[Effect of 30-day space flight and subsequent readaptation on the signaling processes in m. longissimus dorsi of mice].

Some steps of anabolic and catabolic signaling pathways were investigated in postural/tonic m. longissimus dorsi of mice following the 30-day orbital flight of biosatellite "Bion-M1" and 8-day recovery. Western blotting was used for determining insulin receptor substrate 1 (IRS-1) and AMR-activated protein kinase (AMPK) involved in reciprocal regulation of anabolic and catabolic pathways, as well as E3-ligase MURF-1, and elongation factor eEF2. Functioning of the IGF-1-dependent IRS-1 signaling pathway was activated in the recovery period only. Though the content of ubiquitinligase MURF-1 showed an increase after flight, on completion of the recovery period it did not exceed the pre-flight level unambiguously.

[Neuronal NO-synthase as the molecular guard of myofiber stability. NO-dependent signaling pathways in the active and unloaded muscle].

The review is dedicated to the signaling pathways triggered by the nitric oxide II in skeletal muscle. Analysis of the current literature shows that during physical exercise of various intensity and regimen the nitric oxide is an essential trigger of the signaling pathways, leading to the alteration of the structural and metabolic myofiber profile and enhancement of its functional capacity. At the same time during the elevated muscle contractile activity (for instance, eccentric activity), NO serves as a protective and stabilizing agent, preventing from the intensification of the proteolytic processes. Data obtained from the experiments with the modulation of the NO at the background of the functional (gravitational) unloading give evidence that neuronal NO synthase activation in this experimental conditions allows to stabilize the degradation pathways and prevent from disuse atrophy development.

[Role of calcium-dependent mechanisms in the development of atrophy in postural muscle deprived of gravitational loading].

Downsizing of muscle fibers, decline of their contractility and alteration of the myosin phenotype towards fast isoforms prevalence are ranked among the main consequences of gravitational unloading of postural muscles. Role of Ca2+ ions in these processes is the subject of the article. Authors revealed increase in the Ca2+ content in myofibers of resting m. soleus of mice following hindlimb suspension. It has been also reported earlier that nifedipine, a specific blocker of calcium L-channels, can prevent this increase acting on the central and local controls. Therefore, a supposition can be made that activation of the dehydropyridine Ca channels is responsible for Ca2+ rest accumulation during gravitational unloading. The calcium-dependent signaling pathways may be thought of as key players in a number of developments due to gravitational unloading. To begin with, already in early 1990s this role in the hypogravity-induced muscle atrophy was ascribed to calpains, Ca-dependent proteinases which was clearly demonstrated later. We observed maintenance of the relative titin and nebulin contents in unloaded m. soleus associated with the effect of Ca chelators. Also, nifedipine injection reduced significantly the growth of MHC fast isoforms expression during gravitational unloading equally on the RNA and protein levels. To bring to light the place of calcineurin/NFAT signaling system in the MHC phenotype alteration, we undertook inhibition of this pathway by injection of cyclosporine A. This resulted in getting evidence of the stabilizing function of this pathway as it opposes to the myosin phenotype transformation to the fast one.

[Expression of splice-variants of the insulin-like growth factor I and the condition of the cell-myosatellite pool in the rat m. soleus under conditions of gravitation relief and passive stretching].

We suggested that satellite proliferation with subsequent incorporation of their nuclei in the fibres was essential for enhancement of the protein synthesis in the rat relieved m. soleus. In passive stretch of the muscle, the number of labelled cells exceeded 2.5-fold their number in hung out animals and 1.7-fold in the control animals. The expression level of the insulin-like growth factor I (IF-1) was determined in the m. soleus after hanging out with stretching, and no changes were revealed as compared with hanging out without stretching in the control group. We believe that the muscle IF-1 and satellite cell incorporation are not essential for preventing atrophy of relieved soleus muscle in stretching.

[Protective effect of nitric oxide on cytoskeletal proteins in skeletal muscles under eccentric exercise].

The protective effect of nitric oxide (NO) on the cytoskeletal muscle proteins desmin and dystrophin has been studied under eccentric exercise. Experiments were performed on 28 male Wistar rats, which were divided into four groups: cage control (C, n = 7); group of eccentric exercise (running down a motor-driven treadmill, inclination 16 degrees) (20 m/min, 40 min running) (R, n = 7); eccentric exercise + L-arginine group (RA, n = 7) (with a daily supplementation of 500 mg/kg wt L-arginine for 3 days before the running); and eccentric exercise + L-NAME group (RN, n = 7) (with a daily supplementation of 90 mg/kg wt L-NAME (Nomega-nitro-L-arginine methyl ester, nNOS blocker) for 3 days before the running). It was found that increasing the NO concentration (in RA group) prevents the disruption of the dystrophin layer and decreases the loss of desmin in m. soleus under eccentric contraction, whereas in the R and RN groups the level of damage to dystrophin and desmin was significantly higher compared to the control rats. The inhibition of nNOS (by L-NAME) increases the nNOS mRNA level in the m. soleus, whereas increasing the NO concentration in m. soleus (L-arginine administration) does not affect the level of nNOS mRNA during the eccentric running. It was concluded that NO has a protective action on the cytoskeletal proteins desmin and dystrophin in skeletal muscle under eccentric contraction-induced damage.

[The cellular effects of functional unloading and passive stretch on m. soleus of dystrophin-deficient mdx mice].

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to participate in mechanical signal transduction. Recent works testify possible signaling role of this protein to prevent development ofproteolytic processes accompanying muscle fiber atrophy and to stimulate the passive stretch anabolic effects. The experiment was carried out to assess the role of dystrophin in these processes. The study was performed on two months old C57 black and mdx (dystrophin-deficient) mice. Passive stretch resulted in attenuating atrophy development in two fiber types of both C57 black and mdx mice, at the same time fiber type slow-to-fast transformation did not occur in mdx soleus. We established ablatitious effect of chronic hindlimb unloading on SC proliferative activity in soleus muscle and drastic increase of proliferation under effect of passive stretch. We observed no relationship between altered dystrophin synthesis and satellite cell proliferation activity in soleus muscle under conditions of simulated microgravity and concurrent passive stretch. It is concluded that altered dystrophin synthesis partly retarded slow myofibers atrophy and had virtually no effect on passive stretch preventive action. Thus, the hypothesis about dystrophin key role in downregulation of atrophy signaling mechanisms has not found its confirmation concerning gravitational unloading atrophy.

[The contribution of muscle progenitor cells to maintaining morphological characteristics of unweighted rat soleus muscle during passive stretch].

Skeletal muscle work hypertrophy is usually connected with muscle progenitor SC (satellite cells) activation with subsequent incorporation their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and be accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by gamma-irradiation would partly preavent stretched muscle fiber capability to maintain their size under condition of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposure to ionizing radiation (2500 Rad) and then subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myo-nuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We concluded that satellite cells did not make essential contribution to passive stretch preventive action under condition of simulated weightlessness.

[Cytoskeletal proteins and heat shock proteins 27 under eccentric exercise of rats: effects of calcium L-type channel blockade].

It is known that during eccentric exercise the calcium accumulation and degradation of cytoskeletal proteins take place. This study was aimed to investigation into the role of baseline calcium accumulation in cytoskeletal degradation and the way of calcium inflow into skeletal muscle un- der conditions of eccentric exercise. Wister rats were divided into control (C), eccentric exercise (EE) (one set of downhill treadmill running (-16 degrees) at a speed of 20 m. min(-1) for 40 min) and eccentric exercise plus nifedipine administration (EEN) groups (with a daily supplementation of 6.25 mg/kg nifedipine in drinking water during two days). After a 24-hour post-exercise dystrophin, layer integrity and desmin level in m. soleus were declined in EE and didn't change in EEN gr. vs. C (p < 0.05). HSP27 were decreased in EEN in comparison with EE gr. (p < 0.05). Titin and nebullin were not changed after exercise. It seems that calcium L-type channel blocker attenuates or reduces the contraction-induced damage to cytoskeletal proteins.

[Effects of creatine phosphokinase competitive inhibitor on system and tissue energy metabolism in rats in the norm and during unloading].

In an experiment with rats system and intracellular energy metabolism was assessed by cell reactions to chronic injection of beta-guanidine propionic acid (beta-GPA) stimulating AMP-dependent protein kinase (AMPK). Suspension was shown to inhibit the succinate dehydrogenase (SDH) activity, reduce glycogen in both types of muscle fibers, and stimulate the activity of alpha-glycerophosphate dehydrogenase (alpha-GPDH) in fast fibers. Supplementing the rat chow with beta-GPA did not modify these parameters during suspension; however, the blood urea level increased considerably in the suspended and control rats. In the controls, beta-GPA as well as suspension, stimulates growth of the aspartate aminotranspherase activity (AST) in blood. Yet, the suspension and beta-GPA injection had no additive effect. Moreover, their effects were opposite in rats subjected to suspension + beta-GPA. Glucose concentration was observed to become lowered in blood of resting rats treated with beta-GPA. This effect can be associated with a more intensive insulin-dependent glucose transport to muscles. The additional glucose, because of increased demand by fibers, underwent to oxidation and did not replenish the intracellular carbohydrate deposits These data suggest energy metabolism shifting toward activation of the processes of disintegration of substrates for energy production due to a sharp growth of energy demand.

[NFATc1 and slow-to-fast shift of myosin heavy chain isoforms under functional unloading of the rat m. soleus].

It was determined whether the content of NFATc1 (nuclear factor of activation of T cells) in the nuclear and cytoplasmic extracts is related to an increase in the content of fibers containing type IIa myosin heavy chains under gravitational unloading of m. soleus. It was found that three isoforms of NFATc1 with molecular masses of 140, 110, and 86 kDa are present in m. soleus. Under unloading, the translocation of 140 kDa NFATc1 into the nucleus, a decrease in the content of 110 kDa NFATc1 in the cytoplasmic extract of m. soleus, and an increase in the content of 86 kDa NFATc1 in the nuclear extract of m. soleus take place. The content of fibers containing type IIa myosin heavy chains under gravitational unloading increases. The increase in the level of 140 and 86 kDa NFATc1 in the nucleus is accompanied by a decrease in the percentage of fibers containing type I myosin heavy chains and an increase in the percentage of muscle fibers containing type IIa myosin heavy chains.

[Creatine as a metabolic controller of skeletal muscles structure and function in strength exercises in humans. The cellular mechanisms].

The effects of creatine oral supplementation combined with a 10-week resistive training of morphometric, contractile and molecular characteristics of human vast lateral muscle fibers were studied. 2 groups consisting of 9 young healthy men each were involved in resistive training of knee extensors for 10 weeks. Volunteers of the first group received per os 20 g of creatine for the 1st week of training and 5 g for the rest of the experimental training period. We found a significant increase of slow and fast-twitch fiber size in both trained groups and a significant increase of Ca-sensitivity of skinned single fiber contractility in creatine-supplemented group. The serum creatine phosphokinase activity in blood samples taken 24 hours after exercise session increased in all stages of the experimental training in both groups. At the same time, the adaptive decrease of the after-exercise CK concentration was observed in the placebo but not in the creatine-supplemented group. The altered integrity of the subsarcolemmal dystrophin layer was revealed in both groups after training.

[Role of L-type Ca channels in Ca2+ accumulation and changes in distribution of myosin heavy chain and SERCA isoforms in rat M. soleus under gravitational unloading].

It is known that hindlimb unloading brings about the intracellular Ca2+ accumulation and MyHC slow-to-fast shift in m.soleus. SERCA (sarcoendoplasmatic reticulum Ca ATPase) function as a Ca pump to uptake to sarcoendoplasmatic reticulum after skeletal muscle contraction, and can modulate intracellular resting Ca level. The study was aimed at investigation of the role of intracellular Ca2+ level for MyHC and SERCA isoforms transformation in m.soleus under hindlimb unloading. To determine role of intracellular Ca we administrated nifedipin--specific blocker of L-type calcium channel in myofibers. We hypothesized that decrease of intracellular calcium level prevented-NFATc1 nuclear translocation and MyHC slow-to-fast transformation. 42 male Wistar rats (180-200 g) were divided in 3 groups: cage control (C, n = 14), 14 days HU (HU, n = 14), 14 days HU with 7 mg/kg/day of nifedipin administration with water (HUN, n = 14). The study has shown that increase of intracellular Ca2+ level under HU leads to MHC slow-to-fast shift via activation of calcineurin-NFATc1 signaling pathway. Percentage of muscle fibers with SERCA I increased under hindlimb unloading, being dependent of intracellular calcium level, percentage of muscle fibers with SERCA II decreased under hindlimb unloading but did not depend on calcium. We suppose that nifedipin administration decreases intracellular Ca level, prevents MHC slow-to-fast shift via prevention of NFATcl accumulation in nuclear extract of m.soleus, and prevent increase of SERCAI expression. The work was supported by grants RFBR N05-04-49255a, 04-04-49044, 05-04-08200-ofi-a, contract with Federal Agency for Science and Iinnovation N02.467.11.3005, and Presidium of RAS program "Basic sciences for medicine".

[Effect of long-term hypergravitation on the skeletal-muscular tissue in rats].

The space flight or simulated gravitational unloading lead to the muscle atrophy, slow-to-fast transformation of muscle fibers and myofibrillar damages both in humans and animals (1, 7, 13, 17). This process could be prevented by the exercise training during space flight (1), (partly) by periodic weight support during unloading (13). It has been demonstrated in these studies that there is some level of force production necessary for the maintenance of skeletal muscle properties. It is known that adaptation to the physical training frequently induces augmentation in cross-sectional area (CSA) of muscle fibers (MF), transformation of fibers, augmentation of mitochondrial volume density, and increase in absolute volume of myofibrillas. Numerous observations suggest importance of gravitational loading in regulating muscle mass. The centrifuging is believed to be useful for preventing muscle functional and structural losses under microgravity. But there are few studies designed to investigate effect of artificial gravity on the skeletal musculature (2, 7). Our objective was to investigate structural adaptation in slow-twitch soleus muscle (percentage of connective tissue and central nuclei, fiber size, myosin heavy chain isotope, myofibrillar proteins and mitochondria volume density) after 19 and 33 days of hypergravity.

[Human soleus fibers contractile characteristics and sarcomeric cytoskeletal proteins after gravitational unloading. Contribution of support stimulus]. / Sokratitel'nye kharakteristiki i belki sarkomernogo tsitoskeleta volokon m. soleus cheloveka v usloviiakh gravitatsionnoi razgruzki. Rol' opornogo stimula.

The effects of support withdrawal and support stimulation on the contractile characteristics of human soleus fibers and cellular factors which influence them were studied. The experimental model of the "dry" head-out water immersion was used in the study. In this model, the hydrostatic pressure on different sites of the body surface are equal so that the experimental conditions are close to the complete supportlessness. A 7-day exposure to dry immersion resulted in a decrease in the maximal isometric tension of the skinned fibers, a decline in the myofibrillar Ca2+-sensitivity, and the relative loss of the titin and nebulin content. A significant decrease in the percentage of fibers containing slow myosin heavy chains was also observed after dry immersion. The application of the mechanical stimulator influencing the plantar support zones with a pressure of 0.2 +/- 0.15 kg/cm2 6 times a day for 20 minutes of each hour brought about a complete prevention of the above listed effects of dry immersion. The data obtained allow one to conclude that the decline in maximal tension and Ca2+-sensitivity as well as myosin shift and loss of sarcomeric cytoskeletal proteins are associated with the support withdrawal during the exposure to dry immersion.

[Sarcomeric cytoskeletal proteins and myosin phenotype in stretched soleus of hindlimb-suspended rats]. / Belki sarkomernogo tsitoskeleta i miozinovyi fenotip volokon m. soleus pri ee khronicheskom rastiazhenii na vone vyveshivaniia zadnikh konechnostei krysy.

Changes in sarcomeric cytoskeletal proteins of rat m. soleus fibers upon the chronic stretching against the background of gravitational unloading were analyzed and compared with changes in fiber size and myosin phenotype. For rats exposed to gravitational unloading in the usual microgravity-simulating experimental model (hindlimb suspension (HS) according to Morey-Holten), a considerable reduction in the mass of m. soleus (by 54%) and the area of its fibers of both slow-twitch (by 47%) and fast-twitch (37%) types compared with control animals was revealed. Moreover, the percent of fibers containing only slow isoforms of myosin heavy chains (MHC) for suspended animals was slightly smaller and the portion of fibers interacting only with antibodies against fast myosin isoforms was significantly higher than for control animals. For hindlimb-suspended rats, the titin/MHC and nebulin/MHC ratios appeared to be reduced almost by two times as compared with those for the contriol group of animals. Chronic immobilization of m. soleus in stretched state against the background of suspension leads to a partial or complete prevention of the reduction in muscle fiber sizes, the transformation of the myosin phenotype into fast one, and a decrease in relative content of sarcomeric cytoskeletal proteins.

[Contractile characteristics of single skinned rat soleus muscle fibers under gravitational load: effects of a calcium-binding agent]. / Sokratitel'nye kharakteristiki odinochnykh skinirovannykh volokon Musculus soleus krysy v usloviiakh gravitatsionnoi razgruzki: éffekty kal'tsiisviazyvaiushchego agenta.

Excessive intracellular calcium accumulation is believed to trigger the development of functional and structural changes in muscle fibers under microgravity conditions. The hypothesis was testified in the 14-day hindlimb suspension study with the application of a Ca(2+)-binding agent (10% EGTA). Twenty one rats were divided into 3 groups: cage controls (7), hindlimb-suspended rats that received intraperitoneal injections of saline (7), and hindlimb-suspended rats with EGTA treatment. Whereas the diameter of muscle fibers of unloaded rat soleus muscle was 20% less than in the control group (and there were no significant differences between rats with injections of EGTA and without them), the decrease of maximal tension was more pronounced (more than 50%). This discrepancy resulted in a decrease of maximal specific tension. The value of absolute tension in rats treated with placebo was by 52%, and in EGTA-treated rats by 41% less than in the control group. Thus, there were no significant differences in specific tension between this group and the control group. Obviously, the injections of EGTA prevented the effects of those mechanisms that induce a decline of tension in muscle fibers but are not linked with the reduction of fiber size. The Ca/tension curve in hindlimb-suspended saline-treated rats shifted to the right so that the pCa thresholds changed from 6.85 +/- 0.03 in cage controls to 6.70 +/- 0.04 (p < 0.05), which indicates that myofibrils of unloaded soleus are less sensitive to Ca2+. At the same time, the pCa threshold in EGTA-treated hindlimb-suspended rats was 6.93 +/- 0.02. It is concluded that chronic binding of excess calcium results in an increase in Ca sensitivity indices.

[Effect of deafferentation on the size and myosin phenotype of muscle fibers during stretching of rat m. soleus and gravitational unloading]. / Vliianie deafferentatsii na razmery i miozinovyi fenotip myshechnykh volokon pri rastiazhenii m.soleus krysy na fone gravitatsionnoi razgruzki.

The study was purposed to evaluate the contribution of the reflectory and local components during the chronic stretch of the postural muscle to the attenuation of the unloading-induced fiber size reduction and changes in the myosin heavy chain (MHC) profile. The surgical unilateral deafferentation (dorsal rhizotomy) was used. It was shown that unilateral deafferentation didn't influence on the amelioration of unloading-induced fiber size reduction in chronically stretched soleus muscle. Thus, the results obtained in the present study don't confirm the hypothesis, supposing the predominant contribution of the muscle afferent activation to the attenuation of unloading-induced fiber atrophy. Deafferentation of unloaded as well as control rats leads to the increase of the percentage of fibers expressing slow MYC isoforms.

[Changes in structure and metabolism in the m. vastus lateralis in monkeys after a space flight]. / Osobennosti izmeneniia struktury i metabolisma v m. vastus lateralis posle kosmicheskogo poleta.

Monkeys developed a significant reduction in size of the m. vastus lateralis' fast and slow fibres, the amount of protein in them remaining the same.