Marco Caremani

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.

Effect of Inorganic Phosphate on the Force and Number of Myosin Cross-Bridges During the Isometric Contraction of Permeabilized Muscle Fibers from Rabbit Psoas

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12 degrees C, sarcomere length 2.5 mum). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 +/- 1.1 kPa nm(-1). Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 +/- 3.7 kPa nm(-1). An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r(F)) is 23.5 +/- 1.0 s(-1) in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 +/- 0.9 s(-1) at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.

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

•  Force and shortening in muscle are caused by ATP‐driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half‐sarcomere. Crystallographic studies indicate that the working stroke consists in an interdomain movement of the myosin motor associated with the release of inorganic phosphate (Pi). •  Here the coupling of the working stroke with the release of Pi is studied in situ using fast half‐sarcomere mechanics on skinned fibres from rabbit psoas. •  The isotonic velocity transient following stepwise force reductions superimposed on isometric contraction measures the mechanical manifestation of the working stroke and its rate of regeneration. •  The results indicate that the release of Pi from the catalytic site of an actin‐attached myosin motor can occur at any stage of the working stroke, and a myosin motor uses two consecutive actin monomers to maximize the power during shortening.

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

Muscle contraction is driven at the molecular level by a structural working stroke in the head domain of the myosin cross‐bridge linking the thick and thin filaments. Crystallographic models suggest that the working stroke corresponds to a relative movement of 11 nm between the attachments of the head domain to the thin and thick filaments. The molecular mechanism of force generation depends on the relationship between cross‐bridge force and movement, which is determined by cross‐bridge and filament compliances. Here we measured the compliance of the cross‐bridges and of the thin and thick filaments by combining mechanical and X‐ray diffraction experiments. The results show that cross‐bridge compliance is relatively low and fully accounted for by the elasticity of the myosin head, suggesting that the myosin cross‐bridge generates isometric force by a small sub‐step of the 11 nm stroke that drives filament sliding at low load.

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

Significance This paper represents a major advance in understanding intrinsic heart beat regulation, because it provides an integrated view of the Frank–Starling law that combines mechanical, structural, and energetic aspects of heart performance. We show that mechanosensing in the myosin filament adjusts the number of myosin motors recruited from the off, ATP hydrolysis-unavailable state to the systolic force. In this way, the energetic cost of the heart beat is tuned to the end systolic pressure–volume relation. These results elucidate a plausible mechanistic link between the mutations that affect the structure of the myosin filament and cause hypercontractility and/or loss of contractile efficiency and dysregulation of the control by the stress-sensing mechanism on the state of the myosin motors. 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.

Significance To our knowledge, this paper represents a major advancement in the physiology and pathophysiology of the heart as it gives the first quantitative description of the working stroke of the motor protein cardiac myosin II. The experiments demonstrate that our sarcomere-level mechanical methods on trabeculae have the full potential for the in situ investigation of cardiomyopathy-causing mutations in cardiac myosin and tests on specific therapeutic interventions. 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 [Ca2+]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 Ca2+-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.

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

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.

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

ABSTRACT 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

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.