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

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

Effect of dry immersion in combination with stimulation of foot support zones upon muscle force-velocity characteristics

The complex of motor disturbances arising under conditions of real and simulated microgravity that include decrease of contractile characteristics of postural muscles is likely to be a result of withdrawal of support stimuli. Artificial stimulation of support zones of feet is shown to diminish partially or prevent completely the negative effects of microgravity on the motor system. The aim of the study was to evaluate and compare the changes of contractile properties of extensors and flexors of knee joint measured in isokinetic and isotonic regimes under conditions of simulated microgravity (7 days dry immersion).     

Human soleus fibers contractile characteristics and sarcomeric cytoskeletal proteins after gravitational unloading. Contribution of support stimulus

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.     

Biomechanics and regulation of external respiration under conditions of five-day dry immersion

The functional state of external respiration and the features of its regulation in healthy persons were studied under conditions of microgravity simulated using dry immersion. The lung volume, the ratio of thoracic and abdominal components during quiet breathing and performing various respiratory maneuvers, as well as the parameters that characterize the regulation of breathing (the duration of breath holding and the ability to voluntarily control respiratory movements), were recorded during the baseline period, on days 2 and 4 of dry immersion, and after the end of the dry immersion. It has been shown that the breathing pattern did not significantly change under conditions of dry immersion compared to the baseline period; however, the inspiratory reserve volume increased (p < 0.05), while the expiratory reserve volume decreased (p < 0.01). Dry immersion did not alter pulmonary ventilation, yet most of the subjects trended toward an increase in the contribution of the abdominal component of breathing movements during quiet breathing and demonstrated a statistically significant increase in this parameter during the lung vital capacity maneuver. The durations of the inspiratory and expiratory maximal breath holding under conditions of immersion did not differ from the background values. During the immersion, the accuracy of voluntary control of breathing increased. We believe that immersion, similar to microgravity, leads to changes in the reserve lung volume, which are partly because of changes in the body position; changes in relative contributions of the thoracic and abdominal components in the breathing movements; and changes in voluntary breath regulation.     

Craniomandibular System and Postural Balance after 3-Day Dry Immersion

The objective of the study was to determine the influence of simulated microgravity by exposure to dry immersion on the craniomandibular system. Twelve healthy male volunteers participated in a 3-day dry immersion study. Before and immediately after exposure we measured maximal bite force using piezoresistive sensors. The mechanical properties of the jaw and cervical muscles were evaluated before, during, and after dry immersion using MyotonPRO. Because recent studies reported the effects of jaw motor activity on the postural stability of humans, stabilometric measurements of center of pressure were performed before and after dry immersion in two mandibular positions: rest position without jaw clenching, and intercuspidal position during voluntary teeth clenching. Results revealed no significant changes of maximal bite force after dry immersion. All postural parameters were significantly altered by dry immersion. There were however no significant differences in stabilometric data according to mandibular position. Moreover the masseter tonicity increased immediately after the end of dry immersion period. Dry immersion could be used as a valid model for studying the effects of microgravity on human subjects. However, 3 days appear insufficient in duration to evaluate the effects of weightlessness on maximal bite force. Our research suggests a link between postural disturbance after dry immersion and masseter tonicity.     

Muscle transverse stiffness and venous compliance under conditions of simulated supportlessness

Supportlessness arising as a result of 7 hrs dry immersion is accompanied by decreased transverse stiffness and increased calf venous compliance, as well as impaired orthostatic tolerance. Stimulation of support zones of foot is associated with smoothening of these effects. The latter may be considered as an indirect evidence in favor of the concept of support deafferentation triggering role in development of enumerated unfavorable effects of simulated microgravity.     

Muscle atrophy and hormonal regulation in women in 120 day bed rest

It is known, that exposures to real and simulated weightlessness results in pronounced reduction of the cross-sectional area (CSA) of slow-twitch(ST) and fast-twitch(FT) fibers of mammalian muscle. After space flights of various durations, head-down tilt bedrest, and 7-days of dry immersion sufficient [correction of isufficient] reductions of CSA of both fiber types were observed in man and in the majority of these cases the atrophy levels of ST and FT fibers were similar. It is well-known, that elevated contractile activities of muscle system attenuate muscle atrophy development. It remains still unclear which fiber type is more susceptible to training effects. Among physiological mechanisms involved in the process of microgravity-induced atrophy development which are supposed to be the most important are the profound decrease of a mechanical tension of muscle fibers in situ and alterations in hormonal control of muscle protein metabolism. But it is not known yet if the hormonal changes in the course of exposure to gravitational unloading match somehow the time-course of muscle fiber size reduction. The aim of the study was to investigate the time-course of muscle fiber atrophy development and changes in plasma hormone levels in the course of long-duration BR with and without high-intensity locomotor interval physical training.     

Afferent and peripheral control of muscle fiber properties during gravitational unloading

The present paper covers two series of the experiment studies performed in attempt to analyze the support-triggered cellular mechanisms, controlling the maintenance of tonic muscle fiber characteristics. Exposure to 7 day dry immersion induced significant decline of the human soleus single fiber peak isometric tension and the Ca(2+)-sensitivity of myofibrils. 30-40% losses of the relative content of titin and nebulin were found after immersion. The application of the plantar support stimulation device prevented all these alterations. In the second experimental series the treatment of hindlimb suspended rats with the Ca(2+)-binding agent (EGTA) allowed to prevent or attenuate all the above mentioned unloading-induced soleus fiber alterations. Thus it is concluded that resting Ca2+ accumulation in the unloaded fibers may be among the mechanisms involved in the changes of fiber properties during unloading.     

Effects of gravitational unloading on back muscles tone

The study involved 12 healthy volunteers who were exposed to 6-h or 3-day dry immersion (DI). The back muscle tone was recorded by resonance vibrography using parameters of transverse stiffness of the muscles under study. The measurements in 3-day DI were performed twice before DI, daily in the course of DI, and twice after its completion; in the short-term (6-h) DI, the testing was carried out twice before DI, 1 and 4 h after the beginning of DI, and during the first hour after DI completion. It has been shown that the absence of support is followed by a sharp decrease in the back extensor muscles tone, which has the maximal values during the first hours and days of DI. The possible role of back muscle atony in the development of well-known phenomena of the spine length increase and back pain appearance observed at the beginning of space flights and in the first days after landing, as well as under the conditions of simulated microgravity (DI and head-down bed rest), is discussed.     

Contribution of social isolation, restraint, and hindlimb unloading to changes in hemodynamic parameters and motion activity in rats

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient.     

Effects of artificial support stimulation on fiber and molecular characteristics of soleus muscle in men exposed to 7-day dry immersion

Artificial support stimulation is known to attenuate or prevent many motor or skeletal muscle effects of actual or simulated microgravity. The present study was purposed to analyze the effects of artificial support on human soleus fibers after 7-day exposure to supportless environment. 8 healthy male volunteers were exposed to dry immersion in supine position for 7 days according to Shulzhenko and Vil-Villiams (1972). 4 of them worn the support device which provided them with plantar stimulation in regime described elsewhere.     

Relative contribution of articular cartilage’s constitutive components to load support depending on strain rate

Cartilage is considered a biphasic material in which the solid is composed of proteoglycans and collagen. In biphasic tissue, the hydraulic pressure is believed to bear most of the load under higher strain rates and its dissipation due to fluid flow determines creep and relaxation behavior. In equilibrium, hydraulic pressure is zero and load bearing is transferred to the solid matrix. The viscoelasticity of the collagen network also contributes to its time-dependent behavior, and the osmotic pressure to load bearing in equilibrium. The aim of the present study was to determine the relative contributions of hydraulic pressure, viscoelastic collagen stress, solid matrix stiffness and osmotic pressure to load carriage in cartilage under transient and equilibrium conditions. Unconfined compression experiments were simulated using a fibril-reinforced poroviscoelastic model of articular cartilage, including water, fibrillar viscoelastic collagen and non-fibrillar charged glycosaminoglycans. The relative contributions of hydraulic and osmotic pressures and stresses in the fibrillar and non-fibrillar network were evaluated in the superficial, middle and deep zone of cartilage under five different strain rates and after relaxation. Initially upon loading, the hydraulic pressure carried most of the load in all three zones. The osmotic swelling pressure carried most of the equilibrium load. In the surface zone, where the fibers were loaded in tension, the collagen network carried 20 % of the load for all strain rates. The importance of these fibers was illustrated by artificially modifying the fiber architecture, which reduced the overall stiffness of cartilage in all conditions. In conclusion, although hydraulic pressure dominates the transient behavior during cartilage loading, due to its viscoelastic nature the superficial zone collagen fibers carry a substantial part of the load under transient conditions. This becomes increasingly important with higher strain rates. The interesting and striking new insight from this study suggests that under equilibrium conditions, the swelling pressure generated by the combination of proteoglycans and collagen reinforcement accounts cartilage stiffness for more than 90 % of the loads carried by articular cartilage. This finding is different from the common thought that load is transferred from fluid to solid and is carried by the aggregate modulus of the solid. Rather, it is transformed from hydraulic to osmotic swelling pressure. These results show the importance of considering both (viscoelastic) collagen fibers as well as swelling pressure in studies of the (transient) mechanical behavior of cartilage.     

Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts

Prolonged spaceflight gives rise to muscle loss and reduced strength, a condition commonly referred to as space atrophy. During exposure to microgravity, skeletal muscle myoblasts are mechanically unloaded and respond with attenuated cell proliferation, slowed cell cycle progression, and modified protein expression. To elucidate the underlying mechanisms by which muscle mass declines in response to prolonged microgravity exposure, we grew C2C12 mouse muscle cells under conditions of simulated microgravity (SM) and analyzed their proliferative capacity, cell cycle progression, and cyclin B and D expression. We demonstrated that the retarded cell growth observed in SM was correlated with an approximate 16 h delay in G₂/M phase progression, where cells accumulated specifically between the G₂ checkpoint and the onset of anaphase, concomitantly with a positive expression for cyclin B. The effect was specific for gravitational mechanical unloading as cells grown under conditions of hypergravity (HG, 4 g) for similar durations of time exhibited normal proliferation and normal cell cycle progression. Our results show that SM and HG exert phenomenological distinct responses over cell cycle progression. The deficits of SM can be restored by terrestrial gravitational force, whereas the effects of HG are indistinguishable from the 1 g control. This suggests that the mechanotransduction apparatus of cells responds differently to mechanical unloading and loading.     

Cardiovascular responses to apneic facial immersion during altered cardiac filling.

The hypothesis that reduced cardiac filling, as a result of lower body negative pressure (LBNP) and postexercise hypotension (PEH), would attenuate the reflex changes to heart rate (HR), skin blood flow (SkBF), and mean arterial pressure (MAP) normally induced by facial immersion was tested. The purpose of this study was to investigate the cardiovascular control mechanisms associated with apneic facial immersion during different cardiovascular challenges. Six subjects randomly performed 30-s apneic facial immersions in 6.0 +/- 1.2 degrees C water under the following conditions: 1) -20 mmHg LBNP, 2) +40 mmHg lower body positive pressure (LBPP), 3) during a period of PEH, and 4) normal resting (control). Measurements included SkBF at one acral (distal phalanx of the thumb) and one nonacral region of skin (ventral forearm), HR, and MAP. Facial immersion reduced HR and SkBF at both sites and increased MAP under all conditions (P < 0.05). Reduced cardiac filling during LBNP and PEH significantly attenuated the absolute HR nadir observed during the control immersion (P < 0.05). The LBPP condition did not result in a lower HR nadir than control but did result in a nadir significantly lower than that of the LBNP and PEH conditions (P < 0.05). No differences were observed in either SkBF or MAP between conditions; however, the magnitude of SkBF reduction was greater at the acral site than at the nonacral site for all conditions (P < 0.05). These results suggest that the cardiac parasympathetic response during facial immersion can be attenuated when cardiac filling is compromised.     

Simulated microgravity reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts

Calcium homeostasis in osteoblasts plays fundamental roles in the physiology and pathology of bone tissue. Various types of mechanical stimuli promote osteogenesis and increase bone formation elicit increases in intracellular-free calcium concentration in osteoblasts. However, whether microgravity, a condition of mechanical unloading, exerts an influence on intracellular-free calcium concentration in osteoblasts or what mechanisms may underlie such an effect are unclear. Herein, we show that simulated microgravity reduces intracellular-free calcium concentration in primary mouse osteoblasts. In addition, simulated microgravity substantially suppresses the activities of L-type voltage-sensitive calcium channels, which selectively allow calcium to cross the plasma membrane from the extracellular space. Moreover, the functional expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, which mediate the release of calcium from intracellular storage, decreased under simulated microgravity conditions. These results suggest that simulated microgravity substantially reduces intracellular-free calcium concentration through inhibition of calcium channels in primary mouse osteoblasts. Our study may provide a novel mechanism for microgravity-induced detrimental effects in osteoblasts, offering a new avenue to further investigate bone loss induced by mechanical unloading.     

Muscle progenitor cell proliferation during passive stretch of unweighted soleus in dystrophin deficient mice

Dystrophin, subsarcolemmal protein communicating muscle fiber cytoskeleton to extracellular matrix, is believed to be involved in mechanical signal transduction. The experiment was carried out to assess the role of dystrophin in passive stretch-induced preventing unloaded muscle fiber atrophy and possible linkage between this protein and muscle progenitor (satellite cells) proliferation activity. 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. Altered dystrophin synthesis in mdx mice had virtually no effect on passive stretch preventive action. Thus the hypothesis about dystrophin key role in mediating stretch-induced hypertrophy effects didn't find its confirmation concerning gravitational unloading atrophy. Chronic hindlimb unloading downregulated SC proliferative activity in soleus muscle, passive stretch drastically increased proliferation both in C57 and mdx mice. Thus 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.     

The effects of simulated microgravity on characteristics of slow presaccadic potentials

The parameters of saccades and presaccadic slow potentials were studied in seven right-handed male volunteers with a dominant right eye before and after exposure to 6-day dry immersion. Visual stimuli were presented using three light diodes, which were located in the center of the visual field (the central fixation stimulus) and 10° to the right and left of it (peripheral stimuli (PSs)). The subjects performed a test with simple saccades to a PS and a test with antisaccades to the point located symmetrically in the opposite visual field. The EEG (19 monopolar leads) and electrooculogram were recorded. To isolate slow potentials, backward EEG averaging was performed, with the moment of switching on the PS serving as a trigger for the averaging. It was found that the characteristics of saccadic eye movements did not substantially change after exposure to immersion. However, both tests revealed a change in topography and a decrease in the amplitude of presaccadic slow negative potentials (PSNPs) during immersion. Characteristically, the focus of presaccadic negativity shifted to the right hemisphere so that the PSNP amplitude sharply decreased in the left and increased in the right hemisphere. A significant decrease in the PSNP amplitude on day 6 of immersion was found in the midline and left-hemispheric frontal and parietal leads. It may be suggested that, because of support unloading and a decrease in proprioceptive input, exposure to microgravity causes a decrease in the activity of the left hemisphere and prefrontal and parietal cortices, initially involved in preparation and realization of motor responses. The activation of the right hemisphere could be of compensatory character.     

Lipid peroxidation rate and the antioxidant protection system during a three-day dry immersion experiment

The content of lipid peroxidation products (diene conjugates, malondialdehyde, Schiff bases) and antioxidant defense system indices (the main lipid antioxidant tocopherol and the level of general antioxidant activity) were measured in the blood serum of five male volunteers aged 25?C40 years in a three-day dry immersion experiment. During the immersion test, no deviations of indices from the background values were found. An increase in the tocopherol concentration within 2 h after the beginning of the experiment was the only exception. A significant increase in the concentration of lipid peroxidation products, particularly, diene conjugates, was observed 2 h after immersion completion during the reconditioning period. However, the tocopherol content was significantly lower than the background values. It is concluded that the subjects?? adaptation to simulated microgravity conditions displays no pronounced stress component, whereas bringing back to normal vital functions after exposure to immersion induces a pronounced stress reaction illustrated by a significant increase in the lipid peroxidation product levels against a background of a decrease in the functional activity of the antioxidant defense system.     

An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells

The mechanical properties of endothelial cells were measured using the micropipette technique. The cells employed were collected from bovine aortic endothelium and cultured in our laboratory. Endothelial cells from confluent monolayers under no-flow conditions were detached from their substrate by trypsin or by a mechanical method and suspended in modified Dulbecco medium (MDM). In the micropipette technique, a part of the cell is aspirated into the tip of the micropipette under a microscope, and the deformation measured from a photograph. In this study, the data obtained were analyzed using a model where the cytoskeletal elements, which are considered to be the primary stress bearing components, are assumed to reside in a submembranous, cortical layer. Detached cells were found to have almost homogeneous mechanical properties based on measurements from different regions of the surface of a single cell. However, a hysteresis loop was observed in the relation between pressure and cell deformation during the loading and unloading processes. The calculated elastic shear moduli obtained for the trypsin-detached cells were as much as 10-20 times larger than those of a red blood cell. Mechanically-detached cells had moduli approximately twice that of the trypsin detached cells. Passage time, i.e., cell culture age, had no influence on the mechanical properties of the trypsin-detached cells, but did have an effect on the mechanically-detached cells, with both the younger and older cells being somewhat stiffer.     

Serum creatine kinase levels and the number of sarcolemmal dystrophin disruptions in human skeletal muscle fibers under conditions of 7-day "dry" immersion

9 male volunteers took part in the experiment. They were divided in two groups. 5 volunteers (control group) have been in "dry" immersion for 7 days. 4 volunteers (stimulated group) in addition to "dry" immersion were treated with artificial support stimulation. We investigated the number of muscle fibers with the disruptions of sarcolemmal dystrophin and serum creatine kinase levels. 7-day "dry" immersion does not change the mean number of muscle fibers with dystrophin disruptions, it leads to significant decrease of serum creatine kinase levels and does not influence on the sensitivity of sarcolemma to injury. Artificial support stimulation does not influence on these parameters.