Parameters of fiber cell respiration and desmin content in rat soleus muscle at early stages of gravitational unloading

The aim of the work was to study the parameters of fiber cell respiration and desmin content in Wistar rat soleus muscle after 1, 3, 7 and 14 days of gravitational unloading. Gravitational unloading was simulated by antiorthostatic hindlimb suspension. The parameters of cell respiration were determined using polarography, and desmin content was assessed by means of Western blotting. The results showed that the intensity of cell respiration is reduced after three days of gravitational unloading, reaches a minimum level after seven days and slightly increases by the fourteenth day of hindlimb unloading, as well as the content of desmin, which, however, to the fourteenth day returns to the control level. Taking into account that mitochondrial function depends on the state of cytoskeleton, the data allow us to assume that early reduction of the intensity of cell respiration under unloading could be caused by degradation of the protein desmin that determines the intracellular localization of mitochondria.     

Changes in cell respiration of postural muscle fibers under long-term gravitational unloading after addition of succinate into diet

The intensity of cell respiration of the rat m. soleus, gastrocnemius c.m., and tibialis anterior fibers during 35-day gravitational unloading, with the addition of succinate in the diet at a dosage rate of 50 mg per 1 kg animal weight has been investigated. The gravitational unloading was modeled by antiorthostatic hindlimb suspension. The intensity of cell respiration was estimated by polarography. It was shown that the rate of oxygen consumption by soleus and gastrocnemius fibers on endogenous and exogenous substrates and with the addition of ADP decreases after the unloading. This may be associated with the transition to the glycolytic energy pathway due to a decrease in the EMG activity. At the same time, the respiration rate after the addition of exogenous substrates in soleus fibers did not increase, indicating a disturbance in the function of the NCCR-section of the respiratory chain and more pronounced changes in the structure of muscle fibers. In tibialis anterior fibers, no changes in oxygen consumption velocity were observed. The introduction of succinate to the diet of rats makes it possible to prevent the negative effects of hypokinesia, although it reduces the basal level of intensity of cell respiration.     

Changes in cell respiration of postural muscle fibers under long-term gravitational unloading after dietary succinate supplementation

The intensity of cell respiration of the rat m. soleus, m. gastrocnemius c.m. and tibialis anterior fibers during 35-day gravitational unloading, with the addition of succinate in the diet at a dosage rate of 50 mg per 1 kg animal weight has been investigated. The gravitational unloading was modeled by antiorthostatic hindlimb suspension. The intensity of cell respiration was estimated by polarography. It was shown that the rate of oxygen consumption by soleus and gastrocnemius fibers on endogenous and exogenous substrates and with the addition of ADP decreases after the discharge. This may be associated with the transition to the glycolytic energy path due to a decrease in the EMG-activity. At the same time, the respiration rate after the addition of exogenous substrates in soleus fibers did not increase, indicating a disturbance in the function of the NCCR-section of the respiratory chain and more pronounced changes in the structure of muscle fibers. In tibialis anterior fibers, no changes in oxygen consumption velocity were observed. The introduction of succinate to the diet of rats makes it possible to prevent the negative effects of hypokinesia, although it reduces the basal level of intensity of cell respiration.     

Irreversible morphological changes in leg bone following chronic gravitational unloading of growing rats

Effects of gravitational unloading or loading on the growth and development of hindlimb bones were studied in rats. Male Wistar rats were hindlimb-unloaded or loaded at 2-G from the postnatal day 4 to month 3. The morphology and mineral content of tibia and fibula, as well as the mobility of ankle joints, were measured at the end of 3-month suspension or loading, and 1, 2, and 3 months after ambulation recovery. Growth-related increases of bone weight and mineral density were inhibited by unloading. But they were gradually recovered toward the control levels, even though they were still less than those in the age-matched controls after 3 months. None of the parameters were influenced by 2-G loading. However, here we report that chronic unloading causes abnormal morphological development in hindlimb bone of growing rats. Irreversible external bend of the shaft and rotation of the distal end of tibia, which limit the dorsiflexion of ankle joints, were induced following chronic gravitational unloading during developing period. It is also suggested that such phenomena are caused by the abnormal mechanical forces imposed by muscle utilization with altered patterns. The activity of ankle dorsiflexor was increased and that of plantarflexor was inhibited during unloading.     

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.     

Contractile properties, transversal stiffness and cytoskeletal protein content in Mongolian gerbils soleus fibers under long-term hindlimb suspension

Structural and functional changes in Mongolian gerbil soleus fibers were analyzed after a 31-day hindlimb suspension. Contractile properties of muscle fibers were studied by means of tensometry; the transversal stiffness of different parts of the contractile apparatus was measured by atomic force microscopy, resting calcium level was estimated by fluorescence microscopy by using Fluo-4-AM; cytoskeletal protein content was determined by western blotting. It was shown that after gravitational unloading the maximal force of contraction and specific tension of fiber were significantly reduced, as well as calcium sensitivity actually lowered. At the same time, the transversal stiffness of Z-disk in the relaxed and activated state was decreased significantly compared to the control group. Desmin content was at the control level, but alpha-actinin-2, main structural protein of Z-disk, became considerably less after a 31-day hindlimb suspension. Besides, resting calcium level remained at control values during the simulated gravitational unloading. The data suggest that Z-disk destruction, as a result of alpha-actinin-2 content reduction, leads to changes in the lattice spacing and decreases contractile properties.     

Role of gravity in mammalian development: effects of hypergravity and/or microgravity on the development of skeletal muscles.

Effects of hindlimb suspension or exposure to 2-G between postnatal day 4 and month 3 and of 3-month recovery at 1-G environment on the characteristics of rat hindlimb muscles were studied. Pronounced growth inhibition was induced by unloading, but not by 2-G loading. It is suggested that the development and/or differentiation of soleus muscle fibers are closely associated with gravitational loading. The data indicated that gravitational unloading during postnatal development inhibits the myonuclear accretion in accordance with subnormal numbers of both mitotic active and quiescent satellite cells. Even though the fiber formation and longitudinal fiber growth were not influenced, cross-sectional growth of muscle fibers was also inhibited in association with lesser myonuclear domain and DNA content per unit volume of myonucleus. Unloading-related inhibition was generally normalized following the recovery.     

Regulation of the Nuclear–Cytoplasmic Traffic of Class IIa Histone Deacethylases in Rat Soleus Muscle at the Early Stage of Gravitational Unloading

The negative regulation of expression of genes involved in various metabolic pathways in a skeletal muscle is the main function of histone deacetylases 4 and 5 (HDAC4/HDAC5). HDAC4 and HDAC5 seem to be the targets of the AMP-activated protein kinase (AMPK). Earlier, an essential decrease in the level of Thr172-phosphorylated-AMPK in a rat soleus muscle at the first day of gravitational unloading was shown. Possibility of a protein kinase D (PKD) to phosphorylate histone deacetylases 4/5 has been shown, too. We supposed that under the conditions of gravitational unloading, alterations in AMPK phosphorylation level can affect regulation of nuclear-cytoplasmic traffic of class II histone deacetylases and of various skeletal muscle genes expression. To verify the hypothesis, we used administration of an AMPK activator, AICAR, before and during a day-long hindlimb suspension. It was shown that at an early stage of gravitational unloading, HDAC4 is not a PKD target, and its nuclear import is realized due to decrease in AMPK activity. We were the first to show reciprocal relations between AMPK and PKD in a skeletal muscle at early gravitational unloading.     

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 long duration +2G hypergravity on MHC distribution and maximal tension of rat m.soleus fibers

Effects of long duration hypergravity on skeletal muscles are much less studied than effects of microgravity. For instance, it was revealed that hypergravity of 2 week duration induces decrease in cross sectional area (CSA) of slow fibers (SF), while their size remains constant, or increases. Exposure to +2G of 14 day duration results in decreased number of type I fibers, and in changed myosin heavy chain (MHC) profiles of rat hindlimb extensor muscle. It is interesting that gravitational unloading also decreases number of type I fibers. However, while effects of microgravity on relationship between the structural and functional characteristics of skeletal muscles are studied in detail, similar characteristics of skeletal muscles under conditions of gravitational overloading are very much understudied. The aim of our work was to follow dynamics of MHC in rat m.soleus after exposure to 19 and to 33 days of +2G acceleration, and to compare content of contractile proteins in muscle fibers, and their contractile properties.     

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

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

Dynamics of calcium ions accumulation and changes of isoforms of sarcoplasmic reticulum Ca-ATPase (SERCA) in soleus muscle fibers of rats and Mongolian gerbils under gravitational unloading of various duration

The concentration of calcium ions in soleus muscle fibers in Mongolian gerbils and rats has been shown to increase for the first day of gravitational load by 4.5 and 2.8 times, respectively, compared to the control. This increase is preserved for 3 days, while, after 12 days, it decreases, but remains above the control level. The resting calcium level in the control in gerbils is lower than in rats and, as a consequence, its change for the first day of unloading is greater. It is possible that this leads to earlier changes in the muscle fiber isoform composition in gerbils; a statistically significant shift in the ratio of fibers that contain fast and slow calcium pumps towards pumping faster is noted as early as the first day, while that of fibers that express the corresponding isoforms of myosin heavy chains (MHCs) is observed after 3 days of functional unloading. In rats, similar changes are revealed as late as on the 7th day. After 12 days, in both species, a tendency to return to the control level is observed that is more pronounced in gerbil.     

Alternative Splicing of Titin mRNA in Rat Soleus after Seven-Day Gravitational Unloading

Human Physiology - Changes in titin alternative splicing in the rat soleus after seven-day gravitational unloading (the hindlimb unloading model) were studied by long-fragment PCR and nanopore...     

Essential role of satellite cells in the growth of rat soleus muscle fibers

Effects of gravitational loading or unloading on the growth-associated increase in the cross-sectional area and length of fibers, as well as the total fiber number, in soleus muscle were studied in rats. Furthermore, the roles of satellite cells and myonuclei in growth of these properties were also investigated. The hindlimb unloading by tail suspension was performed in newborn rats from postnatal day 4 to month 3 with or without 3-mo reloading. The morphological properties were measured in whole muscle and/or single fibers sampled from tendon to tendon. Growth-associated increases of soleus weight and fiber cross-sectional area in the unloaded group were approximately 68% and 69% less than the age-matched controls. However, the increases of number and length of fibers were not influenced by unloading. Growth-related increases of the number of quiescent satellite cells and myonuclei were inhibited by unloading. And the growth-related decrease of mitotically active satellite cells, seen even in controls (20%, P > 0.05), was also stimulated (80%). The increase of myonuclei during 3-mo unloading was only 40 times vs. 92 times in controls. Inhibited increase of myonuclear number was not related to apoptosis. The size of myonuclear domain in the unloaded group was less and that of single nuclei, which was decreased by growth, was larger than controls. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. It is suggested that the satellite cell-related stimulation in response to gravitational loading plays an essential role in the cross-sectional growth of soleus muscle fibers.     

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

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

Effect of hindlimb unloading on interlimb coordination during treadmill locomotion in the rat

Effects of hindlimb unloading on interlimb coordination were examined in adult rats walking on a treadmill at moderate speed. In the first group of animals, the electromyographic activity (EMG) of soleus muscle of both hindlimbs was recorded after 7 and 14 days of unloading. In the second group, the EMG was recorded daily until the 14th day of unloading. The general organization of locomotion was preserved in the two groups whatever the duration of the unloading. The step cycles of the two hindlimbs were always strictly alternating. However, the locomotor pattern was very irregular. A lateral instability was observed. It was accompanied by an abduction of the hindlimbs, and frequent hyperextensions of the ankle when walking. The EMG analysis showed an increase in step cycle duration and in coactivation duration of the soleus muscles (i.e. in the double stance duration). In the rats recorded daily, mean EMG was dramatically reduced the 1st day of unloading, suggesting a decrease in the neural drive. Taken together, these data indicate that 14 days of hindlimb unloading can alter the neuromuscular pattern during locomotion. It is proposed that these changes are related to changes in the peripheral sensory information. Accepted: 29 June 1998     

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.     

蓝光对绿豆下胚轴愈伤组织生长和呼吸的影响

与白光和黑暗条件相比,蓝光明显促进绿豆(Vigna radiata (Linn.)Wilczek)下胚轴愈伤组织的形成和生长。培养到18d,蓝光下生长的愈伤组织鲜重分别是白光和黑暗下的120％和191％。三种条件下愈伤组织的呼吸速率有明显的不同,培养5d,呼吸速率以蓝光处理的最高,白光处理的次之,黑暗处理的最低;培养14d,与上述相反,呼吸速率由高到低则依次是黑暗、白光和蓝光。应用呼吸抑制剂碘乙酸、丙二酸和正磷酸钠试验及对6-磷酸-葡萄糖脱氢酶活性测定的结果表明,培养5d,蓝光和白光处理时磷酸已糖支路(HMP)明显增加,被碘乙酸 丙二酸抑制后的呼吸占总呼吸的34％～36％,黑暗处理的仅占总呼吸9％～11％ 绿豆下胚轴愈伤组织中亦存在抗氰交替途径,黑暗处理时,此种途径的最大容量明显高于蓝光和白光处理的,培养14d,黑暗处理占总呼吸的31％左右.而蓝光和白光处理的则占20％以下。     

Rat hindlimb unloading: Soleus and Extensor Digitorum Longus histochemistry,mitochondrial DNA content and mitochondrial DNA deletions

Mitochondrial phenotypic alterations, mitochondrial DNA content and mitochondrial DNA deletions in a slow, Soleus, and a fast, Extensor Digitorum Longus, skeletal muscle of 3- and 15-month-old hindlimb suspended rats have been studied. Cytochrome c oxidase-negative fibers appeared after unloading in all examined animals and their percentage increased with increasing unloading time. After 14 days of suspension the mitochondrial DNA content did not change in 3-month-old but decreased significantly in 15-month-old rats. Soleus was much more affected by unloading than Extensor Digitorum Longus. The mitochondrial DNA deletion of 4834 bp as well as other mtDNA deletions, researched with Long Distance-PCR, were absent in both studied muscles before and after unloading.     

Tension- and afferent input-associated responses of neuromuscular system of rats to hindlimb unloading and/or tenotomy

Responses of electromyogram (EMG) in soleus muscle and both afferent and efferent neurograms at the fifth lumbar (L(5)) segmental level of spinal cord were investigated during acute and chronic unloading induced by hindlimb suspension and/or tenotomy in adult rats. The soleus EMG and afferent neurogram decreased 88 and 37%, respectively, relative to those at quadrupedal posture on the floor after acute hindlimb suspension that causes passive shortening of soleus due to ankle plantarflexion. However, the afferent neurogram (P < 0.05) and soleus EMG (P > 0.05) recorded on the floor increased after tenotomy of synergists. Furthermore, the afferent input was inhibited when the soleus EMG disappeared after tenotomy of soleus. The afferent neurogram and EMG of the soleus showed correlated responses to a variety of treatments, suggesting that the afferent neurogram recorded at the L(5) segmental level reflects the neural input associated with the activity level of the soleus predominantly. The level of efferent neurogram decreased after acute hindlimb suspension but was not influenced significantly by tenotomy of synergists and/or soleus itself. The EMG and afferent neurograms remained low up to the 4th day but recovered to the preexperimental levels within 14 days, due to reorganization of sarcomere number and length, as well as the shortening of muscle fiber length and recovery of tension development. It is suggested that the levels of EMG and afferent neurogram associated with antigravity muscle are closely related to the tension development of the muscle.