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.

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.

Straightening Out the Elasticity of Myosin Cross-Bridges.

In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments during cyclical ATP-driven interactions toward the center of the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here, we review some old and new data that confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. The implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in nonmuscle intracellular cargo transport.

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.

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.

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.

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.

Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog.

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 µm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 µm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

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.

The myofilament elasticity and its effect on kinetics of force generation by the myosin motor.

The half-sarcomere is the functional unit of striated muscle, in which, according to a "linear" mechanical model, myosin motors are parallel force generators with an average strain s acting between the opposing myosin and actin filaments that behave as a series elastic element with compliance Cf. Thus the definition of the mechanism of force generation by myosin motors in muscle requires integration of the crystallographic model of the working stroke with the mechanical constraints provided by the organization of motors in the half-sarcomere. The relation between half-sarcomere compliance and force (Chs-T) during the development of isometric contraction deviates, at low forces, from that predicted by the linear model, indicating the presence of an elastic element in parallel with the myosin motors, which may influence the estimate of s. A working stroke model, kinetically constrained by the early phase of the isotonic velocity transient following a force step, predicts that the rate of quick force recovery following a length step is reduced to the observed value by a Cf of 12.6nm/MPa. With this value of Cf, the fit of Chs-T relation during the isometric force rise gives s=1.8-1.9nm, similar to the values estimated using the linear model.

Motion of myosin head domains during activation and force development in skeletal muscle.

Muscle contraction is driven by a change in the structure of the head domain of myosin, the "working stroke" that pulls the actin filaments toward the midpoint of the myosin filaments. This movement of the myosin heads can be measured very precisely in intact muscle cells by X-ray interference, but until now this technique has not been applied to physiological activation and force generation following electrical stimulation of muscle cells. By using this approach, we show that the long axes of the myosin head domains are roughly parallel to the filaments in resting muscle, with their center of mass offset by approximately 7 nm from the C terminus of the head domain. The observed mass distribution matches that seen in electron micrographs of isolated myosin filaments in which the heads are folded back toward the filament midpoint. Following electrical stimulation, the heads move by approximately 10 nm away from the filament midpoint, in the opposite direction to the working stroke. The time course of this motion matches that of force generation, but is slower than the other structural changes in the myosin filaments on activation, including the loss of helical and axial order of the myosin heads and the change in periodicity of the filament backbone. The rate of force development is limited by that of attachment of myosin heads to actin in a conformation that is the same as that during steady-state isometric contraction; force generation in the actin-attached head is fast compared with the attachment step.

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.

Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA-reductase inhibition on skeletal muscle functions.

The rate-limiting step of cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme reductase (HGMR), whose inhibitors, the statins, widely used in clinical practice to treat hypercholesterolemia, often cause myopathy, and rarely rhabdomyolysis. All studies to date are limited to the definition of statin-induced myotoxicity omitting to investigate whether and how HMGR inhibition influences muscle functions. To this end, 3-mo-old male rats (Rattus norvegicus) were treated for 3 wk with a daily intraperitoneal injection of simvastatin (1.5 mg/kg/d), and biochemical, morphological, mechanical, and functional analysis were performed on extensor digitorum longus (EDL) muscle. Our results show that EDL muscles from simvastatin-treated rats exhibited reduced HMGR activity; a 15% shift from the fastest myosin heavy-chain (MHC) isoform IIb to the slower IIa/x; and reduced power output and unloaded shortening velocity, by 41 and 23%, respectively, without any change in isometric force and endurance. Moreover, simvastatin-treated rats showed a decrease of maximum speed reached and the latency to fall off the rotaroad (∼-30%). These results indicate that the molecular mechanism of the impaired muscle function following statin treatment could be related to the plasticity of fast MHC isoform expression.

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.