Background Unloading of skeletal muscles triggers rapid changes in molecular signaling, leading to muscle atrophy and functional alterations. Electrical stimulation of muscles is commonly used to counteract these changes, but the precise molecular mechanisms behind its effects remain unclear. Methods To investigate the early changes in postural soleus muscle under unloading conditions (dry immersion, DI) and the impact of electrical stimulation during unloading, two groups of volunteers (10 men in each) underwent a 6-day DI or a 6-day DI with electrical stimulation (DI ​+ ​ES). Soleus muscle samples were collected 14 days before and 6 days after DI and DI ​+ ​ES. Results Six-day DI did not did not cause atrophy of the soleus myofibers or alter protein synthesis parameters, However, it did lead to an increase in atrogin-1 expression, a downregulation of markers for mitochondrial biogenesis and dynamics, and a decline in the mRNA expression of fast oxidative myosin isoform IIa. It also resulted in the downregulation of microRNAs mir-206 and mir-208b, which support slow fiber types. There was an upregulation of CpG methylation in the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) promoter region and an accumulation of Ca2+/calmodulin-dependent protein kinase (p-CaMK II), indicating an increase in myoplasmic calcium levels. Electrical stimulation during the 6-day disuse period prevented the disuse-induced decreases in mitochondria-related markers and the content of mir-206 and mir-208b. It also induced a shift in myosin mRNA expression from types IId/x to IIa, counteracted the accumulation of p-CaMK II and CpG methylation in the PGC1a promoter region. Conclusions Electrical stimulation upregulated markers of both protein synthesis and proteolysis, as well as resulted in lower cross-sectional area of fast-type fibers compared to pre-DI ​+ ​ES.

Skeletal muscle unloading results in muscle atrophy associated with the upregulation of proteolytic genes and suppression of protein synthesis, often accompanied by altered calcium signaling. Here, we used the inositol trisphosphate receptor (IP3R) inhibitor aminoethoxydiphenyl borate (2-APB) to explore the hypothesis that these changes are mediated by IP3Rs. Male Wistar rats were divided into 4 groups: (i) control, (ii) control with daily injections of 2-APB, (iii) 3 days of hind limb suspension, (iv) 3 days of hind limb suspension with daily administration of 2-APB. At the end-point, soleus muscles from the animals were analyzed by Western blotting for the markers of calcium, anabolic, and catabolic signaling. The 3-day hind limb unloading resulted in a decreased muscle weight index, upregulation of the anabolic suppressor pThr56-eEF2, downregulation of anabolic signaling via the mTOR pathway and rRNA expression, as well as the increase in the content of nuclear pThr286-CaMKII (p < 0.05) and cytosolic calcineurin A. While 2-APB did not affect the mTOR-governed changes in anabolism and catabolism, it significantly attenuated alterations in the calcium-dependent targets, such as CaMKII, calcineurin, and eEF2. By contrast, proteolytic signaling (expression of MuRF1, atrogin-1, Ulk1, and ubiquitin mRNAs) after 3-day hind limb unloading was equally upregulated in the control and 2-APB-treated animals. These results suggest that IP3Rs are involved in the unloading-induced muscle atrophy by controlling the nuclear content of calcium; however, they are dispensable for reduced mTOR activity and altered metabolism.

Muscle atrophy and loss of strength observed in hypokinesia may start soon after functional unloading began. To explore these processes and develop countermeasures, a 3-day suspension experiment was fulfilled with Wistar male rats injected with nifedipine, a blocker of dihydropyridine receptors (DHPR). We used 3 groups with 8 rats in each: suspension (HS), supension + nifedipine (HSN) and control (С). Loss of m. soleus weight by 22 % was found in both suspension groups (p < 0.05). At the same time, DHPR inhibition during 3-day functional unloading prevented strength loss, junctophilin proteolysis, and Са2+ increase in m. soleus myoplasm and mitochondria. Nifedipine injected at the beginning of functional unloading keeps slow postural m. soleus from weakening.

During periods of muscle disuse, calcium accumulates in the myoplasm of slow-tonic muscle fibers, leading to multiple negative consequences for muscle function. Leaky ryanodine receptors (RyRs) could contribute to this excessive accumulation of calcium. We hypothesized that the administration of S-107, a stabilizer of RyRs, would reduce the accumulation of calcium in the myoplasm/mitochondria and improve rat soleus muscle function during disuse. Male Wistar rats underwent 7 days of hindlimb suspension (HS), receiving S-107 in their food daily throughout the experiment. Seven days of HS led to cytosolic and mitochondrial calcium accumulation, enhanced mitochondrial respiration, and reduced mitochondrial biogenesis in the soleus muscle. This was accompanied by reductions in the proportion of slow-type myofibres, maximal isometric force, and fatigue resistance. Administering S-107 during HS prevented the accumulation of calcium in the mitochondria and the overactivation of mitochondrial respiration. It also attenuated the decline in markers of mitochondrial biogenesis and the decrease in fatigue resistance. S-107 treatment also partially prevented the decline in the soleus muscle force production but had only a minor effect on myoplasmic calcium accumulation. Our findings suggest that the destabilization of RyRs during muscle disuse leads to an accumulation of calcium in both the mitochondria and the myoplasm, which in turn causes a decline in muscle strength and resistance to fatigue.

Muscle disuse results in complex signaling alterations followed by structural and functional changes, such as atrophy, force decrease, and slow-to-fast fiber-type shift. Little is known about human skeletal muscle signaling alterations under long-term muscle disuse. In this study, we describe the effects of 21-day dry immersion on human postural soleus muscle. We performed both transcriptomic analysis and Western blots to describe the states of the key signaling pathways regulating soleus muscle fiber size, fiber type, and metabolism. Twenty-one-day dry immersion resulted in both slow-type and fast-type myofibers atrophy, downregulation of rRNA content, and mTOR signaling. Twenty-one-day dry immersion also leads to slow-to-fast fiber-type and gene expression shift, upregulation of p-eEF2, p-CaMKII, p-ACC content and downregulation of NFATc1 nuclear content. It also caused massive gene expression alterations associated with calcium signaling, cytoskeletal parameters, and downregulated mitochondrial signaling (including fusion, fission, and marker of mitochondrial density).NEW & NOTEWORTHY The main findings of our study are as follows: 1) The soleus slow fibers atrophy after 21-day dry immersion (DI) does not exceed that after 7-day DI; 2) The soleus ubiquitin ligases expression after 21-day DI returns to its initial level; 3) The soleus slow fibers atrophy after 21-day DI is accompanied by a mitochondrial apparatus structural markers decrease; 4) The soleus fibers signaling pathways restructuring process during 21-day DI is carried out in a complex manner.

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.

In most mammals, postural soleus muscles are involved in the maintenance of the stability of the body in the gravitational field of Earth. It is well established that immediately after a laboratory rat is exposed to conditions of weightlessness (parabolic flight) or simulated microgravity (hindlimb suspension/unloading), a sharp decrease in soleus muscle electrical activity occurs. However, starting from the 3rd day of mechanical unloading, soleus muscle electrical activity begins to increase and reaches baseline levels approximately by the 14th day of hindlimb suspension. This phenomenon, observed in the course of rat hindlimb suspension, was named the “spontaneous electrical activity of postural muscle”. The present review discusses spinal mechanisms underlying the development of such spontaneous activity of rat soleus muscle and the effect of this activity on intracellular signaling in rat soleus muscle during mechanical unloading.

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.

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.

After the first day of muscle disuse (unloading) mitochondria-derived ROS accumulate in the postural-tonic soleus muscle. It is known that excess of ROS can lead to the accumulation of intramitochondrial calcium and overload of mitochondria with calcium, can negatively affect mitochondrial function and fatigue resistance of soleus muscle. We assumed that the use of mitochondrial ROS scavenger mito-TEMPO will be able to prevent the unloading-induced disruption of mitochondrial functions and will help maintain soleus muscle fatigue resistance. To test this hypothesis, male rats were divided into 3 groups (n = 16 in each): vivarium control with placebo (C), 7-day hindlimb suspension with placebo (7HS) and 7-day hindlimb suspension with intraperitoneal administration of the mimetic superoxide dismutase mito-TEMPO at a dose of 1 mg/kg (7HSM). In the 7HS group, increased fatigue of the soleus muscle was found in the ex vivo test, accompanied with increased activity of ETC complex I and "leak" respiration, as well as a twofold increased content of oxidized tropomyosin (a marker of ROS level in tissues) and increase in intramitochondrial calcium compared to C. In 7HSM, the activity of ETC complex I and "leak" respiration had no significant differences from the control group, and the increase in intramitochondrial calcium and the content of oxidized tropomyosin was partially prevented, however, muscle fatigue was also significantly higher than in the control group. Thus, mitochondrial ROS under 7-day muscle unloading contribute to the accumulation of intramitochondrial calcium and oxidation of tropomyosin, but do not have a significant effect on soleus muscle function.

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.

Background More than half of human protein-coding genes have an alternative transcription start site (TSS). We aimed to investigate the contribution of alternative TSSs to the acute-stress-induced transcriptome response in human tissue (skeletal muscle) using the cap analysis of gene expression approach. TSSs were examined at baseline and during recovery after acute stress (a cycling exercise). Results We identified 44,680 CAGE TSS clusters (including 3764 first defined) belonging to 12,268 genes and annotated for the first time 290 TSSs belonging to 163 genes. The transcriptome dynamically changes during the first hours after acute stress; the change in the expression of 10% of genes was associated with the activation of alternative TSSs, indicating differential TSSs usage. The majority of the alternative TSSs do not increase proteome complexity suggesting that the function of thousands of alternative TSSs is associated with the fine regulation of mRNA isoform expression from a gene due to the transcription factor-specific activation of various alternative TSSs. We identified individual muscle promoter regions for each TSS using muscle open chromatin data (ATAC-seq and DNase-seq). Then, using the positional weight matrix approach we predicted time course activation of "classic" transcription factors involved in response of skeletal muscle to contractile activity, as well as diversity of less/un-investigated factors. Conclusions Transcriptome response induced by acute stress related to activation of the alternative TSSs indicates that differential TSSs usage is an essential mechanism of fine regulation of gene response to stress stimulus. A comprehensive resource of accurate TSSs and individual promoter regions for each TSS in muscle was created. This resource together with the positional weight matrix approach can be used to accurate prediction of TFs in any gene(s) of interest involved in the response to various stimuli, interventions or pathological conditions in human skeletal muscle.

Physical inactivity and disuse lead to a decrease in the functionality of skeletal muscles (oxidative capacity, insulin sensitivity, and performance), which is associated with a change in mitochondrial density. In contrast, aerobic exercise training is effective for maintaining/increasing skeletal muscle mitochondrial density and functionality. The review considers the effect of increasing and decreasing physical activity on the mitochondrial density of human skeletal muscles, as well as the main mechanisms responsible for these changes. It is discussed that the content of mitochondrial proteins can be regulated by changing the content of their mRNAs, changes in the rate of synthesis specific for mitochondrial proteins, as well as changes in the rate of degradation, transport, import, and stability of mitochondrial proteins. It has been shown that the mechanisms of regulation of the mitochondrial proteins content under various interventions are significantly different. At the same time, their contribution to the change in the content of mitochondrial proteins is characterized clearly insufficiently, which emphasizes the relevance of further research in this area.

Inactivity is associated with the development of numerous disorders. Regular aerobic exercise is broadly used as a key intervention to prevent and treat these pathological conditions. In our meta-analysis we aimed to identify and compare (i) the transcriptomic signatures related to disuse, regular and acute aerobic exercise in human skeletal muscle and (ii) the biological effects and transcription factors associated with these transcriptomic changes. A standardized workflow with robust cut-off criteria was used to analyze 27 transcriptomic datasets for the vastus lateralis muscle of healthy humans subjected to disuse, regular and acute aerobic exercise. We evaluated the role of transcriptional regulation in the phenotypic changes described in the literature. The responses to chronic interventions (disuse and regular training) partially correspond to the phenotypic effects. Acute exercise induces changes that are mainly related to the regulation of gene expression, including a strong enrichment of several transcription factors (most of which are related to the ATF/CREB/AP-1 superfamily) and a massive increase in the expression levels of genes encoding transcription factors and co-activators. Overall, the adaptation strategies of skeletal muscle to decreased and increased levels of physical activity differ in direction and demonstrate qualitative differences that are closely associated with the activation of different sets of transcription factors.

Regular low intensity aerobic exercise (aerobic training) provides effective protection against various metabolic disorders. Here, the roles played by transient transcriptome responses to acute exercise and by changes in baseline gene expression during up-regulation of protein content in human skeletal muscle were investigated after 2 months of aerobic training. Seven untrained males were involved in a 2 month aerobic cycling training program. Mass-spectrometry and RNA sequencing were used to evaluate proteome and transcriptome responses to training and acute exercise. We found that proteins with different functions are regulated differently at the transcriptional level; for example, a training-induced increase in the content of extracellular matrix-related proteins is regulated at the transcriptional level, while an increase in the content of mitochondrial proteins is not. An increase in the skeletal muscle content of several proteins (including mitochondrial proteins) was associated with increased protein stability, which is related to a chaperone-dependent mechanism and/or reduced regulation by proteolysis. These findings increase our understanding of the molecular mechanisms underlying regulation of protein expression in human skeletal muscle subjected to repeated stress (long term aerobic training) and may provide an opportunity to control the expression of specific proteins (e.g., extracellular matrix-related proteins, mitochondrial proteins) through physiological and/or pharmacological approaches.

Most studies examining the molecular mechanisms underlying adaptation of human skeletal muscles to aerobic exercise focused on the response to acute exercise. Here, we examined the effect of a 2-month aerobic training program on baseline parameters in human muscle. Ten untrained males performed a one-legged knee extension exercise for 1 h with the same relative intensity before and after a 2-month aerobic training program. Biopsy samples were taken from vastus lateralis muscle at rest before and after the 2 month training program (baseline samples). Additionally, biopsy samples were taken from the exercised leg 1 and 4 h after the one-legged continuous knee extension exercise. Aerobic training decreases baseline phosphorylation of FOXO1(Ser256), increases that of CaMKIIThr286, CREB1(Ser133), increases baseline expression of mitochondrial proteins in respiratory complexes I-V, and some regulators of mitochondrial biogenesis (TFAM, NR4A3, and CRTC2). An increase in the baseline content of these proteins was not associated with a change in baseline expression of their genes. The increase in the baseline content of regulators of mitochondrial biogenesis (TFAM and NR4A3) was associated with a transient increase in transcription after acute exercise. Contrariwise, the increase in the baseline content of respiratory proteins does not seem to be regulated at the transcriptional level; rather, it is associated with other mechanisms. Adaptation of human skeletal muscle to regular aerobic exercise is associated not only with transient molecular responses to exercise, but also with changes in baseline phosphorylation and expression of regulatory proteins.