Expression of cardiac myosin light chain 2 during embryonic heart development in medaka fish,Oryzias latipes,and phylogenetic relationship with other myosin light chains

Cardiac myosin light chain 2 (MLC‐2) plays a key role in heart development, contraction, and embryo and adult heart maintenance. In some animals, defects in the function of cardiac MLC‐2 cause hypertrophic cardiomyopathy. To illuminate the functions of cardiac MLC‐2 in embryonic heart formation and contraction, and into the evolution of MLC‐2, we characterized the expression and requirement for medaka cardiac MLC‐2 gene in the developing heart. Medaka cardiac MLC‐2 cDNA (mcmlc2) was isolated and its gene expression pattern was determined. The mcmlc2 was found to be expressed in the bilateral cardiac mesoderm, the formed heart tube, and in both the differentiated ventricle and atrium. Knockdown of mcmlc2 function caused severe cardiac disorders, including edema in the atrium and sinus venosus. Using phylogenetic analysis, we found that physiological variations in the MLC‐2 molecules evolved due to amino acid changes in the Ca²⁺ binding domain during molecular evolution. Our findings concerning the function and expression of mcmlc2 are nearly identical with those of other MLC‐2 genes, and our phylogenetic analysis suggests that during evolution, the variations in physiological function within the MLC‐2 gene family have arisen from a change in the amino acids in the Ca²⁺ binding domain in the MLC‐2 molecule.     

Alternative splicing of smooth muscle myosin heavy chains and its functional consequences

The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca²⁺/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.     

Distinct interactions between actin and essential myosin light chain isoforms

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein–protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin^ala3) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (K_D = 575 nM) was significantly (p < 0.01) lower compared with the affinity of hVLC-1 to α-actin (K_D = 186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p < 0.01) lower association rate (k_on: 1018 M⁻¹ s⁻¹) compared with k_on of the hVLC-1/α-actin complex interaction (2908 M⁻¹ s⁻¹). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

Truncation of vertebrate striated muscle myosin light chains disturbs calcium-induced structural transitions in synthetic myosin filaments

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca(2+)-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca(2+)-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca(2+)-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.     

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

Phosphorylation of myosin light chains and C-protein in the myocardium of hibernating ground squirrel Citellus undulatus

A comparative study was made of the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of the hibernant ground squirrel Citellus undulatus during the periods of hibernation and activity. During hibernation, the light chains were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averaged 40–45%. The extent of cardiac C-protein phosphorylation in hibernation was about twice higher than in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.     

Design of peptidase‐resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell‐permeant peptide Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys (PIK, P eptide I nhibitor of K inase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L ‐PIK in a biological milieu prompts for development of more stable L ‐PIK analogues for use as experimental tools in basic and drug‐oriented biomedical research. Previously, we designed PIK1, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys‐NH₂, that was 2.5‐fold more resistant to peptidases in human plasma in vitro than L ‐PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site‐protected peptides based on L ‐PIK and PIK1 degradation patterns in human plasma as revealed by ¹H‐NMR analysis. Implemented modifications increased half‐live of the PIK‐related peptides in plasma about 10‐fold, and these compounds retained 25–100% of L ‐PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐D ‐Arg‐Lys‐NH₂, was identified as the most stable and effective L ‐PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin‐induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell‐permeant inhibitors of MLCK in cell culture‐based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction.     

Visualization of head-head interactions in the inhibited state of smooth muscle myosin

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically ‘frozen’ during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca²⁺ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromotography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18?20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3–0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 μM epinephrine.     

Human cardiac myosin light chains: Sequence comparisons between myosin LC1 and LC2 from normal and idiopathic dilated cardiomyopathic hearts

The primary structures of light chains isolated from the human myocardium with idiopathic dilated cardiomyopathy (IDC) were determined and compared with the sequence structures of myosin light chains obtained from control human heart myosin. Sequences were determined by chemical analysis and the identity of N-terminal residues established by mass spectrometry. The N-terminal residues in essential (ELC) and regulatory (RLC) light chains were blocked and were identified to be trimethyl alanine. The amino acid sequences of ELC and RLC from control human myosin revealed a high degree of homology with those purified from rat and chicken cardiac myosin. Comparison with a published partial chemical sequence of the human heart myosin light chains revealed significant variations. However, there was very good agreement with published sequences obtained by molecular biological techniques. Sequences of the light chains from cardiomyopathic myosin revealed no difference in the primary structures when compared with control human heart myosin light chains indicating IDC had no influence on, nor was caused by, altered myosin light chain gene expression.     

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC₁₇), and phosphorylation of the 20 kDa MLC (MLC₂₀) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (35.3/35.3/29.4). In vivo expression of MLC_17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC₂₀ upon maximal Ca²⁰-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm² and 5.6 mN/mm² in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax_app, extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s ¹ (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001). J. Cell. Biochem, 64:171–181. © 1997 Wiley-Liss, Inc.     

Polymorphism of white muscle myosin and parvalbumins in the genus Barbus (Teleostei: Cyprinidae)

Muscle proteins were investigated in two large European barbels, Barbus barbus and B. meridionalis , and in four small tropical barbels native to SE Asia: B. conchonius , B. tetrazona , B. sachsi and B. titteya . Polyacrylamide gel electrophoresis was used to analyse myosin heavy and light chains and parvalbumin isotypes from white trunk muscle. Each species could be biochemically identified. The myosin subunit and parvalbumin isotype patterns obtained for the two European barbels were similar. The Asian barbels, on the other hand, not only differed from the European species but displayed a greater diversity within their group. These biochemical results are largely in agreement with morphological and genetic data, but fail to substantiate suggested close relationships between Asian barbel species.     

Structure and function of the essential light chain of myosin

The review summarizes the recent data on the structure and function of the essential light chain of myosin. It is known that the essential light chain of myosin stabilizes the lever arm. Consistent with the model of the shift of the dynamic population of conformations, the conformational flexibility of the essential light chain is emphasized, which opens the way to determining its new functions. It is proposed that the interaction between the C-terminal domain of the essential light chain and the N-terminal subdomain of the heavy chain of myosin may be involved in the coupling of ATP hydrolysis and rotation of the lever arm. The recent data indicate that the isoforms of the essential light chain with the additional N-terminal peptide are capable of interacting with actin and src-homologous domain 3 of myosin. The structural aspects of these interactions and the modulatory role of the isoforms of the essential light chain of myosin are discussed.     

The complexity of expressed kappa light chains in egg-laying mammals

Complementary DNAs encoding immunoglobulin light chains were isolated from two monotreme species, Ornithorhynchus anatinus (duckbill platypus) and Tachyglossus aculeatus (echidna). The sequences of both the variable and constant regions of these clones had greater similarity to IGK than to other light chain classes and phylogenetic analyses place them squarely within the mammalian IGK group, establishing them as monotreme IGK homologues. The constant region sequences of all clones were essentially identical within each species and, along with Southern blot results, the data are consistent with a single IGKC in each species. The expressed IGKV repertoires from both platypus and echidna were randomly sampled and there appear to be at least four platypus and at least nine echidna IGKV subgroups. The IGKV subgroups are highly divergent within species, in some cases sharing as little as 57% nucleotide identity. Two of the IGKV subgroups are present in both species, so there is some degree of overlap in the germline repertoires of these two monotremes. Overall the complexity seen in platypus and echidna IGK light chains is comparable with that of other mammals considered to have high levels of germline diversity and is in contrast to what has been found so far for monotreme IGL.Electronic Supplementary Material Supplementary material is available for this article at .     

Localization and activity of myosin light chain kinase isoforms during the cell cycle

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.