Rabbit Psoas Muscle Research Articles

Regulation of troponin and myosin conformation in thin- and thick-filament domains in the sarcomere of skeletal muscle myofibrils.

The contractility of striated muscle is controlled by a dual-filament regulatory mechanism which couples calcium-dependent structural changes in thin filaments with those in thick filaments triggering the release of folded-helical myosin motors from the filament surface. The folded-helical conformation of motors in relaxed muscle may be stabilized in the region of the thick filament containing myosin-binding protein-C (C-zone). Moreover, in active muscle actin-bound myosin motors might locally alter the activation level of the thin filament. To test these hypotheses we developed a novel fluorescence-based approach to measure troponin and myosin orientation in subdomains of the sarcomere in isolated myofibrils. Bifunctional rhodamine probes labelling troponin-C (TnC) and the myosin regulatory light chain (RLC) were exchanged into myofibrils from rabbit psoas muscle, and the spatial distribution of probe orientations in the sarcomere was measured by fluorescence polarization microscopy. In the absence of ATP, the RLCs of actin-attached myosin motors were perpendicular to the filament axis and the orientation of the TnC probes in the region of overlap with thick filaments was markedly different from that in the rest of the sarcomere, indicating that myosin binding to actin alters the conformation of troponin in the thin filament. In relaxing conditions all TnC probes had the same conformation which was insensitive to temperature. In contrast, the RLC probes became more parallel to the filament axis as temperature was increased above 12°C, and at 30°C in the presence of 4% Dextran their orientation was more parallel in the C-zone, indicating enrichment of folded myosin motors in this filament domain. These results show directly that troponin and myosin have distinct conformations in different sarcomere subdomains in situ (supported by the Wellcome Trust and the Royal Society, UK).

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 active shortening and stretching on the rate of force re-development in rabbit psoas muscle fibres

The steady-state isometric force produced by skeletal muscle after active shortening and stretching is depressed and enhanced, respectively, compared with purely isometric force produced at corresponding final lengths and at the same level of activation. One hypothesis proposed to account for these force depression (FD) and force enhancement (FE) properties is a change in cross-bridge cycling kinetics. The rate of cross-bridge attachment (f) and/or cross-bridge detachment (g) may be altered following active shortening and active stretching, leading to FD and FE, respectively. Experiments elucidating cross-bridge kinetics in actively shortened and stretched muscle preparations and their corresponding purely isometric contractions have yet to be performed. The aim of this study was to investigate cross-bridge cycling kinetics of muscle fibres at steady-state following active shortening and stretching. This was done by determining muscle fibre stiffness and rate of active force redevelopment following a quick release-re-stretch protocol (kTR). Applying these measures to equations previously used in the literature for a two-state cross-bridge cycling model (attached/detached cross-bridges) allowed us to determine apparent f and g, the proportion of attached cross-bridges, and the force produced per cross-bridge. kTR, apparent f and g, the proportion of attached cross-bridges and the force produced per cross-bridge were significantly decreased following active shortening compared with corresponding purely isometric contractions, indicating a change in cross-bridge cycling kinetics. Additionally, we showed no change in cross-bridge cycling kinetics following active stretch compared with corresponding purely isometric contractions. These findings suggest that FD is associated with changes in cross-bridge kinetics, whereas FE is not.

Fast stretching of skeletal muscle fibres abolishes residual force enhancement.

The steady-state isometric force of a muscle after active stretching is greater than the steady-state force for a purely isometric contraction at the same length and activation level. The mechanisms underlying this property, termed residual force enhancement (rFE), remain unknown. When myofibrils are actively stretched while cross-bridge cycling is inhibited, rFE is substantially reduced, suggesting that cross-bridge cycling is essential to produce rFE. Our purpose was to further investigate the role of cross-bridge cycling in rFE by investigating whether fast stretching that causes cross-bridge slipping is associated with a loss of rFE. Skinned fibre bundles from rabbit psoas muscles were stretched slowly (0.08 µms-1) or rapidly (800 µms-1) while activated, from an average sarcomere length of 2.4 to 3.2 µm. Force was enhanced by 38±4% (mean±s.e.m) after the slow stretches but was not enhanced after the fast stretches, suggesting that proper cross-bridge cycling is required to produce rFE.

Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils.

In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

Sarcomere length non-uniformities dictate force production along the descending limb of the force-length relation.

The force-length relation is one of the most defining features of muscle contraction, and yet a topic of debate in the literature. The sliding filament theory predicts that the force produced by muscle fibres is proportional to the degree of overlap between myosin and actin filaments, producing a linear descending limb of the active force-length relation. However, several studies have shown forces that are larger than predicted, especially at long sarcomere lengths (SLs). Studies have been conducted with muscle fibres, preparations containing thousands of sarcomeres that make measurements of individual SL challenging. The aim of this study was to evaluate force production and sarcomere dynamics in isolated myofibrils and single sarcomeres from the rabbit psoas muscle to enhance our understanding of the theoretically predicted force-length relation. Contractions at varying SLs along the plateau (SL = 2.25-2.39 µm) and the descending limb (SL > 2.39 µm) of the force-length relation were induced in sarcomeres and myofibrils, and different modes of force measurements were used. Our results show that when forces are measured in single sarcomeres, the experimental force-length relation follows theoretical predictions. When forces are measured in myofibrils with large SL dispersions, there is an extension of the plateau and forces elevated above the predicted levels along the descending limb. We also found an increase in SL non-uniformity and slowed rates of force production at long lengths in myofibrils but not in single sarcomere preparations. We conclude that the deviation of the descending limb of the force-length relation is correlated with the degree of SL non-uniformity and slowed force development.

A Myosin II-Based Nanomachine Devised for the Study of Ca2+-Dependent Mechanisms of Muscle Regulation.

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by dual laser optical tweezers. However, the application of the original version of the nanomachine to investigate the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca2+] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca2+ (0.1 mM, the concentration required for actin attachment to the bead with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

Impact of Mavacamten on Force Generation in Single Myofibrils from Rabbit Psoas and Human Cardiac Muscle

Impact of Mavacamten on Force Generation in Single Myofibrils from Rabbit Psoas and Human Cardiac Muscle

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)

The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

Structural Changes in the Thick Filaments during Activation of Demembranated Skeletal Muscle Fibers

We determined the [Ca2+]-dependence of the changes in thick filament structure associated with activation of small bundles of demembranated fibers from rabbit psoas muscle by X-ray diffraction at beamline ID02 of the European Synchrotron Radiation Facility. In relaxing conditions the OFF structure of the thick filament characteristic of resting intact muscle, with a strong first myosin layer line (ML1) corresponding to the quasi-helical arrangement of the myosin motors on the thick filament surface and a short backbone periodicity, measured by the spacing of the myosin-based meridional M6 reflection (SM6; Linari et al. 2015, Nature 528:276-279), was observed at temperatures above 25°C in the presence of 5% Dextran T500. To study the effects of calcium activation in these conditions whilst preserving sarcomere homogeneity and minimising the period of high temperature activation, fiber bundles were equilibrated at the required [Ca2+] at 1°C, and X-ray diffraction data were collected 1.5s after a temperature jump to 25°C. Activation at low [Ca2+], producing only about 20% of the maximal isometric force, was accompanied by large decreases in the intensities of the ML1 and of the myosin-based meridional M2 and M5 X-ray reflections associated with the OFF structure of the thick filament, showing that the quasi-helical arrangement of the myosin motors on the thick filament surface is lost at a relatively low level of isometric force. In contrast the changes in the interference fine structure of the myosin-based meridional M3 reflection, associated with the conformation of the myosin motors during maximal isometric contraction, and the increase in SM6 associated with the longer backbone periodicity require higher [Ca2+].Supported by FIRB-Futuro in Ricerca, PRIN-MIUR and Telethon (Italy), MRC and BHF (UK), and ESRF.

Structural Kinetics of the RLC Domain of Myosin during Activation of Skeletal Muscle Fibers by Photolysis of Caged-Calcium

Recent fluorescence polarization studies in skeletal muscle fibers using bifunctional rhodamine (BSR) probes on the C-lobe of the regulatory light chain (RLC) of myosin showed that the steady-state calcium-sensitivity and co-operativity of the orientation change of the RLC C-lobe are higher than those of force (Fusi et al., Nat Commun 2016,doi:10.1038/ncomms13281). We have now investigated the in situ kinetics of the conformational change of the RLC during isometric contraction of a muscle fiber triggered by a rapid (>104 s−1) increase in [Ca2+] following UV-photolysis of caged-calcium (NP-EGTA). We monitored by polarized fluorescence changes in the orientation of the E-helix of a BSR-labeled RLC exchanged into demembranated fibers from rabbit psoas muscle during activation by photolysis of NP-EGTA, under conditions which preserve the physiological resting structure of the thick filament (T=25°C, 5% Dextran, 2.45 µm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in an intact muscle fiber. The change in the order parameter P2 of the RLC probe, associated with it becoming more perpendicular to the fiber axis, also had a sigmoidal time course. The fractional change in P2 was faster than that of force. The P2 change was 25% complete 20ms after photolysis when activation of the thin filament is already maximal (T=12°C, Fusi et al., PNAS 2014, 111:4626-31) but force had developed to only 10% of its plateau value (T0). The P2 change was essentially complete by ca 70ms after photolysis when force was only 60% T0. These results show that the change in RLC orientation associated with the activation of the myosin motors is slower than thin-filament activation but faster than force generation. Supported by MRC, UK.

A New and Improved View of Force Production

A central question in muscle contraction is “How is the energy release in ATP hydrolysis converted into mechanical force and work?” With the elucidation of the actomyosin ATPase reaction in the 1970s (1,2) and the fact that a large amount of free energy is produced during Pi release from an actomyosin-ADP-Pi species (3), the Pi release step from actomyosin-ADP-Pi must be associated with the generation of force and shortening. It was also known that increases in the [Pi] concentration in isometrically contracting skinned muscle fibers reduced isometric force (4).

The force-generation process in active muscle is strain sensitive and endothermic: a temperature-perturbation study.

In experiments on active muscle, we examined the tension decline and its temperature sensitivity at the onset of ramp shortening and at a range of velocities. A segment (∼1.5 mm long) of a skinned muscle fibre isolated from rabbit psoas muscle was held isometrically (sarcomere length ∼2.5 µm) at 8-9°C, maximally Ca2+-activated and a ramp shortening applied. The tension decline with a ramp shortening showed an early decrease of slope (the P1 transition) followed by a slower decrease in slope (the P2 transition) to the steady (isotonic) force. The tension level at the initial P1 transition and the time to that transition decreased as the velocity was increased; the length change to this transition increased with shortening velocity to a steady value of ∼8 nm half-sarcomere-1 A small, rapid, temperature jump (T-jump) (3-4°C, <0.2 ms) applied coincident with the onset of ramp shortening showed force enhancement by T-jump and changed the tension decline markedly. Analyses showed that the rate of T-jump-induced force rise increased linearly with increase of shortening velocity. These results provide crucial evidence that the strain-sensitive cross-bridge force generation, or a step closely coupled to it, is endothermic.

Energy cost of isometric force production after active shortening in skinned muscle fibres.

The steady-state isometric force after active shortening of a skeletal muscle is lower than the purely isometric force at the corresponding length. This property of skeletal muscle is known as force depression. The purpose of this study was to investigate whether the energy cost of force production at the steady state after active shortening was reduced compared with the energy cost of force production for a purely isometric contraction performed at the corresponding length (same length, same activation). Experiments were performed in skinned fibres isolated from rabbit psoas muscle. Skinned fibres were actively shortened from an average sarcomere length of 3.0 µm to an average sarcomere length of 2.4 µm. Purely isometric reference contractions were performed at an average sarcomere length of 2.4 µm. Simultaneously with the force measurements, the ATP cost was measured during the last 30 s of isometric contractions using an enzyme-coupled assay. Stiffness was calculated during a quick stretch-release cycle of 0.2% fibre length performed once the steady state had been reached after active shortening and during the purely isometric reference contractions. Force and stiffness following active shortening were decreased by 10.0±1.8% and 11.0±2.2%, respectively, compared with the isometric reference contractions. Similarly, ATPase activity per second (not normalized to the force) showed a decrease of 15.6±3.0% in the force-depressed state compared with the purely isometric reference state. However, ATPase activity per second per unit of force was similar for the isometric contractions following active shortening (28.7±2.4 mmol l-1mN-1smm3) and the corresponding purely isometric reference contraction (30.9±2.8 mmol l-1mN-1smm3). Furthermore, the reduction in absolute ATPase activity per second was significantly correlated with force depression and stiffness depression. These results are in accordance with the idea that force depression following active shortening is primarily caused by a decrease in the proportion of attached cross-bridges. Furthermore, these findings, along with previously reported results showing a decrease in ATP consumption per unit of force after active muscle stretching, suggest that the mechanisms involved in the steady-state force after active muscle shortening and active muscle lengthening are of distinctly different origin.

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.

Does Titin Explain The High Passive Force Observed in Frog Tibialis Anterior Muscle?

INTRODUCTIONThe force‐length relationship of various skeletal muscles indicates that passive force is generally much lower than active force [1]. However, a previous study showed that the whole frog tibialis anterior (TA) muscle has significantly higher passive than active forces within its normal range of motion (Figure 1) [2].It is known that titin, the giant protein within the sarcomere, is a main contributor to passive force in skeletal muscle at rest [3]. Furthermore, variations in titin isoforms have been related to different passive forces; the shorter the titin isoform, the higher the passive force [3, 4]. Therefore, the high passive force observed in frog TA muscle could result from the expression of a short titin isoform and thus a high titin‐based passive force.The purpose of this study was to investigate titin isoforms and titin‐based passive force in frog TA muscle. Titin‐based passive force was determined by testing passive force in single skinned muscle fibres in which titin is considered to be the major source of passive force [3].METHODSTitin isoforms: Titin isoforms in frog TA and rabbit psoas muscles were determined using agarose strengthened 2% acrylamide SDS‐PAGE gels.Skinned fibre experiment: Active and passive force‐length relationships were established by stretching skinned frog TA fibres (n=10) to various sarcomere lengths (2.4, 2.6, 2.8, 3.0, 3.2, 3.4 and 3.6 μm), and isometrically activating them after steady state force had been reached.RESULTSThe frog TA expresses two titin isoforms similar to those expressed by rabbit psoas muscle (3.3 and 3.40 MD).The active and passive force‐length relationships in TA skinned fibres indicate that passive force is significantly lower than active force on the descending limb of the force‐length relationship (Figure 2).DISCUSSION AND CONCLUSIONSFrog TA titin isoforms are not different from those of rabbit psoas, which are known to have relatively low passive [3, 4] compared to active force. Single skinned fibres isolated from frog TA muscle do not produce high passive compared to active forces. Therefore, it seems that titin is not responsible for the high passive force observed at the whole frog TA muscle level. It is likely that the high passive forces produced by the frog TA muscle originate from the extracellular matrix rather than structures within the fibres.The high passive forces found for the entire frog TA muscle might have implications for the function of the muscle during swimming. Since frogs have large webbed feet, a high passive force in the TA allows frogs to use their feet like human swimmers use passive fins to increase the speed and decrease the energy expenditure of swimming.Support or Funding InformationCIHR, NSERC, AIHS

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.

The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.

Calcium Sensitivity after Active Shortening in Rabbit Psoas Fibres

Introduction It has been shown that isometric force after active shortening of muscles is lower than the purely isometric force performed at the corresponding lengths. This property is termed force depression. Force depression has been observed in isolated muscle preparations and muscles in situ and in vivo (Rassier et al., J Applied Physiol 2004). The origin of force depression is still unknown. It has been suggested that active shortening may affect calcium sensitivity by changing calcium-troponin binding equilibrium leading to decreased force after active shortening (Ekelund and Edman, Acta Physiol Scand 1982). Our aim in the present study was to test whether calcium sensitivity was decreased after active shortening. Methods Experiments were performed using skinned fibres (n=7) isolated from rabbit psoas muscle. Calcium sensitivity was characterized by establishing the force-pCa curves for isometric reference contractions performed at an average sarcomere length of 2.4 µm and active shortening contractions from an average sarcomere length of 3.0 µm to an average sarcomere length of 2.4 µm. pCa50 and the coefficient of cooperativity (nH) were compared between reference and active shortening contractions. Results and Discussion pCa50 for the reference contractions was 5.99±0.02. No change in pCa50 was observed in the force depressed state (6.00±0.01). Furthermore, the coefficient of cooperativity did not change after active shortening (3.9±0.3 versus 4.3±0.3 for the reference and force depressed states respectively). These results suggest that calcium sensitivity does not decrease after active shortening and that the binding interactions between troponin and calcium are not altered in the force depressed state.