Binding Of Myosin Heads Research Articles

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Troponin-I-induced tropomyosin pivoting defines thin-filament function in relaxed and active muscle.

Regulation of the crossbridge cycle that drives muscle contraction involves a reconfiguration of the troponin-tropomyosin complex on actin filaments. By comparing atomic models of troponin-tropomyosin fitted to cryo-EM structures of inhibited and Ca2+-activated thin filaments, we find that tropomyosin pivots rather than rolls or slides across actin as generally thought. We propose that pivoting can account for the Ca2+ activation that initiates muscle contraction and then relaxation influenced by troponin-I (TnI). Tropomyosin is well-known to occupy either of three meta-stable configurations on actin, regulating access of myosin motorheads to their actin-binding sites and thus the crossbridge cycle. At low Ca2+ concentrations, tropomyosin is trapped by TnI in an inhibitory B-state that sterically blocks myosin binding to actin, leading to muscle relaxation. Ca2+ binding to TnC draws TnI away from tropomyosin, while tropomyosin moves to a C-state location over actin. This partially relieves the steric inhibition and allows weak binding of myosin heads to actin, which then transition to strong actin-bound configurations, fully activating the thin filament. Nevertheless, the reconfiguration that accompanies the initial Ca2+-sensitive B-state/C-state shift in troponin-tropomyosin on actin remains uncertain and at best is described by moderate-resolution cryo-EM reconstructions. Our recent computational studies indicate that intermolecular residue-to-residue salt-bridge linkage between actin and tropomyosin is indistinguishable in B- and C-state thin filament configurations. We show here that tropomyosin can pivot about relatively fixed points on actin to accompany B-state/C-state structural transitions. We argue that at low Ca2+ concentrations C-terminal TnI domains attract tropomyosin, causing it to bend and then pivot toward the TnI, thus blocking myosin binding and contraction.

Dissecting the mechanisms by which a pathogenic variant in cardiac TnT tail domain leads to disturbed myofilament performance.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and high rate of sudden cardiac arrest at juvenile age. cTnT-I79N is located in the cTnT N-terminal (TNT1) loop region and is well-known for its pathological and prognostic relevance. Recent structural study revealed that I79 is part of hydrophobic interface between TNT1 loop and actin which stabilizes the OFF state of the cardiac thin filament. Given the importance of understanding: i) the role of TNT1 loop region on the regulation of the cardiac thin filament, and ii) the underlying mechanisms of cTnT-I79N-linked pathogenesis; we sought to investigate the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased passive force and passive sinusoidal stiffness, which could be due in part to an increase in force-generating cross-bridges during diastole. Tg-I79N also displayed increased myofilament Ca2+-sensitivity, shorter myofilament lattice spacing, and slower cross-bridge kinetics. These findings can be attributed to the destabilization of cardiac thin filament's inhibitory property, and the increase in the overall number of cross-bridges during Ca2+ activation. Finally, we show that cTnT-I79N dysregulates SRX↔DRX equilibrium, which can be explained by the excessive binding of myosin heads to the dysregulated thin filament that causes the SRX↔DRX equilibrium to shift towards the ON state. Altogether, biochemical and functional measurements, and structural data points to the following mechanism: cTnT-I79N weakens the interaction of the TNT1 loop with the actin filament, which in turn destabilizes the OFF state of the cardiac thin filament.

Cooperativity of myosin II motors in the non-regulated and regulated thin filaments investigated with high-speed AFM.

Skeletal myosins II are non-processive molecular motors that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we use high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration in the presence of 2 μM ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of available binding sites at the next half-helical pitch of the filament. The result suggests that myosin cooperativity is observed over five pseudo-repeats and defined by the activation status of the thin filaments.

A Short Review of New Drugs for Heart Failure: Omecamtiv Mecarbil and Vericiguat

Heart failure is associated with increased morbidity and mortality. Although developments of therapeutic agents have shown benefit and have improved the patients’ condition, there is only a slight decreased of mortality rate throughout the years. This condition becomes a reason to develop efficient drugs which can further improve the condition and reducing the mortality significantly. New therapeutic agents have been proposed and studied. Among them is cardiac myosin activator, such as omecamtiv mecarbil. This drug will increase the binding of myosin head to the actin and therefore form a stronger contraction. Omecamtiv mecarbil is beneficial due to its mechanism which not related to higher oxygen consumption and less adverse events. Another promising drug is vericiguat, a soluble guanylate cyclase (sGC) stimulator. This drug enhances the binding of nitric oxide to sGC and therefore increasing cyclic guanosine monophosphate (cGMP) production. cGMP important in regulating physiological processed in the cardiovascular system. In this review, we will discuss about the current evidence about these novel agents.
 Keywords: heart failure, novel agent, omecamtiv mecarbil, vericiguat, management

A Proposed Mechanism for the Initial Myosin Binding Event on the Cardiac Thin Filament: A Metadynamics Study.

The movement of tropomyosin over filamentous actin regulates the cross-bridge cycle of the thick with thin filament of cardiac muscle by blocking and revealing myosin binding sites. Tropomyosin exists in three, distinct equilibrium states with one state blocking myosin-actin interactions (blocked position) and the remaining two allowing for weak (closed position) and strong myosin binding (open position). However, experimental information illuminating how myosin binds to the thin filament and influences tropomyosin's transition across the actin surface is lacking. Using metadynamics, we mimic the effect of a single myosin head binding by determining the work required to pull small segments of tropomyosin toward the open position in several distinct regions of the thin filament. We find differences in required work due to the influence of cardiac troponin T lead to preferential binding sites and determine the mechanism of further myosin head recruitment.

Impact of anti-S2 Peptides on myosin S2 Isoforms and HCM Mutants

Myosin subfragment-2 (S2) is an unstable coiled coil that influences the binding of myosin heads to actin. Instability in the myosin S2 could underlie one of the causes for hyper-contractility in Hypertrophic Cardiomyopathy (HCM). Supporting this hypothesis, HCM mutations E924K and E930del in myosin S2 reduced myosin S2 coiled coil stability compared to wild type as measured by Forester's Resonance Energy Transfer (FRET) and Gravitational Force Spectroscopy (GFS). To modulate the stability of the S2, anti-S2 peptides called stabilizer and destabilizer peptides because they are designed to increase and decrease the stability of the MYH7 S2 coiled coil, were tested on a range of human S2 peptide sequences. The anti-S2 peptides were tested on myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by stabilizer and decreased by destabilizer across the cardiac and skeletal myosin S2 peptides but not for smooth muscle isoform. Dose-response curves for FRET assays indicated that both peptides bound at nanomolar concentrations to wild type cardiac and skeletal, but not smooth muscle isoforms. The wild type cardiac S2 peptide required more force to unzip than skeletal, smooth, or mutant peptides in the absence of stabilizer peptide. Effectively, the stabilizer peptide improved the stability of both HCM mutants (E924K and E930del). The stabilizer peptide enhanced dimer formation in mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. This study demonstrates that the structural stability of myosin S2 can be altered by designed small peptides as well as mutations. These anti-S2 peptides share similar specificities and high affinities for the tested striated muscle isoforms but not the smooth muscle myosin isoform.

The molecular mechanisms of a high Ca2+-sensitivity and muscle weakness associated with the Ala155Thr substitution in Tpm3.12

Substitution of Ala for Thr residue in 155th position in γ-tropomyosin (Tpm3.12) is associated with muscle weakness. To understand the mechanisms of this defect, we studied the Ca2+-sensitivity of thin filaments in solution and multistep changes in mobility and spatial arrangement of actin, Tpm, and myosin heads during the ATPase cycle in reconstituted muscle fibres, using the polarized fluorescence microscopy. It was shown that the Ala155Thr (A155T) mutation increased the Ca2+-sensitivity of the thin filaments in solution. In the absence of the myosin heads in the muscle fibres, the mutation did not alter the ability of troponin to switch the thin filaments on and off at high and low Ca2+, respectively. However, upon the binding of myosin heads to the thin filaments at low Ca2+, the mutant Tpm was found to be markedly closer to the open position, than the wild-type Tpm. In the presence of the mutant Tpm, switching on of actin monomers and formation of the strong-binding state of the myosin heads were observed at low Ca2+, which indicated a higher myofilament Ca2+-sensitivity. The mutation decreased the amount of myosin heads bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation. It is suggested that direct binding of myosin to Tpm may be one оf the reasons for muscle weakness associated with the A155T mutation. The use of reagents that decrease the Ca2+-sensitivity of the troponin complex may not be adequate to restore muscle function in patients with the A155T mutation.

Mechanism of Cardiac Tropomyosin Transitions on Filamentous Actin As Revealed by All-Atom Steered Molecular Dynamics Simulations.

The three-state model of tropomyosin (Tm) positioning along filamentous actin allows for Tm to act as a gate for myosin head binding with actin. The blocked state of Tm prevents myosin binding, while the open state allows for strong binding. Intermediate to this transition is the closed state. The details of the transition from the blocked to the closed state and then finally to the open state by Tm have not been fully accessible to experiment. Utilizing steered molecular dynamics, we investigate the work required to move the Tm strand through the extant set of proposed transitions. We find that an azimuthal motion around the actin filament by Tm is most probable in spite of increased initial energy barrier from the topographical landscape of actin.

C1 IG-Domain of Myosin Binding Protein-C Activates Cardiac Thin Filament by Means of Thethering Tropomyosin to the Subdomain-1 of Actin

Cardiac muscle contraction is based on the interaction between two filamentous systems: the thick filament, which is comprised of myosin and its accessory protein, cardiac myosin binding protein C (cMyBP-C) and the thin filament (TF), which is comprised of troponin (Tn), tropomyosin (Tpm), and filamentous actin (F-actin). Mutations in the gene encoding cMyBP-C are one of the most common causes of hypertrophic cardiomyopathy (HCM), a disease that affects approximately 1 in 500 people. The interaction between thick and thin filaments is regulated through translocation of Tpm cable by the Tn-complex in response to Ca2+ followed by additional Tpm azimuthal movement upon binding of rigor myosin cross-bridges to F-actin. Therefore, activation of the TF is a two-step process which depends on both Ca2+-induced translocation of the Tpm and subsequent binding of rigor myosin heads. The N-terminal domain of cMyBP-C is comprised of three Ig-domains (C0, C1 and C2) and a regulatory linker (M-domain). We show that C1 Ig-domain is the only Ig-domain that can activate the TFs at low Ca2+. 3D-reconstruction of frozen hydrated cardiac TFs decorated with C1 Ig-domain shows that the C1 Ig-domain on its own can translocate the Tpm cable on the surface of F-actin to the same extent as the combination of Ca2+ and rigor myosin by tethering the Tpm cable to the subdomain-1 (SD1) of actin. Disruption of the interaction of the C1 Ig-domain interaction with either the SD1 of actin or Tpm cable by point mutations in C1 Ig-domain results in a complete loss of the C1-induced TF activation at low Ca2+. These data suggest how cMyBP-C can modulate the activation of the TF in heart muscle.

The Closed State of the Thin Filament Is Not Occupied in Fully Activated Skeletal Muscle

Muscle contraction is powered by actin-myosin interaction controlled by Ca2+ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 μm length of a thin filament. In the absence of Ca2+, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca2+ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15–0.275 nm−1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.

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

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

Cloning, molecular characterization, and expression analysis of the unc45 myosin chaperone b(unc45b)gene of grass carp (Ctenopharyngodon idellus).

Unc45 myosin chaperone b(unc45b)gene is a molecular chaperone that mediates the folding, assembly and accumulation of thick-filament myosin in the formation of sarcomere, which plays an important role in the development of striated muscle and the stability of sarcomere. In this study, the complete cDNA sequence of unc45b gene of grass carp was obtained by rapid amplification of cDNA ends (RACE), and the characteristics of the unc45b protein predicted from gene sequence was analyzed by bioinformatics methods. The differential expression pattern in tissues was also detected by quantitative real-time PCR. The results showed that the full-length of unc45b gene of grass carp is 3163bp, which contains a 60bp 5'UTR, a 298bp 3'UTR, and a 2865bp open reading frame (ORF) encoding a 934 amino acid peptide. The deduced unc45b protein exhibits a homology of 92, 86, 86% with the protein of zebrafish (Danio rerio), channel catfish (Ietalurus punctatus) and tilapia (Oreochromis niloticus) respectively, and the protein contains UCS myosin head binding domain and TPR peptide repeat domain. The protein is a hydrophilic and non-secretory protein with a molecular mass and isoeletronic point of 103,699.8 and 7.39Da. The structural elements of the protein includes α-helixes and loops, and the unc45b gene highly expresses in skeletal muscle and heart in grass carp. This study laid a foundation for further research in explaining the myofibril accumulation in crisped grass carp.

Aberrant movement of β-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle

We have investigated the effect of the E41K, R91G, and E139del β-tropomyosin (TM) mutations that cause congenital myopathy on the position of TM and orientation of actin monomers and myosin heads at different mimicked stages of the ATPase cycle in troponin-free ghost muscle fibers by polarized fluorimetry. A multi-step shifting of wild-type TM to the filament center accompanied by an increase in the amount of switched on actin monomers and the strongly bound myosin heads was observed during the ATPase cycle. The R91G mutation shifts TM further towards the inner and outer domains of actin at the strong- and weak-binding stages, respectively. The E139del mutation retains TM near the inner domains, while the E41K mutation captures it near the outer domains. The E41K and R91G mutations can induce the strong binding of myosin heads to actin, when TM is located near the outer domains. The E139del mutation inhibits the amount of strongly bound myosin heads throughout the ATPase cycle.

In vitro Reconstitution of Skeletal Muscle Contraction using Native Thin Filaments

Intracellular Ca2+ concentration regulate the muscle contraction by changing the conformation of Tropomyosin molecule over Actin filament. In this work we have purified native thin filaments (NTF) from Chicken Pectoralis muscle and reconstituted in in vitromotility assay (IVMA) with skeletal muscle myosin II. NTF prepared were 1.67±0.14 µm in length and showed good regulation under different Ca2+ concentrations. At pCa 4, more than 90% of NTF were continuously sliding and when solution changed to pCa 9 all the filaments were rigidly stuck to surface. Sliding velocity of NTF was VF=5.63±1.21 µm/s at 30 oC and 17% higher compared to unregulated actin filaments. Fraction of sliding filament showed a direct correlation with pCa of buffer and pCa50= 6.69 was obtained using Hills equation fit. However the sliding filament velocity was not affected under different pCa concentration. Under varying substrate [MgATP] conditions in IVMA we found k+ATP is 4.3± 0.56 x 106 M−1s−1 and k-ADP is 404.33±2.7 s−1 at 30 oC. These kinetic parameters are comparable to values obtained using unregulated actin filaments, suggesting the detachment kinetics of myosin II is not affected due to Ca2+ regulation. We studied the effect of myosin density on thin filaments activation in IVMA. Even with fully activated thin filament (pCa 4.0), decrease in myosin density leads to increase in fraction of stuck filaments. Decrease in fraction of motile filaments with myosin density in IVMA suggests there is minimum number of myosin head binding required to completely activate the NTF.Supported by Department of Science and Technology, Government of India.

The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.

Tropomyosin dynamics

Tropomyosin is a two chained α-helical coiled coil protein that binds actin filaments and interacts with various actin binding proteins. Tropomyosin function depends on its ability to move to distinct locations on the surface of actin in response to the binding of different thin filament effectors. Tropomyosin dynamics plays an important role in these fluctuating interactions with actin and is thought to be fundamental to many of its biological activities. For example tropomyosin concerted movement on the surface of actin triggered by Ca(2+) binding to troponin or myosin head binding to actin has been argued to be key to the cooperative allosteric regulation of muscle contraction. These large-scale motions are affected by tropomyosin internal dynamics and mechanical properties. Tropomyosin internal dynamics corresponding to smaller and more localised structural fluctuations are increasingly recognised to play an important role in its function. A thorough understanding of the coupling between local and global structural fluctuations in tropomyosin is required to understand how time dependent structural fluctuations in tropomyosin contribute to the overall thin filament dynamics and dictate their various biological activities.

Three-Dimensionally Constrained Actomyosin Motility on Oxide Coated Semiconductor Nanowires

In vitro motility assays with surface-adsorbed myosin motor fragments have given important insights into the molecular physiology and pathology of striated muscle contraction and inspired nanotechnological applications e.g. lab-on-a-chip devices. However, to date neither precise localized control of the motor density nor introduction of nanoscale three-dimensional geometrical constraints has been possible. This hampers studies of cooperative phenomena and realization of three-dimensional (3D) transport systems. Here, we take critical steps to overcome these limitations, using aluminium oxide coated gallium phosphide nanowires as scaffolds for heavy meromyosin (HMM) adsorption. The wires (diameter: 100-200 nm; height < 5 μm) were either positioned vertically in regular arrays or horizontally on surfaces after etching the GaP core to give hollow nanowires. Upon ATP addition, actin filaments were propelled by HMM on top of the arrays, with filaments spanning inter-wire distances up to 1 μm. The filaments also moved up and down vertical nanowires as detected using sub-wavelength light guiding properties of the nanowires. Motility on top of nanowire arrays holds potential for studies of cooperative phenomena e.g. local enhancement of myosin binding along actin filaments upon actomyosin interactions. Here we tested whether the low velocity for long filaments seen at uniformly low motor densities on flat surfaces may be attributed to loss of cooperative enhancement of myosin head binding locally to actin close to an existing actomyosin cross-bridge. Velocity data showing 1/3 the velocity with 1 μm compared to 300 nm inter-wire spacing, in both cases with ∼90 HMM molecules per 150 nm wide wire tip (giving high local motor density), argue against this idea. We further demonstrate HMM propelled transport through hollow nanowires of 80 nm inner diameter. Uses of hollow nanowires in fundamental studies of actomyosin and in nanotechnological applications will be considered.

Phosphorylation of myosin regulatory light chain has minimal effect on kinetics and distribution of orientations of cross bridges of rabbit skeletal muscle

Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to ∼20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges.

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478-44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815-1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca(2+) sensitivity, and contractile defects. The FHC mice die by 6-8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.