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Malcolm Irving - One of the best experts on this subject based on the ideXlab platform.
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dependence of Thick Filament structure in relaxed mammalian skeletal muscle on temperature and interFilament spacing
The Journal of General Physiology, 2021Co-Authors: Marco Caremani, Thomas C Irving, Malcolm Irving, Gabriella Piazzesi, Marco Linari, Massimo Reconditi, Vincenzo Lombardi, Luca Fusi, Theyencheri Narayanan, Elisabetta BrunelloAbstract:Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin Filaments and myosin-containing Thick Filaments, but myosin-based regulation is unlikely to be preserved after Thick Filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the Thick Filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether Thick Filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the Thick Filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar Filament shows that all zones of the Filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the Filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on Thick Filament structure, disrupts the helical order of the myosin motors, and they move out from the Filament backbone. Compression of the Filament lattice of demembranated fibers by 5% Dextran, which restores interFilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the Filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick Filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the Thick Filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.
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site specific phosphorylation of myosin binding protein c coordinates thin and Thick Filament activation in cardiac muscle
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Saraswathi Ponnam, Yinbiao Sun, Malcolm Irving, Ivanka Sevrieva, Thomas KampourakisAbstract:The heart’s response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Ce (PKCe), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C’s multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C’s N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCe, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C’s regulatory N-terminal domains in defined phosphorylation states for protein–protein interaction studies with isolated cardiac native thin Filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing Thick Filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the Thick Filaments and on troponin C in the thin Filaments to monitor structural changes in the myoFilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.
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omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin Filament
The Journal of Physiology, 2018Co-Authors: Thomas Kampourakis, Xuemeng Zhang, Yinbiao Sun, Malcolm IrvingAbstract:Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the Thick Filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the Thick Filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the Thick Filament OFF state and inhibiting acto-myosin ATPase. Thick Filament regulation is a promising target for novel therapeutics in heart disease. Abstract Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin Filaments that allows the head or motor domains of myosin from the Thick Filaments to bind to them and induce Filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the Thick Filaments, although the molecular mechanisms underlying Thick Filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the Thick Filaments and on troponin C in the thin Filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the Thick Filament in which myosin head domains are more parallel to the Filament axis. In contrast, OM stabilizes the ON state of the Thick Filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and Filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and Thick Filaments.
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regulation of contraction by the Thick Filaments in skeletal muscle
Biophysical Journal, 2017Co-Authors: Malcolm IrvingAbstract:Contraction of skeletal muscle cells is initiated by a well-known signaling pathway. An action potential in a motor nerve triggers an action potential in a muscle cell membrane, a transient increase of intracellular calcium concentration, binding of calcium to troponin in the actin-containing thin Filaments, and a structural change in the thin Filaments that allows myosin motors from the Thick Filaments to bind to actin and generate force. This calcium/thin Filament mediated pathway provides the "START" signal for contraction, but it is argued that the functional response of the muscle cell, including the speed of its contraction and relaxation, adaptation to the external load, and the metabolic cost of contraction is largely determined by additional mechanisms. This review considers the role of the Thick Filaments in those mechanisms, and puts forward a paradigm for the control of contraction in skeletal muscle in which both the Thick and thin Filaments have a regulatory function. The OFF state of the Thick Filament is characterized by helical packing of most of the myosin head or motor domains on the Thick Filament surface in a conformation that makes them unavailable for actin binding or ATP hydrolysis, although a small fraction of the myosin heads are constitutively ON. The availability of the majority fraction of the myosin heads for contraction is controlled in part by the external load on the muscle, so that these heads only attach to actin and hydrolyze ATP when they are required. This phenomenon seems to be the major determinant of the well-known force-velocity relationship of muscle, and controls the metabolic cost of contraction. The regulatory state of the Thick Filament also seems to control the dynamics of both muscle activation and relaxation.
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Thick Filament mechano sensing is a calcium independent regulatory mechanism in skeletal muscle
Nature Communications, 2016Co-Authors: Luca Fusi, Elisabetta Brunello, Ziqian Yan, Malcolm IrvingAbstract:Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin Filaments is controlled by the stress in the myosin-containing Thick Filaments. Those results suggested that Thick Filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin Filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of Thick Filament activation depend on Thick Filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by Thick Filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway. Recent data suggest that muscle contraction is regulated by Thick Filament mechano-sensing in addition to the well-known thin Filament-mediated calcium signalling pathway. Here the authors provide direct evidence that myosin activation in skeletal muscle is controlled by Thick Filament stress independently of calcium.
Kenneth A Taylor - One of the best experts on this subject based on the ideXlab platform.
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coupling between myosin head conformation and the Thick Filament backbone structure
Journal of Structural Biology, 2017Co-Authors: Dianne W Taylor, Robert J Edwards, Kenneth A TaylorAbstract:The recent high-resolution structure of the Thick Filament from Lethocerus asynchronous flight muscle shows aspects of Thick Filament structure never before revealed that may shed some light on how striated muscles function. The phenomenon of stretch activation underlies the function of asynchronous flight muscle. It is most highly developed in flight muscle, but is also observed in other striated muscles such as cardiac muscle. Although stretch activation is likely to be complex, involving more than a single structural aspect of striated muscle, the Thick Filament itself, would be a prime site for regulatory function because it must bear all of the tension produced by both its associated myosin motors and any externally applied force. Here we show the first structural evidence that the arrangement of myosin heads within the interacting heads motif is coupled to the structure of the Thick Filament backbone. We find that a change in helical angle of 0.16° disorders the blocked head preferentially within the Lethocerus interacting heads motif. This observation suggests a mechanism for how tension affects the dynamics of the myosin heads leading to a detailed hypothesis for stretch activation and shortening deactivation, in which the blocked head preferentially binds the thin Filament followed by the free head when force production occurs.
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the structure of the relaxed Thick Filaments from lethocerus asynchronous flight muscle
Biophysical Journal, 2016Co-Authors: Dianne W Taylor, Michael K. Reedy, Robert J Perzedwards, Kenneth A TaylorAbstract:The structure of relaxed muscle Thick Filaments is a key element for understanding muscle physiology and the effect of mutations on muscle function. Previous Thick Filament structures by 3D-EM resolved individual myosin heads and revealed an interacting head motif (IHM) first identified in smooth muscle myosin. A later model based on X-ray fiber diffraction of Lethocerus flight muscle showed a different, intermolecular, interaction between myosin heads. Almost nothing is known about the structure of the long α-helical coiled-coil myosin rod in Thick Filaments. Here we show the relaxed structure of Lethocerus Thick Filaments by cryoEM at sufficient resolution to reveal the relative placement of individual myosin heads at ∼20 A resolution, as well as the myosin rod α-helices which can be traced from the head-rod junction all the way to their C-termini at a resolution of 5.5A. In contrast to the model suggested by X-ray diffraction, we find an IHM somewhat different from IHMs of other relaxed Thick Filaments. The myosin rod follows a tortuous path from the outside of the Thick Filament to the inside across a region we call the myosin rod annulus. The pitch of the rod coiled-coil is variable with long pitch regions in the approximate location of skip residues. Surprisingly, threading their way among the forest of myosin rods are four copies, matching the 4-fold symmetry of the Filament, of four polypeptide chains. Their presence in this location could modulate the mechanical properties of the myosin backbone. Our results demonstrate the ubiquity of the IHM in relaxed myosin Filaments but with a variation that might shed light on the mechanism of stretch activation and shortening deactivation. Supported by NIH and AHA.
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the structure of the relaxed Thick Filaments from lethocerus asynchronous flight muscle implications for stretch activation
Biophysical Journal, 2016Co-Authors: Kenneth A Taylor, Michael K. Reedy, Dianne W Taylor, Robert J PerzedwardsAbstract:A general property of striated muscles, particularly rhythmically beating cardiac and fibrillar insect flight muscles, is a delayed tension rise following a rapid stretch, known as stretch activation. The mechanism of stretch activation remains poorly understood. Proposed hypotheses include the match/mismatch between myosin origins and actin targets, changes to thin Filament structure via direct connections between Thick and thin Filaments, or changes in the Thick Filament structure via connecting Filaments. Stretch activation is highly refined in Lethocerus. The cryoEM structure of relaxed Lethocerus Thick Filaments shows myosin heads adopting the same interacting head motif (IHM) seen in smooth muscle myosin. However, the IHM position differs from previously described Thick Filaments in the absence of nearest-neighbor contacts between IHMs. Moreover, myosin's actin-binding region points away from actin; and instead, myosin's SH3 domain is closest to the thin Filament. The presence of extra proteins threaded among the myosin rods may loosen the rod packing density, although no high-resolution structures of coiled-coils of this length exist for comparison. Formation of the IHM is correlated with Filament instability in non-muscle and smooth muscle Filaments. Tama et al. (J Mol. Biol. 345, 837 (2005)) hypothesized that the IHM distorts the coiled-coil rod thereby loosening the packing and destabilizing the Filaments. We reverse that logic, and suggest that lack of nearest neighbor contacts between IHMs, loose myosin rod packing, and the presence of proteins separating the myosin rods within the Lethocerus Thick Filament serve to poise the IHM on the edge of stability, sensitizing it to stretching of the myosin Filament that affects the rod packing. The attraction of this mechanism is simultaneous activation of all heads and continued activation until tension drops. Supported by NIH and AHA.
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electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled s2 domain
Journal of Molecular Biology, 2006Co-Authors: Jun Liu, Michael K. Reedy, Mary C Reedy, Hanspeter Winkler, Carmen Lucaveche, Yifan Cheng, Kenneth A TaylorAbstract:Subfragment 2 (S2), the segment that links the two myosin heads to the Thick Filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interFilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the Thick Filament (except near the tapered ends) is approximately 10 nm, or 16% of S2's total length, with an angular range encompassing 90 degrees axially and 120 degrees azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head-head interactions in IFM relaxed Thick Filaments.
Thomas C Irving - One of the best experts on this subject based on the ideXlab platform.
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dependence of Thick Filament structure in relaxed mammalian skeletal muscle on temperature and interFilament spacing
The Journal of General Physiology, 2021Co-Authors: Marco Caremani, Thomas C Irving, Malcolm Irving, Gabriella Piazzesi, Marco Linari, Massimo Reconditi, Vincenzo Lombardi, Luca Fusi, Theyencheri Narayanan, Elisabetta BrunelloAbstract:Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin Filaments and myosin-containing Thick Filaments, but myosin-based regulation is unlikely to be preserved after Thick Filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the Thick Filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether Thick Filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the Thick Filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar Filament shows that all zones of the Filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the Filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on Thick Filament structure, disrupts the helical order of the myosin motors, and they move out from the Filament backbone. Compression of the Filament lattice of demembranated fibers by 5% Dextran, which restores interFilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the Filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick Filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the Thick Filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.
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the myosin interacting heads motif present in live tarantula muscle explains tetanic and posttetanic phosphorylation mechanisms
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Raul Padron, Thomas C Irving, Sebastian Dunomiranda, Natalia A Koubassova, Kyounghwan Lee, Antonio Pinto, Lorenzo Alamo, Pura Bolanos, Andrey K Tsaturyan, Roger CraigAbstract:Striated muscle contraction involves sliding of actin thin Filaments along myosin Thick Filaments, controlled by calcium through thin Filament activation. In relaxed muscle, the two heads of myosin interact with each other on the Filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing—proposed in vertebrate muscle—is not of primary importance in tarantula. Rather, Thick Filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for Thick Filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca2+-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.
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Thick Filament extensibility in intact skeletal muscle
Biophysical Journal, 2018Co-Authors: Henry Gong, Henk Granzier, Balazs Kiss, Eun Jeong Lee, Thomas C IrvingAbstract:Abstract MyoFilament extensibility is a key structural parameter for interpreting myosin cross-bridge kinetics in striated muscle. Previous studies reported much higher Thick-Filament extensibility at low tension than the better-known and commonly used values at high tension, but in interpreting mechanical studies of muscle, a single value for Thick-Filament extensibility has usually been assumed. Here, we established the complete Thick-Filament force-extension curve from actively contracting, intact vertebrate skeletal muscle. To access a wide range of tetanic forces, the myosin inhibitor blebbistatin was used to induce low tetanic forces in addition to the higher tensions obtained from tetanic contractions of the untreated muscle. We show that the force/extensibility curve of the Thick Filament is nonlinear, so assuming a single value for Thick-Filament extensibility at all force levels is not justified. We also show that independent of whether tension is generated passively by sarcomere stretch or actively by cross-bridges, the Thick-Filament extensibility is nonlinear. Myosin head periodicity, however, only changes when active tension is generated under calcium-activated conditions. The nonlinear Thick-Filament force-extension curve in skeletal muscle, therefore, reflects a purely passive response to either titin-based force or actomyosin-based force, and it does not include a Thick-Filament activation mechanism. In contrast, the transition of myosin head periodicity to an active configuration appears to only occur in response to increased active force when calcium is present.
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Modeling crossbridge structure of the frog skeletal Thick myosin Filament.
2013Co-Authors: Kanji Oshima, Thomas C Irving, Yasunobu Sugimoto, Katsuzo WakabayashiAbstract:(A) Distributions of the perturbed region (blue), the regular region (green) of myosin crossbridge arrays and the C-protein region (red) along the Thick Filament in resting frog muscles. (B) Overview of the Thick Filament model including C-proteins. Members of paired heads constituting a single myosin crossbridge are distinguished by a red or purple color. C-protein is bound to the Thick Filament backbone every at the level of crown 1. Eleven domains of a C-protein having a molecular weight of ∼130 kDa are shown by white spheres, each having a diameter of 4 nm [18]. The backbone of the myosin Filament is shown as a gray cylinder. Upper, a top view and lower, a side view. (C) Parameters describing the arrangement of a two-headed myosin crossbridge in terms of a 68-sphere model on the Filament backbone in ADP and Pi-bound state. The z-axis is parallel to the Filament axis. φ is the rotation angle of crossbridges about the Filament axis and ε is the rotation angle of the two heads of a crossbridge about the z-axis. rh is the average of helical radii of myosin crossbridges. Paired heads of a single crossbridge and the backbone are denoted as in (B).
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myosin binding protein c phosphorylation is the principal mediator of protein kinase a effects on Thick Filament structure in myocardium
Journal of Molecular and Cellular Cardiology, 2012Co-Authors: Brett A Colson, Tanya Bekyarova, Thomas C Irving, Daniel P. Fitzsimons, Mohamed Abdalla, Jitandrakumar R Patel, Peter P Chen, Carl W Tong, Richard L MossAbstract:Abstract Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myoFilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the Thick Filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca2 +-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.
Henk Granzier - One of the best experts on this subject based on the ideXlab platform.
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Thick Filament extensibility in intact skeletal muscle
Biophysical Journal, 2018Co-Authors: Henry Gong, Henk Granzier, Balazs Kiss, Eun Jeong Lee, Thomas C IrvingAbstract:Abstract MyoFilament extensibility is a key structural parameter for interpreting myosin cross-bridge kinetics in striated muscle. Previous studies reported much higher Thick-Filament extensibility at low tension than the better-known and commonly used values at high tension, but in interpreting mechanical studies of muscle, a single value for Thick-Filament extensibility has usually been assumed. Here, we established the complete Thick-Filament force-extension curve from actively contracting, intact vertebrate skeletal muscle. To access a wide range of tetanic forces, the myosin inhibitor blebbistatin was used to induce low tetanic forces in addition to the higher tensions obtained from tetanic contractions of the untreated muscle. We show that the force/extensibility curve of the Thick Filament is nonlinear, so assuming a single value for Thick-Filament extensibility at all force levels is not justified. We also show that independent of whether tension is generated passively by sarcomere stretch or actively by cross-bridges, the Thick-Filament extensibility is nonlinear. Myosin head periodicity, however, only changes when active tension is generated under calcium-activated conditions. The nonlinear Thick-Filament force-extension curve in skeletal muscle, therefore, reflects a purely passive response to either titin-based force or actomyosin-based force, and it does not include a Thick-Filament activation mechanism. In contrast, the transition of myosin head periodicity to an active configuration appears to only occur in response to increased active force when calcium is present.
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the giant protein titin regulates the length of the striated muscle Thick Filament
Nature Communications, 2017Co-Authors: Paola Tonino, Balazs Kiss, Siegfried Labeit, Mei Methawasin, John E Smith, Josh Strom, Justin Kolb, Henk GranzierAbstract:Foundation Leducq [TNE-13CVD04]; National Institutes of Health [HL062881, HL118524, HL115988]
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deleting titin s i band a band junction reveals critical roles for titin in biomechanical sensing and cardiac function
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Henk Granzier, Carol C Gregorio, Kirk R Hutchinson, Paola Tonino, Mei Methawasin, Rebecca E Slater, Mathew M Bull, Chandra Saripalli, Christopher T Pappas, John E SmithAbstract:Titin, the largest protein known, forms a giant Filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the Thick Filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (∼ 40-fold higher) but low, relative to that of the myosin-based Thick Filament (∼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the Thick Filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for Thick Filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.
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Thick Filament strain and interFilament spacing in passive muscle effect of titin based passive tension
Biophysical Journal, 2011Co-Authors: Thomas C Irving, Tanya Bekyarova, Gerrie P Farman, Norio Fukuda, Henk GranzierAbstract:We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myoFilament lattice spacing was measured in the A-band (d1,0) and Z-disk (dZ) regions of the sarcomere. The axial spacing of the Thick-Filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I1,1/I1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial Thick-Filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I1,1/I1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to Thick-Filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the Thick-Filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that Thick Filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.
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Passive tension of rat skeletal soleus muscle fibers: effects of unloading conditions.
Journal of Applied Physiology, 2002Co-Authors: Thierry Toursel, Laurence Stevens, Henk Granzier, Yvonne MounierAbstract:In this work we studied changes in passive elastic properties of rat soleus muscle fibers subjected to 14 days of hindlimb unloading (HU). For this purpose, we investigated the titin isoform expression in soleus muscles, passive tension-fiber strain relationships of single fibers, and the effects of the Thick Filament depolymerization on passive tension development. The myosin heavy chain composition was also measured for all fibers studied. Despite a slow-to-fast transformation of the soleus muscles on the basis of their myosin heavy chain content, no modification in the titin isoform expression was detected after 14 days of HU. However, the passive tension-fiber strain relationships revealed that passive tension of both slow and fast HU soleus fibers increased less steeply with sarcomere length than that of control fibers. Gel analysis suggested that this result could be explained by a decrease in the amount of titin in soleus muscle after HU. Furthermore, the Thick Filament depolymerization was found to similarly decrease passive tension in control and HU soleus fibers. Taken together, these results suggested that HU did not change titin isoform expression in the soleus muscle, but rather modified muscle stiffness by decreasing the amount of titin.
Elisabetta Brunello - One of the best experts on this subject based on the ideXlab platform.
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dependence of Thick Filament structure in relaxed mammalian skeletal muscle on temperature and interFilament spacing
The Journal of General Physiology, 2021Co-Authors: Marco Caremani, Thomas C Irving, Malcolm Irving, Gabriella Piazzesi, Marco Linari, Massimo Reconditi, Vincenzo Lombardi, Luca Fusi, Theyencheri Narayanan, Elisabetta BrunelloAbstract:Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin Filaments and myosin-containing Thick Filaments, but myosin-based regulation is unlikely to be preserved after Thick Filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the Thick Filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether Thick Filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the Thick Filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar Filament shows that all zones of the Filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the Filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on Thick Filament structure, disrupts the helical order of the myosin motors, and they move out from the Filament backbone. Compression of the Filament lattice of demembranated fibers by 5% Dextran, which restores interFilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the Filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick Filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the Thick Filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.
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Thick Filament mechano sensing is a calcium independent regulatory mechanism in skeletal muscle
Nature Communications, 2016Co-Authors: Luca Fusi, Elisabetta Brunello, Ziqian Yan, Malcolm IrvingAbstract:Recent X-ray diffraction studies on actively contracting fibres from skeletal muscle showed that the number of myosin motors available to interact with actin-containing thin Filaments is controlled by the stress in the myosin-containing Thick Filaments. Those results suggested that Thick Filament mechano-sensing might constitute a novel regulatory mechanism in striated muscles that acts independently of the well-known thin Filament-mediated calcium signalling pathway. Here we test that hypothesis using probes attached to the myosin regulatory light chain in demembranated muscle fibres. We show that both the extent and kinetics of Thick Filament activation depend on Thick Filament stress but are independent of intracellular calcium concentration in the physiological range. These results establish direct control of myosin motors by Thick Filament mechano-sensing as a general regulatory mechanism in skeletal muscle that is independent of the canonical calcium signalling pathway. Recent data suggest that muscle contraction is regulated by Thick Filament mechano-sensing in addition to the well-known thin Filament-mediated calcium signalling pathway. Here the authors provide direct evidence that myosin activation in skeletal muscle is controlled by Thick Filament stress independently of calcium.
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force generation by skeletal muscle is controlled by mechanosensing in myosin Filaments
Nature, 2015Co-Authors: Marco Linari, Gabriella Piazzesi, Marco Caremani, Massimo Reconditi, Vincenzo Lombardi, Luca Fusi, Elisabetta Brunello, Theyencheri Narayanan, Malcolm IrvingAbstract:Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin Filaments, which permits the binding of myosin motors from the neighbouring Thick Filaments to drive Filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-Filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the Thick Filament. The resting (switched 'OFF') structure of the Thick Filament is characterized by helical tracks of myosin motors on the Filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside Thick-Filament control. At higher load, these motors generate sufficient Thick-Filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the Thick Filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.
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the contributions of Filaments and cross bridges to sarcomere compliance in skeletal muscle
The Journal of Physiology, 2014Co-Authors: Elisabetta Brunello, Malcolm Irving, Gabriella Piazzesi, Marco Caremani, Marco Linari, Vincenzo Lombardi, Theyencheri Narayanan, Luca Melli, Manuel Fernandezmartinez, Massimo ReconditiAbstract:Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the Thick Filament to form cross-bridges with the actin-containing thin Filament. Following attachment, a structural working stroke in the head pulls the thin Filament towards the centre of the sarcomere, producing, under unloaded conditions, a Filament sliding of ∼ 11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (C(cb)) and Filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined Filament compliance (Cf) of 13.1 ± 1.2 nm MPa(-1), close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa(-1)). C(cb) calculated using these estimates is 0.37 ± 0.12 nm pN(-1), a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN(-1), obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to C(cb) from the myosin tail that joins the head to the Thick Filament is excluded. The low C(cb) value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives Filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle.
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sarcomere length dependence of myosin Filament structure in skeletal muscle fibres of the frog
The Journal of Physiology, 2014Co-Authors: Massimo Reconditi, Malcolm Irving, Marco Linari, Vincenzo Lombardi, Luca Fusi, Elisabetta Brunello, Manuel Fernandez Martinez, Gabriella PiazzesiAbstract:X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the Thick Filament is maintained by an interaction between MyBP-C and the thin Filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the Filaments decreased in proportion to the overlap between Thick and thin Filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the Thick Filaments that do not overlap with thin Filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the Thick Filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin Filaments.
