The molecular basis for sarcomere organization in vertebrate skeletal muscle.

Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 Å further reveal two conformations of the "double-head" myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

Structure of the native myosin filament in the relaxed cardiac sarcomere.

The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-ß chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-ß chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

Myosin filament-based regulation of the dynamics of contraction in heart muscle.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.

The actin polymerization factor Diaphanous and the actin severing protein Flightless I collaborate to regulate sarcomere size.

The sarcomere is the basic contractile unit of muscle, composed of repeated sets of actin thin filaments and myosin thick filaments. During muscle development, sarcomeres grow in size to accommodate the growth and function of muscle fibers. Failure in regulating sarcomere size results in muscle dysfunction; yet, it is unclear how the size and uniformity of sarcomeres are controlled. Here we show that the formin Diaphanous is critical for the growth and maintenance of sarcomere size: Dia sets sarcomere length and width through regulation of the number and length of the actin thin filaments in the Drosophila flight muscle. To regulate thin filament length and sarcomere size, Dia interacts with the Gelsolin superfamily member Flightless I (FliI). We suggest that these actin regulators, by controlling actin dynamics and turnover, generate uniformly sized sarcomeres tuned for the muscle contractions required for flight.

Passive viscoelastic response of striated muscles.

Muscle cells with sarcomeric structure exhibit highly non trivial passive mechanical response. The difficulty of its continuum modeling is due to the presence of long-range interactions transmitted by extended protein skeleton. To build a rheological model for muscle 'material', we use a stochastic micromodel, and derive a linear response theory for a half-sarcomere, which can be extended to the whole fibre. Instead of the first order rheological equation, anticipated by Hill on the phenomenological grounds, we obtain a novel second order equation which shows that tension depends not only on its current length and the velocity of stretching, but also on its acceleration. Expressing the model in terms of elementary rheological elements, we show that one contribution to the visco-elastic properties of the fibre originates in cross-bridges, while the other can be linked to inert elements which move in the sarcoplasm. We apply this model to explain the striking qualitative difference between the relaxation in experiments involving perturbation of length vs. those involving perturbation of force, and we use the values of the microscopic parameters for frog muscles to show that the model is in excellent quantitative agreement with physiological experiments.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding.

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Knockout of MYOM1 in human cardiomyocytes leads to myocardial atrophy via impairing calcium homeostasis.

Myomesin-1 (encoded by MYOM1 gene) is expressed in almost all cross-striated muscles, whose family (together with myomesin-2 and myomesin-3) helps to cross-link adjacent myosin to form the M-line in myofibrils. However, little is known about its biological function, causal relationship and mechanisms underlying the MYOM1-related myopathies (especially in the heart). Regrettably, there is no MYMO1 knockout model for its study so far. A better and further understanding of MYOM1 biology is urgently needed. Here, we used CRISPR/Cas9 gene-editing technology to establish an MYOM1 knockout human embryonic stem cell line (MYOM1-/- hESC), which was then differentiated into myomesin-1 deficient cardiomyocytes (MYOM1-/- hESC-CMs) in vitro. We found that myomesin-1 plays an important role in sarcomere assembly, contractility regulation and cardiomyocytes development. Moreover, myomesin-1-deficient hESC-CMs can recapitulate myocardial atrophy phenotype in vitro. Based on this model, not only the biological function of MYOM1, but also the aetiology, pathogenesis, and potential treatments of myocardial atrophy caused by myomesin-1 deficiency can be studied.

Atrophy, oxidative switching and ultrastructural defects in skeletal muscle of the ataxia telangiectasia mouse model.

Ataxia telangiectasia is a rare, multi system disease caused by ATM kinase deficiency. Atm-knockout mice recapitulate premature aging, immunodeficiency, cancer predisposition, growth retardation and motor defects, but not cerebellar neurodegeneration and ataxia. We explored whether Atm loss is responsible for skeletal muscle defects by investigating myofiber morphology, oxidative/glycolytic activity, myocyte ultrastructural architecture and neuromuscular junctions. Atm-knockout mice showed reduced muscle and fiber size. Atrophy, protein synthesis impairment and a switch from glycolytic to oxidative fibers were detected, along with an increase of in expression of slow and fast myosin types (Myh7, and Myh2 and Myh4, respectively) in tibialis anterior and solei muscles isolated from Atm-knockout mice. Transmission electron microscopy of tibialis anterior revealed misalignments of Z-lines and sarcomeres and mitochondria abnormalities that were associated with an increase in reactive oxygen species. Moreover, neuromuscular junctions appeared larger and more complex than those in Atm wild-type mice, but with preserved presynaptic terminals. In conclusion, we report for the first time that Atm-knockout mice have clear morphological skeletal muscle defects that will be relevant for the investigation of the oxidative stress response, motor alteration and the interplay with peripheral nervous system in ataxia telangiectasia.

Fluid flow in the sarcomere.

A highly organized and densely packed lattice of molecular machinery within the sarcomeres of muscle cells powers contraction. Although many of the proteins that drive contraction have been studied extensively, the mechanical impact of fluid shearing within the lattice of molecular machinery has received minimal attention. It was recently proposed that fluid flow augments substrate transport in the sarcomere, however, this analysis used analytical models of fluid flow in the molecular machinery that could not capture its full complexity. By building a finite element model of the sarcomere, we estimate the explicit flow field, and contrast it with analytical models. Our results demonstrate that viscous drag forces on sliding filaments are surprisingly small in contrast to the forces generated by single myosin molecular motors. This model also indicates that the energetic cost of fluid flow through viscous shearing with lattice proteins is likely minimal. The model also highlights a steep velocity gradient between sliding filaments and demonstrates that the maximal radial fluid velocity occurs near the tips of the filaments. To our knowledge, this is the first computational analysis of fluid flow within the highly structured sarcomere.

Polarization-resolved microscopy reveals a muscle myosin motor-independent mechanism of molecular actin ordering during sarcomere maturation.

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs.

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.

CHD4 and the NuRD complex directly control cardiac sarcomere formation.

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.

Aquatic Training after Joint Immobilization in Rats Promotes Adaptations in Myotendinous Junctions.

The myotendinous junction (MTJ) is the muscle-tendon interface and constitutes an integrated mechanical unit to force transmission. Joint immobilization promotes muscle atrophy via disuse, while physical exercise can be used as an adaptative stimulus. In this study, we aimed to investigate the components of the MTJ and their adaptations and the associated elements triggered with aquatic training after joint immobilization. Forty-four male Wistar rats were divided into sedentary (SD), aquatic training (AT), immobilization (IM), and immobilization/aquatic training (IMAT) groups. The samples were processed to measure fiber area, nuclear fractal dimension, MTJ nuclear density, identification of telocytes, sarcomeres, and MTJ perimeter length. In the AT group, the maintenance of ultrastructure and elements in the MTJ region were observed; the IM group presented muscle atrophy effects with reduced MTJ perimeter; the IMAT group demonstrated that aquatic training after joint immobilization promotes benefits in the muscle fiber area and fractal dimension, in the MTJ region shows longer sarcomeres and MTJ perimeter. We identified the presence of telocytes in the MTJ region in all experimental groups. We concluded that aquatic training is an effective rehabilitation method after joint immobilization due to reduced muscle atrophy and regeneration effects on MTJ in rats.

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin.

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.

The effects of inorganic phosphate on contractile function of slow skeletal muscle fibres are length-dependent.

Muscle operates across a wide range of sarcomere lengths. Inorganic phosphate (Pi) diminishes force output of striated muscle, with greater influence at short relative to long sarcomere lengths in fast skeletal and cardiac muscle fibres. The purpose of this study was to fill a gap in the literature regarding the length-dependent effects of Pi on contractile function of slow skeletal muscle fibres. Permeabilized slow skeletal muscle fibres from rabbit soleus were assessed at average sarcomere lengths of 2.0, 2.4, or 2.8 µm, with and without 20 mM Pi added to activating solutions (22±1 °C). The magnitude of Pi-induced reductions in peak force (43 ± 7% at 2.0 µm, 38 ± 7% at 2.4 µm, and 31 ± 8% at 2.8 µm) and peak stiffness (41 ± 9% at 2.0 µm, 36 ± 8% at 2.4 µm, and 26 ± 9% at 2.8 µm) were length dependent. Peak stiffness was less affected by Pi than peak force. Pi diminished the Ca2+-sensitivity of the force-pCa and stiffness-pCa relationships to a greater extent at 2.8 µm than 2.0 µm. Comparable results were obtained from a cooperative model of Ca2+ and myosin binding to regulated actin. In conclusion, Pi is more detrimental to the peak force output of slow skeletal muscle fibres held at short relative to long sarcomere lengths, whereas Pi has a greater effect on the Ca2+-sensitivity of force production at long relative to short sarcomere lengths. Stiffness data suggest that Pi-induced reductions in force are primarily due to fewer bound cross-bridges, with a lesser contribution attributable to lower average force per cross-bridge.

α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk.

Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.

Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy.

The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.

Structure and Function of Filamin C in the Muscle Z-Disc.

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction.

Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta.

The flight muscle of Manduca sexta (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 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 DLM1 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.

Fine mapping titin's C-zone: Matching cardiac myosin-binding protein C stripes with titin's super-repeats.

Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.