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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.

Background Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. Methods Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. Results MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. Conclusions Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.

The active force of mammalian skeletal muscle is reduced at low temperatures. Caremani et al. reveal that this is due to the rise of a population of myosin motors captured in a refractory state insensitive to muscle 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 (T0) 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. We find that the number of myosin motors in the OFF state at 10°C is half of that at 35°C. At T0, changes in the x-ray signals that report the fraction and conformation of actin-attached motors can be explained if the threefold decrease in force associated with lowering temperature is due not only to a decrease in the force-generating transition in the actin-attached motors but also to a twofold decrease in the number of such motors. Thus, lowering the temperature reduces to the same extent the fraction of motors in the OFF state at rest and the fraction of motors attached to actin at T0, suggesting that motors that leave the OFF state accumulate in a disordered refractory state that makes them unavailable for interaction with actin upon stimulation. This regulatory effect of temperature on the thick filament of mammalian skeletal muscle could represent an energetically convenient mechanism for hibernating animals.

SUMMARYIn contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm−1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.

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.

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.

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.

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.

A dual regulation of contraction operates in both skeletal and cardiac muscles. The first mechanism, based on Ca2+-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. Comparison of the relevant X-ray diffraction signals marking the state of the thick filament demonstrates that the force feedback that controls the regulatory state of the thick filament works in the same way in skeletal as in cardiac muscles: even if in an isometric tetanus of skeletal muscle force is under the control of the firing frequency of the motor unit, while in a heartbeat force is controlled by the afterload, the stress-sensor switching the motors ON plays the same role in adapting the energetic cost of the contraction to the force. A new aspect of the Frank-Starling law of the heart emerges: independent of the diastolic filling of the ventricle, the number of myosin motors switched ON during systole, and thus the energetic cost of contraction, are tuned to the arterial pressure. Deterioration of the thick-filament regulation mechanism may explain the hyper-contractility related to hypertrophic cardiomyopathy, an inherited heart disease that in 40% of cases is due to mutations in cardiac myosin.

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.

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(2+) 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 Ca(2+) 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 (T (p)) of an electrically paced intact trabecula at different extracellular Ca(2+) concentrations ([Ca(2+)](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 T (p) from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca(2+)](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 T (p). Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of T (p) and [Ca(2+)](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.

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.

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.

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.

The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (C(f)) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa(−1). This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa(−1). The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm(−1)) is smaller than that of frog (2.78 ± 0.30 pN nm(−1)). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog.

Measurements of the half-sarcomere stiffness during activation of skinned fibers from rabbit psoas (sarcomere length 2.5 μm, temperature 12°C) indicate that addition of 0.1 mM orthovanadate (Vi) to the solution produces a drop to ∼1/2 in number of force-generating myosin motors, proportional to the drop in steady isometric force (T(0)), an effect similar to that produced by the addition of 10 mM phosphate (Pi). However, in contrast to Pi, Vi does not change the rate of isometric force development. The depression of T(0) in a series of activations in presence of Vi is consistent with an apparent second-order rate constant of ∼1 × 10(3) M(-1) s(-1). The rate constant of T(0) recovery in a series of activations after removal of Vi is 3.5 × 10(-2) s(-1). These results, together with the finding in the literature that the ATPase rate is reduced by Vi in proportion to isometric force, are reproduced with a kinetic model of the acto-myosin cross-bridge cycle where binding of Vi to the force-generating actomyosin-ADP state induces detachment from actin to form a stable myosin-ADP-Vi complex that is not able to complete the hydrolysis cycle and reenters the cycle only via reattachment to actin upon activation in Vi-free solution.

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

Rapid attachment to actin of the detached motor domain of myosin dimers with one motor domain already attached has been hypothesized to explain the stretch-induced changes in X-ray interference and stiffness of active muscle. Here, using half-sarcomere mechanics in single frog muscle fibres (2.15 microm sarcomere length and 4 degrees C), we show that: (1) an increase in stiffness of the half-sarcomere under stretch is specific to isometric contraction and does not occur in rigor, indicating that the mechanism of stiffness increase is an increase in the number of attached motors; (2) 2 ms after 100 micros stretches (amplitude 2-8 nm per half-sarcomere) imposed during an isometric tetanus, the stiffness of the array of myosin motors in each half-sarcomere (e(m)) increases above the isometric value (e(m0)); (3) e(m) has a sigmoidal dependence on the distortion of the motor domains (Delta z) attached in isometric contraction, with a maximum approximately 2 e(m0) for a distortion of approximately 6 nm; e(m) is influenced by detachment of motors at z > 6 nm; (4) at the end of the 100 micros stretch the relation between e(m)/e(m0) and Delta z lies slightly but not significantly above that at 2 ms. These results support the idea that stretch-induced sliding of the actin filament distorts the actin-attached motor domain of the myosin dimers away from the centre of the sarcomere, providing the steric conditions for rapid attachment of the second motor domain. The rate of new motor attachment must be as high as 7.5 x 10(4) s(1) and explains the rapid and efficient increase of the resistance of active muscle to stretch.

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 μm, 4°C were investigated by X-ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X-ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin-attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer-line reflections associated with the quasi-helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half-time of ca 60 ms. Recovery of the equatorial X-ray intensities was largely complete during chaotic relaxation, but the other X-ray signals recovered more slowly than force.

The role of the actin filament-associated protein nebulin on mechanical and kinetic properties of the actomyosin motor was investigated in skeletal muscle of wild-type (wt) and nebulin-deficient (nebulin−/−) mice that were 1 d old, an age at which sarcomeric structure is still well preserved. In Ca2+-activated skinned fibers from psoas muscle, we determined the Ca2+ dependence of isometric force and stiffness, the rate of force redevelopment after unloaded shortening (kTR), the power during isotonic shortening, and the unloaded shortening velocity (V0). Our results show a 65% reduction in isometric force in nebulin−/− fibers at saturating [Ca2+], whereas neither thin-filament length nor the Ca2+ sensitivity of the contractile system is affected. Stiffness measurements indicate that the reduction in isometric force is due to a reduction in the number of actin-attached myosin motors, whereas the force of the motor is unchanged. Furthermore, in nebulin−/− fibers, kTR is decreased by 57%, V0 is increased by 63%, and the maximum power is decreased by 80%. These results indicate that, in the absence of nebulin, the attachment probability of the myosin motors to actin is decreased, revealing a direct role for nebulin in promoting strong actomyosin interactions responsible for force and power production.—Bang, M.-L., Caremani, M., Brunello, E., Littlefield, R., Lieber, R. L., Chen, J., Lombardi, V., Linari, M. Nebulin plays a direct role in promoting strong actin-myosin interactions.

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4 degrees C, 2.1 microm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T(0)) from 3.54 nm in the control solution (T(0,NR)) to approximately 0.5 nm in 1 microM BTS, where T(0) is reduced to approximately 0.15 T(0,NR). The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T(0). The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (approximately 3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T(0) is approximately 6000/s in the absence of filament compliance.

Cardiac performance depends on power developed by the myocardium and mutations of cardiac myosin, which affect power output, have been proposed to be responsible for various forms of cardiomyopathy. Precise measurements of force and filament sliding generated by cardiac myosin are lacking. Here sarcomere-level mechanics are applied for the first time to intact trabeculae from the right ventricle of the rat to determine the size and the speed of the working stroke of cardiac myosin in situ and their dependency on the load. The isotonic shortening transients elicited by stepwise drops in force to 0.2-0.8 of peak force (Tp) are recorded at sarcomere length of 1.9 and 2.2 μm and external Ca2+-concentrations ([Ca2+]o) 1 and 2.5 mM (temperature 27 oC). The size and the speed of the early rapid phase of shortening (the mechanical manifestation of the myosin working stroke) increase with the reduction of the load from approximately 3 nm per half-sarcomere and 1000 s−1 at high load to 6 nm per half-sarcomere and 7000 s−1 at low load, but are not affected by the increase of sarcomere length or [Ca2+]o. Thus the two-fold increase of force and power in the range of sarcomere length and [Ca2+]o explored are solely due to the increase in number of myosin motors. These results demonstrate the possibility for in situ studies of the molecular basis of cardiomyopathies. Supported by MIUR-PRIN and Telethon (Italy).

Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4°C and 2.15 μm sarcomere length, using sarcomere level mechanics and X-ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin-based reflections were recorded with 5 ms frames using the gas-filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T0) with a half-time of 40 ms, and the spacings of the M3 and M6 reflections (SM3 and SM6) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half-sarcomere)−1 shortening at the velocity (V0) that maintains zero force. Shortening at V0 transiently reduces SM3 following the latent period and delays the subsequent increase in SM3, but only delays the SM6 increase without a transient decrease. Shortening at V0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (IM3), whereas the intensity of the M6 reflection (IM6) is only slightly reduced. The changes in half-sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.

X-ray diffraction patterns were recorded from isolated single fibres of frog skeletal muscle during isometric contraction at temperatures between 0 and 17°C. Isometric force was 43 ± 2% (mean ±s.e.m., n= 10) higher at 17°C than 0°C. The intensity of the first actin layer line increased by 57 ± 18% (n= 5), and the ratio of the intensities of the equatorial 1,1 and 1,0 reflections by 20 ± 7% (n= 10), signalling radial or azimuthal motions of the myosin head domains. The M3 X-ray reflection from the axial repeat of the heads along the filaments was 27 ± 4% more intense at 17°C, suggesting that the heads became more perpendicular to the filaments. The ratio of the intensities of the higher and lower angle peaks of the M3 reflection (RM3) was 0.93 ± 0.02 (n= 5) at 0°C and 0.77 ± 0.02 at 17°C. These peaks are due to interference between the two halves of each myosin filament, and the RM3 decrease shows that heads move towards the midpoint of the myosin filament at the higher temperature. Calculations based on a crystallographic model of the heads indicated that the observed RM3 change corresponds to tilting of their light-chain domains by 9 deg, producing an axial displacement of 1.4 nm, which is equal to that required to strain the actin and myosin filaments under the increased force. We conclude that the higher force generated by skeletal muscle at higher temperature can be accounted for by axial tilting of the myosin heads.

The molecular mechanism of muscle contraction was investigated in intact muscle fibres by X–ray diffraction. Changes in the intensities of the axial X–ray reflections produced by imposing rapid changes in fibre length establish the average conformation of the myosin heads during active isometric contraction, and show that the heads tilt during the elastic response to a change in fibre length and during the elementary force generating process: the working stroke. X–ray interference between the two arrays of myosin heads in each filament allows the axial motions of the heads following a sudden drop in force from the isometric level to be measured in situ with unprecedented precision. At low load, the average working stroke is 12 nm, which is consistent with crystallographic studies. The working stroke is smaller and slower at a higher load. The compliance of the actin and myosin filaments was also determined from the change in the axial spacings of the X–ray reflections following a force step, and shown to be responsible for most of the sarcomere compliance. The mechanical properties of the sarcomere depend on both the motor actions of the myosin heads and the compliance of the myosin and actin filaments.

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.5-2 units of pCa are necessary to raise isometric force from its threshold (pCa approximately 6.5) to its maximum (F(0,max)). The amplitude of F(SA) depends only on the preceding baseline level of isometric force, which must reach at least 0.05 F(0,max) to enable stretch-activation. F(SA) rises very steeply to its maximum as F(0) reaches approximately 0.2 F(0,max), then decreases as F(0) increases so as to produce a constant sum (F(0) + F(SA)) = F(max). Thus Ca- and stretch-activation are complementary pathways that trigger a common process of cross-bridge attachment and force production. We suggest that stretch-induced distortion of attached cross-bridges relieves the steric blocking by tropomyosin of additional binding sites on actin, thereby enabling maximum force even at low [Ca(2+)].

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca2+ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0–1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40–70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin–myosin interactions under stretch become independent of the MHC isoform.

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T(0)), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (

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.

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.

Muscles generate force and shortening in a cyclical interaction between the myosin head domains projecting from the myosin filaments and the adjacent actin filaments. Although many features of the dynamic performance of muscle are determined by the rates of attachment and detachment of myosin and actin, the primary event in force generation is thought to be a conformational change or 'working stroke' in the actin-bound myosin head. According to this hypothesis, the working stroke is much faster than attachment or detachment, but can be observed directly in the rapid force transients that follow step displacement of the filaments. Although many studies of the mechanism of muscle contraction have been based on this hypothesis, the alternative view-that the fast force transients are caused by fast components of attachment and detachment--has not been excluded definitively. Here we show that measurements of the axial motions of the myosin heads at ångström resolution by a new X-ray interference technique rule out the rapid attachment/detachment hypothesis, and provide compelling support for the working stroke model of force generation.

Recently, we have shown that the delta subunit of the cGMP phosphodiesterase (PDE delta) interacts with the retinitis pigmentosa guanine regulator (RPGR). Here, using the two-hybrid system, we identify a member of the Arf-like protein family of Ras-related GTP-binding proteins, Arl3, that interacts with PDE delta. The interaction was verified by fluorescence spectroscopy and co-immunoprecipitation. Arl3 features an unusually low affinity for guanine nucleotides, with a KD of 24 nM for GDP and 48 microM for GTP. Fluorescence spectroscopy shows that PDE delta binds and specifically stabilizes the GTP-bound form of Arl3 by strongly decreasing the dissociation rate of GTP. Thus, PDE delta is an effector of Arl3 and could provide a novel nucleotide exchange mechanism by which PDE delta stabilizes Arl3 in its active GTP-bound form.

1. Two-dimensional X-ray diffraction patterns were recorded at the European Synchrotron Radiation Facility from central segments of intact single muscle fibres of Rana temporaria with 5 ms time resolution during the development of isometric contraction. Shortening at ca 0.8 times the maximum velocity was also imposed at the isometric tetanus plateau. 2. The first myosin-based layer line (ML1) and the second myosin-based meridional reflection (M2), which are both strong in resting muscle, were completely abolished at the plateau of the isometric tetanus. The third myosin-based meridional reflection (M3), arising from the axial repeat of the myosin heads along the filaments, remained intense but its spacing changed from 14.34 to 14.56 nm. The intensity change of the M3 reflection, IM3, could be explained as the sum of two components, I14.34 and I14.56, arising from myosin head conformations characteristic of rest and isometric contraction, respectively. 3. The amplitudes (A) of the X-ray reflections, which are proportional to the fraction of myosin heads in each conformation, changed with half-times that were similar to that of isometric force development, which was 33.5 +/- 2. 0 ms (mean +/- s.d., 224 tetani from three fibres, 4 C), measured from the end of the latent period. We conclude that the myosin head conformation changes synchronously with force development, at least within the 5 ms time resolution of these measurements. 4. The changes in the X-ray reflections during rapid shortening have two temporal components. The rapid decrease in intensity of the 14.56 nm reflection at the start of shortening is likely to be due to tilting of myosin heads attached to actin. The slower changes in the other reflections were consistent with a return to the resting conformation of the myosin heads that was about 60 % complete after shortening of 70 nm per half-sarcomere.

Titin, a giant elastomeric muscle protein has been implicated to function as a sensor of sarcomeric stress and strain but with unresolved mechanisms. To gain insight into titin's mechanosensory function here we manipulated single molecules with high-resolution optical tweezers. Discrete, stepwise 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 recovered our experimental results and predicted that structural transitions may begin prior to the complete extension of the PEVK domain. High-resolution AFM of titin extended with meniscus forces 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 intra-PEVK-domain interactions contribute significantly to sarcomere mechanics. Thus, under non-equilibrium conditions across the physiological force range, titin extends via a complex pattern of history-dependent discrete conformational transitions which, by dynamically exposing ligand-binding sites, may set the stage for the biochemical sensing of the sarcomeric mechanical status.

The half-sarcomere, the functional unit of skeletal muscle, is able to produce power at high efficiency or resist a sudden increase in load with low metabolic cost, due to the combination of the function of the myosin II motors in the thick filament overlapped with the thin filament and the meshwork of cytoskeleton proteins acting as a scaffold. The importance of cytoskeleton proteins is illustrated by the identification of mutations in many of the corresponding human genes in patients with skeletal myopathies. Here we studied the role of myopalladin (MYPN), a protein located in the Z-line and the I-band. MYPN gene mutations have been identified in patients with limb-girdle dystrophy as well as dilated, hypertrophic and restrictive cardiomyopathy (Duboscq-Bidot et al. Cardiovasc Res 77:118, 2008; Purevjav et al. Hum Mol Genet 21:2039, 2012). To provide insights into the physiological role of this protein and the mechanisms leading to myopathy, the mechanical performances of skeletal muscles from wt and KO mice were determined. In EDL muscle, the absence of MYPN (i) decreases the isometric force (T0) by 48% and the cross-sectional area (CSA), calculated by the muscle wet weight, by 21%; (ii) decreases the power at any load and (iii) does not affect the curvature of the force-velocity relation. In skinned fibers from the same muscles T0 is reduced in proportion to CSA, indicating that the CSA of EDL muscle is overestimated in KO mice with respect to control. Thus the reduced muscle performance in KO mice is due to the reduction in fiber dimension while the kinetics of actin-myosin interaction is unaffected. Injection of an adeno-associated virus (AAV) vector expressing the wt form of MYPN results in a substantial rescue of muscle performance. Supported by Ministero della Salute, Telethon and MIUR (Italy).

During muscle contraction, the myosin motors emerging from the myosin filament in each half-sarcomere form cross-bridges with the opposing actin filament, pulling it towards the center of the sarcomere through a structural working stroke. Motors are mechanically coupled via their filament attachments, and the co-operative action of this coupled system is the basic functional unit of muscle, so determining the elastic properties of the half-sarcomere components is crucial for understanding the mechanism of this collective motor. To that end we used mechanical and X-ray diffraction measurements on single fibers and whole muscles of the frog. 4kHz length oscillations were imposed on single fibers (4°C, 2.15µm sarcomere length) to determine how the half-sarcomere compliance (Chs) is modulated by force. The results indicate the presence of an elastic element in parallel with the array of cross-bridges with a compliance of 200 nm/MPa, ca20 times larger than that attributed to the cross-bridges. X-ray diffraction from whole frog muscles allows precise measurements of the spacing of the high-order myosin- and actin-based reflections, which report changes in the strain of the two filaments. We found that, at forces >0.4 the isometric tetanic force, the filament compliance is constant and contributes 13.1±1.1 nm/MPa to Chs, from which a cross-bridge compliance of 0.35±0.13 nm/pN is calculated. This value is not significantly different from that of the myosin head alone, 0.38±0.06 nm/pN, estimated from changes in the intensity of the 14.5-nm X-ray reflection from the axial repeat of myosin heads during rapid length oscillations in rigor fibers. We conclude that cross-bridge compliance is low and fully accounted for by the compliance of the myosin head, without a significant contribution from the rod connecting the head to the filament backbone. Supported by MIUR-PRIN and FIRB (ITALY).