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Release of the ATP hydrolysis product inorganic phosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of the production of force and motion by myosin in health and disease and also our understanding of myosin-active drugs. From the 1990s and up to today, models with the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze, three influential alternative models, either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

The interaction between actin and myosin is the basis of contraction and force production in muscle fibers. Studies have shown that actin and myosin oxidation cause myofibrillar weakness in healthy and diseased muscles. The degree to which oxidation of each of these proteins contributes to an attenuated force in myofibrils is unclear. In this study, we show that exposure of actin and myosin to the chemical 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1), an NO and O2•− donor, affected actin-myosin interactions, as shown by a decreased myosin-propelled actin velocity in the in vitro motility assay. We also observed that oxidation of actin and myosin resulted in a decrease in force generated by myosin and actin filaments, as determined by a system of microfabricated cantilevers. Although myosin is more sensitive to oxidative modifications than actin, as indicated by a steeper decrease in velocity and force by the filaments, modifications on actin are sufficient to affect force and velocity and also contribute to a decrease in contractile activity in muscles.

Duchene muscular dystrophy (DMD) is caused by the absence of the protein dystrophin, which leads to muscle weakness, progressive degeneration, and eventually death due to respiratory failure. Low-intensity eccentric training (LIET) has been used as a rehabilitation method in skeletal muscles after disuse. Recently, LIET has also been used for rehabilitating dystrophic muscles, but its effects are still unclear. The purpose of this study was to investigate the effects of 21 days of LIET in dystrophic soleus muscle. Thirty-six male mdx mice were randomized into six groups (n = 6/each): mdx sedentary group; mdx training group-3 days; mdx training group-21 days; wild-type sedentary group; wild-type training group-3 days and wild-type training group-21 days. After the training sessions, animals were euthanized, and fragments of soleus muscles were removed for immunofluorescence and histological analyses, and measurements of active force and Ca2+ sensitivity of the contractile apparatus. Muscles of the mdx training group-21 days showed an improvement in morphological characteristics and an increase of active force when compared to the sedentary mdx group. The results show that LIET can improve the functionality of dystrophic soleus muscle in mice.

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.

Skeletal myosins II are non-processive molecular motors, that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we used high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings to neighboring sites and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration and with 2 μM ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of neighboring available binding sites. The result suggests that myosin cooperativity is defined by the activation status of the thin filaments.eLife digestMuscle contraction is the result of large ensembles of the molecular motor myosin II working in coordination while attached to actin. Myosin II produces the power stroke, responsible for force generation. In this paper, we used High-Speed Atomic Force Microscopy (HS-AFM) to determine the potential cooperativity between myosin motors bound to non-regulated and regulated thin filaments. Based on the direct visualization of myosin-actin interaction, probability of myosin binding, and the myosin fractional occupancy of binding sites along non-regulated and regulated actin filaments, our results show no cooperative effects over ∼100 nm of the actin filament length. In contrast, there is myosin cooperativity within the activated thin filament, that induces a high affinity of myosin heads to the filaments. Our results support the independent behaviour of myosin heads while attached to actin filaments, but a cooperative behavior when attached to regulated thin filaments.

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (

Muscle contraction and a range of critical cellular functions rely on force-producing interactions between myosin motors and actin filaments, powered by turnover of adenosine triphosphate (ATP). The relationship between release of the ATP hydrolysis product ortophosphate (Pi) from the myosin active site and the force-generating structural change, the power-stroke, remains enigmatic despite its central role in energy transduction. Here, we present a model with multistep Pi-release that unifies current conflicting views while also revealing additional complexities of potential functional importance. The model is based on our evidence from kinetics, molecular modelling and single molecule fluorescence studies of Pi binding outside the active site. It is also consistent with high-speed atomic force microscopy movies of single myosin II molecules without Pi at the active site, showing consecutive snapshots of pre- and post-power stroke conformations. In addition to revealing critical features of energy transduction by actomyosin, the results suggest enzymatic mechanisms of potentially general relevance.

Significance The advent of high-speed atomic force microscopy (HS-AFM) changed the field of biology considerably. HS-AFM is the only method where in situ dynamics of biological samples and imaging can be coupled with a spatial resolution of 1 to 5 nm in the horizontal direction. Unlike electron or cryo-electron microscopy, HS-AFM does not require fixation or freezing of the samples, and has the ability to derive kinetic parameters by recording the live movements of single-molecule dynamics. In this paper, we used HS-AFM to investigate directly the mechanisms of cardiac muscle activation. We visualized the muscle regulatory tropomyosin–troponin complex movements during activation by calcium or myosin (motor that drives contraction), and the structural transitions that happen during these events., High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin–troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

The cytoskeleton drives many essential processes in normal physiology, and its impairments underlie many diseases, including skeletal myopathies, cancer, and heart failure, that broadly affect developed countries worldwide. Cytoskeleton regulation is a field of investigation of rapidly emerging global importance and a new venue for the development of potential therapies. This review overviews our present understanding of the posttranslational regulation of the muscle cytoskeleton through arginylation, a tRNA-dependent addition of arginine to proteins mediated by arginyltransferase 1. We focus largely on arginylation-dependent regulation of striated muscles, shown to play critical roles in facilitating muscle integrity, contractility, regulation, and strength.

The goal of this study was to investigate the effects of repetitive stimulation and the oxidant H2O2 on fatigue of diaphragm intact fibers and in myofibrils measured with different Ca2+ concentrations. Intact fibers were isolated from mice diaphragm, and twitch and tetanic contractions (500 ms duration) were performed at different frequencies of stimulation ranging from 15 Hz to 150 Hz to establish a force-frequency relation before and after a fatigue and recovery protocol, without or after a treatment with H2O2. Fatigue was induced with isometric contractions (500 ms, 40 Hz) evoked every 0.8 seconds, with a total of 625 tetani. After the fatigue, the force recovery was followed by invoking tetanic contractions (500 ms, 40 Hz) every 1 min, with a total duration of 30 min. Individual myofibrils were also isolated from the mouse diaphragm and were tested for isometric contractions before and after treatment with H2O2 and NAC. In a second series of experiments, myofibrils were activated at different pCa (pCa = −log10 [Ca2+]), before and after H2O2 treatment. After 15 minutes of H2O2 treatment, the myofibrillar force was decreased to 54 ± 12% of its control, maximal value, and a result that was reversed by NAC treatment. The force was also decreased after myofibrils were treated with H2O2 and activated in pCa ranging between 4.5 and 5.7. These results suggest that fatigue in diaphragm intact fibers and at the myofibrils level is caused partially by oxidation of the contractile proteins that may be responsible for changing the force in various levels of Ca2+ activation.

Background The mechanical work and the actin-activated ATP kinetics in skeletal muscles are closely associated with two surface loops that are present in the myosin molecule: loop 1 and loop 2. They are located close to the ATP-loop (loop 1), and the actin binding domain (loop 2). In this study we investigated the roles of loops 1 and 2 in the regulation of the load-dependent velocity of actin sliding and ATPase activity. Methods Heavy meromyosin (HMM) from rabbit skeletal muscle was subjected to limited tryptic proteolysis to obtain fragments containing different amounts of loops 1 and 2. The amino-acid sequences of these fragments were confirmed with quantitative mass-spectrometry. The velocity of actin motility propelled by the HMM fragments was measured using in-vitro motility assays, with varying loads induced by the addition of different concentrations of α-actinin. Results The load-dependent velocity of the myosin-propelled actin motility, and the fraction of actin filaments motility, were decreased in close association with the depletion of loop 1 in the HMM. The ATPase activity was decreased in close association with depletion of loops 1 and 2. Conclusions Loop 1 is responsible for regulating the load-dependent velocity of actin motility. General significance Myosin-actin interaction is closely regulated by two flexible loops in the structure of myosin. The results of this study are important for the understanding of the molecular mechanisms of contraction, and therefore the most basic functions of life, such as locomotion, heart beating, and breathing.

Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the force-generating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin-myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several non-stereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free Pi increased the short-lived stereospecific binding events in which the power stroke occurred, followed by release of Pi after the power stroke.

The smallest contractile unit in striated muscles is the sarcomere. Although some of the classic features of contraction assume a uniform behavior of sarcomeres within myofibrils, the occurrence of sarcomere length nonuniformities has been well recognized for years, but it is yet not well understood. In the past years, there has been a great advance in experiments using isolated myofibrils and sarcomeres that has allowed scientists to directly evaluate sarcomere length nonuniformity. This review will focus on studies conducted with these preparations to develop the hypotheses that 1) force production in myofibrils is largely altered and regulated by intersarcomere dynamics and that 2) the mechanical work of one sarcomere in a myofibril is transmitted to other sarcomeres in series. We evaluated studies looking into myofibril activation, relaxation, and force changes produced during activation. We conclude that force production in myofibrils is largely regulated by intersarcomere dynamics, which arises from the cooperative work of the contractile and elastic elements within a myofibril.

International audience; The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFPlabeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Objective Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. Methods To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. Results Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. Interpretation Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.

In this study, we measured the stiffness of skeletal muscle myofibrils in rigor. Using a custom-built atomic force microscope, myofibrils were first placed in a rigor state then stretched and shortened at different displacements (0.1–0.3 μm per sarcomere) and nominal speeds (0.4 and 0.8 μm/s). During stretching, the myofibril stiffness was independent of both displacement and speed (average of 987 nN/μm). During shortening, the myofibril stiffness was independent of displacement, but dependent on speed (1234 nN/μm at 0.4 μm/s; 1106 nN/μm at 0.8 μm/s). Furthermore, the myofibril stiffness during shortening was greater than that during stretching and the difference depended on speed (31% at 0.4 μm/s; 8% at 0.8 μm/s). The results suggest that the myofibrils exhibit nonlinear viscoelastic properties that may be derived from myofibril filaments, similar to what has been observed in muscle fibers.

Increased production of reactive oxygen/nitrogen species (ROS) and impaired cellular Ca2+ handling are implicated in the prolonged low-frequency force depression (PLFFD) observed in skeletal muscle...

KEY POINTS When a skeletal muscle is stretched while it contracts, the muscle produces a relatively higher force than the force from an isometric contraction at the same length: a phenomenon referred to as residual force enhancement. Residual force enhancement is puzzling because it cannot be directly explained by the classical force-length relationship and the sliding filament theory of contraction, the main paradigms in the muscle field. We used custom-built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils. Our data report that residual force enhancement is present in skeletal muscles, but not cardiac muscles, and is regulated by the different isoforms of the titin protein filaments. ABSTRACT When a skeletal muscle contracts isometrically, the muscle produces a force that is relative to the final isometric sarcomere length (SL). However, when the same final SL is reached by stretching the muscle while it contracts, the muscle produces a relatively higher force: a phenomenon commonly referred to as residual force enhancement. In this study, we investigated residual force enhancement in rabbit skeletal psoas myofibrils and, for the first time, cardiac papillary myofibrils. A custom-built atomic force microscope was used in experiments that stretched myofibrils before and after inhibiting myosin and actin interactions to determine whether the different cardiac and skeletal titin isoforms regulate residual force enhancement. At SLs ranging from 2.24 to 3.13 μm, the skeletal myofibrils enhanced the force by an average of 9.0%, and by 29.5% after hindering myosin and actin interactions. At SLs ranging from 1.80 to 2.29 μm, the cardiac myofibrils did not enhance the force before or after hindering myosin and actin interactions. We conclude that residual force enhancement is present only in skeletal muscles and is dependent on the titin isoforms.

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein; localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle-type specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that only in the C-zone, MyBP-C Ca2+-sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-C’s N-terminus, as N-terminal proteolysis attenuated MyBP-C’s functional capacities. To determine each MyBP-C isoform’s contribution to thin filament Ca2+-sensitization and slowing in the C-zone, we used a combination ofin vitromotility assays using expressed recombinant N-terminal fragments andin silicomechanistic modeling. Our results suggest that each skeletal MyBP-C isoform’s N terminus is functionally distinct and has modulatory capacities that depend on the muscle-type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.SIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscle’s smallest contractile unit. MyBP-C’s importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-C’s functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscle’s mechanical performance as physiological demands arise.

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO−) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO− donor (SIN-1) or directly with ONOO−, which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO− treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate ( gapp) in combination with a conservation of the force redevelopment rate (kTr) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Diaphragm dysfunction and atrophy develop during prolonged controlled mechanical ventilation. Fiber atrophy has been attributed to activation of the proteasome and autophagy proteolytic pathways. Oxidative stress activates the proteasome during controlled mechanical ventilation, but it is unclear whether it also activates autophagy. This study investigated whether pretreatment with the antioxidant N-acetylcysteine affects controlled mechanical ventilation–induced diaphragm contractile dysfunction, fiber atrophy, and proteasomal and autophagic pathway activation. The study also explored whether proteolytic pathway activity during controlled mechanical ventilation is mediated by microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes. Methods Three groups of adult male rats were studied (n = 10 per group). The animals in the first group were anesthetized and allowed to spontaneously breathe. Animals in the second group were pretreated with saline before undergoing controlled mechanical ventilation for 24 h. The animals in the third group were pretreated with N-acetylcysteine (150 mg/kg) before undergoing controlled mechanical ventilation for 24 h. Diaphragm contractility and activation of the proteasome and autophagy pathways were measured. Expressions of microRNAs that negatively regulate ubiquitin E3 ligases and autophagy-related genes were measured with quantitative polymerase chain reaction. Results Controlled mechanical ventilation decreased diaphragm twitch force from 428 ± 104 g/cm2 (mean ± SD) to 313 ± 50 g/cm2 and tetanic force from 2,491 ± 411 g/cm2 to 1,618 ± 177 g/cm2. Controlled mechanical ventilation also decreased diaphragm fiber size, increased expression of several autophagy genes, and augmented Atrogin-1, MuRF1, and Nedd4 expressions by 36-, 41-, and 8-fold, respectively. Controlled mechanical ventilation decreased the expressions of six microRNAs (miR-20a, miR-106b, miR-376, miR-101a, miR-204, and miR-93) that regulate autophagy genes. Pretreatment with N-acetylcysteine prevented diaphragm contractile dysfunction, attenuated protein ubiquitination, and downregulated E3 ligase and autophagy gene expression. It also reversed controlled mechanical ventilation–induced microRNA expression decreases. N-Acetylcysteine pretreatment had no affect on fiber atrophy. Conclusions Prolonged controlled mechanical ventilation activates the proteasome and autophagy pathways in the diaphragm through oxidative stress. Pathway activation is accomplished, in part, through inhibition of microRNAs that negatively regulate autophagy-related genes.

Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here, we show that oxidative posttranslational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde (MDA) adduct modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located in 3 distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritic mice and patients with RA. Moreover, molecular dynamics simulations revealed that these areas, here coined “hotspots,” are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and may provide novel leads for targeted therapeutic treatment to improve muscle function.

Supplemental Figure S1 and S2, Supplemental Table S1 and S2Supplemental Data for "Force generated by myosin cross-bridges is reduced i myofibrils exposed to ROS/RNS " by Persson et al. (2019) Am J Physiol Cell Physiol.

Background Diaphragm weakness occurs rapidly in adult animals treated with mechanical ventilation (MV), but the effects of MV on the neonatal diaphragm have not been determined. Furthermore, it is unknown whether co-existent lung disease exacerbates ventilator-induced diaphragmatic dysfunction (VIDD). We investigated the impact of MV (mean duration = 7.65 h), either with or without co-existent respiratory failure caused by surfactant deficiency, on the development of VIDD in newborn lambs. Methods Newborn lambs (1–4 days) were assigned to control (CTL, non-ventilated), mechanically ventilated (MV), and MV + experimentally induced surfactant deficiency (MV+SD) groups. Immunoblotting and quantitative PCR assessed inflammatory signaling, the ubiquitin-proteasome system, autophagy, and oxidative stress. Immunostaining for myosin heavy chain (MyHC) isoforms and quantitative morphometry evaluated diaphragm atrophy. Contractile function of the diaphragm was determined in isolated myofibrils ex vivo. Results Equal decreases (25–30%) in myofibrillar force generation were found in MV and MV+SD diaphragms compared to CTL. In comparison to CTL, both MV and MV+SD diaphragms also demonstrated increased STAT3 transcription factor phosphorylation. Ubiquitin-proteasome system (Atrogin1 and MuRF1) transcripts and autophagy indices (Gabarapl1 transcripts and the ratio of LC3B-II/LC3B-I protein) were greater in MV+SD relative to MV alone, but fiber type atrophy was not observed in any group. Protein carbonylation and 4-hydroxynonenal levels (indices of oxidative stress) also did not differ among groups. Conclusions In newborn lambs undergoing controlled MV, there is a rapid onset of diaphragm dysfunction consistent with VIDD. Superimposed lung injury caused by surfactant deficiency did not influence the severity of early diaphragm weakness. Electronic supplementary material The online version of this article (10.1186/s13054-019-2409-6) contains supplementary material, which is available to authorized users.

We tested the hypotheses that 1) a decrease in activation of skeletal muscles at short sarcomere lengths (SLs) is caused by an inhibition of Ca2+release from the sarcoplasmic reticulum (SR), and 2) the decrease in Ca2+would be caused by an inhibition of action potential conduction from the periphery to the core of the fibers. Intact, single fibers dissected from the flexor digitorum brevis from mice were activated at different SLs, and intracellular Ca2+was imaged with confocal microscopy. Force decreased at SLs shorter than 2.1 μm, while Ca2+concentration decreased at SLs below 1.9 μm. The concentration of Ca2+at short SL was lower at the core than at the peripheries of the fiber. When the external concentration of Na+was decreased in the experimental media, impairing action potential conduction, Ca2+gradients were observed in all SLs. When caffeine was used in the experimental media, the gradients of Ca2+were abolished. We concluded that there is an inhibition of Ca2+release from the sarcoplasmic reticulum (SR) at short SLs, which results from a decreased conduction of action potential from the periphery to the core of the fibers.

In this article, we propose the hypothesis that the posttranslational arginylation of proteins, a process catalyzed by the enzyme arginyl-tRNA-transferase, regulates active and passive force generation in striated muscles. Specifically, we propose that proteins essential for muscle contraction and force production are regulated by arginylation, including myosin heavy chain, troponin, actin, and titin filaments.

The production of reactive oxygen/nitrogen species (ROS/RNS) is generally considered to increase during physical exercise. Nevertheless, direct measurements of ROS/RNS often show modest increases in ROS/RNS in muscle fibres even during intensive fatiguing stimulation, and the major source(s) of ROS/RNS during exercise is still being debated. In rested muscle fibres, mild and acute exposure to exogenous ROS/RNS generally increases myofibrillar submaximal force, whereas stronger or prolonged exposure has the opposite effect. Endogenous production of ROS/RNS seems to preferentially decrease submaximal force and positive effects of antioxidants are mainly observed during fatigue induced by submaximal contractions. Fatigued muscle fibres frequently enter a prolonged state of reduced submaximal force, which is caused by a ROS/RNS-dependent decrease in sarcoplasmic reticulum Ca(2+) release and/or myofibrillar Ca(2+) sensitivity. Increased ROS/RNS production during exercise can also be beneficial and recent human and animal studies show that antioxidant supplementation can hamper the beneficial effects of endurance training. In conclusion, increased ROS/RNS production have both beneficial and detrimental effects on skeletal muscle function and the outcome depends on a combination of factors: the type of ROS/RNS; the magnitude, duration and location of ROS/RNS production; and the defence systems, including both endogenous and exogenous antioxidants.

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca2+ activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca2+-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 μm for the psoas, 2.68 and 4.21 μm for the soleus, and 1.51 and 2.86 μm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca2+-dependent change in titin properties and not associated with changes in titin-actin interactions.

The force–length relation is one of the most defining features of muscle contraction, and yet a topic of debate in the literature. The sliding filament theory predicts that the force produced by muscle fibres is proportional to the degree of overlap between myosin and actin filaments, producing a linear descending limb of the active force–length relation. However, several studies have shown forces that are larger than predicted, especially at long sarcomere lengths (SLs). Studies have been conducted with muscle fibres, preparations containing thousands of sarcomeres that make measurements of individual SL challenging. The aim of this study was to evaluate force production and sarcomere dynamics in isolated myofibrils and single sarcomeres from the rabbit psoas muscle to enhance our understanding of the theoretically predicted force–length relation. Contractions at varying SLs along the plateau (SL = 2.25–2.39 µm) and the descending limb (SL > 2.39 µm) of the force–length relation were induced in sarcomeres and myofibrils, and different modes of force measurements were used. Our results show that when forces are measured in single sarcomeres, the experimental force–length relation follows theoretical predictions. When forces are measured in myofibrils with large SL dispersions, there is an extension of the plateau and forces elevated above the predicted levels along the descending limb. We also found an increase in SL non-uniformity and slowed rates of force production at long lengths in myofibrils but not in single sarcomere preparations. We conclude that the deviation of the descending limb of the force–length relation is correlated with the degree of SL non-uniformity and slowed force development.

Skeletal muscle weakness is a comorbidity in patients with rheumatoid arthritis (RA), which impairs the ability to work and leads to reduced quality of life for the afflicted patients. However, little molecular insight is available on RA-induced muscle weakness. The reactive oxygen/nitrogen species peroxynitrite (ONOO-) can induce oxidative post-translational protein modifications by nitrating tyrosine residues (3-NT) and facilitating of malondialdehyde (MDA) adduct formation on basic amino acids, e.g. histidine. Increased 3-NT and MDA levels have been observed in rodent models with arthritis and in patients with RA. The skeletal muscles depend on interaction between actin and myosin as the main constituents for force production. Previous work in rodents by others, and us, indicate that arthritis-induced muscle weakness is associated with higher levels of oxidative modifications on actin. However, it has not yet been known how oxidative modification interferers with actin and the force producing machinery in RA. Here we show that oxidative post-translational modifications directly introduced on the contractile machinery and actin lead to impaired actin polymerization and reduced force production. Using mass spectrometry (MS), we identified which actin residues that were targeted by 3-NT or MDA modifications in weakened skeletal muscle from mice with arthritis (3-NT 3; MDA 10) and patients with RA (3-NT 2; MDA 9). The residues were primarily located to three hotspots within the three-dimensional structure of actin. Intriguingly, the identified actin residues from mice with arthritis matched the ones identified from patients with RA. Moreover, structural analysis together with molecular dynamic simulations provided atomistic details on actin and highlighted that four of the residues were sited at locations important for filament stability, and intersubdomain and myosin interaction. In summary, we have identified specific skeletal muscle actin residues that are 3-NT or MDA modified in weakened RA patients and provided molecular insight how this promotes muscle weakness.

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human β-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999–1006.]. Since then, more than 300 mutations in the β-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or β-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a β-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force–velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in β-cardiac myosin.

Cyclic interactions between myosin II motor domains and actin filaments that are powered by turnover of ATP underlie muscle contraction and have key roles in motility of nonmuscle cells. The elastic characteristics of actin-myosin cross-bridges are central in the force-generating process, and disturbances in these properties may lead to disease. Although the prevailing paradigm is that the cross-bridge elasticity is linear (Hookean), recent single-molecule studies suggest otherwise. Despite convincing evidence for substantial nonlinearity of the cross-bridge elasticity in the single-molecule work, this finding has had limited influence on muscle physiology and physiology of other ordered cellular actin-myosin ensembles. Here, we use a biophysical modeling approach to close the gap between single molecules and physiology. The model is used for analysis of available experimental results in the light of possible nonlinearity of the cross-bridge elasticity. We consider results obtained both under rigor conditions (in the absence of ATP) and during active muscle contraction. Our results suggest that a wide range of experimental findings from mechanical experiments on muscle cells are consistent with nonlinear actin-myosin elasticity similar to that previously found in single molecules. Indeed, the introduction of nonlinear cross-bridge elasticity into the model improves the reproduction of key experimental results and eliminates the need for force dependence of the ATP-induced detachment rate, consistent with observations in other single-molecule studies. The findings have significant implications for the understanding of key features of actin-myosin-based production of force and motion in living cells, particularly in muscle, and for the interpretation of experimental results that rely on stiffness measurements on cells or myofibrils.