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Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca 2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H + ) act to directly and indirectly (via decreased Ca 2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases.

Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients ( N = 54) to those of non-ICU patients undergoing thoracic surgery ( N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.

Studies of muscle structure and function can be traced to at least 2,000 years ago. However, the modern era of muscle contraction mechanisms started in the 1950s with the classic works by AF Huxley and HE Huxley, both born in the United Kingdom, but not related and working independently. HE Huxley was the first to suggest that muscle contraction occurred through the sliding of two sets of filamentous structures (actin or thin filaments and myosin or thick filaments). AF Huxley then developed a biologically inspired mathematical model suggesting a possible molecular mechanism of how this sliding of actin and myosin might take place. This model then evolved from a two-state to a multi-state model of myosin-actin interactions, and from one that suggested a linear motor causing the sliding to a rotating motor. This model, the cross-bridge model of muscle contraction, is still widely used in biomechanics, and even the more sophisticated cross-bridge models of today still contain many of the features originally proposed by AF Huxley. In 2002, we discovered a hitherto unknown property of muscle contraction that suggested the involvement of passive structures in active force production, the so-called passive force enhancement. It was quickly revealed that this passive force enhancement was caused by the filamentous protein titin, and the three-filament (actin, myosin, and titin) sarcomere model of muscle contraction evolved. There are many suggestions of how these three proteins interact to cause contraction and produce active force, and one such suggestion is described here, but the molecular details of this proposed mechanism still need careful evaluation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Molecular motors such as kinesin and myosin often work in groups to generate the directed movements and forces critical for many biological processes. Although much is known about how individual motors generate force and movement, surprisingly, little is known about the mechanisms underlying the macroscopic mechanics generated by multiple motors. For example, the observation that a saturating number, N, of myosin heads move an actin filament at a rate that is influenced by actin-myosin attachment and detachment kinetics is accounted for neither experimentally nor theoretically. To better understand the emergent mechanics of actin-myosin mechanochemistry, we use an in vitro motility assay to measure and correlate the N-dependence of actin sliding velocities, actin-activated ATPase activity, force generation against a mechanical load, and the calcium sensitivity of thin filament velocities. Our results show that both velocity and ATPase activity are strain dependent and that velocity becomes maximized with the saturation of myosin-binding sites on actin at a value that is 40% dependent on attachment kinetics and 60% dependent on detachment kinetics. These results support a chemical thermodynamic model for ensemble motor mechanochemistry and imply molecularly explicit mechanisms within this framework, challenging the assumption of independent force generation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Myofibroblasts are contractile cells found in multiple tissues. They are physiological cells as in the human placenta and can be obtained from bone marrow mesenchymal stem cells after differentiation by transforming growth factor-β (TGF-β). They are also found in the stroma of cancerous tissues and can be located in non-muscle contractile tissues. When stimulated by an electric current or after exposure to KCl, these tissues contract. They relax either by lowering the intracellular Ca 2+ concentration (by means of isosorbide dinitrate or sildenafil) or by inhibiting actin-myosin interactions (by means of 2,3-butanedione monoxime or blebbistatin). Their shortening velocity and their developed tension are dramatically low compared to those of muscles. Like sarcomeric and smooth muscles, they obey Frank-Starling's law and exhibit the Hill hyperbolic tension-velocity relationship. The molecular motor of the myofibroblast is the non-muscle myosin type IIA (NMIIA). Its essential characteristic is the extreme slowness of its molecular kinetics. In contrast, NMIIA develops a unitary force similar to that of muscle myosins. From a thermodynamic point of view, non-muscle contractile tissues containing NMIIA operate extremely close to equilibrium in a linear stationary mode.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles., Competing Interests: CH, EB, LF, JO, MI No competing interests declared, (© 2021, Hill et al.)

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca 2+ -sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin's ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca 2+ . These changes in the behavior of tropomyosin and the troponin-tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca 2+ -sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca 2+ -dependent movement and the ability of the troponin-tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.

Striated muscle contraction is initiated by Ca 2+ binding to, and activating, thin filament regulatory units (RU) within the sarcomere, which then allows myosin cross-bridges from the opposing thick filament to bind actin and generate force. The amount of overlap between the filaments dictates how many potential cross-bridges are capable of binding, and thus how force is generated by the sarcomere. Myopathies and atrophy can impair muscle function by limiting cross-bridge interactions between the filaments, which can occur when the length of the thin filament is reduced or when RU function is disrupted. To investigate how variations in thin filament length and RU density affect ensemble cross-bridge behavior and force production, we simulated muscle contraction using a spatially explicit computational model of the half-sarcomere. Thin filament RUs were disabled either uniformly from the pointed end of the filament (to model shorter thin filament length) or randomly throughout the length of the half-sarcomere. Both uniform and random RU 'knockout' schemes decreased overall force generation during maximal and submaximal activation. The random knockout scheme also led to decreased calcium sensitivity and cooperativity of the force-pCa relationship. We also found that the rate of force development slowed with the random RU knockout, compared to the uniform RU knockout or conditions of normal RU activation. These findings imply that the relationship between RU density and force production within the sarcomere involves more complex coordination than simply the raw number of RUs available for myosin cross-bridge binding, and that the spatial pattern in which activatable RU are distributed throughout the sarcomere influences the dynamics of force production., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2 , are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2 -/- ) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2 -/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2 -/- mice. Small-angle X-ray diffraction of C2 -/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2 -/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca 2+- activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2 -/- mice. Finally, C2 -/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle., Competing Interests: Competing interest statement: S.S. provided consulting and collaborative research studies to the Leducq Foundation, Red Saree Inc., Greater Cincinnati Tamil Sangam, AstraZeneca, MyoKardia, Merck, and Amgen, but such work is unrelated to the content of this manuscript.

The origin of the incessant rhythmical heartbeat and the mechanism of muscle contraction have fascinated scientists over centuries. This short review outlines physiological explanations that were discussed in the 19th century starting with Albrecht von Haller (1708-1777), an 18th century physiologist who proposed that the heart has an intrinsic irritability. He argued that under normal conditions the inflow of blood stimulates the heart muscle to contract by mechanical touch and distension. Johannes Müller (1800-1858, physiologist in Bonn and Berlin) contended that the influence of the sympathetic nerve, specifically the activity of intracardiac ganglia, is the foremost cause of the heartbeat. Walter H. Gaskell and Theodor Engelmann (physiologists in Cambridge and Utrecht, respectively) independently criticized this neurogenic theory. They reported experimental evidence that supported the myogenic theory of the origin of the heartbeat, which has been accepted since about 1900. The concept of cardiac mechano-sensitivity, which can be traced back to A. von Haller, is currently resurging. Concerning mechanisms of contraction, Edward A. Schäfer (1850-1935), histologist and physiologist in Edinburgh, described differences between cardiac and skeletal muscle and coined the term sarcomere. Based on microscopic studies of cross-striated muscle, Schäfer outlined a detailed and plausible mechanism of muscle contraction in 1892. He put forward that during muscle shortening the "clear part of the muscle substance" (actin) might pass into longitudinal canals, which exist between the "sarcous elements" (myosin). His model foresaw fundamental elements of the sliding filament model, which was discovered by the Huxleys about 60 years later., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Vertebrate striated muscle thin filaments are thought to be thermodynamically activated in response to an increase in Ca 2+ concentration. We tested this hypothesis by measuring time intervals for gliding runs and pauses of individual skeletal muscle thin filaments in cycling myosin motility assays. A classic thermodynamic mechanism predicts that if chemical potential is constant, transitions between runs and pauses of gliding thin filaments will occur at constant rate as given by a Poisson distribution. In this scenario, rate is given by the odds of a pause, and hence, run times between pauses fit an exponential distribution that slopes negatively for all observable run times. However, we determined that relative density of observed run times fits an exponential only at low Ca 2+ levels that activate filament gliding. Further titration with Ca 2+ , or adding excess regulatory proteins tropomyosin and troponin, shifted the relative density of short run times to fit the positive slope of a gamma distribution, which derives from waiting times between Poisson events. Events that arise during a run and prevent the chance of ending a run for a random interval of time account for the observed run time distributions, suggesting that the events originate with cycling myosin. We propose that regulatory proteins of the thin filament require the mechanical force of cycling myosin to achieve the transition state for activation. During activation, combinations of cycling myosin that contribute insufficient activation energy delay deactivation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Zot et al.)

Muscle contraction results from cyclic interactions between myosin II motors and actin with two sets of proteins organized in overlapping thick and thin filaments, respectively, in a nearly crystalline lattice in a muscle sarcomere. However, a sarcomere contains a huge number of other proteins, some with important roles in muscle contraction. In particular, these include thin filament proteins, troponin and tropomyosin; thick filament proteins, myosin binding protein C; and the elastic protein, titin, that connects the thin and thick filaments. Furthermore, the order and 3D organization of the myofilament lattice may be important per se for contractile function. It is possible to model muscle contraction based on actin and myosin alone with properties derived in studies using single molecules and biochemical solution kinetics. It is also possible to reproduce several features of muscle contraction in experiments using only isolated actin and myosin, arguing against the importance of order and accessory proteins. Therefore, in this paper, it is hypothesized that "single molecule actomyosin properties account for the contractile properties of a half sarcomere during shortening and isometric contraction at almost saturating Ca concentrations". In this paper, existing evidence for and against this hypothesis is reviewed and new modeling results to support the arguments are presented. Finally, further experimental tests are proposed, which if they corroborate, at least approximately, the hypothesis, should significantly benefit future effective analysis of a range of experimental studies, as well as drug discovery efforts.

We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln 147 Pro substitution in β-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca 2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln 147 Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca 2+ .

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

Molecular motors have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction of these motors is a root cause of many pathologies necessitating the need for intrinsic control over molecular motor function. Herein, we demonstrate that positional isomerism can be used as a simple and powerful tool to control the molecular motor of muscle, myosin. Using three isomers of a synthetic non-nucleoside triphosphate, we demonstrate that myosin's force- and motion-generating capacity can be dramatically altered at both the ensemble and single-molecule levels. By correlating our experimental results with computation, we show that each isomer exerts intrinsic control by affecting distinct steps in myosin's mechanochemical cycle. Our studies demonstrate that subtle variations in the structure of an abiotic energy source can be used to control the force and motility of myosin without altering myosin's structure., (Copyright © 2020. Published by Elsevier Inc.)

Among patients with intensive care unit-acquired weakness (ICUAW), skeletal muscle strength often decreases significantly. The present study aimed to explore the effects of testosterone propotionate on skeletal muscle using rat model of sepsis. Male SD rats were randomly divided into experimental group, model control group, sham operation group and blank control group. Rats in experimental group were given testosterone propionate two times a week, 10 mg/kg for 3 weeks. Maximal contraction force, fatigue index and cross-sectional area of the extensor digitorum longus (EDL) were measured. Myosin, IGF-1, p-AKT and p-mTOR levels in EDL were detected by Western blot. Histological changes of the testis and prostate were detected by hematoxylin and eosin staining. We found that maximal contraction force and fatigue index of EDL in experimental group were significantly higher than in model control group. Cross-sectional area of fast MHC muscle fiber of EDL in group was significantly higher than in model control group. The levels of myosin, IGF-1, p-AKT and p-mTOR of EDL in experimental group were significantly higher than in model control group. In addition, no testicle atrophy and prostate hyperplasia were detected in experimental group. In conclusion, these results suggest that testosterone propionate can significantly improve skeletal muscle strength, endurance and volume of septic rats, and the mechanism may be related to the activation of IGF-1/AKT pathway. Moreover, testosterone propionate with short duration does not cause testicular atrophy and prostate hyperplasia in septic rats. Therefore, testosterone propionate is a potential treatment for muscle malfunction in ICUAW patients., (© 2020 The Author(s).)

In the present study we evaluated the load dependence of force produced by isolated muscle myosin filaments interacting with fluorescently labeled actin filaments, using for the first time whole native myosin filaments. We used a newly developed approach that allowed the use of physiological levels of ATP. Single filaments composed of either skeletal or smooth muscle myosin and single filaments of actin were attached between pairs of nano-fabricated cantilevers of known stiffness. The filaments were brought into contact to produce force, which caused sliding of the actin filaments over the myosin filaments. We applied load to the system by either pushing or pulling the filaments during interactions and observed that increasing the load increased the force produced by myosin and decreasing the load decreased the force. We also performed additional experiments in which we clamped the filaments at predetermined levels of force, which caused the filaments to slide to adjust the different loads, allowing us to measure the velocity of length changes to construct a force-velocity relation. Force values were in the range observed previously with myosin filaments and molecules. The force-velocity curves for skeletal and smooth muscle myosins resembled the relations observed for muscle fibers. The technique can be used to investigate many issues of interest and debate in the field of muscle biophysics.

Muscle contraction is initiated by the interaction between actin and myosin filaments. The sliding of actin filaments relative to myosin filaments is produced by cross-bridge cycling, which is governed by the theoretical framework of the cross-bridge theory. The cross-bridge theory explains well a number of mechanical responses, such as isometric and concentric contractions. However, some experimental observations cannot be explained with the cross-bridge theory; for example, the increased isometric force after eccentric contractions. The steady-state, isometric force after an eccentric contraction is greater than that attained in a purely isometric contraction at the same muscle length and same activation level. This well-acknowledged and universally observed property is referred to as residual force enhancement (rFE). Since rFE cannot be explained by the cross-bridge theory, alternative mechanisms for explaining this force response have been proposed. In this review, we introduce the basic concepts of sarcomere length non-uniformity and titin elasticity, which are the primary candidates that have been used for explaining rFE, and discuss unresolved problems regarding these mechanisms, and how to proceed with future experiments in this exciting area of research.

Although there is good evidence to indicate a major role of intrinsic impairment of the contractile apparatus in muscle weakness seen in several pathophysiological conditions, the factors responsible for control of myofibrillar function are not fully understood. To investigate the role of mechanical load in myofibrillar function, we compared the skinned fiber force between denervated (DEN) and dexamethasone-treated (DEX) rat skeletal muscles with or without neuromuscular electrical stimulation (ES) training. DEN and DEX were induced by cutting the sciatic nerve and daily injection of dexamethasone (5 mg/kg/day) for 7 days, respectively. For ES training, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. In situ maximum torque was markedly depressed in the DEN muscles compared to the DEX muscles (-74% vs. -10%), whereas there was not much difference in the degree of atrophy in gastrocnemius muscles between DEN and DEX groups (-24% vs. -17%). Similar results were obtained in the skinned fiber preparation, with a greater reduction in maximum Ca2+-activated force in the DEN than in the DEX group (-53% vs. -16%). Moreover, there was a parallel decline in myosin heavy chain (MyHC) and actin content per muscle volume in DEN muscles, but not in DEX muscles, which was associated with upregulation of NADPH oxidase (NOX) 2, neuronal nitric oxide synthase (nNOS), and endothelial NOS expression, translocation of nNOS from the membrane to the cytosol, and augmentation of mRNA levels of muscle RING finger protein 1 (MuRF-1) and atrogin-1. Importantly, mechanical load evoked by ES protects against DEN- and DEX-induced myofibrillar dysfunction and these molecular alterations. Our findings provide novel insights regarding the difference in intrinsic contractile properties between DEN and DEX and suggest an important role of mechanical load in preserving myofibrillar function in skeletal muscle., Competing Interests: The authors have declared that no competing interests exist.

The stiffness of the myosin cross-bridges is a key factor in analysing possible scenarios to explain myosin head changes during force generation in active muscles. The seminal study of Huxley and Simmons (1971: Nature 233 : 533) suggested that most of the observed half-sarcomere instantaneous compliance (=1/stiffness) resides in the myosin heads. They showed with a so-called T 1 plot that, after a very fast release, the half-sarcomere tension reduced to zero after a step size of about 60Å (later with improved experiments reduced to 40Å). However, later X-ray diffraction studies showed that myosin and actin filaments themselves stretch slightly under tension, which means that most (at least two-thirds) of the half sarcomere compliance comes from the filaments and not from cross-bridges. Here we have used a different approach, namely to model the compliances in a virtual half sarcomere structure in silico. We confirm that the T 1 curve comes almost entirely from length changes in the myosin and actin filaments, because the calculated cross-bridge stiffness (probably greater than 0.4 pN/Å) is higher than previous studies have suggested. Our model demonstrates that the formulations produced by previous authors give very similar results to our model if the same starting parameters are used. However, we find that it is necessary to model the X-ray diffraction data as well as mechanics data to get a reliable estimate of the cross-bridge stiffness. In the light of the high cross-bridge stiffness found in the present study, we present a plausible modified scenario to describe aspects of the myosin cross-bridge cycle in active muscle. In particular, we suggest that, apart from the filament compliances, most of the cross-bridge contribution to the instantaneous T 1 response may come from weakly-bound myosin heads, not myosin heads in strongly attached states. The strongly attached heads would still contribute to the T 1 curve, but only in a very minor way, with a stiffness that we postulate could be around 0.1 pN/Å, a value which would generate a working stroke close to 100 Å from the hydrolysis of one ATP molecule. The new model can serve as a tool to calculate sarcomere elastic properties for any vertebrate striated muscle once various parameters have been determined (e.g., tension, T 1 intercept, temperature, X-ray diffraction spacing results).

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes 1,2 by assembling into various architectures with distinct force generation properties 3,4 . While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems 3 , it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis 5-7 . We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments 8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis 7 . Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.

Rapid sarcomere lengthening waves propagate along a single muscle myofibril during spontaneous oscillatory contraction (SPOC). In asynchronous insect flight muscles, SPOC is thought to be almost completely synchronized over the entire myofibril. This phenomenon does not require Ca 2+ regulation of the dynamics of the motor proteins, and cannot be explained simply by the longitudinal mechanical equilibrium among sarcomeres in the myofibril. In the present study, we rationalize these phenomena by considering the lateral mechanical equilibrium, in which two tensions originating from the inverse relationship between sarcomere length and lattice spacing, along with the lattice alignment, play important roles in the mechanical communication between motor proteins on adjacent filaments via the Z-disc. The proposed model is capable of explaining various SPOC phenomena based on the stochastic power-stroke mechanism of motor proteins, which responds to temporal changes in longitudinal mechanical load.

The relation between the force (load) and the velocity of shortening ( V ) in contracting skeletal muscle is part of a rectangular hyperbola: ( P + a) V = b( Po - P ); where Po is the maximum isometric force and a and b are constants. The force-velocity ( P-V ) relation suggests that muscle can regulate its energy output depending on the load imposed on it (Hill, 1938). After the establishment of the sliding filament mechanism (H.E. Huxley and Hanson, 1954), the P - V relation has been regarded to reflect the cyclic interaction between myosin heads in myosin filaments and the corresponding myosin-binding sites in actin filaments, coupled with ATP hydrolysis (A.F. Huxley, 1957). In single skeletal muscle fibers, however, the P - V relation deviates from the hyperbola at the high force region, indicating complicated characteristics of the cyclic actin-myosin interaction. To correlate the P - V relation with kinetics of actin-myosin interaction, skinned muscle fibers have been developed, in which the surface membrane is removed to control chemical and ionic conditions around the 3D lattice of actin and myosin filaments. This article also deals with experimental methods with which the structural instability of skinned fibers can be overcome by applying parabolic decreases in fiber length.

Some vertebrate muscles (e.g. those in bony fish) have a simple lattice A-band which is so well ordered that low-angle X-ray diffraction patterns are sampled in a simple way amenable to crystallographic techniques. Time-resolved X-ray diffraction through the contractile cycle should provide a movie of the molecular movements involved in muscle contraction. Generation of 'Muscle-The Movie' was suggested in the 1990s and since then efforts have been made to work out how to achieve it. Here we discuss how a movie can be generated, we discuss the problems and opportunities, and present some new observations. Low angle X-ray diffraction patterns from bony fish muscles show myosin layer lines that are well sampled on row-lines expected from the simple hexagonal A-band lattice. The 1st, 2nd and 3rd myosin layer lines at d-spacings of around 42.9 nm, 21.5 nm and 14.3 nm respectively, get weaker in patterns from active muscle, but there is a well-sampled intensity remnant along the layer lines. We show here that the pattern from the tetanus plateau is not a residual resting pattern from fibres that have not been fully activated, but is a different well-sampled pattern showing the presence of a second, myosin-centred, arrangement of crossbridges within the active crossbridge population. We also show that the meridional M3 peak from active muscle has two components of different radial widths consistent with (i) active myosin-centred (probably weak-binding) heads giving a narrow peak and (ii) heads on actin in strong states giving a broad peak.

The hyperbolic shape of the muscle force-velocity relationship (FVR) is characteristic of all muscle fiber types. The degree of curvature of the hyperbola varies between muscle fiber types and is thought to be set by force-dependent properties of different myosin isoforms. However, the structural elements in myosin and the mechanism that determines force dependence are unresolved. We tested our hypothesis that the myosin converter domain plays a critical role in the force-velocity relationship (FVR) mechanism. Drosophila contains a single myosin heavy chain gene with five converters encoded by alternative exons. We measured FVR properties of Drosophila jump muscle fibers from five transgenic lines each expressing a single converter. Consistent with our hypothesis, we observed up to 2.4-fold alterations in FVR curvature. Maximum shortening velocity ( v 0 ) and optimal velocity for maximum power generation were also altered, but isometric tension and maximum power generation were unaltered. Converter 11a, normally found in the indirect flight muscle (IFM), imparted the highest FVR curvature and v 0 , whereas converter 11d, found in larval body wall muscle, imparted the most linear FVR and slowest v 0 . Jump distance strongly correlated with increasing FVR curvature and v 0 , meaning flies expressing the converter from the IFM jumped farther than flies expressing the native jump muscle converter. Fitting our data with Huxley's two-state model and a biophysically based four-state model suggest a testable hypothesis that the converter sets muscle type FVR curvature by influencing the detachment rate of negatively strained myosin via changes in the force dependence of product release.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease characterized by cortical and spinal motor neuron degeneration, some inherited cases of which are caused by mutations in the gene coding for copper-zinc superoxide dismutase-1 (SOD1). The SOD1 G93A mutant model mouse, which expresses large amounts of mutant SOD1, develops adult-onset neurodegeneration of spinal motor neurons and progressive motor deficits leading to paralysis. We used the Tandem Mass Tag technique to investigate the proteome profile of hippocampus, cerebral cortex, and medulla oblongata of the SOD1 G93A mutant model mice as compared with that of wild-type (WT) mice. Fifteen proteins were significantly increased or decreased (i.e., changed) in all three tissues. Gene ontology analysis revealed that the changed proteins were mainly enriched in negative regulation of reactive oxygen species, myosin complex and copper ion binding. In the Striated Muscle Contraction Pathway, most of the identified proteins were decreased in the SOD1 G93A mice compared with the WT mice. Myosin-1 (MYH1), fructose-2,6-bisphosphatase TIGAR (TIGAR), and sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1) were significantly reduced in mutant vs WT mice, as confirmed by Western blot analysis. Since myosins and tropomyosins are specific for synapse function and drive actin dynamics in the maturation of dendritic spines, changes in these proteins may contribute to perturbations of brain neuronal circuitry in addition to spinal motor neuron disease.

The actomyosin cytoskeleton is responsible for most force-driven processes in cells and tissues. How it assembles into the necessary structures at the right time and place is an important question. Here, we focus on molecular mechanisms of actomyosin regulation recently elucidated in animal models, and highlight several common principles that emerge. The architecture of the actomyosin network - an important determinant of its function - results from actin polymerization, crosslinking and turnover, localized myosin activation, and contractility-driven self-organization. Spatiotemporal regulation is achieved by tissue-specific expression and subcellular localization of Rho GTPase regulators. Subcellular anchor points of actomyosin structures control the outcome of their contraction, and molecular feedback mechanisms dictate whether they are transient, cyclic, or persistent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cell adhesion, morphogenesis, mechanosensing, and muscle contraction rely on contractile actomyosin bundles, where the force is produced through sliding of bipolar myosin II filaments along actin filaments. The assembly of contractile actomyosin bundles involves registered alignment of myosin II filaments and their subsequent fusion into large stacks. However, mechanisms underlying the assembly of myosin II stacks and their physiological functions have remained elusive. Here, we identified myosin-18B, an unconventional myosin, as a stable component of contractile stress fibers. Myosin-18B co-localized with myosin II motor domains in stress fibers and was enriched at the ends of myosin II stacks. Importantly, myosin-18B deletion resulted in drastic defects in the concatenation and persistent association of myosin II filaments with each other and thus led to severely impaired assembly of myosin II stacks. Consequently, lack of myosin-18B resulted in defective maturation of actomyosin bundles from their precursors in osteosarcoma cells. Moreover, myosin-18B knockout cells displayed abnormal morphogenesis, migration, and ability to exert forces to the environment. These results reveal a critical role for myosin-18B in myosin II stack assembly and provide evidence that myosin II stacks are important for a variety of vital processes in cells., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Myofilament extensibility is a key structural parameter for interpreting myosin cross-bridge kinetics in striated muscle. Previous studies reported much higher thick-filament extensibility at low tension than the better-known and commonly used values at high tension, but in interpreting mechanical studies of muscle, a single value for thick-filament extensibility has usually been assumed. Here, we established the complete thick-filament force-extension curve from actively contracting, intact vertebrate skeletal muscle. To access a wide range of tetanic forces, the myosin inhibitor blebbistatin was used to induce low tetanic forces in addition to the higher tensions obtained from tetanic contractions of the untreated muscle. We show that the force/extensibility curve of the thick filament is nonlinear, so assuming a single value for thick-filament extensibility at all force levels is not justified. We also show that independent of whether tension is generated passively by sarcomere stretch or actively by cross-bridges, the thick-filament extensibility is nonlinear. Myosin head periodicity, however, only changes when active tension is generated under calcium-activated conditions. The nonlinear thick-filament force-extension curve in skeletal muscle, therefore, reflects a purely passive response to either titin-based force or actomyosin-based force, and it does not include a thick-filament activation mechanism. In contrast, the transition of myosin head periodicity to an active configuration appears to only occur in response to increased active force when calcium is present., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The loss of muscle force and power during fatigue from intense contractile activity is associated with, and likely caused by, elevated levels of phosphate ([Formula: see text]) and hydrogen ions (decreased pH). To understand how these deficits in muscle performance occur at the molecular level, we used direct measurements of mini-ensembles of myosin generating force in the laser trap assay at pH 7.4 and 6.5. The data are consistent with a mechanochemical model in which a decrease in pH reduces myosin's detachment from actin (by slowing ADP release), increases non-productive myosin binding (by detached myosin rebinding without a powerstroke), and reduces myosin's attachment to actin (by slowing the weak-to-strong binding transition). Additional support of this mechanism is found by incorporating it into a branched pathway model for the effects of [Formula: see text] on myosin's interaction with actin. Including pH-dependence in one additional parameter (acceleration of [Formula: see text]-induced detachment), the model reproduces experimental measurements at high and low pH, and variable [Formula: see text], from the single molecule to large ensemble levels. Furthermore, when scaled up, the model predicts force-velocity relationships that are consistent with muscle fiber measurements. The model suggests that reducing pH has two opposing effects, a decrease in attachment favoring a decrease in muscle force and a decrease in detachment favoring an increase in muscle force. Depending on experimental details, the addition of [Formula: see text] can strengthen one or the other effect, resulting in either synergistic or antagonistic effects. This detailed molecular description suggests a molecular basis for contractile failure during muscle fatigue.

Shape transitions in developing organisms can be driven by active stresses, notably, active contractility generated by myosin motors. The mechanisms generating tissue folding are typically studied in epithelia. There, the interaction between cells is also coupled to an elastic substrate, presenting a major difficulty for studying contraction induced folding. Here we study the contraction and buckling of active, initially homogeneous, thin elastic actomyosin networks isolated from bounding surfaces. The network behaves as a poroelastic material, where a flow of fluid is generated during contraction. Contraction starts at the system boundaries, proceeds into the bulk, and eventually leads to spontaneous buckling of the sheet at the periphery. The buckling instability resulted from system self-organization and from the spontaneous emergence of density gradients driven by the active contractility. The buckling wavelength increases linearly with sheet thickness. Our system offers a well-controlled way to study mechanically induced, spontaneous shape transitions in active matter.

Tropomyosin, one of the major actin filament-binding proteins, regulates actin-myosin interaction and actin-filament stability. Multicellular organisms express a number of tropomyosin isoforms, but understanding of isoform-specific tropomyosin functions is incomplete. The nematode Caenorhabditis elegans has a single tropomyosin gene, lev-11, which has been reported to express four isoforms by using two separate promoters and alternative splicing. Here, we report a fifth tropomyosin isoform, LEV-11O, which is produced by alternative splicing that includes a newly identified seventh exon, exon 7a. By visualizing specific splicing events in vivo, we find that exon 7a is predominantly selected in a subset of the body wall muscles in the head, while exon 7b, which is the alternative to exon 7a, is utilized in the rest of the body. Point mutations in exon 7a and exon 7b cause resistance to levamisole--induced muscle contraction specifically in the head and the main body, respectively. Overexpression of LEV-11O, but not LEV-11A, in the main body results in weak levamisole resistance. These results demonstrate that specific tropomyosin isoforms are expressed in the head and body regions of the muscles and contribute differentially to the regulation of muscle contractility.

Sympathetic tone may influence force potentiation, that is, the stimulation-induced increase in skeletal muscle mechanical function associated with myosin phosphorylation, although the mechanism for this effect remains unknown. The purpose of this study was to examine the influence of epinephrine on concentric twitch force potentiation of wild-type and skeletal myosin light-chain kinase devoid mouse muscle (skMLCK -/- ). To this end, concentric twitch force was assessed before and after a potentiating stimulus (PS) to determine the peak and the duration of potentiation in the absence (-EPI) and presence (+EPI) of 1 μmol/L epinephrine in both genotypes. Twitch force of wild-type and skMLCK -/- muscles was increased by up to 31 and 35% and 18 and 23% in the -EPI and EPI conditions, respectively (all data n = 8, P < 0.05). In wild-type muscles, the PS increased RLC phosphorylation from 0.14 ± 0.05 (rest) to 0.66 ± 0.08 mol phos mol RLC; by 480 sec RLC phosphorylation had returned to baseline (all data n = 4 each time point, P < 0.05). Neither resting nor peak levels of RLC phosphorylation were altered by +EPI, although the duration of RLC phosphorylation was prolonged. In skMLCK -/- muscles, RLC phosphorylation was not elevated above constituent levels by stimulation in either the -EPI or +EPI condition. Thus, given the similarity in potentiation responses between genotypes our data suggest that the influence of epinephrine on potentiation was independent of skMLCK catalyzed phosphorylation of the RLC, although the clinical significance of this pathway for skeletal muscle function remains to be identified., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Mechanical properties are cues for many biological processes in health or disease. In the heart, changes to the extracellular matrix composition and cross-linking result in stiffening of the cellular microenvironment during development. Moreover, myocardial infarction and cardiomyopathies lead to fibrosis and a stiffer environment, affecting cardiomyocyte behavior. Here, we identify that single cardiomyocyte adhesions sense simultaneous (fast oscillating) cardiac and (slow) non-muscle myosin contractions. Together, these lead to oscillating tension on the mechanosensitive adaptor protein talin on substrates with a stiffness of healthy adult heart tissue, compared with no tension on embryonic heart stiffness and continuous stretching on fibrotic stiffness. Moreover, we show that activation of PKC leads to the induction of cardiomyocyte hypertrophy in a stiffness-dependent way, through activation of non-muscle myosin. Finally, PKC and non-muscle myosin are upregulated at the costameres in heart disease, indicating aberrant mechanosensing as a contributing factor to long-term remodeling and heart failure., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK -/- ) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK -/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK -/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK -/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK -/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK -/- muscles without RLC phosphorylation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Skeletal myosin light chain kinase (skMLCK)-catalyzed phosphorylation of the myosin regulatory light chain (RLC) increases (i.e. potentiates) mechanical work output of fast skeletal muscle. The influence of this event on contractile economy (i.e. energy cost/work performed) remains controversial, however. Our purpose was to quantify contractile economy of potentiated extensor digitorum longus (EDL) muscles from mouse skeletal muscles with (wild-type, WT) and without (skMLCK ablated, skMLCK -/- ) the ability to phosphorylate the RLC. Contractile economy was calculated as the ratio of total work performed to high-energy phosphate consumption (HEPC) during a period of repeated isovelocity contractions that followed a potentiating stimulus (PS). Consistent with genotype, the PS increased RLC phosphorylation measured during, before and after isovelocity contractions in WT but not in skMLCK -/- muscles (i.e. 0.65 and 0.05 mol phosphate mol -1 RLC, respectively). In addition, although the PS enhanced work during repeated isovelocity contractions in both genotypes, the increase was significantly greater in WT than in skMLCK -/- muscles (1.51±0.03 versus 1.10±0.05, respectively; all data P <0.05, n =8). Interestingly, the HEPC determined during repeated isovelocity contractions was statistically similar between genotypes at 19.03±3.37 and 16.02±3.41 μmol P; respectively ( P <0.27). As a result, despite performing significantly more work, the contractile economy calculated for WT muscles was similar to that calculated for skMLCK -/- muscles (i.e. 5.74±0.67 and 4.61±0.71 J kg -1  μmol -1 P, respectively ( P <0.27). In conclusion, our results support the notion that myosin RLC phosphorylation enhances dynamic contractile function of mouse fast skeletal muscle but does so without decreasing contractile economy., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Contraction of skeletal muscle cells is initiated by a well-known signaling pathway. An action potential in a motor nerve triggers an action potential in a muscle cell membrane, a transient increase of intracellular calcium concentration, binding of calcium to troponin in the actin-containing thin filaments, and a structural change in the thin filaments that allows myosin motors from the thick filaments to bind to actin and generate force. This calcium/thin filament mediated pathway provides the "START" signal for contraction, but it is argued that the functional response of the muscle cell, including the speed of its contraction and relaxation, adaptation to the external load, and the metabolic cost of contraction is largely determined by additional mechanisms. This review considers the role of the thick filaments in those mechanisms, and puts forward a paradigm for the control of contraction in skeletal muscle in which both the thick and thin filaments have a regulatory function. The OFF state of the thick filament is characterized by helical packing of most of the myosin head or motor domains on the thick filament surface in a conformation that makes them unavailable for actin binding or ATP hydrolysis, although a small fraction of the myosin heads are constitutively ON. The availability of the majority fraction of the myosin heads for contraction is controlled in part by the external load on the muscle, so that these heads only attach to actin and hydrolyze ATP when they are required. This phenomenon seems to be the major determinant of the well-known force-velocity relationship of muscle, and controls the metabolic cost of contraction. The regulatory state of the thick filament also seems to control the dynamics of both muscle activation and relaxation., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Muscles convert chemical free energy into mechanical work. The energy conversion occurs in 2 steps. First, free energy obtained from oxidation of metabolic substrates (ΔG S ) is transferred to ATP and, second, free energy from ATP hydrolysis (ΔG ATP ) is converted into work by myosin cross-bridges. The fraction of ΔG S transferred to ATP is called mitochondrial efficiency (η M ) and the fraction of ΔG ATP converted into work is called cross-bridge efficiency (η CB ). Overall cross-bridge efficiency varies among muscles from ~20% and 35% and the analysis presented in the current studies shows that this variation is largely due to differences in η CB whereas η M is similar (~80%) in all the muscles assessed. There is an inverse, linear relationship between maximum normalised power output and η CB ; that is, more efficient muscles tend to be less powerful than less efficient muscles. It is proposed that differences in cross-bridge efficiency reflect the extent to which cross-bridges traverse the force-length relationship for attached cross-bridges. In this framework, cross-bridges from tortoise muscle (η CB  = 45%) produce close to the maximum possible work a cross-bridge can perform in a single attachment cycle., (© 2017 John Wiley & Sons Australia, Ltd.)

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. In this study, we explored transcriptional alterations that ensue from the absence of the two major muscle proteins - myosin and actin - using the Drosophila indirect flight muscles. Our aim was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. Our results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies., Competing Interests: The authors declare no conflict of interest.

Myosin-powered force generation and contraction in nonmuscle cells underlies many cell biological processes and is based on contractility of random actin arrays. This contractility must rely on a microscopic asymmetry, the precise mechanism of which is not completely clear. A number of models of mechanical and structural asymmetries in actomyosin contraction have been posited. Here, we examine a contraction mechanism based on a finite size of myosin clusters and anisotropy of force generation by myosin heads at the ends of the myosin clusters. We use agent-based numerical simulations to demonstrate that if average lengths of actin filaments and myosin clusters are similar, then the proposed microscopic asymmetry leads to effective contraction of random 1D actomyosin arrays. We discuss the model's implication for mechanics of contractile rings and stress fibers., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Stress fibers-contractile actomyosin bundles-are important for cellular force production and adaptation to physical stress and have been well studied within the context of cell migration. However, less is known about actomyosin bundle formation and organization in vivo and in specialized contractile cells, such as smooth muscle and myoepithelial cells. The Caenorhabditis elegans spermatheca is a bag-like organ of 24 myoepithelial cells that houses the sperm and is the site of fertilization. During ovulation, spermathecal cells are stretched by oocyte entry and then coordinately contract to expel the fertilized embryo into the uterus. Here we use four-dimensional confocal microscopy of live animals to observe changes to spermathecal actomyosin network organization during cell stretch and contraction. Oocyte entry is required to trigger cell contraction and concomitant production of parallel actomyosin bundles. Actomyosin bundle size, connectivity, spacing, and orientation are regulated by myosin activity. We conclude that myosin drives actomyosin bundle production and that myosin activity is tightly regulated during ovulation to produce an optimally organized actomyosin network in C. elegans spermathecae., (© 2017 Wirshing and Cram. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Contraction of skeletal and cardiac muscle is controlled by Ca 2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca 2+ , Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca 2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3 nm -1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.

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 0 ) 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 0 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 V 0 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., Abstract: The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V 0 ) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca 2+ -induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin-attached motors per half-thick filament during V 0 shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 μm), small 4 kHz oscillations and determining the relation between half-sarcomere (hs) compliance and force during the force development following V 0 shortening. When V 0 shortening is superimposed on the maximum isometric force T 0 , n decreases progressively with the increase of shortening (range 30-80 nm per hs) and, when V 0 shortening is imposed at the end of LR, n can be as low as 1-4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V 0 shortening., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar-like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical-chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress-generating conformational changes in the myosin cross bridge, and relief of built-up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin-myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin-myosin engagement during the weak-to-strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin-myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre-power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle., (© 2016 Wiley Periodicals, Inc.)

Bridging the gaps between experimental systems on different hierarchical scales is needed to overcome remaining challenges in the understanding of muscle contraction. Here, a mathematical model with well-characterized structural and biochemical actomyosin states is developed to that end. We hypothesize that this model accounts for generation of force and motion from single motor molecules to the large ensembles of muscle. In partial support of this idea, a wide range of contractile phenomena are reproduced without the need to invoke cooperative interactions or ad hoc states/transitions. However, remaining limitations exist, associated with ambiguities in available data for model definition e.g.: (1) the affinity of weakly bound cross-bridges, (2) the characteristics of the cross-bridge elasticity and (3) the exact mechanistic relationship between the force-generating transition and phosphate release in the actomyosin ATPase. Further, the simulated number of attached myosin heads in the in vitro motility assay differs several-fold from duty ratios, (fraction of strongly attached ATPase cycle times) derived in standard analysis. After addressing the mentioned issues the model should be useful in fundamental studies, for engineering of myosin motors as well as for studies of muscle disease and drug development.

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers., Competing Interests: The authors have declared that no competing interests exist.

Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 μm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca 2+ sensitivity for 2.5 vs. 2.0 μm sarcomere length fibers (pCa 50  = 5.68 ± 0.01 vs. 5.60 ± 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca 2+ sensitivity of force was attenuated, though the Ca 2+ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by ∼15% as sarcomere length increased, due to a slower MgADP release rate (11.2 ± 0.5 vs. 13.5 ± 0.7 s -1 ). In the presence of dextran, cross-bridge detachment was ∼20% slower at 2.5 vs. 2.0 μm sarcomere length due to a slower MgADP release rate (10.1 ± 0.6 vs. 12.9 ± 0.5 s -1 ). However, osmotic compression of fibers at either 2.5 or 2.0 μm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)