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.

Cryo-EM structure of the human cardiac myosin filament.

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly1. Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years2. Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin's motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state3; how titin and cMyBP-C may contribute to length-dependent activation4; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease5,6. The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.

When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein-protein interactions and protein stability.

Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing obscurin's Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/obscurin interaction, the PLN/obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these obscurin variants as pathogenic.

Estimation of Forces on Actin Filaments in Living Muscle from X-ray Diffraction Patterns and Mechanical Data.

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.

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.

Titin and Nebulin in Thick and Thin Filament Length Regulation.

In this review we discuss the history and the current state of ideas related to the mechanism of size regulation of the thick (myosin) and thin (actin) filaments in vertebrate striated muscles. Various hypotheses have been considered during of more than half century of research, recently mostly involving titin and nebulin acting as templates or 'molecular rulers', terminating exact assembly. These two giant, single-polypeptide, filamentous proteins are bound in situ along the thick and thin filaments, respectively, with an almost perfect match in the respective lengths and structural periodicities. However, evidence still questions the possibility that the proteins function as templates, or scaffolds, on which the thin and thick filaments could be assembled. In addition, the progress in muscle research during the last decades highlighted a number of other factors that could potentially be involved in the mechanism of length regulation: molecular chaperones that may guide folding and assembly of actin and myosin; capping proteins that can influence the rates of assembly-disassembly of the myofilaments; Ca2+ transients that can activate or deactivate protein interactions, etc. The entire mechanism of sarcomere assembly appears complex and highly dynamic. This mechanism is also capable of producing filaments of about the correct size without titin and nebulin. What then is the role of these proteins? Evidence points to titin and nebulin stabilizing structures of the respective filaments. This stabilizing effect, based on linear proteins of a fixed size, implies that titin and nebulin are indeed molecular rulers of the filaments. Although the proteins may not function as templates in the assembly of the filaments, they measure and stabilize exactly the same size of the functionally important for the muscles segments in each of the respective filaments.

Computing Average Passive Forces in Sarcomeres in Length-Ramp Simulations.

Passive forces in sarcomeres are mainly related to the giant protein titin. Titin's extensible region consists of spring-like elements acting in series. In skeletal muscles these elements are the PEVK segment, two distinct immunoglobulin (Ig) domain regions (proximal and distal), and a N2A portion. While distal Ig domains are thought to form inextensible end filaments in intact sarcomeres, proximal Ig domains unfold in a force- and time-dependent manner. In length-ramp experiments of single titin strands, sequential unfolding of Ig domains leads to a typical saw-tooth pattern in force-elongation curves which can be simulated by Monte Carlo simulations. In sarcomeres, where more than a thousand titin strands are arranged in parallel, numerous Monte Carlo simulations are required to estimate the resultant force of all titin filaments based on the non-uniform titin elongations. To simplify calculations, the stochastic model of passive forces is often replaced by linear or non-linear deterministic and phenomenological functions. However, new theories of muscle contraction are based on the hypothesized binding of titin to the actin filament upon activation, and thereby on a prominent role of the structural properties of titin. Therefore, these theories necessitate a detailed analysis of titin forces in length-ramp experiments. In our study we present a simple and efficient alternative to Monte Carlo simulations. Based on a structural titin model, we calculate the exact probability distributions of unfolded Ig domains under length-ramp conditions needed for rigorous analysis of expected forces, distribution of unfolding forces, etc. Due to the generality of our model, the approach is applicable to a wide range of stochastic protein unfolding problems.

Smooth muscle titin forms in vitro amyloid aggregates.

Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700-4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.

Nanopuller-open data acquisition platform for AFM force spectroscopy experiments.

Atomic Force Microscope (AFM) is a widely used tool in force spectroscopy studies. Presently, this instrument is accessible from numerous vendors, albeit commercial solutions are expensive and almost always hardware and software closed. Approaches for open setups were published, as with modern low cost and readily available piezoelectric actuators, data acquisition interfaces and optoelectronic components building such force spectroscopy AFM is relatively easy. However, suitable software to control such laboratory made instrument was not released. Developing it in the lab requires significant time and effort. Our Nanopuller software described in this paper is intended to eliminate this obstacle. With only minimum adjustments this program can be used to control and acquire data with any suitable National Instruments universal digital/analog interface and piezoelectric actuator analog controller, giving significant freedom and flexibility in designing force spectroscopy experiment. Since the full code, written in a graphical LabVIEW environment is available, our Nanopuller can be easily customized. In this paper we describe the program and test its performance in controlling different setups. Successful and accurate force curve acquisition for standard samples (single molecules of I27O reference titin polyprotein and DNA as well as red blood cells) is shown.

Fibre typing of intrafusal fibres.

The first descriptions of muscle spindles with intrafusal fibres containing striated myofibrils and nervous elements were given approximately 150 years ago. It took, however, another 100 years to establish the presence of two types of intrafusal muscle fibres: nuclear bag and nuclear chain fibres. The present paper highlights primarily the contribution of Robert Banks in fibre typing of intrafusal fibres: the confirmation of the principle of two types of nuclear bag fibres in mammalian spindles and the variation in occurrence of a dense M-band along the fibres. Furthermore, this paper summarizes how studies from the Umeå University group (Laboratory of Muscle Biology in the Department of Integrative Medical Biology) on fibre typing and the structure and composition of M-bands have contributed to the current understanding of muscle spindle complexity in adult humans as well as to muscle spindle development and effects of ageing. The variable molecular composition of the intrafusal sarcomeres with respect to myosin heavy chains and M-band proteins gives new perspectives on the role of the intrafusal myofibrils as stretch-activated sensors influencing tension/stiffness and signalling to nuclei.

The crystal structure of the human titin:obscurin complex reveals a conserved yet specific muscle M-band zipper module.

M10 is the most C-terminal immunoglobulin (Ig) domain of the giant protein titin and a frequent target of disease-linked mutations. Currently, it is the only known muscle Ig domain able to interact with two alternative ligands-obscurin and obscurin-like-1 (Obsl1)-in different sarcomeric subregions. Obscurin and Obsl1 use their homologous N-terminal Ig domain (O1 in obscurin and OL1 in Obsl1) to bind M10 in a mutually exclusive manner. We present here the X-ray structure of the human titin:obscurin M10:O1 complex extending our previous work on the M10:OL1 interaction. Similar to M10:OL1, the M10:O1 complex displays a chevron-shaped antiparallel Ig-Ig architecture held together by a conserved molecular interface, which we validated by isothermal titration calorimetry and sorting experiments in neonatal rat cardiomyocytes. O1, although structurally related to OL1 and M10, both members of the intermediate set (I-set) Ig family, presents an intriguing switch of its ßA' strand. This leads to structural differences between the complexes, particularly for the "open side" of the chevron-shaped assembly. A bioinformatics analysis reveals that the ßA'-switch observed for O1 is rare and that it is involved in mediating protein-protein interactions. Molecular dynamics simulations also suggest that this topological alteration substantially increases local flexibility compared to the conventional I-set Ig domains. The O1/OL1 Ig domains are candidate discriminatory structural modules potentially directing the binding of specific additional partners at the M-band. Cellular sorting experiments in neonatal rat cardiomyocytes are consistent with the view that the titin:obscurin/Obsl1 complexes might be a platform for higher-order interactions.

Zebrafish cardiac muscle thick filaments: isolation technique and three-dimensional structure.

To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes.

Gigantic business: titin properties and function through thick and thin.

The giant protein titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signaling functions of the heart. TTN, which encodes titin, is also a major human disease gene. In this review, we cover the roles of cardiac titin in normal and failing hearts, with a special emphasis on the contribution of titin to diastolic stiffness. We provide an update on disease-associated titin mutations in cardiac and skeletal muscles and summarize what is known about the impact of protein-protein interactions on titin properties and functions. We discuss the importance of titin-isoform shifts and titin phosphorylation, as well as titin modifications related to oxidative stress, in adjusting the diastolic stiffness of the healthy and the failing heart. Along the way we distinguish among titin alterations in systolic and in diastolic heart failure and ponder the evidence for titin stiffness as a potential target for pharmacological intervention in heart disease.

Acute change of titin at mid-sarcomere remains despite 8 wk of plyometric training.

The purpose of this study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise (PlyEx) both before and after PlyEx training, to understand if titin is affected differently after PlyEx training. Healthy untrained individuals (N = 11) completed the 1stPlyEx (10 × 10 squat-jumps, 1-min rest). Thereafter, six subjects completed 8 wk of PlyEx, while five controls abstained from any jumping activity. Seven days after the last training session, all subjects completed the 2ndPlyEx. Blood samples were collected before and 6 h and 1, 2, 3, and 4 days after each acute bout of PlyEx, and muscle biopsies 4 days before and 3 days after each acute bout of PlyEx. The 1stPlyEx induced an increase in circulating myoglobin concentration. Muscle sample analysis revealed Z-disk streaming, a stretch or a fragmentation of titin (immunogold), and increased calpain-3 autolysis. After training, 2ndPlyEx did not induce Z-disk streaming or calpain-3 activation. The previously observed post-1stPlyEx positional change of the titin COOH terminus was still present pre-2ndPlyEx, in all trained and all control subjects. Only two controls presented with Z-disk streaming after 2ndPlyEx, while calpain-3 activation was absent in all controls. Eccentric explosive exercise induced a stretch or fragmentation of titin, which presented as a positional change of the COOH terminus. Calpain-3 activation does not occur when titin is already stretched before explosive jumping. Enzymatic digestion results in titin fragmentation, but since an increase in calpain-3 autolysis was visible only after the 1stPlyEx acute bout, fragmentation cannot explain the prolonged positional change.