Three-dimensional structure of vertebrate cardiac muscle myosin filaments

Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the cardiac modulatory protein, myosin binding protein C (MyBP-C). Inherited hypertrophic cardiomyopathy (HCM) is a disease caused mainly by mutations in these proteins. The structure of cardiac myosin filaments and the alterations caused by HCM mutations are unknown. We have used electron microscopy and image analysis to determine the three-dimensional structure of myosin filaments from wild-type mouse cardiac muscle and from a MyBP-C knockout model for HCM. Three-dimensional reconstruction of the wild-type filament reveals the conformation of the myosin heads and the organization of titin and MyBP-C at 4 nm resolution. Myosin heads appear to interact with each other intramolecularly, as in off-state smooth muscle myosin [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366], suggesting that all relaxed muscle myosin IIs may adopt this conformation. Titin domains run in an elongated strand along the filament surface, where they appear to interact with part of MyBP-C and with the myosin backbone. In the knockout filament, some of the myosin head interactions are disrupted, suggesting that MyBP-C is important for normal relaxation of the filament. These observations provide key insights into the role of the myosin filament in cardiac contraction, assembly, and disease. The techniques we have developed should be useful in studying the structural basis of other myosin-related HCM diseases.     

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.     

Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.     

Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ∼20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.     

Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone.     

Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2

Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically "blocked" because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.     

Charge replacement near the phosphorylatable serine of the myosin regulatory light chain mimics aspects of phosphorylation.

Phosphorylation of the myosin regulatory lightchains (RLCs) activates contraction in smooth muscle and modulates forceproduction in striated muscle. RLC phosphorylation changes the net charge in acritical region of the N terminus and thereby may alter interactions between theRLC and myosin heavy chain. A series of N-terminal charge mutations in the humansmooth muscle RLC has been engineered, and the mutants have been evaluated fortheir ability to mimic the phosphorylated form of the RLC when reconstitutedinto scallop striated muscle bundles or into isolated smooth muscle myosin.Changing the net charge in the region from Arg-13 to Ser-19 potentiates force inscallop striated muscle and maintains smooth muscle myosin in an unfoldedfilamentous state without affecting ATPase activity or motility of smooth musclemyosin. Thus, the effect of RLC phosphorylation in striated muscle and itsability to regulate the folded-to-extended conformational transition in smoothmuscle may be due to a simple reduction of net charge at the N terminus of thelight chain. The ability of phosphorylation to regulate smooth musclemyosin's ATPase activity and motility involves a more complexmechanism.     

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber-associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells.     

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.     

Cloning and characterization of a vertebrate cellular myosin regulatory light chain complementary DNA

We have isolated two series of complementary DNAs (cDNAs) from a chicken gizzard cDNA library encoding two isoforms of phosphorylatable myosin regulatory light chain (RLC). One of the cDNAs encodes a previously isolated smooth muscle myosin RLC (also referred to as LC20-A); the other encodes a protein that shares 92% homology with the LC20-A isoform. The phosphorylatable threonine and serine residues at positions 18 and 19 of the two myosin RLC sequences are conserved. The two cDNAs are 81% homologous at the nucleotide level over the coding region; the 5' and 3' untranslated regions are divergent. Most of the DNA nonhomology in the coding region does not affect the protein sequence, indicating strong evolutionary conservation pressure to maintain the myosin RLC structure. Northern blot analysis using 3' untranslated region probes reveals restrictive tissue specific expression of one myosin RLC isoform (LC20-A) in smooth muscle tissue and not in other tissues examined. In contrast, the novel myosin RLC isoform messenger RNA (mRNA) is uniformly expressed in all smooth and nonmuscle tissues examined and is designated as cellular myosin RLC for this reason. Our results indicate that cellular and smooth muscle myosin RLC isoforms are distinct and are encoded by separate genes. This report describes the cloning of a novel vertebrate cellular myosin RLC mRNA that differs from previously characterized smooth muscle RLC isoform mRNAs in both primary sequence and expression pattern.     

Spare the rod, spoil the regulation: Necessity for a myosin rod

Regulation of a variety of cellular contractile events requires that vertebrate smooth and non-muscle myosin II can achieve an “off” state. To examine the role of the myosin rod in this process, we determined the minimal size at which a myosin molecule is capable of regulation via light chain phosphorylation. Expressed smooth muscle myosin subfragments with as many as 100 amino acids of the coiled-coil rod sequence did not dimerize and were active independently of phosphorylation. To test whether dimerization per se restores regulation of ATPase activity, mutants were expressed with varying lengths of rod sequence, followed by C-terminal leucine zippers to stabilize the coiled-coil. Dimerization restored partial regulation, but the presence of a length of rod approximately equal to the myosin head was necessary to achieve a completely off state. Partially regulated short dimers could be converted into fully regulated molecules by addition of native rod sequence after the zipper. These results suggest that the myosin rod mediates specific interactions with the head that are required to obtain the completely inactive state of vertebrate smooth and non-muscle myosins. If these interactions are prohibited under cellular conditions, unphosphorylated crossbridges can slowly cycle.     

Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments

Dictyostelium actin was shown to become phosphorylated on Tyr-53 late in the developmental cycle and when cells in the amoeboid stage are subjected to stress but the phosphorylated actin had not been purified and characterized. We have separated phosphorylated and unphosphorylated actin and shown that Tyr-53 phosphorylation substantially reduces actin's ability to inactivate DNase I, increases actin's critical concentration, and greatly reduces its rate of polymerization. Tyr-53 phosphorylation substantially, if not completely, inhibits nucleation and elongation from the pointed end of actin filaments and reduces the rate of elongation from the barbed end. Negatively stained electron microscopic images of polymerized Tyr-53-phosphorylated actin show a variable mixture of small oligomers and filaments, which are converted to more typical, long filaments upon addition of myosin subfragment 1. Tyr-53-phosphorylated and unphosphorylated actin copolymerize in vitro, and phosphorylated and unphosphorylated actin colocalize in amoebae. Tyr-53 phosphorylation does not affect the ability of filamentous actin to activate myosin ATPase.     

The effect of myosin regulatory light chain phosphorylation on the frequency-dependent regulation of cardiac function

Although it has been suggested that in cardiac muscle the phosphorylation level of myosin regulatory light chain (RLC) correlates with frequency of stimulation, its significance in the modulation of the force-frequency and pressure-frequency relationships remains unclear. We examined the role of RLC phosphorylation on the force-frequency relation (papillary muscles), the pressure-frequency relation (Langendorff perfused hearts) and shortening-frequency relation (isolated cardiac myocytes) in nontransgenic (NTG) and transgenic mouse hearts expressing a nonphosphorylatable RLC protein (RLC(P-)). At 22 degrees C, NTG and RLC(P-) muscles showed a negative force-frequency relation. At 32 degrees C, at frequencies above 1 Hz, both groups showed a flat force-frequency relation. There was a small increase in RLC phosphorylation in NTG muscles when the frequency of stimulation was increased from 0.2 Hz to 4.0 Hz. However, the level of RLC phosphorylation in these isolated muscles was significantly lower compared to samples taken from NTG intact hearts. In perfused hearts, there was no difference in the slope of pressure-frequency relationship between groups, but the RLC(P-) group consistently developed a reduced systolic pressure and demonstrated a decreased contractility. There was no difference in the level of RLC phosphorylation in hearts paced at 300 and 600 bpm. In RLC(P-) hearts, the level of TnI phosphorylation was reduced compared to NTG. There was no change in the expression of PLB between groups, but expression of SERCA2 was increased in hearts from RLC(P-) compared to NTG. In isolated cardiac myocytes, there was no change in shortening-frequency relationship between groups. Moreover, there was no change in Ca(2+) transient parameters in cells from NTG and RLC(P-) hearts. Our data demonstrate that in cardiac muscle RLC phosphorylation is not an essential determinant of force- and pressure-frequency relations but the absence of RLC phosphorylation decreases contractility in force/pressure developing preparations.     

Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.     

Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.

Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin.     

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

The contraction of cardiac muscle is generated by reciprocal sliding of actin-containing thin filaments and myosin-containing thick filaments in the sarcomere driven by myosin motors. The interaction of the myosin motors with the overlapping thin filament is primarily controlled by calcium-induced structural changes in the thin filament linked to the intracellular calcium transient (1). Calcium ions released in the cytoplasm following an action potential bind to troponin, triggering the movement of tropomyosin around the filament, which uncovers actin sites to which the motors can bind and power contraction (2). However, some of the myosin motors may not be available for actin binding, as they are folded onto the thick filament surface in relaxing conditions (3). Thick filament–based regulatory mechanisms control the release of the myosin motors from the folded conformation and contribute to the regulation of contractility of striated muscle (3–6).Electron microscopy (EM) studies on isolated thick filaments from vertebrate heart muscle showed that the myosin motors in the region of the filament that contains myosin-binding protein C (MyBP-C), the C-zone, are sequestered in helical tracks on the thick filament surface and are folded back onto their tails in an asymmetric conformation called the interacting-heads motif, or IHM (7, 8), originally identified in two-dimensional crystals of dephosphorylated smooth muscle myosin (9). The IHM has also been associated with a biochemical state of myosin with a very low adenosine triphosphate (ATP)-ase rate, called the “super-relaxed” state (10), which is considered to be an OFF state of myosin. A recent X-ray diffraction study of cardiac muscle (11) extended that concept and suggested that in diastole, the resting phase of the cardiac cycle, three distinct motor conformations coexist in the thick filament in roughly equal numbers: folded helical, folded nonhelical, and disordered. The folded helical motors are likely to correspond to the IHM conformation and are confined to the C-zone. All the folded motors would be unavailable for actin binding and therefore OFF, but the disordered motors would constitute a population of constitutively ON motors that are immediately available for actin binding upon activation of the thin filament.Stress sensing in the thick filament can control the release of the myosin motors from the folded states and might be responsible for modulating the strength of contraction of cardiac muscle in response to changes in the afterload (i.e., the arterial pressure) (12). Moreover, the transitions between these motor conformations, together with the calcium-induced structural changes in the thin filament, control the speed of contraction and relaxation (11). According to this mechanosensing paradigm of thick filament regulation, the constitutively ON motors play a fundamental role in the activation of cardiac muscle, as the force generated by these motors immediately after the electrical stimulus triggers a positive mechanosensing feedback loop that controls the number of active motors and the dynamics of contraction. Destabilization of the folded conformations by mutations in myosin and other thick filament proteins can alter the equilibrium between these motor conformations, leading to a hypercontractile phenotype in some hypertrophic cardiomyopathies (HCM) (13, 14). Pharmacological therapies targeting thick filament proteins to treat HCM (15–17) have been aimed at reversing the destabilization of the folded states caused by these mutations.The contractility of the heart is also controlled by phosphorylation of the myosin regulatory light chain (RLC) (18) and by β-adrenergic signaling pathways mediated by phosphorylation of MyBP-C in the thick filament (19) as well as troponin in the thin filament, which are also likely to alter the equilibrium between regulatory conformations of the motors. RLC phosphorylation is essential for the normal function of the heart. The pattern of contraction of the heart may depend on a spatial gradient of RLC phosphorylation across the ventricle wall (20), and a decrease in the level of RLC phosphorylation is associated with heart failure (21, 22). RLC phosphorylation potentiates the contractility of vertebrate and invertebrate striated muscle, an effect that is generally thought to be mediated by disrupting the folded helical conformation of the myosin motors on the thick filament and increasing the number of motors available for interaction with actin during contraction at a given [Ca²⁺] (23, 24). Disordering of the myosin motors on the thick filament by RLC phosphorylation has been shown in in vitro studies on isolated thick filaments (23) and is the main mechanism of thick filament activation in intact striated muscle of tarantula during contraction (25). However, the effect of RLC phosphorylation on the structure of the cardiac thick filament in diastole is unclear. More generally, the large changes in force and the speed of contraction and relaxation of cardiac muscle produced by β-adrenergic agonists are not associated with significant changes in the diastolic structure of the thick filament (26), so they are not simply mediated by increasing the number of ON motors in diastole. Similarly, length-dependent activation (LDA), the cellular correlate of the Frank–Starling law of the heart and a key autoregulatory mechanism that adjusts the cardiac output in response to different extents of diastolic filling (27, 28), seems not to be simply mediated by a stretch-induced change in the structure of the thick filament in diastole (11, 26). Conflicting results have been reported (29), however, and mathematical models have suggested that activation of myosin motors induced by the passive tension transmitted to the thick filament by titin might contribute to LDA in cardiac muscle (30, 31).Here, we investigated the in situ conformation of the myosin motors and its dependence on temperature, RLC phosphorylation, [Ca²⁺], and sarcomere length (SL) in demembranated cardiac trabeculae from rat hearts using the polarized fluorescence from probes on the N- and C-lobes of the RLC (18, 32). We show that, at the low [Ca²⁺] values that maintain the relaxed state and at near-physiological temperature and lattice spacing, the RLC-lobe orientations are consistent with about one-third of the myosin motors being in the folded helical conformation corresponding to the IHM, likely stabilized by MyBP-C in the C-zone of the filament. At the low [Ca²⁺] values that maintain the relaxed state, the folded conformation of the myosin motors is disrupted by cooling but not by RLC phosphorylation or stretch. However, stretching cardiac muscle at higher [Ca²⁺] that partially activates the thin filament triggers a stress-dependent activation of the thick filament and a force increase that is potentiated by RLC phosphorylation. This increase in contractility, induced by RLC phosphorylation and stretch, can be explained by an interfilament signaling pathway that links the stress-dependent activation of the thick filament to the activation state of the thin filament.     

Holding two heads together: stability of the myosin II rod measured by resonance energy transfer between the heads

Myosin, similar to many molecular motors, is a two-headed dimer held together by a coiled-coiled rod. The stability of the coiled coil has implications for head-head interactions, force generation, and possibly regulation. Here we used two different resonance energy transfer techniques to measure the distances between probes placed in the regulatory light chain of each head of a skeletal heavy meromyosin, near the head-rod junction (positions 2, 73, and 94). Our results indicate that the rod largely does not uncoil when myosin is free in solution, and at least beyond the first heptad, the subfragment 2 rod remains relatively intact even under the relatively large strain of two-headed myosin (rigor) binding to actin. We infer that uncoiling of the rod likely does not play a role in myosin II motility. To keep the head-rod junction intact, a distortion must occur within the myosin heads. This distortion may lead to different orientations of the light-chain domains within the myosin dimer when both heads are attached to actin, which would explain previously puzzling observations and require reinterpretation of others. In addition, by comparing resonance energy transfer techniques sensitive to different dynamical time scales, we find that the N terminus of the regulatory light chain is highly flexible, with possible implications for regulation. An intact rod may be a general property of molecular motors, because a similar conclusion has been reached recently for kinesin, although whether the rod remains intact will depend on the relative stiffness of the coiled coil and the head in different motors.     

Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.     

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

We have determined the 2.3-Å-resolution crystal structure of a myosin light chain domain, corresponding to one type found in sea scallop catch (“smooth”) muscle. This structure reveals hinges that may function in the “on” and “off” states of myosin. The molecule adopts two different conformations about the heavy chain “hook” and regulatory light chain (RLC) helix D. This conformational change results in extended and compressed forms of the lever arm whose lengths differ by 10 Å. The heavy chain hook and RLC helix D hinges could thus serve as a potential major and localized source of cross-bridge compliance during the contractile cycle. In addition, in one of the molecules of the crystal, part of the RLC N-terminal extension is seen in atomic detail and forms a one-turn alpha-helix that interacts with RLC helix D. This extension, whose sequence is highly variable in different myosins, may thus modulate the flexibility of the lever arm. Moreover, the relative proximity of the phosphorylation site to the helix D hinge suggests a potential role for conformational changes about this hinge in the transition between the on and off states of regulated myosins.     

Myosin phosphorylation decreases the ATPase activity of cardiac myofibrils

Our previous work showed that myosin phosphorylation decreased the ATPase activity of skeletal muscle myofibrils that were lightly fixed with glutaraldehyde. The fixation process prevented sarcomere shortening and destruction of the ordered filament array upon the addition of ATP. We have now extended these results to myofibrils prepared from hearts of rabbits, dogs and rats. Myofibrils were phosphorylated by incubation with myosin light chain kinase, calmodulin and either ATP-gamma s or ATP, for 15 minutes at 25 degrees C. The extent of myosin light chain phosphorylation was 50% to 80%. The ATPase activity of unphosphorylated myofibrils was not altered by reaction with 0.01% glutaraldehyde for 5 minutes at 0 degrees C, and the ATPase activity of unfixed myofibrils was not changed by phosphorylation. However, phosphorylation decreased the ATPase activity of fixed myofibrils by 50%. The effect on myocardial myofibrillar ATPase activity of phosphorylation was similar in the three animal species. These results suggest that in both skeletal and cardiac muscle, myosin phosphorylation decreases the rate of cross-bridge cycling resulting in decreased energy expenditure. It also appears that the effect of myosin light chain phosphorylation on ATPase activity requires an ordered myofilament structure.