An integrated picture of the structural pathways controlling the heart performance.

The regulation of heart function is attributed to a dual filament mechanism: i) the Ca-dependent structural changes in the regulatory proteins of the thin, actin-containing filament making actin available for myosin motor attachment, and ii) the release of motors from their folded (OFF) state on the surface of the thick filament allowing them to attach and pull the actin filament. Thick filament mechanosensing is thought to control the number of motors switching ON in relation to the systolic performance, but its molecular basis is still controversial. Here, we use high spatial resolution X-ray diffraction data from electrically paced rat trabeculae and papillary muscles to provide a molecular explanation of the modulation of heart performance that calls for a revision of the mechanosensing hypothesis.

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

The medaka fish () 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.

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.

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.

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 Ca 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[Ca]), estimated by Hill coefficient n.

Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle.

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament.

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing.

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies.

Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of omecamtiv mecarbil.

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM).

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.

Contracting striated muscle has a dynamic I-band spring with an undamped stiffness 100 times larger than the passive stiffness.

Key Points: Fast sarcomere-level mechanics in contracting intact fibres from frog skeletal muscle reveal an I-band spring with an undamped stiffness 100 times larger than the known static stiffness. This undamped stiffness remains constant in the range of sarcomere length 2.7-3.

Low temperature traps myosin motors of mammalian muscle in a refractory state that prevents activation.

Myosin motors in the thick filament of resting striated (skeletal and cardiac) muscle are trapped in an OFF state, in which the motors are packed in helical tracks on the filament surface, inhibiting their interactions with actin and utilization of ATP. To investigate the structural changes induced in the thick filament of mammalian skeletal muscle by changes in temperature, we collected x-ray diffraction patterns from the fast skeletal muscle extensor digitorum longus of the mouse in the temperature range from near physiological (35°C) to 10°C, in which the maximal isometric force ( ) shows a threefold decrease. In resting muscle, x-ray reflections signaling the OFF state of the thick filament indicate that cooling produces a progressive disruption of the OFF state with motors moving away from the ordered helical tracks on the surface of the thick filament.

Inotropic interventions do not change the resting state of myosin motors during cardiac diastole.

When striated (skeletal and cardiac) muscle is in its relaxed state, myosin motors are packed in helical tracks on the surface of the thick filament, folded toward the center of the sarcomere, and unable to bind actin or hydrolyze ATP (OFF state). This raises the question of whatthe mechanism is that integrates the Ca-dependent thin filament activation, making myosin heads available for interaction with actin. Here we test the interdependency of the thin and thick filament regulatory mechanisms in intact trabeculae from the rat heart.

Thick Filament Mechano-Sensing in Skeletal and Cardiac Muscles: A Common Mechanism Able to Adapt the Energetic Cost of the Contraction to the Task.

A dual regulation of contraction operates in both skeletal and cardiac muscles. The first mechanism, based on Ca-dependent structural changes of the regulatory proteins in the thin filament, makes the actin sites available for binding of the myosin motors. The second recruits the myosin heads from the OFF state, in which they are unable to split ATP and bind to actin, in relation to the force during contraction.

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 Ca 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.

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).

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 (V ) 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 V 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.

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.

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.

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.

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.

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 non-linear elasticity of the muscle sarcomere and the compliance of myosin motors.

Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s.