The electromyographic activity of the soleus muscle is a reliable indicator of its functional status. Support unloading causes an immediate cessation of electrical activity of the soleus muscle, which resumes upon restoration of the support load. Prolonged support unloading, however, results in the emergence of spontaneous electrical activity of the soleus muscle. Previous research has established a correlation between this activity and the presence of the potassium-chloride cotransporter (KCC2) on the membranes of spinal cord motor neurons. It has also been demonstrated that the administration of the KCC2 activator prochlorperazine can eliminate spontaneous soleus muscle activity. Here, we aimed to investigate the effect of CLP290, an alternative KCC2 activator, on the spontaneous tonic activity of the rat soleus muscle. The results indicated that daily administration of CLP290 to rats during a 14-day period of hindlimb suspension prevented the reduction in KCC2 levels in lumbar spinal cord motor neurons and the increase in soleus muscle spontaneous tonic activity. Notably, there were no significant differences in the cross-sectional area of slow-type fibers between the antiorthostatic suspension groups with and without CLP290 administration.

The highly specialized mechanically activated (MA) Piezo1 channel of the Piezo ion channel family has recently aroused increased interest among researchers because of its involvement in mammalian mechanotransduction. However, nothing has been known until now about the role of this channel in the regulation of passive muscle stiffness and contraction. We hypothesized that both passive deformation of an isolated muscle and evoked single (twitch) and tetanic muscle contractions would lead to the activation of Piezo1 channels whose activity may increase passive stiffness and muscle contraction amplitude. The study was aimed to assess the possible contribution of Piezo1 channels to the mechanical response of an isolated slow (soleus) muscle to passive deformation and evoked twitch and tetanic contractions using Dooku1, a specific Piezo1 channel inhibitor, and gadolinium chloride (GdCl3), a nonspecific MA channel inhibitor. The experimental results showed that the use of GdCl3 during passive stretching led to a significant decrease in the indices of passive muscle stiffness. Interestingly, Dooku1-induced specific blockade of Piezo1 channels did not affect these indices. At the same time, when the isolated muscle was treated with Dooku1 solution, the maximum tension of evoked twitch and tetanic contractions decreased by 21 and 25 respectively. Also, incubation of muscles in GdCl3 solution led to no significant changes in the indices of evoked contraction. Thus, we demonstrated that Piezo1 channels, which can be blocked by Gd3+, are stimulated by passive muscle stretching and are involved in the regulation of passive muscle stiffness. At the same time, Piezo1 channels are activated during muscle contraction and participate in its regulation. It can be assumed that the involvement of MA calcium channels in maintaining muscle stiffness and the implementation of muscle contraction can be achieved via the additional activation of myofibrils by calcium ions whose concentration in muscle fibers increases due to activity of these channels.

Physical activity is well known to have a beneficial effect on whole body functions, whereas a sedentary lifestyle contributes to the development of metabolic and other diseases and can lead to cognitive decline and increased risk of dementia. The hippocampus mainly controls cognitive performance and the hippocampal neurodegeneration is directly correlated with dementia progression. Hindlimb unloading (HU) is a widely used method to simulate microgravity in rodents and can be used as a model of mobility restriction since one of the main factors of HU is muscle disuse. Additionally, rodents show impaired learning and memory after long-term HU. Here, we explored whether HU would affect the survival or death of the hippocampal cells. Our data demonstrated that after 3-day HU, both apoptosis and autophagy were activated in the hippocampus, as evidenced by the activation of caspase 3 and 9 and an increase in the number of Cathepsin D and LC3b double-positive cells correspondently. Our data indicated that HU has no deleterious effects leading to neurodegeneration for up to 14 days. Moreover, our results also showed that the activation of autophagy during short-term HU had a protective effect, as we did not observe any cell loss or damage.

The electromyographic activity of the soleus muscle is a reliable indicator of its functional status. Unloading of support causes an immediate cessation of electrical activity in the soleus muscle, which resumes upon restoration of the support load. Prolonged support unloading, however, results in the emergence of spontaneous electrical activity in the soleus muscle. Previous research has established a correlation between this activity and the presence of the potassium-chloride cotransporter (KCC2) on the membranes of spinal cord motor neurons. Additionally, it has been demonstrated that the introduction of the KCC2 activator prochlorperazine can eliminate spontaneous muscle activity. This study aimed to investigate the impact of CLP290, an alternative KCC2 activator, on the spontaneous tonic activity of the rat soleus muscle. The results indicated that daily administration of CLP290 to rats during a 14-day period of hindlimb suspension prevented the reduction in KCC2 levels in the motor neurons of the lumbar spinal cord and the increase in spontaneous tonic activity in the soleus muscle. Notably, there were no significant differences in the cross-sectional area of slow-type fibers between the antiorthostatic suspension groups with and without CLP290 administration.

At present, the role of mechanically-activated (МА) channels in realization of anabolic processes in the anti-gravity postural muscles in response to passive stretching remains poorly explored. The purpose of our investigation was to define a potential role of МА-channels in activation of ribosome biogenesis and the ERK1/2-dependent pathway in isolated rat soleus muscle in response to mechanical loading induced by passive stretching. Wistar rats were divided into 3 groups: 1) Control (m. soleus was not subjected to the action of a MA-channels inhibitor or MA-channels activator Piezo1); 2) Gd3+ (m. soleus was incubated in the presence of MA-channels inhibitor gadolinium chloride); 3) Yoda1 (m. soleus was incubated with MA-channels activator Piezo1 – Yoda1). In each group, m. soleus from the left leg was incubated in solution without mechanical loading and m. soleus from the right leg was exposed to passive stretching. The content or expression of anabolic markers was determined using the western-blotting or real-time PCR. In an hour after completion of the m. soleus passive stretching, we discovered a reliable increase in c-Myc and 45S pre-rRNA expression, as well as increases in ribosomal protein S6 (rpS6) and phospho-p90RSK when compared to the resting muscle. After incubation with Gd3+, no difference was found in с-Myc expression and levels of rpS6 and phospho-p90RSK between the resting and stimulated m. soleus. In the Yoda1 group, no difference was noted between the resting and stimulated m. soleus in such parameters as phospho-p90RSK, c-Myc, 45S pre-rRNA and rpS6. Exposure of the resting muscle in the Yoda1 solution resulted in a significant increase in expression of c-Myc, 45S pre-rRNA, and rise in the content of rpS6 in comparison with the control muscle. Incubation of antigravity m. soleus together with the MA-channels inhibitor (Gd3+) attenuates the mechano-dependent response of a number of anabolic markers (phospho-r90RSK, c-Myc, 45S pre-rRNA, rpS6). Chemical activation of the Piezo1 channels in resting muscles produced activation of the markers of ribosomal biogenesis.

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used β-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.

Critical illness myopathy (CIM) is a primary myopathy that develops in critically ill patients. Histological features of CIM include a general reduction in muscle fiber cross-sectional area and a predominant loss of the motor protein myosin in the absence of inflammatory infiltrates but with detectable cytokine activation. This study was aimed to examine the state of the soleus muscle extracellular matrix in patients with CIM caused by chronic disorders of consciousness. Incisional needle biopsies of the soleus muscle were taken from 6 patients with a chronic (≥ 2 months) disorder of consciousness, undergoing treatment at the Polenov Neurosurgical Institute (Almazov National Medical Research Center, St. Petersburg), and healthy men (control). Histological staining of soleus muscle sections in patients with CIM revealed a significantly increased collagen area that exceeded control values by 82 along with type I and III collagen content, were increased. No changes were found in fibronectin and extracellular tissue growth factor mRNA levels. However, integrin α7 mRNA levels were elevated. Our results indicate significant skeletal muscle fibrosis in CIM, requiring further studies on the signaling pathways that regulate this process.

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.

The soleus is one of the key muscles for stability of the majority mammals in Earth's gravity. It is well known that as soon as a laboratory animal (rat) is put in a real or modeled weightlessness (loss of the hindlimb contact with substrate due to tail-suspension) the electrical activity in m. soleus decreases sharply. However, starting on day 3 of the functional unloading this activity renews and grows to the level characteristic of control animals (approximately by day 14 of suspension). The phenomenon was termed "the spontaneous activity of unloaded postural muscle". The review discusses spinal mechanisms of the spontaneous postural muscle activity, the input of ion co-transporters in marrow motoneurons specifically, and effect of this activity on intra-cell signaling in fibers of unloaded m. soleus.

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.

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.

Mechanical unloading of skeletal muscles leads to the developmentof atrophic processes and a decrease in the total number of myosatellitecells involved in muscle regeneration. In vitro studies revealedan accelerated differentiation of myoblasts derived from the rat m. soleus after an unloading-induceddecrease in AMP-activated protein kinase (AMPK). AMPK is requiredfor the activation of SCs and also participates in the regulationof myoblast proliferation and differentiation. It can be hypothesizedthat a decrease in AMPK activity after mechanical unloading promotesthe acceleration of myoblast differentiation. The main purpose ofthis study was to elucidate a possible role of AMPK in the regulationof differentiation of myoblasts isolated from the rat m. soleus after mechanical unloading.To test this hypothesis, a specific AMPK activator, AICAR, was usedto prevent a decrease in AMPK activity during differentiation of myoblastsisolated from the rat m. soleus after7-day unloading. Immunocytochemistry, PCR-RT and Western blottingwere used to assess changes during myoblast differentiation. Indifferentiating myoblasts derived from the unloaded m. soleus, there was a significantdecrease in AMPK (Thr172) and ACC (Ser79) phosphorylation levels,an increase in myotube differentiation index, as well as in theexpression of myoblast fusion factors and myogenic regulatory factors(MRF). Furthermore, there was a decrease in the expression of slowmyosin heavy chains (MyHC) and an increase in the expression offast MyHC isoforms. AICAR treatment of differentiating myoblasts derivedfrom the unloaded m. soleus preventeda decrease in AMPK and ACC phosphorylation, returned the expressionlevels of MRF and fast MyHC isoforms to the control levels, as wellas maintained the expression of slow MyHC. Thus, abnormally accelerateddifferentiation of myoblasts isolated from the atrophied rat m. soleus can be compensated by theAICAR-assisted maintenance of control AMPK activity levels.

The ability of skeletal muscles to sense mechanical stimuli and respond to them by changing intracellular electrochemical and biochemical processes (mechanotransduction) is of crucial importance for the regulation of physiological processes in muscle fibers. This review describes the main sarcolemmal, sarcomeric, and cytoskeletal mechanosensitive structures and analyzes mechano-dependent signaling pathways and mechanisms involved in the regulation of gene expression, as well as muscle protein synthesis and degradation. The final part of the review formulates specific questions in the field of muscle mechanotransduction that need to be addressed in future studies. The understanding of skeletal muscle mechanotransduction is required for the development of effective measures aimed at the treatment of muscular dystrophies, sarcopenia, and prevention of disuse-induced muscle atrophy.

Unloading of slow-twitch muscles results in increased muscle fatigue and the mechanisms of this effect are poorly studied. We aimed to analyze the role of high-energy phosphates accumulation during the first week of rat hindlimb suspension plays in a fiber-type phenotype shift towards fast-type fatigable muscle fibers. Male Wistar rats were divided into 3 groups (n = 8): C -vivarium control; 7HS -7-day hindlimb suspension; 7HB -7-day hindlimb suspension with intraperitoneal injection of beta-guanidine propionic acid (8-GPA, 400 mg/kg b w). 8-GPA is a competitive inhibitor of creatine kinase and it reduces concentrations of ATP and phosphocreatine. In the 7HB group, 8-GPA treatment protected a slow-type signaling network in an unloaded soleus muscle, including MOTS-C, AMPK, PGC1 alpha and micro-RNA-499. These signaling effects resulted in a preserved soleus muscle fatigue resistance, slow-type muscle fibers percentage and mitochondrial DNA copy number under muscle unloading.

Excessive long-term accumulation of calcium ions in the myoplasmof skeletal muscles can negatively affect mitochondria and leadto muscle dysfunction. The aim of our study was to identify therole of L-type calcium channels in the development of increasedfatigue of the rat soleus muscle under functional unloading. Youngmale Wistar rats were divided into three groups of 8 animals each:vivarium control group (C), group exposed to 7-day hindlimb unloading(7HS), and group exposed to 7-day hindlimb unloading with dailyintraperitoneal injections of nifedipine (7 mg/kg body weight) (7HS+N).Nifedipine administration during hindlimb unloading prevented the upregulationof calcium-dependent phosphorylation of calcium-calmodulin kinaseII (CaMKII) and an increase in fatigue, as well as promoted thepreservation of mitochondrial proteins, mtDNA, and mRNA expressionof a number of regulatory genes of mitochondrial biogenesis in the ratsoleus muscle.

Apoptosis and its control during differentiation of primary myoblasts isolated from rat soleus muscle after 7-d tail suspension were investigated. Primary myoblasts were first subjected to myogenic differentiation. TUNEL labeling of double-strand DNA breaks was applied to detect apoptotic cells during myoblast differentiation. Western blot was used to determine the apoptosis markers and a number of signaling molecules, i.e. protein BAX, activated caspase-3, phospho-AMPK (Thr172), phospho-AKT (Ser 437), phospho-p27Kip1 (Thr198), phospho-p27Kip1 (Thr157). PCR analysis was used to investigate the expression of pro-apoptotic markers in myoblasts. The investigation showed enhancement of apoptosis as well as АМРК and p27Kip1 dephosphorylation in the process of differentiation of myoblasts taken from m. soleus of tail-suspended rats.

It is well known that the inactivity of mammalian skeletal muscles leads to the cessation of their electrical activity and is accompanied by atrophic changes in muscle fibers. However, it has been repeatedly noted that starting from the 3rd day of functional unloading, spontaneous rhythmic neuromuscular activity appears, which is the result of a decrease in the expression of the potassium chloride co-transporter KCC-2 in neurons of the lumbar spinal cord. A decrease in the expression of KCC-2 and the onset of autonomous electrical activity of the unloaded muscle can be prevented by the administration of the neuroleptic prochlorperazine. Thus, the aim of this study was to evaluate the structural and signaling effects of the reduced spontaneous activity of the unloaded m.soleus. It was found that daily administration of prochlorperazine to rats under conditions of 7-day simulated gravitational unloading prevented a decrease in the content of the main markers of ribosome biogenesis (c-Myc, 18S rRNA and 28S rRNA), and also partially prevented a decrease in the cross-sectional area of fast and slow muscle fibers in the m.soleus. Morphofunctional changes caused by a decrease of spontaneous activity of the unloaded muscle were accompanied by complete or partial prevention of activation of key proteolytic markers expression (MuRF-1, MAFbx/atrogin-1, ubiquitin). Thus, we assume that spontaneous neuromuscular activity may be a factor that augments muscle atrophy during the first week of functional unloading.

Under the initial stage of muscle mechanical unloading, the skeletal muscle undergo accumulation of high-energy phosphates followed by AMP-dependent proteinkinase (AMPK) inactivation. Since AMPK is known to activate mitochondrial biogenesis, it cannot be excluded that AMPK inactivation results in oxidative potential decrease at the later stages of muscle unloading. We decided to test the role of the accumulation of high-energy phosphates in skeletal muscle fibers in the inactivation of mitochondrial biogenesis regulators at an early stage of muscle unloading. To reduce the ATP/ADP ratio, we used beta-guanidine propionic acid, and the obtained data indicating that already during the first day of simulated microgravity, the accumulation of high-energy phosphates can reduce the expression level of mRNA of the key regulator of mitochondrial biogenesis PGC-1α, the transcription factor TFAM, as well as the mitochondrial fusion regulator - mitofusin-1. A number of other parameters of mitochondrial signaling were not subject to changes at this time-point. Thus, we demonstrated the role of the ATP/ADP ratio in the inactivation of several regulators of mitochondrial biogenesis in the postural soleus muscle at an early stage of functional unloading.

The mechanoelectrical feedback in the heart is based on the work of mechanically gated (MGCs) and mechanosensitive (MSCs) channels. Since microgravity alters the heart’s morphological and physiological properties, we hypothesized that the expression of both MGCs and MSCs would be affected. We employed RNA transcriptome sequencing to investigate changes in the gene transcript levels of MGCs and MSCs in isolated rat ventricular cardiomyocytes under control conditions and in a simulated microgravity environment. For the first time, our findings demonstrated that simulated microgravity induces alterations in the gene transcript levels of specific MGCs, such as TRPM7, TRPV2, TRPP1, TRPP2, Piezo1, TMEM63A, TMEM36B, and known MSCs, including K 2P 2.1, K 2P 3.1, Kir6.1, Kir6.2, Na V 1.5, Ca V 1.2, K V 7.1. However, other voltage-gated channels and channels lacking a voltage sensor remained unaffected. These findings suggest that the altered expression of MGCs and MSCs could lead to changes in the net currents across the membrane, ultimately impacting the heart’s function.