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.

Both research conducted under microgravity conditions and ground-based space analog studies have shown that air pump-based plantar mechanical stimulation (PMS) of cutaneous mechanoreceptors of the sole of the foot is able to increase neuromuscular activity in the musculature of the lower limbs. This type of stimulation is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. The aim of the present study was to evaluate the effects of PMS on anabolic signaling pathways in rat soleus muscle following 7-day hindlimb suspension (HS) and to elucidate if the effects of PMS on anabolic processes would be NO-dependent. The soles of the feet were stimulated with a frequency of 1-s inflation/1-s deflation with a total of 20 min followed by 10 min rest. This cycle was repeated for 4 h each day. We observed a decrease in the soleus muscle mass after 7-day HS, which was not prevented by PMS. We also observed a decrease in slow-type fiber cross-sectional area (CSA) by 56%, which significantly exceeded a decrease (–22%) in fast-type fiber CSA. PMS prevented a reduction in slow-twitch fiber CSA, but had no effect on fast-twitch fiber CSA. PMS prevented a 63% decrease in protein synthesis after 7-day HS as well as changes in several key anabolic signaling regulators, such as p70S6k, 4E-BP1, GSK3β, eEF-2, p90RSK. PMS also prevented a decrease in the markers of translational capacity (18S and 28S rRNA, c-myc, 45S pre-rRNA). Some effects of PMS on anabolic signaling were altered due to NO-synthase inhibitor (L-NAME) administration. Thus, PMS is able to partially prevent atrophic processes in rat soleus muscle during 7-day HS, affecting slow-type muscle fibers. This effect is mediated by alterations in anabolic signaling pathways and may depend on NO-synthase activity.

The study was aimed at investigating the mechanisms and structures which determine mechanical properties of skeletal muscles under gravitational unloading and plantar mechanical stimulation (PMS). We hypothesized that PMS would increase NO production and prevent an unloading-induced reduction in skeletal muscle passive stiffness. Wistar rats were hindlimb suspended and subjected to a daily PMS and one group of stimulated animals was also treated with nitric oxide synthase (NOS) inhibitor (L-NAME). Animals received mechanical stimulation of the feet for 4 h a day throughout 7-day hindlimb suspension (HS) according to a scheme that mimics the normal walking of the animal. Seven-day HS led to a significant reduction in soleus muscle weight by 25%. However, PMS did not prevent the atrophic effect induced by HS. Gravitational unloading led to a significant decrease in maximum isometric force and passive stiffness by 38% and 31%, respectively. The use of PMS prevented a decrease in the maximum isometric strength of the soleus muscle. At the same time, the passive stiffness of the soleus in the PMS group significantly exceeded the control values by 40%. L-NAME (NOS inhibitor) administration attenuated the effect of PMS on passive stiffness and maximum force of the soleus muscle. The content of the studied cytoskeletal proteins (alpha-actinin-2, alpha-actinin-3, desmin, titin, nebulin) decreased after 7-day HS, but this decrease was successfully prevented by PMS in a NOS-dependent manner. We also observed significant decreases in mRNA expression levels of alpha-actinin-2, desmin, and titin after HS, which was prevented by PMS. The study also revealed a significant NOS-dependent effect of PMS on the content of collagen-1a, but not collagen-3a. Thus, PMS during mechanical unloading is able to maintain soleus muscle passive tension and force as well as mRNA transcription and protein contents of cytoskeletal proteins in a NOS-dependent manner.

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.

It was observed that gravitational unloading during space missions and simulated microgravity in ground-based studies leads to both transformation of slow-twitch muscle fibers into fast-twitch fibers and to the elimination of support afferentation, leading to the "switching-off" of postural muscle motor units electrical activity. In recent years, plantar mechanical stimulation (PMS) has been found to maintain the neuromuscular activity of the hindlimb muscles. Nitric oxide (NO) was shown to be one of the mediators of muscle fiber activity, which can also promote slow-type myosin expression. We hypothesized that applying PMS during rat hindlimb unloading would lead to NO production upregulation and prevention of the unloading-induced slow-to-fast fiber-type shift in rat soleus muscles. To test this hypothesis, Wistar rats were hindlimb suspended and subjected to daily PMS, and one group of PMS-subjected animals was also treated with nitric oxide synthase inhibitor (L-NAME). We discovered that PMS led to sustained NO level in soleus muscles of the suspended animals, and NOS inhibitor administration blocked this effect, as well as the positive effects of PMS on myosin I and IIa mRNA transcription and slow-to-fast fiber-type ratio during rat hindlimb unloading. The results of the study indicate that NOS activity is necessary for the PMS-mediated prevention of slow-to-fast fiber-type shift and myosin I and IIa mRNA transcription decreases during rat hindlimb unloading.

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.

Unloading leads to skeletal muscle atrophy via the upregulation of MuRF-1 and MAFbx E3-ligases expression. Reportedly, histone deacetylases (HDACs) 4 and 5 may regulate the expression of MuRF1 and MAFbx. To examine the HDAC-dependent mechanisms involved in the control of E3-ubiquitin ligases expression at the early stages of muscle unloading we used HDACs 4 and 5 inhibitor LMK-235 and HDAC 4 inhibitor Tasqinimod (Tq). Male Wistar rats were divided into four groups (eight rats per group): nontreated control (C), three days of unloading/hindlimb suspension (HS) and three days HS with HDACs inhibitor LMK-235 (HSLMK) or Tq (HSTq). Treatment with LMK-235 diminished unloading-induced of MAFbx, myogenin (MYOG), ubiquitin and calpain-1 mRNA expression (p < 0.05). Tq administration had no effect on the expression of E3-ligases. The mRNA expression of MuRF1 and MAFbx was significantly increased in both HS and HSTq groups (1.5 and 4.0 folds, respectively; p < 0.05) when compared with the C group. It is concluded that during three days of muscle unloading: (1) the HDACs 4 and 5 participate in the regulation of MAFbx expression as well as the expression of MYOG, ubiquitin and calpain-1; (2) the inhibition of HDAC 4 has no effect on MAFbx expression. Therefore, HDAC 5 is perhaps more important for the regulation of MAFbx expression than HDAC 4.

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.

Muscle unloading leads to its atrophy. The expression of E3 ligases (MuRF1 and MAFbx) increases under these conditions. We hypothesized that HDAC 4 and 5 may regulate the E3-ligase expression in the early stages of muscle unloading and that myogenin may be involved in this process. To check this hypothesis, we administered trichostatin A (inhibitor of HDAC 4 / 5 ) to male Wistar rats (180-200 g) upon 3-day suspension of the hindlimb. Twenty four animals were divided into 3 groups (n = 8 in each): C, control; HST, hindlimb suspension with Trichostatin A (i.p., 0.6 mg/kg); HS, hindlimb suspension with placebo administration. The content of the HDAC4 protein in soleus of group HST was decreased in the nuclear fraction as compared to groups HS and C (by 88 +/- 6% and 86 +/- 7%, respectively) and increased in the cytoplasmic fraction in both suspended groups HST and HS (by 33 +/- 10% and 21 +/- 9%, respectively, vs. group C (p < 0.05). In contrast, the nuclear content of protein HDAC5 in soleus of the HST group did not differ from that of group C, while in group HS this parameter was significantly reduced (by 41 +/- 10%, p < 0.05). The myogenin content in the HST group did not differ from that of the C group, while its amount in the HS group was significantly higher (by 30 +/- 6% vs. C, p < 0.05). M for the E3-ligases mRNA expression, MAFbx level in the HST group did not differ from that of control, while in the HS group it increased 2.5-fold (from 1.75- to 3.5-fold, p < 0.05). MuRFI mRNA expression was equally elevated in both suspended groups, HS and HST, relative to the control (p < 0.05). The results suggest that HDAC 4/5 regulates the expression of myogenin and controls the atrogin-1 expression during a 3-day of unloading. The inhibition of HDAC 4 and 5 does not affect the regulation of the expression of E3 ligase MuRF1.

To test the hypothesis that p38α-MAPK plays a critical role in the regulation of E3 ligase expression and skeletal muscle atrophy during unloading, we used VX-745, a selective p38α inhibitor. Three groups of rats were used: non-treated control (C), 3 days of unloading/hindlimb suspension (HS), and 3 days HS with VX-745 inhibitor (HSVX; 10 mg/kg/day). Total weight of soleus muscle in HS group was reduced compared to C (72.3 ± 2.5 vs 83.0 ± 3 mg, respectively), whereas muscle weight in the HSVX group was maintained (84.2 ± 5 mg). The expression of muscle RING-finger protein-1 (MuRF1) mRNA was significantly increased in the HS group (165%), but not in the HSVX group (127%), when compared with the C group. The expression of muscle-specific E3 ubiquitin ligases muscle atrophy F-box (MAFbx) mRNA was increased in both HS and HSVX groups (294% and 271%, respectively) when compared with C group. The expression of ubiquitin mRNA was significantly higher in the HS (423%) than in the C and HSVX (200%) groups. VX-745 treatment blocked unloading-induced upregulation of calpain-1 mRNA expression (HS: 120%; HSVX: 107%). These results indicate that p38α-MAPK signaling regulates MuRF1 but not MAFbx E3 ligase expression and inhibits skeletal muscle atrophy during early stages of unloading.