Sarcomeric Filaments Research Articles

Interacting myosin head dynamics and their modification by 2’-deoxy-ADP

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are inhibited from performing motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state can pathologically disrupt IHM structure and the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2’-deoxy-ATP (dATP) is a potent myosin activator that destabilizes the IHM. Here, we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations of the IHM state containing ADP.Pi in both nucleotide binding pockets revealed dynamic motions of the blocked head – free head interface, light chain binding domain, and S2 in this ‘inactive’ state of myosin. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that increased heterogeneity among residue contact pairs at the blocked head – free head interface and a 14% decrease in the interaction energy at the interface. Dynamic changes to this interface were accompanied by dynamics in the light chain binding region. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.

An atomistic model of myosin interacting heads motif dynamics and their modification by 2'-deoxy-ADP.

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are unable to perform motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state via missense mutations can pathologically disrupt the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2'-deoxy-ATP (dATP) is a potent myosin activator which destabilizes the IHM. Here we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations with IHM containing ADP.Pi in both nucleotide binding pockets revealed residual dynamics in an otherwise 'inactive' and 'sequestered' state of a motor protein. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that modify the protein-protein interface that stabilizes the sequestered state, and changes to this interface were accompanied by allosteric changes in remote regions of the protein complex. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.

Myosin folding boosts solubility in cardiac muscle sarcomeres.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Gaining new understanding of sarcomere length non-uniformities in skeletal muscles.

Sarcomere lengths are non-uniform on all structural levels of mammalian skeletal muscle. These non-uniformities have been associated with a variety of mechanical properties, including residual force enhancement and depression, creep, increased force capacity, and extension of the plateau of the force-length relationship. However, the nature of sarcomere length non-uniformities has not been explored systematically. The purpose of this study was to determine the properties of sarcomere length non-uniformities in active and passive muscle. Single myofibrils of rabbit psoas (n = 20; with 412 individual sarcomeres) were subjected to three activation/deactivation cycles and individual sarcomere lengths were measured at 4 passive and 3 active points during the activation/deactivation cycles. The myofibrils were divided into three groups based on their initial average sarcomere lengths: short, intermediate, and long average sarcomere lengths of 2.7, 3.2, and 3.6µm. The primary results were that sarcomere length non-uniformities did not occur randomly but were governed by some structural and/or contractile properties of the sarcomeres and that sarcomere length non-uniformities increased when myofibrils went from the passive to the active state. We propose that the mechanisms that govern the systematic sarcomere lengths non-uniformities observed in active and passive myofibrils may be associated with the variable number of contractile proteins and the variable number and the adjustable stiffness of titin filaments in individual sarcomeres.

PO-04-073 ELECTRON MICROSCOPIC INSIGHTS FROM AN ACUTE PULSED FIELD MYOCARDIAL LESION

Pulsed field ablation (PFA) is a nonthermal ablative energy modality that increases membrane permeability by creating pores and causing chemical changes to the lipids and proteins on the cell membrane. However, little is known about its effect on cell membrane, organelle structure and time course of lesion formation. To evaluate cell membrane and organelle structure after PFA and assess lesion differences between 1- and 4-hour after PFA. Healthy swine underwent endocardial PFA using an 11 Fr circular, multielectrode catheter (CellFX, Pulse Biosciences) in nanosecond pulsing mode. Pulses were delivered to the left ventricle under general anesthesia with fluoroscopic and intracardiac echocardiography guidance. Two discrete PFA lesions were sampled 1 and 4 hours after PFA. Transmission electron microscopy assessment was performed after fixation using paraformaldehyde and glutaraldehyde. At 1-hour post-PFA, healthy myocytes with normal sarcomeric structure, clear Z band, and A band were noted adjacent to ablated myocytes (Figure). Mitochondria inside the healthy myocyte were aligned in parallel with sarcomere filaments. Ablated myocytes on the other hand demonstrated disrupted sarcomeric structures and were randomly clustered and misaligned. Cell membranes of the ablated myocyte at the lesion border were relatively preserved compared to ablated myocytes from within the core of the PFA lesion. At 4-hour post-PFA, the PF lesion was noticeably more edematous and areas of coagulum could be seen adjacent to the capillaries. Alignment of cell membranes at the 4-hour timepoint was further disorganized. Fewer mitochondria per section were observed at 4-hour post-PFA, and noted to be randomly scattered amongst damaged myocytes. There were no normal sarcomeres noted anywhere within the lesion at 4 hours. This electron microscopy study demonstrates significant rapid disappearance of myocytes after PFA (∼ 1 hour). The cell membrane structure and organelle structure progressively deteriorate by 4 hours post ablation.

Molecular Dynamics Assessment of Mechanical Properties of the Thin Filaments in Cardiac Muscle

Contraction of cardiac muscle is regulated by Ca2+ ions via regulatory proteins, troponin (Tn), and tropomyosin (Tpm) associated with the thin (actin) filaments in myocardial sarcomeres. The binding of Ca2+ to a Tn subunit causes mechanical and structural changes in the multiprotein regulatory complex. Recent cryo-electron microscopy (cryo-EM) models of the complex allow one to study the dynamic and mechanical properties of the complex using molecular dynamics (MD). Here we describe two refined models of the thin filament in the calcium-free state that include protein fragments unresolved by cryo-EM and reconstructed using structure prediction software. The parameters of the actin helix and the bending, longitudinal, and torsional stiffness of the filaments estimated from the MD simulations performed with these models were close to those found experimentally. However, problems revealed from the MD simulation suggest that the models require further refinement by improving the protein-protein interaction in some regions of the complex. The use of relatively long refined models of the regulatory complex of the thin filament allows one to perform MD simulation of the molecular mechanism of Ca2+ regulation of contraction without additional constraints and study the effects of cardiomyopathy-associated mutation of the thin filament proteins of cardiac muscle.

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.

The effect of variable troponin C mutation thin filament incorporation on cardiac muscle twitch contractions

One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k−Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k−Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SummaryA challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.

Direct visualization of human myosin II force generation using DNA origami-based thick filaments

The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highly-structured lattice. This complex imposes a geometric restriction on myosin in force generation. However, how single myosins generate force within the restriction remains elusive and conventional synthetic filaments do not recapitulate the symmetric bipolar filaments in sarcomeres. Here we engineered thick filaments using DNA origami that incorporate human muscle myosin to directly visualize the motion of the heads during force generation in a restricted space. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. Our results illustrate the usefulness of our DNA origami-based assay system to dissect the mechanistic details of motor proteins.

Association of single nucleotide polymorphisms in the 3'UTR region of TPM1 gene with dilated cardiomyopathy: A case-control study.

Tropomyosin 1 (TPM1) is a protein that constitutes the sarcomere filaments and is encoded by the TPM1 gene. The aim of the present study is to investigate the correlation between the 3′ untranslated region (3′UTR) single nucleotide polymorphisms (SNPs) of the TPM1 gene and dilated cardiomyopathy (DCM).A total of 245 patients with DCM and 245 healthy controls were recruited with 5 ml of venous blood. Genomic DNA was extracted to analyze the TPM1 gene rs12148828, rs11558748, rs707602, rs6738, rs7178040 loci genotypes, and the plasma miR-21 level was analyzed by reverse transcription-PCR (RT-PCR).The risk of DCM development in the rs6738 locus G allele carriers were 1.69 times more than A allele carriers (95% CI: 1.22-2.33, P = .001). Age and gender had no effect on the association of TPM1 gene SNPs with DCM risk (P > .05). The plasma miR-21 level of TPM1 gene rs6738 locus AA carriers was significantly higher than that of the AG and GG genotypes (P < .001).The SNPs of TPM1 gene rs6738 locus is associated with the risk of DCM, which may be related to the abnormal increase of miR-21 level in DCM patients, but further research is needed to prove the causal relationship between miR-21 level and DCM risk.

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.

C 2]Daisy Chain Rotaxanes as Molecular Muscles

Bistable [ c2]daisy chain rotaxanes represent a particularly intriguing class of interlocked molecules that can produce internal sliding movements with a net contraction or extension at the single-molecule level. These nanometric motions show some analogies with the sliding motions of actin and myosin filaments in sarcomeres, and this is why [ c2]daisy chain rotaxanes have been also named as “molecular muscles,” as their first synthesis in 2000. In this minireview, the authors discuss the recent history of these molecules, their modular chemical structures, and the various synthetic pathways described in the literature to access them. The authors also detail how their internal motions can be controlled and characterized by a number of chemical and physical tools. The authors finally show that their integration within polymers and materials can give access to synchronized motions and amplifications up to the macroscopic scale. Overall, the numerous examples that have been described in the literature to date demonstrate that this family of molecules has already strongly influenced the entire field of research on artificial molecular machines, and has the potential to be implemented as actuators working at all scales, from nanometric-switchable devices to mechanically active soft matter materials.

Leiomodin and Tropomyosin, Binding at the Pointed End of the Thin Filaments

Leiomodin is an important actin-binding protein responsible for the thin filament formation. Mutations in leiomodins were demonstrated to cause myopathies. The spectrum of leiomodin functions remains a subject of an intensive debate. One of the models proposes that leiomodin and its homolog tropomodulin modulate the length of the thin filaments in muscle sarcomeres. The modulation is thought to function through competition between leiomodin and tropomodulin for the pointed end of the thin filaments in a tropomyosin-dependent manner. We studied complexes formed by peptides representing the tropomyosin-binding site of leiomodin 2 (Lmod2s1) and the N-terminal coiled coil fragment of tropomyosin. By using a combination of NMR and MDS methods, we determined the mode of Lmod2s1 binding to the N-terminus of tropomyosin. The structure places leiomodin at the pointed end of thin filaments. This finding provides compelling evidence in favor of the competition hypothesis and has important implications for the role of leiomodin in forming thin filaments in muscle sarcomeres.

Titin-based mechanosensing modulates muscle hypertrophy.

BackgroundTitin is an elastic sarcomeric filament that has been proposed to play a key role in mechanosensing and trophicity of muscle. However, evidence for this proposal is scarce due to the lack of appropriate experimental models to directly test the role of titin in mechanosensing.MethodsWe used unilateral diaphragm denervation (UDD) in mice, an in vivo model in which the denervated hemidiaphragm is passively stretched by the contralateral, innervated hemidiaphragm and hypertrophy rapidly occurs.ResultsIn wildtype mice, the denervated hemidiaphragm mass increased 48 ± 3% after 6 days of UDD, due to the addition of both sarcomeres in series and in parallel. To test whether titin stiffness modulates the hypertrophy response, RBM20ΔRRM and TtnΔIAjxn mouse models were used, with decreased and increased titin stiffness, respectively. RBM20ΔRRM mice (reduced stiffness) showed a 20 ± 6% attenuated hypertrophy response, whereas the TtnΔIAjxn mice (increased stiffness) showed an 18 ± 8% exaggerated response after UDD. Thus, muscle hypertrophy scales with titin stiffness. Protein expression analysis revealed that titin‐binding proteins implicated previously in muscle trophicity were induced during UDD, MARP1 & 2, FHL1, and MuRF1.ConclusionsTitin functions as a mechanosensor that regulates muscle trophicity.

A1603P and K1617del, Mutations in β-Cardiac Myosin Heavy Chain that Cause Laing Early-Onset Distal Myopathy, Affect Secondary Structure and Filament Formation In Vitro and In Vivo

Over 20 mutations in β-cardiac myosin heavy chain (β-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase–light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged β-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged β-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease.

Mechanical responses of the mechanosensitive unstructured domains in cardiac titin.

Titin is one of the three main filaments in cardiac sarcomere. Besides a chain of Ig domains, cardiac titin also contains a proline (P), glutamate (E), valine (V), lysine (K) (PEVK) domain and a cardiac-specific N2B domain, both are largely unstructured. While they are believed to be involved in the elastic (PEVK and N2B) and the trophic (N2B) functions of the heart, their mechanical responses in physiological level of forces remains poorly understood. In order to gain understanding on their mechanical responses, we used magnetic tweezers to investigate their force responses from 1 to 30pN. We confirmed that in vitro the PEVK domain is intrinsically disordered within the force range. Surprisingly, we discovered a mechanosensitive folded element in the disordered region of N2B, ∼84 amino acids in length, which has a large folding energy of approximately -10 kB T. Based on the force responses of PEVK and N2B domains, as well as an approximated force-dependent unfolding and refolding rates of titin Ig domains, we show that the tension in cardiac titin fluctuates within 5pN during cardiac contraction and extension cycle using Gillespie simulation algorithm. Exceptionally, the simulation shows that deletion of N2B domain results in 10-fold increase in peak force. Our results highlight a critical role that N2B may potentially play in regulating tension on cardiac titin. The study provides new insights into the tension regulatory role of unstructured domains in the elastic function of the heart, which has broad implication in diastolic dysfunction and cardiac trophic mechanisms. In addition, the method can be applied to probing other unstructured mechanosensitive proteins/domains.

RBM20, a potential target for treatment of cardiomyopathy via titin isoform switching

Cardiomyopathy, also known as heart muscle disease, is an unfavorable condition leading to alterations in myocardial contraction and/or impaired ability of ventricular filling. The onset and development of cardiomyopathy have not currently been well defined. Titin is a giant multifunctional sarcomeric filament protein that provides passive stiffness to cardiomyocytes and has been implicated to play an important role in the origin and development of cardiomyopathy and heart failure. Titin-based passive stiffness can be mainly adjusted by isoform switching and post-translational modifications in the spring regions. Recently, genetic mutations of TTN have been identified that can also contribute to variable passive stiffness, though the detailed mechanisms remain unclear. In this review, we will discuss titin isoform switching as it relates to alternative splicing during development stages and differences between species and muscle types. We provide an update on the regulatory mechanisms of TTN splicing controlled by RBM20 and cover the roles of TTN splicing in adjusting the diastolic stiffness and systolic compliance of the healthy and the failing heart. Finally, this review attempts to provide future directions for RBM20 as a potential target for pharmacological intervention in cardiomyopathy and heart failure.

Titin Gene and Protein Functions in Passive and Active Muscle.

The thin and thick filaments of muscle sarcomeres are interconnected by the giant protein titin, which is a scaffolding filament, signaling platform, and provider of passive tension and elasticity in myocytes. This review summarizes recent insight into the mechanisms behind how titin gene mutations cause hereditary cardiomyopathy and how titin protein is mechanically active in skeletal and cardiac myocytes. A main theme is the evolving role of titin as a modulator of contraction. Topics include strain-sensing via titin in the sarcomeric A-band as the basis for length-dependent activation, titin elastic recoil and refolding of titin domains as an energy source, and Ca2+-dependent stiffening of titin stretched during eccentric muscle contractions. Findings suggest that titin stiffness is a principal regulator of the contractile behavior of striated muscle. Physiological or pathological changes to titin stiffness therefore affect contractility. Taken together, titin emerges as a linker element between passive and active myocyte properties.

Tropomyosin movement is described by a quantitative high-resolution model of X-ray diffraction of contracting muscle.

Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.