Cloning of the cDNA encoding the myosin heavy chain of a vertebrate cellular myosin.

The complete amino acid sequence of a vertebrate cellular myosin heavy chain (MHC; 1,959 amino acids, 226 kDa) has been deduced by using cDNA clones from a chicken intestinal epithelial cell library. RNA blot analysis of kidney, spleen, brain, liver, and intestinal epithelial cells as well as smooth muscle cells from the aorta and gizzard indicates the presence of a 7.3-kilobase (kb) message that is larger than the message for chicken smooth and striated muscle MHC. The chicken intestinal epithelial cell MHC shows overall similarity in primary structure to other MHCs in the areas of the reactive thiol residues and in areas contributing to the ATP binding site and actin binding site. The globular head domain is followed by an alpha-helical coiled-coil region, and as in smooth muscle MHC there is a short uncoiled sequence at the carboxyl terminus of the molecule. Comparison of amino acid sequences in the rod regions between human and chicken cellular MHCs shows a remarkable 92% identity.     

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q- and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered load-dependent kinetics that may explain the observed mutant-induced FHC phenotypes.     

Cloning and characterization of a nonmuscle myosin heavy chain cDNA.

Despite many biochemical and structural similarities between muscle and nonmuscle myosins, their genes appear to have completely diverged, since muscle myosin molecular clones will not hybridize to RNA from nonmuscle sources. Here we report the isolation and characterization of a partial myosin heavy chain (MHC) cDNA clone from the slime mold Dictyostelium discoideum. We have isolated this clone from a lambda gt11 expression cDNA library by antibody screening. In contrast to the highly conserved sarcomeric muscle MHC multigene families in other organisms, there appears to be only one gene encoding MHC in the Dictyostelium genome. The cloned portion of this gene does not hybridize to the genomic DNAs of other eukaryotic organisms. Analysis of the predicted amino acid sequence of the partial Dictyostelium MHC clone shows that while there is no sequence homology to known striated muscle MHCs, the structure- and coiled-coil-forming capacities have been conserved.     

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.     

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.     

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.     

Coexpression and assembly of myosin heavy chain and myosin light chain in Escherichia coli.

A fragment of the Dictyostelium discoideum myosin heavy chain gene representing heavy meromyosin was coexpressed in Escherichia coli with the entire essential myosin light chain from the scallop. The expressed myosin heavy chain and essential myosin light chain copurify through ammonium sulfate fractionation, anion exchange, and gel filtration chromatography. The purified complex consists of about 1 mol of light chain per mol of heavy chain. This stoichiometry, which is that of native myosin, suggests that no special eukaryotic machinery is required for coassembly of these two proteins. By coexpressing different myosin heavy chain and myosin light chain combinations, it should be possible to study various isoforms of these two proteins, which are both products of multigene families in mammals. E. coli is thus an ideal system in which to study expression and multimeric assembly of individual components of the eukaryotic contractile apparatus.     

Analysis of the upstream regulatory region of human ventricular myosin light chain 1 gene.

To explore the mechanisms regulating expression of ventricular myosin light chain 1, the human gene including 5'-flanking DNA was cloned and characterized by Southern blot and restriction mapping. A 2 kb 5'-flanking DNA was sequenced and linked to a chloramphenicol acetyltransferase reporter gene. The constructs then were transfected into cultured human and rat cardiomyocytes as well as rat aortic endothelial cells. Deletion analysis of constructs revealed that the basal promoter sequences, which were located within 62 base pairs of the cap site, could direct high levels of chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. The region between -62 to -312 base pairs strongly repressed the chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. Positive elements were found between -312 and -2000 base pairs of the cap site. These results are indicative, among other possibilities, that the human ventricular myosin light chain 1 gene is turned on in cardiomyocytes by the presence of trans-acting factors that are bound to upstream positive elements and is turned off in non-muscle cells by the presence of repressor-binding proteins. But this mechanism remains to be established.     

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.     

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 →Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca²⁺ sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.Hypertrophic cardiomyopathy (HCM) is a complex and heterogeneous disorder with extensive diversity in the course of the disease, age of onset, severity of symptoms, and risk for sudden cardiac death (SCD) (1). HCM is the most common cause of SCD among young people, particularly in athletes (2). The characteristic pathologic features of HCM include cardiac hypertrophy with disproportionate involvement of the ventricular septum and extensive disorganization of the myocyte structure and myocardial fibrosis (3). At present, there is no cure for HCM, and it is now becoming evident that any effective therapy must target the underlying mechanisms involved in the pathogenesis of the disease.The D166V (aspartic acid replaced by valine) mutation in the ventricular myosin regulatory light chain (RLC) was reported by Richard et al. to cause HCM and SCD (4). Our extensive study of the mechanisms underlying the D166V phenotype revealed that the severity of mutation-induced effects in transgenic (Tg) mice correlated with reduced in situ RLC phosphorylation (5, 6). Tg-D166V papillary muscle preparations demonstrated a significantly lower contractile force and abnormally increased myofilament Ca²⁺ sensitivity compared with Tg-WT, expressing a nonmutated human ventricular RLC (5). Likewise, single molecule detection applied to fluorescently labeled cardiac myofibrils from Tg-D166V mice showed slower rates of cross-bridge cycling, and, as in Kerrick et al. (5), these functional abnormalities were paralleled by a low level of RLC phosphorylation compared with Tg-WT hearts (7).Cardiac myosin RLC is a major regulatory subunit of muscle myosin and a modulator of the troponin and Ca²⁺-controlled regulation of muscle contraction (8). It is localized at the head-rod junction of the myosin heavy chain (MHC), and, in addition to the N-terminal Ca²⁺-Mg²⁺ binding site, it also contains the myosin light chain kinase (MLCK)-specific phosphorylatable Serine15. Phosphorylation of Ser15 has been recognized to play an important role in cardiac muscle contraction under normal and disease conditions (9). Significantly reduced RLC phosphorylation was reported in patients with heart failure (10, 11) and observed in animal models of cardiac disease (5, 12–14). Attenuation of RLC phosphorylation in cardiac MLCK knockout mice led to ventricular myocyte hypertrophy, with histological evidence of necrosis and fibrosis, and to mild dilated cardiomyopathy (15, 16). These results and our decade-long investigation of the RLC mutant-induced pathology of the heart suggest that RLC phosphorylation may have an important physiological role in the heart and serve as a rescue tool to mitigate detrimental disease phenotypes.We first addressed this hypothesis in vitro and pursued the MLCK-induced phosphorylation studies on Tg-D166V mice (6), followed by the use of the pseudophosphorylation mimetic proteins exchanged in porcine myosin or skinned muscle fibers (17). Results from both lines of investigation showed that phosphorylation of RLC could counterbalance the adverse contractile effects of an HCM-causing mutation in vitro. In this report, we aimed to test the idea of pseudophosphorylation-induced prevention of a deleterious phenotype in HCM mice. Transgenic S15D-D166V mice were generated expressing the pseudophosphorylated Ser15 (S15D) in the background of the disease-causing D166V mutation. Functional, structural, and morphological assessments were conducted on Tg-S15D-D166V mice, and the results were compared with those of previously generated Tg-D166V (5) and Tg-WT (18) mice. Our findings indicate that myosin phosphorylation may have an important translational application and be used to prevent the development of a severe RLC mutant-induced HCM phenotype.     

Identification of cardiac-specific myosin light chain kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca(2+)/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.     

Construction of recombinant plasmids containing rat muscle actin and myosin light chain DNA sequences.

The construction and partial characterization of recombinant bacterial plasmids carrying DNA sequences that hybridize with rat skeletal muscle actin and a myosin light chain mRNA is described. DNA of one clone hybridizes specifically with the muscle-specific alpha-actin mRNA. Three plasmid clones contain DNA inserts that hybridize with muscle as well as with nonmuscle actin mRNA. A fifth plasmid contains sequences complementary to mRNA coding for myosin light chain 2. DNA of this plasmid hybridizes specifically with RNA extracted from muscle and differentiated muscle cultures but not with RNA extracted from proliferating mononucleated myogenic cells.     

Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain.

Microtubules have been proposed to function as rigid struts which oppose cellular contraction. Consistent with this hypothesis, microtubule disruption strengthens the contractile force exerted by many cell types. We have investigated alternative explanation for the mechanical effects of microtubule disruption: that microtubules modulate the mechanochemical activity of myosin by influencing phosphorylation of the myosin regulatory light chain (LC20). We measured the force produced by a population of fibroblasts within a collagen lattice attached to an isometric force transducer. Treatment of cells with nocodazole, an inhibitor of microtubule polymerization, stimulated an isometric contraction that reached its peak level within 30 min and was typically 30-45% of the force increase following maximal stimulation with 30% fetal bovine serum. The contraction following nocodazole treatment was associated with a 2- to 4-fold increase in LC20 phosphorylation. The increases in both force and LC20 phosphorylation, after addition of nocodazole, could be blocked or reversed by stabilizing the microtubules with paclitaxel (former generic name, taxol). Increasing force and LC20 phosphorylation by pretreatment with fetal bovine serum decreased the subsequent additional contraction upon microtubule disruption, a finding that appears inconsistent with a load-shifting mechanism. Our results suggest that phosphorylation of LC20 is a common mechanism for the contractions stimulated both by microtubule poisons and receptor-mediated agonists. The modulation of myosin activity by alterations in microtubule assembly may coordinate the physiological functions of these cytoskeletal components.     

A point mutation in the regulatory light chain reduces the step size of skeletal muscle myosin

Current evidence favors the theory that, when the globular motor domain of myosin attaches to actin, the light chain binding domain or "lever arm" rotates, and thereby generates movement of actin filaments. Myosin is uniquely designed for such a role in that a long alpha-helix (approximately 9 nm) extending from the C terminus of the catalytic core is stabilized by two calmodulin-like molecules, the regulatory light chain (RLC) and the essential light chain (ELC). Here, we introduce a single-point mutation into the skeletal myosin RLC, which results in a large (approximately 50%) reduction in actin filament velocity (V(actin)) without any loss in actin-activated MgATPase activity. Single-molecule analysis of myosin by optical trapping showed a comparable 2-fold reduction in unitary displacement or step size (d), without a significant change in the duration of the strongly attached state (tau(on)) after the power stroke. Assuming that V(actin) approximately d/tau(on), we can account for the change in velocity primarily by a change in the step size of the lever arm without incurring any change in the kinetic properties of the mutant myosin. These results suggest that a principal role for the many light chain isoforms in the myosin II class may be to modulate the flexural rigidity of the light chain binding domain to maximize tension development and movement during muscle contraction.     

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.     

Effect of phosphorylation of myosin light chain by myosin light chain kinase and protein kinase C on conformational change and ATPase activities of human platelet myosin.

Human platelet myosin forms 10S and 6S conformations, and its Ca(2+)- and Mg(2+)-ATPase activities are parallel with the transition between 10S and 6S conformation, as judged by the gel filtration, intrinsic fluorescence, and viscosity methods. The 20,000-dalton myosin light chain (LC20) is phosphorylated by both myosin light chain kinase (MLC kinase) and Ca2+, phospholipid-dependent protein kinase (protein kinase C [PKC]). The phosphorylation (1 mol of phosphate/mol of LC20) by MLC kinase shifts the equilibrium toward the 6S conformation, but that by PKC does not. The prephosphorylation of myosin by PKC prevents the effect of phosphorylation by MLC kinase on actin-activated Mg(2+)-ATPase activity, but not the effect on conformational change. Inhibition of actin-activated ATPase activity by PKC is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. These results suggest that sequential phosphorylation of myosin by both kinases plays an important role in the ATPase activities of human platelet myosin.