Molecular Mechanisms of Muscle Tone Impairment under Conditions of Real and Simulated Space Flight.

Kozlovskaya et al. [1] and Grigoriev et al. [2] showed that enormous loss of muscle stiffness (atonia) develops in humans under true (space flight) and simulated microgravity conditions as early as after the first days of exposure. This phenomenon is attributed to the inactivation of slow motor units and called reflectory atonia. However, a lot of evidence indicating that even isolated muscle or a single fiber possesses substantial stiffness was published at the end of the 20th century. This intrinsic stiffness is determined by the active component, i.e. the ability to form actin-myosin cross-bridges during muscle stretch and contraction, as well as by cytoskeletal and extracellular matrix proteins, capable of resisting muscle stretch. The main facts on intrinsic muscle stiffness under conditions of gravitational unloading are considered in this review. The data obtained in studies of humans under dry immersion and rodent hindlimb suspension is analyzed. The results and hypotheses regarding reduced probability of cross-bridge formation in an atrophying muscle due to increased interfilament spacing are described. The evidence of cytoskeletal protein (titin, nebulin, etc.) degradation during gravitational unloading is also discussed. The possible mechanisms underlying structural changes in skeletal muscle collagen and its role in reducing intrinsic muscle stiffness are presented. The molecular mechanisms of changes in intrinsic stiffness during space flight and simulated microgravity are reviewed.

Early Deсline in Rat Soleus Passive Tension with Hindlimb Unloading: Inactivation of Cross-bridges or Activation of Calpains?

The study was aimed at testing the hypotheses about the role of cross-bridges and calpains in reduction of rat soleus passive tension under conditions of hindlimb unloading. For this purpose, we used an inhibitor of µ-calpain PD 150606 as well as a blocker of actomyosin interaction (blebbistatin). It was found for the first time that a decrease in passive tension of rat soleus after 3-day hindlimb unloading is associated with the activity of µ-calpain and does not depend on the processes of cross-bridges formation.

[Mathematical Modelling of the Dependence of the Performance of the Left Ventricle of the Heart on Preload and Afterload].

The results of the numerical simulation of the end-diastolic, end-systolic and stroke volumes of the left ventricle of the heart are presented. The simulation was based on a published simple kinetic model of cardiac muscle and approximation of the ventricle geometry with thick-wall cylinder where the fibre orientation varied linearly from sub-epicardium towards sub-endocardium. Blood flow was modelled with a liner compartment model. This simplified approach provides correct dependencies of the stroke volume on the pre- and afterload, namely end-diastolic pressure and peripheral resistance. The calculations show that the stroke volume is independent of arterial compliance and blood inertia.

Stabilization of the Central Part of Tropomyosin Molecule Alters the Ca2+-sensitivity of Actin-Myosin Interaction.

We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са(2+) concentrations and the Са(2+)-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin-Tm-myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.

Molecular mechanism of actin-myosin motor in muscle.

The interaction of actin and myosin powers striated and smooth muscles and some other types of cell motility. Due to its highly ordered structure, skeletal muscle is a very convenient object for studying the general mechanism of the actin-myosin molecular motor. The history of investigation of the actin-myosin motor is briefly described. Modern concepts and data obtained with different techniques including protein crystallography, electron microscopy, biochemistry, and protein engineering are reviewed. Particular attention is given to X-ray diffraction studies of intact muscles and single muscle fibers with permeabilized membrane as they give insight into structural changes that underlie force generation and work production by the motor. Time-resolved low-angle X-ray diffraction on contracting muscle fibers using modern synchrotron radiation sources is used to follow movement of myosin heads with unique time and spatial resolution under near physiological conditions.

Direct modeling of x-ray diffraction pattern from skeletal muscle in rigor.

Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a "principle of minimal elastic distortion energy" so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the "lever arm" region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and "beating" actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction.

Structural responses to the photolytic release of ATP in frog muscle fibres, observed by time-resolved X-ray diffraction.

1. Structural changes following the photolytic release of ATP were observed in single, permeabilised fibres of frog skeletal muscle at 5-6 C, using time-resolved, low-angle X-ray diffraction. The structural order in the fibres and their isometric function were preserved by cross-linking 10-20 % of the myosin cross-bridges to the thin filaments. 2. The time courses of the change in force, stiffness and in intensity of the main equatorial reflections (1,0) and (1,1), of the third myosin layer line (M3) at a reciprocal spacing of (14.5 nm)-1 on the meridian and of the first myosin-actin layer line (LL1) were measured with 1 ms time resolution. 3. In the absence of Ca2+, photolytic release of ATP in muscle fibres initially in the rigor state caused the force and stiffness to decrease monotonically towards their values in relaxed muscle fibres. 4. In the presence of Ca2+, photolytic release of ATP resulted in an initial rapid decrease in force, followed by a slower increase to the isometric plateau. Muscle fibre stiffness decreased rapidly to approximately 65 % of its value in rigor. 5. In the absence of Ca2+, changes on the equator, in LL1 and in M3 occurred with a time scale comparable to that of the changes in tension and stiffness. 6. In the presence of Ca2+, the changes on the equator and LL1 occurred simultaneously with the early phase of tension decrease. The changes in the intensity of M3 (IM3) occurred on the time scale of the subsequent increase in force. The time courses of the changes in tension and IM3 were similar following the photolytic release of 0. 33 or 1.1 mM ATP. However the gradual return towards the rigor state began earlier when only 0.33 mM ATP was released. 7. In the presence of Ca2+, the time course of changes in IM3 closely mimicked that of force development following photolytic release of ATP. This is consistent with models that propose that force development results from a change in the average orientation of cross-bridges, although other factors, such as their redistribution, may also be involved.

Structural changes in the actin-myosin cross-bridges associated with force generation induced by temperature jump in permeabilized frog muscle fibers.

Structural changes induced by Joule temperature jumps (T-jumps) in frog muscle fibers were monitored using time-resolved x-ray diffraction. Experiments made use of single, permeabilized fibers that were fully activated after slight cross-linking with 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide to preserve their structural order. After T-jumps from 5-6 to approximately 17 degrees C and then on to approximately 30 degrees C, tension increased by a factor of 1.51 and 1.84, respectively, whereas fiber stiffness did not change with temperature. The tension rise was accompanied by a decrease in the intensity of the (1, 0) equatorial x-ray reflection by 15 and 26% (at approximately 17 and approximately 30 degrees C) and by an increase in the intensity of the M3 myosin reflection by 20% and 41%, respectively. The intensity of the (1,1) equatorial reflection increased slightly. The peak of the intensity on the 6th actin layer line shifted toward the meridian with temperature. The intensity of the 1st actin layer line increased from 12% (of its rigor value) at 5-6 degrees C to 36% at approximately 30 degrees C, so that the fraction of the cross-bridges labeling the actin helix estimated from this intensity increased proportionally to tension from approximately 35% at 5-6 degrees C to approximately 60% at approximately 30 degrees C. This suggests that force is generated during a transition of nonstereo-specifically attached myosin cross-bridges to a stereo-specific binding state.

Muscle force is generated by myosin heads stereospecifically attached to actin.

Muscle force is generated by myosin crossbridges interacting with actin. As estimated from stiffness and equatorial X-ray diffraction of muscle and muscle fibres, most myosin crossbridges are attached to actin during isometric contraction, but a much smaller fraction is bound stereospecifically. To determine the fraction of crossbridges contributing to tension and the structural changes that attached crossbridges undergo when generating force, we monitored the X-ray diffraction pattern during temperature-induced tension rise in fully activated permeabilized frog muscle fibres. Temperature jumps from 5-6 degrees C to 16-19 degrees C initiated a 1.7-fold increase in tension without significantly changing fibre stiffness or the intensities of the (1,1) equatorial and (14.5 nm)(-1) meridional X-ray reflections. However, tension rise was accompanied by a 20% decrease in the intensity of the (1,0) equatorial reflection and an increase in the intensity of the first actin layer line by approximately 13% of that in rigor. Our results show that muscle force is associated with a transition of the crossbridges from a state in which they are nonspecifically attached to actin to one in which stereospecifically bound myosin crossbridges label the actin helix.

Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature.

Tension responses to joule temperature jump in skinned rabbit muscle fibres.

1. Joule temperature jumps (T-jumps) from 5-9 degrees C up to 40 degrees C were used to study the cross-bridge kinetics and thermodynamics in skinned rabbit muscle fibres. To produce a T-jump, an alternating current pulse was passed through a fibre 5 s after removing the activating solution (pCa congruent to 4.5) from the experimental trough. The pulse frequency was congruent to 30 kHz, amplitude less than or equal to 3 kV, and duration 0.2 ms. The pulse energy liberated in the fibre was calculated using a special analog circuit and then used for estimation of the T-jump amplitude. 2. The T-jump induced a tri-exponential tension transient. Phases 1 and 2 had rate constants k1 = 450-1750 s-1 and k2 = 60-250 s-1 respectively, characterizing the tension rise, whereas phase 3 had a rate constant k3 = 5-10 s-1 representing tension recovery due to the fibre cooling. 3. An increase from 13 to 40 degrees C for the final temperature achieved by the T-jump led to an increase in the amplitudes of phases 1 and 2. After T-jumps to 30-40 degrees C during phase 1, tension increased by 50-80%. During phase 2 an approximately 2-fold tension increase continued. Rate constants k1 and k2 increased with temperature and temperature coefficients (Q10) were 1.6 and 1.7, respectively. 4. To study which processes in the cross-bridges are involved in phases 1 and 2, a series of experiments were made where step length changes of -9 to +3 nm (hs)-1 (nanometres per half-sarcomere length) were applied to the fibre 4 ms before the T-jump. 5. After the step shortening, the rate constant of phase 1 increased, whereas its amplitude decreased compared to those without a length change. This indicates that phase 1 is determined by some force-generating process in the cross-bridges attached to the thin filaments. This process is, most probably, the same as that producing the early tension recovery following the length change. The enthalpy change (delta H) associated with the reaction controlling this process was estimated to be positive (15-30 kJ mol-1). 6. Both the rate constant k2 and the maximal tension achieved at the end of phase 2 were practically independent of the preceding length changes. This means that phase 2 is accompanied by the cross-bridge detachment and reattachment to new sites on the thin filaments.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of joule temperature jump on tension and stiffness of skinned rabbit muscle fibers.

The effects of a temperature jump (T-jump) from 5-7 degrees C to 26-33 degrees C were studied on tension and stiffness of glycerol-extracted fibers from rabbit psoas muscle in rigor and during maximal Ca2+ activation. The T-jump was initiated by passing an alternating current pulse (30 kHz, up to 2.5 kV, duration 0.2 ms) through a fiber suspended in air. In rigor the T-jump induces a drop of both tension and stiffness. During maximal activation, the immediate stiffness dropped by (4.4 +/- 1.6) x 10(-3)/1 degree C (mean + SD) in response to the T-jump, and this was followed by a monoexponential stiffness rise by a factor of 1.59 +/- 0.14 with a rate constant ks = 174 +/- 42 s-1 (mean +/- SD, n = 8). The data show that the fiber stiffness, determined by the cross-bridge elasticity, in both rigor and maximal activation is not rubber-like. In the activated fibers the T-jump induced a biexponential tension rise by a factor of 3.45 +/- 0.76 (mean +/- SD, n = 8) with the rate constants 500-1,000 s-1 for the first exponent and 167 +/- 39 s-1 (mean +/- SD, n = 8) for the second exponent. The data are in accordance with the assumption that the first phase of the tension transient after the T-jump is due to a force-generating step in the attached cross-bridges, whereas the second one is related to detachment and reattachment of cross-bridges.

Extracellular fluid filtration as the reason for the viscoelastic behaviour of the passive myocardium.

The experimental results are described confirming the hypothesis that the viscous properties of the passive cardiac muscle are connected with the extracellular fluid filtration in the elastic medium formed by the connective tissue network and myocytes. It is shown that the relaxation properties are more pronounced in cold-blooded animals myocardium (frog, turtle) than in that of warm-blooded (cat, rabbit), which correlates with the smaller connective tissue content and larger porosity of myocardium in cold-blooded animals. The decrease in porosity of the cardiac muscle samples by reducing the osmosis of the surrounding solution or squeezing the fluid out of the samples by mechanical torsion results in the slowing down of the stress relaxation, the increase in porosity of the muscle in the hyperosmotic solution results in its speeding up. The increase of the surrounding solution viscosity by adding saccharose to it leads to the proportional stress relaxation deceleration, which agrees quantitatively and qualitatively with the advanced hypothesis.