Constitutive Phosphorylation of Cardiac Myosin Regulatory Light Chain in Vivo

In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca²⁺ sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.     

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head–head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head–head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837–854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.     

Modification of interface between regulatory and essential light chains hampers phosphorylation-dependent activation of smooth muscle myosin

We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, V(max), and K(ATPase) of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca(2+) binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218-8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.     

Essential light chain modulates phosphorylation-dependent regulation of smooth muscle myosin

To examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH(4)(+)/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin subfragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Multiple regulatory steps control mammalian nonmuscle myosin II assembly in live cells

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (ΔIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Δtailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Δtailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.     

Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy

Hyperphosphorylation of myosin regulatory light chain (RLC) in cardiac muscle is proposed to cause compensatory hypertrophy. We therefore investigated potential mechanisms in genetically modified mice. Transgenic (TG) mice were generated to overexpress Ca2+/calmodulin-dependent myosin light chain kinase specifically in cardiomyocytes. Phosphorylation of sarcomeric cardiac RLC and cytoplasmic nonmuscle RLC increased markedly in hearts from TG mice compared with hearts from wild-type (WT) mice. Quantitative measures of RLC phosphorylation revealed no spatial gradients. No significant hypertrophy or structural abnormalities were observed up to 6 months of age in hearts of TG mice compared with WT animals. Hearts and cardiomyocytes from WT animals subjected to voluntary running exercise and isoproterenol treatment showed hypertrophic cardiac responses, but the responses for TG mice were attenuated. Additional biochemical measurements indicated that overexpression of the Ca2+/calmodulin-binding kinase did not perturb other Ca2+/calmodulin-dependent processes involving Ca2+/calmodulin-dependent protein kinase II or the protein phosphatase calcineurin. Thus, increased myosin RLC phosphorylation per se does not cause cardiac hypertrophy and probably inhibits physiological and pathophysiological hypertrophy by contributing to enhanced contractile performance and efficiency.     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-Δ43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (≈ 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I(1,1)/I(1,0), indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-Δ43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-Δ43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.     

Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain.     

Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle

The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (β) of 120-125° with the filament axis. This is ∼30° larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with β ∼70° and ∼110°, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at β ∼105°, similar to that determined previously for the RLC region.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

The role of super-relaxed myosin in skeletal and cardiac muscle

The super-relaxed (SRX) state of myosin was only recently reported in striated muscle. It is characterised by a sub-population of myosin heads with a highly inhibited rate of ATP turnover. Myosin heads in the SRX state are bound to each other along the thick filament core producing a highly ordered arrangement. Upon activation, these heads project into the interfilament space where they can bind to the actin filaments. Thus far, the population and lifetimes of myosin heads in the SRX state have been characterised in rabbit cardiac, and fast and slow skeletal muscle, as well as in the skeletal muscle of the tarantula. These studies suggest that the role of SRX in cardiac and skeletal muscle regulation is tailored to their specific functions. In skeletal muscle, the SRX modulates the resting metabolic rate. Cardiac SRX represents a “reserve” of inactive myosin heads that may protect the heart during times of stress, e.g. hypoxia and ischaemia. These heads may also be called up when there is a sustained demand for increased power. The SRX in cardiac muscle provides a potential target for novel therapies.     

Intradomain distances in the regulatory domain of the myosin head in prepower and postpower stroke states: fluorescence energy transfer.

The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle [Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65]. To test this model we have measured, using frequency-modulated FRET, three distances between the catalytic domain and regulatory domains and within the regulatory domain of myosin. The donor/acceptor pairs included MHC cys707 and ELC cys177; ELC cys177 and RLC cys154; and ELC cys177 and gizzard RLC cys108. The IAEDANS (donor) or acceptor (DABMI or IAF) labeled light chains (ELC and RLC) were exchanged into monomeric myosin and the distances were measured in the putative prepower stroke states (in the presence of MgATP or ADP/AlF(4-)) and the postpower stroke states (ADP and the absence of nucleotides). For each of the three distances, the donor/acceptor pairs were reversed to minimize uncertainty in the distance measured, arising from probe orientational factors. The distances obtained from FRET were in close agreement with the distances in the crystal structure. Importantly, none of the measured distances varied by more than 2 A, putting a strong constraint on the extent of conformational changes within S1. The maximum axial movement of the distal part of myosin head was modeled using FRET distance changes within the myosin head reported here and previously. These models revealed an upper bound of 85 A for a swing of the regulatory domain with respect to the catalytic domain during the power stroke. Additionally, an upper bound of 22 A could be contributed to the power stroke by a reorientation of RLC with respect to the ELC during the power stroke.     

Direct detection of the myosin super-relaxed state and interacting-heads motif in solution

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.     

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca²⁺-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca²⁺ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca²⁺]_i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca²⁺ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca²⁺]_i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.     

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch,slow-twitch,and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s⁻¹) and super-relaxed states (SRX, 0.005 s⁻¹) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969–1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C–30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.     

Molecular mechanism regulating myosin and cardiac functions by ELC

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM^hVLC-1) or E56G-mutated hVLC-1 (hVLC-1^E56G; TgM^E56G). hVLC-1 or hVLC-1^E56G expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM^hVLC-1 (1.67 pN/nm and 2.3 μm/s, respectively) were significantly higher than myosin with hVLC-1^E56G prepared from TgM^E56G (1.25 pN/nm and 1.7 μm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 μm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM^hVLC-1 (80.0 mmHg) were significantly higher than hearts from TgM^E56G (66.2 mmHg) or C57/BL6 (59.3 ± 3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1 > hVLC-1^E56G ≈ mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.     

The On-Off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca²⁺ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca²⁺. Our results indicate that loss of Ca²⁺ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.