Rigor Cross-bridges Research Articles

Troponin C modulates the activation of thin filaments by rigor cross-bridges

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.

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.

Electron Tomography of Insect Flight Muscle in Rigor and AMPPNP at 23°C

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23°C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23°C reveal many double-headed and some single-headed “lead” bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situcrossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar compari son using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.

P-F1-23 Strain dependent behaviour of rigor cross bridges: Ketelaars M 1,2 van der Heide U.A., 1,2 Treijtel B.W., 1 de Beer E.L., 2 Blangé T. 1 1 Dep. of Physiology, Univ. of Amsterdam (NL), 2 Dep. of Medical Physiology, Univ. Utrecht (NL)

P-F1-23 Strain dependent behaviour of rigor cross bridges: Ketelaars M 1,2 van der Heide U.A., 1,2 Treijtel B.W., 1 de Beer E.L., 2 Blangé T. 1 1 Dep. of Physiology, Univ. of Amsterdam (NL), 2 Dep. of Medical Physiology, Univ. Utrecht (NL)

Thin filament regulation of force activation is not essential in single vascular smooth muscle cells.

To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized single smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.

Rigor cross-bridges bind to two actin monomers inthin filaments of rabbit psoas muscle

The mode of binding of myosin subfragment-1 (S1) to actin is known to depend on their molar ratio: when actin is in excess, S1 binds to two actin monomers within the actin filament, and when S1 is in excess or is equimolar with actin, each S1 binds to one actin monomer. Since in vertebrate striated muscle actin is in molar excess over myosin, we expect that in fibers each myosin head binds to two actin monomers. To test this idea, we compared the conformation of the heads in native muscle with the conformation of S1 in fibers that were loaded with either high (S1 equimolar with actin) or low (excess of actin over S1) concentration of extrinsic S1. Conformation was assessed by the accessibility of heads to trypsin (measured by the rate of trypsinolysis) and by their orientation with respect to the muscle axis (measured by a combination of polarization of fluorescence and linear dichroism). In muscle fibers loaded with a high concentration of S1, the region of the heavy chain of the myosin head at the junction of 20 and 50 kDa proteolytic fragments was readily digested by trypsin and its orientation was approximately perpendicular to an axis of a thin filament. In contrast, when muscle fibers were loaded with a low concentration of S1, the 20/50 kDa junction of S1 was protected from trypsinolysis and its orientation was more parallel with a filament axis. Native muscle in rigor behaved like muscle irrigated with a low concentration of S1, i.e. the 20/50 kDa junction of the myosin head was protected from trypsinolysis and the orientation of the heads was parallel to the filament axis. We conclude that in rigor rabbit psoas muscle each myosin head binds to two actin monomers in a thin filament, and that this binding is different from the binding of S1 to actin in equimolar solutions.

Gold/Fab immuno electron microscopy localization of troponin H and troponin T in Lethocerus flight muscle.

The position of the large troponin complex relative to myosin crossbridges in Lethocerus flight muscle (IFM) has been probed by electron microscopy (EM) using monoclonal antibodies against troponin T (TnT) and troponin H (TnH), a heavy troponin component found in several insect muscles. Infiltration of gold-tagged and plain Fab fragments into glycerinated IFM before fixation established in non-overlap fibers that the beads every 38.7 nm along thin filaments are troponin. Original and optically filtered EM images from 25 nm longitudinal and 15 nm cross-sections of partially overlapped fibers suggests that epitopes on both TnT and TnH are very close to the rear crossbridge of the rigor double chevron. When Fab was infiltrated into relaxed fibers and ATP was subsequently removed, the resulting rigor crossbridge lattice was disrupted by antibody labeling of the troponin. The results confirm that the lattice of rigor crossbridges and troponin are congruent and suggest that crossbridges may interact with troponin in IFM, possibly serving as a partial basis for the stretch activation characteristic of this muscle.

High frequency characteristics of elasticity of skeletal muscle fibres kept in relaxed and rigor state.

The viscoelastic properties of crossbridges in rigor state are studied by means of application of small length changes, completed within 30 microseconds, to isometric skinned fibre segments of the iliofibularis muscle of the frog in relaxed and rigor state and measurement of the tension response. Results are expressed as a complex Young's modulus, the real part of which denotes normalized stiffness, while the imaginary part denotes normalized viscous mechanical impedance. Young's modulus was examined over a wide frequency range varying from 5 Hz up to 50 kHz. Young's modulus can be interpreted in terms of stiffness and viscous friction of the half-sarcomere or in terms of elastic changes in tension and recovery upon a step length change. The viscoelastic properties of half-sarcomeres of muscle fibre segments in rigor state showed strong resemblance to those of activated fibres in that shortening a muscle fibre in rigor state resulted in an immediate drop in tension, after which half of the drop in tension was recovered. The following slower phases of tension recovery--a subsequent drop in tension and slow completion of tension recovery--as seen in the activated state, do not occur in rigor state. The magnitude of Young's moduli of fibres in rigor state generally decreased from a value of 3.12 x 10(7) N m-2 at 40 kHz to 1.61 x 10(7) N m-2 at about 100 Hz. Effects of increased viscosity of the incubation medium, decreased interfilament distance in the relaxed state and variation of rigor tension upon frequency dependence of complex Young's modulus have been investigated. Variation of tension of crossbridges in rigor state influenced to some extent the frequency dependence of the Young's modulus. Recovery in relaxed state is not dependent on the viscosity of the medium. Recovery in rigor is slowed down at raised viscosity of the incubation medium, but less than half the amount expected if viscosity of the medium would be the cause of internal friction of the half-sarcomere. Internal friction of the half-sarcomere in the relaxed fibre at the same interfilament distance as in rigor is different from internal friction in rigor. It will be concluded that time necessary for recovery in rigor cannot be explained by friction due to the incubation medium. Instead, recovery in rigor expressed by the frequency dependence of the Young's modulus has to be due to intrinsic properties of crossbridges. These intrinsic properties can be explained by the occurrence of state transitions of crossbridges in rigor.(ABSTRACT TRUNCATED AT 400 WORDS)

Relaxation from rigor of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP

The kinetics of ATP-induced rigor cross-bridge detachment were studied by initiating relaxation in chemically skinned trabeculae of the guinea pig heart using photolytic release of ATP in the absence of calcium ions (pCa > 8). The time course of the fall in tension exhibited either an initial plateau phase of variable duration with little change in tension or a rise in tension, followed by a decrease to relaxed levels. The in-phase component of tissue stiffness initially decreased. The rate then slowed near the end of the tension plateau, indicating transient cross-bridge rebinding, before falling to relaxed levels. Estimates of the apparent second-order rate constant for ATP-induced detachment of rigor cross-bridges based on the half-time for relaxation or on the half-time to the convergence of tension records to a common time course were similar at 3 x 10(3) M-1 s-1. Because the characteristics of the mechanical transients observed during relaxation from rigor were markedly similar to those reported from studies of rabbit psoas fibers in the presence of MgADP (Dantzig, J. A., M. G. Hibberd, D. R. Trentham, and Y. E. Goldman. 1991. Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J. Physiol. 432:639-680), direct measurements of MgADP using [3H]ATP in cardiac tissue in rigor were made. Results indicated that during rigor, nearly 18% of the cross-bridges in skinned trabeculae had [3H]MgADP bound. Incubation of the tissue during rigor with apyrase, an enzyme with both ADPase and ATPase activity, reduced the level of [3H]MgADP to that measured following a 2-min chase in a solution containing 5 mM unlabeled MgATP. Apyrase incubation also significantly reduced the tension and stiffness transients, so that both time courses became monotonic and could be fit with a simple model for cross-bridge detachment. The apparent second-order rate constant for ATP-induced rigor cross-bridge detachment measured in the apyrase treated tissue at 4 x 10(4) M-1 s-1 was faster than that measured in untreated tissue. Nevertheless, this rate was still over an order of magnitude slower than the analogous rate measured in previous studies of isolated cardiac actomyosin-S1. These results are consistent with the hypothesis that the presence of MgADP bound cross-bridges suppresses the inhibition normally imposed by the thin filament regulatory system in the absence of calcium ions and allows cross-bridge rebinding and force production during relaxation from rigor.

Structural change of crossbridges of rabbit skeletal muscle during isometric contraction

Structural changes of crossbridges during isometric contraction have been studied by electron microscopy. Chemically skinned rabbit fibres were rapidly frozen either in activating solution or in ATP-free (rigor) solution, freeze-substituted and embedded. Longitudinal sections of muscle fibres show that the number of crossbridges in active fibres (isometric contraction) is approximately the same as in rigor fibres. Crossbridges of the active and rigor states differ in their shapes, angles and manner of arrangement on the thin filaments. In rigor many crossbridges are wide near the thin filaments and narrow near the thick filament shafts; in active fibres they have more uniform width along their length. The angle of the crossbridges in active fibres is somewhat variable. The average angle is approximately 90 degrees to the filament axis. The crossbridges are arranged on the thin filament retaining the 14.3 nm thick filament periodicity. The crossbridges in rigor are tilted and their arrangement near the thin filament reveals the 36 nm actin periodicity. The variability in the shapes of the crossbridges in active fibres is still higher when we look at them in cross-sections of muscle fibres. The crossbridge shapes in the cross-sections were classified and the relative frequency of different shapes was determined. The shapes that are commonly observed in active fibres are similar in that the majority of the mass of the crossbridges is farther away from the thin filament than the crossbridges in rigor fibres.

Filament overlap affects TnC extraction from skinned muscle fibres.

Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca2+/Mg2+ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 microns were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 microns with 20 mM Tris, pH 7.8, 5 mM EDTA. The decrease in the post-extraction maximum Ca2+ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40-120 microns, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca2+ binding and that under the conditions used here, they retard its extraction.

Structural changes in the actomyosin cross-bridges associated with force generation.

It is generally thought that to generate active force in muscle, myosin heads (cross-bridges) that are attached to actin undergo large-scale conformational changes. However, evidence for conformational changes of the attached cross-bridges associated with force generation has been ambiguous. In this study, we took advantage of the recent observation that cross-bridges that are weakly attached to actin in a relaxed muscle are apparently in attached preforce-generating states. The experimental conditions were chosen such that there were large fractions of cross-bridges attached under relaxing and activating conditions, and high-resolution equatorial x-ray diffraction patterns obtained under these conditions were compared. Changes brought about by activation in the two innermost intensities, I10 and I11, did not follow the familiar reciprocal changes. Instead, there was almost no change in I11, whereas I10 decreased by 34%. Together with the changes found in the higher-order reflections, the results suggest that the structure of the attached force-generating cross-bridges differs from that of the weakly bound, preforce-generating cross-bridges and possibly also differs from that of the cross-bridges in rigor. These observations support the concept that force generation involves a transition between distinct structural states of the actomyosin cross-bridges.

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. 2. The single muscle fibres were subjected to compression by 0-8% of dextran T500. The changes in lattice spacings by dextran compression were compared with changes induced by cross-bridge attachment to actin. Based on this comparison, the magnitude and the direction of the radial force generated by the attached cross-bridges were estimated. The radial cross-bridge force varied with filament separation, and the magnitude of the radial cross-bridge force reached as high as the maximal axial force produced during isometric contraction. 3. One key parameter of the radial elasticity, i.e. the equilibrium spacing where the radial force is zero, was found to depend on the ligand bound to the myosin head. In the presence of ADP, the equilibrium spacing was 36 nm. In the presence of MgPPi the equilibrium spacing shifted to 35 nm and Ca2+ had little effect on the equilibrium spacing. 4. The equilibrium spacing was independent of the fraction of cross-bridges attached to actin. The fraction of cross-bridges attached in rigor was modulated from 100% to close to 0% by adding up to 10 mM of ATP gamma S in the rigor solution. The lattice spacing remained at 38 nm, the equilibrium spacing for nucleotide-free cross-bridges at mu = 170 mM. 5. Radial force generated by cross-bridges in rigor at large lattice spacings (38 nm < or = d10 < or = 46 nm) appeared to vary linearly with lattice spacing. 6. The titration of ATP gamma S to fibres in rigor provided a correlation between the radial stiffness of the nucleotide-free cross-bridges and the equatorial intensities. The relation between the equatorial intensity ratio I11/I10 and radial stiffness appeared to be approximately linear. 7. The fibres under different conditions showed a wide range of radial stiffness, which was not proportional to the apparent axial stiffness of the fibre. If the apparent axial stiffness is a measure of the fraction of cross-bridges bound to actin, it follows that the radial elastic constant is state dependent; or vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanical response to photolytic ATP pulses of skinned muscle fibres pre-activated with a small pulse of ATP.

(1) Skinned fibres from rat psoas muscle were placed in oil and activated at 10 degrees C by pulse photolysis of caged ATP. From the force and stiffness transients on small ATP pulses, we estimated the concentration and rate of ATP hydrolysis of the crossbridges in the fibres. They were 0.14 mM and 0.16 mMs-1, respectively, and thus the turnover rate was 1.1 s-1. (2) To examine the mechanical properties of the physiological ADP-bound crossbridges, we took a double-pulse protocol: the skinned fibre was first activated with a conditioning ATP pulse (0.2-0.5 mM). Several (3-5) seconds later, when almost all the ATP should have been hydrolysed into ADP+P(i), we applied another ATP pulse (0.2-1 mM) to test the fibre. (3) An analysis of the transients on the test pulses indicated that the conditioning pulse slowed the rate of crossbridge detachment. The time course of force development was thus expected to be also delayed, but was not. (4) We suggested that the rigor crossbridge might differ from the nucleotide-free intermediate in the physiological reaction cycle.

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 (PP(i)), 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.2. The single muscle fibres were subjected to compression by 0-8% of dextran T(500). The changes in lattice spacings by dextran compression were compared with changes induced by cross-bridge attachment to actin. Based on this comparison, the magnitude and the direction of the radial force generated by the attached cross-bridges were estimated. The radial cross-bridge force varied with filament separation, and the magnitude of the radial cross-bridge force reached as high as the maximal axial force produced during isometric contraction.3. One key parameter of the radial elasticity, i.e. the equilibrium spacing where the radial force is zero, was found to depend on the ligand bound to the myosin head. In the presence of ADP, the equilibrium spacing was 36 nm. In the presence of MgPP(i) the equilibrium spacing shifted to 35 nm and Ca(2+) had little effect on the equilibrium spacing.4. The equilibrium spacing was independent of the fraction of cross-bridges attached to actin. The fraction of cross-bridges attached in rigor was modulated from 100% to close to 0% by adding up to 10 mM of ATPgammaS in the rigor solution. The lattice spacing remained at 38 nm, the equilibrium spacing for nucleotide-free cross-bridges at mu = 170 mM.5. Radial force generated by cross-bridges in rigor at large lattice spacings (38 nm </= d(10) </= 46 nm) appeared to vary linearly with lattice spacing.6. The titration of ATPgammaS to fibres in rigor provided a correlation between the radial stiffness of the nucleotide-free cross-bridges and the equatorial intensities. The relation between the equatorial intensity ratio I(11)/I(10) and radial stiffness appeared to be approximately linear.7. The fibres under different conditions showed a wide range of radial stiffness, which was not proportional to the apparent axial stiffness of the fibre. If the apparent axial stiffness is a measure of the fraction of cross-bridges bound to actin, it follows that the radial elastic constant is state dependent; or vice versa.8. Differences in equilibrium lattice spacing and in radial elastic constant, most probably reflect differences in the molecular structure of the acto-myosin complex and there is more than one single conformation of the various strongly bound cross-bridge states.9. Determining equilibrium spacings of the radial elasticity appears to be an effective new approach in detecting structural differences among the attached cross-bridges, since this approach is independent of the fraction of cross-bridges attached, a factor that frequently encumbers the interpretation of structural studies of attached cross-bridge states.

Kinetic and physical characterization of force generation in muscle: a laser temperature-jump and length-jump study on activated and contracting rigor fibers.

Experiments are presented that probe the mechanism of contraction in normal activated muscle fibers and in heated rigor fibers. In activated fibers we subdivide the partial recovery of isometric tension during the Huxley-Simmons phase 2 into temperature-independent and temperature-dependent steps termed, respectively, phase 2fast and phase 2slow. Evidence is presented to show that phase 2fast arises from the perturbation of a damped elastic element in the cross-bridge and that phase 2slow is the manifestation of an endothermic, order-disorder transition responsible for de novo tension generation. These responses are common to both frog and rabbit fibers. The only difference between animals is that the kinetics of phase 2slow appears to scale with the working temperature of the muscle and not absolute temperature. Rigor fibers heated above the working temperature of the muscle contract. Tension generation is, as with activated fibers, endothermic. Tension transients following a laser temperature-jump of activated and heated rigor fibers are virtually indistinguishable on the basis of either the form or magnitude of the response. In length-jump experiments, tension recovery by heated rigor fibers consists of three exponentials with a tension-dependent rate for the medium speed step. Preliminary data indicate that the rigor cross-bridge operates over a distance of between 13.5 and 18 nm. Collectively, these data imply that tension generation in muscle arises from accessible conformational states in the proteins of the cross-bridge alone. ATP hydrolysis in active fibers and the heating of rigor fibers simply serve to shift these intrinsic conformational equilibria towards tension generation.

Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle

Direct measurements were made of the Ca distribution within sarcomeres of glycerinated rabbit psoas muscle fibers in rigor using electron probe x-ray microanalysis. Both analogue raster analysis and digital x-ray imaging were used to quantitate the Ca distribution along thick and thin filaments as a function of the concentration of free Ca2+. Even when corrected for the estimated contribution of Ca bound to thick filaments, the Ca measured in the region of overlap between thick and thin filaments significantly exceeded the Ca in the I-band at subsaturating concentrations of free Ca2+. At saturating levels of free Ca2+, the excess Ca in the overlap region was diminished but still statistically significant. The data thus suggest that the formation of rigor linkages exerts multiple effects on the binding of Ca2+ to thin filaments in the overlap region by increasing the affinity of troponin C for Ca2+ and possibly by unmasking additional Ca2+ binding sites. The data also show that the cooperativity invested in the thin filaments is insufficient to permit the effects of rigor cross-bridge formation on Ca2+ binding to propagate far along the thin filaments into the I-band.

Inferences concerning crossbridges from work on insect muscle.

This paper presents a number of separate results concerning crossbridge attachment: [1] X-ray diffraction from live bumble bee flight muscle shows a set of layer lines distinct from that of relaxed Lethocerus, in which the apparent myosin helix is shorter than that of the actin. [2] Rigor crossbridges of Lethocerus are not rotatable by stretch. [3] Rabbit and Lethocerus fibres in rigor relaxed by ATP at -35 degrees C show evidence of non-rigor crossbridge attachment.

Time course of rise of muscle stiffness at onset of contraction induced by photorelease of ATP.

Isometric contraction (9-10 degrees C) of skinned fibres from rat psoas muscle was elicited by photorelease of ATP (1.3-1.4 mM), and force (F), in-phase (I) and quadrature (Q) stiffness were monitored. The sinusoidal length change for the stiffness measurement was approximately 0.09% fibre length and 200-1000 Hz. On photolysis, F and I initially fell, and then rose. The half-time of the rise of I was shorter than that of F. Contrary to the previous assumption, Q rose even earlier than I. Although the time courses of the rise of F and I were greatly affected by the presence of phosphate ion (Pi; 4 mM), its effect on Q was modest. We propose the following hypothesis: the rigor crossbridges, after binding ATP, initially enter a state characterized by high Q with low I and none or little F, and then, releasing Pi during passing the state with the increased I, reach the final force generating state.

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.