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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 2+ -sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca 2+ . 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). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency. Omecamtiv mecarbil is a small molecule effector under clinical trial for the treatment of systolic heart failure. Here the authors define the molecular mechanisms of its inotropic action and find it can increase the efficiency of contraction in muscle fibres when the orthophosphate concentration rises with the beat frequency.

The stiffness of the single myosin motor ( ɛ) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force ( T 0) modulated by changing either [Ca 2+] or temperature. The hs compliance is 43.0 ± 0.8 nm MPa −1 in isometric contraction at saturating [Ca 2+], whereas in rigor it is 28.2 ± 1.1 nm MPa −1. The equivalent compliance of myofilaments is 21.0 ± 3.3 nm MPa −1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5 ± 1.7 kPa nm −1 in isometric contraction at saturating [Ca 2+] ( e 0), and in rigor ( e r) it rises to 138.9 ± 21.2 kPa nm −1. ɛ, calculated from e r and the lattice molecular dimensions, is 1.21 ± 0.18 pN nm −1. ɛ estimated, using a thermodynamic approach, from the relation of T 0 at saturating [Ca 2+] versus the reciprocal of absolute temperature is 1.25 ± 0.14 pN nm −1, similar to that estimated for fibers in rigor. Consequently, the ratio e 0/ e r (0.33 ± 0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca 2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e 0 and e r increase by ∼40%. ɛ becomes ∼1.7 pN nm −1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.

Creatine (Cr) is essential for cellular energy homeostasis, particularly in muscle and brain tissues. Creatine Transporter Deficiency (CTD), an X-linked disorder caused by mutations in the SLC6A8 gene, disrupts Cr transport, leading to intellectual disability, speech delay, autism, epilepsy, and various non-neurological symptoms. In addition to neurological alterations, Creatine Transporter knockout (CrT −/y ) mice exhibit severe muscle atrophy and functional impairments. This study provides the first characterization of the skeletal muscle phenotype in CrT −/y mice, revealing profound ultrastructural abnormalities accompanied by reduced fiber cross-sectional area and muscle performance. Notably, mitochondria are involved, as evidenced by disrupted cristae, increased mitochondrial size, impaired Ca 2+ uptake, reduced membrane potential and ATP production. Mechanistically, the expression of atrophy-specific E3 ubiquitin ligases and suppression of the IGF1-Akt/PKB pathway, regulated by mitochondrial Ca 2+ levels, further support the atrophic phenotype. These findings highlight the profound impact of Cr deficiency on skeletal muscle, emphasizing the need for targeted therapeutic strategies to address both the neurological and peripheral manifestations of CTD. Understanding the underlying mechanisms, particularly mitochondrial dysfunction, could lead to novel interventions for this disorder.

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 T P to a value T (0.8–0.2 T P). 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 T P, do not affect the dependence on T, normalized for T P, of the size and speed of the working stroke. Thus, length- and Ca2+-dependent increase of T P 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.

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 2+ 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 2+ ]), estimated by Hill coefficient n H . We use sarcomere-level mechanics in demembranated fibres of rabbit skeletal muscle activated by Ca 2+ at different temperatures (12–35 °C) to show that n H 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. Sarcomeres of Ca 2 +  -activated demembranated fibres of rabbit soleus muscles show that the stress exerted by each motor protein on the thin filament determines the cooperativity in thin filament activation. 

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 output of a nanomachine powered by an array of myosin molecules purified from the skeletal muscle is fed to a stochastic model, which provides a self-consistent estimate of all the mechanokinetic parameters of the motor ensemble.

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.

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.

A shortening muscle is a machine that converts metabolic energy into mechanical work, but, when a muscle is stretched, it acts as a brake, generating a high resistive force at low metabolic cost. The braking action of muscle can be activated with remarkable speed, as when the leg extensor muscles rapidly decelerate the body at the end of a jump. Here we used time-resolved x-ray and mechanical measurements on isolated muscle cells to elucidate the molecular basis of muscle braking and its rapid control. We show that a stretch of only 5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere is sufficient to double the number of myosin motors attached to actin within a few milliseconds. Each myosin molecule has two motor domains, only one of which is attached to actin during shortening or activation at constant length. A stretch strains the attached motor domain, and we propose that combined steric and mechanical coupling between the two domains promotes attachment of the second motor domain. This mechanism allows skeletal muscle to resist external stretch without increasing the force per motor and provides an answer to the longstanding question of the functional role of the dimeric structure of muscle myosin.

Muscle contraction is driven by the motor protein myosin II, which binds transiently to an actin filament, generates a unitary filament displacement or ‘working stroke’, then detaches and repeats the cycle. The stroke size has been measured previously using isolated myosin II molecules at low load, with rather variable results 1 , 2 , 3 , 4 , but not at the higher loads that the motor works against during muscle contraction. Here we used a novel X-ray-interference technique 5 , 6 to measure the working stroke of myosin II at constant load 7 in an intact muscle cell, preserving the native structure and function of the motor. We show that the stroke is smaller and slower at higher load. The stroke size at low load is likely to be set by a structural limit 8 , 9 ; at higher loads, the motor detaches from actin before reaching this limit. The load dependence of the myosin II stroke is the primary molecular determinant of the mechanical performance and efficiency of skeletal muscle.