Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.     

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

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.     

BDM compared with P(i) and low Ca2+ in the cross-bridge reaction initiated by flash photolysis of caged ATP.

Using flash photolysis of caged ATP in skinned muscle fibers from rat psoas, we examined the inhibitory effects of 2,3-butanedione monoxime (BDM) on the contraction kinetics and the rate of ATP hydrolysis of the cross-bridges at approximately 10 degrees C. The hydrolysis rate was estimated from the stiffness records. The effects of BDM were compared with those of orthophosphate (P(i)) and of reduction in [Ca2+] (low Ca2+), and it was found that i) BDM and low Ca2+ inhibited ATPase activity to the same extent as they inhibited the steady tension, whereas P(i) inhibited ATPase activity much less than tension; ii) BDM and P(i) decreased tension per stiffness during the steady contraction more than did low Ca2+; iii) neither BDM nor low Ca2+ affected the initial relaxation of the fiber on release of ATP, but P(i) slightly slowed it; and iv) BDM hardly influenced the rate of contraction development after relaxation, although P(i) and low Ca2+ accelerated it. We concluded that BDM inhibits the Ca(2+)-regulated attachment of the cross-bridges and force-generation of the attached cross-bridges.     

Activation of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP.

The kinetics of force production in chemically skinned trabeculae from the guinea pig were studied by laser photolysis of caged ATP in the presence of Ca2+. Preincubation of the tissue during rigor with the enzyme apyrase was used to reduce the population of MgADP-bound cross-bridges (Martin and Barsotti, 1994). In untreated tissue, tension remained constant or dipped slightly below the rigor level immediately after ATP release, before increasing to the maximum measured in pCa 4.5 and 5 mM MgATP. The in-phase component stiffness, which is a measure of cross-bridge attachment, exhibited a large decrease before increasing to 55% of that measured in rigor. Neither the rate of the decline nor of the rise in tension was sensitive to the concentration of photolytically released ATP. The rate of the decline in stiffness was found to be dependent on [ATP]: 1.8 x 10(4) M-1/s-1, a value more than four times higher than that previously measured in similar experiments in the absence of Ca2+. The rate of tension development averaged 14.9 +/- 2.5 s-1. Preincubation with apyrase altered the mechanical characteristics of the early phase of the contraction. The rate and amplitude of the initial drop in both tension and stiffness after caged ATP photolysis increased and became dependent on [ATP]. The second-order rate constants measured for the initial drop in tension and stiffness were 8.4 x 10(4) M-1 s-1 and 1.5 x 10(5) M-1 s-1. These rates are more than two times faster than those previously measured in the absence of Ca2+. The effects of apyrase incubation on the time course of tension and stiffness were consistent with the hypothesis that during rigor, skinned trabeculae retain a significant population of MgADP-bound cross-bridges. These in turn act to attenuate the initial drop in tension after caged ATP photolysis and slow the apparent rate of rigor cross-bridge detachment. The results also show that Ca2+ increases the rate of cross-bridge detachment in both untreated and apyrase-treated tissue, but the effect is larger in untreated tissue. This suggests that in cardiac muscle Ca2+ modulates the rate of cross-bridge detachment.     

Cross-bridge scheme and force per cross-bridge state in skinned rabbit psoas muscle fibers.

The rate and association constants (kinetic constants) which comprise a seven state cross-bridge scheme were deduced by sinusoidal analysis in chemically skinned rabbit psoas muscle fibers at 20 degrees C, 200 mM ionic strength, and during maximal Ca2+ activation (pCa 4.54-4.82). The kinetic constants were then used to calculate the steady state probability of cross-bridges in each state as the function of MgATP, MgADP, and phosphate (Pi) concentrations. This calculation showed that 72% of available cross-bridges were (strongly) attached during our control activation (5 mM MgATP, 8 mM Pi), which agreed approximately with the stiffness ratio (active:rigor, 69 +/- 3%); active stiffness was measured during the control activation, and rigor stiffness after an induction of the rigor state. By assuming that isometric tension is a linear combination of probabilities of cross-bridges in each state, and by measuring tension as the function of MgATP, MgADP, and Pi concentrations, we deduced the force associated with each cross-bridge state. Data from the osmotic compression of muscle fibers by dextran T500 were used to deduce the force associated with one of the cross-bridge states. Our results show that force is highest in the AM*ADP.Pi state (A = actin, M = myosin). Since the state which leads into the AM*ADP.Pi state is the weakly attached AM.ADP.Pi state, we confirm that the force development occurs on Pi isomerization (AM.ADP.Pi --> AM*ADP.Pi). Our results also show that a minimal force change occurs with the release of Pi or MgADP, and that force declines gradually with ADP isomerization (AM*ADP -->AM.ADP), ATP isomerization (AM+ATP-->AM*ATP), and with cross-bridge detachment. Force of the AM state agreed well with force measured after induction of the rigor state, indicating that the AM state is a close approximation of the rigor state. The stiffness results obtained as functions of MgATP, MgADP, and Pi concentrations were generally consistent with the cross-bridge scheme.     

Stiffness and fraction of Myosin motors responsible for active force in permeabilized muscle fibers from rabbit psoas

The stiffness of the single myosin motor (epsilon) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T0) modulated by changing either [Ca2+] or temperature. The hs compliance is 43.0+/-0.8 nm MPa-1 in isometric contraction at saturating [Ca2+], whereas in rigor it is 28.2+/-1.1 nm MPa-1. The equivalent compliance of myofilaments is 21.0+/-3.3 nm MPa-1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5+/-1.7 kPa nm-1 in isometric contraction at saturating [Ca2+] (e0), and in rigor (er) it rises to 138.9+/-21.2 kPa nm-1. Epsilon, calculated from er and the lattice molecular dimensions, is 1.21+/-0.18 pN nm-1. epsilon estimated, using a thermodynamic approach, from the relation of T0 at saturating [Ca2+] versus the reciprocal of absolute temperature is 1.25+/-0.14 pN nm-1, similar to that estimated for fibers in rigor. Consequently, the ratio e0/er (0.33+/-0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e0 and er increase by approximately 40%. Epsilon becomes approximately 1.7 pN nm-1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.     

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.     

Cross-bridge kinetics, cooperativity, and negatively strained cross- bridges in vertebrate smooth muscle. A laser-flash photolysis study

The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig protal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an approximately 45 degree angle. In the absence of Ca2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some cross-bridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM Pi, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca2+, was estimated to be 0.1-2.5 X 10(5) M-1s-1, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca2+, force development occurred at a rate (0.4 s-1) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP gamma S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca2+, consistent with cooperative attachment of cross-bridges. Cooperative reattachment of dephosphorylated cross-bridges may contribute to force maintenance at low energy cost and low cross-bridge cycling rates in smooth muscle.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

A birefringence study of changes in myosin orientation during relaxation of skinned muscle fibers induced by photolytic ATP release.

The birefringence of isolated skinned fibers from rabbit psoas muscle was measured continuously during relaxation from rigor produced by photolysis of caged ATP at sarcomere length 2.8-2.9 microns, ionic strength 0.1 M, 15 degrees C. Birefringence, the difference in refractive index between light components polarized parallel and perpendicular to the fiber axis, depends on the average degree of alignment of the myosin head domain with the fiber axis. After ATP release birefringence increased by 5.8 +/- 0.7% (mean +/- SE, n = 6) with two temporal components. A small fast component had an amplitude of 0.9 +/- 0.2% and rate constant of 63 s-1. By the completion of this component, the instantaneous stiffness had decreased to about half the rigor value, and the force response to a step stretch showed a rapid (approximately 1000 s-1) recovery phase. Subsequently a large slow birefringence component with rate constant 5.1 s-1 accompanied isometric force relaxation. Inorganic phosphate (10 mM) did not affect the fast birefringence component but accelerated the slow component and force relaxation. The fast birefringence component was probably caused by formation of myosin.ATP or myosin.ADP.Pi states that are weakly bound to actin. The average myosin head orientation at the end of this component is slightly more parallel to the fiber axis than in rigor.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.     

Behavior of N-phenylmaleimide-reacted muscle fibers in magnesium-free rigor solution.

Using x-ray diffraction and mechanical stiffness, the response of N-phenylmaleimide (NPM)-reacted cross-bridges to solutions containing different amounts of ATP and Mg2+ has been studied. In relaxing solution containing greater than millimolar amounts of ATP and Mg2+, NPM-treated muscle fibers give x-ray diffraction patterns and stiffness records, which are nearly indistinguishable from those of untreated relaxed fibers. In a solution devoid of added ATP, but with Mg2+ (rigor(+Mg) solution), the muscle fibers still give x-ray diffraction patterns and mechanical responses characteristic of relaxed muscle. The new finding reported here is that in a solution devoid of both ATP and Mg2+ (rigor(-Mg) solution containing EDTA with no added ATP), NPM-reacted cross-bridges do give rigor-like behavior. This is the first report that NPM-reacted cross-bridges, at least in the presence of EDTA, are capable of going into a strongly binding conformation.     

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.     

Evidence for cooperative interactions of myosin heads with thin filament in the force generation of vertebrate skeletal muscle fibers

To examine the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA-and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-phenylenedimaleimide (p-PDM)-treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one, and two of their heads inactivated, and the relationships among these values (expressed relative to the control values) were studied. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, muscle fiber stiffness is determined by the total number of native heads, and there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.     

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.     

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.     

Elastic properties of relaxed, activated, and rigor muscle fibers measured with microsecond resolution.

Tension responses due to small and rapid length changes (completed within 40 microseconds) were obtained from skinned single-fiber segments (4- to 7-mm length) of the iliofibularis muscle of the frog incubated in relaxing, rigor, and activating solution. The fibers were skinned by freeze-drying. The first 500 microseconds of the responses for all three conditions could be described with a linear model, in which the fiber is regarded as a rod composed of infinitesimally small identical segments, containing an undamped elastic element, two damped elastic elements and a mass in series. An additional damped elastic element was needed to describe tension responses of activated fibers up to the first 5 ms. Consequently phase 1 and phase 2 of activated fibers can be described with four apparent elastic constants and three time constants. The results indicate that fully activated fibers and fibers in rigor have similar elastic properties within the first 500 microseconds of tension responses. This points either to an equal number of attached cross-bridges in rigor and activated fibers or to a different number of attached cross-bridges in rigor and activated fibers and nonlinear characteristics in rigor cross-bridges. Mass-shift measurements obtained from equatorial x-ray diffraction patterns support the latter possibility.     

Radial stiffness of frog skinned muscle fibers in relaxed and rigor conditions.

Radial stiffness in various conditions of mechanically skinned fibers of semitendinosus muscle of Rana catesbeiana was determined by compressing the fiber with polyvinylpyrrolidone (PVP K-30, Mr = 40,000) in incubating solution. The change in width (D) of fibers with increasing and decreasing PVP concentrations was highly reproducible at a range 0-6% PVP. Radial stiffness of relaxed fibers was almost independent of the sarcomere length. On the other hand, radial stiffness of rigor fibers showed a linear relation against the sarcomere length. These results indicate that cross-bridge attachment would be a major factor in the increase of the radial stiffness. Radial stiffness of relaxed and rigor fibers was (2.14 +/- 0.52) X 10(4) N/m2 (mean +/- SD) and (8.76 +/- 2.04) X 10(4) N/m2, respectively, at the relative fiber width (D/D0) of 0.92, where D0 denotes the fiber width in the rigor solution at 0% PVP. Radial stiffness of a fiber in a rigor solution containing pyrophosphate (PPi) was between those of relaxed and rigor fibers, i.e., (4.76 +/- 0.86) X 10(4) N/m2 at D/Do of 0.92. In PPi and rigor solutions, radial stiffness reversibly increased to around 150 and 130%, respectively, in the presence of 10(-6) M Ca2+. To explain these results, especially the Ca2+-induced change in the radial stiffness, some factor in addition to the number of attached cross-bridges has to be taken into account. The variation of radial stiffness under various conditions will be discussed in relation to the possible manner of cross-bridge attachment.