This collection showcases the journal articles and other publications authored by researchers in the Padrón-Craig Lab at UMass Chan Medical School. The Craig Lab joined the Department of Radiology in 2017 from the former Department of Cell and Developmental Biology.

Recently Published

  • Interacting-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.; Padron, Raul A.; Craig, Roger W. (2022-03-19)
    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.
  • Structural basis of the super- and hyper-relaxed states of myosin II

    Craig, Roger W.; Padron, Raul A. (2021-12-10)
    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.
  • Fast 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; et al. (2021-04-27)
    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.
  • Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

    Lynch, Thomas L. 4th; Lee, Kyounghwan; Craig, Roger W.; Sadayappan, Sakthivel (2021-03-26)
    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.
  • Relaxed tarantula skeletal muscle has two ATP energy-saving mechanisms

    Ma, Weikang; Duno-Miranda, Sebastian; Irving, Thomas; Craig, Roger W.; Padron, Raul A. (2021-03-01)
    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.
  • The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

    Rahmanseresht, Sheema; Lee, Kyoung H.; O'Leary, Thomas S.; McNamara, James W.; Sadayappan, Sakthivel; Robbins, Jeffrey; Warshaw, David M.; Craig, Roger W.; Previs, Michael J. (2021-02-01)
    Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility.
  • Cryo-EM structure of the inhibited (10S) form of myosin II

    Yang, Shixin; Tiwari, Prince; Lee, Kyounghwan; Sato, Osamu; Ikebe, Mitsuo; Padron, Raul A.; Craig, Roger W. (2020-12-02)
    Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape(1). The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients(2,3). The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail(3-7), creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited(7,8). This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells(9-12), which can be activated to form functional filaments as needed(13)-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.
  • The myosin interacting-heads motif present in live tarantula muscle explains tetanic and posttetanic phosphorylation mechanisms

    Padron, Raul; Ma, Weikang; Duno-Miranda, Sebastian; Koubassova, Natalia; Lee, Kyounghwan; Pinto, Antonio; Alamo, Lorenzo; Bolanos, Pura; Tsaturyan, Andrey; Irving, Thomas; et al. (2020-06-02)
    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 Ca(2+)-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.
  • The mesa trail and the interacting heads motif of myosin II

    Woodhead, John Leslie; Craig, Roger (2019-12-13)
    Myosin II molecules in the thick filaments of striated muscle form a structure in which the heads interact with each other and fold back onto the tail. This structure, the "interacting heads motif" (IHM), provides a mechanistic basis for the auto-inhibition of myosin in relaxed thick filaments. Similar IHM interactions occur in single myosin molecules of smooth and nonmuscle cells in the switched-off state. In addition to the interaction between the two heads, which inhibits their activity, the IHM also contains an interaction between the motor domain of one head and the initial part (subfragment 2, S2) of the tail. This is thought to be a crucial anchoring interaction that holds the IHM in place on the thick filament. S2 appears to cross the head at a specific location within a broader region of the motor domain known as the myosin mesa. Here, we show that the positive and negative charge distribution in this part of the mesa is complementary to the charge distribution on S2. We have designated this the "mesa trail" owing to its linear path across the mesa. We studied the structural sequence alignment, the location of charged residues on the surface of myosin head atomic models, and the distribution of surface charge potential along the mesa trail in different types of myosin II and in different species. The charge distribution in both the mesa trail and the adjacent S2 is relatively conserved. This suggests a common basis for IHM formation across different myosin IIs, dependent on attraction between complementary charged patches on S2 and the myosin head. Conservation from mammals to insects suggests that the mesa trail/S2 interaction plays a key role in the inhibitory function of the IHM.
  • Lattice arrangement of myosin filaments correlates with fiber type in rat skeletal muscle

    Ma, Weikang; Lee, Kyounghwan; Yang, Shixin; Irving, Thomas C.; Craig, Roger (2019-12-02)
    The thick (myosin-containing) filaments of vertebrate skeletal muscle are arranged in a hexagonal lattice, interleaved with an array of thin (actin-containing) filaments with which they interact to produce contraction. X-ray diffraction and EM have shown that there are two types of thick filament lattice. In the simple lattice, all filaments have the same orientation about their long axis, while in the superlattice, nearest neighbors have rotations differing by 0 degrees or 60 degrees . Tetrapods (amphibians, reptiles, birds, and mammals) typically have only a superlattice, while the simple lattice is confined to fish. We have performed x-ray diffraction and electron microscopy of the soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat and found that while the EDL has a superlattice as expected, the SOL has a simple lattice. The EDL and SOL of the rat are unusual in being essentially pure fast and slow muscles, respectively. The mixed fiber content of most tetrapod muscles and/or lattice disorder may explain why the simple lattice has not been apparent in these vertebrates before. This is supported by only weak simple lattice diffraction in the x-ray pattern of mouse SOL, which has a greater mix of fiber types than rat SOL. We conclude that the simple lattice might be common in tetrapods. The correlation between fiber type and filament lattice arrangement suggests that the lattice arrangement may contribute to the functional properties of a muscle.
  • The central role of the tail in switching off 10S myosin II activity

    Yang, Shixin; Lee, Kyounghwan; Woodhead, John L.; Sato, Osamu; Ikebe, Mitsuo; Craig, Roger (2019-09-02)
    Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule is not fully understood. We have performed a 3-D reconstruction of negatively stained 10S myosin from smooth muscle in the inhibited state using single-particle analysis. The reconstruction reveals multiple interactions between the tail and the two heads that appear to trap ATP hydrolysis products, block actin binding, hinder head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function could explain the high degree of inhibition of the folded form of myosin thought to underlie its energy-conserving function in cells. The reconstruction also suggests a mechanism for unfolding when myosin is activated by phosphorylation.
  • Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy

    Kuster, Diederik W.D.; Lynch, Thomas L.; Barefield, David Y.; Sivaguru, Mayandi; Kuffel, Gina; Zilliox, Michael J.; Lee, Kyounghwan; Craig, Roger; Namakkal-Soorappan, Rajasekaran; Sadayappan, Sakthivel (2019-05-03)
    AIMS: A 25-base pair (bp) deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CDeltaC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CDeltaC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CDeltaC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. METHODS AND RESULTS: To determine whether expression of cMyBP-CDeltaC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CDeltaC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CDeltaC10mut (heart weight/body weight ratio: 4.43+/-0.11 mg/g nontransgenic (NTG) vs. 5.34+/-0.25 mg/g cMyBP-CDeltaC10mut, P < 0.05). Furthermore, hematoxylin and eosin, Masson's trichrome staining, as well as second harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CDeltaC10mut hearts compared to NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%+/-2.9% NTG vs. 66.4%+/-4.7% cMyBP-CDeltaC10mut; P < 0.05) and early diastolic dysfunction (E/E': 28.7+/-3.7 NTG vs. 46.3+/-8.4 cMyBP-CDeltaC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CDeltaC10mut mice compared to NTG controls (EC50: 3.6+/-0.02 microM NTG vs. 2.90+/-0.01 microM cMyBP-CDeltaC10mut; P < 0.0001). CONCLUSIONS: Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.
  • Getting into the thick (and thin) of it

    Irving, Thomas C.; Craig, Roger (2019-02-21)
    The heart is a highly regulated system in which a combination of mechanisms work together to match cardiac output to the needs of the body. Loss of this regulation characterizes many cardiomyopathies, where muscle is either hypo- or hypercontractile. For many years, x-ray diffraction has been used to study the structural basis of myofilament length-dependent activation...
  • Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca(2+)-dependent manner

    Lin, Brian Leei; Li, Amy; Mun, Ji Young; Previs, Michael J.; Previs, Samantha Beck.; Campbell, Stuart G.; Dos Remedios, Cristobal G.; P. Tombe, Pieter de; Craig, Roger; Warshaw, David M.; et al. (2018-02-08)
    Muscle contraction, which is initiated by Ca(2+), results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca(2+). Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca(2+), addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca(2+) cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca(2+). However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.
  • Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals

    Lee, Kyounghwan; Sulbaran, Guidenn; Yang, Shixin; Mun, Ji Young; Alamo, Lorenzo; Pinto, Antonio; Sato, Osamu; Ikebe, Mitsuo; Liu, Xiong; Korn, Edward D.; et al. (2018-02-01)
    Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca(2+) binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.
  • Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

    Kensler, Robert W.; Craig, Roger; Moss, Richard L. (2017-02-21)
    Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of beta1-adrenergic receptors in myocardium.
  • Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function

    Previs, Michael J.; Mun, Ji Young; Michalek, Arthur J.; Previs, Samantha Beck; Gulick, James; Robbins, Jeffrey; Warshaw, David M.; Craig, Roger (2016-03-22)
    During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state.
  • The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position

    Mun, Ji Young; Kensler, Robert W.; Harris, Samantha P.; Craig, Roger (2016-02-01)
    Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca(2+)-sensitivity by displacement of tropomyosin from its blocked (low Ca(2+)) to its closed (high Ca(2+)) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca(2+)-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca(2+), tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca(2+)-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.
  • An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits

    Wang, Shixia; Woodhead, John L.; Zhao, Fa-Qing; Sulbaran, Guidenn; Craig, Roger (2016-01-01)
    Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size. Here, we describe an approach that improves the resolution in such cases. In filaments with a large axial rise, many segments must be shifted a long distance along the filament axis to match with a reference projection, potentially causing loss of alignment accuracy and hence resolution. In our study of myosin filaments, we overcame this problem by pre-determining the axial positions of myosin head crowns within segments to decrease the alignment error. In addition, homogeneous, well-ordered segments were selected from the raw data set by checking the assigned azimuthal rotation angle of segments in each filament against those expected for perfect helical symmetry. These procedures improved the resolution of the filament reconstruction from 30 A to 13 A. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits.
  • Pacemaker-induced transient asynchrony suppresses heart failure progression

    Lee, Kyounghwan; Craig, Roger; Kass, David A. (2015-12-23)
    Uncoordinated contraction from electromechanical delay worsens heart failure pathophysiology and prognosis, but restoring coordination with biventricular pacing, known as cardiac resynchronization therapy (CRT), improves both. However, not every patient qualifies for CRT. We show that heart failure with synchronous contraction is improved by inducing dyssynchrony for 6 hours daily by right ventricular pacing using an intracardiac pacing device, in a process we call pacemaker-induced transient asynchrony (PITA). In dogs with heart failure induced by 6 weeks of atrial tachypacing, PITA (starting on week 3) suppressed progressive cardiac dilation as well as chamber and myocyte dysfunction. PITA enhanced beta-adrenergic responsiveness in vivo and normalized it in myocytes. Myofilament calcium response declined in dogs with synchronous heart failure, which was accompanied by sarcomere disarray and generation of myofibers with severely reduced function, and these changes were absent in PITA-treated hearts. The benefits of PITA were not replicated when the same number of right ventricular paced beats was randomly distributed throughout the day, indicating that continuity of dyssynchrony exposure is necessary to trigger the beneficial biological response upon resynchronization. These results suggest that PITA could bring the benefits of CRT to the many heart failure patients with synchronous contraction who are not CRT candidates.

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