ObjectiveSkeletal muscle weakness is a prominent clinical feature in patients with rheumatoid arthritis (RA), but the underlying mechanism(s) is unknown. Here we investigate the mechanisms behind arthritis-induced skeletal muscle weakness with special focus on the role of nitrosative stress on intracellular Ca2+ handling and specific force production.MethodsNitric oxide synthase (NOS) expression, degree of nitrosative stress and composition of the major intracellular Ca2+ release channel (ryanodine receptor 1, RyR1) complex were measured in muscle. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) and force production were assessed in single-muscle fibres and isolated myofibrils using atomic force cantilevers.ResultsThe total neuronal NOS (nNOS) levels were increased in muscles both from collagen-induced arthritis (CIA) mice and patients with RA. The nNOS associated with RyR1 was increased and accompanied by increased [Ca2+]i during contractions of muscles from CIA mice. A marker of peroxynitrite-derived nitrosative stress (3-nitrotyrosine, 3-NT) was increased on the RyR1 complex and on actin of muscles from CIA mice. Despite increased [Ca2+]i, individual CIA muscle fibres were weaker than in healthy controls, that is, force per cross-sectional area was decreased. Furthermore, force and kinetics were impaired in CIA myofibrils, hence actin and myosin showed decreased ability to interact, which could be a result of increased 3-NT content on actin.ConclusionsArthritis-induced muscle weakness is linked to nitrosative modifications of the RyR1 protein complex and actin, which are driven by increased nNOS associated with RyR1 and progressively increasing Ca2+ activation.

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca(2+) activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca(2+)-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 Ca(2+)-dependent change in titin properties and not associated with changes in titin-actin interactions.

When activated muscle fibers are stretched, there is a long-lasting increase in the force. This phenomenon, referred to as "residual force enhancement," has characteristics similar to those of the "static tension," a long-lasting increase in force observed when muscles are stretched in the presence of Ca(2+) but in the absence of myosin-actin interaction. Independent studies have suggested that these two phenomena have a common mechanism and are caused either by 1) a Ca(2+)-induced stiffening of titin or by 2) promoting titin binding to actin. In this study, we performed two sets of experiments in which activated fibers (pCa(2+) 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 μm at a speed of 40 L(o)/s, first, after partial extraction of TnC, which inhibits myosin-actin interactions, or, second, after treatment with gelsolin, which leads to the depletion of thin (actin) filaments. We observed that the static tension, directly related with the residual force enhancement, was not changed after treatments that inhibit myosin-actin interactions or that deplete fibers from troponin C and actin filaments. The results suggest that the residual force enhancement is caused by a stiffening of titin upon muscle activation but not with titin binding to actin. This finding indicates the existence of a Ca(2+)-regulated, titin-based stiffness in skeletal muscles.

Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by α-myosin heavy chain promoter (αMHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6 months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover, Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects.

Key pointsr 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.r 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.r We used custom-built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils.r 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.

Protein arginylation is a post-translational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 knockout driven by skeletal muscle-specific creatine kinase (Ckmm) promoter. Such Ckmm-Ate1 mice were viable and outwardly normal, however their skeletal muscle strength was significantly reduced compared to the control. Mass spectrometry of the isolated skeletal myofibrils showed a limited set of proteins arginylated on specific sites, including myosin heavy chain. Atomic force microscopy measurements of the contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of the myosin filaments could be fully rescued by re-arginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in the muscle and exerts a direct effect on muscle strength through arginylation of myosin.

Prolonged CMV in humans triggers significant decreases in active and passive diaphragm myofibrillar force generation. This response is mediated, in part, by impaired myosin cross-bridge kinetics and decreased myofibrillar protein levels.

This study evaluated two different stretching protocols employed during a period of hind-limb immobilization in terms of their effects on muscle morphology. Quantitative data regarding the soleus muscle were obtained based on the clinical hypothesis that a high frequency of this exercise would improve the recovery of muscle structure.Twenty-four male Wistar rats were divided into four groups ( n = 6 each): the control group (C); the immobilized group, in which the left hind limb was immobilized in order to maintain the soleus muscle in a fully shortened position for 3 weeks (I); the 'immobilized and stretched every 3 days' group, in which the left hind limb was immobilized as in the immobilized group, but with the soleus muscle stretched every 3 days for 40 min (Ist3); and the 'immobilized (as in the immobilized group) and stretched every 7 days' group (ISt7). All soleus muscles were excised 21 days after the beginning of the experiment, and were processed for (1) haematoxylin and eosin and myosin ATPase to evaluate muscle morphology and cross-sectional area and the proportions of the different fibre types, and (2) ultrastructural analysis. The cross-sectional area was found to have decreased in all fibre types (I, II and C), mainly in ISt7, when compared with the C group and ISt3 group. The proportion of the different fibre types did not show statistical difference between groups. Light and electron microscopy examination revealed signs of cell degeneration that was more intense in the group immobilized and stretched three times a week. In conclusion, sessions of passive stretching applied to the soleus during immobilization induce muscle fibre injury, suggesting that this therapeutic tool should be applied carefully to disused muscles.