Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated ß-myosin activity.

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 ß-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.

Simulated Microgravity Changes the Number of Mechanically Gated and Mechanosensitive Ion Channels Genes Transcripts in Rat Ventricular Cardiomyocytes.

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 K2P2.1, K2P3.1, Kir6.1, Kir6.2, NaV1.5, CaV1.2, KV7.1. 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.

Accumulation of high-energy phosphates blocks the expression of mitochondrial biogenesis markers and slow-type myosin in soleus muscle under 24 hours of rat hindlimb suspension.

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.

Β-GPA administration activates slow oxidative muscle signaling pathways and protects soleus muscle against the increased fatigue under 7-days of rat hindlimb suspension.

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 (ß-GPA, 400 mg/kg b w). ß-GPA is a competitive inhibitor of creatine kinase and it reduces concentrations of ATP and phosphocreatine. In the 7HB group, ß-GPA treatment protected a slow-type signaling network in an unloaded soleus muscle, including MOTS-C, AMPK, PGC1 α 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.

Calpain-dependent degradation of cytoskeletal proteins as a key mechanism for a reduction in intrinsic passive stiffness of unloaded rat postural muscle.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

Molecular Mechanisms of Muscle Tone Impairment under Conditions of Real and Simulated Space Flight.

Kozlovskaya et al. [1] and Grigoriev et al. [2] showed that enormous loss of muscle stiffness (atonia) develops in humans under true (space flight) and simulated microgravity conditions as early as after the first days of exposure. This phenomenon is attributed to the inactivation of slow motor units and called reflectory atonia. However, a lot of evidence indicating that even isolated muscle or a single fiber possesses substantial stiffness was published at the end of the 20th century. This intrinsic stiffness is determined by the active component, i.e. the ability to form actin-myosin cross-bridges during muscle stretch and contraction, as well as by cytoskeletal and extracellular matrix proteins, capable of resisting muscle stretch. The main facts on intrinsic muscle stiffness under conditions of gravitational unloading are considered in this review. The data obtained in studies of humans under dry immersion and rodent hindlimb suspension is analyzed. The results and hypotheses regarding reduced probability of cross-bridge formation in an atrophying muscle due to increased interfilament spacing are described. The evidence of cytoskeletal protein (titin, nebulin, etc.) degradation during gravitational unloading is also discussed. The possible mechanisms underlying structural changes in skeletal muscle collagen and its role in reducing intrinsic muscle stiffness are presented. The molecular mechanisms of changes in intrinsic stiffness during space flight and simulated microgravity are reviewed.

Inhibition of Histone Deacetylases 4 and 5 Reduces Titin Proteolysis and Prevents Reduction of TTN Gene Expression in Atrophied Rat Soleus Muscle after Seven-Day Hindlimb Unloading.

The effect of HDACs 4 and 5 on the level of atrophy, calpain-1 and titin content, and TTN gene expression in rat soleus after 7-day gravitational unloading (hindlimb suspension model) was studied. The development of atrophic changes induced by gravitational unloading in rat soleus was accompanied by an increase in the calpain-1 content, an increase in titin proteolysis, and a decrease in the mRNA content of the protein. Inhibition of HDACs 4 and 5 did not eliminate the development of unloading-induced atrophy but significantly prevented proteolysis of titin and the decrease in the TTN gene expression.

The role of MAP-kinase p38 in the m. soleus slow myosin mRNA transcription regulation during short-term functional 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 slow-type 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 slow-tonic 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.

Inhibition of Histone Deacetylase 1 Prevents the Decrease in Titin (Connectin) Content and Development of Atrophy in Rat m. soleus after 3-Day Hindlimb Unloading.

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.

Low Ouabain Doses and AMP-Activated Protein Kinase as Factors Supporting Electrogenesis in Skeletal Muscle.

Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.

Regulation of Protein Synthesis in Inactivated Skeletal Muscle: Signal Inputs, Protein Kinase Cascades, and Ribosome Biogenesis.

Disuse atrophy of skeletal muscles is characterized by a significant decrease in the mass and size of muscle fibers. Disuse atrophy develops as a result of prolonged reduction in the muscle functional activity caused by bed rest, limb immobilization, and real or simulated microgravity. Disuse atrophy is associated with the downregulation of protein biosynthesis and simultaneous activation of protein degradation. This review is focused on the key molecular mechanisms regulating the rate of protein synthesis in mammalian skeletal muscles during functional unloading.

Early Deсline in Rat Soleus Passive Tension with Hindlimb Unloading: Inactivation of Cross-bridges or Activation of Calpains?

The study was aimed at testing the hypotheses about the role of cross-bridges and calpains in reduction of rat soleus passive tension under conditions of hindlimb unloading. For this purpose, we used an inhibitor of µ-calpain PD 150606 as well as a blocker of actomyosin interaction (blebbistatin). It was found for the first time that a decrease in passive tension of rat soleus after 3-day hindlimb unloading is associated with the activity of µ-calpain and does not depend on the processes of cross-bridges formation.

In vitro characterization of MOD-5014, a novel long-acting carboxy-terminal peptide (CTP)-modified activated FVII.

INTRODUCTION: Recombinant FVIIa (rFVIIa) is an effective treatment for haemophilia through frequent administration. However, the short half-life of rFVIIa decreases its prophylactic ability to reduce bleeding. Carboxy-terminal peptide (CTP)-modified FVIIa (MOD-5014) is a long-acting rFVIIa developed for on-demand treatment of haemophilia using either an intravenous or subcutaneous injection with the aim of less frequent administrations, as well as for prophylactic use. AIM: The comprehensive evaluation of the activity MOD-5014 vs commercially available rhFVIIa, as well as their interaction with cofactors and inhibitors. METHODS: The in vitro characterization included clotting activity, affinity by surface plasmon resonance, cleavage of synthetic substrates, thrombin generation (TG) and rotation thromboelastometry. RESULTS: Reduced specific activity was obtained for MOD-5014 compared to rhFVIIa, while both compounds demonstrated comparable affinity to tissue factor (TF). MOD-5014 showed reduced TG when spiked at a similar concentration as rhFVIIa, suggesting that an increased concentration might be needed in a clinical setting to provide initial haemostatic effect. MOD-5014 demonstrated a slightly lower affinity for binding to activated platelets and slightly lower proteolytic activity on the platelet surface, possibly as the fusion of CTP has the potential to sterically hinder binding to both the platelet membrane and to protein substrates. Both compounds showed a similar dose-dependent stimulatory effect on clot formation, and both showed a similar deactivation pattern following incubation with TF pathway inhibitor (TFPI), antithrombin and heparin. CONCLUSION: The comparable in vitro activity of MOD-5014 and rhFVIIa paves the way for in vivo pharmacology evaluations of MOD-5014 in preparation for clinical studies.

Eukaryotic elongation factor 2 kinase activation in M. soleus under 14-day hindlimb unloading of rats.

Functional unloading of m. soleus of male Wistar rats was found to cause a reduction in protein synthesis. The level of phosphorylation of the translation elongation factor 2 (eEF2) and the eEF2 kinase (eEF2k) activity in m. soleus after 14 days of unloading were assessed. Rats were divided into the control group (C) and the group with hindlimb unloading for 14 days (HU14). The level of eEF2 phosphorylation in group HU14 was 80%, whereas in the control is was 40%. The indices of eEF2k expression and protein content in group HU14 increased compared to group C.

Signaling targets of alcoholic intoxication in human skeletal muscle.

Intracellular signaling pathways were investigated in skeletal muscle cells at the early stages of alcohol addiction manifestations. No muscle fiber atrophy was observed in m. vastus lateralis of male patients. No significant changes in the signaling mechanisms that control protein degradation were detected as well. However, the concentration of the insulin-like growth factor (IGF-1) in blood plasma as well as the content of markers of intracellular signaling pathways regulating protein synthesis were significantly reduced compared to the control group.

[EFFECT OF SHORT-TERM DRY IMMERSION ON PROTEOLYTIC SIGNALING IN HUMAN SOLEUS MUSCLE].

The signaling processes initiating proteolytic events in m. soleus of humans during short-term exposure in the non-weight bearing conditions were analyzed. Dry immersion (DI) was used to induce weight deprivation over 3 days. Western blotting was used to define the IRS-1 content, total and phosphorylated neuronal NO-synthase (nNOS), AMP-activated protein kinase (AMPK) that control the anabolic and catabolic pathways, and concentrations of cytoskeletal protein desmin and Ca²âº-activated protease calpin. Already on day-3 of DI calpain-dependent proteolysis manifests itself by reductions in both the total content and level of nNOS phosphorilation. Moreover, AMPK phosphorilation was decreased drastically.

From Slow to Fast: Hypogravity-Induced Remodeling of Muscle Fiber Myosin Phenotype.

Skeletal muscle consists of different fiber types arranged in a mosaic pattern. These fiber types are characterized by specific functional properties. Slow-type fibers demonstrate a high level of fatigue resistance and prolonged contraction duration, but decreased maximum contraction force and velocity. Fast-type fibers demonstrate high contraction force and velocity, but profound fatigability. During the last decades, it has been discovered that all these properties are determined by the predominance of slow or fast myosin-heavy-chain (MyHC) isoforms. It was observed that gravitational unloading during space missions and simulated microgravity in ground-based experiments leads to the transformation of some slow-twitch muscle fibers into fast-twitch ones due to changes in the patterns of MyHC gene expression in the postural soleus muscle. The present review covers the facts and mechanistic speculations regarding myosin phenotype remodeling under conditions of gravitational unloading. The review considers the neuronal mechanisms of muscle fiber control and molecular mechanisms of regulation of myosin gene expression, such as inhibition of the calcineurin/NFATc1 signaling pathway, epigenomic changes, and the behavior of specific microRNAs. In the final portion of the review, we discuss the adaptive role of myosin phenotype transformations.

[Physiological basis of alcohol-induced skeletal muscle injury].

Alcohol-induced muscle damage (AIMD) - an umbrella term that includes all forms of alcoholic myopathy developing in acute or chronic alcohol intoxication. The most common form of destruction of skeletal muscle in alcoholism is a chronic alcoholic myopathy, which develops independently of other manifestations of alcoholism, such as polyneuropathy, malabsorption syndrome, liver damage, but can be combined with them. The basis of the destruction of skeletal muscle in chronic AIPM is atrophy of muscle fibers. Mainly affects muscle fiber type II with less destruction of type ! fibers. Currently, the pathogenesis of chronic alcoholic myopathy is studied. The imbalance of protein synthesis and proteolysisand increased apoptosis rate are discussed.

[REARRANGEMENT OF THE LYMPH TISSUE IN THE MICE SPLEEN AND JEJUNUM WALL DURING THE GROUND-BASED REPRODUCTION OF THE CONDITIONS OF ANIMAL MAINTENANCE IN THE BIOSATTELITE BION-M1 MISSION].

Purpose of the investigation was microscopic examination of changes in cyto architectonics of the spleen and jejunum lymph (immune) tissue in 19-20-week C57BL/6N male mice exposed to some conditions their counterparts had lived in during the 30-d Bion-M1 mission (ground experiment). Local deviations in reactions of the morphofunctional zones of these organs were found. In the spleen, reaction in the centers of lymph nodules generation or the B-lymphocytes maturation zone grows strong. Changes in the cell composition of periarterial lymph sheaths that constitute the morphological site of T-lymphocytes accumulation suggest inhibition of its functional activity. Cell composition of the jejunum wall structure implies a decline of the jejunal immune activity. Our investigation of the organs taken from the ground control mice maintained in the flight BIOS-MLZh module evidences that unceasing noise, hypokinesia, isolation, and paste-like feed weaken general immunity of laboratory animals.

Human muscle signaling responses to 3-day head-out dry immersion.

To date little is known about catabolic NO-dependent signaling systems in human skeletal muscle during early stages of gravitational unloading. The goal of the study was to analyze signaling pathways that determine the initial development of proteolytic events in human soleus muscle during short-term gravitational unloading (simulated microgravity). Gravitational unloading was simulated by 3-day head-out dry immersion. Before and after the immersion the samples of soleus muscle were taken under local anesthesia, using biopsy technique. The content of desmin, IRS-1, phospho-AMPK, total and phospho-nNOS in soleus of 6 healthy men was determined using Western-blotting before and after the dry-immersion. Three days of the dry immersion resulted in a significant decrease in desmin, phospho-nNOS and phospho-AMPK as compared to the pre-immersion values. The results of the study suggest that proteolytic processes in human soleus at the early stage of gravitational unloading are associated with inactivation of nNOS. Reduction in AMPK phosphorylation could serve as a trigger event for the development of primary atrophic changes in skeletal muscle.