Padrón-Craig Labhttp://hdl.handle.net/20.500.14038/2012024-03-28T12:26:42Z2024-03-28T12:26:42ZCryo-EM structure of the human cardiac myosin filamentDutta, DebabrataNguyen, VuCampbell, Kenneth SPadrón, RaúlCraig, Rogerhttp://hdl.handle.net/20.500.14038/527702023-12-04T15:23:16Z2023-11-01T00:00:00ZCryo-EM structure of the human cardiac myosin filament
Dutta, Debabrata; Nguyen, Vu; Campbell, Kenneth S; Padrón, Raúl; Craig, Roger
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.
2023-11-01T00:00:00ZThe structural and functional integrities of porcine myocardium are mostly preserved by cryopreservationMa, WeikangLee, Kyoung HwanDelligatti, Christine EDavis, M ThereseZheng, YahanGong, HenryKirk, Jonathan ACraig, RogerIrving, Thomashttp://hdl.handle.net/20.500.14038/524452023-08-22T05:40:17Z2023-07-03T00:00:00ZThe structural and functional integrities of porcine myocardium are mostly preserved by cryopreservation
Ma, Weikang; Lee, Kyoung Hwan; Delligatti, Christine E; Davis, M Therese; Zheng, Yahan; Gong, Henry; Kirk, Jonathan A; Craig, Roger; Irving, Thomas
Structural and functional studies of heart muscle are important to gain insights into the physiological bases of cardiac muscle contraction and the pathological bases of heart disease. While fresh muscle tissue works best for these kinds of studies, this is not always practical to obtain, especially for heart tissue from large animal models and humans. Conversely, tissue banks of frozen human hearts are available and could be a tremendous resource for translational research. It is not well understood, however, how liquid nitrogen freezing and cryostorage may impact the structural integrity of myocardium from large mammals. In this study, we directly compared the structural and functional integrity of never-frozen to previously frozen porcine myocardium to investigate the consequences of freezing and cryostorage. X-ray diffraction measurements from hydrated tissue under near-physiological conditions and electron microscope images from chemically fixed porcine myocardium showed that prior freezing has only minor effects on structural integrity of the muscle. Furthermore, mechanical studies similarly showed no significant differences in contractile capabilities of porcine myocardium with and without freezing and cryostorage. These results demonstrate that liquid nitrogen preservation is a practical approach for structural and functional studies of myocardium.
2023-07-03T00:00:00ZCryo-EM structure of the human cardiac myosin filament [preprint]Dutta, DebabrataNguyen, VuCampbell, Kenneth SPadrón, RaúlCraig, Rogerhttp://hdl.handle.net/20.500.14038/520212023-11-21T15:52:25Z2023-04-12T00:00:00ZCryo-EM structure of the human cardiac myosin filament [preprint]
Dutta, Debabrata; Nguyen, Vu; Campbell, Kenneth S; Padrón, Raúl; Craig, Roger
Pumping of the heart is powered by filaments of the motor protein myosin, which 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 assembly 1 . 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 years 2 . Here, we have solved the structure of the main (cMyBP-C-containing) region of the human cardiac filament to 6 Å resolution by cryo-EM. The reconstruction reveals the architecture of titin and cMyBP-C for the first time, and shows how myosin's motor domains (heads) form 3 different types of motif (providing functional flexibility), which interact with each other and with specific domains of titin and cMyBP-C to dictate filament architecture and regulate function. A novel packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps generate the cardiac super-relaxed state 3 , how titin and cMyBP-C may contribute to length-dependent activation 4 , and how mutations in myosin and cMyBP-C might disrupt interactions, causing disease 5, 6 . A similar structure is likely in vertebrate skeletal myosin filaments. 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.
This article is a preprint. Preprints are preliminary reports of work that have not been certified by peer review.
2023-04-12T00:00:00ZDilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosinRasicci, David VTiwari, PrinceBodt, Skylar M LDesetty, RohiniSadler, Fredrik RSivaramakrishnan, SivarajCraig, RogerYengo, Christopher Mhttp://hdl.handle.net/20.500.14038/514162022-12-09T11:03:59Z2022-11-24T00:00:00ZDilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin
Rasicci, David V; Tiwari, Prince; Bodt, Skylar M L; Desetty, Rohini; Sadler, Fredrik R; Sivaramakrishnan, Sivaraj; Craig, Roger; Yengo, Christopher M
The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.
2022-11-24T00:00:00ZVariants of the myosin interacting-heads motifPadrón, RaúlDutta, DebabrataCraig, Rogerhttp://hdl.handle.net/20.500.14038/514202022-12-09T11:04:08Z2022-11-08T00:00:00ZVariants of the myosin interacting-heads motif
Padrón, Raúl; Dutta, Debabrata; Craig, Roger
Under relaxing conditions, the two heads of myosin II interact with each other and with the proximal part (S2) of the myosin tail, establishing the interacting-heads motif (IHM), found in myosin molecules and thick filaments of muscle and nonmuscle cells. The IHM is normally thought of as a single, unique structure, but there are several variants. In the simplest ("canonical") IHM, occurring in most relaxed thick filaments and in heavy meromyosin, the interacting heads bend back and interact with S2, and the motif lies parallel to the filament surface. In one variant, occurring in insect indirect flight muscle, there is no S2-head interaction and the motif is perpendicular to the filament. In a second variant, found in smooth and nonmuscle single myosin molecules in their inhibited (10S) conformation, S2 is shifted ∼20 Å from the canonical form and the tail folds twice and wraps around the interacting heads. These molecule and filament IHM variants have important energetic and pathophysiological consequences. (1) The canonical motif, with S2-head interaction, correlates with the super-relaxed (SRX) state of myosin. The absence of S2-head interaction in insects may account for the lower stability of this IHM and apparent absence of SRX in indirect flight muscle, contributing to the quick initiation of flight in insects. (2) The ∼20 Å shift of S2 in 10S myosin molecules means that S2-head interactions are different from those in the canonical IHM. This variant therefore cannot be used to analyze the impact of myosin mutations on S2-head interactions that occur in filaments, as has been proposed. It can be used, instead, to analyze the structural impact of mutations in smooth and nonmuscle myosin.
2022-11-08T00:00:00ZInteracting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscleKoubassova, Natalia A.Tsaturyan, Andrey K.Bershitsky, Sergey Y.Ferenczi, Michael A.Padrón, RaúlCraig, Roger W.http://hdl.handle.net/20.500.14038/486152023-12-04T15:39:43Z2022-03-19T00:00:00ZInteracting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle
Koubassova, Natalia A.; Tsaturyan, Andrey K.; Bershitsky, Sergey Y.; Ferenczi, Michael A.; Padrón, Raúl; Craig, Roger W.
Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This "interacting-heads motif" (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3-4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3-4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.
2022-03-19T00:00:00ZStructural basis of the super- and hyper-relaxed states of myosin IICraig, Roger W.Padrón, Raúlhttp://hdl.handle.net/20.500.14038/485712023-12-04T15:41:36Z2021-12-10T00:00:00ZStructural basis of the super- and hyper-relaxed states of myosin II
Craig, Roger W.; Padrón, Raúl
Super-relaxation is a state of muscle thick filaments in which ATP turnover by myosin is much slower than that of myosin II in solution. This inhibited state, in equilibrium with a faster (relaxed) state, is ubiquitous and thought to be fundamental to muscle function, acting as a mechanism for switching off energy-consuming myosin motors when they are not being used. The structural basis of super-relaxation is usually taken to be a motif formed by myosin in which the two heads interact with each other and with the proximal tail forming an interacting-heads motif, which switches the heads off. However, recent studies show that even isolated myosin heads can exhibit this slow rate. Here, we review the role of head interactions in creating the super-relaxed state and show how increased numbers of interactions in thick filaments underlie the high levels of super-relaxation found in intact muscle. We suggest how a third, even more inhibited, state of myosin (a hyper-relaxed state) seen in certain species results from additional interactions involving the heads. We speculate on the relationship between animal lifestyle and level of super-relaxation in different species and on the mechanism of formation of the super-relaxed state. We also review how super-relaxed thick filaments are activated and how the super-relaxed state is modulated in healthy and diseased muscles.
2021-12-10T00:00:00ZFast skeletal myosin-binding protein-C regulates fast skeletal muscle contractionSong, TaejeongMcNamara, James W.Ma, WeikangLandim-Vieira, MaiconLee, KyounghwanMartin, Lisa A.Heiny, Judith A.Lorenz, John N.Craig, Roger W.Pinto, Jose RenatoIrving, ThomasSadayappan, Sakthivelhttp://hdl.handle.net/20.500.14038/485332022-12-29T13:42:45Z2021-04-27T00:00:00ZFast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction
Song, Taejeong; McNamara, James W.; Ma, Weikang; Landim-Vieira, Maicon; Lee, Kyounghwan; Martin, Lisa A.; Heiny, Judith A.; Lorenz, John N.; Craig, Roger W.; Pinto, Jose Renato; Irving, Thomas; Sadayappan, Sakthivel
Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2(-/-)) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2(-/-) mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2(-/-) mice. Small-angle X-ray diffraction of C2(-/-) EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2(-/-) EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca(2+-)activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2(-/-) mice. Finally, C2(-/-) muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.
2021-04-27T00:00:00ZAmino terminus of cardiac myosin binding protein-C regulates cardiac contractilityLynch, Thomas L. 4thLee, KyounghwanCraig, Roger W.Sadayappan, Sakthivelhttp://hdl.handle.net/20.500.14038/485072022-12-29T13:49:53Z2021-03-26T00:00:00ZAmino terminus of cardiac myosin binding protein-C regulates cardiac contractility
Lynch, Thomas L. 4th; Lee, Kyounghwan; Craig, Roger W.; Sadayappan, Sakthivel
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C(C0-C1f)), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.
<p>Full author list omitted for brevity. For the full list of authors, see article.</p>
2021-03-26T00:00:00ZRelaxed tarantula skeletal muscle has two ATP energy-saving mechanismsMa, WeikangDuno-Miranda, SebastianIrving, ThomasCraig, Roger W.Padrón, Raúlhttp://hdl.handle.net/20.500.14038/484852023-12-04T15:40:54Z2021-03-01T00:00:00ZRelaxed tarantula skeletal muscle has two ATP energy-saving mechanisms
Ma, Weikang; Duno-Miranda, Sebastian; Irving, Thomas; Craig, Roger W.; Padrón, Raúl
Myosin molecules in the relaxed thick filaments of striated muscle have a helical arrangement in which the heads of each molecule interact with each other, forming the interacting-heads motif (IHM). In relaxed mammalian skeletal muscle, this helical ordering occurs only at temperatures > 20 degrees C and is disrupted when temperature is decreased. Recent x-ray diffraction studies of live tarantula skeletal muscle have suggested that the two myosin heads of the IHM (blocked heads [BHs] and free heads [FHs]) have very different roles and dynamics during contraction. Here, we explore temperature-induced changes in the BHs and FHs in relaxed tarantula skeletal muscle. We find a change with decreasing temperature that is similar to that in mammals, while increasing temperature induces a different behavior in the heads. At 22.5 degrees C, the BHs and FHs containing ADP.Pi are fully helically organized, but they become progressively disordered as temperature is lowered or raised. Our interpretation suggests that at low temperature, while the BHs remain ordered the FHs become disordered due to transition of the heads to a straight conformation containing Mg.ATP. Above 27.5 degrees C, the nucleotide remains as ADP.Pi, but while BHs remain ordered, half of the FHs become progressively disordered, released semipermanently at a midway distance to the thin filaments while the remaining FHs are docked as swaying heads. We propose a thermosensing mechanism for tarantula skeletal muscle to explain these changes. Our results suggest that tarantula skeletal muscle thick filaments, in addition to having a superrelaxation-based ATP energy-saving mechanism in the range of 8.5-40 degrees C, also exhibit energy saving at lower temperatures ( < 22.5 degrees C), similar to the proposed refractory state in mammals.
2021-03-01T00:00:00Z