Skinned Rabbit Psoas Muscle Fibers Research Articles

Fitting a model to multiscale data suggests thick filament activation can produce force depression in muscle fibers.

Multiscale muscle models aim to connect muscle's molecular and organ-scale function to, e.g., inform the treatment of muscle diseases. Current muscle models have not quantitatively fit data spanning the molecular to cellular scales due, in part, to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single skinned rabbit psoas muscle fibers to ramp shortenings (5% amplitude) and step stretches (0.25%, 0.5%, 0.75% amplitude), performed on the plateau region of the force-length relationship. We isolated myosin from the same muscle source and, under similar conditions, performed single molecule and mini ensemble measurements of event duration and displacement using optical trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of the superrelaxed state, a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force following stretch (residual force enhancement). By optimizing the model fit to fiber measurements, we specified seven unknown parameters. The model then successfully predicted our molecular measurements from the optical trap and in vitro motility at different ATP concentrations. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale modeling efforts. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combined with thick filament activation serve to decrease the number of cycling cross-bridges.

Effect of Inorganic Phosphate and Low pH on the Force-Velocity Relation of Single Skinned Skeletal Muscle Fibers Studied by Applying Parabolic Fiber Length Changes

Although force-velocity (P-V) relation is important to obtain insights into kinetics of actin-myosin interaction in muscle, determination of P-V relation in skinned muscle fibers are difficult mainly due to gradual deterioration at the two cut fiber ends, which makes it impossible to obtain enough data points to construct P-V curve over the whole range of forces. In addition, determination of the maximum shortening velocity Vmax by back extrapolation of limited data points to velocity axis is ambiguous because of the steep force versus velocity relation. To overcome these difficulties, we developed a novel method to obtain continuous P-V curves over the whole range of forces from 0 to the maximum isometric force Po, by applying length changes, with time course L=-kt2, to Ca2+-activated skinned fibers, so that time derivative of length change, i.e. shortening velocity V=-dL/dt=-2kt; namely, V increased linearly with time t. By recording the resulting chane in force P, we could obtain continuous P-V curve in one shot. Using the above method, we examined effect of high inorganic phosphate (Pi) concentration and low pH (6.5) on P-V relation of skinned rabbit psoas muscle fibers at 20°C and at 5°C with the following results: (1) The P-V curves obtained exhibited a distinct hump at high force region >0.5-0.6Po. The P-V curves, deviated from rectangular hyperbola with forces > ~0.2Po, did not change their shape appreciably by Pi and low pH.; (2) Pi (30 mM) reduced Po by ~30% at 20°C and ~45% at 5°C; (3) Low pH (6.5) showed no significant effect on Vmax at both 20 and 5°C; and (4) Pi (30 mM) at low pH (6.5) reduced Po by ~43% at 20°C and ~45% at 5°C. These results are discussed in relation of other published results.

Tension Recovery following Ramp-Shaped Release in High-Ca and Low-Ca Rigor Muscle Fibers: Evidence for the Dynamic State of AMADP Myosin Heads in the Absence of ATP.

During muscle contraction, myosin heads (M) bound to actin (A) perform power stroke associated with reaction, AMADPPi → AM + ADP + Pi. In this scheme, A • M is believed to be a high-affinity complex after removal of ATP. Biochemical studies on extracted protein samples show that, in the AM complex, actin-binding sites are located at both sides of junctional peptide between 50K and 20K segments of myosin heavy chain. Recently, we found that a monoclonal antibody (IgG) to the junctional peptide had no effect on both in vitro actin-myosin sliding and skinned muscle fiber contraction, though it covers the actin-binding sites on myosin. It follows from this that, during muscle contraction, myosin heads do not pass through the static rigor AM configuration, determined biochemically and electron microscopically using extracted protein samples. To study the nature of AM and AMADP myosin heads, actually existing in muscle, we examined mechanical responses to ramp-shaped releases (0.5% of Lo, complete in 5ms) in single skinned rabbit psoas muscle fibers in high-Ca (pCa, 4) and low-Ca (pCa, >9) rigor states. The fibers exhibited initial elastic tension drop and subsequent small but definite tension recovery to a steady level. The tension recovery was present over many minutes in high-Ca rigor fibers, while it tended to decrease quickly in low-Ca rigor fibers. EDTA (10mM, with MgCl2 removed) had no appreciable effect on the tension recovery in high-Ca rigor fibers, while it completely eliminated the tension recovery in low-Ca rigor fibers. These results suggest that the AMADP myosin heads in rigor muscle have long lifetimes and dynamic properties, which show up as the tension recovery following applied release. Possible AM linkage structure in muscle is discussed in connection with the X-ray diffraction pattern from contracting muscle, which is intermediate between resting and rigor muscles.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Enhancement of Force Generated by Individual Myosin Heads in Skinned Rabbit Psoas Muscle Fibers at Low Ionic Strength

Although evidence has been presented that, at low ionic strength, myosin heads in relaxed skeletal muscle fibers form linkages with actin filaments, the effect of low ionic strength on contraction characteristics of Ca2+-activated muscle fibers has not yet been studied in detail. To give information about the mechanism of muscle contraction, we have examined the effect of low ionic strength on the mechanical properties and the contraction characteristics of skinned rabbit psoas muscle fibers in both relaxed and maximally Ca2+-activated states. By progressively decreasing KCl concentration from 125 mM to 0 mM (corresponding to a decrease in ionic strength μ from 170 mM to 50 mM), relaxed fibers showed changes in mechanical response to sinusoidal length changes and ramp stretches, which are consistent with the idea of actin-myosin linkage formation at low ionic strength. In maximally Ca2+-activated fibers, on the other hand, the maximum isometric force increased about twofold by reducing KCl concentration from 125 to 0 mM. Unexpectedly, determination of the force-velocity curves indicated that, the maximum unloaded shortening velocity Vmax, remained unchanged at low ionic strength. This finding indicates that the actin-myosin linkages, which has been detected in relaxed fibers at low ionic strength, are broken quickly on Ca2+ activation, so that the linkages in relaxed fibers no longer provide any internal resistance against fiber shortening. The force-velocity curves, obtained at various levels of steady Ca2+-activated isometric force, were found to be identical if they are normalized with respect to the maximum isometric force. The MgATPase activity of muscle fibers during isometric force generation was found not to change appreciably at low ionic strength despite the two-fold increase in Ca2+-activated isometric force. These results can be explained in terms of enhancement of force generated by individual myosin heads, but not by any changes in kinetic properties of cyclic actin-myosin interaction.

Effect of Antibodies to Myosin Head Reveals Definite Difference between In Vitro Actin-Myosin Sliding and Muscle Contraction

Mechanism of myosin head power stroke, responsible for muscle contraction, still remains to be a matter of debate and speculation. Despite considerable progress in studying actin filament sliding over myosin fixed on a glass surface, it is not clear whether the in vitro actin-myosin sliding takes place by a mechanism similar to contraction in muscle, consisting of three-dimensional myofilament lattice structures. To make this point clear, we prepared two different monoclonal antibodies, one directed to reactive lysine residue close to the myosin head converter region (anti-RLR antibody) while the other directed to two peptides of regulatory light chain in the myosin head lever arm region (anti-LD antibody). We compared the effect of these antibodies on in vitro actin- myosin sliding and contraction of skinned rabbit psoas muscle fibers with the following results: (1) anti-RLR antibody completely inhibited in vitro actin-myosin sliding without changing actin-activated myosin head ATPase activity, while it showed no effect on Ca2+-activated contraction of muscle fibers; (2) anti-LD antibody had no effect on in vitro actin-myosin sliding, but suppressed Ca2+-activated muscle fiber contraction without changing Mg-ATPase activity. These results indicate definite difference between in vitro actin-myosin sliding and muscle contraction.

Sarcomere length-dependent Ca2+ activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca2+ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca2+ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca2+ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca2+ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca2+ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca2+ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.Electronic supplementary materialThe online version of this article (doi:10.1007/s12576-011-0173-8) contains supplementary material, which is available to authorized users.

A Second Look at the Two Phases of Ca2+ Binding to Fast Skinned Fibers

Experiments that replace native TnC with TnCdanz in skinned rabbit psoas muscle fibers generate pCa/Ca2+ binding curves that are left of and roughly parallel to the pCa/force curve (Guth & Potter, 1987). The fluorescence curve best fits two binding phases, #1 with a slope of about one and #2 with a slope of about 3 or more, a cooperative binding that is associated with major force development (Allen et al, 1992, Huang et al, 2001). Phase 1 is also accompanied with subtle increments in force. The force, fluorescence, phase 1 & 2 curves synthesized from mean fitted parameters for 22 experiments are shown. To demonstrate the difference between force and binding, we subtract the force from the fluorescence. The difference curve indicates that phase 1 Ca2+ binding dominates at high pCa then decreases as cooperative binding increases. We argue that the regulatory sites are shifting from normal to cooperative binding and this is why the phase 2 parameters can only approximate force. Because phase 2 binding begins on top of phase 1 the fluorescence curve is shifted left away from force.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Measuring Myosin Light Chain Domain Orientation in the Pre-Power Stroke AlF4 States with a Bifunctional Spin Label in Skinned Muscle Fibers

We are using electron paramagnetic resonance and site-directed spin labeling to measure the orientation of the light chain domain of skeletal muscle myosin in pre-power stroke states trapped by the phosphate analog AlF4. Previous work (Kraft et al. (2005) Proc. Natl. Acad. Sci. USA 102:13861-66) has shown via single-fiber X-ray diffraction that AlF4 traps two distinct pre-powerstroke myosin states in activated muscle. The first state (ADP.AlF4-I) produces a weak actin binding, disordered myosin whereas the second state (ADP.AlF4-II) produces a strong binding, stereospecific actomyosin complex. This weak-to-strong transition is a clear indication of the initiation of the power stroke before phosphate release, but it does not reveal the state of the light chain domain (LCD), which undergoes a rotation during the power stroke. We measured the orientation of the light chain domain by exchanging the native regulatory light chain (RLC) of skinned rabbit psoas muscle fiber bundles with a Di-Cys mutant RLC labeled with a bifunctional spin label. Our group has shown previously (Thompson and Naber et al. 2008 Biophys J, in press) that a bifunctional methanethiosulfonate spin label binds rigidly to myosin and reports protein orientation accurately. EPR spectra of oriented fibers show that the LCD produces a distinct orientation from rigor and relaxation in the ADP.AlF4-II state whereas the ADP.AlF4-I state is indistinguishable from relaxed muscle, supporting the hypothesis that the II state represents a state early in the power stroke with a distinct LCD orientation. This work was supported by NIH (AR32961, AR007612). We thank Bernhard Brenner and Theresia Kraft for guidance.

Changes in Actin Structural Transitions Associated with Oxidative Inhibition of Muscle Contraction

We have used transient phosphorescence anisotropy (TPA) to detect changes in actin structural dynamics associated with oxidative inhibition of muscle contraction. Contractility of skinned rabbit psoas muscle fibers was inhibited by treatment with 50 mM H 2O 2, which induced oxidative modifications in the myosin head and in actin, as previously reported. Using proteins purified from oxidized and unoxidized muscle, we used TPA to measure the effects of weakly (+ATP) and strongly (no ATP) bound myosin heads (S1) on the microsecond dynamics of actin labeled at Cys374 with erythrosine iodoacetamide. Oxidative modification of S1 had no effect on actin dynamics in the absence of ATP (strong binding complex), but restricted the dynamics in the presence of ATP (weakly bound complex). In contrast, oxidative modification of actin did not have a significant effect on the weak-to-strong transitions. Thus, we concluded that (1) the effects of oxidation on the dynamics of actin in the actomyosin complex are predominantly determined by oxidation-induced changes in S1, and (2) changes in weak-to-strong structural transitions in actin and myosin are coupled to each other and are associated with oxidative inhibition of muscle contractility.

Thin filament activation and unloaded shortening velocity of rabbit skinned muscle fibres.

The unloaded shortening velocity of skinned rabbit psoas muscle fibres is sensitive to [Ca2+]. To determine whether Ca2+ affects the unloaded shortening velocity via regulation of crossbridge kinetics or crossbridge number, the shortening velocity was measured following changes in either [Ca2+] or the number of active thin filament regulatory units. The native troponin C (TnC) was extracted and replaced with either cardiac TnC (cTnC) or a mixture of cTnC and an inactive mutant cardiac TnC (CBMII TnC). The unloaded shortening velocity of the cTnC-replaced fibres was determined at various values of [Ca2+] and compared with different cTnC:CBMII TnC ratios at a saturating [Ca2+]. If Ca2+ regulates the unloaded shortening velocity via kinetic modulation, differences in the velocity-tension relationship between the cTnC fibres and the cTnC:CBMII TnC fibres would be apparent. Alternatively, Ca2+ control of the number of active crossbridges would yield similar velocity-tension relationships when comparing the cTnC and cTnC:CBMII TnC fibres. The results show a decline in the unloaded shortening velocity that is determined by the relative tension, defined as the level of thin filament activation, rather than the [Ca2+]. Furthermore, at lower levels of relative tension, the reduction in unloaded shortening is not the result of changes in any cooperative effects of myosin on Ca2+ binding to the thin filament. Rather, it may be related to a decrease in crossbridge-induced activation of the thin filament at the level of the individual regulatory unit. In summary, the results suggest that Ca2+ regulates the unloaded shortening velocity in skinned fibres by reducing the number of crossbridges able to productively bind to the thin filament without affecting any inherent property of the myosin.

Mechanical Defects of Muscle Fibers with Myosin Light Chain Mutants that Cause Cardiomyopathy

Familial hypertrophic cardiomyopathy is a disease caused by single mutations in several sarcomeric proteins, including the human myosin ventricular regulatory light chain (vRLC). The effects of four of these mutations (A13T, F18L, E22K, and P95A) in vRLC on force generation were determined as a function of Ca 2+ concentration. The endogenous RLC was removed from skinned rabbit psoas muscle fibers, and replaced with either rat wildtype vRLC or recombinant rat vRLC (G13T, F18L, E22K, and P95A). Compared to fibers with wildtype rat vRLC, the E22K mutant increased Ca sensitivity of force generation, whereas the G13T and F18L mutants decreased the Ca sensitivity, and the P95A mutant had no significant effect. None of the RLC mutants affected the maximal tension (observed at saturating Ca 2+ concentrations), except for F18L, which decreased the maximal tension to 69 ± 10% of the wildtype value. Of the mutant RLCs, only F18L decreased the cooperativity of activation of force generation. These results suggest that the primary cause of familial hypertrophic cardiomyopathy, in some cases, is perturbation in the Ca sensitivity of force generation, in which Ca-sensitizing or Ca-desensitizing effects can lead to similar disease phenotypes.

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 MgATP γS at saturating [Ca 2+] 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.

A Model of Cross-Bridge Attachment to Actin in the A·M·ATP State Based on X-Ray Diffraction from Permeabilized Rabbit Psoas Muscle

A model of cross-bridges binding to actin in the weak binding A·M·ATP state is presented. The modeling was based on the x-ray diffraction patterns from the relaxed skinned rabbit psoas muscle fibers where ATP hydrolysis was inhibited by N-phenylmaleimide treatment (S. Xu, J. Gu, G. Melvin, L. C. Yu. 2002. Biophys. J. 82:2111–2122). Calculations included both the myosin filaments and the actin filaments of the muscle cells, and the binding to actin was assumed to be single headed. To achieve a good fit, considerable flexibility in the orientation of the myosin head and the position of the S1-S2 junction is necessary, such that the myosin head can bind to a nearby actin whereas the tail end was kept in the proximity of the helical track of the myosin filament. Hence, the best-fit model shows that the head binds to actin in a wide range of orientations, and the tail end deviates substantially from its lattice position in the radial direction (∼60 Å). Surprisingly, the best fit model reveals that the detached head, whose location thus far has remained undetected, seems to be located close to the surface of the myosin filament. Another significant requirement of the best-fit model is that the binding site on actin is near the N terminus of the actin subunit, a position distinct from the putative rigor-binding site. The results support the idea that the essential role played by the weak binding states M·ATP ↔ A·M·ATP for force generation lies in its flexibility, because the probability of attachment is greatly increased, compared with the weak binding M·ADP·P i ↔ A·M·ADP·P i states.

The M·ADP·P i State Is Required for Helical Order in the Thick Filaments of Skeletal Muscle

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49–59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M·ATP (disordered) being favored at 0°C and M·ADP·P i (ordered) at 20°C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M·ADP·P i state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M·ADP·P i state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358–367) so that the myosin was exclusively in the M·ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M·NDP·P i state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M·NDP·P i state is 143.4 Å, but in the disordered state, it is 144.2 Å. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.

Image-analysis-based assessment of the effects of the "Ca2+-jump" technique on sarcomere uniformity.

A new image analysis-based technique was used to quantitatively examine the effects of the "Ca2+-jump" activation protocol on the maintenance of fiber quality in skinned rabbit psoas muscle fiber segments. Specifically, contractions in pCa 4.6 were preceded by short-duration "preactivation" soaks in a solution in which EGTA was replaced with the low-Ca2+ buffering capacity analog hexamethylenediamine-N, N, N', N'-tetraacetate, which facilitated rapid Ca2+ equilibration within the fiber segments. Fiber quality was assessed by examining the Fourier spectra of the muscle fiber images before, during, and after activation. Segment lengths were typically below 500 micrometer, thus allowing the majority of the sarcomeres to be visualized in the field of view (x200 and x400 magnification). The preactivation protocol resulted in less deterioration of fiber quality with repetitive activation. In addition, there was also a significant reduction in the time required to reach the 50% level of maximum tension, with no significant change in the maximum tension level.

ATP Analogs and Muscle Contraction: Mechanics and Kinetics of Nucleoside Triphosphate Binding and Hydrolysis

The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin ( V f) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis. Whereas all NTPs studied support force production and stiffness that vary by a factor 2 or less, the unloaded shortening velocity ( V u) of muscle fibers varies by almost 10-fold. 2-Deoxy ATP (dATP) was unique in that V u was 30% greater than with ATP. Parallel behavior was observed between V f and the steady-state maximum actin-activated HMM ATPase rate. Further comparisons suggest that the variation in force correlates with the rate and equilibrium constant for NTP cleavage; the variations in V u or V f are related to the rate of cross-bridge dissociation caused by NTP binding or to the rate(s) of product release.

X-ray diffraction studies of the cross-bridge intermediate states.

Two dimensional x-ray diffraction was obtained from skinned rabbit psoas muscle fibers. The goal is to correlate structures of the cross-bridge population with various intermediate states in the cross-bridge cycle by using nucleotides and their analogs. It was found that in a relaxed muscle in ATP containing solutions, cross-bridges are distributed in three populations in equilibrium: those detached from actin and ordered on the myosin helix, those that are detached and disordered, and those weakly attached to actin in random orientations. The distribution among the three populations is highly dependent on temperature. Those that are detached and yet ordered in a helical structure surrounding the myosin backbone are very likely in the M.ADP.Pi state, supporting an earlier suggestion by Wray (1987). It was also found that the attached cross-bridges with bound MgADP are structurally distinct from those without nucleotide, in agreement with one of our earlier findings by osmotic compression (Xu et al., 1993). Another finding of interest is that the analog AMP-PNP was found to be an ATP analog, rather than an ADP analog as it has been reported previously by many research groups.

Length-dependence of actin-myosin interaction in skinned cardiac muscle fibers in rigor.

It has been suggested that the length dependence of myofilament Ca2+ sensitivity and of Ca2+ binding to troponin C, observed over the ascending limb of the cardiac force-length curve, is based on variation in the number of interacting cross-bridges. This interaction would be reduced at short sarcomere length as a consequence of double overlap of oppositely polarized actin filaments and increased lateral separation of actin and myosin filaments. Based on current evidence, it is not clear to what extent the actin-myosin interaction is hindered at sarcomere lengths where Ca2+ sensitivity is reduced. We have used two biochemical assays to assess cross-bridge attachment in rigor muscle at sarcomere lengths corresponding to the ascending limb of the cardiac force-length curve. These are based on (1) the inhibition of K+-activated myosin ATPase by the complexation of actin with myosin, and (2) the enhancement of Ca2+ binding to troponin C by rigor bridge attachment to actin. Measurements were made with skinned fibers from bovine ventricle. As a check on our method, measurements were also made with skinned rabbit psoas muscle fibers. With both muscle types, a reduction in sarcomere length along the ascending limb of the force-length curve was associated with an increase in K+-activated ATPase activity and a reduction in Ca2+ binding to the regulatory sites of troponin C. These results indicate that actin-myosin interaction is significantly reduced at short sarcomere length.

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.