Abstract: Objective To determine the effects of 4-week hindlimd unweighting (HU) with and without 1h/day standing (STD) on myotonic-tone and vasoconstrictor responses of rats mesenteric arteries. Method Male Sprague-Dawley rats were subjected to 4-week hindlimb unweighting to simulate cardiovascular deconditioning due to microgravity, 1h/day standing as the countermeasure. The diameter of isolated mesenteric resistance artery (diameter from 180-220:Im) were recorded with Pressure myograph system in both Physiology salt solution PSS (Pa) and calcium-free Physiology salt solution (Pd), the myogenic-tone were obtained from (Pd-Pa)/Pd*100%. The responses of these arteries to vasoconstrictors Chloratum Kalium and Phenylephrine were also detected.

Results 1) The myogenic tone of arteries was significantly attenuated by 4-week hindlimb unweighting in HU group compared to control (CON) group(P<0.05); With 1h/day standing, the attenuated myogenic tone was recovered completely (P<0.05). 2) The vasoconstrictor responses of arteries to KCL and PE was decreased by 4-week hindlimb unweighting, and 1h/day standing can also prevent the attenuation (P<0.05). Conclusion Both the myogenic tone and vasoconstrictor responses of rats mesenteric arteries were significantly attenuated by 4-week hindlimb unweighting, 1h/d standing can prevent the attenuation completely, which could give us a better understanding of the underlying mechanism of orthostatic-intolerance post-flight and can also provide us new countermeasures to avoid it.

Keyword: hindlimd unweighting; myogenic-Tone; vasoconstrictor responses

Vascular tone of cerebral arteries regulates the needs of energetic and food of the central nervous system. Endothelial, glial and neuronal factors modulate vascular functions in cerebral area. The vascular tone is assumed by smooth muscle cells of cerebral arteries. In these cells, the propagated calcium signals lead to the contraction and an increase of blood pressure whereas localized calcium signals (calcium sparks) activate hyperpolarization and vasodilatation. Confocal microscopy coupled with Fluo-8 calcium probe allows evaluating calcium concentration and vascular contraction. Microgravity induces cerebral hypertension. This effect is also measured in hindlimb suspended rat but the molecular mechanism of this hypertension is not known. Our work wants to characterize the modification of the calcium signalling induced by hindlimb suspension in isolated rat cerebral arteries. In suspended rat, the frequency of spontaneous calcium waves inducing vascular contraction is increased. Secondly, the expression of ryanodine receptor RYR1, revealed by western blot, is also increased. These first results suggest that the vascular reactivity is increased in simulated microgravity model.

Our previous studies on vascular adaptation to microgravity have been focused mostly on medium- or small-sized muscular arteries (1). However, the structural adaptive remodeling in large elastic arteries has received little attention, which cannot be simply extrapolated from the data obtained from muscular arteries. Moreover, our previous work has suggested that the vascular local renin-angiotensin system (L-RAS) might play an important role in microgravity-induced vascular adaptation (2). In the present study, we examined the differential remodeling of rat common carotid artery and superior mesenteric artery after a 28-d simulated microgravity (SUS). Furthermore, the expression of two key components of vascular L-RAS, angiotensinogen (Ao) and angiotensin ¢òreceptor type 1 (AT1R) in the wall tissue were also examined by immunohistochemistry. The results showed that, in the common carotid artery, a 28-d SUS induced an increase in the media thickness and total cross-sectional area (CSA) due to the hypertrophy of the smooth muscle (SM) layers (P<0.01), but the lumen diameter and mean number of SM layers and elastic laminae did not show significant changes. On the contrary, in the superior mesenteric artery, a 28-d SUS induced a decrease in the total CSA of SM layers (P<0.05), whereas the lumen diameter, media thickness, and mean number of SM layers and elastic laminae did not show significant changes. Furthermore, we found that the the most profound changes were in the innermost layers of the two kinds of arteries, which is consistent with the state of stress concentration under an intravascular pressure load. Immunohistochemistry revealed the presence of protein expression of Ao and AT1R in the media, adventitia, and perivascular tissues. A 28-d SUS induced an up- and down-regulation in Ao and AT1R expression in the common carotid artery and superior mesenteric artery, respectively.

Reference: (1) Zhang LF. Vascular adaptation to microgravity: what have we learned? J Appl physiol. 2001;91: 2415-2430. (2) Zhang LF, Bao JX. Vascular adaptation to microgravity: role and implications of local vascular renin-angiotensin system. In: Hargen A,et al eds. Adaptation biology and medicine (Volume 4). Narosa Publishing House; 2005: 329-339.

Several studies revealed changes in gastrointestinal secretion, motility, evacuation and absorption in humans and animals after space missions. Among biological alterations, low gravity generates modifications of gastrointestinal function as enzyme activity, intestinal mucosa integrity, colonic microflora and liver activity. The main goal of this study was to determine whether modeled microgravity conditions, performed by the Random Positioning Machine (RPM), influence the rat intestinal transit as well as the expression of enzymes involved in this process and in gastrointestinal homeostasis as ciclooxygenase (COX-1 and COX-2), the inducibile isoform of nitric oxide synthase (iNOS), ICAM-1 and heat shock proteins HSP70 and HSP90. Rats were individually kept for 48 hours in RPM. In order to rule out a possible effect of immobilization stress we included 3 groups of rats: RPM and two control groups on the floor of RPM with rats being individually kept either in a standard cage (CC) or immobilized in the semicylinder (CG).

Our results indicate that modeled microgravity conditions significantly reduce rat intestinal transit. Western blot analysis on small intestine tissues of RPM rats revealed a significant increase in iNOS expression respect to CC group. Furthermore, while COX-1 expression remained unaltered, a significant reduction in COX-2 levels was observed both in RPM and CG, respect to CC group. Significant changes in intestinal homeostasis in RPM rats was also confirmed by a significant increase in ICAM-1 and HSP70 expression, indicating a modification of their basal levels induced by RPM. Moreover, a significant effect of stress was observed in stomach of immobilized rats: indeed, a significant reduction in COX-2 expression, both in RPM and in CG groups respect to CC was observed. On the other hand a significant increase in HSP90 stomach expression, indicates a strong effect of simulated low gravity. The exact cellular mechanisms by which microgravity produces alteration of intestinal function remains to be better understood. This experimental model offers the opportunity to explore gastrointestinal protein expression changes and may provide new insights into the adaptive mechanisms that take place during space missions. In the next future, MHOR (Mouse Habitat On RPM), a system mounted on the inner frame of our RPM, will permit longer duration (15-20 days) rat experiments providing water delivery ad libitum, food delivery (given quantity/day), waste management (faeces and urine). Research supported by Italian Space Agency (ASI).

Abstract: AIM To investigate alterations of CaL in cerebral vascular smooth muscle cells isolated from rats subjected to a two-week simulated weightlessness, and influence of Bay K 8644 (an agonist of CaL) to the channel currents. METHODS Tail-suspended rat model was used to simulate the effects of microgravity. Whole-cell patch-clamp technique was used to record CaL currents before and after Bay K 8644 treatment, with intracellular Ca2+ concentration maintained physiological level. The corresponding parameters such as steady state activation and inactivation curves were also recorded. RESULTS Whole-cell CaL current densities increased obviously, and sensitivity of CaL to Bay K 8644 also increased in cerebral vascular smooth muscle cells from suspension group. But membrane capacitance (Cm), access resistance (Ra), and other parameters of CaL such as steady state activation / inactivation curves have no significant changes compared with those of control group. CONCLUSION These results suggest that enhanced CaL function of cerebrovascular smooth muscle cells induced by simulated microgravity may be one of the electrophysiological mechanisms that mediate enhanced vasoreactivity of cerebrovascular smooth muscle cells during adaptation to simulated weightlessness in rats.

In 2009 the WOSP (WOrms in SPace) experiment will be performed on the ISS in the European Modular Cultivation System (EMCS) to study the effects of space conditions, microgravity mainly, on development, morphology and fitness of a free living nematode, Panagrolaimus rigidus.The distribution of yolk granules and of cytoskeleton components is known to determine the spindle orientation and the body pattern of the embryo. Thus we expect that eggs produced by mothers under microgravity will reflect the perturbations occurred in the spatial arrangement of microfilaments during the cytoplasm synthesis, and the orientation of asymmetric cleavage should affect the cytoskeleton arrangement of the newborns. As all nematodes, P. rigidus is eutelic (fixed number of cells) and develops through a 'spiral' cleavage, i.e. the fate of its cells is fixed during early embryogenesis. In addition it is a benthic species and moves only through ondulations.

To test the response of the nematode to microgravity, we checked its capacity to feed and to reproduce. Nematodes do not have adhesive glands or suckers and are not suspension feeders. In absence of gravity, nematodes do not settle at any bottom and their food is suspended in the cell. Experiments were run cultivating nematodes in a Random Positioning Clinostat (desktop RPM). Parallel cells were set up with and without a filter net on RPM (µg) and on desk (1g). Results showed that the filter support is necessary for breeding the nematodes in RPM. Embryo development was also studied comparing RPM (µg) and desk (1g) condition. Developmental stages were analysed at light and confocal laser microscopy.

Murine pre-adipocite cells (3T3-L1) have the peculiarity to differentiate into adipocyte cells if properly stimulated by the presence of insulin, dexamethazone and isobuthyl-methyl-xantine (DMI protocol) in the culture medium. To investigate the effects of microgravity on cellular differentiation rate, the entire program of differentiation has been conducted on two groups of 3T3-L1 cells, in microgravity and normal gravity conditions respectively.

The entity of differentiation with the time has been partially assessed through the evaluation of the production of lipid drops and enlightening the expression of specific positive (c/EBP-β and PPAR-ã) and negative (tubulin) differentiation markers through Western and Immuno-blotting experiments.

Preliminary data obtained suggest that cells in microgravity condition differentiate sooner in respect to cells kept in normogravity condition. To validate these results we are performing FACS experiments to evaluate the distribution of differentiating cell in the cell cycle to correlate microgravity, differentiation and growth.

Although two sets of independent experiments have been collected up to date, the differences observed in the rate of differentiation still remain of questionable significance. To give a final answer to this important question we are planning Band-Shift experiments to monitor the early appearance of a key regulator of differentiation program transcriptional cascade, namely c/EBP-β DNA binding activity.

If the data, in whole, will be independently confirmed, our final aim will be the identification of the specific molecular targets through which microgravity affects the rate of differentiation.

Two strains of Pseudomonas aeruginosa, PA01 and PA103, were flown on NASA Shuttle Endeavour mission STS-123 in March, 2008, aiming to determine if spaceflight affects virulence gene regulation and expression in a Microbial Drug Resistance and Virulence (MDRV) experiment. Samples were contained in Fluid Processing Apparatus (FPA; BioServe, Boulder, CO) tubes with a gas-exchange insert adjacent to the growth media and silicone rubber septa separating the media from the microbial inoculum suspensions and fixative solutions. Each FPA was placed in a Lexan sheath with a plunger to move the septa for activation and termination, and these were placed in sets of 8 in Group Activation Packs. Three sets of 4 FPAs were prepared for each organism: activation (T0) cell counts and toxin production, with 0.2% formalin in the medium chamber A, cells suspended in water in the inoculum chamber B, and 0.2% formalin in the fixative chamber C; termination (T2) cell counts and toxin production, with modified simple defined medium 2 (MSDM2) in chamber A, cell suspension in chamber B, and 0.4% formalin in chamber C; and, for RNA and protein analysis at termination (R2), MSDM2 in chamber A, cell suspension in chamber B, and RNA Later II in chamber C. An additional set of 4 FPAs was prepared for a comparative study of gene regulation in PA01, with Lennox broth (LB) in chamber A, bacterial inoculum in chamber B, and RNA Later II in chamber C. An identical set of FPAs was prepared as ground controls.

The flight samples were kept at ambient temperatures for ca. 60h before loading onto the shuttle, and were then at mid-deck temperature (ca. 25oC). Ground control temperatures followed mid-deck readings with a 1h delay. T0, T48, and R2 samples were activated after 10d on orbit, incubated for 2d, then terminated. LB samples were activated after 11d on orbit, incubated 1d, then terminated. Preflight trials showed that under these incubation conditions, both strains of P. aeruginosa grew to ca. 109 cells/ml when inoculated with ca. 107 cells/ml. Inocula were stable for more than the ca. 14d from preparation to activation, and fixed cultures were stable for more than the 3-4d from termination to return. Post-flight analyses include T0 and T2 cell counts to determine growth, T0 and T2 production of exotoxins by ELISA and other virulence proteins by MALDI-TOF/MS, and expression of virulence genes using quantitative RT-PCR. Virulence gene regulation, particularly via the hfq regulon, will be examined using quantitative RT-PCR and Affymetrix gene arrays, and results for P. aeruginosa compared with those for Salmonella typhimurium and Streptococcus pneumoniae being flown for other experiments in the same package. The results will extend our understanding on the effects of spaceflight on the virulence of pathogenic bacteria.

Rationale. Microgravity and cosmic irradiations are the main new physical conditions accounting for human space exploration-related damages. Scarce knowledge is available on angiogenesis alterations under space conditions. While simulated hypogravity has been shown to impair the angiogenic healing response of endothelium by up-regulating apoptotic signals, low-dose irradiation under ground conditions results in angiogenesis promotion. Angiogenesis is a process of new blood capillaries formation, which consists of several steps, where degradation of the anatomical barriers (ECM) by proteolytic enzymes, such as uPA (urokinase plasminogen activator), required to open a path to migrating endothelial cells (EC), is critical. Plasminogen activation system regulates angiogenesis by both uPA-driven ECM degradation and uPA receptor (uPAR) adhesion to ECM. In the present work we isolated microvascular endothelial cells (EC) from human skin biopsies and evaluated their angiogenic properties in vitro under control ground conditions, as dependent on uPAR expression

Results. EC migration was studied by the Boyden chamber assay. Cells were applied to the upper well of the migration apparatus on a Matrigel-coated porous membrane. Cell migration was measured by counting cells on the lower side of the membrane after 6 h incubation at 37°C. Capillary morphogenesis, characterized by the formation of endothelial tubular-like structures, was studied in a Matrigel bed, after 6 and 24 hours incubation. Both migration and capillary morphogenesis were evaluated under control conditions and in the presence of an uPAR-aODN (ISIS product 17916, International Patent of our laboratory). The aODN treatment caused a 75% inhibition of MVEC migration and a complete inhibition of capillary morphogenesis, paralleled by a strong reduction of uPAR mRNA and uPAR protein.

Conclusions. The efficacy of uPAR-aODN in preventing angiogenesis deserves an in depth study under microgravity and low-irradiation conditions. However, our results suggest its applicability as a countermeasure to prevent and cure eye hyper-angiogenesis during long-term interplanetary space explorations.

Acknowledgements. We thank Ministero dell'Universita e della Ricerca, Ente Cassa di Risparmio di Firenze and Fondazione Cassa di Risparmio di Lucca for their contributions to our research.

Insects spread their wings at the last step of metamorphosis. The wings attain full size immediately after emergence from the pupae. Rapid extension of wings may involve two mechanisms, influx of body fluid into wing veins and gravity on wings. To examine the relative importance of these two mechanisms, we compared wing shapes of adults that emerged at a horizontal plane, at a vertical plane, or in the vertical but centrifugally accelerated conditions using a turntable.

In Hagenomyia micans (antlions), Myrmeleon bore (antlions), Parachauliodes continentalis (fishflies), Eterusia aedea (moths), and Papilio memnon (butterflies), wings were crumpled when they emerged at the horizontal plane, whereas in Lucilia cuprina (flies), Tenebrio molitor (beetles), and Zophobas atratus (beetles), wings extended fully even in horizontal emergence. In the field, the former five species spread their wings hanging from the substrate such as plant stems and tree branches and, thus, gravity may be used for their wing extension. The effects of gravity seemed to be greater in the species with larger wings. In the latter three species, however, adults do not climb substrate after eclosion to hang from it, and the main force to extend their wings may be influx of body fluid into wings. In this group of insects, the species possessing large wings were able to expand wings similarly to those possessing small wings.

The prediction that wings would be more elongated if centrifugal acceleration is given, was not evident in our experiment under about 3 G, excluding the moth E. aedea in which the forewings became slightly but significantly elongated. In the future, observation of insect emergence at zero gravity in space will be needed to clarify the effects of gravity on their wing extension. Moreover, the wing extension patterns of insects may be used as a model system of deployable membrane structures in space in respects of high redundancy, sequential deployment, and utilization of gravity force.

Human melanocytes, which are located in the epidermis, are responsible for skin pigmentation by producing the pigment melanin. The melanins play a key role in the protection against harmful effects of UV radiation and oxidative stress. Nitric oxide (NO) is an endogenous molecule with multiple functions involved in UV-induced melanogenesis, inflammation, and skin cancer. Its cellular signaling is mainly mediated by activation of the soluble guanylyl cyclase isoforms resulting in an increased formation of guanosine 3',5'-cyclic monophosphate (cGMP). In previous studies we found that long-time exposure to hypergravity (5xg for 24h) stimulates the cGMP efflux in normal human melanocytes under the conditions of inhibited degradation or stimulated synthesis of cGMP (e.g., by NO). These effects were not observed in the presence of highly selective inhibitors of cGMP exporters (e.g., the multidrug resistance proteins, MRP4 and 5) as well as of de novo protein synthesis indicating that their expression is increased by hypergravity. In the present study we therefore investigated the effects of hypergravity on the expression of MRP4 and 5 at mRNA levels in NO-stimulated normal human epidermal melanocytes. The NONOate DETA-NO (0.1mM) was used as a direct donor of NO. The mRNA levels were measured by real-time polymerase chain reaction with a calibrator-normalized and efficiency-corrected quantification method (LightCycler^®, Roche). Our results show that hypergravity (5xg for 24 h) induces an increase in the mRNAs for MRP4 and 5 in NO-stimulated melanocytes of about 30% over 1xg controls. In addition, hypergravity did not affect the mRNA levels for MRP8 known as another cGMP transporter. These results show that the previously reported elevation of cGMP export in NO-stimulated human melanocytes is most probably due to hypergravity-induced mRNA synthesis of MRP4 and 5. Thus, altered gravity conditions associated with an increased endogenous production of NO (e.g., during inflammation) may induce changes in the MRP expression profile which could lead to complications in the medical care in space.

The SOX (Sry-related HMG box containing) family of transcription factors contains, in mammals, at least 30 members classified into 8 groups according to their sequence similarity. They all contain a HMG-box containing DNA-binding domain, they are often widely expressed and play important roles in embryo development. Among these, the Sox4 gene, belonging to the C group, is involved in development of endocardial crests, brain, lung, teeth, gonads and lymphocytes. Recently, heterozygous KO mice for Sox4 were shown to present a decrease in bone mass and mineralization, its presence is required for expression of specific markers in osteoblast cultures and its expression increases in differentiating chondrocytes. In zebrafish, two homologs for the mammalian Sox4 are present, sox4a and sox4b. Sox4b was shown to be required for formation of glucagon cells in the zebrafish pancreas.

Here, we investigate the role of the sox4a and sox4b genes in cartilage and bone development in zebrafish. Therefore, we focus our attention on the first bone structures to be formed, the head skeleton and more precisely the pharyngeal cartilage. We show that both genes are expressed in the pharyngeal region, albeit at different time points during development. Double in situ hybridization experiments are used to exactly define the particular tissues where they are expressed. Furthermore, microinjection experiments of antisense oligonucleotides are used to block translation of these specific genes and to define their precise function during cartilage and bone development.

The functional properties of neuronal tissue critically depend on intracellular communication.
A basic principal of such communication found in various types of neurons is the generation of action potentials. These action potentials depend on the presence and the properties of voltage gated ion channels.
Earlier studies in parabolic flights and the drop tower have shown that action potentials and ion channel parameters do depend on gravity. In these experiments a reduced open state probability under ìg conditions has been proven with the technique of planar lipid bilayers.

Furthermore first experiments with the conventional patch clamp technique (2003) could deliver some data about the gravity dependence of neuronal cells. These experiments demonstrate that patch-clamping is possible even under the particular conditions in parabolic flights.
But with the conventional technique the experiments did not deliver sufficient amount of data to allow a complete interpretation of the previously found gravity dependent changes of the properties of neuronal cells and tissues. However, the data obtained within these experiments were compatible with the bilayer experiments.

In a new attempt, the gravity dependence of voltage gated ion channel parameters will be investigated in detail with an improved, partially automated, modern patch clamp technique within a DLR-funded project.
A first flight in November 2007 showed that this technique has a much better handling under the difficult parabolic flight experiment conditions. Both, the amount of data and the quality of the collected data was notably increased.
In the upcoming flights (2008-2010), the following parameters of voltage gated ion channels will be mainly determined under the given different gravity conditions:

- open state probability
- current /voltage characteristics
- voltage dependent desensitising
For an easy portability, primary cultures of different human tissue is used for the experiments.

The results of of these experiments should give the possibility of a mechanistic interpretation of the gravity dependence of neuronal cells and tissue and action potentials generated there that could have been observed in earlier experiments.

Radiation and microgravity are amongst the most important stresses in space conditions. Gravity is universally present on Earth. It determines the vertical orientation of all living organisms, thus their proper development. As a matter of fact, the presence of gravity at critical developmental moments of life is likely to determine the morphology that subsequently develops. It has a major impact on many biological functions, from muscle properties and contractile function to primary gravity transducer, in passing by neuronal signals and controls, behaviour, etc.

The purpose of this study is to decipher the effects of simulated space conditions (for microgravity and irradiation) on the morphology, physiology and gene expression of the mouse embryo and fetus. The mouse has been chosen since it is one of the major models used in genomic studies and that it provides the most applicable animal model for human pathology studies. Two developmental periods have been selected within this study: the preimplantation period (from fertilisation up to the blastula stage) and late organogenesis (with fetal fibroblastic cells harvested at day 19 post conception). To simulate microgravity, the Rotation Wall Vessel (RWV) and the desktop Random Positioning Machine (RPM) are being used. The radiation conditions on board of the International Space Station are simulated by applying similar dose rates using a mixture of gamma (Cs-137) and neutron (Cf-252) sources and compared to the effect of X-irradiation. With the microgravity and radiation facilities available at SCK

The Bacillus cereus group contains six species including the three genetically closely related Bacillus thuringiensis, Bacillus sensu stricto and Bacillus anthracis. The phenotypical features that enable to discriminate these bacteria are mainly conferred by plasmids. The understanding of the mechanisms of plasmid horizontal transfer by conjugation and mobilisation is then very important, also in extreme environments like in space. This study focuses on the kinetics of conjugation of our system and on the transfer behaviour of plasmids in simulated microgravity.

B. thuringiensis serovar israelensis strains GBJ002 (pAW63, pUB110) and GBJ001 were used as donor and recipient bacteria, respectively. The pUB110 was chosen as mobilisable plasmid, and pAW63 as conjugative. Frequencies of conjugation were obtained in rich medium, at 30°C, after 4h. The kinetics of conjugation was studied by plating bacteria every 10 minutes. Magnetic levitation, Random Positioning Machine and Rotating Wall Vessel were used as devices to simulate the microgravity.

The transfer of the conjugative was detected after 50 minutes of mating experiment, following 30 minutes later by the mobilisable plasmid. Maximum frequencies were reached after 3 hours of conjugation. Differences in transfer frequencies of the conjugative plasmid can be noticed among the different microgravity conditions. Only slightly differences were observed for the mobilisable plasmid and for both plasmids.

This study highlights the requirement of a first transfer of the conjugative plasmid to allow the migration of the mobilisable plasmid. A microgravity environment modifies the mechanisms of plasmid horizontal transfer by conjugation. Moreover this study highlights the different behaviour between a conjugative and a mobilisable plasmid.

Previous experiments in space (unmanned satellites, space shuttle or the International Space Station, ISS), have shown that adult Drosophila flies change their motile behaviour in microgravity. A consistent increase in motility was found in these experiments, but more mature flies (two weeks old) showed less increase in motility than recently hatched flies. In the case of relatively long exposures to microgravity, male fly aging, measured upon return to Earth was increased, with flies dying earlier than the corresponding in-flight 1g centrifuge or ground controls. The older flies, which experienced a smaller increase in motility, did not show this acceleration in the aging process. More recently we have started running experiments using ground simulation facilities. Preliminary experiments in the RPM indicate that the effects of this simulation approach on the behaviour of Drosophila are of smaller magnitude than the corresponding real microgravity exposure.

Further experiments are in progress to confirm this effect. However, when exposed to magnetic levitation, flies exposed to simulated weightlessness increased markedly their motile behavior compared with 1g controls inside and outside the magnet. This gravity-related increase in motility is also less pronounced in more mature flies. So far, the exposure has been only for a few days, not sufficient to have an observable impact on the lifespan of the flies, and thus, on the aging process. This motility effect at the levitation position reproduces the results in real microgravity indicating the interest for space science of this simulation approach. Similar experiments are being performed in the LDC located in ESTEC (the Netherlands) and indicate that 6g, 12g and 20g are key points in the analysis of hypergraviresponse in the flies. Our previous experiments have shown that developmental processes from embryo to adult proceeded normally in the magnet, the RPM and the LDC. In terms of gene expression, preliminary results indicate that the affected set of genes under hypergravity responds in general in an opposite direction than that induced by the real or simulated microgravity exposure. The interest in conducting comparative parallel experiments in the complete spectrum of ground simulation methods is shown in the above studies and related experiments that will be presented in the symposium.

Gravity changes are known to alter the behaviour of mammalian cells. Important functions in immune cells are severely affected; in particular a dramatic depression of the mitogenic "in vitro" activation was observed in space. The mechanism of T-cell activation is very complex. The hypothesis of experiment MIA (Motion and InterAct) flown on ISS is that a reduced interaction between T lymphocytes and monocytes might be one of the reasons for the depression of the "in vitro" activation. This interaction is a prerequisite for the co-stimulatory transduction pathway via B7/CD28 in the T cell activation process. Cell motility and a continuous rearrangement of the cytoskeletal network are essential for cell-cell contacts. Earlier investigations have shown that T lymphocytes are highly motile in microgravity. The goal of experiment MIA was to determine in space the ability of adherent monocytes to migrate (Motion), as well as to interact with T-cells (Interact). In the "Motion" experiment monocytes (J-111 cells) were incubated on a colloid gold substrate attached to a cover slide. Migrating cells remove the colloid gold, leaving a track recording cell motility. Control experiments were performed simultaneously. Postfligth, the migration tracks and the cytoskeletal structures of tubulin, F-actin and vinculin were analysed. In "Interact" the interaction of monocytes and lymphocytes was investigated by analysing the cap formation of the adhesion proteins LFA-1 on T cells and ICAM-1 on monocytes.

A normal pattern of cell migration was observed after 24h of incubation at 1xg in flight and on ground, with areas around monocytes completely cleaned out of gold particles. Conversely, in microgravity, very short migration tracks were observed and cell shape was more contracted with short protrusion reaching the neighbouring cells, indicating a severe reduction in the locomotion ability of monocytes. We also observed marked changes in the cytoskeletal network of monocytes exposed to microgravity conditions compared to 1xg controls, showing fewer filamentous biopolymers of F-actin, a reduced and incomplete arborisation of â-tubulin and vinculin appearing not evenly spread but thickened close to the cell membrane as globular clusters. Moreover, LFA-1/ICAM-1 interactions occur in space but are dependent on activation time; they show differences in number, arrangement and fluorescence intensity. Thus, LFA-1 and ICAM-1 adhesion proteins seem to be sensitive to real microgravity, without being altered their interaction. Therefore, the reason for the reduced lymphocyte activation has to be investigated on downstream molecules of signal transduction pathway. This research was supported by Italian Space Agency (ASI)

The immune system is an important regulatory mechanism affected by space flight. As plan for long-term missions continue to develop, alterations of immunity could seriously impair the ability of the host to deal with infections. In a previous work, we studied the humoral immune response induced by the immunization of Pleurodeles waltl (urodele amphibian) on Earth or during a 5 months stay on board the Mir space station (Perseus mission) and showed that the heavy chain variable domains (VH domains) of specific IgM are encoded by genes of the VHII and VHVI families. However, the VHII and VHVI families are found, respectively, in 28% and 58% of the antibodies of animals immunized on Earth and in 61% and 24% of the antibodies of animals immunized onboard Mir (Boxio et al., 2005).

We have now determined how these animals have used their individual VH genes to build their antibody binding sites in response to the antigenic stimulation. To do this, we sequenced the 5' untranslated regions and leader sequences of Ig heavy chains transcripts and aligned them because these sequences are specific markers of each VH gene. Our data indicate that animals immunized on Earth and in space used the same VHII gene to build their Ig heavy chains. The expression of this VHII gene is therefore increased by a factor 2 in space. Concerning the use of the VHVI genes, we observed that animals immunized on Earth used 4 different VHVI genes to build their Ig heavy chains while animals immunized onboard Mir used only 2 VHVI genes. These observations confirm that a spaceflight might affect the humoral immune response.

This work is supported by the Centre National d'Etudes Spatiales, the European Space Agency, the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, Nancy-University and the Région Lorraine. Keywords: antibody; VH genes; spaceflight, Pleurodeles waltl

Murine fetal thymus organ cultures used within a novel microgravity organ culture dish system (MOCDS) developed in our lab, has revealed a microgravity-induced block in normal T-cell development. This developmental impedance occurs during the double negative (DN) and double positive (DP) transition, specifically the latter part of the DN4 to DP transition. This is evident in the relatively increased accumulation of immature single positive (ISP) cells in clinorotated cultures. Functionally significant signaling molecules for this stage in thymocyte development include CD3, IL-7R (CD127), and most notably pT-α. We show that CD3 levels are decreased in the DN and ISP populations. However, anti-CD3 was able to restore early DP populations suggesting some other signaling pathway as the source of the developmental block, possibly pT-α signaling. In addition, a significant reduction in IL-7R positive ISP cells is also likely due to a problem with pT-α signaling. While we show that pT-α levels actually increase on DN cells, this may be a compensatory mechanism indicating a defect in downstream signaling. Since the pT-α signaling mechanism is poorly defined our current efforts include microarray analysis of clinorotated and recently acquired space-flown thymocytes from STS-118. Subsequent molecular analysis of significant changes in gene expression may not only lead to an explanation for this microgravity-induced block but also help to reveal unknown components of pT-α signaling.

It is generally known that gravitational unloading causes atrophy of antigravity skeletal muscles. There was, however, no evidence regarding the effects of unloading on the regenerative potential of injured skeletal muscle. Therefore, the current study was performed to investigate the influence of unloading on the regenerative process of injured skeletal muscle. Male mice (C57BL/6J), aged 8 weeks, were randomly divided into 4 groups; normal cage control (CC), cardiotoxin (CTX)-injected (CX), hindlimb suspended (HS), and HS+CX (SX) groups. HS, as the preconditioning, was performed for 2 weeks in group HS and SX. The animals in group CC and CX were maintained in 1-G environment. And then, CTX was injected into soleus muscles bilaterally in CX and SX groups. HS was continued for additional 6 weeks in group HS and SX. Soleus muscles were dissected after 2, 4, and 6 weeks.

Wet weight and protein content of soleus in group CX decreased, but recovered to the level of group CC after 6 weeks. Atrophy, caused by 2-week HS, in group HS and SX was maintained throughout the experimental period. The numbers of satellite cells in HS and SX groups after 2, 4, and 6 weeks were lower than those in group CC. The number of satellite cells in CX group was increased by the CTX-injection compared with group CC. On the other hand, satellite cell number in CTX-injected group SX after 2, 4, and 6 weeks were lower than that in group HS. Percentage of fibers with central nuclei, relative to the total muscle fibers, in HS and SX groups at week 6 was higher than that in group CC. That in group CX was also increased at 2nd and 4th week, but was lowered toward the control level after 6 week. It was suggested that loading plays a key role for the activation of the regenerating potential of injured skeletal muscle. This study was supported, in part, by Grant-in-Aid for Scientific Research (C, 17500444, KG; A, 18200042, TY; S, 19100009, YO) from Japan Society for the Promotion of Science, Ground-based Research Program for Space Utilization from Japan Space Forum (KG), and Research Grant from KAO Health Science Research (KG).

Exposure to simulated microgravity induces a disuse atrophy of postural muscles which results in a decrease in muscle volume and strength and in a compromised ability to deal with physical work capacity. Among the signaling pathways involved in skeletal muscle atrophy, oxidative stress might contribute to muscle mass regulation via proteolysis activation. Vitamin E supplementation partly counteracts unloading-induced soleus muscle atrophy via muscle proteolysis-related gene regulation. The present work was designed to study oxidative stress and soleus muscle proteolysis during early (1 and 5 days) and later (14 days) stages of the regrowth process subsequent to 14 days of hindlimb suspension. Five days of reloading were sufficient to recover soleus muscle mass.

Biomarkers of oxidative stress (glutathione vs glutathione disulfide ratio, level of thiobarbituric acid-reactive substance), antioxidant enzyme activities (superoxide dismutase, catalase) returned to control values at day 14 while the upregulation of uncoupling protein 3 transcripts was reversed within 5 days of reloading (increased demand in ATP synthesis). The increased mRNA levels of genes involved in pro-apopototic and proteolysis pathways (caspases - 3, - 9, - 12, µ - calpain, ubiquitin ligases MAFbx and MuRF1) either regained or were below baseline values within 1 day of reloading. In conclusion, muscle regrowth is facilitated during the early stages of recovery by transcriptional reprogramming within cellular pathways involved in muscle proteolysis. In the later stage of recovery, ROS generation might be due the release of substantial amount of ROS from the macrophages and other phagocytic cells, essential to allow effective regeneration.

Several reports showed that an atrophy of anti-gravity skeletal muscle in tail-suspended rat was related to the increase in intracellular calcium ion concentration. So the purpose of this study was to observe the effects of nifedipine, a blocker of L-type Ca2+ channel, on soleus muscle weight and expression of myosin heavy chain(MHC) isoforms in control and tail-suspended rats. Animals were treated with nifedipine received by water at a dose of 10 mg/kg per day for 1 or 2 weeks. The expression of MHC isoform protein was observed by SDS-polyacrylamide gel electrophoresis at 4 degree centigrade. The expression of MHC mRNA was detected by RT-PCR. The relative weight(muscle weight/Body weight) of soleus muscle was decreased by 39.5 % or 36.6 % in 1 or 2 weeks of tail-suspended group, respectively, but no changes in EDL(extensor digitorum longus) weight. The relative weight of soleus and EDL in 1 or 2 weeks of nifedipine treated control group was not changed as compared with those of control group. The relative weight of soleus in 1 or 2 weeks of nifedipine treated tail-suspension group was decreased by 51.7 % or 52.0 %, respectively, as compared with those of control, but there was no difference in relative weight of soleus between tail-suspension group with and without nifedipine. The expression of MHC I, IIa mRNA and protein could be detected in control soleus with and without nifedipine. The expression MHC I, IIa, IIx and IIb mRNA was detected in 1 or 2 weeks of unloaded soleus with and without nifedipine. Nifedipine inhibited the expression of MHC IIa, IIx and IIb mRNA and of MHC IIa preotein in soleus muscle of control and tail-suspended rats. Nifedipine can't resist the atrophy of unloaded soleus, but inhibit the transition of MHC from slow to fast isoforms at the transcription level.

Effects of gravitational loading on the growth-associated increase in the soleus muscle mass of rats. Particular attention was paid on the recruitment of satellite cells in response to loading or unloading. New-born pups with the age of 4 days were hindlimb-unloaded by tail suspension until 3 months old. Although the pups were returned to their dam for 1 hour after every 5 hours for nursing, they were isolated from their dam during suspension. Such treatment was repeated until day 21 after birth, but the suspension was continuously performed thereafter. Pups in the control group were also separated from their dam and followed the same feeding schedule. After 3 months of suspension, the suspension was terminated and ambulation recovery was allowed in the unloaded rats for 3 months. The sampling of soleus muscle was performed before and immediately, 1, 2, and 3 months after suspension.

The fiber cross-sectional area and number of total muscle fibers, myonuclei, and satellite cells were measured in whole single muscle fibers sampled from tendon-to-tendon or cross-section of the muscle. The total fiber number was ~800 at day 4. The number was increased to ~2,500 after 3 months in both control and unloaded rats, suggesting that fiber formation is not load-dependent. Increase of fiber cross-sectional area during the first 3 months was ~69% less in the unloaded than the age-matched controls. Growth-related increases of the number of quiescent and mitotic active satellite cells were inhibited by unloading. The number of myonuclei was also less in the unloaded rats. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. These results suggest that the adhesion of satellite cells to growing muscle fibers and subsequent proliferation of satellite cells and accretion of myonuclei are load-dependent, although the formation of fibers might be genetically programmed.

The main objective of the study was to evaluate glycogen and triglyceride content in the fast- and slow-twitch fibers of m. soleus and m. tibialis anterior under conditions of simulated microgravity. In experiment performed on Wistar rats muscle unloading was simulated by means of hindlimb suspension for 3 and 14 days. Muscle samples were taken from left m. soleus or m. tibialis anterior. The corresponding muscle sections were double stained for different types of myosin heavy chains (MHC-I and MHC-II) and triglycerides (Oil Red O) or glycogen. The quantitative evaluation of the energy substrate content was done by means of the computerized fluorescence microscope with different filters. Rat hindlimb suspension led to increase of triglyceride content in the fast-twitch fibers of m. tibialis anterior and in both type fibers of m. soleus after 3 days. The same tendency was revealed in the fast-twitch fibers of m. tibialis anterior after 14 days of unloading. Glycogen content decreased in the fast- and slow-twitch fibers of m. soleus and in fast-twitch fibers of m. tibialis anterior after 3 days of unloading, bat returned to the control level on 14th day of hindlimb suspension. So we for the first time analyzed glycogen and triglyceride content in the fast- and slow-twitch fibers of rat m. soleus and m. tibialis anterior on the different durations of unloading.

Structure alterations of postural muscles of animals, subjected to weightlessness, have been studied for several years. In the scope of space project Foton M-3 the structure characteristics of m. soleus of Mongolian Gerbils (Meriones unguiculatus) after 12-days spaceflight were measured. These data were compared to those, obtained in previous studies on space flown rats.

The cross-section area of slow- and fast twitch fibers in space-flown gerbils were reduced 29,7% and 28,8%, respectively, being much lower, than in rat soleus after 14-days spaceflight aboard of SLS-2 (50% reduction of CSA). [Ilyina-Kakueva et al., 1995] In contrast to rats, Mongolian Gerbils did not demonstrate significant slow-to-fast transition of myosin heavy chain fiber.

Increased percentage of total collagen amount by 31,1% was observed on the 2 day after spaceflight, without significant changes in I and III isoform ratio. Data, obtained on rat soleus after 7-days on board NASA SL-3 also displayed significant increase of total collagen amount (estimated by the level of hydroxyproline). [Taylor W. E. et al, 2002]

After 12 days spaceflight the amount of satellite cells, expressing m-cadherin in m. soleus of Mongolian Gerbils decreased by 59% as compared to the synchronous control group. It is interesting that the amount of m-cadherin expressing satellite-cells decreased by 33% in hindlimb suspended rats. [Tarakina et al., 2008] We observed no changes in myonuclei number after the spaceflight. 14-days gravitational unloading of rats was accompanied by the decrease in myonuclei number by 23 %. [Tarakina et al., 2008]

Thus, the trend of changes in Gerbils soleus structure after the spaceflight generally corresponds to the changes in rat soleus muscle after spaceflight of similar duration. However, the changes in myonuclei number, extracellular matrix and myosin heavy chain composition were lacked or much less marked than in rat soleus muscle.

This work was supported by Russian Federal Space Agency. Authors express acknowledgements to the team, involved in preparation and realization of space mission Foton-M3.

Changes in muscle fibres under the conditions of real or simulated microgravity are associated not only with in myofiber protein depletion. Some authors have shown that loss of postural muscle wet weight during gravitational unloading accounts to water loss to great extent. To reveal the influence of systems dehydration process which, accompanie the gravitational unloading, on muscle water loss a new experimental approach was used. The hormonal antidiuretic drug - desmopressin was administered in hundlimb suspended rats. In desmopressin administered group soleus muscle water loss was partially prevented (26,2 % in comparison with 44,9 % in hindlimb suspension group). As a result of desmopressin application soleus muscle dry weight decreased by 20,3 % as compared with 53 % decrease in hindlimb suspension group. We observed no significant difference in myofiber cross-sectional area in desmopressin administered group as compared to the control group. It was shown earlier the possibility to maintain postural muscle mass in unloading conditions by concomitant passive stretching. We show that passive stretching prevents atrophic changes and keeps unchanged soleus muscle hydration (no differences from control group were find). During hindlimb suspension this effect is more expressed in group only with stretching than with only demopressin administration application. The simultaneous application of system is impact (desmopressin) and passive stretching also preserved muscle fibers hydration and prevented atrophy, but didn’t differed from the preventive effect of passive stretching alone. Research is supported by grant of the Russian Foundation for Bassic Research 06-04-49264-à.

We have shown initial exposure to hindlimb unloading (HLU), used as a model of disuse, causes a moderate reduction (5-15%) in body mass and food consumption during the acute exposure period. These changes are caused by an initial reduction in activity and food consumption, thus shifting energy balance. In addition, hormonal changes have been observed to be related to the changes in body mass and food consumption. After the first week, HLU rats begin to gain body mass at a similar rate with an increase in food intake as compared to controls. To compare metabolic changes over time, we exposed groups of mature rats to HLU for 15d, 30d, 60d and 90d.

Body mass and food consumption data were collected throughout the study and urine samples of each group were collected during the final 10 days of each stage and samples analyzed for catecholamines (CATS; epinephrine (E), norepinephrine (NE), dopamine (D)) were pooled together. Tissue and blood samples were collected and stored at -80C. Initial HLU exposure resulted in an acute reduction in body mass gain with an increase of body mass in HLU groups beginning at around day 8 and which continued for 90d. Absolute food consumption was reduced after initial exposure as compared to controls, but showed similar absolute consumption rates by day 15. When corrected for body mass, HLU animals had significantly greater food consumption at each time point (p<0.001). Plasma leptin, as well as epididymal fat showed a significant decrease in HLU at the end of each time point (p<0.001). Plasma insulin and glucose were significantly increased at all time points, indicating insulin resistance. These data, along with the increase in corrected food consumption, indicates that the animals to be in a hypermetabolic state. Urinary CATS (E,D) were significantly reduced over time in HLU (p<0.02). NE showed no significant change. HLU results in a chronic hypermetabolic state in which increased sympathetic activity is not a factor. (NASA grant 121-10-30/40/50 & NNJ05H037G; Juvenile Diabetes Foundation).

Background: There is little knowledge about effects of spaceflight, especially long-term spaceflight on body¡¯s antioxidant defense system. And antioxidant defense system plays an important role of protecting body against free radical attack. There are some reasons for suspecting that antioxidant defense system may be damaged with spaceflight.

Purpose: The purpose of the present study was to investigate effects of weightlessness included in long-term spaceflight on antioxidant defense system and briefly to evaluate its potent consequences. Methods: Our experiment was designed on basis of summarizing and analyzing a few literatures in this field. Tail-suspension for three months was used to simulate long-term weightlessness. Thirty-two male Sprague-Dawley rats were randomly divided into control, 30 d suspension, 1 d recovery and 7 d recovery group and each group included 8 rats. Lipid peroxidation, contents of antioxidants and activities of antioxidant enzymes in rat liver were measured. Abundances of mRNA for some antioxidant enzymes were quantificationally determined by Northern blot.

Results: Compared with control group, liver malondialdehyde (MDA) and hydroperoxide (LOOH) contents showed an increasing trend (P<0.10) or significantly increased (P<0.05), while glutathione (GSH) and total GSH concentration dramatically decreased (P<0.05 or 0.01) in suspension group and 1 d recovery group. Activities of glutathione peroxidase (GSH-Px) and catalase showed a downtrend (P<0.10) or significantly decreased (P<0.05). The relative abundance of mRNA for Cu-Zn superoxide dismutase (Cu-Zn SOD) and GSH-Px was markedly decreased (P<0.05). These changes induced by suspension did not completely come back to control level in 7 d recovery group. In addition, the changing magnitude of above indexes was largest for 1 d recovery group among experimental groups.

Conclusion: Long time weightlessness may injure antioxidant defense system and caused oxidative stress and damage. These effects are more significant when rats move from weightlessness to gravity environment. It is suggested that potent harm to astronauts¡¯ health induced by wakened antioxidant defense system should be considered in long-term spaceflight, especially in future trip to Mars. At last, author think it is of importance to deepen concerned research and bring forward attempt directions.

The aim of this study was to compare the changes in soleus wet weight, fiber cross-sectional area (CSA) and percentage of MHC(myosin heavy chain) I/II fibers between the tail-suspended and aged rats, then to get some evidence for accelerative aging in unloaded soleus. The CSA and percentage of MHC I/II fibers were measured on the immunohistochemical staining sections and CSA was normalized by body weight. The wet weight and relative wet weight of unloaded soleus decreased significantly by 23.89 %(P<0.01) and 21.48 %(P<0.05), respectively at the fifth day of tail-suspension, as compared with their synchronous control. The CSA in 5-day of unloaded soleus decreased by 14.1 % in MHC I (P<0.01) and 13.4 % in MHC II fibers(P<0.01). The normalized CSA in 5-day of unloaded soleus also decreased by 19.4% in MHC I (P<0.01) and 12.1 % in MHC II fibers(P<0.01). The above parameters decreased further at the seventh and fourteenth day of tail-suspension.

The wet weight and CSA of soleus in 30-month-old rats increased, but the relative wet weight and normalized CSA reduced significantly as compared with 3-month-old control group. The relative wet weight and normalized CSA of soleus in 30-month-old rats were decreased to a similar extent with that of 14-day tail-suspended group. The percentage of MHC I/II fibers in 5-, 7-, and 14-day synchronous control soleus kept a constant value: MHC I 82.4%-88.3%, MHC II 11.7%-17.6%. The percentage of MHC I fibers of 5-, 7-, and 14-day of unloaded soleus decreased by 24.4%, 14.7%, and 26.4%, respectively. The above results suggest that the atrophic process of soleus is slower in the aged rat than that in the tail-suspended rat. The reduction of relative wet weight and normalized CSA of soleus in aged rats is earlier than those of absolute wet weight and CSA, but the absolute and relative wet weight of soleus decrease simultaneously in tail-suspended rats. So the atrophic soleus only shows partially senescent characteristics in unloaded rat.

The main objective of the study was to develop and apply the experimental approach to estimate the functional activity of motoneurons innervating distinct muscle under conditions of rat hindlimb suspension. For this purpose we performed a retrograde labeling of tibialis anterior and soleus motoneurons by using a neuronal tracer carbocyanine (Molecular Probes, D7756) and then evaluated c-fos protein expression, which is a marker of neuron functional activity. Muscle unloading was simulated by means of standard procedure of rat hindlimb suspension for 3 days. Spinal cord sections were stained with monoclonal antibodies against c-fos protein.

We counted the percentage of labeled motoneurons containing c-fos proteins and measured soma size of all labeled motoneurons. It was shown that percentage of tibialis anterior motoneurons containing c-fos proteins significantly increased and mean soma size of all labeled motoneurons decreased after 3 days of hindlimb suspension. At the same time percentage of soleus motoneurons containing c-fos proteins and mean soma size of all labeled motoneurons after 3 days of unloading did not differ from control group. The obtained data correspond to increased EMG activity of m. tibialis anterior under conditions of hindlimb suspension. To conclude whether the c-fos proteins content and motoneuron soma size may be used as a marker of decreased functional activity it is necessary to complete this investigation with evaluation of these parameters on the other durations of unloading. We gratefully acknowledge Tavitova M.G. for participation. The study was supported by grant from Russian Foundation of basic research 07-04-01608.

It is known that hindlimb unloading produces atrophy and suppresses proliferative processes in postural muscles. Muscle stretch applied concomitantly with hindlimb suspension is known to prevent soleus muscle atrophy. We established drastic increase in BrdU incorporation along with attenuation of atrophy in both fiber types during gravitational unloading with concomitant passive stretch (fivefold as compared to hindlimb suspension group).

In rat experiment we assessed the number of m-cadherin (satellite cell marker molecule)-labelled cells per one myofiber cross-section (in control group it is about 0.3). After 2-week hindlimb suspension the number of m-cadherin expressing cells decreases 33% as compared to control group. The amount of m-cadherin-labelled cells was 2.5 times more in passive stretch and hindlimb suspension group in comparison with hindlimb suspended animals and 1.7 times more as compared to control group.

We suppose that that proliferation of satellite cells with subsequent incorporation of their nuclei in myofiber is sufficient to increase protein synthesis in unloaded soleus. Different factors play a crucial role in satellite cell activation or inhibition. The insulin-like growth factor (IGF)-I is implicated in regulation of protein turnover and exerts potent mitogenic and differentiating effects on most cell types. Thus passive stretch preventive action might be realized by IGF-1-dependent signaling pathway. We investigate the level of IGF-1 expression in soleus muscle tissue after 14 day hindlimb suspension with stretch and observed no changes as compared to control or 14 day hindlimb suspended group.

To assess the role of SC in development of passive stretch preventive effect, SC proliferation was suppressed by local exposing to ã ionized radiation (2500 Rad), subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myonuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We conclude that muscle IGF-1 and satellite cells incorporation don't make essential contribution to passive stretch preventive action under the conditions of simulated weightlessness. The question remains undecided about the importance of satellite cell proliferation in unloaded and concomitantly stretched soleus muscle.

Atrophy and shift of phenotype toward fast-twitch types are induced adductor longus (AL) muscle fibers in response to gravitational unloading by actual spaceflight and/or simulation model such as hindlimb suspension with unknown mechanism. Therefore, the responses of electromyogram (EMG) activities of the rostral and caudal side of rat AL muscle to altered gravity level during parabolic flight of a jet airplane were investigated using 10-wk old male Wistar Hannover rats. Bipolar constantan-wire electrodes were implanted into the caudal and rostral regions of the left AL muscle in anesthetized rat. The section of the wire, with the insulation removed (~2 mm), was implanted into the mid-belly of the muscle. The experiment was performed in conscious rats after, at least, 2-days recovery from the surgery and anesthesia. The EMGs were recorded using a wireless telemetry system. The EMG levels at each G level were analyzed by comparing with the joint angles of hindlimbs monitored by video recording simultaneously. Further, AL muscle was fixed at various joint angles by using formaldehyde and the responses of fiber length at rostral and caudal side were analyzed.

Tonic EMGs in both regions were noted most of the times, when the rats were exposed to hyper-G, as well as 1-G. The hip joints were adducted and the sedental quadrupedal position was maintained at these G levels. However, the EMG activities in these regions became silent and/or phasic activity was observed sometimes, when the hip joints were abducted and extended backward in microgravity environment. Such changes of joint angles caused passive shortening of sarcomeres in both regions of AL. The data may suggest that the atrophy and shift of fiber phenotype caused by gravitational unloading may be related to the decreased muscle activity. Fiber-type transformation toward fast-twitch type may be also related to the change of muscle activity from tonic to phasic patterns, which are the typical characteristics of fast-twitch muscle.

Effects of mechanical overloading on the characteristics of soleus muscle fibers were studied. Overloading was applied for 14 days to the left soleus muscle in mdx and wild type mice by removing the tendons of plantaris and gastrocnemius muscles, keeping the neural-vascular supplies in soleus intact. Contralateral muscle served as the normal control. These animals were then allowed ambulation recovery in the cage. Following the samplings after 14 days of treatments, soleus muscle was treated with collagenase and entire single muscle fibers were isolated from tendon-to-tendon using fine needles. The muscle satellite cells, neuromuscular junctions (NMJ), and myonuclei were stained. Most of the properties, such as number of myonuclei and satellite cells, size of NMJ, and fiber length, were not influenced by mechanical overloading in both mdx and wild type mice. Approximately 0.6% branched fibers were seen in the right soleus of mdx mice, although these fibers were not detected in wild type mice. However, the percentage of these fibers was increased by overloading especially in mdx mice (~50% vs. ~2.5% in wild type). The fiber cross sectional area in both mdx and wild type mice was insignificantly increased by overloading. It was suggested that application of mechanical overloading causes similar compensatory adaptation in soleus muscle fibers of both types of mice. However, marked fiber splitting was induced in the muscle fibers of mdx mice.

Introduction. Stay in conditions of real and modeled weightlessness is accompanied by decrease of force-velocity properties of skeletal muscles. The most pronounce changes are revealed in extensors, in particular in the m.triceps surae. Recently it has been shown that low-frequency electromiostimulation have training effects when it used in rehabilitation and sports medicine. However the effect of low-frequency electromiostimulation in microgravity condition is not examined. The aim of investigations was estimation of effects of low-frequency electromiostimulation upon force-velocity properties of m.triceps surae during 6 days dry immersion.

Methods. 12 volunteers took part in experiment. As a model of microgravity the dry immersion (6 day) was used. 6 volunteers (experimental group - E-group) undergone of low-frequency electromiostimulation during 3 hours per day. Other 6 volunteers served as a control group (C-group). Before and after 6-day dry immersion the force-velocity properties were evaluated using Biodex system. Testing of m.triceps was carried out in isokinetic regime at angular velocities of 150,90,30, 0 deg/s.

Results. One of participant of E-group demonstrated great significant increase of muscle torque in all range of tested angular velocity (20-40%). In other hand in isometric regime the changes was not found. 4 participants of E-group demonstrated not significant decrease of torque (2-5 %) at angular velocities of 150, 30, 0 deg/s. At the same time on angular velocity of 90 deg/s the decrease was significant (11 %). At one of participants the most pronounced decrease (30-40%) of dynamics torque was found except isometric regime in which the decrease was noticeably smaller (6 %). In C-group the significant decrease (approximately 20%) of force-velocity characteristics in whole range of angular velocity was found. Thus, except for one person the low-frequency electromiostimulation of m.triceps surae prevent negative effects of microgravity.

Exposure of humans to microgravity condition resulted in body fluid shift and myatrophy. To examine the effectiveness of artificial gravity and ergometric exercise as countermeasure to these changes, 12 healthy young men were exposed to stimulated microgravity for 20 days by head-down bed rest (HDBR). 6 subjects randomly selected were subjected to 1.4G of artificial gravity with 60W of ergometric workload everyday for 30 minutes (CM group). The rest of the subjects served as the control (control group). Head-up tilt test and Anti-G test were used to measure the effects of countermeasure. In anti-G test, anti-G score was significantly decreased by HDBR, and this improved in CM group. Body fluids of upper body were shifted to downward by HDBR, and the changes were prevented in CM group. In head-up tilt test, body fluid shift of upper body were the same responses as in anti-G test, and were also prevented by countermeasure. On the contrary, body fluid shift of lower body did not show markedly changes both control group and CM group. The myatrophy measured by MRI in the thigh was smaller in CM group than in control group. These suggest that artificial gravity with exercise appeared to be effective in preventing changes in body fluid shift of upper body and myatrophy of thigh due to microgravity exposure.

A 3D-clinostat is a multi-directional gravity device, by controlled rotation of two axes, and makes 10^-3 G average over time. We previously reported that microgravity inhibited stem cell and bone marrow-derived cell differentiation. In this study, the effects of microgravity on myoblast differentiation were examined.

L6 rat myoblast cells were seeded in OptiCell and propagated in normal 1G environment. When L6 cells reached confluence on culture day 4, they were induced differentiation and then exposed to 10^-3G environment (group CL). Control cells were cultured in normal 1G environment (group 1G). After 5 days induced differentiation, cells were divided into two more groups and cultured for another 5 days. Subject was continued to culture in same environment (group CL/CL or group 1G/1G), the other subject was exchanged culture environment of the cells (group CL/1G or group 1G/CL), thus cells in group CL were transferred to 1G, and cells in group C were transferred to 10^-3G.

It was shown that microgravity inhibited myoblast differentiation by morphological observation and molecular biological analysis. The cells in group CL/1G began differentiation when cells in normal 1G environment, while the cells in group 1G/CL supressed differentiation, when cells in 10^-3G environment. In group 1G/CL, myotubes seemed to turn back to myoblasts. Moreover, these gravitational environmental changes became activated or inactivated p38^MAPK cascade to control myoblast differentiation. Here we show that gravity truly influence cultured myoblast differentiation.

Across phyla, spaceflight induces changes in muscle, including decreased mass and strength. C. elegans returning from 10 days in space onboard the Dutch DELTA mission displayed uncoordinated movement and decreased expression for roughly 100 "muscle" genes. The expression of many of these genes is likewise decreased in mammals returning from space. However, in flight, C. elegansdisplayed normal movement over ten generations of growth during Expeditions 14 and 15. To determine the possible significance of decreased expression of transcripts encoding proteins that make up a muscle attachment complex we conducted a series of experiments on the ground.

We find that acute treatment of adult C. eleganswith RNAi against one of these genes, unc-97 (PINCH/LIM-domain), causes degradation of a reporter protein in muscle cytosol. Acute RNAi treatment against any of another eight genes, whose products are likewise members of an integrin-containing muscle adhesion complex, also causes degradation. Suggesting specificity in the regulation of degradation, we find that RNAi against the gene for another complex member, unc-95 (LIM-domain), fails to cause muscle protein degradation. Experiments using temperature-sensitive mutations in two of these genes, either unc-112 (MIG-2) or unc-52 (Perlican), confirm that disruption of this complex causes degradation, and further show that the extramuscular ligand (UNC-52/Perlican) is required to prevent degradation. In these mutants movement becomes uncoordinated and muscle structure is disrupted following temperature shift. In C. elegans, AChR-Ca++ and IGFR-FGFR signalling networks control muscle protein degradation via proteasome and non-proteasome dependent mechanisms, respectively. Drugs, mutations, and RNAi treatments targeted at these networks, and known to block degradation in C. elegans muscle, fail to block degradation triggered by acute loss of members of the muscle attachment complex. Current goals include determining the identity and regulation of the relevant protease(s).

The Na,K-ATPase is critically important for excitability, electrogenesis and contractility of skeletal muscle expressing α1 and α2 isoforms of the enzyme. It is well known that disuse induced by hind limb unloading (HU) leads to progressive atrophy of skeletal muscle and a number of dramatic remodeling events. Decrease of resting membrane potential (RMP) and membrane resistance, changes of ionic permeability during 7-28 days of HU was reported. In the present study, the effects of 3 days of HU on electrogenic contribution of the Na,K-ATPase isoforms, excitability and contractility of isolated rat soleus muscle were studied. The RMP was recorded using standard microelectrode techniques.

The electrogenic contribution of high ouabain-sensitive α2 isoform was tested as depolarization due to administration of 1 µM ouabain (a concentration selectively blocking α2 isoform in rodents). The contribution of low ouabain-sensitive α1 isoform was estimated as additional depolarization after 500 µM ouabain administration. Isometric twitch tension, excitability and dynamics of fatigue of the whole muscle were recorded. To estimate intracellular Ca²⁺ content, we measured fluorescence intensity of Fluo-4-AM in isolated single muscle fibers. After HU the RMP was depolarized from -71.0±0.5 mV in control to -66.8±0.7 mV (p<0.01). In control, electrogenic contributions of α2 and α1 isoforms were 6.2±0.6 mV and 4.6±0.6 mV, respectively. The total Na,K-ATPase contribution was 10.8±0.6 mV. After HU, contribution of α2 isoform was absent (0.5±0.8 mV); the contribution of α1 isoform was decreased (p<0.01) to 2.6±0.6 mV. The total Na,K-ATPase contribution was 3.1±0.9 mV i.e. 3 times (p<0.01) less than in control. The HU-induced depolarization was accompanied by decrease in excitability whereas the maximal contraction force was increased. A trend to fatigue acceleration was observed. Fluorescence intensity of single muscle fibers loaded with Fluo-4-AM increased more than 4 times indicating an increase in intracellular resting Ca²⁺ concentration. It is suggested that 3-day HU brings about to significant decrease of RMP, which in turn might activate the dihydropyridine channels resulting in intracellular Ca²⁺ accumulation. Supported by RFBR #07-04-01027; #07-04-00763 and #08-04-00923.