Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 µm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 µM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Sarcomere level mechanics of the fast skeletal muscle of the medaka fish larva.

The medaka fish (Oryzias latipes) is a vertebrate model used in developmental biology and genetics. Here we explore its suitability as a model for investigating the molecular mechanisms of human myopathies caused by mutations in sarcomeric proteins. To this end, the relevant mechanical parameters of the intact skeletal muscle of wild-type medaka are determined using the transparent tail at larval stage 40. Tails were mounted at sarcomere length of 2.1 µm in a thermoregulated trough containing physiological solution. Tetanic contractions were elicited at physiological temperature (10°C-30°C) by electrical stimulation, and sarcomere length changes were recorded with nanometer-microsecond resolution during both isometric and isotonic contractions with a striation follower. The force output has been normalized for the actual fraction of the cross section of the tail occupied by the myofilament lattice, as established with transmission electron microscopy (TEM), and then for the actual density of myofilaments, as established with X-ray diffraction. Under these conditions, the mechanical performance of the contracting muscle of the wild-type larva can be defined at the level of the half-thick filament, where ∼300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates. The results of this study point out that the medaka fish larva is a suitable model for the investigation of the genotype/phenotype correlations and therapeutic possibilities in skeletal muscle diseases caused by mutations in sarcomeric proteins.NEW & NOTEWORTHY The suitability of the medaka fish as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the tail of medaka larva. The mechanical performance of the medaka muscle, scaled at the level of the myosin-containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.

Matching Mechanics and Energetics of Muscle Contraction Suggests Unconventional Chemomechanical Coupling during the Actin-Myosin Interaction.

The mechanical performances of the vertebrate skeletal muscle during isometric and isotonic contractions are interfaced with the corresponding energy consumptions to define the coupling between mechanical and biochemical steps in the myosin-actin energy transduction cycle. The analysis is extended to a simplified synthetic nanomachine in which eight HMM molecules purified from fast mammalian skeletal muscle are brought to interact with an actin filament in the presence of 2 mM ATP, to assess the emergent properties of a minimum number of motors working in ensemble without the effects of both the higher hierarchical levels of striated muscle organization and other sarcomeric, regulatory and cytoskeleton proteins. A three-state model of myosin-actin interaction is able to predict the known relationships between energetics and transient and steady-state mechanical properties of fast skeletal muscle either in vivo or in vitro only under the assumption that during shortening a myosin motor can interact with two actin sites during one ATP hydrolysis cycle. Implementation of the molecular details of the model should be achieved by exploiting kinetic and structural constraints present in the transients elicited by stepwise perturbations in length or force superimposed on the isometric contraction.

Anisotropic Elasticity of the Myosin Motor in Muscle.

To define the mechanics and energetics of the myosin motor action in muscles, it is mandatory to know fundamental parameters such as the stiffness and the force of the single myosin motor, and the fraction of motors attached during contraction. These parameters can be defined in situ using sarcomere-level mechanics in single muscle fibers under the assumption that the stiffness of a myosin dimer with both motors attached (as occurs in rigor, when all motors are attached) is twice that of a single motor (as occurs in the isometric contraction). We use a mechanical/structural model to identify the constraints that underpin the stiffness of the myosin dimer with both motors attached to actin. By comparing the results of the model with the data in the literature, we conclude that the two-fold axial stiffness of the dimers with both motors attached is justified by a stiffness of the myosin motor that is anisotropic and higher along the axis of the myofilaments. A lower azimuthal stiffness of the motor plays an important role in the complex architecture of the sarcomere by allowing the motors to attach to actin filaments at different azimuthal angles relative to the thick filament.

Allosteric modulation of cardiac myosin mechanics and kinetics by the conjugated omega-7,9 trans-fat rumenic acid.

KEY POINTS: Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of ß-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the ß-cardiac myosin-2 motor domain with an EC50 of 200 nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. ABSTRACT: Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine ß-cardiac myosin, the EC50 for rumenic acid varies from 10.5 µM in the absence of actin to 200 nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of ß-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5 µM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

Myosin filament activation in the heart is tuned to the mechanical task.

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.

A mechanical model of the half-sarcomere which includes the contribution of titin.

The evidence, in both resting and active muscle, for the presence of an I-band spring element like titin that anchors the Z line to the end of the thick filament did not yet produce a proper theoretical treatment in a complete model of the half-sarcomere. The textbook model developed by A. F. Huxley and his collaborators in 1981, which provides that the half-sarcomere (hs) compliance is due to the contribution of the compliances of the thin and thick filaments and actin-attached myosin motors, predicts that at any sarcomere length (SL) the absence of attached motors results in an infinite half-sarcomere compliance, in contrast with the observations. Growing evidence for the presence of a titin-like I-band spring urges the 1981 model to be implemented to include the contribution of this element in the mechanical model of the half-sarcomere. The model described here represents a tool for the interpretation of measurements of hs stiffness at increasing SL, which is important either in relation to the mechanism of stabilisation of SL against the consequence of sarcomere inhomogeneity in active force generation, or for investigations on the role of titin as mechano-sensor in thick filament regulation. Moreover the model opens the possibility for understanding the functional differences related to the titin isoform of various muscle types and the mechanism by which mutations in titin gene lead to myopathies.

Size and speed of the working stroke of cardiac myosin in situ.

The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 µm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.

Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit.

KEY POINTS: The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them. In this study fast sarcomere-level mechanics has been applied to Ca2+ -activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano-kinetics of force generation by slow and fast myosin isoforms in situ. The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform-dependent functional diversity between skeletal muscles. These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle. ABSTRACT: The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4 µm, temperature 12°C, 4% dextran T-500) from slow (soleus, containing the MHC-1 isoform) and fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca2+ -activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56 ± 0.04 and 1.70 ± 0.37 pN nm-1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0 ) was similar (∼3.3 nm) in both isoforms. Consequently the force per motor (F0  = Îµs0 ) was three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 versus 5.35 ± 1.51 pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5 ) was 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.

The force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

KEY POINTS: Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. ABSTRACT: The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+ ]o from 1 to 2.5 mm at sarcomere length ∼2.2 µm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

Minimum number of myosin motors accounting for shortening velocity under zero load in skeletal muscle.

KEY POINTS: Myosin filament mechanosensing determines the efficiency of the contraction by adapting the number of switched ON motors to the load. Accordingly, the unloaded shortening velocity (V0 ) is already set at the end of latency relaxation (LR), ∼10 ms after the start of stimulation, when the myosin filament is still in the OFF state. Here the number of actin-attached motors per half-myosin filament (n) during V0 shortening imposed either at the end of LR or at the plateau of the isometric contraction is estimated from the relation between half-sarcomere compliance and force during the force redevelopment after shortening. The value of n decreases progressively with shortening and, during V0 shortening starting at the end of LR, is 1-4. Reduction of n is accounted for by a constant duty ratio of 0.05 and a parallel switching OFF of motors, explaining the very low rate of ATP utilization found during unloaded shortening. ABSTRACT: The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V0 ) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca2+ -induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin-attached motors per half-thick filament during V0 shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 µm), small 4 kHz oscillations and determining the relation between half-sarcomere (hs) compliance and force during the force development following V0 shortening. When V0 shortening is superimposed on the maximum isometric force T0 , n decreases progressively with the increase of shortening (range 30-80 nm per hs) and, when V0 shortening is imposed at the end of LR, n can be as low as 1-4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V0 shortening.

Low-force transitions in single titin molecules reflect a memory of contractile history.

Titin is a giant elastomeric muscle protein that has been suggested to function as a sensor of sarcomeric stress and strain, but the mechanisms by which it does so are unresolved. To gain insight into its mechanosensory function we manipulated single titin molecules with high-resolution optical tweezers. Discrete, step-wise transitions, with rates faster than canonical Ig domain unfolding occurred during stretch at forces as low as 5 pN. Multiple mechanisms and molecular regions (PEVK, proximal tandem-Ig, N2A) are likely to be involved. The pattern of transitions is sensitive to the history of contractile events. Monte-Carlo simulations of our experimental results predicted that structural transitions begin before the complete extension of the PEVK domain. High-resolution atomic force microscopy (AFM) supported this prediction. Addition of glutamate-rich PEVK domain fragments competitively inhibited the viscoelastic response in both single titin molecules and muscle fibers, indicating that PEVK domain interactions contribute significantly to sarcomere mechanics. Thus, under non-equilibrium conditions across the physiological force range, titin extends by a complex pattern of history-dependent discrete conformational transitions, which, by dynamically exposing ligand-binding sites, could set the stage for the biochemical sensing of the mechanical status of the sarcomere.

Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products.

KEY POINTS: Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin-actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration. A model simulation of the myosin-actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation. These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction. The chemo-mechanical cycle of the myosin II--actin reaction in situ has been investigated in Ca(2+)-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi ), shortening at V ≤ 350 nm s(-1) per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0 ), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mM, which reduces T0 and the number of motors by 40-50%, does not influence the dependence on V of number and strain. A model simulation of the myosin-actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction.

The contributions of filaments and cross-bridges to sarcomere compliance in skeletal muscle.

Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ∼ 11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (C(cb)) and filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa(-1), close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa(-1)). C(cb) calculated using these estimates is 0.37 ± 0.12 nm pN(-1), a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN(-1), obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to C(cb) from the myosin tail that joins the head to the thick filament is excluded. The low C(cb) value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle.

Force and kinetics of fast and slow muscle myosin determined with a synthetic sarcomere-like nanomachine.

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP-driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform-dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self-consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin-attached motors and the rate of transition through the attachment-detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site.

Skeletal muscle shortens faster against a lower load. This force-velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre-microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere.

Dependence of myosin filament structure on intracellular calcium concentration in skeletal muscle.

Contraction of skeletal muscle is triggered by an increase in intracellular calcium concentration that relieves the structural block on actin-binding sites in resting muscle, potentially allowing myosin motors to bind and generate force. However, most myosin motors are not available for actin binding because they are stabilized in folded helical tracks on the surface of myosin-containing thick filaments. High-force contraction depends on the release of the folded motors, which can be triggered by stress in the thick filament backbone, but additional mechanisms may link the activation of the thick filaments to that of the thin filaments or to intracellular calcium concentration. Here, we used x-ray diffraction in combination with temperature-jump activation to determine the steady-state calcium dependence of thick filament structure and myosin motor conformation in near-physiological conditions. We found that x-ray signals associated with the perpendicular motors characteristic of isometric force generation had almost the same calcium sensitivity as force, but x-ray signals associated with perturbations in the folded myosin helix had a much higher calcium sensitivity. Moreover, a new population of myosin motors with a longer axial periodicity became prominent at low levels of calcium activation and may represent an intermediate regulatory state of the myosin motors in the physiological pathway of filament activation.

The force of the myosin motor sets cooperativity in thin filament activation of skeletal muscles.

Contraction of striated muscle is regulated by a dual mechanism involving both thin, actin-containing filament and thick, myosin-containing filament. Thin filament is activated by Ca2+ binding to troponin, leading to tropomyosin displacement that exposes actin sites for interaction with myosin motors, extending from the neighbouring stress-activated thick filaments. Motor attachment to actin contributes to spreading activation along the thin filament, through a cooperative mechanism, still unclear, that determines the slope of the sigmoidal relation between isometric force and pCa (-log[Ca2+]), estimated by Hill coefficient nH. We use sarcomere-level mechanics in demembranated fibres of rabbit skeletal muscle activated by Ca2+ at different temperatures (12-35 °C) to show that nH depends on the motor force at constant number of attached motors. The definition of the role of motor force provides fundamental constraints for modelling the dynamics of thin filament activation and defining the action of small molecules as possible therapeutic tools.