Leepo C. Yu

Equatorial x-ray diffraction from single skinned rabbit psoas fibers at various degrees of activation. Changes in intensities and lattice spacing

Equatorial x-ray diffraction patterns were obtained from single skinned rabbit psoas fibers during various degrees of activation under isometric conditions at ionic strength 170 mM and 6-9 degrees C. By direct calcium activation, contraction was homogeneous throughout the preparation, and by using a cycling technique (Brenner, 1983) integrity of the fiber was maintained even during prolonged steady activation. The intensity ratio of the two innermost reflections I11/I10, and the normalized intensities I*10 and I*11 varied linearly with increasing force. Thus the result agreed qualitatively with an earlier finding, obtained from the whole sartorius muscle, that intensity changes in 10 and 11 are directly correlated with isometric force level (Yu et al., 1979). Spacing of the myofilament lattice (d10) was found to decrease with increasing isometric tension. With the filaments in full overlap, maximum shrinkage was 14%. The lattice spacing started to level off when the degree of calcium activation was greater than or equal to 50%, approaching a limit approximately at 380-360 A. This decrease of the lattice spacing indicates that there is a radial force produced by force generating cross-bridges, but the net radial force appears to become insignificant as lattice spacing approaches 380-360 A.

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.

Distribution of mass in relaxed frog skeletal muscle and its redistribution upon activation.

Five orders of equatorial reflection were recorded from both relaxed and fully activated intact frog sartorius muscle using synchrotron x-ray radiation. Electron density maps of the myofilament lattice in axial projection were calculated from the integrated intensities by Fourier synthesis, using all possible phase combinations. These maps were evaluated systematically in terms of their compatibility with electron microscopically and biochemically derived properties of the lattice structure and with the minimum wavelength principle. For the relaxed state, one phase combination emerged as most consistent with these constraints: it shows a thick filament with a compact core surrounded by an annular shell of density. The distribution of mass suggests that the S-2 moiety of the myosin molecule is an integral part of the thick-filament backbone and the S-1 moiety makes up the shell and is tilted or slewed around the backbone. For the active state, there are two feasible maps, which differ according to whether or not the activation process is associated with phase inversion in two of the reflections. Both maps represent patterns of redistribution of mass upon activation in which the thick-filament backbone is practically unaffected and there is movement of density from the annular shell towards the thin filaments. In addition to this outward radial flux of density from the thick-filament periphery, the pattern of net mass transfer involves a pronounced azimuthal component in both cases. The total net mass transfer is equivalent to approximately 20% (no phase change) or approximately 40% (with phase change) of the S-1 mass. From the observed systematic increase in peak widths of the higher orders, the size of the crystalline domain in the myofilament lattice in the relaxed sartorius is estimated to be greater than 650 nm and the variations in myofilament lattice spacing among different myofibrils to be about +/- 3%. Furthermore, in the activated state, the equilibrium positions of the myofilaments are no longer well ordered, but are distributed statistically about the lattice points with a standard deviation of approximately 3 nm.

Characterization of a non-indexible equatorial x-ray reflection from frog sartorius muscle

Abstract X-ray equatorial reflections from frog sartorius muscle were studied using a position sensitive detector. A weak reflection appeared between the 10 and 11 peaks which did not index on the hexagonal filament lattice. This reflection, first reported by Elliott et al . (1967), was further characterized. The spacing of the reflection varied in direct proportion to that of the 10 peaks for sarcomere lengths between 2·0 μm and 3·0 μm. Its intensity appeared relatively insensitive to length changes. Optical diffraction patterns from electron micrographs of oblique sections through muscle gave ratios for the spacings of the myosin filaments and the Z -disc lattice that correlated very closely with the X-ray results. It is suggested that the Z -disc structure is the major source of this nonindexible reflection.

Equatorial x-ray intensities and isometric force levels in frog sartorius muscle.

Abstract Isometric force levels, ranging between 0 and 100% of maximal force P 0 at 2 to 3 °C, were elicited in frog sartorius muscle by means of rapidly cooling a Ringer solution containing 1·25 to 2·0 m m -caffeine. Equatorial X-ray diffraction patterns were obtained in the resting state and during contraction. The ratio of the intensities I 11 I 10 increased with force almost linearly, with a slight upward curvature. The individual intensities for the contracting state were normalized relative to both the intensity of the undiffracted beam and the intensity of each reflection in the resting state. These normalized intensities were found to vary in a reciprocal way: I 10 decreased while I 11 increased throughout the range of forces studied. The gradual change in I 11 I 10 with force level indicates that this ratio is a sensitive measure of the number of cross-bridges in the isometric state. A two-state model in which myosin projections are either in a resting or attached state and in which force is proportional to the fraction of projections in the attached state was applied to the experimental data of the individual reflections. I 10 deviates from this model in a way that suggests that formation of the first few cross-bridges may decrease the regularity of the remaining unattached myosin projections.

Factors Contributing to Troponin Exchange in Myofibrils and in Solution

The troponin complex in a muscle fiber can be replaced with exogenous troponin by using a gentle exchange procedure in which the actin–tropomyosin complex is never devoid of a full complement of troponin (Brenner et al. (1999) Biophys J77: 2677–2691). The mechanism of this exchange process and the factors that influence this exchange are poorly understood. In this study, the exchange process has now been examined in myofibrils and in solution. In myofibrils under rigor conditions, troponin exchange occurred preferentially in the region of overlap between actin and myosin when the free Ca2+ concentration was low. At higher concentrations of Ca2+, the exchange occurred uniformly along the actin. Ca2+ also accelerated troponin exchange in solution but the effect of S1 could not be confirmed in solution experiments. The rate of exchange in solution was insensitive to moderate changes in pH or ionic strength. Increasing the temperature resulted in a two-fold increase in rate with each 10°C increase in temperature. A sequential two step model of troponin binding to actin–tropomyosin could simulate the observed association and dissociation transients. In the absence of Ca2+ or rigor S1, the following rate constants could describe the binding process: k1 = 7.12 μM−1s−1, k−1 = 0.65 s−1, k2 = 0.07 s−1, k−2 = 0.0014 s−1. The slow rate of detachment of troponin from actin (k−2) limits the rate of exchange in solution and most likely contributes to the slow rate of exchange in fibers.

State-dependent radial elasticity of attached cross-bridges in single skinned fibres of rabbit psoas muscle

1. In a single skinned fibre of rabbit psoas muscle, upon attachment of cross‐bridges to actin in the presence of ADP or pyrophosphate (PPi), the separation between the contractile filaments, as determined by equatorial X‐ray diffraction, is found to decrease, suggesting that force is generated in the radial direction.

Radial equilibrium lengths of actomyosin cross-bridges in muscle.

Radial equilibrium lengths of the weakly attached, force-generating, and rigor cross-bridges are determined by recording their resistance to osmotic compression. Radial equilibrium length is the surface-to-surface distance between myosin and actin filaments at which attached cross-bridges are, on average, radially undistorted. We previously proposed that differences in the radial equilibrium length represent differences in the structure of the actomyosin cross-bridge. Until now the radial equilibrium length had only been determined for various strongly attached cross-bridge states and was found to be distinct for each state examined. In the present work, we demonstrate that weakly attached cross-bridges, in spite of their low affinity for actin, also exert elastic forces opposing osmotic compression, and they are characterized by a distinct radial equilibrium length (12.0 nm vs. 10.5 nm for force-generating and 13.0 nm for rigor cross-bridge). This suggests significant differences in the molecular structure of the attached cross-bridges under these conditions, e.g., differences in the shape of the myosin head or in the docking of the myosin to actin. Thus, the present finding supports our earlier conclusion that there is a structural change in the attached cross-bridge associated with the transition from a weakly bound configuration to the force-generating configuration. The implications for imposing spatial constraints on modeling actomyosin interaction in the filament lattice are discussed.

Involvement of weak binding crossbridges in force production in muscle

Many recent studies on the regulation of striated muscle contraction have shown that weak binding myosin crossbridges (containing ATP or ADP + Pi af their active sites) can attach to actin even in relaxed muscle. The smooth muscle protein, caldesmon, has been shown fo selectively inhibit binding of these weak binding crossbridges and has been used as a probe of the function of attachment in the weak binding configuration. Inhibition of attachment of the weak binding crossbridges is sufficient to inhibit active force production. Caldesmon causes no change in crossbridge cycling kinetics. We conclude that attachment in the weak binding states is an essential step in the crossbridge cycle and it follows that the state preceding force generation is a weak binding state. It has long been recognized that the binding of myosin fo actin is sensitive to the nucleotide bound to myosin. In the absence of nucleotide, or in the presence of ADP, this binding is very strong while in the presence of ATP this binding is weak. If was actually thought that myosin was irreversibly dissociated from actin by ATP during the initial stage of ATP hydrolysis. Only following cleavage of the terminal phosphate anhydride linkage was the myosin thought to bind fo actin. This view was held in the model of Lymn and Taylor (1971) and subsequently in the Refractory State model of Eisenberg and coworkers (Eisenberg et al., 1972). This mandatory dissociation by ATP was attractive because it provided a simple way of resetting the crossbridges following the power stroke of contraction fo allow motion. This simple picture ended when Sleep & Hutton (1978) and Stein and coworkers (1979) obtained evidence that af high protein concentrations myosin could remain bound fo actin throughout the process of ATP hydrolysis. This binding of myosin fo actin, in the presence of ATP, was shown fo have about 0.07% of the affinity as in the presence of ADP. Because of this difference in affinity the terms weak binding states and strong binding states were employed.

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium (weak-binding) state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.