14.60 ± 0.97, day 7: 11.03 ± 0.81, and day 14: 10.13 ± 0.55 | Deg/MHz|, p < 0.0001), and was associated with a significant decrease in muscle grip force. Although EIM parameters such as 50 kHz phase, reactance, and resistance remained variable over 14 days in PWB animals, we identified major PWB-dependent effects at 7 days. Moreover, the data at both 7 and 14 days correlated to previously observed changes in rear paw grip force using the same PWB model. In conclusion, our data suggest that EIM has the potential to serve as biomarker of muscle deconditioning during exposure to both micro- and partial- gravity during future human space exploration.

Physiological adaptations to microgravity involve alterations in cardiovascular systems. These adaptations result in cardiac remodeling and orthostatic hypotension. However, the response of the left ventricle (LV) and right ventricle (RV) following hindlimb unloading (HU) and hindlimb reloading (HR) is not clear and the underlying mechanism remains to be understood. In this study, three groups of mice were subjected to HU by tail suspension for 28 days. Following this, two groups were allowed to recover for 7 or 14 days. The control group was treated equally, with the exception of tail suspension. Echocardiography was performed to detect the structure and function changes of heart. Compared with the control, the HU group of mice showed reduced LV-EF (ejection fraction), and LV-FS (fractional shortening). However, mice that were allowed to recover for 7 days after HU (HR-7d) showed increased LVIDs (systolic LV internal diameter) and LV Vols (systolic LV volume). Mice that recovered for 14 days (HR-14d) returned to the normal state. In comparison, RV-EF and RV-FS didn't recover to the normal conditions till being reloaded for 14 days. Compared with the control, RVIDd (diastolic RV internal diameter), and RV Vold (diastolic RV volume) were reduced in HU group and recovered to the normal conditions in HR-7d and HR-14d groups, in which groups RVIDs (systolic RV internal diameter) and RV Vols (systolic RV volume) were increased. Histological analysis and cardiac remodeling gene expression results indicated that HU induces left and right ventricular remodeling. Western blot demonstrated that the phosphorylation of HDAC4 and ERK1/2 and the ratio of LC3-II / LC3-I, were increased following HU and recovered following HR in both LV and RV, and the phosphorylation of AMPK was inhibited in both LV and RV following HU, but only restored in LV following HR for 14 days. These results indicate that simulated microgravity leads to cardiac remodeling, and the remodeling changes can be reversed. Furthermore, in the early stages of recovery, cardiac remodeling may be intensified. Finally, compared with the LV, the RV is not as easily reversed. Cardiac remodeling pathways, such as, HDAC4, ERK1/2, LC3-II, and AMPK were involved in the process.

It is known that plantar mechanical stimulation (PMS) is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. However, molecular mechanisms underlying the effect of PMS on skeletal muscle during unloading remain undefined. The aim of the study was to evaluate the effects of PMS on anabolic and catabolic signaling pathways in rat soleus at the early stages of mechanical unloading. Wistar rats were randomly assigned to ambulatory control, hindlimb suspension (HS) for 1 or 3 days, and HS for 1 or 3 days with PMS. The key anabolic and catabolic markers were assessed by western blotting and RT-PCR. Protein synthesis (PS) rate was estimated using SUnSET technique. PMS attenuated a 1-day HS-induced decrease in 4E-BP1, GSK-3β, and AMPK phosphorylation. PMS also partially prevented a decrease in PS, phosphorylation of GSK-3β, nNOS, and an increase in eEF2 phosphorylation after 3-day HS. PMS during 1- and 3-day HS prevented MuRF-1, but not MAFbx, upregulation but did not affect markers of ribosome biogenesis (18S + 28S rRNA, c-myc) as well as AKT phosphorylation. Thus, PMS during 3-day HS partially prevented a decrease in the global rate of PS in rat soleus muscle, which was accompanied by attenuation of MuRF-1 mRNA expression as well as changes in GSK-3β, nNOS, and eEF2 phosphorylation.

Bones are made of complex material comprising organic components and mineral hydroxyapatite, both of which formulate the unique hierarchical structure of bone and its mechanical properties. Bones are capable of optimizing their structure and mechanical properties according to the mechanical environment. Mineral loss is a well-known consequence of skeleton disuse. By contrast, the response of the non-mineral phase of bone, i.e., the collagen network, during disuse remain largely unknown. In this study, a tail-suspension mice model was used to induce bone loss. Atomic force microscopy-based imaging and indentation approaches were adopted to investigate the influence of disuse on the morphology and in situ mechanical behavior of the collagen fibrils, under both non-loaded and load-bearing conditions, in the cortical tibia of mice. The results indicate that disuse induced by hindlimb unloading did not alter the orientation and D-periodic spacing of the collagen fibril, but results in decreased collagen crosslinking which correlates with decreased elasticity and increased susceptibility to mechanical damage. More concretely, the collagen fibrils in the disused tibia were misaligned under mechanical loading. It therefore indicates that the disordered arrangement of the mineralized collagen fibrils is one of the characteristics of the weakened bone during elastic deformation. These findings reveals the unique adaptation regimes of the collagen fibrils in the cortical bone to disuse, as well as the deformation mechanisms of bone in the relevant pathological process at different scales.

Skeletal muscle disuse rapidly decreases muscle mass. Resistance training (RT) is believed as the most effective way to gain muscle mass via an increase in mTORC1 activity and muscle protein synthesis (MPS). However, it remains unclear whether muscle atrophy by disuse alters the mTORC1 activation and MPS response to an acute resistance exercise (RE) and chronic RT-mediated skeletal muscle hypertrophy. This study investigated the influence of disuse muscle atrophy on the response of mTORC1 activation and MPS to an acute RE. We also evaluated whether disuse muscle atrophy affects the response of RT-induced muscle mass gain. Thirty male Sprague-Dawley rats were randomly divided into control (CON) or hindlimb suspension (HS) groups. A 14-day HS via the tail was used as the model for gastrocnemius muscle disuse in the HS group. Unilateral lower limb muscle contraction using by percutaneous electrical stimulation was used to mimic the stimuli of RE. Ten bouts of RE were performed in 3-week as chronic RT. Our results showed that MPS and mTORC1 activity was unchanged after HS at basal state. However, the ribosomal RNA (rRNA) level was reduced in HS rats compared to that in CON rats at basal state. MPS and rRNA increased in both HS and CON rats in response to acute RE to the same extent. However, the level of mTORC1 activation in response to an acute RE was significantly higher in HS than that in the CON group at 12 h after exercise, even though no difference was observed at 3 h after exercise. The 10-bout RT significantly increased gastrocnemius muscle mass in both CON and HS rats. The response of muscle hypertrophy did not differ between the groups. Therefore, MPS in response to acute RE and muscle hypertrophy in response to chronic RT were unaltered after disuse muscle atrophy.

The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219-225 deleted, the TtnΔ219-225 model (MGI: TtnTM 2.1Mgot ). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219-225 muscles: a larger isoform that represents the TtnΔ219-225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219-225 soleus muscle (WT 240 ± 9; TtnΔ219-225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219-225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219-225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin's PEVK exons 219-225 have high passive tension, due in part to alterations elsewhere in splicing of titin's spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin's C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.