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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.

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.

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.

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.

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.

When skeletal muscles are stretched during activation in the absence of myosin-actin interactions, the force increases significantly. The force remains elevated throughout the activation period. The mechanism behind this non-crossbridge force, referred to as static tension, is unknown and generates debate in the literature. It has been suggested that the static tension is caused by Ca(2+)-induced changes in the properties of titin molecules that happens during activation and stretch, but a comprehensive evaluation of such possibility is still lacking. This paper reviews the general characteristics of the static tension, and evaluates the proposed mechanism by which titin may change the force upon stretch. Evidence is presented suggesting that an increase in intracellular Ca(2+) concentration leads to Ca(2+) binding to the PEVK region of titin. Such binding increases titin stiffness, which increases the overall sarcomere stiffness and causes the static tension. If this form of Ca(2+)-induced increase in titin stiffness is confirmed in future studies, it may have large implications for understating of the basic mechanisms of muscle contraction.

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.

Objective Skeletal 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. Methods Nitric 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. Results The 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. Conclusions Arthritis-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.

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 6months 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.

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 Ca2+ 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 Ca2+-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 (pCa2+ 4.5) treated with the myosin inhibitor blebbistatin were stretched from 2.7 to 2.8 μm at a speed of 40 Lo/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 Ca2+-regulated, titin-based stiffness in skeletal muscles.

We analyzed lesser diameter and distribution of fiber types in different skeletal muscles from female Wistar rats using a histoenzymology Myofibrillar Adenosine Tri-phosphatase (mATPase) method. Fragments from muscles were frozen and processed by mATPase in different pH. Adult and weanling rat soleus muscles presented a predominance of type I fibers and larger fiber diameters. In the plantar muscle in adult rats, the type IIB fibers demonstrated greater lesser diameter while in the weanling animals, types I and IIB fibers were larger. The plantar muscle of animals of both ages was composed predominantly of the type IID fibers. The type IID fibers were observed in similar amounts in the lateral gastrocnemius and the medial gastrocnemius muscles. Type IIB fibers showed predominance and presented higher size in comparison with other types in the EDL muscle. The present study shows that data on fiber type distribution and fiber lesser diameter obtained in adult animals cannot always be applied to weanling animals of the same species. Using the mATPase, despite the difficult handling, is an important tool to determine the different characteristics of the specific fibers in the skeletal muscle tissue.

The aim of this study was to evaluate and compare the efficacy of different remobilization protocols in different skeletal muscles considering the changes induced by hindlimb suspension of the tail. Thirty-six female Wistar rats were divided into six groups: control I, control II, suspended, suspended free, suspended trained on a declined treadmill and suspended trained on a flat treadmill. Fragments of soleus and tibialis anterior (TA) muscle were frozen and processed by different histochemical methods. The suspended soleus showed a significant increase in the proportional number of intermediate/hybrid fibers and a decrease in the number of type I fibers. Some of these changes proved to be reversible after remobilization. The three remobilization programs led to the recovery of both the proportional number of fibers and their size. The TA muscle presented a significant increase in the number and size of type I fibers and a cell size reduction of type IIB fibers, which were recovered after training on a declined treadmill and free movement. Especially regarding the soleus, the present findings indicate that, among the protocols, training on a declined treadmill was found to induce changes of a more regenerative nature, seemingly indicating a better tissue restructuring after the suspension procedure.

Background Prolonged controlled mechanical ventilation (CMV) in humans and experimental animals results in diaphragm fibre atrophy and injury. In animals, prolonged CMV also triggers significant declines in diaphragm myofibril contractility. In humans, the impact of prolonged CMV on myofibril contractility remains unknown. The objective of this study was to evaluate the effects of prolonged CMV on active and passive human diaphragm myofibrillar force generation and myofilament protein levels. Methods and results Diaphragm biopsies were obtained from 13 subjects undergoing cardiac surgery (control group) and 12 brain-dead organ donors (CMV group). Subjects in each group had been mechanically ventilated for 2–4 and 12–74 h, respectively. Specific force generation of diaphragm myofibrils was measured with atomic force cantilevers. Rates of force development (K act ), force redevelopment after a shortening protocol (K tr ) and relaxation (K rel ) in fully activated myofibrils (pCa 2+ =4.5) were calculated to assess myosin cross-bridge kinetics. Myofilament protein levels were measured with immunoblotting and specific antibodies. Prolonged CMV significantly decreased active and passive diaphragm myofibrillar force generation, K act , K tr and K rel . Myosin heavy chain (slow), troponin-C, troponin-I, troponin-T, tropomyosin and titin protein levels significantly decreased in response to prolonged CMV, but no effects on α-actin, α-actinin or nebulin levels were observed. Conclusions 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.

Eccentric exercise is an essential resource for skeletal muscle rehabilitation following muscle disuse however, abnormalities linked to the tissue recuperation require further research. Our aim was analyze the adaptation ability of rehabilitated muscular tissue in rats during different periods of eccentric training after 10 days of limb immobilization. Twenty-seven Wistar rats were divided into six groups: immobilized 10 days, immobilized and eccentric trained for 10 days, immobilized and eccentric trained for 21 days, and three age-matched control groups. After sacrifice, soleus and plantaris muscles were frozen, cut and stained for general histology using hematoxylin and eosin and Gomori trichrome methods and immunohistochemical methods for fiber typing (mATPase, NADH2-TR), for capillaries (CD31) and intermediate filaments (desmin, vimentin) and high resolution microscopy of resin embedded material. Immobilization resulted in more intense morphological alterations in soleus muscles such as formation of target fibers, nuclear centralization, a reduction in the number of type I fibers, diameter of type I, IIA, IIAD fibers, and capillaries. After 10 days of eccentric training, increases in the nuclear centralization and the number of lobulated fibers were observed. This period was insufficient to reestablish the capillary/fiber (C/F) ratio and distribution of fiber types as that observed in the control group. However, 21 days of rehabilitation allowed the reversal of all morphological and quantitative abnormalities. For the plantaris muscles, 10-days of training restored their basic characteristics. Despite the fact that immobilization affected soleus and plantaris muscles, 10 days of eccentric training was insufficient to restore the morphological characteristics of soleus muscles, which was not the case observed in plantaris muscle.

Stretching is frequently used in physiotherapy to minimize or even reverse the alterations that occur after muscle disuse. Alterations that occur after 10 and 21 days of maintained stretch in soleus and plantaris muscles post-disuse were evaluated in the present study in experimental rats. Thirty adult female Wistar rats were divided into seven groups: hindlimb immobilization for 10 days; immobilization and 10 days stretched; immobilized and 21 days stretched; three control groups consisting of animals of different ages and anesthetized group. The right hindlimb was immobilized using a lightweight apparatus composed of two sections: (i) upper part: a small cotton T-shirt and, and (ii) lower part: a steel mesh to fix the ankle in plantar-flexion-shortened position. Fragments of the soleus and plantaris muscle were frozen and processed using histochemical and immunohistochemical methods. Limb immobilization caused important morphological alterations in skeletal muscle including: reduction in the number of type I fibers and an increase in type IIC fibers, reduction in the lesser diameter of type I, IIA and IIAD fibers and in the number of capillaries in soleus muscle. The stretching program applied for 10 days was insufficient to allow recovery from the disuse alterations in both muscles. However, after 21 days there were improved morphological characteristics, size and distribution of the different fibers.

We studied the effects of different protocols of post-disuse rehabilitation on angiogenesis and myosin heavy chain (MHC) content in rat hindlimb muscles after caudal suspension. Thirty female Wistar rats were divided into five groups: (1) Control I, (2) Control II, (3) Suspended, (4) Suspended trained on declined treadmill, and (5) Suspended trained on flat treadmill. Fragments of the soleus and tibialis anterior (TA) muscles were frozen and processed by electrophoresis and immunohistochemistry (CD31 antibody). Hindlimb suspension caused reduction of capillary/fiber (C/F) ratios and contents of MHC type I (MHCI) in the soleus in parallel to increased capillary density. Flat treadmill protocols increased the content of the MHCI isoform. The C/F ratio was increased by concentric training after hypokinesis, but was not modified by eccentric training, which caused a greater reduction of capillary density compared to the other protocols. In the TA muscle, hindlimb suspension caused a non-significant increase in capillary density and C/F ratio with limited changes in MHC. The present data demonstrate that the different training protocols adopted and the functional performance of the muscles analyzed caused specific changes in capillarization and in the content of the various MHC types.

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.