Millisecond-scale biochemical response to change in strain

Muscle fiber contraction involves the cyclical interaction of myosin cross-bridges with actin filaments, linked to hydrolysis of ATP that provides the required energy. We show here the relationship between cross-bridge states, force generation, and Pi release during ramp stretches of active mammalian skeletal muscle fibers at 20°C. The results show that force and Pi release respond quickly to the application of stretch: force rises rapidly, whereas the rate of Pi release decreases abruptly and remains low for the duration of the stretch. These measurements show that biochemical change on the millisecond timescale accompanies the mechanical and structural responses in active muscle fibers. A cross-bridge model is used to simulate the effect of stretch on the distribution of actomyosin cross-bridges, force, and Pi release, with explicit inclusion of ATP, ADP, and Pi in the biochemical states and length-dependence of transitions. In the simulation, stretch causes rapid detachment and reattachment of cross-bridges without release of Pi or ATP hydrolysis.     

Changes in orientation of actin during contraction of muscle

It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.     

Correlation between mechanical and enzymatic events in contracting skeletal muscle fiber

The conventional hypothesis of muscle contraction postulates that the interaction between actin and myosin involves tight coupling between the power stroke and hydrolysis of ATP. However, some in vitro experiments suggested that hydrolysis of a single molecule of ATP caused multiple mechanical cycles. To test whether the tight coupling is present in contracting muscle, we simultaneously followed mechanical and enzymatic events in a small population of cross-bridges of glycerinated rabbit psoas fibers. Such small population behaves as a single cross-bridge when muscle contraction is initiated by a sudden release of caged ATP. Mechanical events were measured by changes of orientation of probes bound to the regulatory domain of myosin. Enzymatic events were simultaneously measured from the same cross-bridge population by the release of fluorescent ADP from the active site. If the conventional view were true, ADP desorption would occur simultaneously with dissociation of cross-bridges from thin filaments and would be followed by cross-bridge rebinding to thin filaments. Such sequence of events was indeed observed in contracting muscle fibers, suggesting that mechanical and enzymatic events are tightly coupled in vivo.     

Rigor-force producing cross-bridges in skeletal muscle fibers activated by a substoichiometric amount of ATP

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of approximately 100 microM ATP, approximately 2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (approximately 50% of the maximal active force), which gradually recovered to the original force level with a time constant of approximately 4 s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1-1.9 s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.     

Cleavage of DNA-binding loops of actin by subtilisin prevent formation of a strong type of myosin binding with actin

In order to elucidate the role of DNA-binding loop of actin (amino acid residues 38-52) in mechanisms of muscle contraction, polarizational fluorimetry and ghost muscle fibers, containing thin filaments reconstructed by intact and subtilisin-cleaved G-actin were used. The thin filaments were modified by fluorescent probes rhodamin-phalloidin and 1,5-IAEDANS. Changes in orientation and mobility of the probes were considered as an indication of changes in actin conformation. The stage AM of ATP hydrolysis cycle was simulated. For this purpose, thin filaments were decorated by myosin subfragment-1 (S1) in the absence of nucleotide. It has been shown that S1 binding to actin is accompanied by changes in orientation and mobility of the fluorescent probes. For intact filaments, the changes of these parameters indicate the formation of a strong binding between S1 and actin. Cleavage of DNA-binding loop by subtilisin markedly inhibits this effect. The cleavage of actin by subtilisin has also been shown to diminish the changes in fiber birefringence, which takes place at the formation of F-actin-S1 complex in the muscle fiber. The spatial organization of the actin DNA-binding loop is suggested to play an important role in determining the character of myosin interaction with actin in the ATP hydrolysis cycle.     

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.     

Sliding distance per ATP molecule hydrolyzed by myosin heads during isotonic shortening of skinned muscle fibers.

We measured isotonic sliding distance of single skinned fibers from rabbit psoas muscle when known and limited amounts of ATP were made available to the contractile apparatus. The fibers were immersed in paraffin oil at 20 degrees C, and laser pulse photolysis of caged ATP within the fiber initiated the contraction. The amount of ATP released was measured by photolyzing 3H-ATP within fibers, separating the reaction products by high-pressure liquid chromatography, and then counting the effluent peaks by liquid scintillation. The fiber stiffness was monitored to estimate the proportion of thick and thin filament sites interacting during filament sliding. The interaction distance, Di, defined as the sliding distance while a myosin head interacts with actin in the thin filament per ATP molecule hydrolyzed, was estimated from the shortening distance, the number of ATP molecules hydrolyzed by the myosin heads, and the stiffness. Di increased from 11 to 60 nm as the isotonic tension was reduced from 80% to 6% of the isometric tension. Velocity and Di increased with the concentration of ATP available. As isotonic load was increased, the interaction distance decreased linearly with decrease of the shortening velocity and extrapolated to 8 nm at zero velocity. Extrapolation of the relationship between Di and velocity to saturating ATP concentration suggests that Di reaches 100-190 nm at high shortening velocity. The interaction distance corresponds to the sliding distance while cross-bridges are producing positive (working) force plus the distance while they are dragging (producing negative forces). The results indicate that the working and drag distances increase as the velocity increases. Because Di is larger than the size of either the myosin head or the actin monomer, the results suggest that for each ATPase cycle, a myosin head interacts mechanically with several actin monomers either while working or while producing drag.     

Mechanical and structural properties underlying contraction of skeletal muscle fibers after partial 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide cross-linking.

We show prolonged contraction of permeabilized muscle fibers of the frog during which structural order, as judged from low-angle x-ray diffraction, was preserved by means of partial cross-linking of the fibers using the zero-length cross-linker 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide. Ten to twenty percent of the myosin cross-bridges were cross-linked, allowing the remaining 80-90% to cycle and generate force. These fibers displayed a well-preserved sarcomeric order and mechanical characteristics similar to those of intact muscle fibers. The intensity of the brightest meridional reflection at 14.5 nm, resulting from the projection of cross-bridges evenly spaced along the myofilament length, decreased by 60% as a relaxed fiber was deprived of ATP and entered the rigor state. Upon activation of a rigorized fiber by the addition of ATP, the intensity of this reflection returned to 97% of the relaxed value, suggesting that the overall orientation of cross-bridges in the active muscle was more perpendicular to the filament axis than in rigor. Following a small-amplitude length step applied to the active fibers, the reflection intensity decreased for both releases and stretches. In rigor, however, a small stretch increased the amplitude of the reflection by 35%. These findings show the close link between cross-bridge orientation and tension changes.     

A physical model of ATP-induced actin-myosin movement in vitro.

The nature of the mechanism limiting the velocity of ATP-induced unidirectional movements of actin-myosin filaments in vitro is considered. In the sliding process two types of "cyclic" interactions between myosin heads and actin are involved, i.e., productive and nonproductive. In the productive interaction, myosin heads split ATP and generate a force which produces sliding between actin and myosin. In the nonproductive interaction "cycle," on the other hand, myosin heads rapidly attach to and detach from actin "reversibly," i.e., without splitting ATP or generating an active force. Such a nonproductive interaction "cycle" causes irreversible dissipation of sliding energy into heat, because the myosin cross-bridges during this interaction are passive elastic structures. This consideration has led us to postulate that such cross-bridges, in effect, exert viscous-like frictional drag on moving elements. Energetic considerations suggest that this frictional drag is much greater than the hydrodynamic viscous drag. We present a model in which the sliding velocity is limited by the balance between the force generated by myosin cross-bridges in the productive interaction and the frictional drag exerted by other myosin cross-bridges in the nonproductive interaction. The model is consistent with experimental findings of in vitro sliding, including the dependence of velocity on ATP concentration, as well as the sliding velocity of co-polymers of skeletal muscle myosin and phosphorylated and unphosphorylated smooth muscle myosins.     

Activating efficiency of Ca2+ and cross-bridges as measured by phosphate analog release

To assess the activating efficiency of Ca2+ and cross-bridges, the release rates of phosphate analogs from skinned fibers were estimated from the recovery of contractility and that of stiffness. Estimations were performed based on the assumptions that contractility was indicative of the population of analog-free myosin heads and that stiffness reflected the population of formed cross-bridges. Aluminofluoride (AlFx) and orthovanadate (Vi) were used as phosphate analogs with mechanically skinned fibers from rabbit psoas muscle. The use of the analogs enabled the functional assessment of activation level in the total absence of ATP. Fibers loaded with the analogs gradually recovered contractility and stiffness in normal plain rigor solution. The addition of Ca2+ to the plain rigor solution significantly accelerated their recovery, whereas ADP had no appreciable effect. ATP plus Ca2+(contracting condition) accelerated the recovery by several tens of times. These results indicate that the cross-bridges formed during contraction have prominent activating efficiency, which is indispensable to attain full activation. A comparison between the activating efficiency evaluated from stiffness and that from contractility suggested that Ca2+ is more potent in accelerating the binding of actin to analog-bound myosin heads whereas cross-bridges mainly accelerate the subsequent analog-releasing step.     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.     

Calcium regulates scallop muscle by changing myosin flexibility

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called “thick filament regulation” is quite different to vertebrate striated muscle which is switched on and off via “thin filament regulation” whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation. This paper is not being submitted elsewhere and the authors have no competing financial interests     

Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding

Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.     

GFP-tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber

Myosin is the molecular motor in muscle-binding actin and executing a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. A green fluorescent protein (GFP)-tagged human cardiac myosin regulatory light chain (HCRLC) was constructed to study in situ lever arm orientation one molecule at a time by polarized fluorescence emitted from the GFP probe. The recombinant protein physically and functionally replaced the native RLC on myosin lever arms in the thick filaments of permeabilized skeletal muscle fibers. Detecting single molecules in fibers where myosin concentration reaches 300 microM is accomplished using total internal reflection fluorescence microscopy. With total internal reflection fluorescence, evanescent field excitation, supercritical angle fluorescence detection, and CCD detector pixel size limits detection volume to just a few attoliters. Data analysis manages both the perturbing effect of the TIR interface on probe emission and the effect of high numerical aperture collection of light. The natural myosin concentration gradient in a muscle fiber allows observation of fluorescence polarization from C-term GFP-tagged HCRLC exchanged myosin from regions in the thick filament containing low and high myosin concentrations. In rigor, cross-bridges at low concentration at the end of the thick filament maintain GFP dipole moments at two distinct polar angles relative to the fiber symmetry axis. The lower angle, where the dipole is nearly parallel to fiber axis, is more highly populated than the alternative, larger angle. Cross-bridges at higher concentration in the center of the thick filament are oriented in a homogeneous band at approximately 45 degrees to the fiber axis. The data suggests molecular crowding impacts myosin conformation, implying mutual interactions between cross-bridges alter how the muscle generates force. The GFP-tagged RLC is a novel probe to assess single-lever-arm orientation characteristics in situ.     

Structural features of cross-bridges in isometrically contracting skeletal muscle

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1 nm and of the first actin layer line at 37 nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPgammaS at saturating [Ca2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.     

Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers

This paper presents the results of simultaneous measurements of the electron paramagnetic resonance signal of spin-label bound to myosin cross-bridges and the mechanical response of glycerol-treated rabbit psoas fibers under isometric contraction. No observable change has been detected in vitro in the local motion of spin-label bound to myosin-ATP with conventional electron paramagnetic resonance techniques when F-actin is added, even under conditions where more than 30% of the myosin is expected to be in an attached state. In contrast, a clear change in the spin-label mobility is observed when cross-bridges are attached to thin filaments. Similar spectra are also observed when cross-bridges are in the rigor state or in an attached state in the presence of 5′-adenylyl imidodiphosphate in place of ATP. A good proportionality is found between the change in the electron paramagnetic resonance signal and the tension when substrate concentration is varied under conditions where no appreciable amount of rigor complex is present. Thus, by assuming 0 and 100% attachment in the relaxed and rigor states, respectively, the extent of cross-bridge attachment can be estimated; it is about 80% at a relatively low ATP concentration where the maximum tension is observed, while it is about 35% in the millimolar range of ATP concentration. A consistent explanation can be given for the spectra obtained both in solution and in the fiber, provided that two distinct states, the preactive and active states, exist in cross-bridges attached to thin filaments. The contribution of intermediate complexes to the force generation is discussed. The effect of Ca²⁺ control on cross-bridge attachment is also studied at various concentrations of substrate.     

Myosin regulatory domain orientation in skeletal muscle fibers: application of novel electron paramagnetic resonance spectral decomposition and molecular modeling methods

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.     

Conformation of the myosin motor during force generation in skeletal muscle

Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide.     

Effects of hindlimb unweighting and aging on rat semimembranosus muscle and myosin.

We tested the hypothesis that lower specific force (force/cross-sectional area) generated by type II fibers from hindlimb-unweighted rats resulted from structural changes in myosin (i.e., a change in the ratio of myosin cross bridges in the weak- and strong-binding state during contraction). In addition, we determined whether those changes were age dependent. Permeabilized semimembranosus muscle fibers from young adult and aged rats, some of which were hindlimb unweighted for 3 wk, were studied for Ca(2+)-activated force generation and maximal unloaded shortening velocity. Fibers were also spin labeled specifically at myosin Cys707 to assess the structural distribution of myosin during maximal isometric contraction using electron paramagnetic resonance spectroscopy. Myosin heavy chain isoform (MHC) expression and the ratio of MHC to actin were evaluated in each fiber. Fibers from the unweighted rats generated 34% less specific force than fibers from weight-bearing rats (P < 0.001), independent of age. Electron paramagnetic resonance analyses showed that the fraction of myosin heads in the strong-binding structural state during contraction was 11% lower in fibers from the unweighted rats (P = 0.019), independent of age. More fibers from unweighted rats coexpressed MHC IIB-IIX compared with fibers from weight-bearing rats (P = 0.049). Unweighting induced a slowing of maximal unloaded shortening velocity and an increase in the ratio of MHC to actin in fibers from young rats only. These data indicate that altered myosin structural distribution during contraction and a preferential loss of actin contribute to unweighting-induced muscle weakness. Furthermore, the age of the rat has an influence on some parameters of changes in muscle contractility that are induced by unweighting.