Cross-bridges In Muscle Research Articles

Oscillatory work and the step that generates force in single myofibrils from rabbit psoas.

The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (Pi) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the Pi release step. The question asked here is whether the force generation occurs before Pi release or after release. We investigated the effect of Pi on oscillatory work production in single myofibrils and found that Pi-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of Pi in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have Pi attached. With sinusoidal analysis, we obtained the kinetic constants around the Pi release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before Pi is released, and the same force is maintained after Pi is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

History-dependent muscle resistance to stretch remains high after small, posturally-relevant pre-movements.

The contributions of intrinsic muscle fiber resistance during mechanical perturbations to standing and other postural behaviors are unclear. Muscle short-range stiffness is known to vary depending on the current level and history of the muscle's activation, as well as the muscle's recent movement history; this property has been referred to as history dependence or muscle thixotropy. However, we currently lack sufficient data about the degree to which muscle stiffness is modulated across posturally-relevant characteristics of muscle stretch and activation. We characterized the history dependence of muscle's resistance to stretch in single, permeabilized, activated, muscle fibers in posturally-relevant stretch conditions and activation levels. We used a classic paired muscle stretch paradigm, varying the amplitude of a "conditioning" triangular stretch-shorten cycle followed by a "test" ramp-and-hold imposed after a variable inter-stretch interval. We tested low (<15%), intermediate (15-50%) and high (>50%) muscle fiber activation levels, evaluating short-range stiffness and total impulse in the test stretch. Muscle fiber resistance to stretch remained high at conditioning amplitudes of <1% L0 and inter-stretch intervals of >1 s, characteristic of healthy standing postural sway. A ∼70% attenuation of muscle resistance to stretch was reached at conditioning amplitudes of >3% L0 and inter-stretch intervals of <0.1s, characteristic of larger, faster postural sway in balance-impaired individuals. The thixotropic changes cannot be predicted solely on muscle force at the time of stretch. Consistent with the disruption of muscle cross-bridges, muscle resistance to stretch during behavior can be substantially attenuated if the prior motion is large enough and/or frequent enough.

Intrafusal cross-bridge dynamics shape history-dependent muscle spindle responses to stretch.

Computational models can be critical to linking complex properties of muscle spindle organs to the sensory information that they encode during behaviours such as postural sway and locomotion where few muscle spindle recordings exist. Here, we augment a biophysical muscle spindle model to predict the muscle spindle sensory signal. Muscle spindles comprise several intrafusal muscle fibres with varied myosin expression and are innervated by sensory neurons that fire during muscle stretch. We demonstrate how cross-bridge dynamics from thick and thin filament interactions affect the sensory receptor potential at the spike initiating region. Equivalent to the Ia afferent's instantaneous firing rate, the receptor potential is modelled as a linear sum of the force and rate change of force (yank) of a dynamic bag1 fibre and the force of a static bag2/chain fibre. We show the importance of inter-filament interactions in (i) generating large changes in force at stretch onset that drive initial bursts and (ii) faster recovery of bag fibre force and receptor potential following a shortening. We show how myosin attachment and detachment rates qualitatively alter the receptor potential. Finally, we show the effect of faster recovery of receptor potential on cyclic stretch-shorten cycles. Specifically, the model predicts history-dependence in muscle spindle receptor potentials as a function of inter-stretch interval (ISI), pre-stretch amplitude and the amplitude of sinusoidal stretches. This model provides a computational platform for predicting muscle spindle response in behaviourally relevant stretches and can link myosin expression seen in healthy and diseased intrafusal muscle fibres to muscle spindle function.

The role of myosin loop-4 in localizing tropomyosin on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, namely myosin loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct. In the mutant, the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366 to 372. Cryo-EM analysis of fully-decorated actin-tropomyosin filaments with either wild-type or mutant S1, yielded 3.4- to 3.6-Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum site, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the relatively high potential energy associated with wild-type tropomyosin positioning favors an effective transition to B- and C-states following release of myosin from thin filaments during relaxation.

Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366-372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4-3.6 Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B- and C-states following release of myosin from the thin filaments during relaxation.

The Location and Rate of the Phosphate Release Step in the Muscle Cross-Bridge Cycle

It is controversial whether the phosphate (Pi) release step in the cross-bridge cycle occurs before or after the first tension-generating step and whether it is fast or slow. We have therefore modified our previous model of the frog cross-bridge cycle by including a Pi release step either before (model A) or after (model B) the first tension-generating step and refined the two models by downhill simplex runs against experimental data for the force-velocity relation and the tension transients after length steps. Pi release step was initially made slow (70 s−1), but after refinement, it became fast (∼500 s−1 for model A and ∼6000 s−1 for model B). The two models gave similar fits to the experimental tension transients after length steps, but model A gave a better fit to the lengthening limb of the force-velocity relation than model B. 50 mM Pi inhibited the isometric tension of model A by ∼50% but that of model B by only ∼25%. The half-inhibition was at 6.0 mM Pi for model A and at 1.6 mM Pi for model B. The values for model A were consistent with experimental data. We also simulated the effect Pi jump as in caged Pi experiments. For model A, a Pi jump induced a tension fall at a rate similar to the experimental phase II. There was then a small rise in tension to the steady state mimicking the experimental phase III. The initial tension fall was caused by detachment of M⋅ADP⋅Pi myosin heads from actin and reversal of the first tension-generating step. For model B, the fall in tension was more rapid and due to reversal of the first tension-generating step, and phase III was not observed. We conclude that, as in model A, the Pi release step is before the first tension-generating step and is moderately fast.

An in silico study examining the role of airway smooth muscle dynamics and airway compliance on the rate of airway re-narrowing after deep inspiration

Deep inspirations are a widely studied topic due to their varied effectiveness as a bronchodilator in asthmatic and non-asthmatic patients. Specifically, they are known to be effective at reversing bronchoconstriction in non-asthmatic patients but may fail to prevent bronchoconstriction in asthmatic patients. Inspired by a recent study on the effect of deep inspirations on the rate of re-narrowing of an isolated airway, we investigate whether the latch-bridge dynamics of smooth muscle cross-bridge theory, coupled with non-linear compliance of the airway wall, can account for the reported results: namely that only the rate of renarrowing after DI is sensitive to the interval between deep inspirations, while other measures are unaffected. We develop and present length- and pressure-controlled protocols which mimic both the experiments performed in the study, as well as simulate in vivo conditions respectively. Both protocols are simulated and show qualitative agreement with the results reported by the experiments, suggesting that latch-bridge dynamics coupled with airway wall non-compliance may be sufficient to explain these results. Moreover pressure- and length-controlled protocols show important differences which should be considered when designing in vitro experiments to mimic in vivo conditions.

Feedforward Motion Control With a Variable Stiffness Actuator Inspired by Muscle Cross-Bridge Kinematics

High mechanical impedance, sensor resolution, and computing bandwidth are desirable for achieving stability in feedback control. In contrast, although the human body is physically inferior to man-made systems in terms of feedback control due to signal transmission delay, humans can achieve not only stable but also robust and adaptive control ability. For these reasons, the significance of the feedforward control of the human has been emphasized in neuroscience. In previous studies, virtual trajectory control and internal model hypotheses were used to explain the principle of human feedforward control in view of the mechanical stiffness of joints and the internal model of the brain, respectively. Inspired by these insights, in this paper, we focus on the relationship between the joint stiffness and inverse model accuracy and attempted to apply it to the field of robotics. We present a variable stiffness actuator developed using variable radius gear transmission inspired by muscle cross-bridge kinematics. The developed mechanism allows the joint stiffness to be accurately controlled without any sensor-based feedback control. Using the developed actuator, we conduct a feedforward motion generating experiment with respect to variations in the inverse dynamics model uncertainty and verified that joint stiffness can compensate for inverse model uncertainty and external disturbances. These results indicate that the developed variable stiffness actuator can be applied to the robotics field for feedforward applications and support the hypothesis that humans may utilize joint stiffness to compensate for the inverse dynamics model uncertainty.

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Force encoding in muscle spindles during stretch of passive muscle.

Muscle spindle proprioceptive receptors play a primary role in encoding the effects of external mechanical perturbations to the body. During externally-imposed stretches of passive, i.e. electrically-quiescent, muscles, the instantaneous firing rates (IFRs) of muscle spindles are associated with characteristics of stretch such as length and velocity. However, even in passive muscle, there are history-dependent transients of muscle spindle firing that are not uniquely related to muscle length and velocity, nor reproduced by current muscle spindle models. These include acceleration-dependent initial bursts, increased dynamic response to stretch velocity if a muscle has been isometric, and rate relaxation, i.e., a decrease in tonic IFR when a muscle is held at a constant length after being stretched. We collected muscle spindle spike trains across a variety of muscle stretch kinematic conditions, including systematic changes in peak length, velocity, and acceleration. We demonstrate that muscle spindle primary afferents in passive muscle fire in direct relationship to muscle force-related variables, rather than length-related variables. Linear combinations of whole muscle-tendon force and the first time derivative of force (dF/dt) predict the entire time course of transient IFRs in muscle spindle Ia afferents during stretch (i.e., lengthening) of passive muscle, including the initial burst, the dynamic response to lengthening, and rate relaxation following lengthening. Similar to acceleration scaling found previously in postural responses to perturbations, initial burst amplitude scaled equally well to initial stretch acceleration or dF/dt, though later transients were only described by dF/dt. The transient increase in dF/dt at the onset of lengthening reflects muscle short-range stiffness due to cross-bridge dynamics. Our work demonstrates a critical role of muscle cross-bridge dynamics in history-dependent muscle spindle IFRs in passive muscle lengthening conditions relevant to the detection and sensorimotor response to mechanical perturbations to the body, and to previously-described history-dependence in perception of limb position.

Effects of Thermal Tension Transients on the Muscle Crossbridge

The transverse thermal fluctuations of the myosin molecule are significant. This paper explores the contribution of lateral myosin bending to the developed crossbridge force and power stroke. The equipartition theorem is used to calculate the mode amplitudes for myosin bending. Crossbridge axial force [Formula: see text] and power stroke [Formula: see text] are developed by transverse in-plane fluctuations along the [Formula: see text]- and [Formula: see text]-axes. Practical applications include the effects of temperature on the flexibility of the myosin molecule stiffness and tension, relevant to man-made fabrication of synthetic muscle using micromachines and nanowires. Scaling laws for the [Formula: see text] bending amplitude depend on filament length, mode number, and stiffness, as [Formula: see text], and (EI)[Formula: see text]. This paper quantifies the effects of thermal motion on the mechanics of miniature molecular motors, including the muscle crossbridge.

S2 Diode Model of Muscle Crossbridge Dynamics

This report explores the contribution of lateral myosin bending to the developed crossbridge force and power stroke. The equipartition theorem and Boltzmann distribution are used to calculate crossbridge force and displacement, consistent with experimental values. Negligible buckling strength of the S2-myosin link means that the muscle crossbridge is effectively a one-way force transducer, a mechanical diode, transmitting axial tension forces only. Crossbridge stiffness surfaces as an important factor. Power-stroke displacement is found to decrease with increasing stiffness, whereas axial force increases. The transverse thermal fluctuations of the myosin molecule are significant. Equipartition is used to calculate the mode amplitudes for myosin bending. Crossbridge axial force Fx and power stroke Δx develop from transverse in-plane fluctuations along the y and z axes. Single and doubleheaded actin-myosin attachment configurations are calculated in detail. Practical applications include the effects of temperature on the flexibility of the myosin molecule stiffness and tension, relevant to man-made fabrication of synthetic muscle using micro-machines. Scaling laws for the S2 bending amplitude depend on mode number, filament length, and stiffness, as (n)-2, (L)2, and (EI)-1.

Is There A Performance Advantage To Internal Femoral Rotation During A Broad Jump?

Internal femoral rotation, resulting in greater knee valgus angle, occurs in some individuals during broad jumps. Fitness professionals consistently train athletes away from this movement prior to jumping for fear of injury. However, given the knowledge regarding optimal cross-bridge potential and stimulation of the stretch shortening cycle, it is theorized that internal femoral rotation during jumping may recruit additional hip muscle cross-bridges and utilize the stretch shortening cycle resulting in better performance. PURPOSE:To investigate the effect of knee valgus angle on broad jump performance. METHODS:A sample of 18 males aged 20-29, recreationally trained jumpers performed a brief warm-up followed by 3 control jumps (C) and 3 internally rotated jumps (IR). During the IR trial, participants were given a ball to hold between their knees. The ball was then removed. The participants were then instructed to focus on keeping their knees closer together as if squeezing the ball at the bottom of the countermovement while attempting to jump as far as possible. During the C trial, participants were instructed to jump as far as possible. Fourteen individual retro-reflective markers and rigid body markers were applied to track kinematic data. Jump distance was measured from the starting line to the nearest 1 cm increment of participant’s heels. False step trials were not used. RESULTS:Knee valgus angle was greater (p<0.05) during the IR trials for both the right (C= 4.9±4.0o, IR= 6.4±4.0o) and left (C= 5.7±4.3o, IR= 8.9±5.1o) leg. Furthermore, C jump distance (2.27±0.25m) was significantly greater (p<0.05) than IR jump distance (2.17±0.25m). CONCLUSIONS:This data suggests that avoiding valgus knee collapse during broad jumping may be beneficial for injury prevention and performance; therefore fitness professionals should teach clients to avoid hip internal rotation during jumping.

Dynamics of cross-bridge cycling, ATP hydrolysis, force generation, and deformation in cardiac muscle

Despite extensive study over the past six decades the coupling of chemical reaction and mechanical processes in muscle dynamics is not well understood. We lack a theoretical description of how chemical processes (metabolite binding, ATP hydrolysis) influence and are influenced by mechanical processes (deformation and force generation). To address this need, a mathematical model of the muscle cross-bridge (XB) cycle based on Huxley's sliding filament theory is developed that explicitly accounts for the chemical transformation events and the influence of strain on state transitions. The model is identified based on elastic and viscous moduli data from mouse and rat myocardial strips over a range of perturbation frequencies, and MgATP and inorganic phosphate (Pi) concentrations. Simulations of the identified model reproduce the observed effects of MgATP and MgADP on the rate of force development. Furthermore, simulations reveal that the rate of force re-development measured in slack–restretch experiments is not directly proportional to the rate of XB cycling. For these experiments, the model predicts that the observed increase in the rate of force generation with increased Pi concentration is due to inhibition of cycle turnover by Pi. Finally, the model captures the observed phenomena of force yielding suggesting that it is a result of rapid detachment of stretched attached myosin heads.

Computational Model of Cross-Bridge Cycling and Force Generation to Explain the Effect of Metabolites on Cardiac Muscle Mechanics

Despite extensive study over the past six decades the coupling of chemical reaction and mechanical processes in muscle dynamics is not well understood. We lack a theoretical description of how chemical processes (metabolite binding, ATP hydrolysis) influence and are influenced by mechanical processes (deformation and force generation). To address this need, a mathematical model of the muscle crossbridge (XB) cycle based on Huxley's sliding filament theory is developed that explicitly accounts for the chemical transformation events and the influence of strain on state transitions. The model is identified based on elastic and viscous moduli data from mouse and rat myocardial strips over a range of perturbation frequencies, and MgATP and Pi concentrations. Simulations of the identified model reproduce the observed effects of MgATP and MgADP on the rate of force development. Furthermore, simulations reveal that the rate of force re-development measured in slack-restretch experiments is not directly proportional to the rate of XB cycling. For these experiments, the model predicts that the observed increase in the rate of force generation with increased Pi concentration is due to inhibition of cycle turnover by Pi. Finally, the model captures the observed phenomena of force yielding suggesting that it is a result of rapid detachment of stretched attached myosin heads.

Correction: Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction

Correction: Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca(2+) binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33-45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430-435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969-1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca(2+) saturation. A simple theory was developed to account for this fact.

Relaxed Skeletal Muscle Crossbridges Containing Dephosphorylated Regulatory Light Chain are Better Oriented Than Phosphorylated Ones

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca2+ binding, such as phosphorylation of myosin RLC also controls contraction of vertebrate striated muscle [1]. Electron Paramagnetic Resonance (EPR) studies using nucleotide-analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC [2]. The slower ATPase activity in the dephosphorylated moiety has been defined as a new super-relaxed state (SRX) [1]. It can be observed in both skeletal [3] and cardiac muscle fibers [4]. Given the importance of the SRX which suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (1) relaxed cross-bridges are statistically better ordered in muscle where the RLC is dephosphorylated compared to phosphorylated RLC; (2) during relaxation, phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (3) during isometric contraction, phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones.1. Cooke, R. Biophys Rev, 2011. 3(1): p. 33-45.2. Naber et al. Biophysical Soc 55-th Annual Meeting, 2011: p. 26.3. Stewart, M.A., et al. Proc Natl Acad Sci USA, 2010. 107(1): p. 430-5.4. Hooijman, et al, Biophys J, 2011. 100(8): p. 1969-76.

Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.