The aim of this research was the analysis of structural changes in various parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy under gravitational unloading. Soleus, medial gastrocnemius, and tibialis anterior muscles of Wistar rats were the objects of the study. Gravitational unloading was carried out by antiorthostatic suspension of hindlimbs for 1, 3, 7, and 12 days. It was shown that the transversal stiffness of different parts of the contractile apparatus of soleus muscle fibers decreases during gravitational unloading in the relaxed, calcium-activated, and rigor states, the fibers of the medial gastrocnemius show no changes, whereas the transversal stiffness of tibialis anterior muscle increases. Thus the transversal stiffness of the sarcolemma in the relaxed state is reduced in all muscles, which may be due to the direct action of gravity as an external mechanical factor that can influence the tension on a membrane. The change of sarcolemma stiffness in activated fibers, which is due probably to the transfer of tension from the contractile apparatus, correlates with the dynamics of changes in the content of desmin.
- musculus soleus
microgravity conditions lead to a decrease in muscle functionality realized at the cellular level (21). This situation may be a consequence of initiation of the atrophic program, particularly activation of the calpain system (13) as a result of accumulation of calcium ions. It has been shown that accumulation of calcium ions in the soleus muscle of mice occurs already on the second day of gravitational unloading (17, 18). Rats and Mongolian gerbils show an increase in the basal content of calcium ions after a day of gravitational unloading in the soleus, medial gastrocnemius, and tibialis anterior muscles (24). These muscles have a fundamentally different level of neural activation under microgravity. Alford et al. (2) showed that electromyographic (EMG) activity of musculus soleus (Sol) was reduced almost completely (up to 9% compared with control level) even at the early stages of gravitational unloading (1 and 3 days under microgravity conditions), while the activity in its synergist, musculus gastrocnemius c.m. (medial gastrocnemius, MG), does not decrease so significantly, whereas its antagonist, musculus tibialis anterior (TA), shows an increase in activity. As shown by the data of the same authors, after ∼10 days of unloading, EMG activity of Sol and MG did not change compared with control level. Thus it remains unclear whether the fibers of MG and TA undergo any structural changes.
The structural integrity of muscle fibers, as well as any other cell, can be measured in different ways, in particular by analyzing their mechanical properties. Muscle fiber can be considered as a compound mechanical system in which the mechanical stress is transduced not only in the axial but also in the lateral direction (4). One can expect that changes in the mechanical properties of each of the sarcolemma and contractile apparatus would result in changes in the transversal stiffness of a muscle fiber. The transversal stiffness of muscle fibers in the relaxed state allows evaluation of the continuity of structural organization. Such studies in activated and rigor states will provide the possibility to determine the effectiveness of muscle fiber contractility activation. The development of methods of atomic force microscopy allows measurement of single cells in the native state. Because such methods have become widespread quite recently, only few data on transversal stiffness of muscle fibers are found in the literature. Thus Collinsworth et al. (9) have shown in undifferentiated muscle cells of mice that the Young's modulus is 11.5 ± 1.3 kPa and that on the eighth day of differentiation it increases four times and reaches 45.3 ± 4.0 kPa. Mathur et al. (20) defined the Young's modulus of C2C12 myoblasts of mice of the C3H line as equal to 24.7 ± 3.5 kPa. The same value for differentiated muscle fibers of C3H mice is 61 ± 5 kPa (11). Comparison of the results of such studies (9, 11, 20) supports the concept that Young's modulus increases in the transversal direction with the development of the cytoskeleton during differentiation of muscle fibers. Nyland and Maughan (23) studied bundles of myofibrils of flying muscles of Drosophila, using a rigor solution (without ATP), an activating solution (pCa 4.5), and a relaxing solution (pCa 8.0). The authors obtained the following results: in rigor solution, the transversal stiffness is 10.3 ± 5.0 pN/nm and Young's modulus is 94 ± 41 kPa; in activating solution, the transversal stiffness is 5.9 ± 3.1 pN/nm and Young's modulus is 55 ± 29 kPa; and in relaxing solution, the transversal stiffness is 4.4 ± 2.0 pN/nm and Young's modulus is 40 ± 17 kPa. Unlike Nyland and Maughan, Akiyama et al. (1) studied the structure and the transversal stiffness of separate myofibrils isolated from muscle fibers of cardiac and skeletal muscle of young rabbits and neonatal rats and showed that the Z disk of myofibrils of skeletal muscles in rigor solution was 100 nm in width and had a stiffness of 7.7 pN/nm and for the cardiac muscle was 320 nm thick with a stiffness of 25.8 pN/nm. Myofibril stiffness of cardiac muscle in rigor solution in the area of the M band, whose width was ∼200 nm, was 11 pN/nm.
Therefore, the determination of values of transversal stiffness or Young's modulus by atomic force microscopy is an effective method to analyze the structural state of muscle fibers in native conditions. This is why the performance of measurements of such parameters after gravitational unloading is of particular interest.
Previously, we proposed (25) a method for differentiated estimation of transversal stiffness values for various sections of both sarcolemma and contractile apparatus of muscle fibers. A similar approach can be applied in this case since it will permit localizing the changes in the structure of muscle fibers more precisely, which can be of significance in the analysis of signaling pathways for formation of an adaptive pattern of key protein expression.
MATERIALS AND METHODS
Experiments were carried out on fibers of Sol, MG, and TA muscles of Wistar rats weighing 190–210 g. To simulate gravitational unloading, we used antiorthostatic suspension of the hindlimbs, using the Morey-Holton modification of the Ilyin-Novikov method (22). Gravitational unloading lasted for 1, 3, 7, and 12 days. Thus the following groups were formed: control group and 1-HS, 3-HS, 7-HS, and 12-HS groups (each group contained 7 animals).
All procedures with animals were approved by the Biomedical Ethics Committee of the State Scientific Center of the Russian Federation Institute for Biomedical Problems of the Russian Academy of Sciences.
Atomic Force Microscopy
To determine the transversal stiffness of the different compartments of muscle fibers we used atomic force microscopy, which we described in detail previously (25).
The muscles were cut from tendon to tendon and were treated by the method of chemical skinning described by Stevens et al. (31) for partial destruction of cell membranes. Before the experiments the samples were stored at −20°C in a buffer containing relaxing solution R (in mM: 20 MOPS, 170 potassium propionate, 2.5 magnesium acetate, 5 K2EGTA, 2.5 ATP) and glycerol in equal parts by volume. On the day of the experiment the samples were transferred to solution R, in which the permeabilized single muscle fibers were isolated.
To obtain demembranated fibers, single permeabilized muscle fibers from solution R were incubated with the detergent Triton X-100 (2% final concentration by volume) within 12 h at a temperature of +4°C. After processing with the detergent the Triton-treated fibers were washed with solution R.
To measure transversal stiffness the isolated fibers were attached to the liquid cell bottom of the atomic force microscope by fixing their tips with a special Fluka shellac wax-free glue (Sigma).
Depending on the series of experiments, a cell was filled with relaxing solution R, activating solution A (in mM: 20 MOPS, 172 potassium propionate, 2.38 magnesium acetate, 5 CaEGTA, 2.5 ATP), or rigor solution Rg (in mM: 20 MOPS, 170 potassium propionate, 2.5 magnesium acetate, 5 K2EGTA). All experiments were carried out at a temperature of +16°C.
Measurements of transversal stiffness both of permeabilized and Triton-treated fibers were carried out with the Solver-P47-Pro platform (NT-MDT), with previous scanning of the surface (25).
Surface scan of the permeabilized muscle fibers in the relaxed state shows protuberances (referred to here as “humps”) near both the Z-disk and M-band projections (Fig. 1). The hump near the Z disk is probably due to the presence of the costameres, submembrane structures between the membrane and the contractile apparatus. A costamere-like structure that connects the M band to sarcolemma probably exists near the M band. Triton-treated fibers show smaller humps and similar heights near the Z disks and M bands (Fig. 2), which can support the total removal of membrane and related structures. Using the atomic force microscopy surface images, we were able to measure transverse stiffness of specific regions of the fiber such as projections of the M band and Z disks (Fig. 3). Because treatment with Triton X-100 destroys sarcolemma and sarcolemma-associated cytoskeleton, comparison of stiffness of permeabilized and Triton-treated fibers allows one to dissect the mechanical properties of myofibrils and extrasarcomere cytoskeleton. At least 49 fibers in each group and for each muscle were used for obtaining the results of the transversal stiffness for each analyzed point.
The change of the applied force ΔF (in N) was determined at a depth of bursting Δh =150 nm, and the stiffness of the sample (in N/m) was defined by the formula k = ΔF/Δh.
The above-described results were analyzed in a program specially designed for this case under MatLab 6.5.
For determination of desmin content variation in total protein of the muscle, muscle was frozen at the temperature of liquid nitrogen. Denaturing electrophoresis in polyacrylamide gel was performed according to the method of Laemmli on the Bio-Rad system, with a two-component system consisting of 6% concentrating gel and 12% separating gel. Based on the carried measurement of the total protein concentration the identical amount of protein was loaded in each hole. The transfer to nitrocellulose membrane (Bio-Rad) was carried out by the method of Towbin et al. (33). To detect desmin, a protein with a molecular mass of 55 kDa, we used monoclonal antibodies based on mouse immunoglobulins (Sigma) diluted to 1:200 as primary antibodies. The biotinylated goat antibodies against mouse IgG (Sigma) were used as secondary antibodies diluted to 1:6,000. Membranes were further processed with a solution of streptavidin conjugated with horseradish peroxidase (Sigma) diluted to 1:10,000. Protein bands were revealed by 3,3′-diaminobenzidine (Merck).
The obtained results were statistically analyzed with ANOVA and the post hoc t-test, with a significance level of P < 0.05 for estimation of significant differences between groups. The results are represented as means ± SE, where the mean is an average index of the estimated value and SE is an error of average value.
In the control group, transversal stiffness of the contractile apparatus (Table 1) in the area of half-sarcomere, i.e., between the Z disk and M band, increased along the relaxed-activated-rigor sequence. Moreover, the increase of this parameter was significant in both cases—from relaxed state to activated state and from activated state to rigor. The described situation did not change after 1 day of gravitational unloading. However, after 3 days there was a significant decrease of the stiffness of the contractile apparatus in the area of half-sarcomere in relaxed, activated, and rigor states compared with similar states in the control group. This decrease became even more evident at 7 days of antiorthostatic suspension. The minimal value of stiffness relative to the control level took place after 12 days of gravitational unloading. Nevertheless, after 3, 7, and 12 days of suspension, the stiffness of the half-sarcomere increased significantly during the activation of contraction, as in the rigor condition compared with the calcium-activated state.
In the M-band area, the stiffness of the contractile apparatus was higher than the stiffness of the half-sarcomere. The tendency in the dynamics of gravitational unloading described for the area of the half-sarcomere is valid for the M band, although the increase of its stiffness at activation and in rigor was not so high as in the half-sarcomere.
The transversal stiffness of the Z disk was significantly higher than that of the half-sarcomere and M band. It increased in the same way as described above, at activation and in rigor, although less intensively. Also, the change of transversal stiffness of the Z disk within the gravitational unloading occurred the same way in 3 days of suspension. However, these changes were more significant than those in the half-sarcomere and M band.
The transversal stiffness of different sites of permeabilized fibers (Table 2) in the control group increased by the activation of contraction compared with the relaxed state and at the transition to rigor compared with the activated state. However, the intensity of this increase was not so significant concerning the Triton-treated fiber, which was especially evident after a slight increase of stiffness of sarcolemma between the projections of the Z disk and M band upon activation of contraction compared with an increase of stiffness in area of the half-sarcomere in a similar situation. Moreover, it is necessary to note that the stiffness of this part of the sarcolemma and the half-sarcomere in the relaxed state were comparable. Activation of contraction led to a more considerable increase of sarcolemma stiffness at the projections of the Z disk and M band; in addition, the stiffness of these membrane sites of muscle fiber in the relaxed state was significantly less than that of parts of the Z disk and M band of the contractile apparatus.
After a day of gravitational unloading the stiffness of all parts of the sarcolemma in both relaxed and activated states was reduced compared with the same state in the control group; it reached its minimum after 7 days of suspension and showed a tendency to increase to the 12th day of gravitational unloading.
However, at the activation of contraction and at rigor the transversal stiffness of sarcolemma between the projections of the Z disk and M band after a day of gravitational unloading were not significantly different. A similar situation occurred up to the 12th day of antiorthostatic suspension. Thus the transversal stiffness of the sarcolemma at the projection of the Z disk and M band increased at the activation of contraction within the gravitational unloading, although it was not as significant as in the control group.
Medial gastrocnemius muscle.
The transversal stiffness of different parts of the MG contractile apparatus (Table 3) was almost two times less than the values of similar parameters in Sol fibers, not only in the relaxed but also in the activated and rigor states. However, the increase of transversal stiffness in the line of half-sarcomere-M band-Z disk was similar to that in Sol fibers.
During the gravitational unloading, except for the early periods, the values of the transversal stiffness of half-sarcomere, Z disk, and M bands did not undergo any significant changes in all investigated conditions. At early stages (1st day and 3rd day) there was a significant increase in transversal stiffness of the half-sarcomere and Z disk that was expressed more in the first day of suspension. Also, an increase of the transversal stiffness of the M band occurred only in the first day.
Unlike the contractile apparatus, the transversal stiffness of the MG sarcolemma (Table 4) in the area between the projections of the Z disk and M band started to decrease already after a day of gravitational unloading and continued throughout the period of suspension. The increase of the stiffness of the sarcolemma upon activation and at rigor took place after 1 day of functional unloading, but after 3 days change in the stiffness of this part of the sarcolemma upon activation of contraction and transition to rigor condition compared with the stiffness in the relaxed state was not observed. The transversal stiffness of the sarcolemma in the projection of the M band increased in the first day of gravitational unloading in both the relaxed and activated states compared with the fibers of the control group in a similar condition, although there was no significant difference between the parameters in the relaxed and activated states. After 7 days of unloading the stiffness underwent no changes in different conditions compared with the control group.
A similar situation was observed for the sarcolemma in the area at the projection of the Z disk. However, after 12 days of suspension the transversal stiffness of this site of sarcolemma was significantly reduced compared with the control group.
Tibialis anterior muscle.
The transversal stiffness of different parts of the TA contractile apparatus (Table 5) upon activation of contraction and transition to rigor state changed as well as the Sol did in the control group. However, after 1 day of gravitational unloading the transversal stiffness essentially increased in the area of the half-sarcomere and then started to decrease a little but could not reach the value of the control group, and so it remained increased through all conditions (activation, relaxing, rigor) by the 12th day. A similar situation was observed in the area of the M band. At the same time, the stiffness of the Z disk exceeded the control level after 1 and 3 days of gravitational unloading, but in the seventh day it did not differ from the controls.
The transversal stiffness of the TA sarcolemma (Table 6) in the area between the projections of the Z disk and M band did not change by activation of contraction and rigor, unlike the Sol. During gravitational unloading the stiffness of this site of the sarcolemma significantly reduced with a day of antiorthostatic suspension, returning to the control level in 7 days of gravitational unloading. At the same time, the transversal stiffness of the sarcolemma at the projection of the M band did not change during the suspension, although differences in values in the activated and rigor states were not observed. The increase of the sarcolemma transversal stiffness at the area of Z-disk projection was observed in 3 and 7 days of the functional unloading, while it reached control level by the 12th day.
The content of desmin in rat Sol in the 1-HS group did not change compared with the control group (Fig. 4). The content of desmin was significantly reduced in the 3-HS group by 25% compared with the control group and in the 7-HS group by 43% and restored to the control level in the 12-HS group.
The content of desmin in rat MG in the control group was less than in the Sol by 62.9%. Desmin increased in the 1-HS group by 50% and in the 3-HS group by 67% compared with the control group. In the 7-HS group desmin decreased compared with the 3-HS group but remained 35% higher compared with the control group. In the 12-HS group desmin was restored to the control level.
The content of desmin in the control group in rat TA was higher than in the Sol by 67.9%. In the 1-HS group desmin content increased by 100%, in the 3-HS group by 153%, in the 7-HS group by 75%, and in the 12-HS group by 87% compared with the control group.
The obtained experimental data show that stiffness in the different parts of muscle fibers of different muscles changes differently.
The significant increase in transversal stiffness between the M bands and Z disks was possibly caused by myosin cross bridges between the thin and thick filaments upon activation and in rigor. Xu et al. (34) showed that one of the key parameters of radial elasticity is the equilibrium spacing, while the fibers in different states exhibited a broad range of radial stiffness coefficients depending, in the view of the authors, on the state of the cross bridges rather than on the fraction of the myosin heads bound to actin. Ranatunga et al. (26) showed that isometric tension of muscle fibers in the relaxed state (passive tension) was insensitive to increased pressure, whereas the muscle fiber tension in the rigor state increased linearly with pressure. They could not exclude that changes in rigor tension are induced by hydrostatic compression of a specific elastic component in a cross bridge. It is possible that the increase in transversal stiffness of the Z disks and M bands is probably a second-order effect caused by cross-bridge formation in the overlap zone. At the same time, the transversal stiffness of Z disks and M bands in the relaxed state can reflect the state of their structures and in the activated and rigor states the effectiveness of tension transfer along the sarcomere. The stiffness of the contractile apparatus of relaxed Sol fibers of rats on the first day of gravitational unloading does not change compared with control. By the third day the stiffness of the Z disk of the relaxed fiber essentially reduces, reaching its minimum, and remains the same throughout the entire duration of the unloading. We have previously shown (24) that after a day of gravitational unloading the resting calcium level increases in Sol fibers of the rat. In addition, it is known that accumulation of calcium ions in the Sol fibers of the mouse occurs on the second day of gravitational unloading (17, 18). This may lead to the activation of calpains, calcium-dependent proteases (13). The structure of the Z disk includes a number of proteins that are substrates of calpains (29). Perhaps their destruction leads to early reduction of the stiffness of the Z disk that was observed in the experiments. At the same time change of Z disk stiffness in the activated state is less apparent: there is a more gradual decrease, although the difference in stiffness between the activated and rigor states significantly decreases.
This stiffness of the M band and half-sarcomere of Sol fibers gradually reduces from the third day and reaches a minimum by the 12th day, although these changes of the M band and half-sarcomere develop much faster in the activated state. A similar effect can be caused by several factors.
First, titin is a substrate of calpains, and its degradation is observed to the seventh day of functional unloading (19, 15, 30). In addition, sarcomere lesions, which are a characteristic feature of the slow-twitch fibers (I type), first shown by Riley et al. (28), can also lead to a decrease of the transversal stiffness in the area of the half-sarcomere and reduce the transversal stiffness. We did not determine the fiber type; however, we supposed that the use of a large statistical sampling and the fact that Sol is mostly slow-twitch muscle allow us to state that our results characterize the state of slow-twitch fibers mostly.
Second, reduction of the basal number of cross bridges can also lead to a decrease in transversal stiffness of the contractile apparatus in both relaxed and activated states. During gravitational unloading different data on the reduction of calcium sensitivity of muscle fibers show the reduction of the basal number of cross bridges and reflect the number of bound cross bridges. Therefore, by the seventh day of suspension a parameter such as pCa0 (21) decreases, reflecting the concentration of calcium that leads to a significant contraction of muscle fiber initiated by a certain number of bound cross bridges. In addition, it can also be the reason for the reduction of stiffness in the activated state, as pca50 during gravitational unloading is also reduced (21).
The probability of the formation of cross bridges depends on a number of factors, including the efficiency of tension transfer in the longitudinal direction, which means the consistency of cross-bridge formation at the neighboring sarcomeres, the mobility of myosin heads, and lattice spacing. The first of these factors can be reduced by calcium-dependent degradation of titin, damage of the sarcomere (see above), and destruction of the Z disk. The mobility of myosin heads may increase faster as a result of increased phosphorylation of myosin light chains (32) that is promoted by the increase of phosphokinase activity within the gravitational unloading (3). In addition, the increase of resting calcium level also increases the probability of forming cross bridges, although the sensitivity of the “fast” myosin isoforms to calcium is lower and a significant slow-to-fast shift is observed by the seventh day of unloading (5, 12, 6). The effect of different values of lattice spacing on transversal stiffness was shown first by Xu et al. (34) and Ranatunga et al. (26) in experiments with osmotic agents. But the question is whether the lattice spacing undergoes changes within the gravitational unloading. One can only assume that the destruction of the Z disk and M band can lead to an increase of equilibrium spacing. This hypothesis is supported by the fact that the decrease of Z-disk and contractile apparatus stiffness develops by the third day of hypokinesia, whereas the other changes develop later, by the seventh day of unloading.
At the same time, the significant decrease in sarcolemma transversal stiffness in the relaxed state is already observed by the first day of gravitational unloading, and this fact is not related to the stiffness decrease of the contractile apparatus since its stiffness is not changed by the first day of unloading. According to the data of Collinsworth et al. (9), the transversal stiffness of muscle fiber sarcolemma can decrease because of the destruction of submembrane actin cytoskeleton. We suppose that such destruction can be related both to destruction of actin fibers and to dissociation of actin-binding proteins, particularly α-actinin 1, filamin, and Arp protein family. We could not find any data on the state of subsarcolemmal actin cytoskeleton of muscle fibers after being in microgravitational conditions, although data have been obtained in human endothelial cells (16) and human A431 carcinoma cells (27), indicating the destruction of actin cytoskeleton of such cells within the conditions of gravitational unloading. Moreover, using cells from human aorta, Costa et al. (10) showed that destruction of submembrane actin cytoskeleton leads to destruction of the stiffness of such cells (the destruction was measured by atomic force microscopy). Therefore we suppose that the decrease found in sarcolemma stiffness within the conditions of gravitational unloading can be related to the destruction of submembrane actin cytoskeleton of muscle fibers. This can lead to the activation of various signal pathways related to G-actin, but it will probably not have any crucial significance in the decrease of functional possibilities of fiber to contribute to atrophy.
In rats, changes in the area of the costamere (sarcolemma at the Z-disk projection) appear after a day of unloading, but by the 12th day of suspension this parameter is restored. Perhaps the reduction of stiffness of the costamere is associated with its destruction as a result of loss of desmin, which is the basic protein of the costamere that determines the transmission of tension from the contractile apparatus to the sarcolemma (7, 14). The destruction of the costamere can have not only structural but also signal meaning because of the bounding of the costamere with the number of signaling molecules. Moreover, the desmin loss and, as a result, the decrease in effectiveness of tension transfer to the membrane can reflect the decrease in intensity of growth of the transversal sarcolemma stiffness in activated and rigor states after gravitational unloading. Desmin is a substrate of a specific calcium-dependent proteolysis system—the calpain system. Enns et al. (13) showed that the desmin content in a mix of Sol and MG of mice decreased at the first day of unloading, dropped significantly at the third day, and was almost completely restored at the ninth day. Chopard et al. (8) showed that a long period of antiorthostatic suspension in rats does not change the content of desmin in Sol. The experimental data obtained in this study show that the concentration of desmin in the Sol decreases after 1 day of suspension, reliably decreases after 3 days, and reaches a minimum after 7 days; after 12 days it does not differ from the control level. That is, these results are quite consistent with the data of Enns et al. (13), although our study showed that the restoration of desmin concentration occurs somewhat later.
A different picture is observed in the study of MG and TA fibers. By the first day of gravitational unloading the stiffness of the contractile apparatus of the relaxed fibers of these muscles reliably increases; however, the increase is much higher in the area of the half-sarcomere than in the area of the Z disk. Furthermore, during unloading in MG fibers the stiffness of the contractile apparatus returns to the control level and that of TA fibers remains above the initial level. On the basis of arguments explaining the changes of the stiffness of the Sol contractile apparatus, we can assume that on the first day of gravitational unloading either an increase of the mobility of myosin heads or a decrease of lattice spacing occurs, and it is the area of the half-sarcomere where the changes occur, since the changes of stiffness of the Z disk and M band are not so expressed. In this case the change of physicochemical factors that remained constant within the experiment could lead to the modification of lattice spacing. This is why we can assume that an increase of the transversal stiffness of MG and TA fibers leads to an increase of the mobility of myosin heads due to a possible rise of the general level of phosphorylation in cells observed during gravitational unloading (3).
In this case the transversal stiffness of the fiber sarcolemma of both MG and TA muscles, as well as Sol fibers, significantly reduces at the first day of gravitational unloading, which may be associated with the destruction of the cortical cytoskeleton as it happens in the Sol fibers. Nevertheless, after 7 days of unloading transversal stiffness of sarcolemma of MG fibers increases and reaches the control level, and the stiffness of TA fibers even slightly exceeds the control level. In this case, the stiffness of the MG and TA fiber costamere increases slightly after 3 and 7 days of suspension but returns to the initial level by the 12th day, which allows us to assume different dynamics of desmin content change compared with the Sol.
In the MG the desmin content remains almost the same during gravitational unloading; there is only a slight increase by the seventh day of suspension. Probably this can explain the results received by Enns et al. (13): they had no significant differences when working with a mixture of Sol and MG, because the dynamics of these muscles are different and the mixture is affected by superposition. The desmin content in the TA significantly increases by the first day of unloading, reaches its minimum by the third day, and then decreases a little but remains significantly higher compared with the control level. It should be noted that our results show that in the control batch the content of desmin in the Sol is three times higher than in the MG and TA, which coordinates with the fact that the concentration of desmin in slow-twitch muscles is more than two times higher than in fast-twitch muscles (7, 8).
The main difference between the examined types of muscles is the change of the level of their neural activation within the gravitational unloading, apart from the fact that Sol is predominantly slow and MG and TA are predominantly fast. Thus, according to Alford et al. (2), who used electromyography of rat leg muscles, the activity of Sol is 9% of the control level during the first day, 67% after 3 days, and 81% after 7 days, and after 12 days it does not differ from the control level. At the same time EMG activity of the MG does not change during the unloading except for a slight decrease at the first day, and that of TA significantly increases within the first 3 days, exceeding the control level by three times, then somewhat decreases but remains increased during the entire experiment.
Comparing the data of Alford et al. (2), Riley et al. (28), and our results, we can make the following assumptions. The reduction of neural activation leads to a decrease of stiffness of the contractile apparatus, possibly due to the changes in efficiency of tension transmission in the longitudinal direction caused by changes in lattice spacing. However, considering that the reduced level of activation is typical for Sol predominantly consisting of type I fibers, where the damage of sarcomere is observed according to Riley et al. (28), it also can lead to a decrease of the transversal stiffness. Increased activity may lead to the dominance of the factors that increase the mobility of myosin heads. In addition, directly proportional dependence is observed between the level of activation and the content of desmin, a protein that provides the tension transmission from the contractile apparatus to the sarcolemma.
In this case, the observed functional characteristics of the sarcolemma do not depend on the level of activity and content of type I and II muscle fibers. Probably the direct reduction of gravity as a mechanical factor influencing any cell may lead to a decrease in the stiffness of the cell membrane. The mechanism of this reduction is not clear yet; however, one may assume that the change in tension of the submembrane cytoskeleton leads to deformation and subsequently either to depolymerization of submembrane actin filaments or to its destruction because of the dissociation of actin-binding proteins. The decrease of stiffness of submembrane cytoskeleton may, for example, modulate the activity of membrane channels and may play a key role in the initiation of the signaling processes, providing for mechanosensitivity of the cells, which are probably not only muscle cells.
The financial support of the Russian Fund for Basic Research (RFBR Grant 10-04-00106-a) is gratefully acknowledged.
No conflicts of interest, financial or otherwise, are declared by the author(s).
The author expresses gratitude to V. A. Kurushin, E. V. Ponomareva, and E. G. Altaeva for their assistance in carrying out the experiments.
- Copyright © 2010 the American Physiological Society