Weakly attached cross-bridges in relaxed frog muscle fibers.

Tension responses due to small, rapid length changes (completed within 40 microseconds) were obtained from skinned single frog muscle fiber segments (4-10 mm length) incubated in relaxing and rigor solutions at various ionic strengths. The first 2 ms of these responses can be described with a linear model in which the fiber is regarded as a rod, composed of infinitesimally small, identical segments, containing one undamped elastic element and two or three damped elastic elements and a mass in series. Rigor stiffness changed less than 10% in a limited range, 40-160 mM, of ionic strength conditions. Equatorial x-ray diffraction patterns show a similar finding for the filament spacing and intensity ratio I(11)/I(10). Relaxed fibers became stiffer under low ionic strength conditions. This stiffness increment can be correlated with a decreasing filament spacing and (an increased number of) weakly attached cross-bridges. Under low ionic strength conditions an additional recovery (1 ms time constant) became noticeable which might reflect characteristics of weakly attached cross-bridges.     

X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths

Equatorial x-ray diffraction patterns from single skinned rabbit psoas fibers were studied at various ionic strengths to obtain structural information regarding cross-bridge formation in relaxed muscle fibers. At ionic strengths between 20 and 50 mM, the intensity of the 11 reflection, I11, of the relaxed state was close to that of the rigor state, whereas the intensity of the 10 reflection, I10, was approximately twice that of rigor reflection. Calculations by two-dimensional Fourier synthesis indicated that substantial extra mass was associated with the thin filaments under these conditions. With increasing ionic strength between 20 and 100 mM, I10 increased and I11 decreased in an approximately linear way, indicating net transfer of mass away from the thin filaments towards the thick filaments. These results provided evidence that cross-bridges were formed in a relaxed fiber at low ionic strengths, and that the number of cross-bridges decreased as ionic strength was raised. Above mu = 100 mM, I10 and I11 both decreased, indicating the onset of increasing disorder within the filament lattice.     

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.     

Modulation of cross-bridge affinity for MgGTP by Ca2+ in skinned fibers of rabbit psoas muscle.

Previously we reported that saturation of cross-bridges with MgATP gamma S in skinned muscle fibers was calcium sensitive. In the present study we investigate whether this observation can be generalized to other nucleotides by studying saturation of cross-bridges with MgGTP. In solution, myosin-subfragment 1 (S1) in the presence of 10 mM MgGTP was found to bind to actin with low affinity, similar to that in the presence of MgATP and MgATP gamma S. In EGTA buffer, the equatorial x-ray diffraction intensity ratio I11/I10 recorded in single skinned fibers decreased upon increasing MgGTP concentration from 0 to 10 mM (1 degree C and 170 mM ionic strength). The I11/I10 ratio leveled off at 10 mM MgGTP, indicating full saturation of cross-bridges with the nucleotide. Under these conditions, the value of I11/I10 is indistinguishable from that obtained in the presence of saturating [MgATP]. In CaEGTA buffer, however, the decrease in I11/I10 occurs over a wider range of concentrations, and there is no indication of I11/I10 leveling off at 10 mM MgGTP, suggesting that full saturation is not reached. The Ca2+ dependence of GTP binding appears to be a direct consequence of the differences in the affinities of the strongly bound cross-bridges to actin versus weakly bound cross-bridges to actin. A biochemical scheme that could qualitatively explain the titration behavior of ATP gamma S and GTP is presented.     

Cross-bridge attachment during high-speed active shortening of skinned fibers of the rabbit psoas muscle: implications for cross-bridge action during maximum velocity of filament sliding

To characterize the kinetics of cross-bridge attachment to actin during unloaded contraction (maximum velocity of filament sliding), ramp-shaped stretches with different stretch-velocities (2-40,000 nm per half-sarcomere per s) were applied to actively contracting skinned fibers of the rabbit psoas muscle. Apparent fiber stiffness observed during such stretches was plotted versus the speed of the imposed stretch (stiffness-speed relation) to derive the rate constants for cross-bridge dissociation from actin. The stiffness-speed relation obtained for unloaded shortening conditions was shifted by about two orders of magnitude to faster stretch velocities compared to isometric conditions and was almost identical to the stiffness-speed relation observed in the presence of MgATPgammaS at high Ca(2+) concentrations, i.e., under conditions where cross-bridges are weakly attached to the fully Ca(2+) activated thin filaments. These data together with several control experiments suggest that, in contrast to previous assumptions, most of the fiber stiffness observed during high-speed shortening results from weak cross-bridge attachment to actin. The fraction of strongly attached cross-bridges during unloaded shortening appears to be as low as some 1-5% of the fraction present during isometric contraction. This is about an order of magnitude less than previous estimates in which contribution of weak cross-bridge attachment to observed fiber stiffness was not considered. Our findings imply that 1) the interaction distance of strongly attached cross-bridges during high-speed shortening is well within the range consistent with conventional cross-bridge models, i.e., that no repetitive power strokes need to be assumed, and 2) that a significant part of the negative forces that limit the maximum speed of filament sliding might originate from weak cross-bridge interactions with actin.     

X-ray diffraction studies of the thick filament in permeabilized myocardium from rabbit

Low angle x-ray diffraction patterns from relaxed permeabilized rabbit cardiac trabeculae and psoas muscle fibers were compared. Temperature was varied from 25 degrees C to 5 degrees C at 200 mM and 50 mM ionic strengths (mu), respectively. Effects of temperature and mu on the intensities of the myosin layer lines (MLL), the equatorial intensity ratio I(1,1)/I(1,0), and the spacing of the filament lattice are similar in both muscles. At 25 degrees C, particularly at mu = 50 mM, the x-ray patterns exhibited up to six orders of MLL and sharp meridional reflections, signifying that myosin heads (cross-bridges) are distributed in a well-ordered helical array. Decreasing temperature reduced MLL intensities but increased I(1,1)/I(1,0). Decreases in the MLL intensities indicate increasing disorder in the distribution of cross-bridges on the thick filaments surface. In the skeletal muscle, order/disorder is directly correlated with the hydrolysis equilibrium of ATP by myosin, [M.ADP.P(i)]/[M.ATP]. Similar effects of temperature on MLL and similar biochemical ATP hydrolysis pathway found in both types of muscles suggest that the order/disorder states of cardiac cross-bridges may well be correlated with the same biochemical and structural states. This implies that in relaxed cardiac muscle under physiological conditions, the unattached cross-bridges are largely in the M.ADP.P(i) state and with the lowering of the temperature, the equilibrium is increasingly in favor of [M.ATP] and [A.M.ATP]. There appear to be some differences in the diffraction patterns from the two muscles, however. Mainly, in the cardiac muscle, the MLL are weaker, the I(1,1)/I(1,0) ratio tends to be higher, and the lattice spacing D(10), larger. These differences are consistent with the idea that under a wide range of conditions, a greater fraction of cross-bridges is weakly bound to actin in the myocardium.     

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)     

Structures of actomyosin crossbridges in relaxed and rigor muscle fibers.

It was shown previously that a significant fraction of the myosin crossbridges is attached to actin in the skinned rabbit psoas fibers under relaxed conditions at low ionic strength and low temperature (Brenner, B., M. Schoenberg, J. M. Chalovich, L. E. Greene, and E. Eisenberg. 1982. Proc. Natl. Acad. Sci. USA. 79:7288-7291; Brenner, B., L. C. Lu, and R. J. Podolsky. 1984. Biophys. J. 46:299-306). In the present work, the structure of the attached crossbridges in the relaxed state between ionic strengths of 20 and 100 mM, as compared with that in the rigor state, is further examined by equatorial x-ray diffraction. Mass distributions projected along the fiber axis are reconstructed based on the first five equatorial reflections such that the spatial resolution is 128 A. The fraction of crossbridges attached under relaxed conditions are estimated to be in the range of 30% (at 100 mM ionic strength) and 60% (at 20 mM). The reconstructed density maps suggest that in the relaxed state, upon attachment the part of the crossbridge that centers around the thin filament is small, and the attachment does not significantly alter the center of mass of the myosin head distribution around the thick filament backbone. In contrast, accretion of mass in the rigor state occurs in a wider region surrounding the thin filament. In this case, mass in the surface region of the thick filament backbone is shifted slightly outward, probably by approximately 10 A. A schematic model for interpreting the present data is presented.     

Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.     

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.     

Structural characterization of weakly attached cross-bridges in the A*M*ATP state in permeabilized rabbit psoas muscle

It is well established that in a skeletal muscle under relaxing conditions, cross-bridges exist in a mixture of four weak binding states in equilibrium (A*M*ATP, A*M*ADP*P(i), M*ATP, and M*ADP*P(i)). It has been shown that these four weak binding states are in the pathway to force generation. In the past their structural, biochemical, and mechanical properties have been characterized as a group. However, it was shown that the myosin heads in the M*ATP state exhibited a disordered distribution along the thick filament, while in the M*ADP*P(i) state they were well ordered. It follows that the structures of the weakly attached states of A*M*ATP and A*M*ADP*P(i) could well be different. Individual structures of the two attached states could not be assigned because protocol for isolating the two states has not been available until recently. In the present study, muscle fibers are reacted with N-phenylmaleimide such that ATP hydrolysis is inhibited, i.e., the cross-bridge population under relaxing conditions is distributed only between the two states of M*ATP and A*M*ATP. Two-dimensional x-ray diffraction was applied to determine the structural characteristics of the attached A*M*ATP state. Because the detached state of M*ATP is disordered and does not contribute to layer line intensities, changes as a result of increasing attachment in the A*M*ATP state are attributable to that state alone. The equilibrium toward the attached state was achieved by lowering the ionic strength. The results show that upon attachment, both the myosin and the first actin associated layer lines increased intensities, while the sixth actin layer line was not significantly affected. However, the intensities remain weak despite substantial attachment. The results, together with modeling (see J. Gu, S. Xu and L. C. Yu, 2002, Biophys. J. 82:2123-2133), suggest that there is a wide range of orientation of the attached A*M*ATP cross-bridges while the myosin heads maintain some degree of helical distribution on the thick filament, suggesting a high degree of flexibility in the actomyosin complex. Furthermore, the lack of sensitivity of the sixth actin layer line suggests that the binding site on actin differs from the putative site for rigor binding. The significance of the flexibility in the A*M*ATP complex in the process of force generation is discussed.     

X-ray diffraction studies on muscle regulation

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca²⁺ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca²⁺ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case.Troponin molecules also change their conformation upon activation depending on both Ca²⁺ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle.Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle.Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges.Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.     

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.     

Distributions of calcium in A and I bands of skinned vertebrate muscle fibers stretched to beyond filament overlap.

Measurements were made of the distributions of total calcium along the length of A and I bands in skinned frog semitendinosus muscles using electron probe x-ray microanalysis. Since calcium in the water space was kept below the detection limit of the technique, the signal was assumed to reflect the distribution of calcium bound to myofilament proteins. Data from sarcomeres with overlap between thick and thin filaments showed enhancement of calcium in this region, as previously demonstrated in rabbit psoas muscle fibers in rigor (Cantino, M. E., T. S. Allen, and A. M. Gordon. 1993. Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle. Biophys. J. 64:211-222). Such enhancement could arise from intrinsic non-uniformities in calcium binding to either thick or thin filaments or from enhancement of calcium binding to either filament by rigor cross-bridge attachment. To test for intrinsic variations in calcium binding, calcium distributions were determined in fibers stretched to beyond filament overlap. Calcium binding was found to be relatively uniform along both thick and thin filaments, and therefore cannot account for the increased calcium observed in the overlap region. From these results it can be concluded that the observed enhancement of calcium is due to an increase in calcium binding to myofilaments as a result of rigor attachment of cross-bridges to actin. The source of the enhancement is most likely an increase in calcium binding to troponin, although enhancement of calcium binding to myosin light chains cannot be ruled out.     

Unfixed cryosections of striated muscle to study dynamic molecular events.

The structures of the actin and myosin filaments of striated muscle have been studied extensively in the past by sectioning of fixed specimens. However, chemical fixation alters molecular details and prevents biochemically induced structural changes. To overcome these problems, we investigate here the potential of cryosectioning unfixed muscle. In cryosections of relaxed, unfixed specimens, individual myosin filaments displayed the characteristic helical organization of detached cross-bridges, but the filament lattice had disintegrated. To preserve both the filament lattice and the molecular structure of the filaments, we decided to section unfixed rigor muscle, stabilized by actomyosin cross-bridges. The best sections showed periodic, angled cross-bridges attached to actin and their Fourier transforms displayed layer lines similar to those in x-ray diffraction patterns of rigor muscle. To preserve relaxed filaments in their original lattice, unfixed sections of rigor muscle were picked up on a grid and relaxed before negative staining. The myosin and actin filaments showed the characteristic helical arrangements of detached cross-bridges and actin subunits, and Fourier transforms were similar to x-ray patterns of relaxed muscle. We conclude that the rigor structure of muscle and the ability of the filament lattice to undergo the rigor-relaxed transformation can be preserved in unfixed cryosections. In the future, it should be possible to carry out dynamic studies of active sacromeres by cryo-electron microscopy.     

Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle.

By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowy's hypothesis of coexistence of ordered and disordered cross-bridge populations in muscle (Poulsen, F. R., and J. Lowy. 1983. Small angle scattering from myosin heads in relaxed and rigor frog skeletal muscle. Nature (Lond.). 303:146-152.). However, our results added new insights into the disordered population. Present modeling together with data analysis (Xu, S., S. Malinchik, Th. Kraft, B. Brenner, and L. C. Yu. 1997. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys. J. 73:000-000) indicate that in a relaxed muscle, cross-bridges are distributed in three populations: those that are ordered on the thick filament helix and those that are disordered; and within the disordered population, some cross-bridges are detached and some are weakly attached to actin. One critical conclusion of the present study is that the apparent order <--> disorder transition as a function of temperature is not due to an increase/decrease in thermal motion (temperature factor) for the entire population, but a redistribution of cross-bridges among the three populations. Changing the temperature leads to a change in the fraction of cross-bridges located on the helix, while changing the ionic strength at a given temperature affects the disordered population leading to a change in the relative fraction of cross-bridges detached from and weakly attached to actin. Since the redistribution is reversible, we suggest that there is an equilibrium among the three populations of cross-bridges.     

Interplay between passive tension and strong and weak binding cross- bridges in insect indirect flight muscle. A functional dissection by gelsolin-mediated thin filament removal

The interplay between passive and active mechanical properties of indirect flight muscle of the waterbug (Lethocerus) was investigated. A functional dissection of the relative contribution of cross-bridges, actin filaments, and C filaments to tension and stiffness of passive, activated, and rigor fibers was carried out by comparing mechanical properties at different ionic strengths of sarcomeres with and without thin filaments. Selective thin filament removal was accomplished by treatment with the actin-severving protein gelsolin. Thin filament, removal had no effect on passive tension, indicating that the C filament and the actin filament are mechanically independent and that passive tension is developed by the C filament in response to sarcomere stretch. Passive tension increased steeply with sarcomere length until an elastic limit was reached at only 6-7% sarcomere extension, which corresponds to an extension of 350% of the C filament. The passive tension-length relation of insect flight muscle was analyzed using a segmental extension model of passive tension development (Wang, K, R. McCarter, J. Wright, B. Jennate, and R Ramirez-Mitchell. 1991. Proc. Natl. Acad. Sci. USA. 88:7101-7109). Thin filament removal greatly depressed high frequency passive stiffness (2.2 kHz) and eliminated the ionic strength sensitivity of passive stiffness. It is likely that the passive stiffness component that is removed by gelsolin is derived from weak-binding cross-bridges, while the component that remains is derived from the C filament. Our results indicate that a significant number of weak-binding cross-bridges exist in passive insect muscle at room temperature and at an ionic strength of 195 mM. Analysis of rigor muscle indicated that while rigor tension is entirely actin based, rigor stiffness contains a component that resists gelsolin treatment and is therefore likely to be C filament based. Active tension and active stiffness of unextracted fibers were directly proportional to passive tension before activation. Similarly, passive stiffness due to weak bridges also increased linearly with passive tension, up to a limit. These correlations lead us to propose a stress-activation model for insect flight muscle in which passive tension is a prerequisite for the formation of both weak-binding and strong-binding cross-bridges.     

Force-Generating Cross-Bridges during Ramp-Shaped Releases: Evidence for a New Structural State

Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5°C, force transients were slowed sufficiently to record diffraction patterns in several 2-4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATPγS] and at 1°C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.     

Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.     

Characterizations of cross-bridges in the presence of saturating concentrations of MgAMP-PNP in rabbit permeabilized psoas muscle.

Several earlier studies have led to different conclusions about the complex of myosin with MgAMP-PNP. It has been suggested that subfragment 1 of myosin (S1)-MgAMP-PNP forms an S1-MgADP-like state, an intermediate between the myosin S1-MgATP and myosin S1-MgADP states or a mixture of cross-bridge states. We suggest that the different states observed result from the failure to saturate S1 with MgAMP-PNP. At saturating MgAMP-PNP, the interaction of myosin S1 with actin is very similar to that which occurs in the presence of MgATP. 1) At 1 degrees C and 170 mM ionic strength the equatorial x-ray diffraction intensity ratio I11/I10 decreased with an increasing MgAMP-PNP concentration and leveled off by approximately 20 mM MgAMP-PNP. The resulting ratio was the same for MgATP-relaxed fibers. 2) The two dimensional x-ray diffraction patterns from MgATP-relaxed and MgAMP-PNP-relaxed bundles are similar. 3) The affinity of S1-MgAMP-PNP for the actin-tropomyosin-troponin complex in solution in the absence of free calcium is comparable with that of S1-MgATP. 4) In the presence of calcium, I11/I10 decreased toward the relaxed value with increasing MgAMP-PNP, signifying that the affinity between cross-bridge and actin is weakened by MgAMP-PNP. 5) The degree to which the equatorial intensity ratio decreases as the ionic strength increases is similar in MgAMP-PNP and MgATP. Therefore, results from both fiber and solution studies suggest that MgAMP-PNP acts as a non hydrolyzable MgATP analogue for myosin.