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Introduction: Increases in skeletal muscle size occur in response to prolonged exposure to resistance training that is typically ascribed to increased muscle fiber size. Whether muscle fiber number also changes remains controversial, and a paucity of data exists about myofibrillar structure. This cross-sectional study compared muscle fiber and myofibril characteristics in long-term resistance-trained (LRT) versus untrained (UNT) individuals., Methods: The maximal anatomical cross-sectional area (ACSAmax) of the biceps brachii muscle was measured by magnetic resonance imaging in 16 LRT (5.9 ± 3.5 yr' experience) and 13 UNT males. A muscle biopsy was taken from the biceps brachii to measure muscle fiber area, myofibril area, and myosin spacing. Muscle fiber number, and myofibril number in total and per fiber were estimated by dividing ACSAmax by muscle fiber area or myofibril area, and muscle fiber area by myofibril area, respectively., Results: Compared with UNT, LRT individuals had greater ACSAmax (+70%, P < 0.001), fiber area (+29%, P = 0.028), fiber number (+34%, P = 0.013), and myofibril number per fiber (+49%, P = 0.034) and in total (+105%, P < 0.001). LRT individuals also had smaller myosin spacing (-7%, P = 0.004; i.e., greater packing density) and a tendency toward smaller myofibril area (-16%, P = 0.074). ACSAmax was positively correlated with fiber area ( r = 0.526), fiber number ( r = 0.445), and myofibril number (in total r = 0.873 and per fiber r = 0.566), and negatively correlated with myofibril area ( r = -0.456) and myosin spacing ( r = -0.382) (all P < 0.05)., Conclusions: The larger muscles of LRT individuals exhibited more fibers in cross-section and larger muscle fibers, which contained substantially more total myofibrils and more packed myofilaments than UNT participants, suggesting plasticity of muscle ultrastructure., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Sports Medicine.)

The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (P i ) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the P i release step. The question asked here is whether the force generation occurs before P i release or after release. We investigated the effect of P i on oscillatory work production in single myofibrils and found that P i -attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of P i in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have P i attached. With sinusoidal analysis, we obtained the kinetic constants around the P i release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before P i is released, and the same force is maintained after P i is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The molecular mechanisms of sarcomere proteins underlie the contractile function of the heart. Although our understanding of the sarcomere has grown tremendously, the focus has been on ventricular sarcomere isoforms due to the critical role of the ventricle in health and disease. However, atrial-specific or -enriched myofilament protein isoforms, as well as isoforms that become expressed in disease, provide insight into ways this complex molecular machine is fine-tuned. Here, we explore how atrial-enriched sarcomere protein composition modulates contractile function to fulfill the physiological requirements of atrial function. We review how atrial dysfunction negatively affects the ventricle and the many cardiovascular diseases that have atrial dysfunction as a comorbidity. We also cover the pathophysiology of mutations in atrial-enriched contractile proteins and how they can cause primary atrial myopathies. Finally, we explore what is known about contractile function in various forms of atrial fibrillation. The differences in atrial function in health and disease underscore the importance of better studying atrial contractility, especially as therapeutics currently in development to modulate cardiac contractility may have different effects on atrial sarcomere function.

Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle., (© 2023. The Author(s).)

Residual force enhancement (RFE), an increase in isometric force after active stretching of a muscle compared with the purely isometric force at the corresponding length, has been consistently observed throughout the structural hierarchy of skeletal muscle. Similar to RFE, passive force enhancement (PFE) is also observable in skeletal muscle and is defined as an increase in passive force when a muscle is deactivated after it has been actively stretched compared with the passive force following deactivation of a purely isometric contraction. These history-dependent properties have been investigated abundantly in skeletal muscle, but their presence in cardiac muscle remains unresolved and controversial. The purpose of this study was to investigate whether RFE and PFE exist in cardiac myofibrils and whether the magnitudes of RFE and PFE increase with increasing stretch magnitudes. Cardiac myofibrils were prepared from the left ventricles of New Zealand White rabbits, and the history-dependent properties were tested at three different final average sarcomere lengths (n = 8 for each), 1.8, 2, and 2.2 μm, while the stretch magnitude was kept at 0.2 μm/sarcomere. The same experiment was repeated with a final average sarcomere length of 2.2 μm and a stretching magnitude of 0.4 μm/sarcomere (n = 8). All 32 cardiac myofibrils exhibited increased forces after active stretching compared with the corresponding purely isometric reference conditions (p < 0.05). Furthermore, the magnitude of RFE was greater when myofibrils were stretched by 0.4 compared with 0.2 μm/sarcomere (p < 0.05). We conclude that, like in skeletal muscle, RFE and PFE are properties of cardiac myofibrils and are dependent on stretch magnitude., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The cardiac sarcomere is a triumph of biological evolution wherein myriad contractile and regulatory proteins assemble into a quasi-crystalline lattice to serve as the central point upon which cardiac muscle contraction occurs. This review focuses on the many signaling components and mechanisms of regulation that impact cardiac sarcomere function. We highlight the roles of the thick and thin filament, both as necessary structural and regulatory building blocks of the sarcomere as well as targets of functionally impactful modifications. Currently, a new focus emerging in the field is inter-myofilament signaling, and we discuss here the important mediators of this mechanism, including myosin-binding protein C and titin. As the understanding of sarcomere signaling advances, so do the methods with which it is studied. This is reviewed here through discussion of recent live muscle systems in which the sarcomere can be studied under intact, physiologically relevant conditions., Competing Interests: The authors declare no conflicts of interest.

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis., Competing Interests: QM, AA, AR, FM, BD, ZA, JR, MD, AM, EW, CC, VG, PL, BH, OT, OP, FS No competing interests declared, (© 2022, Mao, Acharya et al.)

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca 2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca 2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca 2+ sensitivity as shown by a change in the force-pCa 2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.

Desmin is the primary intermediate filament (IF) of cardiac, skeletal, and smooth muscle. By linking the contractile myofibrils to the sarcolemma and cellular organelles, desmin IF contributes to muscle structural and cellular integrity, force transmission, and mitochondrial homeostasis. Mutations in desmin cause myofibril misalignment, mitochondrial dysfunction, and impaired mechanical integrity leading to cardiac and skeletal myopathies in humans, often characterized by the accumulation of protein aggregates. Recent evidence indicates that desmin filaments also regulate proteostasis and cell size. In skeletal muscle, changes in desmin filament dynamics can facilitate catabolic events as an adaptive response to a changing environment. In addition, post-translational modifications of desmin and its misfolding in the heart have emerged as key determinants of homeostasis and disease. In this review, we provide an overview of the structural and cellular roles of desmin and propose new models for its novel functions in preserving the homeostasis of striated muscles., (© 2021 Federation of European Biochemical Societies.)

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly β-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & β-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles., (© 2021. The Author(s).)

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete's heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin's role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete's heart. The athlete's heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete's heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

We investigated the mechanisms associated with E22K mutation in myosin regulatory light chain (RLC), found to cause hypertrophic cardiomyopathy (HCM) in humans and mice. Specifically, we characterized the mechanical profiles of papillary muscle fibers from transgenic mice expressing human ventricular RLC wild-type (Tg-WT) or E22K mutation (Tg-E22K). Because the two mouse models expressed different amounts of transgene, the B6SJL mouse line (NTg) was used as an additional control. Mechanical experiments were carried out on Ca 2+ - and ATP-activated fibers and in rigor. Sinusoidal analysis was performed to elucidate the effect of E22K on tension and stiffness during activation/rigor, tension-pCa, and myosin cross-bridge (CB) kinetics. We found significant reductions in active tension (by 54%) and stiffness (active by 40% and rigor by 54%). A decrease in the Ca 2+ sensitivity of tension (by ∆pCa ~ 0.1) was observed in Tg-E22K compared with Tg-WT fibers. The apparent (=measured) rate constant of exponential process B (2πb: force generation step) was not affected by E22K, but the apparent rate constant of exponential process C (2πc: CB detachment step) was faster in Tg-E22K compared with Tg-WT fibers. Both 2πb and 2πc were smaller in NTg than in Tg-WT fibers, suggesting a kinetic difference between the human and mouse RLC. Our results of E22K-induced reduction in myofilament stiffness and tension suggest that the main effect of this mutation was to disturb the interaction of RLC with the myosin heavy chain and impose structural abnormalities in the lever arm of myosin CB. When placed in vivo, the E22K mutation is expected to result in reduced contractility and decreased cardiac output whereby leading to HCM., Sub-Discipline: Bioenergetics., Database: The data that support the findings of this study are available from the corresponding authors upon reasonable request., Animal Protocol: BK20150353 (Soochow University)., Research Governance: School of Nursing: Hua-Gang Hu: seuboyh@163.com; Soochow University: Chen Ge chge@suda.edu.cn., (© 2021 Federation of European Biochemical Societies.)

Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on in vitro substrate stiffness, which is an analog for in vivo tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also investigated two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both substrate stiffness and contractile phenotype, with intermediate contractility and high substrate stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of substrate stiffness and cell contractility on FN fibril assembly., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine the role of estrogens in myofiber cross-sectional area (CSA) of the pubococcyegeus (Pcm) and iliococcygeus muscles (Icm)., Methods: In Experiment 1, we excised the Pcm and Icm during the metestrus and proestrus stages of the estrous cycle to measure the myofiber CSA. In Experiment 2, we allocated other rats into the following groups: sham (Sh), ovariectomized (OVX), OVX plus 1,4,6-androstatriene-3,17-dione (ATD; OVX + ATD), an aromatase inhibitor, and OVX plus estradiol benzoate (OVX + EB). We carried out appropriate statistical tests to determine significant differences ( p  ≤ 0.05) in variables measured for both Experiments., Results: The Pcm myofiber CSA at proestrus was higher than at metestrus, while the Icm myofiber CSA did not change. Ovariectomy increased the Pcm myofiber CSA, which was exacerbated with the ATD administration. The EB supplementation successfully reversed the ovariectomy-induced enlargement of the CSA. No significant changes were detected for the Icm myofiber CSA., Conclusions: Fluctuating ovarian steroid levels at the estrus cycle significantly influence the CSA myofiber of the Pcm but not that of the Icm. Estrogen actions, having a gonadal or extragonadal origin, influence importantly the CSA of the Pcm.

The volitional movement of skeletal is controlled by the motor neuron at the site of neuromuscular junction (NMJ) where the retrograde signals are also passed back from muscle to the motor neuron. As the normal function of muscle largely depends on mitochondria that determine the fate of a skeletal muscle myofiber, there must exist a fine-controlled functional coupling between NMJ and mitochondria in myofibers. This mini-review discusses recent publications that reveal how spatiotemporal profiles of intracellular free Ca 2+ could couple mitochondrial function with the activity of NMJ in skeletal muscle myofibers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.

Two groundbreaking papers published in 1954 laid out the theory of the mechanism of muscle contraction based on force-generating interactions between myofilaments in the sarcomere that cause filaments to slide past one another during muscle contraction. The succeeding decades of research in muscle physiology have revealed a unifying interest: to understand the multiscale processes-from atom to organ-that govern muscle function. Such an understanding would have profound consequences for a vast array of applications, from developing new biomimetic technologies to treating heart disease. However, connecting structural and functional properties that are relevant at one spatiotemporal scale to those that are relevant at other scales remains a great challenge. Through a lens of multiscale dynamics, we review in this article current and historical research in muscle physiology sparked by the sliding filament theory.

Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch. Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period. Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min -1 ). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca 2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes. Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement., Competing Interests: Competing Interests: AD and TS are shareholders of InVitroSys GmbH. Other authors declare no conflict of interest., (© The author(s).)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Disease modeling and pharmaceutical testing using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) requires accurate assessment of contractile function. Micropatterning iPSC-CMs on elastic substrates controls cell shape and alignment to enable contractile studies, but determinants of intrinsic variability in this system have been incompletely characterized. The objective of this study was to determine the impact of myofibrillar structure on contractile function in iPSC-CMs. Automated analysis of micropatterned iPSC-CMs labeled with a cell-permeant F-actin dye revealed that myofibrillar abundance is widely variable among iPSC-CMs and strongly correlates with contractile function. This variability is not reduced by subcloning from single iPSCs and is independent of the iPSC-CM purification method. Controlling for myofibrillar structure reduces false-positive findings related to batch effect and improves sensitivity for pharmacologic testing and disease modeling. This analysis provides compelling evidence that myofibrillar structure should be assessed concurrently in studies investigating contractile function in iPSC-CMs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

When a muscle is stretched during a contraction, the resulting steady-state force is higher than the isometric force produced at a comparable sarcomere length. This phenomenon, also referred to as residual force enhancement, cannot be readily explained by the force-sarcomere length relation. One of the most accepted mechanisms for the residual force enhancement is the development of sarcomere length non-uniformities after an active stretch. The aim of this study was to directly investigate the effect of non-uniformities on the force-producing capabilities of isolated myofibrils after they are actively stretched. We evaluated the effect of depleting a single A-band on sarcomere length non-uniformity and residual force enhancement. We observed that sarcomere length non-uniformity was effectively increased following A-band depletion. Furthermore, isometric forces decreased, while the percent residual force enhancement increased compared to intact myofibrils (5% vs. 20%). We conclude that sarcomere length non-uniformities are partially responsible for the enhanced force production after stretch.

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.

Background Detection of the fiber orientation pattern of the myocardium using diffusion tensor magnetic resonance imaging lags ≈12 weeks of gestational age (WGA) behind fetal myocardial remodeling with invasion by the developing coronary vasculature (8 WGA). We aimed to use diffusion tensor magnetic resonance imaging tractography to characterize the evolution of fiber architecture in the developing human heart from the later embryonic period. Methods and Results Twenty human specimens (8-24 WGA) from the Kyoto Collection of Human Embryos and Fetuses, including specimens from the embryonic period (Carnegie stages 20-23), were used. Diffusion tensor magnetic resonance imaging data were acquired with a 7T magnetic resonance system. Fractional anisotropy and helix angle were calculated using standard definitions. In all samples, the fibers ran helically in an organized pattern in both the left and right ventricles. A smooth transmural change in helix angle values (from positive to negative) was detected in all 16 directions of the ventricles. This feature was observed in almost all small (Carnegie stage 23) and large samples. A higher fractional anisotropy value was detected at the outer side of the anterior wall and septum at Carnegie stage 20 to 22, which spread around the ventricular wall at Carnegie stage 23 and in the early fetal samples (11-12 WGA). The fractional anisotropy value of the left ventricular walls decreased in samples with ≥13 WGA, which remained low (≈0.09) in larger samples. Conclusions From the human late embryonic period (from 8 WGA), the helix angle arrangement of the myocardium is comparable to that of the adult, indicating that the myocardial structure blueprint, organization, and integrity are already formed.

X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 10 13  vg kg -1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.

Low-load blood flow restricted resistance exercise (BFRRE) can stimulate whole-muscle growth and improve muscle function. However, limited knowledge exists on the effects at the myocellular level. We hypothesize that BFRRE has the ability to produce concurrent skeletal muscle myofibrillar, mitochondrial, and microvascular adaptations, thus offering an alternative strategy to counteract decay in skeletal muscle health and function in clinical populations.

Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog -/- zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs, we found that myog -/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, Pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog -/- myofibres instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an 'alerted' state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life., Competing Interests: MG, SB, KW, PZ, SH No competing interests declared, (© 2020, Ganassi et al.)

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

The flight muscle of Manduca sexta (DLM 1 ) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila , the DLM 1 of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM 1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system.

Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils.

Forces generated by heart muscle contraction must be balanced by adhesion to the extracellular matrix (ECM) and to other cells for proper heart function. Decades of data have suggested that cell-ECM adhesions are important for sarcomere assembly. However, the relationship between cell-ECM adhesions and sarcomeres assembling de novo remains untested. Sarcomeres arise from muscle stress fibers (MSFs) that are translocating on the top (dorsal) surface of cultured cardiomyocytes. Using an array of tools to modulate cell-ECM adhesion, we established a strong positive correlation between the extent of cell-ECM adhesion and sarcomere assembly. On the other hand, we found a strong negative correlation between the extent of cell-ECM adhesion and the rate of MSF translocation, a phenomenon also observed in nonmuscle cells. We further find a conserved network architecture that also exists in nonmuscle cells. Taken together, our results show that cell-ECM adhesions mediate coupling between the substrate and MSFs, allowing their maturation into sarcomere-containing myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-Pérot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles., Competing Interests: The authors have declared that no competing interests exist.

We compare steps observed during the fibrillogenesis of myofibrils with the sequence of steps predictable by a recent analysis of the structurization and functioning of striated muscles. The predicted assembly steps are based solely on fundamental equilibrium processes, particularly supramolecular interactions and liquid crystalline alignment of the rigid thick and thin filaments hosted within the sarcomer. Satisfactory agreement is obtained between several of the observed and the predicted fibrillogenesis steps. In several cases, however, the actual steps appear to be more complex than expected, evidencing the occurrence of transport and kinetic pathways that may assist the attainment of the equilibrium structure. The memory of the order of a precursor mesophase is imprinted during the remodeling of the surfaces at which the two sets of filaments are anchored. The relevance of the present analysis to the functioning of the myofibril is considered.

Aerobic exercise capacity is critical to bodily health. As a model to investigate the mechanisms that determine health and disease, we employed low (LCR) and high (HCR) capacity running rat models selectively bred to concentrate the genes responsible for divergent aerobic running capacity. To investigate the skeletal muscle contribution to this innate difference in running capacity we employed an approach combining examination of the myofilament protein composition and contractile properties of the fast fiber extensor digitorum longus (EDL) and slow fiber soleus (SOL) muscles from LCR and HCR rats. Intact muscle force experiments demonstrate that SOL, but not EDL, muscles from LCR rats exhibit a three times greater decrease in fatigued force. To investigate the mechanism of this increased fatigability in the LCR SOL muscle, we determined the myofilament protein composition and functional properties. Force-Ca 2+ measurements demonstrate decreased Ca 2+ sensitivity of single skinned SOL muscle fibers from LCR compared with that of HCR rats. Segregating SOL fibers into fast and slow types demonstrates that the decreased Ca 2+ sensitivity in LCR SOL results from a specific decrease in slow-type SOL fiber Ca 2+ sensitivity such that it was similar to that of fast-type fibers. These results identify that the altered myofilament contractile properties of LCR SOL slow-type fibers result in a fast muscle type Ca 2+ sensitivity and the LCR muscle phenotype. Overall our findings demonstrate alterations of the myofilament proteins could contribute to fatigability of the SOL muscle and the decreased innate aerobic running performance of LCR compared with HCR rats.

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.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca 2+ -activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca 2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium., (© 2019 Johnston et al.)

The anabolic action of "fast" whey protein on the regulation of postprandial muscle protein synthesis has been established to be short-lived in healthy young adults. We assessed the time course of anabolic signaling activation and stimulation of myofibrillar protein synthesis rates (MPS) after ingestion of a food source that represents a more typical meal-induced pattern of aminoacidemia. Seven young men (age: 22 ± 1 y) underwent repeated blood and biopsy sampling during primed, continuous l-[ ring - 2 H 5 ]phenylalanine and l-[1- 13 C]leucine tracer infusions and ingested 38 g of l-[1- 13 C]phenylalanine- and l-[1- 13 C]leucine-labeled milk protein concentrate. A total of ∼27 ± 4 (∼10 g) and ∼31 ± 1% (∼12 g) of dietary protein-derived amino acids were released in circulation between 0 and 120 min and 120-300 min, respectively, of the postprandial period. l-[ ring - 2 H 5 ]phenylalanine-based MPS increased above basal (0.025 ± 0.008%/h) by ∼75% (0.043 ± 0.009%/h; P = 0.05) between 0 and 120 min and by ∼86% (0.046 ± 0.004%/h; P = 0.02) between 120 and 300 min, respectively. l-[1- 13 C]leucine-based MPS increased above basal (0.027 ± 0.002%/h) by ∼72% (0.051 ± 0.016%/h; P = 0.10) between 0 and 120 min and by ∼62% (0.047 ± 0.004%/h; P = 0.001) between 120 and 300 min, respectively. Myofibrillar protein-bound l-[1- 13 C]phenylalanine increased over time ( P < 0.001) and equaled 0.004 ± 0.001, 0.008 ± 0.002, 0.017 ± 0.004, and 0.020 ± 0.003 mole percent excess at 60, 120, 180, and 300 min, respectively, of the postprandial period. Milk protein ingestion increased mTORC1 phosphorylation at 120, 180, and 300 min of the postprandial period (all P < 0.05). Our results show that ingestion of 38 g of milk protein results in sustained increases in MPS throughout a 5-h postprandial period in healthy young men. NEW & NOTEWORTHY The stimulation of muscle protein synthesis after whey protein ingestion is short-lived due to its transient systemic appearance of amino acids. Our study characterized the muscle anabolic response to a protein source that results in a more gradual release of amino acids into circulation. Our work demonstrates that a sustained increase in postprandial plasma amino acid availability after milk protein ingestion results in a prolonged stimulation of muscle protein synthesis rates in healthy young men.

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 ( g app ) in combination with a conservation of the force redevelopment rate (k Tr ) 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.

Sarcomere length (SL) instability and SL non-uniformity have been used to explain fundamental properties of skeletal muscles, such as creep, force depression following active muscle shortening and residual force enhancement following active stretching of muscles. Regarding residual force enhancement, it has been argued that active muscle stretching causes SL instability, thereby increasing SL non-uniformity. However, we recently showed that SL non-uniformity is not increased by active muscle stretching, but it remains unclear if SL stability is affected by active stretching. Here, we used single myofibrils of rabbit psoas muscle and measured SL non-uniformity and SL instability during isometric contractions and for isometric contractions following active stretching at average SLs corresponding to the descending limb of the force-length relationship. We defined isometric contractions as contractions during which mean SL remained constant. SL instability was quantified by the rate of change of individual SLs over the course of steady-state isometric force and SL non-uniformity was defined as deviations of SLs from the mean SL at an instant of time. We found that whereas the mean SL remained constant during isometric contraction, by definition, individual SLs did not. SLs were more stable in the force-enhanced, isometric state following active stretching compared with the isometric reference state. We also found that SL instability was not correlated with the rate of change of SL non-uniformity. Also, SL non-uniformity was not different in the isometric and the post-stretch isometric contractions. We conclude that since SL is more stable but similarly non-uniform in the force-enhanced compared with the corresponding isometric reference contraction, it appears unlikely that either SL instability or SL non-uniformity contribute to the residual force enhancement property of skeletal muscle., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (<48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (>48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

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.

Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future., (© 2019 Ravichandran et al.)

Aim: Cardiovascular disease (CVD) risk is lower in pre-menopausal females vs age matched males. After menopause risk equals or exceeds that of males. CVD protection of pre-menopausal females is ascribed to high circulating oestrogen levels. Despite experimental evidence that oestrogen are cardioprotective, oestrogen replacement therapy trials have not shown clear benefits. One hypothesis to explain the discrepancy proposed hearts remodel during peri-menopause. Peri-menopasual myocardial changes have never been investigated, nor has the ability of oestrogen to regulate heart function during peri-menopause., Methods: We injected female mice with 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg/d IP) to cause gradual ovarian failure over 120d and act as a peri-menopausal model RESULTS: Left ventricular function assessed by Langendorff perfusion found no changes in VCD-injected mice at 60 or 120 days compared to intact mice. Cardiac myofilament activity was altered at 60 and 120 days indicating a molecular remodelling in peri-menopause. Myocardial TGF-β1 increased at 60 days post-VCD treatment along with reduced Akt phosphorylation. Acute activation of oestrogen receptor-α (ERα) or -β (ERβ) depressed left ventricular contractility in hearts from intact mice. ER-regulation of myocardial and myofilament function, and myofilament phosphorylation, were disrupted in the peri-menopausal model. Disruption occurred without alterations in total ERα or ERβ expression., Conclusions: This is the first study to demonstrate remodelling of the heart in a model of peri-menopause, along with a disruption in ER-dependent regulation of the heart. These data indicate that oestrogen replacement therapy initiated after menopause affects a heart that is profoundly different from that found in reproductively intact animals., (© 2019 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Objective: Motor Unit Number Estimation (MUNE) methods, such as the recently developed MScanFit MUNE (MScan), may be valuable in tracking motor unit loss in ALS. Muscle Velocity Recovery Cycles (MVRCs) provide information about muscle membrane properties and can reveal disease-related changes. This study was undertaken to test the applicability of MScan to the anterior tibial muscle (TA) and to test whether the MVRCs could improve understanding of ALS pathophysiology., Methods: Twenty-six ALS patients and 25 healthy controls were evaluated by quantitative electromyography, nerve conduction study and the two novel methods: MScan and MVRC; all in the TA and peroneal nerve., Results: The estimated number of motor units for ALS patients (Median: 45, interquartile range: 28.5-76.5) was significantly lower than for the controls (117, 96.0-121.0) (P = 2.19 × 10 -7 ). Unit size was increased only when amplitudes were expressed as percentage of CMAP. Of MVRC measurements, only relative refractory period was significantly abnormal in patients., Conclusion: MScanFit MUNE gives a sensitive and quantitative measure of loss of TA motor units in ALS. Muscle fiber membrane properties are mostly unaffected, despite substantial denervation, presumably due to collateral reinnervation., Significance: MScan is suitable for detecting motor unit loss in TA. MVRCs do not provide new insights in ALS., (Copyright © 2019 International Federation of Clinical Neurophysiology. All rights reserved.)

Deer mice, Peromyscusmaniculatus, live at high altitudes where limited O 2 represents a challenge to maintaining oxygen delivery to tissues. Previous work has demonstrated that hypoxia acclimation of deer mice and low altitude white-footed mice (P. leucopus) increases the force generating capacity of the diaphragm. The mechanism behind this improved contractile function is not known. Within myocytes, the myofilament plays a critical role in setting the rate and level of force production, and its ability to generate force can change in response to changes in physiological conditions. In the current study, we examined how chronic hypobaric hypoxia exposure of deer mice and white-footed mice influences the Ca 2+ activation of force generation by skinned diaphragmatic myofilaments, and the phosphorylation of myofilament proteins. Results demonstrate that myofilament force production, and the Ca 2+ sensitivity of force generation, were not impacted by acclimation to hypobaric hypoxia, and did not differ between preparations from the two species. The cooperativity of the force-pCa relationship, and the maximal rate of force generation were also the same in the preparations from both species, and not impacted by acclimation. Finally, the relative phosphorylation of TnT, and MLC was lower in deer mice than white-footed mice, but was not affected by acclimation. These results indicate that species differences in diaphragm function, and the increase in force production with hypoxia acclimation, are not due to differences, or changes, in myofilament function. However, it appears that diaphragmatic myofilament function in these species is not affected by chronic hypobaric hypoxia exposure.

This document is the consensus of international experts on the current status of Single Fiber EMG (SFEMG) and the measurement of neuromuscular jitter with concentric needle electrodes (CNE - CN-jitter). The panel of authors was chosen based on their particular interests and previous publications within a specific area of SFEMG or CN-jitter. Each member of the panel was asked to submit a section on their particular area of interest and these submissions were circulated among the panel members for edits and comments. This process continued until a consensus was reached. Donald Sanders and Erik Stålberg then edited the final document., (Copyright © 2019. Published by Elsevier B.V.)

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 emergence of a mature nervous system requires a significant refinement of the synaptic connections initially formed during development. Redundant synaptic connections are removed in a process known as synapse elimination. Synapse elimination has been extensively studied at the rodent neuromuscular junction (NMJ). Although several axons initially converge onto each postsynaptic muscle fiber, all redundant inputs are removed during early postnatal development until a single motor neuron innervates each NMJ. Neuronal activity as well as synaptic glia influence the course of synapse elimination. It is, however, unclear whether target muscle fibers are more than naïve substrates in this process. I examined the influence of target myofiber contractile properties on synapse elimination. The timing of redundant input removal in muscles examined correlates strongly with their proportion of slow myofibers: muscles with more slow fibers undergo elimination more slowly. Moreover, this intermuscular difference in the timing of synapse elimination appears to result from local differences in the rate of elimination on fast versus slow myofibers. These results, therefore, imply that differences in the constituent fiber types help account for the variation in the timing of the developmental synapse elimination between muscles and show that the muscle plays a role in the process.

Understanding and predicting the mechanical behavior of myocardium under healthy and pathophysiological conditions are vital to developing novel cardiac therapies and promoting personalized interventions. Within the past 30 years, various constitutive models have been proposed for the passive mechanical behavior of myocardium. These models cover a broad range of mathematical forms, microstructural observations, and specific test conditions to which they are fitted. We present a critical review of these models, covering both phenomenological and structural approaches, and their relations to the underlying structure and function of myocardium. We further explore the experimental and numerical techniques used to identify the model parameters. Next, we provide a brief overview of continuum-level electromechanical models of myocardium, with a focus on the methods used to integrate the active and passive components of myocardial behavior. We conclude by pointing to future directions in the areas of optimal form as well as new approaches for constitutive modeling of myocardium.