Prolonged bed rest can have a significant negative effect on skeletal muscles, leading to muscle wasting and reduced strength. This process can take as little as 10 days in healthy individuals, with the loss of muscle mass and strength being particularly pronounced during the first week of immobilization. Head-down tilt bed rest (HDTBR) is a method used to simulate physiological changes that occur in weightlessness during spaceflight. This technique involves lying in bed with the head tilted downward. Here, we analyze the key anabolic markers of the human m. soleus during 21 days of HDTBR. Six healthy male volunteers, aged 25–35 years, were exposed to 21 days of strict bed rest at a tilt angle of –6°. A needle biopsy of the m. soleus was performed via the Bergström method before the onset of HDTBR and on day 21 after that. The biopsy material was immediately frozen in liquid nitrogen for further Western blot and PCR analysis. A study of mTORC1 substrates showed a significant decrease in p70 and 4EBP1 phosphorylation after HDTBR. We also observed a significant decrease in the phosphorylation of another ribosomal kinase, p90RSK, a significant increase in eEF2 phosphorylation, and an increase in eEF2k mRNA expression. In addition, the phosphorylation of AMPK and its substrate ACC decreased after HDTBR. The data obtained in this work support the hypothesis that a decrease in protein synthesis, together with an increase in proteolysis, contributes to the development of human m. soleus atrophy after 21 days of HDTBR.

Phosphoinositide-3-kinase (PI3K) (PI3Kγ, specifically) can be activated by a change in the membrane potential due to muscle functional unloading; as a result, IP3 enhances Ca2+ entry in the nucleus via IS3R. This benefits activation of the transcriptional factors that trigger atrophy. Inhibitor LY294002 was used to explore the PI3K role in ATP-dependent regulation of signaling in rat's muscles after 3-day suspension. PI3K inhibition after functional unloading slows down m. soleus atrophy, prevents ATP deposition, and expression of Е3-ubuquitine ligase MuRF1 and ubuquitine, prevents rise in expression of IP3-receptors, regulates the activity of Ca-dependent signaling pathways by reducing expression of Ca-dependent мРНК CaN markers, and phosphorylation of CaMKII (Са/calmodulin kinase II); the inhibitor modulates also regulation of the anabolic signaling markers IRS1 and 4E-BP in unloaded muscles.

Critical illness myopathy (CIM) is a primary myopathy that develops in critically ill patients. Histologic features of CIM include a general decrease in muscle fiber cross-sectional area and a predominant loss of the motor protein myosin. These features are observed in the absence of inflammatory infiltrates but with detectable cytokine activation. The purpose of this study was to examine the state of the extracellular matrix of the human soleus muscle under conditions of CIM caused by chronic impairment of consciousness. Incisional muscle biopsies were taken from the soleus muscle of 6 patients who were in a chronic critical condition and were treated in the Department of Anesthesiology and Reanimation at the A.L. Polenov Russian Research Institute - branch of the Almazov National Medical Research Center. The study included patients with a chronic impairment of consciousness lasting at least 2 months. Muscle biopsies taken from healthy men were used as controls. The biopsies were obtained using needle biopsy under local anesthesia. Using histological staining of tissue sections, it was determined that patients with CIM exhibited a significant increase in collagen area, surpassing the control value by 82%. An increased mRNA content of collagens I, III, and VIa was also observed, along with an increase in the protein content of collagen I and III. At the same time, we did not observe any changes in the content of fibronectin and extracellular tissue growth factor mRNA. However, we did observe an increase in the mRNA of the integrin A7 subunit. The results obtained indicate significant skeletal muscle fibrosis under CIM conditions. Further studies on the signaling pathways that regulate this process are needed.

Membrane IP3 receptors (IP3Rs) abound in the sarcoplasmic reticulum, nucleus and mitochondria of muscle fibers. We hypothesized that IP3R activation during muscle unloading may elicit a weak Ca2+ release signal, both cytosolic and nucleoplasmic, which promotes (perhaps in cooperation with other signaling cascades) the activation of transcription factors and thus leads to the expression or repression of genes associated with muscle phenotypes. Here, we tested this hypothesis by blocking IP3Rs with 2-APB (2-aminoethoxydiphenyl borate, 10 mg/kg in 5 of soleus muscle unloading. The blocking of IP3Rs prevented a decrease in the cross-sectional area of both slow and fast soleus muscle fibers and thus slowed down the development of atrophic processes in this postural muscle during 7-day hindlimb suspension. Such an effect of blocking IP3Rs during rat soleus muscle unloading may be due to preventing a decrease in ribosomal biogenesis and an increase in the expression of autophagy markers ULK-1 and IL-6.

IP3 receptors are found in significant quantities in muscle fibers in the sarcoplasmic reticulum, nucleus and mitochondria. We hypothesized that activation of IP3 receptors (IP3Rs) during muscle unloading may induce a weak calcium release signal, both cytosolic and nucleoplasmic, that promotes (possibly with other signaling cascades) the activation of transcription factors, leading to the expression or repression of genes involved in muscle phenotype. This hypothesis was tested by blocking IP3R during unloading of rat muscles by administering 2-APB (2-aminoethoxydiphenyl borate). Wistar rats were administered intraperitoneally at a dose of 10 mg/mg in 5 % DMSO daily. We found that the IP3R state influences the development of atrophic processes in the postural m. soleus during unloading. Administration of the IP3R blocker 2-APB to animals successfully prevented a decrease in m. soleus cross-sectional area (CSA) of both fast and slow muscle fibers. The slowdown in CSA decrease upon administration IP3R inhibitor during 7 days m. soleus unloading is associated with the prevention of a decrease in ribosomal biogenesis and an increase in the expression of autophagy markers ULK-1 and IL-6.

Functional unloading of skeletal muscles enhances proteasome degradation in which the key role is played by Е3-ligases atrogin-1/MAFbx and MuRF-1. Their expression starts growing on the first day of unloading. There is actually little literature on expression of ubiquitin ligases in dry immersion or bedrest. Our purpose was to investigate MuRF-1 and MAFbx expression and control in postural m. soleus and locomotor m. vastus lateralis during a 21-day bedrest study. The observed reduction of cross-section areas in slow fibers of m. soleus and m. vastus lateralis was accompanied by activation of MuRF-1 and MAFbx in m. soleus and MAFbx in m. vastus lateralis, respectively. The investigation of E3 ligases signal pathways showed an increased expression of myogenin and IL-6 receptors in both muscles, whereas transcriptional activation of FoxO3 was seen in m. soleus only. These results suggest elevated expression of Е3-ubiquitin ligases MuRF-1 and MAFbx in m. soleus and MAFbx in m. vastus lateralis, as well as their key signal pathways in bedrested human subjects. Also, our findings argue against the previously stated notion that degradation of protein has no influence on skeletal muscles atrophy due to hypokinesia.

Dysfunction of skeletal muscles and their atrophy during unloading are accompanied by excess calcium accumulation in the myoplasm of muscle fibers. We hypothesized that calcium accumulation may occur, among other reasons, due to inhibition of SERCA activity under muscle unloading. In this case, the use of a SERCA activator will reduce the calcium level in the myoplasm and prevent the consequences of unloading. Male Wistar rats were divided into 3 groups: vivarium control with placebo administration (C, n = 8), 7-day suspension group with placebo administration (7HS, n = 8) and 7-day suspension group with intraperitoneal administration of SERCA CDN1163 activator (50 mg/kg (7HS + CDN), n = 8). One m. soleus of each rat was frozen in liquid nitrogen, the second was tested for functional properties. In the 7HS group, increased soleus fatigue was found in the ex vivo test, a significant increase in mRNA and the number of fast muscle fibers, an increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein. All these changes were prevented in the SERCA CDN1163 activator group. Conclusion: 7-day SERCA activator administration does not delay of soleus atrophy, but prevents the development of its fatigue, probably by preventing a decrease in the number of type I fibers and markers of mitochondrial biogenesis.

Dysfunction of skeletal muscles and their atrophy under unloading are accompanied by excess calcium accumulation in the myoplasm of muscle fibers. We hypothesized that calcium accumulation may occur, among other reasons, due to inhibition of SERCA activity under muscle unloading. In this case, the use of a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activator would reduce the calcium level in the myoplasm and prevent the consequences of unloading. Male Wistar rats were divided into 3 groups: vivarium control with placebo administration (C, n = 8), 7-day suspension group with placebo administration (7HS, n = 8), and 7-day suspension group with intraperitoneal administration of the SERCA activator CDN1163 (7HS + CDN, 50 mg/kg, n = 8). One m. soleus of each rat was frozen in liquid nitrogen, the second was tested for functional properties. 7HS rats demonstrated an increased m. soleus fatigue in the ex vivo test, a significant increase in mRNA and the number of fast-twitch muscle fibers, an increase in the level of calcium-dependent CaMK II phosphorylation and tropomyosin oxidation, as well as a decrease in mitochondrial DNA and protein levels. All these changes were prevented in the 7HS + CDN group administered with SERCA activator. Conclusion: 7-day SERCA activator administration does not delay m. soleus atrophy but prevents the development of its fatigue, probably due to prevention of a reduction in the number of type I fibers and mitochondrial biogenesis markers.

We tested the hypothesis that in unloaded skeletal muscles the calcium-dependent signaling pathways and E3-ligase expression are controlled by regulation of phosphorylation of AMP-activated protein kinase (AMPK). For a 3-d experiment 32 Wister male rats were divided into 4 groups: placebo control (С), metformin control (300 mg/kg of body, per oral, MC), suspension and placebo (SP), suspension and metformin (SM). Object of the investigation was m. soleus. In comparison to group C, rAMPK in group MC reduced 46 % and ATP increased 49 % (p < 0.05); pCaMK II showed an increase and expression of mRNA CaN, SERCA2a and Calpain 1 grew 483 %, 87 %, 41 % and 62 %, respectively; p < 0.05). In group SP, MuRF1, MAFbx E3-ligase expression and ubiquitin increased 167 %, 146 % and 191 %, respectively (p < 0.05). Per oral metformin prevented these changes in the suspended rats. During 3 days of suspension, metformin prevented changes in rAMK and ATP; also, it influenced regulation of the calcium-dependent signaling pathways through expression and phosphorylation of such markers as CaMK, CaN, SERCA2a and Calpain-1, and prevented to a degree growth in expression of key markers of ubuquitin-proteasome pathway – MuRF1, MAFbx, and uniquitin.

Skeletal muscle atrophy during their unloading is due to a decrease in protein synthesis and an increase in proteolysis. The accumulation of ATP in muscle during unloading, detected at its early stages, may be one of the stimuli triggering this process. It has been shown that pannexin channels allow ATP efflux from the cytoplasm to the extracellular space during muscle unloading. Extracellular ATP can be sensed by P2Y2 receptors. To test the hypothesis on the involvement of P2Y2 receptors in the regulation of signaling processes in skeletal muscles at early stages of unloading, they were inhibited by their selective inhibitor AR-C 18925XX. The inhibition of P2Y2 receptors during 3-day unloading attenuated m. soleus atrophy, prevented ATP accumulation therein, downregulated the expression of MAFbx E3-ligase mRNA, ubiquitin and IL6 receptors, upregulated the level of AMPK phosphorylation and intensity of protein synthesis.

Active mechanical properties of postural and locomotor muscles were investigated in Wistar rats after 21-d motor constraint in small cages. Muscles Soleus and EDL of the right leg were tested mechanically ex vivo; RNA obtained from the left legs was used to determine concentrations of various type myosin. Motor constraint caused a significant degradation of active mechanical properties (peak strength of tetanic contraction and intensity) and reliable mass loss in both muscles. Enhanced expression of myosin IIa, IIb and IId/x was revealed in m. soleus only. Also, motor constraint affected functional properties of equally fast and slow muscles. We anticipate that further investigations will shed light on the molecular mechanisms underlying degradation of the skeletal muscles activity.

Due to urbanization, automation and mechanization of labor, as well as difficult epidemiological situation and self-isolation, the level of daily physical activity in a modern man decreases. The aim of the study was to study the effect of restricted activity on protein synthesis in postural and locomotor muscles. An experiment with activity restricted for 21 days was carried out on Wistar rats. The intensity of protein synthesis and anabolic signaling pathways was studied in the soleus muscle ( m. soleus ) mainly consisting of slow fibers, and the extensor digitorum longus muscle ( m. EDL ), mainly consisting of fast fibers. The mass of m. soleus and m. EDL was reduced, while a significant decrease in protein synthesis was observed only in m. EDL . There was also a decrease in the phosphorylation of S6 ribosomal protein only in the fast muscle. At the same time, in contrast to m. EDL , а GSK3β phosphorylation was decreased in m. soleus . Additionally, the markers of proteolysis were studied. In the experiment, a decrease in the expression of MuRF-1 in m. soleus and Atrogin-1/MAFbx in m. EDL was observed, as well as an increase in the expression of calpains in m. soleus . Thus, atrophic processes caused by restricted activity in fast and slow muscles are determined by different signaling mechanisms.

Changes in titin alternative splicing in the rat soleus after seven-day gravitational unloading (the hindlimb unloading model) were studied by long-fragment PCR and nanopore sequencing. An increase in the proportion of shorter titin mRNA transcripts was found in m. soleus of the hindlimb suspended rats, which may have led to the translation of shorter variants of the N2A isoform of this protein. In the nuclear fraction of m. soleus of control and hindlimb suspended rats, no significant differences were found in the content of Rbm20, an RNA-binding protein involved, as is shown earlier, in the regulation of titin alternative splicing in mammalian cardiac muscle. It can be assumed that Rbm20 is not involved in the regulation of titin alternative splicing in the rat soleus muscle.

With urbanization, automation and mechanization of labor, a difficult epidemiological situation and self-isolation, the level of daily physical activity in a modern person decreases. The aim of the study was to study the effect of restricted activity on protein synthesis in postural and locomotor muscles. An experiment with restricted activity for 21 days was carried out on Wistar rats. The intensity of protein synthesis and anabolic signaling pathways were studied in the soleus muscle (m. soleus), which mainly consists of slow fibers, and the long extensor digitorum of the fingers (m. EDL), mainly consists of fast fibers. The mass of m. soleus and m. EDL was reduced, and a significant decrease in protein synthesis was observed only in m. EDL. There was also a decrease in the phosphorylation of S6 ribosomal protein only in the fast muscle. At the same time, а GSK3β phosphorylation was decreased in m. soleus, in contrast to m. EDL. Markers of proteolysis have been studied. In the experiment, a decrease in the MuRF-1 expression in m. soleus and Atrogin-1/MAFbx expression in m. EDL was observed, as well as an increase in the expression of calpains in m. soleus. Thus, atrophic processes are associated with restricted activity in fast and slow muscle by different signaling mechanisms.

The unloading of postural muscles leads to the changes in myosins heavy chains isoforms (MyHCs) mRNAs transcription pattern, that cause severe alterations of muscle functioning. Several transcription factors such as NFATc1 and TEAD1 upregulate slow MyHC mRNA transcription, and p38 MAP kinase can phosphorylate NFAT and TEAD1, causing their inactivation. However, the role p38 MAP kinase plays in MyHCs mRNAs transcription regulation in postural soleus muscle during unloading remains unclear. We aimed to investigate whether pharmacological inhibition of p38 MAPK during rat soleus unloading would prevent the unloading-induced slowtype MyHC mRNA transcription decrease by affecting calcineurin/NFATc1 or TEAD1 signaling. Male Wistar rats were randomly assigned to three groups: cage control (C), 3-day hindlimb suspended group (3HS) and 3-day hindlimb suspended group with the daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS + VX-745). 3 days of hindlimb suspension caused the significant decreases of slow MyHC and slowtonic myh7b mRNAs transcription as well as the decrease of NFATc1-dependent MCIP1.4 mRNA transcription in rat soleus muscles compared to the cage control. P38 MAP-kinase inhibition during hindlimb suspension completely prevented slow MyHC mRNA content decrease and partially prevented slow-tonic myh7b and MCIP1.4 mRNAs transcription decreases compared to the 3HS group. We also observed NFATc1 and TEAD1 myonuclear contents increases in the 3HS + VX-745 group compared to both 3HS and C groups (p < 0.05). Therefore, we found that p38 inhibition counteracts the unloading-induced slow MyHC mRNA transcription downregulation and leads to the activation of calcineurin/NFAT signaling cascade in unloaded rat soleus muscles.

Molecular mechanisms initiating the development of atrophic changes in a mammalian postural muscle remain unclear. We have suggested that AMP-activated protein kinase (AMPK) may play an important role in the regulation of anabolic and catabolic signaling pathways in postural muscle at the initial stage of mechanical unloading, since skeletal muscle inactivation can lead to changes in the balance of intramuscular high-energy phosphates. Beta-guanidinopropionic acid (β-GPA) can act as a modulator of the balance of high-energy phosphates, application of which can reduce the content of intracellular high-energy phosphates. The purpose of this study was to assess signaling pathways of protein synthesis and degradation in rat soleus muscle after preliminary treatment with β-GPA followed by 1-day hindlimb unloading. The experiments were performed on male Wistar rats, which were divided into the following groups: (1) vivarium control (C); (2) vivarium control + β-GPA (C + GPA); (3) 1-day hindlimb unloading (HU); and (4) 1-day hindlimb unloading + β-GPA (HU + GPA). β-GPA was administered daily by intraperitoneal injections (400 mg/kg). The content of the key signaling proteins (AMPK, ACC, p70S6K, 4E-BP1, and FOXO3) was determined by gel electrophoresis followed by immunoblotting. The expression of mRNA of ubiquitin ligases (MuRF1 and MAFbx) was evaluated by real-time PCR. A prevention of reduction in phosphorylation of AMPK, ACC and 4E-BP1 and the return to control values of the increased level of p70S6K phosphorylation were observed in the group with 1-day unloading and β-GPA pretreatment. The phosphorylation of AKT and FOXO3, as well as the expression levels of MuRF1 and MAFbx in the HU + GPA group did not differ from the HU group. The obtained data indicate that the AMPK activity may play an important role in the modulation of anabolic mTORC1-signaling in rat soleus muscle at the early stage of simulated microgravity.

We studied the effect of histone deacetylase 1 (HDAC1) inhibition on titin content and expression of TTN gene in rat m. soleus after 3-day gravitational unloading. Male Wistar rats weighing 210±10 g were randomly divided into 3 groups: control, 3-day hindlimb suspension, and 3-day hindlimb suspension and injection of HDAC1 inhibitor CI-994 (1 mg/kg/day). In hindlimb-suspended rats, the muscle weight/animal body weight ratio was reduced by 13.8% (p<0.05) in comparison with the control, which attested to the development of atrophic changes in the soleus muscle. This was associated with a decrease in the content of NT-isoform of intact titin-1 by 28.6% (p˂0.05) and an increase in TTN gene expression by 1.81 times (p˂0.05) in the soleus muscle. Inhibition of HDAC1 by CI-994 during 3-day hindlimb suspension prevented the decrease in titin content and development of atrophy in rat soleus muscle. No significant differences in the TTN gene expression from the control were found. These results can be used when finding the ways of preventing or reducing the negative changes in the muscle caused by gravitational unloading.

The role of histone deacetylases 4/5 (HDAC4/5) in the activation of myogenin and E3 ligases (MuRF1 and MAFbx) under functional unloading of m. soleus was studied. For this purpose, trichostatin A, a histone deacetylase inhibitor, was used in suspended rats. Wistar rats (24 three-month-old males) were divided into three groups (eight in each). One group served as a control (C), the second (HS) and third groups were suspended for 3 days (the rats of group 3 were intraperitoneally administered trichostatin A at a dose of 0.6 mg/kg of body mass per day (HST)). The rats were killed with an overdose of nembutal (75 mg/kg of mass), and m. soleus was immediately frozen in liquid nitrogen. In the HST group, a significant decrease in the HDAC4 protein content in the nuclear fraction (in contrast to the HS group) and its increase in the cytoplasmic fraction (relative to the control (p < 0.05)) were found. The administration of trichostatin A prevented changes in the HDAC4 content in the nucleus (relative to the C group), whereas the HDAC5 content was significantly reduced (p < 0.05) in the HS group. Thus, the drug prevented the effect of functional unloading on the nuclear localization of HDAC 4/5. The level of myogenin transcription factor in the HST group of rats did not differ from that in the control, while it was significantly higher in the HS group (p < 0.05). The expression of mRNA of E3 ligase atrogin-1/MAFbx in the HST rats was the same as in the control group and was significantly higher in the HS group (p < 0.05). The expression of mRNA of E3 ligase MuRF1in both groups was increased compared to the controls (regardless of the drug administration). Conclusion: HDAC4/5 regulates the expression of myogenin and E3 ligase atrogin-1/MAFbx. Inhibition of HDAC4/5 does not affect the regulation of the expression of E3 ligase MuRF1.